Foodborne Disease Handbook, Volume 4: Seafood and Environmental Toxins

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Foodborne Disease Handbook Second Edition, Revised and Expanded Volume 4: Seafood and Environmental Toxins

edited by

Y. H. Hui Science Technology System West Sacramento, California

David Kitts University of British Columbia Vancouver, British Columbia, Canada

Peggy S. Stanfield Dietetics Resources Twin Falls, Idaho

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D E K K E R

MARCEL DEKKER, INC.

NEWYORK BASEL

ISBN: 0-8247-0344-8

This book is printed on acid-free paper. Headquarters Marcel Dekker, Inc. 270 Madison Avenue, New York. NY 10016 tell 212-696-9000; fax: 212-685-4540 Eastern Hemisphere Distribution Marcel Dekkcr AG Hutgasse 4, Postfach 812, CH-4001 Basel, Switzcrland tel: 41-61-261-8482: fax: 41-61-261-8896 World Wide Web http:llwww.dekker.com

The publisher offers discountson this book when ordered in bulk quantities. For more information, write to Special SaleslProfessional Marketing at the headquarters address above. Copyright 0 2001 by Marcel Dekker, Inc. All Rights Reserved.

Neither this book nor any part may be reproduced or transmitted in any form or byanymeans, electronic or mechanical, including photocopying, microfilming, and recording,or by any infortnain writing from the publisher. tion storage and retrieval system, without permission Current printing (last digit): 1 0 9 8 7 6 5 4 3 2 1 PRINTED IN THE UNITED STATES OF AMERICA

Introduction to the Handbook

The Foodborne Disease Handbook, Second Edition, Revised and Expanded, could not be appearing at a more auspicious time. Never before has the campaign for food safety been pursued so intensely on so many fronts in virtually every country around the world. This new edition reflects at least one of the many aspects of that intense and multifaceted campaign: namely, that research on food safety has been very productive in the years since the first edition appeared. The Handbook is now presented in four volumes instead of the three of the 1994 edition. The four volumes are composed of 86 chapters, a 22% increase over the 67 chapters of thefirstedition. Much of the information in thefirst edition has been carried forward to this new edition because that information is still as reliable and pertinent as it was in 1994. This integration of the older data with the latest research findings gives the readera secure scientific foundation on which to base important decisions affecting the public’s health. We are not so naive as to think that only scientific facts influence decisions affecting food safety. Political and economic factors and compelling national interests may carry greater weight in the minds of decision-makers than the scientific findings offered in this new edition. However,if persons in the higher levelsof national governments and international agencies, such as the Codex Alimentarius Commission, the World Trade Organization, the World Health Organization, and the Food and Agriculture Organization, who must bear the burden of decision-making need and are willingto entertain scientific findings, then the information in these four volumes will serve them well indeed. During the last decade of the previous century, we witnessed an unprecedentedly intense and varied program of research on food safety, as we have already noted. There are compelling forces driving these research efforts. The traditional food-associated pathogens, parasites, and toxins of forty years ago still continue to cause problems today, but newer or less well-known species and strains present extraordinary challenges to human health. These newer threats may be serious even for the immunocompetent, but for the immunocompromised they can be devastating. The relative numbers of the immunocompromised in the world population are increasing daily. We include here not just those also the elderly; thevery young; affected by the human inmunodeficiency virus (HIV), but the recipients of radiation treatments, chemotherapy, and immunosuppressive drugs; paiii

iV

Introduction to the Handbook

tients undergoing major invasive diagnostic or surgical procedures; and sufferers of debilitating diseases such as diabetes.To this daunting list of challenges must be added numerous instances of microbial resistance to antibiotics. Moreover, it is not yet clear how the great HACCP experiment will play out on the worldwide stage of food safety. Altruism and profit motivation have always made strange bedfellows in the food industry. It remains to be seen whether HACCP will succeed in wedding these two disparate motives into a unifying force for the benefitofall concerned-producers, manufacturers, retailers, and consumers. That HACCP shows great promise is thoroughly discussed in Volume 2, with an emphasis on sanitation in a public eating place. All the foregoing factors lend a sense of urgency to the task of rapidly identifying toxins, species, and strainsof pathogens and parasites as etiologic agents, and of determining their roles in the epidemiology and epizootiology of disease outbreaks, which are described in detail throughout the Foodborne Diseuse Handbook. It is very fortunate for the consumerthat there existsin the food industry a dedicated cadre of scientific specialists who scrutinize all aspects of food production and bring their expertise to bear on the potential hazards they know best. A good sampling of the kinds of work they do is contained in these four new volumes of the Hmdbook. And the benefits of their research are obvious to the scientific specialist who wants to learn even more about food hazards, to the scientific generalist who is curious about everything and who will be delighted to find a good sourceof accurate, up-to-date information, and to consumers who care about what they eat. We are confident that these four volumes will provide competent, trustworthy, and timely information to inquiring readers, no matter what roles they may play in the global campaign to achieve food safety.

Y. H. Hui J. Richard Gorhrrm David Kitts K. D. Murre11 Wui-Kit Nip Merle D. Pierson Syed A. Scrttar R. A. Smith David G. Spoerke, Jr. Peggy S. Stnnjield

Preface

Most people prefer to think of marine environments farthest removed from human settlements as being pristine and pure; however, much of the ocean is under attack on several fronts. One of the foci of this fourth volume of the Foodborne Disease Hundbook is the topic of pollutants, which are being deposited in the oceansin unprecedented quantitiesas raw sewage (often with viable pathogens and parasites), industrial effluents containing toxic or radioactive chemicals, trash and garbage, pesticides in runoff from crop lands, and top soil, one of our most valuable natural resources. Some of these potentially harmful organisms and chemicals enter the food webs of freshwater and marine organisms. Someof these organisms are harvested for human food and have the capacity and tendency to concentrate and sequester in their bodies many of the chemicals and pathogens present in the aquatic environments. The methods of how harmful organisms or hazardous chemicals are detected, analyzed, and identified, and how they can affect human health, are thoroughly reviewed in this volume. In contrast, some marine organisms do not collect toxins from the environment but rather produce their own toxins as a part of normal metabolic processes. When such fish are usedas human food, the result can belife-threatening.Thisvolumediscussesthe species and toxins of importance, analytical methods, and epidemiological aspects of intoxication. The seafood processor was one of the first food industries required to implement the HACCP principles. The development of the seafood HACCP program and its benefits to the consumer are discussed in this volume. A cloud of controversy hovers over the concept of food irradiation. In this volume we present informative facts needed for the reader to come to an enlightened conclusion about the safety of food irradiation. We caution that no matter how successful irradiation might be, mishandling after irradiation treatment makes the product unsafe. We also discuss the continuing need to adhere to HACCP principles and essential sanitary standards that make it possible for HACCP to work. Many, perhaps even most, of the food toxicity issues addressed in this volume are subtle and unknown to most of us. For example, the use of food additives, radioactive isotopes, pesticide chemicals applied to our crops, toxicants occurring naturally in some of our foods, and the therapeutic and growth-promoting drugs fed to domestic animals V

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Preface

are important topics of food toxicology. In addition, plasticizers i n many kinds of vessels used for food storage and handling mayin some instances be a source of toxic chemicals. Most consumers are quite unaware of rat hairs, beetle setae, and fly eggs in their food, and less aware of what effect, if any, such adulterants might have on their health. The editors and contributors to the fourth volume of the Foodborne Disease H L U I ~ book have provided an abundance of facts and supporting explanatory information enabling readers to make confident decisions about their health. Moreover, all sectors of the food industry have the tools needed to apply the sanitary practices and HACCP-driven safeguards that will result in the prevention of foodborne diseases and helpto make available wholesome foods for all consumers around the world. Volume 4 is a composite of current information and policies that enable proper risk-assessment decisions to be made regarding potential food toxicants derived naturally in the environment or through agricultural production and food processing practices.

Y. H . Hui David Kitts Peggy S. Stcrt1jieId

Contents

Itltroduction to the Handbook Pwfrtce Contributors Contents of Other Vnlurnes

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I’

Xi

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.v11 l

I. Poison Centers 1.

SeafoodandEnvironmentalToxicantExposures:TheRoleofPoisonCenters David G. Spoerke, Jr.

11.

Seafood Toxins

2.

Fish Toxins Bruce W. Hctlstertd

1

23

3. Other Poisonous Marine Animals Bruce W. Hdstead

51

4. Shellfish Chemical Poisoning L w d m E. Llewellyn

77

5. Pathogens Transmitted by Seafood

109

Russell P. Herwig

6. Laboratory Methodology for Shellfish Toxins Dcwid Kitts

183

Vii

Viii

7.

Contents

Ciguatera Fish Poisoning Yoshitsugi Hokama and Joanne S. M. YoshikLrwa-Ebesu

209

8. Tetrodotoxin Joanne S. M. Yoshikawa-Ebesu, Yoshitsugi Hokama, and Tamao Noguchi

253

9.

287

Epidemiology of Seafood Poisoning b r a E. Fleming, Dolores Katz, Judy A. Bean, and Roberta Hammond

10. The Medical Management of Seafood Poisoning Donna Glcrd Blythe. Eileen Hack, Giavanni Washington, and b r a E. Fleming

31 1

11. The U S . National Shellfish Sanitation Program

32 l

Rebecca A. Reid and Timothy D. Durance 12. HACCP, Seafood, and the U.S. Food and Drug Administration Kim R. Young, Miguel Rodrigues Kamat, and George Perry Hoskin

339

111. Environmental Toxins

13. Toxicology and Risk Assessment Donald J. Ecobichon

347

14. Nutritional Toxicology David Kitts

379

15. Food Additives Laszlo P. Somogyi

447

16. Analysis of Aquatic Contaminants Joe W. Kiceniuk

517

17. Agricultural Chemicals Debra L. Browning and Carl K. Winter

537

18. Radioactivity in Food and Water Hank Kocol

557

19. Food Irradiation Hank Kocol

57 1

20.

Drug Residues in Foods of Animal Origin Austin R. L m g and Jose E. Roybol

579

21.

Migratory Chemicals from Food Containers and Preparation Utensils Yvonne V. Yutrn

599

Contents

ix

22. FoodandHardForeignObjects:AReview J. Richard Gorham

617

23.Food,Filth,andDisease:AReview J. Richard Gor-ham

627

24.

Ides

Food FilthandAnalyticalMethodology:ASynopsis J. Richnrd Gorhnm

639

645

This Page Intentionally Left Blank

Contributors

Judy A. Bean Children's Hospital of Cincinnati, Cincinnati, Ohio,and National Institute of Environmental Health Sciences (NIEHS) Marine and Freshwater Biomedical Sciences Center, University of Miami Rosensteil School of Marine and Atmospheric Sciences, Miami, Florida

Donna Glad Blythe Department of Epidemiology and Public Health, University of Miami School of Medicine, Miami, Florida Debra L. Browning Food Safe Program, Department of Food Science and Technology, University of California-Davis, Davis, California Timothy D. Durance Food, Nutrition, and Health, University of British Columbia, Vancouver, British Columbia, Canada Donald J. Ecobichon Department of Pharmacology and Toxicology, Queen's

Univer-

sity, Kingston, Ontario, Canada

Lora E. Fleming Department of Epidemiology and Public Health, University of Miami School of Medicine, and National Institute of Environmental Health Sciences (NIEHS) MarineandFreshwaterBiomedicalSciencesCenter,University of MiamiRosensteil School of Marine and Atmospheric Sciences, Miami, Florida

J. Richard Gorham

Department of Preventive Medicine, Uniformed Services University of the Health Sciences, Bethesda, Maryland

Eileen Hack Department of Epidemiology andPublicHealth,University School of Medicine, Miami, Florida Bruce W. Halstead

of Miami

InternationalBiotoxicologicalCenter,WorldLifeResearchInsti-

tute, Colton, California

Roberta Hammond State of Florida Department of Health, Tallahassee, Florida Russell P. Herwig School of Fisheries, University of Washington, Seattle, Washington xi

xii

Contributors

Yoshitsugi Hokama Pathology Department, JohnA. Burns School of Medicine, University of Hawaii, Honolulu, Hawaii George Perry Hoskin Office of Seafood, Division of Science and Applied Technology, Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, Washington, D.C.

Miguel Rodrigues Kamat Office of Seafood, Division of Programs and Enforcement Policy, Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, Washington, D.C.

Dolores Katz Department of EpidemiologyandPublicHealth,UniversityofMiami School of Medicine,Miami,andState of FloridaDepartment ofHealth,Tallahassee, Florida

Joe W. Kiceniuk Department of Chemistry, Dalhousie University, Halifax, Nova Scotia, Canada

David Kitts

Food, Nutrition, and Health, University British Columbia, Canada

of British Columbia, Vancouver,

Hank Kocol* HealthPhysicist,Roseville,California Lyndon E. Llewellyn Australian Institute of Marine Science, Townsville, Queensland, Australia

Austin R. Long

Pacific Regional Laboratory Northwest, U.S. Food and Drug Administration, Bothell, Washington

TamaoNoguchi RebeccaA.Reid

Laboratory of Food Hygiene, Nagasaki University, Nagasaki, Japan Department of Fisheries and Oceans, Vancouver, British Columbia,

Canada

Jose E. Roybal AnimalDrugsResearchCenter,

U.S. Food and DrugAdministration,

Denver, Colorado

Laszlo P. Somogyi ConsultingFoodScientist,Kensington,California DavidG. Spoerke, Jr. BristleconeEnterprises,Denver,Colorado Giavanni Washington Department of Epidemiology and Public Health, University Miami School of Medicine, Miami, Florida

of

Carl K. Winter Department of Food Science and Technology, Universityof CaliforniaDavis, Davis, California

Joanne S. M. Yoshikawa-Ebesu Oceanit Test Systems, Inc., Honolulu, Hawaii Kim R. Young Office of Seafood, Divisionof Programs and Enforcement Policy, Center for Food Safety and Applied Nutrition,U.S. Food and Dmg Administration, Washington, D.C. Yvonne V. Yuan School of Nutrition,RyersonPolytechnicUniversity,Toronto,Ontario, Canada

* F o n w r c!ffi/itrtiorz: California Department of Health Services, Roseville, California.

Contents of Other Volumes

VOLUME 1: BACTERIAL PATHOGENS

I. Poison Centers 1.

The Role of U.S. Poison Centers in Bacterial Exposures David G. Spoerke, Jr.

11. Bacterial Pathogens 2 . Bacterial Biota (Flora) in Foods Jut1re.s M. Joy 3. Aerotmtzns hydrophiltr Carlos Aheytcr, Jr., Srmuel A. Palumbo, cltrd Gertrrd N. Steltnrr, Jr.

4. Update: Food Poisoning and Other Diseases Induced Kcrthleetl T. Rtrjkowski cwd Jcrtnes L. Smith

by Bacillus cereus

5. B r u c e l l ~ ~ Shirley M. Hullitrg nrtd Edward J. Youtrg 6.

Campylobncter jejutri Dorz A. Frtrtzco ntzd Charles E. Williatm

7.

Clostritliunr botulirwn Johtr W. Austitl erne1 Karen L. Dodds

8.

Clostridium pelfiingetzs Dorotl1.v M. Wrigley xiii

xiv

Contents of Other Voolumes

9. Eschericlritr coli Mcrrguerite A. Neil, Phillip I. Tarr, David N. Toylor, arld Mtrrcio Wolf 10. Listerirr rrrormytogerres

Cutherirre W. Dortnelly 11.

Bacteriology of Salrnorwllcr Robill C. Anderson crnd Richlrrd L. Ziprirl

12. Salmonellosis i n Animals Dmid J. Nisbet nrrd Richrrrd L. Zipritr

13. Human Salmonellosis: General Medical Aspects Richcrrd L. Zipritr trtrd Michrrel H. Hrme 14. Shigelltr

Allthotry T. Muurelli rrr~dKeith A. Lntrrpel 15.

Strrl~hyloc,occust1ureu.s Scott E. Mcrrtirr, Eric R. Myers, artd Jokrr J. I m d o l o

16. Vibrio cholertre Charles A. Kaysner rrnd June H. Wetherirlgtorl 17.

Vibrio prrrtrhtremolytic~~.s TuuTjyi Clmi t r r d Johrr L. Pace

18.

Vilwio vulnificus Arlc1er.v Dtrlsgntrr-d,Lise H@i,Debi Lirtkous, turd Jcmes D. Oliver

19.

Yersirritr Scott A. Mirlrrick, M i c h e l J. Smith, Steverr D. Wengant, mrd Peter Ferrg

111. Disease Surveillance, Investigation, and Indicator Organisms

20. Surveillance of Foodborne Disease EW[JII C. D. Todd 21.

22.

Investigating Foodborne Disease D d r L. Morse, Grrtllrie S. Birkhecrd, crrrd Jock J. Guwvich Indicator Organisms in Foods Jtrrtlrs

M. Jcry

xv

Contents of Other Volumes

VOLUME 2: VIRUSES, PARASITES, PATHOGENS, AND HACCP

I. Poison Centers 1.

The Role of Poison Centers in the United States Dmid G. Spoerke, Jr.

11. Viruses 2. Hepatitis A and E Viruses There,sa L. Cromearls, Michael 0. Favoro1~,Omnntr V. Naitrcrtr, Mtrrgo1i.s

trtld

Htrrold S.

3. Norwalk Virus and the Small Round Viruses Causing Foodborne Gastroenteritis Hrrzel Appleton

4. Rotavirus Syed A. Strttw, V. Sustrrl Spritrgthorpe, raid Jmorl A. Tetro 5.

Other Foodborne Viruses Syecl A. Sattrrr trtrci Jcrsotr A. Tetra

6. Detection of Human Enteric Viruses in Foods Lec~-A~Irr JtrYkus 7. Medical Management of Foodborne Viral Gastroenteritis and Hepatitis Su:trnne M. Matsui trnd Rtrmsey C. Cheurrg 8. Epidemiology of Foodborne Viral Infections Thomas M. Liithi

9. Environmental Considerations in Preventing the Foodborne Spread of Hepatitis A Syed A. Srrttar a t d Scrbtrh Bidawid 111. Parasites 10. Taeniasis and Cysticercosis Zbigrriew S. Pawlowski m d K. D. MLrrrell

11.

12.

Meatborne Helminth Infections: Trichinellosis William C. Crrnlpbell Fish- and Invertebrate-Borne Helminths

John H . Cross

xvi

Contents of Other Volumes

13. Waterborne and Foodborne Protozoa Rotlrrld Frryer. 14. Medical Management Prrul Pro& 15. Inmunodiagnosis of Infections with Cestodes Bruno Gottsteitl

16. Immunodiagnosis: Nematodes H . Ray Gamble

17. Diagnosis of Toxoplasmosis A I m M.Johnsow rrtld J. P. DuDey 18. Seafood Parasites: Prevention, Inspection, and HACCP Atw M.Adams n t d Debra D. DeVlieger

IV.

HACCP and the Foodservice Industries

19. Foodservice Operations: HACCP Principles 0. Peter Stlyder, Jr.

20. Foodservice Operations: HACCP Control Program 0. Peter Snyder, Jr. It1de.v

VOLUME 3: PLANT TOXICANTS

I. Poison Centers 1.

U.S. Poison Centers for Exposures to Plant and Mushroom Toxins Dovid G. Spoerke, Jr.

11. Selected Plant Toxicants

2. Toxicology of Naturally Occurring Chemicals in Food Ross C. Beier crnd Herbert N. Nigg 3. Poisonous Higher Plants Doreen Grcrce L m g crttd R. A. Smith 4. Alkaloids R . A. Smith

Contents of Other Volumes

5.

Antinutritional Factors Related to Proteins and Amino Acids It-vitl E. Liener

6. Glycosides W d t r r Mujcrk und Miclruel H . Berm

7. Analytical Methodology for Plant Toxicants Alister David Muir 8. Medical Management and Plant Poisoning Robert H. Poppetlgcr 9. Plant Toxicants and Livestock: Prevention and Management Michcrel H. R d p k s 111. Fungal Toxicants 10. Aspergillus

.&$a Kozakiewicz 11.

Clnviceps and Related Fungi Gretchen A. Kuldau and Charles W. Bacotl

12. Fusarium Walter F. 0. Murcrsrrs 13.

Perricilliutu John 1. Pitt

14.

Foodborne Disease and Mycotoxin Epidemiology Sara H d e Hen? and F. Xavier Bosclr

15.

Mycotoxicoses: The Effects of Interactions with Mycotoxins Heather A. Kosllitlsky. Adrietrtre Woytowich, crtrci George G. Kl1rrchatourirrn.s

16.

Analytical Methodology for Mycotoxins James K. Porter

17. Mycotoxin Analysis: Immunological Techniques Futl S. QILI 18.

Mushroom Biology: General Identification Features Drrvid G. Spoerke, Jr.

xvii

xviii

Contents of Other Volumes

19. Identification of MushroomPoisoning(Mycetismus),Epidemiology, and Medical Management David G. Spoerke, Jr. 20. FungiinFolkMedicineandSociety David G. Spoerke, Jr.

Seafood and Environmental Toxicant Exposures: The Role of Poison Centers

Epidemiology 1 AAPCC A. 2 B. Who staffs poison a center'? 3 C. Whattypes of callsarereceived? 4 D. How are calls handled? 5 E. What references are used'! 6 F. How are poisoncentersmonitoredfor quality? 7 G. Professional and public education programs 7 Related H. toxicology organizations 7 I. International affiliations IO J. Toxicology and poison center Web sites IO 11. PoisonInformationCenters intheUnitedStates 10 References 2 l I.

1.

EPIDEMIOLOGY

Epidemiological studies aid treatment facilities in determining risk factors and who becomes exposed, and in establishing the probable outcomes of various treatments. A few organizations have attemptedto gather such information and organizeit into yearly reports. The American Associationof Poison Control Centers (AAPCC) and some federal agencies work toward obtaining epidemiological information, while the AAPCC has an active role in assisting with the treatment of exposures Epidemiological studies assist government and industry in determining package safety, effective treatment measures, conditions of exposure, and frequency of exposure. Studies on seafood and environmental exposures provide information on the type of people most commonly involved in exposures. Are they children, adults at home, outdoorsmen, industrial workers, or blue-collar workers. Studies can also tell us which bacterial species are most commonly involved. What symptoms are seenfirst, what is the onset

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of symptoms, and are theirany sequalae may also be determined and compared to current norms. A.

AAPCC

1. What Are Poison Centers and the AAPCC? The group in the United States concerned, on a daily basis, with potential poisonings due to household agents, industrial agents, biologics, and food poisoning (including seafood poisoning) is the AAPCC. This is an affliliation of local and regional centers that provides information to health care professionals and the lay public concerning all aspects of poisoning. These centers also refer patients to treatment centers. This group of affiliated centers is often supported by government and private funds, as well as industrial sources. in the late 1950s; the first is thought to have been in Poison centers were started the Chicago area. The idea caught on quickly andat the peak of the movement there were hundreds of centers throughout the United States. Unfortunately there were few or no standards for what could be called a poison center, including the type of staff, hours of operation, or information resources. One center may have hada dedicated staff of doctors, pharmacists, and nurses trained specifically in handling poison cases, while another had nothing but a book on toxicology in the emergency room or hospital library. In 1993 the Health and Safety Code (Sec. 777.002) specified that poison centers provide 24-hour service for public and health care professionals and meet the requirements established by the AAPCC. This action helped the AAPCC standardize the staffs and activities of the various poison centers. The federal government does not fund poison centers, even though for every dollar spent on poison centers there is a savings of $2-$9 in unnecessary expenses (1,2). The federal agency responsible for the Poison Prevention Packaging Act is the U.S. Consumer Product Safety Commission (CPSC). The National Clearinghouse for Poison Control Centers initially collected data on poisonings and information on commercial product ingredients and toxic biological agents. For several years the National Clearinghouse provided product and treatment information to the poison centers. At first most poison centers were fundedby the hospital in which they were located. As the centers grew in size and the numberof calls increased, both city and state governments took on the responsibility of contributing funds. In recent years local governments have found it very difficult to fund such operations and centers havehad to look to private industry for additional funding. Government funding may take several forms, either as a line-item on a state’s budget, as a direct grant, or moneys distributed on a per call basis. Some stateswith fewer residents may contractwith a neighboring state to provide services to its residents. Some states are so populous that more than one center is funded by the state.Industrialfunding also varies,sometimes as a grant,sometimes as paymentfor handling the company’s poison and drug information-related calls,and sometimes as payment for collection of data regarding exposure to the company’s product. Every year the AAPCC reports a summary of all kinds of exposures.

2. Regional Centers The number of listed centers has dropped significantly since its peak of more than 600. Many centers have been combined into regional organizations. These regional poison centers provide poison information, telephone management, and consultation, collect pertinent data, and deliver professional and public education. Cooperation between regional poison

The

of Poison Centers

3

centers and poison treatment facilities is crucial. Regional poison information centers, in cooperation with local hospitals, should determine the treatment capabilitiesof the hospia working relationship with their analytical toxicoltals in the region and identify and have ogy, emergency and critical care, medical transportation, and extracorporeal services. This evaluation should be done for both adults and children. A“region” isusually determined by stateauthorities in conjunctionwith local health agencies and health care providers. Documentation of these state designations must be in writing unless a state chooses (in writing) not to designate any poison center or accepts a designation by other political or health jurisdictions. Regional poison information centers should serve a population base of more than one million people and must receive at least 10,000 human exposure calls per year. The number of certified regional centers in the United States is now less than 50. Certification as a regional center requires the following: Maintain a 24 hourslday, 365 dayslyear service. Provide service to both health care professionals and the public. Have available in the center at all times at least one specialistin poison information. Have on call by telephone at all times a medical director or qualified designee. Readily accessible service by telephone from all areas within the region. Comprehensive poison information resources and comprehensive toxicology information covering both general and specific aspectsof acute and chronic poisoning should be available. A list of on-call poison center specialty consultants. Writtenoperationalguidelines that provideaconsistentapproachtoevaluation, follow-up, and management of toxic exposures should be obtained and maintained. These guidelines must be approved in writing by the medical director of the program. A staff of certified professionals answering the phones (at least one of the persons on the phone has to bea pharmacist or nurse with 2000 hours and 2000 cases of supervised experience). A 24 hourdday physician (board certified) consultation service. An ongoing quality assurance program. Othercriteria,determined bythe AAPCCandestablished with membershipapproval. The regional poison information center must be an institutional lnember ingood standing of the AAPCC. Many hospital emergency rooms still maintain a toxicology reference such as the POISINDEXB system to handle routine exposure cases, but they rely on regional poison centers to handle most of the calls in their area.

B. Who Staffs a Poison Center? The staff of poison centers varies considerably from center to center. The three professional groups most often involved are physicians, nurses, and pharmacists. Who answers the phones is somewhat dependent on thelocallaborpool, moneys available, andthe types of calls being received. Other persons who answer the phone include students in medically related fields, toxicologists, and biologists. Persons responsible for answering the phones are either certifiedby the AAPCC or arein the process of obtaining the certifi-

4

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cation. Passage of an extensive examination on toxicology is required for initial certification, with periodic recertification required. Regardless of who takes the initial call, there is a medical director and other physician backup available. These physicians have specialized training or experience in toxicology and are able to provide in-depth consultations for health care professionals calling the center.

1. Medical Director A poison center medical director should be board certified in medical toxicology or in internal medicine, pediatrics, family medicine, or emergency medicine. The medical director should be ableto demonstrate ongoing interest and expertise in toxicology as evidenced by publications, research, and meeting attendance. The medical director must have a medical staff appointment at a comprehensive poison treatment facility and must be involved in the management of poisoned patients.

2. ManagingDirector The managing director must be a registered nurse, pharmacist, or physician, or hold a degree in a health science discipline. The individual should be certified by the American Board of Medical Toxicology (for physicians) orby the American Board of Applied Toxicology (for nonphysicians). The director must be able to demonstrate ongoing interest and expertise in toxicology.

3. Specialists in PoisonInformation These individuals must be registered nurses, pharmacists, or physicians, or be certified by the AAPCC as a specialist in poison information. Specialists in poison information must complete a training program approved by the nledical director and must be certified by the AAPCC as a specialist in poison information within two examination administrations of their initial eligibility. Specialists not currently certified by the associqt' L Ion must spend an annual average of no less than 16 h o u d w e e k in poison center-related activities. Specialists currently certified by the AAPCC must spend an annual average of no less than 8 hourdweek. Other poison information providers must have sufficient background to understand and interpret standard poison information resources and to transmit that information understandably to both health professionals and the public.

4. Consultants I n addition to physicians specializing in toxicology, most centers also have listsof experts by in many other fields as well. Poison center specialty consultants should be qualified training or experience to provide sophisticated toxicology or patient care information in their area(s) of expertise. In regard to seafoodor environmental toxins, this would include specialists in pesticides, heavy metals, botanical exposures, marine toxins, and hydrocarbons, just to name a few. These experts should be willing to donate their expertise in identifying and handling cases within their specialty. Most poison centers do not have the money to pay a wide variety of consultants.

C. What Types of Calls Are Received? All types of calls are received by poison centers, most of which are handled immediately, while others are referred to more appropriate agencies. Which calls are referred depends

The Role of Poison Centers

5

on the center, its expertise, and the appropriateness of a referral. Below are lists of calls which generally fall into each group Remember, there is considerable variation between poison centers; if there is doubt, call the poison center and they will tell you if your case is more appropriately referred. Poison centers do best on calls regarding acute exposures. Complicated calls regarding exposure to several agents over a long period of time which produce nonspecific symptoms are often referred to other medical specialists, to the toxicologist associated with the center, or to an appropriate government agency. The poison center will often follow-up on these cases to track outcome and the type of service given. Types of calls usually accepted Drug identification Actual acute exposure to a drug or chemical Actual acute exposure to a biological agent (plants, mushrooms, various animals) Information regarding the toxic potential of an agent Possible food poisonings (including seafood poisoning) Exposure to environmental toxins Types of calls often referred Questions regarding treatment of a medical condition (not poisoning) Questions on common bacterial, viral, or parasitic infections General psychiatric questions Proper disposal of household agents such as batteries, bleach, insecticides Use of insecticides (e.g., which insecticide to use, how to use it) unless related to a health issue (e.g., a person allergic to pyrethrins wanting to know which product does not contain pyrethrins)

1. Data Collection Records of all calls/cases handledby the center should bekept in a form that is acceptable as a medical record. The regional poison information center should submit all its human exposure data to the association’s National Data Collection System. The regional poison information center shall tabulate its experience for regional program evaluation on at least an annual basis. N. AAPCC Toxic Exposure Surveillmce Systern (TESS) In 1983theAAPCC formed TESS from the former National Data Collection System. Currently TESS contains nearly 16.2 million human poison exposure cases. Sixty-five poison centers, representing 181 3 million people, participate in the data collection. The information has various uses for both governmental agencies and industry, providing data for product reformulations, repackaging, recall, bans, injury potential, and epidemiology. The summation of each year’s surveillance is published i n the late summer or fall in the Arnericm Jolrrrlal of E~nerge~lcy Mecliciwe.

D.

How Are Calls Handled?

Most poison centers receive requests for information via the telephone. Calls come from both health care professionals and consumers. Only a few requests are received by mail or in person, these are often medicolegal or complex cases. Most centers can be reached as well as by a local number. Busy by a toll-free phone number in the areas they serve, centers have a single number that rings on several lines. Calls are often direct referrals from the 91 1 system.

Spoerke

6

Poison information specialists listen to the caller, recording the history of the case on a standardized form developed by the AAPCC. Basic information such as the agent involved, the amount of the agent, time of ingestion, symptoms, previous treatment, and currentconditionarerecorded, as well as patient information such as sex, age, phone number, who is with the patient, relevant medical history, and sometimes patient address. All information is considered a medical record, and is therefore confidential. The case is evaluated (using various references) as Infonnation only, no patient involved Harmless and not requiring follow-up a follow-up call is given Slightly toxic, no treatment necessary but Potentially toxic, treatment given at home and follow-up given to case resolution Potentially toxic, treatment may or may not be given at home, but it is necessary for the patient to be referred to a medical facility Emergency, an ambulance and/or paramedics are dispatched to the scene. Cases are usually followed until symptoms have resolved. In cases where the patient is referred to a health care facility, the receiving agency is notified. The history is relayed, toxic potential discussed, and suggestion for treatment given.

E. What ReferencesAreUsed? References used also vary from center to center, but virtually all centers use a toxicology system called POISINDEXB which contains lists of products, their ingredients, and suggestions for treatment. The systeln is compiled using medical literature and toxicology specialists throughout the world. Biological products such as plants, insects, mushrooms, and animal bites etc are handled similarly. There is an entry for each individual plant containing a description, the toxic agent present, potential toxic amounts, and so forth. The physician or poison information specialist is then referred to a treatment protocol that may be for a general class of agents; for example, exposure to malathion is referred to a protocol on organophosphate pesticides. An unknown skin irritation or potential infection would deserve a consult with an infectious disease specialist. Questions involving specific agents, such as lead or mercury, are directed to individual treatment protocols. POISINDEXB is available on microfiche, CD-ROM, over a network, or on a mainframe. It is updated every 3-months. Various texts are also used, but much of this infomation is already i n POISINDEXB. It is often difficult to identify some potentially toxic marine animals using a description given over the phone, so often the assistance of a marine biologist is used. If a type of marine food poisoning is involved, the help of an infectious disease consultant and an epidemiologist may be requested. Some poison centers have more experiencewith certain types of poisonings than do others, so often one center will consult another. These are often more complex cases, or cases involving centers in the same region. For example, a poison center in Utah may consult with one in California or Hawaii concerning a lionfish envenomization. A recent trend has been for manufacturers to contract with one poison center to provide poison information services for the whole country. Product information is given

The Role of Poison Centers

7

to only that center and exposures throughout the country can only be handled effectively in that one center.

F. How Are Poison Centers Monitored for Quality? Most poison centers havea system of peer review in place. One person takes a call, another reviews it. Periodic spot reviews are done by supervisory and physician staff. General competence is ensured by certification and recertification via examination of physicians and poison infonnation specialists.

G. Professional and Public Education Programs The regional poison information center provides information on the management of poiat soning to health professionals throughout the region. Public eduction programs aimed educating both children and adults about poison safety and potential dangers should be provided. In thepast, some centers provided stickers or logos suchasOfficer Ugh, Safety Sadie, and Mr. Yuck that could be placed on or near potentially toxic substances. While the intent was to identify potentially toxic substances that children should stay away from, the practice has been much curtailed because in some cases the stickers actually attracted children. Poison prevention week is held each yearin the spring. National attention is focused on the problem of potentially toxic exposures. During this week many centers run special programs for the public. This may include lectures on prevention, potentially toxic agents in the home, potentially toxic biological agents, or general first aid methods. Although an important time for poison centers, public and professional education is a year-round commitment. Physicians are frequently involved in medical toxicology rounds, journal clubs, and lectures by specialty consultants. Health fairs, school programs, and various men’s and women’s clubs are used to educate the public. The extent of these activities is often determined by the amount of funding from government, private organizations, and public donations.

H. Related Toxicology Organizations ACGIH American Conference of Governmentaland IndustrialHygienists Address: Kemper Woods Center; Cincinnati, OH 45240 Phone: 5 13-742-2020 FAX: 5 13-742-3355 ABAT American Board of AppliedToxicology Address: Truman Medical Center, West, 2301 Holmes St., Kansas City, MO 64 108

Phone: 8 16-556-3 1 I 2 FAX: 8 16-88 1-6282 AACT AmericanAssociation of ClinicalToxicologists Address: c/o Medical Toxicology Consultants, Four Columbia Dr., Suite810, Tampa, FL 33606 AAPCC AmericanAssociation of PoisonControlCenters

8

Spoerke

Address: 3201 New Mexico Ave. NW, Washington, DC 20016 Phone: 202-362-72 17 FAX. 202-362-8377 ABEM AmericanBoard of EmergencyMedicine Address: 300 Coolidge Rd., East Lansing, MI 48823 Phone: 5 17-332-4800 FAX. 5 11-332-2234 ACEP American College of Emergency Physicians (Toxicology Section) Address: P.O. Box 6 I99 1 I , Dallas, TX 7526 1-991 1 Phone 800-798- 1822 FAX: 214-580-2816 ACMT American College of Medical Toxicology (formerly ABMT) Address: 777 E. Park Dr., P.O. Box 8820, Harrisburg, PA 17105-8820 Phone: 717-558-7846 FAX: 7 17-558-7841 e-mail: [email protected] (Linda L. Koval) ACOEM AmericanCollege of OccupationalandEnvironmentalMedicine Address: 55 West Seegers Rd., Arlington Heights, IL 60005 Phone: 708-228-6850 FAX: 708-228- 1856 ACS Association of ClinicalScientists Address: Dept. of Laboratory Medicine, University of Connecticut Medical School, 263 Fannington Ave., Farmington, CT 06030-2225 Phone: 203-679-2328 FAX: 203-679-2328 ACT AmericanCollege of Toxicology Address: 9650 Rockville Pike, Bethesda, MD 20814 Phone: 301 -57 1 - 1 840 FAX: 301-571-1852 AOEC Association of OccupationalandEnvironmentalClinics Address: IO10 Vermont Ave. NW, #513, Washington, DC 20005 Phone: 202-347-4976 FAX: 202-347-4950 e-mail: [email protected] ASCEPT AustralianSociety of ClinicalandExperimentalPharmacologistsand Toxicologists Address: 145 Macquarie St., Sydney N.S.W 2000 Australia Phone: 61-2-256-5456 FAX: 6 1-2-252-33I O BTS BritishToxicologySociety Address: MJ Tucker,ZenecaPharmaceuticals,22B 1 1 Mareside,Alderley Park, Macclesfield, Cheshire, SKI0 4TG UK Phone: 0428 65 5041 CAPCC CanadianAssociation of PoisonControlCenters Address: Hopital Sainte-Justine, 3 175 Cote Sainte-Catherine, Montreal, Quebec H3T 1 C5, Canada Phone: 5 14-345-4675 FAX: 5 14-345-4822

The Role of Poison Centers

9

CSVVA (CEVAP) Center for the Study of Venoms and Venomous Animals Address: UNESP, Alarneda Santos, N 647, CEP 01419-901, Sa0 Paulo. sp, Brazil Phone 55-0 1 1-252-0233 FAX: 55-01 1-252-0200 EAPCCT EuropeanAssociation of PoisonControlCenters Address: J. Vale, National Poisons Infornlation Centre, P 0 Box 81898 Dep, N-0034 Oslo, Norway Phone: 47-260-8460 HPS HungarianPharrnacologicalSociety Address: Central Research Institute for Chemistry, Hungarian Academy of Sciences, H- 1525 Budapest, P.O. Box 17, Pusztaszeri ut 59-67 Phone: 36- 1- 135-21 12 ISOMT International Society of Occupational Medicine and Toxicology Address:USCSchool of Medicine,222OceanviewAve.,Suite 100, Los Angeles, CA 90057 Phone: 213-365-4000 JSTS JapaneseSociety of ToxicologicalSciences Address: Gakkai Center Building, 4-16, Yayoi 2-chome, Bunkyo-ku, Tokyo 113, Japan Phone: 3-38 12-3093 FAX: 3-3812-3552 SOT Society of Toxicology Address: 1101 14th St., Suite 1100, Washington, DC 20005-5601 Phone: 202-371 - 1393 FAX: 202-37 1- 1090 e-mail: [email protected] SOTC Society of Toxicology of Canada Address: P.O. Box 517, Beaconsfield, Quebec H9W 5V1, Canada Phone: 5 14-428-2676 FAX: 5 14-482-8648 STP Society of ToxicologicPathologists Address: 875 Kings Hw., Suite 200, Woodbury, NJ 08096-3172 Phone: 609-845-7220 FAX: 609-853-041 1 SSPT Swiss Society of PharmacologyandToxicology Address: Peter Donatsch, Sandoz Pharma AG, Toxicologue 88 1/130, CH4132 Muttenz, Switzerland Phone: 41-61-469-5371 FAX: 41-6 1-469-6565 WFCT WorldFederation of Associations of ClinicalToxicologyCenters and Poison Control Centers Address:CentreAnti-Poisons,HopitalEdonardHerriot, 5 p1 d’Arsonva], 69003 Lyon, France Phone: 33 72 54 80 22 FAX: 33 72 34 55 67

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10

1.

InternationalAffiliations

The AAPCC and its members attend various world conferences to learn of toxicology problems and new methods used by other agencies. An especially close relationship has formed between the American and Canadian poison center associations. Once a year the AAPCC and CAPCC hold a joint scientific meeting and invite speakers and other toxicology specialists from throughout the world to attend.

J. Toxicology and Poison Center Web Sites Association of Occupational ctnd Environmental Clinics: This group is dedicated to higher standards of patient-centered, multidisciplinary care emphasizing prevention and total health through information sharing, quality service, and collaborative research. Address: [email protected] Finger Lrrkes Regional Poison Center. Address: [email protected] Mediccll/Clinical/Occupntional Toxicology Professionctl Groups A list of primarily U.S. professional groups interested in toxicology. There is a description of each group, addresses, phone numbers, and contact names. Keyword: poison centers, toxicology Address: http://www.pitt.edu/-martint/pages/motoxorg.htm Poison Net A mailing list dedicated to sharing information, problem solving, and networking in the areas of poisoning, poison control centers, hazardous materinot als, and related topics. The list is intended for health care professionals, the lay public. The moderators do not encourage responses to individual poisoning cases from the public. Keywords: poisoning, poison control centers

II. POISON INFORMATION CENTERS IN THE UNITED STATES The Poison Control Center telephone numbers and addresses listed below are thought to be accurate as of the date of publication. Poison Control Center telephone numbers and addresses may change. The address and phone number of the Poison Control Center nearest you should be frequently checked.If the number listed doesnot reach thepoison center, contact the nearest emergency service, such as9 l 1 or your local hospital emergency room. The author disclaims any liability resulting from or relating to any inaccuracies or changes in the phone numbers provided below. This information should NOT be used as a substitute for seeking professional medical diagnosis, treatment, and care. (* Indicates a regional center designated by the American Association of Poison Control Centers.)

11

The Role of Poison Centers ALABAMA

ARKANSAS

Birrningharrr

Little Rock

Regional Poison Control Center* Children's Hospital of Alabama 1600 Seventh Ave., South Birmingham, AL 35233-171 1 800-292-6678 (AL only) 205-933-4050

Arkansas Poison and Drug Information Center University of Arkansas College of Pharmacy 4301 West Markham, Slot 522 Little Rock, AR 77205 800-482-8948 (AR only) 501-661-6161

Tusccrloosu

Alabama Poison Control System, Inc. 408A Paul Bryant Dr. East Tuscaloosa, AL 3540 1 800-462-0800 (AL only) 205-345-0600 ALASKA Arlchorqe

Anchorage Poison Center Providence Hospital P.O. Box 196604 3200 Providence Dr. Anchorage, AK 995 19-6604 800-478-3193 (AK ody) Ftrirbartks

Fairbanks Poison Center Fairbanks Memorial Hospital 1650 Cowles St. Fairbanks, AK 99701 907-456-7 I X2 ARIZONA Pkoetl~s

Samaritan Regional Poison Center'@ Good Samaritan Medical Center 1130 East McDowell Rd., Suite A-5 Phoenix, AZ 85006 602-253-3334 7irc.son

Arizona Poison and Drug Information Center* Arizona Health Sciences Center, Room 1 l56 1501 N. CampbellAve. Tucson, AZ 85724 800-362-0101 (AZ only) 602-626-6016

CALIFORNIA Fresrlo

Frcsno Regional Poison Control Center" Fresno Community Hospital and Medical Center 2823 Fresno St. Fresno, CA 93721 800-346-5922 (CA 011ly) 209-445- 1222 Los Angeles

Los Angeles County University of Southern California Regional Poison Center* 1200 North State. Room I 107 Los Angeles, CA 90033 800-825-2722 2 13-222-3212 Orarlge

University of California lrvine Medical Center Regional Poison Center* 101 The City Dr. South Route 78 Orange, CA 92668-3298 800-544-4404 (CA only) 7 14-634-5988 Richmorld

Chevron Emergency Information Center 15299 San Pablo Ave. P 0. Box 4054 Richmond, CA 94804-0054 800-457-2202 510-233-3737 or 3738

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72 S~lc.rtrrnento

Regional Poison Control Center* University of California at Davis Medical Center 2315 Stockton Blvd.. Rm. HSF-124 Sacramento. CA 95817 800-342-3293 (northern CA only) 9 16-734-3692 Scrn Diexo

San Diego Rcgional Poison Center* University of California at San Diego Medical Center 225 Wcst Dickinson St. San Diego, CA 92013-8925 800-876-4766 (CA only) 6 19-543-6000 Son Frt~nci.sc.o

San Francisco Bay Area Poison Center* San Francisco General Hospital 1001 PotreroAve., Rm. IE86 San Francisco, CA 94122 800-523-2222 4 15-476-6600 Son Jose

San Jose Regional Poison Center Santa Clara Valley Medical Center 751SouthBascomAve. San Jose, CA 95128 800-662-9886, 9887 (CA only) 408-299-51 12,51 13, 51 14 COLORADO Den\vr

Rocky Mountain Poison Center* 1010 Yosernite Circle Denver. CO 80230 800-332-3073 (CO only) 303-629- 1 123 CONNECTICUT

DELAWARE Wilnrington

Poison Information Center Medical Center of Delaware Wilmington Hospital 501 West14thSt. Wilmington, DE19899 302-655-3389

DISTRICT OF COLUMBIA Wt~S1Iill~tOll

National Capital Poison Center* Georgetown University Hospital 3800 Reservoir Rd. NW Washington, DC 20007 202-625-3333

FLORIDA Jtrcksonville

Florida Poison Information Center University Medical Center 655 West Eighth St. Jacksonville, FL 32209 904-549-4465 or 764-7667 Tnllohtrssee

Tallahassee Memorial Regional Medical Center 1300 Miccosukk Rd. Tallahassee, FL 32308 904-68 1-541 1 Tu~npo

Tampa Poison Information Center* Tampa General Hospital Davis Islands P.O. Box1289 Tampa, FL 33601 800-282-3171 (FL only) 813-253-4444

~tJ'Llllill,~fOII

Connecticut Poison Control Center University of Connecticut Health Center 263 Farmington Ave. Farmington, CT 06030 800-343-2722 (CT only) 203-679-3456

GEORGIA Atlontcr

Georgia Regional Poison Control Center* Cerady Memorial Hospital

13

The Role of Poison Centers

80 Butler St. SE Box 26066 Atlanta, C A 30335-3801 800-282-5846 (CA only) 404-61 6-9000 Macon

Regional Poison Control Center Medical Center of Central Georgia 777 Hemlock St. Macon, CA 3 I208 912-744-1 146, 1100, 1427 Savannah

Savannah Regional Poison Control Center Memorial Medical Center Inc. 4700 Waters Ave. Savannah, GA 31403 912-355-5228 or 356-5228

Chicago, IL 606 12 800-942-5969 (Northeast IL only) 3 12-942-5969 Normal

Bronlenn Hospital Poison Center Virginia at Franklin Normal, IL 61761 309-454-6666 Springfield

Central and Southern Illinois Poison Resource Center St John’s Hospital 800 East Carpenter St. Springfield, IL 62769 800-252-2022 (IL only) 217-753-3330 Urlmna

HAWAII

Honolulu

Kapiolani Women’s and Children’s Medical Center 1319 Punahou St. Honolulu, HI 96826 800-362-3585, 3586 (HI only) 808-941 -4411

IDAHO Boi.W?

Idaho Poison Center St Alphonsus Regional Medical Center 1055 North Curtis Rd. Boise, ID 83706 800-632-8000 (ID only) 208-378-2707

National Animal Poison Control Center University of Illinois Department of Veterinary Biosciences 2001 South Lincoln Ave., 1220 VMBSB Urbana, IL 61801 800-548-2423 (Subscribers only) 217-333-2053

INDIANA

Indinnapolis

Indiana Poison Center* Methodist Hospital 1701 North Senate Blvd. Indianapolis, IN 46202-1 367 800-382-9097 3 17-929-2323

IOWA ILLINOIS Chiccrgo

Chicago and NE Illinois Regional Poison Control Center Rush Presbyterian-St. Luke’s Medical Center 1653 West Congress Pky.

Des Moines

Variety Club Drug and Poison Information Center Iowa Methodist Medical Center 1200 Pleasant St. Des Moines, IA 50309 800-362-2327 5 15-24 1-6254

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14 IOM'U

Cih

University of Iowa Hospitals and Clinics 200 Hawkins Dr. Iowa City, IA 52246 800-272-6477 or 800-362-2327 (IA only) 3 19-356-2922 Cih St. Luke's Poison Center St. Luke's Regional Medical Center 2720 Stone Park Blvd. Sioux City, IA 51 104 800-352-2222 (IA, NE, SD) 712-277-2222

Si0lt.X

KANSAS

Lolii.s\~ille

Kentucky Poison Control Center of Kosair Children's Hospital 315 East Broadway P.0 Box 35070 Louisville, KY 40232 800-722-5725 (KY only) 502-589-8222

LOUISIANA Hou~ncc

Terrebonne General Medical Center Drug and Poison Information Center 936 East Main St. Houma, LA 70360 504-873-4069

Karrscrs C i h

Monroe

Mid America Poison Center Kansas University Medical Center 39th and Rainbow Blvd., Rm. B-400 Kansas City, KS 66160-7231 800-332-6633 (KS only) 9 13-588-6633

Louisiana Drug and Poison Information Center Northeast Louisiana University School of Pharmacy, Sugar Hall Monroe, LA 7 1209-6430 800-256-9822 (LA only) 3 18-362-5393

7opeka

Stormont Vail Regional Medical Center Emergency Department 1500 West 10th Topeka, KS 66604 9 13-354-6100 Wicllitcl

Wesley Medical Center 550 North Hillside Ave Wichita, KS 67214 3 16-688-2222

MAINE

Portlnnd

Maine Poison Control Center Maine Medical Center 22 Bramhall St. Portland, ME 04102 800-442-6305 (ME only) 207-87 1-2950

MARYLAND KENTUCKY Baltinrore

Ft. Tllovra.7

Northern Kentucky Poison Information Center St Luke Hospital 85 North Grand Ave. Ft. Thomas, KY 41075 5 13-872-51 1 1

Maryland Poison Center* University of Maryland School of Pharmacy ' 20 North Pine St. Baltimore, MD 21201 800-492-2414 (MD only) 410-528-7701

75

The Role of Poison Centers MASSACHUSElTS Boston

St. Pa111

Center

Massachusetts Poison Control System* The Children’s Hospital 300 1222 800-222Longwood Ave. Boston, MA 021 15 800-682-9211 (MA only) 617-232-2120 or 735-6607

Minnesota1 Regional Poison Center* Medical St Paul-Ramsey 640 Jackson St. St Paul, MN 55101 (MN only) 612-221-2113

MISSISSIPPI MICHIGAN Adriurr

Bixby Hospital Poison Center Emma L. Bixby Hospital 818 Riverside Ave. Adrian, MI 4922 1 517-263-2412 Detroit

Poison Control Center Children’s Hospital of Michigan 3901 Beaubien Blvd. Detroit, MI 48201 800-462-6642 (outside metropolitan Detroit) 3 13-745-571 1

Juck~ort

University of Mississippi Medical Center 2500 North State St. Jackson, MS 39216 60 1-354-7660 Huttieshrg

Forrest General Hospital 400 S 28th Ave. Hattiesburg, MS 39402 601-288-4235

MISSOURI Kcrrtsus City

Blodgett Regional Poison Center 1840 Wealthy St. S.E. Grand Rapids, MI 49506 800-632-2727 (MI only)

Poison Control Center Children’s Mercy Hospital 2401 Gillharn Rd. Kansas City, MO 64108-9898 8 16-234-3000 or 234-3430

Kolonltrzoo

St. Louis

Grmd Rupids

Bronson Poison Information Center 252 East Lovell St. Kalamazoo, MI 49007 800-442-41 12616 (MI only) 6 16-341 -6409

Regional Poison Center* Cardinal Glennon Children’s Hospital 1465 South Grand Blvd. St. Louis, MO 63104 800-392-9111 (MO only) 800-366-8888 (MO, West IL) 3 14-772-5200

MINNESOTA Minneupolis

Hennepin Regional Poison Center* 701ParkAve. S. Minneapolis, MN 55415 612-347-3144 612-347-3141 (Petline)

MONTANA Derwer

Rocky Mountain Poison and Drug Center Denver, CO 80204 800-525-5042 (MT only)

16

Spoerke

NEBRASKA

NEW MEXICO

Olll(1hLI

Albuquerque

The Poison Center* Children's Memorial Hospital 8301Dodge St. Omaha, NE 681 14 800-955-9119 (WY, NE) 402-390-5400, 5555

New Mexico Poison and Drug Information Center* University of New Mexico Albuquerque, NM 87 13I 800-432-6866 (NM only) 505-843-2551

NEVADA NEW YORK Lcrs Vrp1.s

Hurnana Hospital-Sunrise" 3 l86 Maryland Pky. Las Vcgas, NV 89109 800-446-6I79 (NV only) RPilO

Washoe Medical Center 77 Pringle Way Rcno, NV 89520 702-328-4 144 NEW HAMPSHIRE Lellailol~

New Hampshire Poison Center Dartmouth-Hitchcock Medical Center 1 Medical Center Dr. Lebanon, NH 03756 800-562-8236 (NH only) 603-650-5000 NEW JERSEY

Newcrrk New Jersey Poison Information and Education Systems* 201 LyonsAve. Newark, NJ 071 12 800-962-1253 (NJ only) 20 1 -923-0764 Phi1lip.shur.g

Warren Hospital Poison Control Center 185 Rosberg St. Phillipsburg. NJ 08865 800-962- 1253 008-859-6768

Buffalo

Western New York Poison Control Center Children's Hospital of Buffalo 219 Bryant St. Buffalo, N Y 14222 800-888-7655 ( N Y only) 7 16-878-7654 Mineolu

Long Island Regional Poison Control Center" Winthrop University Hospital 259 First St. Mineola, NY 11501 5 16-542-2323, 2324, 2325 New York C i v

Ncw York City Poison Control Center* 455 First Ave., Rm. 123 New York, NY 10016 2 12-340-4494 2 12-764-7667 Npck Hudson Valley Regional Poison Center Nyack Hospital 160 North Midland Ave. Nyack, NY 10920 800-336-6997 (NY only) 914-353-1000 Rochester

Finger Lakes Regional Poison Control Center University of Rochester Medical Center 601 Eltnwood Ave. Rochester, NY 14642 800-333-0542 (NY only) 7 16-275-515 1

17

The Role of Poison Centers Syrmuse

DAKOTA

Central New York Poison Control Center SUNY Health Science Center 750 E Adams St. Hospital Luke’s St NY 13210 Syracuse, 800-252-5655 Fargo, 3 15-476-4766

NORTH CAROLINA Ashevillc

Western North Carolina Poison Control Center Memorial Mission Hospital 509 Biltmore Ave. Asheville, NC 28801 800-542-4225 (NC only) 704-255-4490 or 258-9907 Chcrrlotte

Carolinas Poison Center Carolinas Medical Center 100 Blythe Blvd. Charlotte, NC 28232-2861 800-848-6946 704-355-4000 Durlzom

Duke Regional Poison Control Center P.O. Box3007 Durham, NC 27710 800-672-1697 (NC only) 919-684-811 1 Greensboro Triad Poison Center Moses H Cone Memorial Hospital 1200 North Elm St. Greensboro, NC 2740 l - 1020 800-953-4001 (NC only) 919-574-8105

NORTH

F0 rg o Poison Dakota North

Center

720 North 4th St. ND 58122 800-732-2200 (ND only) 701-234-5575

OHIO Akron

Akron Regional Poison Center 281 Locust St. Akron, OH 44308 800-362-9922 (OH only) 216-379-8562 C~lrlron

Stark County Poison Control Ccnter Timken Mercy Medical Center 1320 Timken Mercy Dr. NW Canton, OH 44667 800-722-8662 (OH only) 2 16-489- 1304 Cincinrlari

South West Ohio Regional Poison Control System and Cincinnati Drug and Poison Information Center* University of Cincinnati College of Medicine 231 Bethesda Ave., ML #l44 Cincinnati, OH 45267-0144 800-872-51 1 1 (Southwest OH only) 5 13-558-51 1 1 Cleveland

Greater Cleveland Poison Control Center 2074 Abington Rd. Cleveland, OH 44106 2 16-23 1-4455

Hickory

Colundm.7

Catawba Memorial Hospital Poison Control Center 810 Fairgrove Church Rd. SE Hickory, NC 28602 704-322-6649

Central Ohio Poison Center* 700 Children’s Dr. Columbus, OH 43205 800-682-7625 (OH only) 614-228-1323

Spoerke

18 Dayton

West Ohio Regional Poison and Drug Information Center Children’s Medical Center One Children’s Plaza Dayton, OH 45404- 18 15 800-762-0727 (OH only) 5 13-222-2227 Loruin

County Poison Control Center Lorain Community Hospital 3700 Kolbe Rd. Lorain, OH 44053 800-821-8972 (OH only) 216-282-2220

940 NE 13th St. Oklahoma City, OK 73 104 800-522-4611 (OK only) 405-27 1-5454

OREGON Portland

Oregon Poison Center Oregon Health Sciences University 3 181 SW Sam Jackson Park Rd. Portland, OR 97201 800-452-7165 (OR only) 503-494-8968

Sandusky

Firelands Community Hospital Poison Information Center 1101 Decatur St. Sandusky, OH 44870 419-626-7423 Toledo

Poison Information Center of Northwest Ohio Medical College of Ohio Hospital 3000 Arlington Ave. Toledo, OH 49614 800-589-3897 (OH only) 419-381-3897 Yuungsfown

Mahoning Valley Poison Center St Elizabeth Hospital Medical Center 1044 Belmont Ave. Youngstown, OH 44501 800-426-2348 (OH only) 21 6-746-2222 Zanesville

Bethesda Poison Control Center Bethesda Hospital 2951 Maple Ave. Zanesville, OH 43701 800-686-4221 (OH only) 614-454-4221 OKLAHOMA

PENNSYLVANIA Hershey

Central Pennsylvania Poison Center* Milton Hershey Medical Center Pennsylvania State University P.O. Box 850 Hershey, PA 17033 800-521-6110 717-531-611 1 Lancaster

Poison Control Center St. Joseph Hospital and Health Care Center 250 College Ave. Lancaster, PA 17604 7 17-299-4546 Philadelphia

Philadelphia Poison Control Center” One Children’s Center 34th and Civic Center Blvd. Philadelphia, PA 19104 215-386-2100 Pittsburgh

Pittsburgh Poison Center* One Children’s Place 3705 Fifth Ave. at DeSoto St. Pittsburgh, PA 15213 41 2-68 1-6669

Oklahonlu C i h

Williamsport

Oklahoma Poison Control Center Children’s Memorial Hospital

The Williamsport Hospital Poison Control Center

79

The Role of Poison Centers

777 Rural Ave. Williamsport, PA 1770I 717-321-2000

RHODE ISLAND

800 East 21st St. P.O. Box 5045 Sioux Falls, SD 57 117-5045 800-952-0123 (SD only) 800-843-0505 (IA, MN, NE) 605-336-3894

Providence

Rhode Island Poison Center* 593 Eddy St. Providence, RI 02903 40 144-5727

SOUTH CAROLINA Charlotte

Carolinas Poison Center Carolinas Medical Center 1000 Blythe Blvd. Charlotte, NC 28232-2861 800-848-6946 Cohonhia

Palmetto Poison Center University of South Carolina College of Pharmacy Columbia, SC 29208 800-922-11 17 (SC only) 803-765-7359

SOUTH DAKOTA Aberdeen

Poison Control Center St Luke’s Midland Regional Medical Center 305 S. State St. Aberdeen, SD 57401 800-592-1889 (SD, MN, ND, WY) 605-622-5678 Rapid Cif?, Rapid City Regional Poison Control Center 835 Fairmont Blvd. P.O. Box 6000 Rapid City, SD 57709 605-341-3333 Sioux Fcrlls McKennan Poison Center McKennan Hospital

TENNESSEE Knoxville

Knoxville Poison Control Center University of Tennessee Memorial Research Center and Hospital 1924 Alcoa Hwy. Knoxville, TN 37920 615-544-9400 Memphis

Southern Poison Center, Inc. Lebanheur Children’s Medical Center 848 Adams Ave. Memphis, TN 38103-2821 901 -528-6048 Nashville

Middle Tennessee Regional Poison Center, Inc. 501 Oxford House 1161 21st Ave. S, B-IOIVUII Nashville, TN 37232-4632 800-288-9999 (TN only) 615-322-6435

TEXAS Conroe

Montgomery County Poison Information Center Medical Center Hospital 504 Medical Center Blvd. Conroe, TX 77304 409-539-7700 Dallas

North Central Texas Poison Center* Parkland Memorial Hospital 5201 Harry Hines Blvd. P.O. Box 35926 Dallas, TX 75235 800-441-0040 (TX only) 214-590-5000

Spoerke

20

El Paso El Paso Poison Control Center

Thomas General Hospital 4815 Alameda Ave. El Paso, TX 79905 915-533-1244 Gnlveston

Texas State Poison Control Center University of Texas Medical Branch 8th and Mechanic St. Galveston, TX 77550-2780 800-392-8548 (TX only) 7 13-654-1701 (Houston) 409-765-1420 (Galveston) Lubbock

Methodist Hospital Poison Control 3615 19th St. Lubbock, TX 79413 806-793-4366

Box 67 Charlottesville, VA 22901 800-45 1- 1428 (VA only) 804-924-5543 Richmond

Virginia Poison Center Virginia Commonwealth University MCV Station Box 522 Richmond, VA 23298-0522 800-552-6337 (VA only) 804-786-9123

WASHINGTON Washington

P.O. Box 5371 Seattle, WA 98105-0371 800-732-6985 (Within WA) 206-526-2121

UTAH Salt Lake C i h

Utah Poison Control Center* Intermountain Regional Poison Control Center 410 Chipeta Way, Suite 230 Salt Lake City, UT 84108 800-456-7707 (UT only) 801-581-2151

VERMONT Burlington

Vermont Poison Center Medical Center Hospital of Vermont 11 1 Colchester Ave. Burlington, VT 05401 802-658-3456

WEST VIRGINIA Chcrrleston

West Virginia Poison Center* West Virginia University 31 I O MacCorkle Ave. SE Charleston, WV 25304 800-642-3625 (WV only) 304-348-4211 Porkershurg

St. Joseph’s Hospital Center 19th St. and Murdoch Ave. Parkersburg, WV 26 101 304-424-4222

WISCONSIN VIRGINIA Cl~trlorte.sville

Blue Ridge Poison Center* University of Virginia Health Sciences Center

Mndisorr

Regional Poison Control Center University of Wisconsin Hospital 600 Highland Ave. Madison, WI 53792 608-262-3702

21

The Role of Poison Centers Milwaukee

Poison Center of Eastern Wisconsin Children’s Hospital of Wisconsin Poison Wisconsin The West Ave. 9000 Hospital Memorial Children’s Box 1997 P.O. Milwaukee, Dodge8301 W1 53201 414-266-2222

WYOMING Omoha

Center” St.

681 Omaha, NE 14 800-955-91 19 (WY, NE) 402-390-5400. 5555

REFERENCES DL Harrison, JR Draugalis, MK Slack, PC Langly. Cost effectiveness of regional poison control centers.ArchlnternMed156:2601-2608,1996 2. CPSC. CPSC Chairman Ann Brown suggests information technology study to support work of poison ccnters. News release 94-047, March 15, 1994. 1.

This Page Intentionally Left Blank

2 Fish Toxins

I. 11.

111.

1V.

V. VI. VI1. VIIl.

1.

Introduction 23 Toxigcnesis 24 DystrophicationandToxigenesis 24 Icthyosarcotoxic Fish 25 A. Lampreys and hagfish: cyclostome fish 25 B. Sharks,skates,rays,andchimaeras:elasmobranchfish C. Ciguatoxic fish 28 D. Clupeotoxic fish 33 E. Gempylotoxic fish 35 Scombrotoxic F. fish 35 G. Tetrodotoxic 37 fish Icthyootoxic Fish 40 Icthyohcmotoxic Fish 42 Icthyohepatotoxic Fish 43 Icthyoallyeinotoxic(Hallucinogenic)Fish 44 Acknowledgment 45 References 45

26

INTRODUCTION

Fish toxins are of two types: the small, molecular oral biotoxins that are poisonous to eat, and the large molecular venoms that are injected into the body by means of a specialized device known as the venom apparatus. Thus all venoms are poisons, but not all poisons are venoms. In this chapter, only the oral biotoxins are discussed in regard to the names of thetransvectors,theirgeographicaldistribution,clinicalcharacteristics,andabrief statement of the toxin involved. Reports of food poisoningof marine origin are increasing in frequency and outbreaks appear to be spreading geographically. Part of this increase may be credited to heightened awareness, more travel to areas of the world where marine toxicity is endemic, and a greater opportunity for exposure to oral fish toxins, There also has been an increase in

23

Halstead

24

the importation of toxic marine food products into North America, Europe, Russia, Taiwan, Japan, and elsewhere. However, travel and awareness are only two facets of the overall epidemiology of the marine food biotoxication problem. There is evidence that suggests that pollution may be an added factor that needs to be taken into consideration.

II. TOXIGENESIS Toxicity in marine organisms is the result of a progression of biochemical events taking place in the bodyof the target organism. Various combinations of atoms of carbon, hydrogen, oxygen, nitrogen, chlorine, sulfur, and phosphorus are synthesized by the organism into complex biotoxin molecules thatmay have extreme complexity and toxicity. The process by which this is accomplished is referred to as biogenesis, biosynthesis, or, more specifically, as toxigenesis. In actuality, very little is known about the precise chemical processes involved. There is a growing amountof chemical data that suggests that in some instances marine bacteria play a role in toxin biosynthesis. Oral marine biotoxins may develop in the bodies of marine organisms as a result of naturallyoccurringprecursorchemicalagents, or theymay develop as a resultof human-induced chemical pollutants. In either case, the resulting biotoxins are capable of producing serious public health problems. These problems may occur in endemic areas wherever marine biotoxins exist or in far-removed areas to which toxic marine products have been transported. The increase of marine biotoxications worldwide are of grave economic and public health significance.

111.

DYSTROPHICATION AND TOXIGENESIS

There is an aspect of ecotoxicology that appears to have a bearing on the topic of foodborne marine intoxicants, that is, the matter of ocean eutrophication and its resultant dystrophication due to pollution. The process of ocean eutrophication is a phenomenon that has been well documented (1 - 3 ) . Eutrophication is a process of nutrient enrichment involving ocean ecosystems. Dystrophication generally is looked upon as a posteutrophication process in which there is oxygen depletion resulting from the actionof aerobic bacteria upon organic matter accompanied by other poorly defined chemical alterations in the marine environment. These two processes generally are looked upon as an aging activity in a body of water. A vast array of chemical agents, military and industrial pollutants, pesticides, and heavy metals are entering the marine environment and contributing to the eutrophication process. This ocean enrichment process is takingon global proportions and is of growing concern to marine toxicologists. More detailed information on this subject has been published in Ref. 4. Current evidence suggests thatthe combined onslaught of all of these chemical substances entering the ocean environment undoubtedly contributes to the degradative enrichment process. This involves a series of chemical and physical vector forces that presently appearto defy analysis. The ever-increasing chemical contamination of the ocean environment strongly suggests that the growing number of outbreaks of oral intoxications may be related events.

Fish Toxins

25

The bacterial degradation of organic matter by proteolytic bacteria produces a decrease in dissolved oxygen and may increase the growth of sulfate bacteria and the production of hydrogen sulfate and sulfur. Pollutants of various types may upset the phytoplanktoniccycleandaltertheworkofchemicalmediators,resulting ineutrophicationand dystrophication. All of these factors may cause an increasein toxic phytoflagellate blooms and various forms of bacterial toxigenesis. Yasumoto et al. (5,6)and Noguchi etal. (7) reported that the bacterial Psc~1rrlorr1orrcrs and Vibrio species were found in association with toxic pufferfish and toxic phytoflagellates. The investigators concluded that the bacteria were responsible for the production of tetrodotoxin and saxitoxin. Kotaki et al. (8) isolated the bacteriunl Mornellrr species and concluded that it was responsible for the biosynthesis of saxitoxin in cultures of the toxic dinoflagellate P r [ ) t ~ ) ~ o t ~ (Gorryauleu) ~ ~ l l l ~ r . ~ tcrn~crrensisis. The studies cited above provide substantial evidence that microbial organistus are responsible for the production of such toxins as tetrodotoxin, saxitoxin, and some of their congeners. It is possible that the ciguatoxin complex, palytoxin, and probably other marine toxins yet to be identified may also be the products of bacterial activity acting in association with a variety of marine plants and animals (5,6,9-21); Aubert feels that it is difficult to explainthetoxigenesis of phytoplankton by any other means than by bacteria (M. Aubert, personal communication, 1991). Bacterial toxigenesis has now become an area of major epidemiological concernin dealing with outbreaksof organic marine biotoxications. There are various waysof classifying food-borne outbreaks resulting from the ingestion of toxic fishes. Here the outbreaks are arranged phylogenetically accordingto the fish transvectors involved. All of the fish presented in the sections below are members of the phylum Chordata.

IV.

ICHTHYOSARCOTOXIC FISH

Ichthyosarcotoxic fish are those that contain a poison within the flesh of the fish (i.e., the musculature, viscera, skin, or slime) which, when ingested, causes a biotoxication. This category should not be confused with that which causes ichthyootoxism, in which the poison is restricted to the gonads or roe of the fish.

A.

LampreysandHagfish:CyclostomeFish

The cyclostome fish are members of the class Agnatha. The cyclostomes, which include thelampreysand hagfish,are a group of fishlikevertebrateshavinganeel-likeform, cartilaginous or fibrous skeleton, no definite jaws or bony teeth, and a primitive type of cranium. There are no pelvic girdles, paired limbs or true ribs. There are 6-14 pairs of gill pouches opening either directly into the pharynx or into a separate respiratory tube. Only a single nostril is present. Because of their structural simplicity, cyclostomes generally are considered the most primitive of true vertebrates. The hagfish are strictly inhabitants of temperate and subtropical inshore marine waters of the Atlantic and Pacific Oceans. Hagfish are members of the family Myxinidae. The skin of the hagfish is richly supplied with large mucous cells. A large hagfish is said to be capable of filling a twogallon bucket with slime. The slime is reputed to be toxic. Hagfish are rarely eaten as food.

26

Halstead

Representative Species Family: Myxinidae (hagfish) Species: Myxiwe glutinosa Linnaeus. Atlantic hagfish. Length 31 in. (79 cm). Distribution: North Atlantic. Family: Petromyzonidae (lampreys) Species: Petmtnyzon rnarinus Linnaeus. Sea lamprey. Length 33 in. (84 cm). Distribution: Coasts and rivers of both sides of the Atlantic, rivers of the Mediterranean.

Cyclostomc Poisoning Clinicd Chrrrrrcteristics: Poisoning from cyclostomes is rare because they seldom are eaten. The slime is said to be toxic to eat. Symptoms consist of nausea, vomiting, dysenteric diarrhea, tenesmus, abdominal pain, and weakness (22-26). There are no recent accounts of cyclostome poisoning. Treetrttnent:Treatment is symptomatic. See Refs. 27-29. Prevetltion: Most cyclostome poisonings are said to be caused because of failure to deslime the fish. For prevention, some authors claim that if the fresh fish is covered with salt and left in a concentrated brine solution for several hours prior to cooking, the fish is safe to eat (30,31).

B. Sharks, Skates, Rays, and Chimaeras: Elasmobranch Fish The elasmobranch fishes include the sharks, skates, rays, and chimaeras, all of which are members of the class Chondrichthyes. The poisoning is referred to as elasmobranch poisoning when it involves sharks, skates, or rays. When involving chimaeras, also known as the elephantfish or ratfish, the intoxication is known as chimaera poisoning. The sharks, skates, and rays are fishlike vertebrateswith well-developed lower jaws and bony teeth; two pairs of appendages supported by pectoral and pelvic girdles; a cartilaginous skeleton that, while more or less calcified, lacks any true bone; scales, essentially toothlikeinstructure,known as placoidscales;twonostrils,eachsingleandpartially subdivided; and blind olfactory sacs, not opening into the mouth. The posterior end of the vertebral column is either straight or heterocercal. A sympathetic nervous system, pancreas, spleen, and contractile arterial cone are present. There is a series of two, three, or more heart valves and a swim bladder is present. There are five to seven pairs of gills and five to seven gill clefts, each of the latter opening separately to an exterior dorsal fin or fins, and spines, if present, are rigid and not erectile.The skin is with or without dermal denticles, teeth are numerous, and the upper jaw or palatoquadrate cartilage is not fused to the cranium, although it may be attached locally to it. The nostril cartilage is fused to the cranium. At least some of the vertebrae of the trunk region have transverse ribs and the two halves of the pelvic girdle are fused into a single bar. The anus and urogenital canals discharge into a common cloaca and the males are without prepelvic or frontal tenacula.

1, Poisonous Sharks Representrrtive Species Family: Isuridae (mackerel sharks) Species: Crr~-chcrrodoncxrrchrirrs (Linnaeus). Great white shark. Length 20 f t (6 m).

Fish Toxins

27

Distribution: Cosmopolitan: tropical, subtropical, and warm temperate seas worldwide. Family: Carcharhinidae (requiem sharks) Species: Ccrrchcrrhinus me1mopteru.s (Quoy and Gaimard).Black-tip reef shark. Length 6.5 ft (2 m). Distribution: Tropical-Indo Pacific region. Species: Carchnrhitrus amboinet?.sisMuller and Henle. Pig-nosed Shark. Length 82 in. (2.8 m). Distribution: Eastern North Atlantic, Indo-Pacific oceans, South Africa, Madagascar, Gulf of Aden: Indian Ocean, and Australia. 12 f t Species: Cerrcherr11inu.s Ieucas (Valenciennes).Bullheadshark.Length (3.6 m). Distribution: Warm waters of the Atlantic, Pacific, and Indian Oceans. Family: Hexanchidae (cow sharks) Species: Heptranc1riLr.s perlo (Bonnatere). Seven-gilled shark. Length 6.3 ft (2 m). Distribution: Atlantic, Mediterranean, South Africa, and Japan. Family: Dalatidae (sleeper sharks). Species: Sormiosus tnicrocephcrlus (Bloch and Schneider). Greenland shark. Length 6.3 ft (2 m). Distribution: Arctic Atlantic, North Sea. east to the White Sea, and west to the Gulf of St. Lawrence. Family: Sphyrnidae (hammerhead sharks). Species: Sphyrmr ;ygaetrcr (Linnaeus). Scalloped hammerhead shark. Length 14 ft (4 m). Distribution: Tropical to warm temperate belt of the Atlantic and Pacific Oceans.

The elasmobranch form of icthyosarcotoxism is most commonly caused by eating sharkliversandtheflesh of some of the tropical sharks. Skates and rays are seldom involved in food poisoning. Clinicd Characteristics: The ingestion of fresh Greenland shark flesh is toxic to both dogs and man. The symptoms consist of nausea, vomiting, diarrhea, difficulty in walking, convulsions, respiratory distress, and muscular twitching. The local natives gradually build up a tolerance to the poison. The toxicity of Greenland shark poisoning is believed to be due to large amounts of trimethylamine oxide (32). Serious intoxications have resulted from eating the tropical sharks Ctrrchcrroclon curcherrim, C(rrcharhinus tnekrnopterus, Ccwcharhinus amboinensis, Ccrrckrrrhitrus leuc m , He~)trernchinsperlo,and Sphyrnrr zygtrerrcr. The symptoms resulting from the ingestion of shark liver may be severe, developing within 30 minutes after eating. The symptoms consist of nausea, vomiting, diarrhea, abdominal pain, headache, joint aches, tingling about the mouth, and a burning sensation of the tongue, throat, and esophagus. As time progresses, the symptoms involving the nervous system may worsen, resulting in muscular incoordination and difficulty in breathing due to muscular paralysis, followed by coma, and finally death. Ingestion of the flesh of certain species of tropical and arctic sharks may be dangerous to eat, butthe symptoms usually aremild,consistingmainlyof a gastrointestinal upset.

Halstead

28

Trecrtnw1t: Treatment is symptomatic. See ciguatera fish poisoning. See Refs. (2729).

Prevetrtion: Avoid eating the liver of any shark unless it is known for certain to be edible. The livers of tropical and arctic sharks are known to be especially dangerous to eat. The flesh of tropical and arctic sharks is potentially poisonous and should be eaten with caution.

2. Poisonous Chimaeras The chimaeroids-elephantfish or ratfish-differ from the sharks, skates, and raysin that they have only four pairs of gills and four pairs of gill clefts, with only one opening to the exterior on each side of the head. The dorsal fin and spine are erectile. In the adult, the skin is naked, without dermal denticles. Teeth are represented by six pairs of grinding plates; the upper jaw or platorate cartilage is fused with the cranium and the rostral cartiof the lage is articulated to the cranium, not fused. Ribs are lacking and the two halves pelvic girdle separate. There is no cloaca, and the urogenital aperture is distinct from the anus and posterior to it. The males have an erectile prepelvic tenaculum, usually with a frontal tenaculum on the head (33).

Represewtative Species Family: Chimaeridae (Chimaeras) Species: Chirnrrercr ttwt1stro.w Linnaeus. Length 39 in. European chimaera. Distribution: North Atlantic and Mediterranean.

( 1 m).

Clrimcrerrr Poisotlirzg The musculature and visceraof some of the elephantfish and ratfish have been found to be toxic, but the nature of the chimaera poison is unknown. Cliwiccrl Chcrructeristics: The symptomatology of chimaera poisoning in humans is unknown. Treatmetzt: Treatment is symptomatic. Prevention: Chimaeras should not be used for human consumption.

C. Ciguatoxic Fish Ciguatoxic fish cause oneof the most widespread and seriousforms of ichthyosarcotoxism known. More than 400 species of fish are alleged to have transvectored the ciguatoxin complex poisons that serve as the causative toxins in ciguatera fish poisoning. The fish involved are for the most part tropical or warm, temperate zone reef or inshore species found between 35"N latitude and 34"s latitude in the Caribbean and tropical Pacific and Indian Oceans. Occasionally, offshore fish may be involved, but by far the bulk of the outbreaks have occurred in insular areas. Historically, ciguatera-like symptoms have resulted from eating marine turban shells (Turbo picrr) in the Caribbean (25,34,35), and similar outbreaks have been caused by eating coconut crabs in Tahiti and in the Ryukyu Islands (36,37). No freshwater fish have been incriminated. Many of the ciguatoxic fish are valuable food fish that on occasion become toxic within a few hours of ingesting toxic dinoflagellates or algae in association with toxic dinoflagellates. Carnivorous fish may become toxic as a result of ingesting toxic herbivores. Thus ciguatera is a toxic food chain problem. Once a fish becomes poisonous, the

Fish Toxins

29

toxicity within the body of the fish may continue for a period of many years. One of the species of dinoflagellates .that has been incriminated in ciguatera fish poisoning is Gambierdiscus tosicus Adachi and Fukuyo. Several other dinoflagellate species are highly suspect as causative agents of ciguatera fish poisoning, including Ampl~idiniutncnr'tertre Hulbert, Ostreopsis ovtrt~Fukuyo, Prorocentrum C ' O I ~ C L I V U IFukuyo, I? P. limtr (Ehrenberg) Dodge, and P. me.~iccrnu~r~ Tafall (38). There is growing evidence that suggests that the in a symbiotic primary causative agent in this toxicity cycle may be bacteria that live relationship with dinoflagellates or possibly macroalgae (see Sec. 111). Ciguatoxic fish are a group of phylogentically diversified species, most of which are members of theclassOsteichthyes,thebonyfish.Thebiology of thesefishis as diversified in habitat, habits, feeding, and reproduction as it is in morphology. Consequently, it is impossible to present a stereotyped characterization of a ciguatoxic fish. Moreover, you cannot detect a ciguatoxic fish by its appearance. In one part of an island, any given fish species may be edible, whereas on the opposite side of the island or on an adjacent reef, the same species may be deadly poisonous. This is the major problem in ciguatera fish poisoning. The members of the class Osteichthyes are characterized as having a skeleton, in part or all with true bone; the skull has sutures; and the teeth are fused to the bones. The soft fin rays usually are segmented. Nasal openings on each side usually are double and more or less dorsal in position. The biting edge of the upper jaw usually is formed by a functional lung dermal bones, the premaxillae and the maxillae. A swim bladder or usually is present. There is an intestinal spiral valve in only a few lower groups. Internal fertilization is relatively rare, and there is a pelvic copulation device in only one group (phallosthoids). The embryos are not encapsulated in a case (39).

Representdve Species An attempt to list all of the ciguatoxic fish species that have been incriminated to date would not be feasible; consequently, only a small representative group of species is listed below. The fish are arranged in alphabetical order according to their family names and within the family by their generic names. Family: Acanthuridae (surgeonfish) Species: A c c r t h m s lineatus (Linnaeus). Striped surgeonfish. Length 7 in. (18 cm). Distribution: Indo-Pacific. Family: Balistidae (leatherjackets, filefish, triggerfish) Species: AIutera scriljttr (Osbeck). Scribbled filefish. Length 19 in. (50 cm). Distribution: All warm seas. Species: Btrlistoitfes conspicillurn Bloch and Schneider. Clown triggerfish (Fig. 1 1). Length 13.7 in. (35 cm). Distribution: Tropical Pacific from Polynesia to Madagascar, China, Japan. Family: Carangidae (jacks, pompanos) Species: Ctrrtrn.~ hi[)i)os(Linnaeus). Jack, crevalle. Length 29.5 in. (75 cm). Distribution: Tropical Atlantic. Cuvier. Blue jack. Length 25.5 in. (65 cm). Species: ccrrcrnx melatn~~pgus Distribution: Tropical Pacific. Family: Lutjanidae (snappers) 35.5 in. Species: Lutjarzr4.s bohtrr (Forskil).Redtwo-spottedsnapper.Length (90 cm).

30

Halstead

Distribution: Tropical Indo-Pacific. Species: Lutjrrnus gibbus (Forskil). Humpback snapper. Length 15.5 in. (40 cm). Distribution: Tropical Indo-Pacific. 19.5 in. Species: Lutjatzus vaigiensis (Quoy and Gaimard). Redsnapper.Length (50 cm). Distribution: Indo-Pacific. Family: Mugilidae (mullets). Species: CIlelow vcligiensis (Quoy and Gaimard). Mullet. Length 12 in. (30.5 cm). Distribution: Indo-Pacific. Species: Mugil cepkrr1u.s Linnaeus. Mullet. Length 12 in. (30.5 cm). Distribution: Cosmopolitan in warm temperate seas. Family: Mullidae (goatfish, surmullets) Species: Mulloidicl~thysc/ur(jIatrrma (Forskil). Goatfish. Length 13 in. (35 cm). Distribution: Indo-Pacific. Species: Parupet~euschtyser:\ldros (Lacepide). Goatfish. Length 13 in. (33 cm). Distribution: Indo-Pacific, East Africa. Family: Muraenidae (moray eels) Species: Gytnnothortr.u javcrrlicus (Bleeker). Giant brown moray eel. Length 5 ft (1.5 m). Distribution: Indo-Pacific. Species: Gymtlothorc/.r rnelecrgris (Shaw and Nodder). White-mouthed moray eel. Length 39 in. ( I m). Distribution: Indo-Pacific, Japan. Family: Scombridae (tunas, mackerels, albacore) solrrttdri (Cuvier). Wahoo. Length 78 in. (2 m). Species: Acc~t~thocybiun~ Distribution: Circumtropical. Species: Scombemmorus crrvcrllrr (Cuvier). King mackerel. Length 59 in. (1.5 m). Distribution: Tropical Atlantic. Family: Serranidae (sea basses, grouper) Species: Cephnlopholis nrgus (Bloch and Schneider). Peacock grouper. Length 20 in. (51 cm). Distribution: Indo-Pacific. (Forskil). Brown nnarbled grouper. Length 23.5 Species: E~~ine~~halus.fu.scoglrttr~tu,s in. (60 cm). Distribution: Indo-Pacific. Species: Mycterol~erccr vetzertow (Linnaeus).Poisonousgrouper.Length35 in. (90 cm). Distribution: Western tropical Atlantic. Species: Plectroponrrrs olipccrrlthus Bleeker. Blue-lined coral grouper. Length 2 1 in. (55 cm). Distribution: Indo-Pacific. Species: Vrrriola louti (Forskil). Lyretail grouper. Length 23 in. (60 cm). Distribution: Indo-Pacific. Family: Siganidae (rabbitfish). Species: Sig~rnlrslinearus (Valenciennes). Rabbitfish. Length 1I in. (30 ~111). Distribution: Indo-Pacific. Species: Sigrrrrrrs prrellus (Schlegel). Rabbitfish. Length 10 in. (27 cm). Distribution:Indo-Pacific.

Fish Toxins

31

Family: Sphyraenidae (Barracuda) Species: Sphyraetu~hnrrczcudcl (Walbaum). Great barracuda. Length 5.2 ft (1.6 m). Distribution: All warm seas, except eastern Pacific.

Cigutrtern Fish Poisotlitlg Ciguatera fish poisoning results from the ingestionof any of a large variety of shore and reef fish which are usually subtropical or tropical in their distribution. The degree of freshness of the fish has no bearing on its toxicity. The victim becomes poisoned as a result of ingesting a toxin within the flesh of the fish. Ciguatera is not a form of ordinary bacterial food poisoning. Ciguatera fish poisoning involves a complex of poisons: ciguatoxin, molecular formula CI,,,HXhOll,, molecular weight 1 1 12, median lethal dose (LDS,,) 0.45g/kg mouse intraperitoneal (IP) (40-45); maitotoxin, molecular formula ClhlhH2?cS?07J, approximate molecular weight 3396.1, LDcotoxicity 0.13 g/kg mouse IP (46); scaritoxin, molecular formula and toxicity unknown (47). Ciguatoxin and maitotoxin are two of the most toxic marine poisons known. Ciguatoxin acts by increasing the membrane permeability to sodium ions of excitableneurons by openingthevoltage-dependentsodiumchannels(43,48,49).Repeated doses of ciguatoxin to mice on an experimental basis have been found to produce severe ultrastructural morphological changes in the cardiac muscle cells and endothelial lining cells of blood capillaries in the heart. Damage to the capillaries was followed by effusion of serum and erythrocytes into the interstitial spaces of the myocardium. Swelling of the endothelial lining cells of capillaries caused narrowingof the lumen and accumulation of of cardiac blood platelets in capillaries, which resulted in multiple single-cell necrosis muscle cells (50). These experimental findings in mice may possibly explain some of the clinical cardiac findings in individuals that have suffered multiple exposures to ciguatera fish poisoning. The most toxic part of the fish is usually the liver, followed by the intestines, then the testes, ovaries, and the muscle. As noted in Sec. IV.C, the toxin originates in the food web of the fish. There is no evidence of a seasonalincidenceinciguatoxicity,butthespawning season in some of the larger predacious fish may be a more dangerous period than the other seasons of the year. There is no way to detect a ciguatoxic fish by its appearance. Clitliccrl Chcrrcrcteristics: Ciguatera fish poisoning produces a constellation of 175 gastrointestinal, cardiovascular, and neurological symptoms, some of which are pathognomonic for the disease. The onset of the poisoning may occur within minutes and up to 48 hours after the fish is ingested. The initial symptoms generally consist of paresthesias and tingling or numbness of the lips, tongue, and extremities. The neurosensory symptoms may be accompanied by nausea, abdominal pain, vomiting, diarrhea, salivation, general malaise, and muscle and joint pain. The gastrointestinal symptoms are present in about 75% of cases, but usually resolve within 24 hours. There is a neurosensory symptom that is of diagnostic importance in ciguatera poisoning: the reversal of temperature sensation in which cold objects (water, ice, etc.) feel hot, produce a stinging sensation, or are painful upon contact. Warm objects may feel cold. This temperature-reversal sensation appears in more than 89% of cases. Cardiovascular symptoms usuallyconsist of bradycardia and hypotension, which later may change to

32

Halstead

tachycardiaandhypertension.Cardiovascularsymptomsgenerallyresolvewithin 48 hours, but may persist for several weeks. Neurological symptoms of perioral and extremity paresthesias, ataxia, pruritus, mental depression, hysteria, maculopapular skin eruptions, blisters, desquamation, loss of hair and nails, cranial nerve palsy, vertigo, tremors, chills, headache, sweating, dysurea, hiccups, visual blurring, superficial hyperesthesia, motor weakness, respiratory distress, myalgia, arthralgia, temperature reversal, hyporeflexia, metallic taste, loose or painful sensation of the teeth, and extraocular muscle pain Inay be present. Their occurrence and duration vary with the individual. Seldom is fever present. Extreme muscle weakness is a common complaint. Paralysis of the extremities may occur. Physical findings are variable and nonspecific in ciguatera poisoning. The severity of the case varies withthe individual’s sensitivity to the toxin, the toxicity of the fish, and the amount of toxic fish eaten. Recovery varies greatly from case to case, and can be from 48 hours to several days, weeks, months, and, in some cases, years. Fatalities are uncommon, but do occur. Fatalities may be due to cardiovascular collapse or respiratory paralysis. Exposure to the ciguatoxin complex does notconfer immunity. There are no accurate statistics worldwide as to the incidence or mortality rate of ciguatera fish poisoning. There is a gross underreporting of outbreaks even in regions in which ciguatera is endemic. The subject of ciguatera fish poisoning has been under intensive investigation by numerous workers over an extended period of time (25,483 190). Treatment: The treatment of ciguatera fish poisoning is largely symptomatic, but thereare a fewspecificmodalitiesthatmaybeespeciallyhelpful.Theidentity of the fish is of minor value because about 300 different species of tropical reef fish have been incriminated thus far and the amount of toxin in the fish varies greatly from one specimen to the next. The diagnosis of ciguatera fish poisoning is based on the symptomatology presented. Gastric lavage or vomiting induced by sticking ones finger down the throat, or using apomorphine or ipecac, should be done as soon as possible. This should be followed by the administration of a slurry of charcoal to absorb the poison in the intestinal tract. Nausea and vomiting can be controlledby using an antiemetic drug such as prochlorperazine. Hypotension can usually be helped with the useof a pressor drug such as dopamine or dobutamine. Calcium gluconate may also be helpful in treating the hypotension and myocardial insufficiency. Bradycardia may be controlled with the use of atropine. Cool showers and the use of hydroxyzine may be helpful in relieving the pruritus. Intravenous sodium ascorbate (25 g diluted in 250 1111 of normal saline per day for10 days) and vitamin B complex have been ernployed in relieving someof the toxic effectsof ciguatoxin. Mannitol has been found to provide symptornatic relief in many cases (121,130). The fruit of the nono tree (Morinda citrijiolia Linnaeus) has been used for centuries by South Pacific islanders to treat the symptoms of ciguatera fish poisoning ( 2 ) . The juice of this fruit is now sold in the United States and elsewhere throughout the world under the trade name “Noni.” Theusual dosage is 3-4 ounces of the juice per day. The product is nontoxic and should be tried. A variety of other therapies have been employed, but none of them have been shown to be completely effective. See Refs. 27-29. Preverlrion: There is no reliable methodof detecting a ciguatoxic fish by its appearance. However, there are a few basic guidelines that are helpful. Such large, predacious reef fish as snapper, barracuda, grouper, and jacks should be eaten with caution. The larger the fish is the greater is the potentialof ciguatoxication if the fish iscaptured in an endemic

Fish Toxins

33

region. Large fish generally accumulate the toxic products fromthe various trophic levels in the food chain below them. Ciguatera is essentially the end resultof a toxic biochemical magnification problem in which humans are the final recipient of the toxic agents from all of the marine organisms in the trophic levels below them. When catching fish in a suspected ciguatoxic region, it is always advisable to seek the advice of the locals as to the edibility of the fish. The viscera (i.e., liver, gonads, and intestines) of many tropical reef fish are toxic and should not be eaten. Such ordinary cooking procedures as frying, baking, boiling, or drying do not render a fish safe to eat. Ifin doubt concerning the toxicity of the fish, eat only a small amount and wait for a period of several hours before eating additional quantities of the fish. Tropical moray eels frequently are toxic, maybe deadly, and should not be eaten. Offshore fish generally are safer to eat than inshore reef species. Bioassay methods have been used to detect ciguatoxic fish utilizing a variety of test animals,includingbrineshrimp,ants, flies, cats,dogs,mongooses,rats,guineapigs, plants, chickens, frogs, and so on. See Ref. 48 for a review of these bioassay methods. Cats have been found to be extremely sensitive to ciguatoxin. All of these bioassay methods tend to be cumbersome, time consuming, and, in most instances, expensive. Several immunoassay methods have been developed for the detection of ciguatoxic fish. One of thefirst of these techniques utilized a radioimmunoassay method for the detection of minute quantities of antigens and antibodies (91-94). This test was refined further into an enzyme immunoassay method (95) that was found to be easier to run, less expensive, more feasible for screening all sizes and varieties of fish, and could be used to test liver and musculature. A more inexpensive, but still reliable assay is the stick test developed by Hokama (79,96,97) and Hokama et al. (98). The stick test measures ciguatoxin and polyether compounds, including okadaic acid. The stick test has been used commercially to a limited extent in Hawaii. The stick test appears to be the most reliable and practical assay method currently available for the screening of ciguatoxic fish. Unfortunately the stick test is generally not available in most endemic ciguatoxic regions of the world.

D. Clupeotoxic Fish Clupeotoxic fishare members of theorderClupeiformeswhichincludes theherrings, anchovies, and related species. Clupeiform fishes become poisonous after eating toxic dinoflagellates such as Osrreopsis siarnensis Schmidt (99). Undoubtedly other species of dinoflagelllates are also involved but have yet to be incriminated. This fom1 of poisoning is rare and resembles ciguatera, but it is very rapid in its action and has a high mortality rate. The membersof the order Clupeiformes include the families Clupeidae, the herrings; Engraulidae, the anchovies; Elopidae, the tarpons; Albulidae, the bonefish; Pterothrissidae, the deep-sea bonefish; and Alepocephalidae, the deep-sea slickheads. However, the families most commonly incriminated in human clupeotoxications are members of the Clupeidae and Engraulidae. The clupeiform fish are characterized as follows. They are softrayed fish with the anterior vertebrae simple, unmodified, and without auditory ossicles; symplectic bone is present and there are no interclavicles.The recessus lateralis is present, and the infraorbital canal merges with the preopercular canal within a chamber of the neurocranium. Parasphenoid teeth are absent. There is no large foramen on the anterior ceratohyal, the parietals are separated by the supraoccipital, and the opercular bones are

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distinct. The pharyngeal bones are simple above and below,with the lower not falciform. The bones of the jaws are developed; the maxillary is broad, always distinct from the premaxillary, and forms part of the margin of the upper jaw. There are no barbs. The shoulder girdle is well developed and connected withthe cranium by a bony posttemporal. a slit behind the fourth. The air bladder,if present, hasa pneumatic There are four gills with duct. The dorsal and anal fins are without true spines; the ventral fins are abdominal and the adipose fin can be present or absent. This is a large group of fish comprising most of themarinesoft-rayedfish.Mostareplanktonfeederswithnumerous,longgillrakers (39.100).

Representcltive Species Family: Clupeidae (sardines, herrings) Species: Clupmodon thrissa (Linnaeus). Thread herring. Length 9 in. (25 cm). Distribution: Indo-Pacific, China, Japan, Korea. Species: Cluperr sprnttus Linnaeus. Sprat. Length 6 in. ( 1 S cm). Distribution: Northeastern Atlantic, Mediterranean. Species: Hcrrewgulrr ovnlis (Bennett). Sardine. Length 6 in. ( I S cm). Distribution: Indo-Pacific, Red Sea. Species: Opisthot?etncr oglinum (LeSueur). Atlantic thread herring. Length 9 in. (25 cm). Distribution: West Indies. north to Cape Cod. Family: Engraulidae Species: Etrgrcrulis et~crnsicholus(Linnaeus). Anchovy. Length 7 in. (20 cm). Distribution: Eastern Atlantic and Mediterranean. Species: Thrissincr baelmrcr (Forskil). Anchovy. Length 4 in. ( 12 cm). Distribution: Indo-Pacific, Red Sea, enters river mouths.

Clupeotoxications result from eating clupeiform fish such as sardines, herring, and anchovies. Most poisonings have occurred in tropical island areas and were caused by eating fish captured close to shore. It is believed that clupeotoxism is seasonal and most likely to occur during the summer months. Clupeotoxin has been isolated (99), but its molecular structure has not been elucidated. Clirzicul Clrcrrrrc.ter-istics: The symptoms and signs of clupeotoxism are distinct and usually violent. The first indication of an intoxication is a sharp metallic taste that may be present immediately following ingestion of the fish, followed by nausea, dryness of the mouth, vomiting, malaise, abdominal pain, and diarrhea. The gastrointestinal upset may be accompanied by a feeble pulse, tachycardia, chills, cold and clammy skin, vertigo, a drop in blood pressure, cyanosis, and other evidences of vascular collapse. Within a very short period of time, or concurrently, a variety of neurological disturbances ensue, such as nervousness, dilated pupils, violent headaches, numbness, tingling, hypersalivation,musclecramps,respiratorydistress,progressivemuscularparalysis,convulsions, coma, and death. Death may occur in less than IS minutes. Ferguson (101) claimed that the poison was so rapid in its action that natives died while in the very act of eating the yellow-billed sprat (Clulwn tlrrissrr). Pruritus and various typesof skin eruptions, including desqualnation and ulcerations, have been reported in v i c t i m that have survived. There are no accurate statistics available regarding the mortality rateof clupeotoxism, but judg-

Fish Toxins

35

ing from the documented case reports, the fatality rateis very high and the victims generally die within minutes to hours. It is believed that clupeotoxisln in some instances may be related to ciguatera poisoning, but this has not been documented. Some cases appearto exhibit ciguatera-like symptoms. Since clupeoid fish are primarily plankton feeders, it is likely that some of these fish are ingesting highly toxic dinoflagellates. Tretrtnzent: Follow thetreatmentrecommendedforciguaterafishpoisoning.See Refs. 27-29. Prcventiorr: There are no reliable methods of detecting a clupeotoxic fish and preventing intoxication. Outbreaks of this intoxication are rare and there are insufficient data concerning the nature of the poison. No screening methods have been developed for the testing of clupeotoxic fish. Most of the clupeotoxic fish are generally valuable food fish.

E. Gempylotoxic Fish The gempylids, escolars, or pelagic mackerels are a small group of predacious oceanic fish. They have a band-shaped body, large. sharp teeth, and are distinguished from the true mackerels by the complete absence of a later keel or ridge on the caudal peduncle. Two dorsal finsarepresent,thefirst of which is spinous and longer thanthesecond. Gempylids produce an oil that has a pronounced purgative effect.

Representcrtive Species Family: Gempylidae (castor oil fish) Species: Rrrvcttus pr-etiosus Cocco. Castor oil fish. Length 4.2 ft (1.3 m). Distribution: Tropical Atlantic and Indo-Pacific.

Gernpylicl Poisonirrg The gempylid poisoning formof ichthyosarcotoxism is caused by ingesting the flesh or sucking the rich, oily bones of the fish. People suffering from constipation in the South Pacific islands use gempylid fish for its relief. Clinical Clrnrcrcteristics: Ingestion of theoil contained i n theflesh and bones of gempylid fish causes diarrhea, which, although pronounced, is generally without pain or cramping (26,102,103). No other untoward effects have been reported. Gempylotoxisln has also been referred to as gempylid diarrhea. The oil is similar to castor oil, comprised mainly of oleic acid, but it has different pharmacodynamic properties ( 104,105). Treutmerlt: No treatment is required. Prew1tiotl: Avoid eating gempylid fish.

F.Scombrotoxic

Fish

Scombrotoxism,orscombroidpoisoning, is caused by perciform fish of thesuborder Scombroidei, all of which are members of the single family Scombridae, the tunas and related species. One of the members of the order Beloniformes, family Scomberesocidae, the Japanese saury (Cololabis s a i r . c ~ )has also been incriminated in sombroid poisoning. Recently, such otherfish species as mahi-mahi, jack, bluefish, herring, sardines, and anchovies have been reportedto cause scombroid poisoning (87). Scombroid poisoning accounts for about 5% of food-borne poisonings i n the United States. Scombroid fish are characterized by their adaptation for swift locomotion, having

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a sharp profile anteriorly and a slender tail with a widely forked caudal. There is a series of detached finlets on the back behind the second dorsalfin and on the undersurface behind the anal fin, a feature that distinguishes them from most other fish. Scombroids have two dorsal fins, of which the first is composed of spines and the second of soft rays. The fins fit into grooves or depressions on the body, the bones of the head lie flat, and the gill covers fit tightlyagainstthesides. Thescalesare usuallysmall,thin, andmetallicin appearance, offering a minimum of friction in the water. Scombroids are the epitome of grace, form, and speed. Tunas and related species are largely oceanic, migrating great distances through the open seas. Mackerels are largely inhabitantsof littoral waters. Scombroids are distributed widely throughout all temperate and tropical seas, and some occasionally are found in arctic and antarctic waters. They usually swim in large schools. Scombroids generally swim near the surface of the water during spawning season. During the warmer months they approach the shore, but retire to deeper water during the cold months. They feed on to the surface at night. the plankton swimmingin the deeper water during the day, and rise Scombroids are predatory and voracious feeders. In addition to plankton, they also feed on a wide variety of moderate-size fish. Some of the tunas can be found at depths of 200 m or more, whereas other scombroids seldom descend below 40 m . The sauries of the family Scomberesocidae resemble the needlefish, but have short jaws and are identified by a series of five to seven finlets following both the dorsal and anal fins. There are four species and none attain a length much beyond 35 cm. Sauries inhabit temperate and tropical waters. They arevery abundant in some regions and constitute an important foodfisheryin Japan. Sauries feed on planktonandsmall fish. The sauries also have been incriminated in scombroid fish poisoning, but outbreaks have been limited to Japan. Repr~ser~tcrtive Species Family: Scomberesocidae (sauries) Species: Cololabis saircr (Brevoort). Saury. Length 12 in. (30 cm). Distribution: Japan. Family: Scombridae (tunas. nmckerels, albacore) Species: Euthgnm~.s peltrrtis (Linnaeus). Skipjack. Length 19 in. (50 cm). Distribution: Cosmopolitan in warm seas. Species: Scolllber. jcrpotzic~~ Houttuyn. Pacific mackerel. Length 15 in. (40 cm). Distribution: Cosmopolitan in warm seas. Species: Thunnlr.~tl1gtIr~u.s(Linnaeus). Bluefin tuna. Length I O ft (3 m). Distribution: Cosmopolitan in subtropical and temperate seas.

Sconlhroicl (Histtmirle) PoisorlirlR Scombroid poisoning is caused by the improper preservation of scombroid fish or other fish species that results in certain bacteria, mainly species of the family Enterobacteriaceae ( C l o . ~ f r i d i wLrrctobmillus, ~, Prote~rs,Vibrio), acting on histidine in the muscle of the fish convertingit to histamine.The toxicity of histamine is enhanced by the presence of certain potentiators (e.g., cadaverine and putrescine) that act by inhibiting intestinal histamine-metabolizing enzymes. The enzyme inhibition increases the intestinal uptake of unmetabolized histamine (106-108). Histamine ingested by itself generally is much less toxic. Scombroid poisoning is the most common form of ichthyosarcotoxism and occurs

Fish Toxins

37

throughout the world wherever scombroid fish are eaten. Moreover, it is the only form of ichthyosarcotoxism in which bacteria play an active role i n toxin production within the body of the fish. Clirlical Chrrracteristics: The symptoms of acutescombroidpoisoningresemble those of histamine intoxication. The symptoms are characteristic and appear with almost monotonous consistency. Toxic scombroid fish frequently can be detected immediately upon tasting the fish. Victims state that the fish has a sharp or peppery taste. Symptoms usually occur within a few minutes after ingestion of thetoxin and consist of intense headaches, dizziness, throbbing of the carotid and temporal vessels, epigastric pain, burning of the throat, cardiac palpitation, rapid and weak pulse, dryness of the mouth, thirst, inability to swallow, nausea, vomiting, diarrhea, and abdominal pain. Within a short time a generalized erythema and an urticarial eruption may develop, covering the entire body and accompanied by a severe pruritus. The face of the individual becomes swollen and flushed, the eyes become injected, and coryza develops. In severe cases there may be bronchospasm, suffocation, and severe respiratory distress. Various other minor discomforts such as fever, chills, malaise, tremors, metallic taste, and cyanosis of the gums and tongue may occur. There is a danger of shock, and deaths have been reported. However, the acute symptoms generally are transient, lasting only 8-12 hours (48,108-1 1l ) . Treatment: The treatment of scombroid poisoning is largely directed toward relieving the symptoms of the histamine reaction. This form of poisoning is generally selflimiting and fatalities are rare. Minor intoxications can usually be treated with diphenhydramine (Benedryl). Scombroid poisoning may cause respiratory distress, in which case the victim should be taken immediately to an emergency treatment center. Epinephrine is the treatment of choice for respiratory problems in this instance, but it should be used withcaution in olderindividuals with a historycardiacproblems.SeeRefs.(27-29, 92,l 12-1 15). Prevenrion: Scombroid fish and other related species believed to cause this form of ichthyosarcotoxism should be refrigerated promptly or eaten soon after capture. It has been shown that the histamine content in some of the scombroid fish increases from 0.09 mg/100 g of tissue to about 95 mg/ 100 g of tissue when kept at room temperature (20°C25°C) for about10 hours ( 1 16).Toxic scombroid fish cannot always be detectedby appearance or odor. The histamine content in the flesh may be very high with little or no evidence of putrefaction. Scombroid or any other fish having a sharp or peppery taste should be discarded. Scombroid fish with histamine levels greater than 20 mg/ 100 g should be discarded.

G. Tetrodotoxic Fish Tetrodotoxications constitute one of the most violent formsof marine biotoxications. This type of ichthyosarcotoxism commonly is designated as tetrodon or puffer poisoning. The causative transvectors of the poison tetrodotoxin are members of the order Tetraodontiformes, formerly the Plectognathi, which includes the families Tetraodontidae, the puffers; Diodontidae, the porcupinefish; Canthigasteridae, the sharpnosed puffers; and Molidae, the molasor ocean sunfish. The order also includes such other fish families asthe spikefish, filefish, trunkfish, three-toothed fish, and triggerfish, but these are not included in any fish mayalsotransvectany or all of the degree of detail in this chapter. These same ciguatera complex of poisons (e.g., ciguatoxin, maitotoxin, or possibly palytoxin). For

38

Halstead

the 111ost part, this section focuses on the fish families Tetraodontidae, Diodontidae, and Canthigasteridae. Tetrodotoxications are the resultof ingesting a poison known as tetrodotoxin, molecular formula C,,H,,N,O,, molecular weight 319, and LDS,, toxicity in mice is I O pg/kg ( 1 17,118). Tetrodotoxin actsby preventing nerve conductionby an extremely specific and reversible blockage of the inward movement of sodium ions through the cell membrane of an activated neuron (48,119-121). The tetraodontiform fish are characterized by the absence of parietal, nasal, or infraorbital bones, and usually have no lower ribs; the posttenlporal region is present and is siniple and fused with the pteroticof the skull; the hyomandibular and palatine are firmly attached to the skull. Gill openings are restricted. The maxilla is usually firmly united or fused with the premaxilla. Scales are usually modified as shields or plates. The lateral line may be present or absent, and sometimes is multiple. The swim bladder is present except in molids, and there are 16-30 vertebrae. Tetraodontifornles can produce sounds by grinding the jaw teeth or the pharyngeal teeth or by vibrating the swim bladder. The stomach of some of these fish is highly modified to permit inflation to an enormous size. Fish with this ability are commonly called puffers. Inflation is caused by gulping large quantitiesof water into aventral diverticulum of the stomach when the fish is frightened or annoyed. Deflation occurs by expelling the water. If the fish is removed from the water, inflation can occur with air (39,122).Puffers feed mainly on corals and mollusks, but tend to be omnivorous. A wide range of phylogenetically unrelated aquatic organisms are now known to transvect tetrodotoxin, including starfish, gastropod mollusks, the Australian blue-ringed octopus, tropical reef crabs, gobyfish, and a variety of freshwater amphibians. This subject has been reviewed in greater depth elsewhere (48).

Representcrtive Species There are a large numberof fish species incriminated in transvectoring tetrodotoxin, but only a few representative fish are listed below. Family: Canthigasteridae (sharp-nosed puffers) Species: Canthigaster rivuhtus (Temminck and Schlegel). Rivulated Goby. Length 3 in. ( I O cm). Distribution: Indo-Pacific, Japan. Family: Diodontidae (porcupinefish) Species: Chilonzycteru.7 c!jjitziirlis Giinther. Porcupinefish. Length 6 in. ( 1 7 cm). Distribution: Southern California, Galapagos Islands, Hawaii, Japan. Species: Diodorl hystri.r Linnaeus. Porcupinefish. Length 35 in. (90 ctn). Distribution: All warm seas. Family: Tetraodontidae (puffers, blowfish, fugu) Species: Arothron lzisjdus (Linnaeus). White-spotted puffer. Length21 in. (53 cm). Distribution: Indo-Pacific, Panama, Japan, Australia, South Africa, Red Sea. Species: Arothron meleagris (LacCpkde). Guineafowl puffer. Length 13 in. (33 cm). Distribution: West coast of Central America, Indo-Pacific. Species: Fugu Imrddis (Temminck and Schlegel). Fugu. Length 14 in. (35 cm). Distribution: China, Japan. Species: kcgocephcc/u.s luncrris (Bloch and Schneider).Puffer.Length 12 in. (35 Clll).

Fish Toxins

39

Distribution: Indo-Pacific, Red Sea, China, Japan, eastern coast of Africa. Species: Sldlaeroides umul~/tu.s(Jenyns). Puffer. Length 1 I in. (28 cIl1). Distribution: California to Peru, Galapagos Islands. Species: S~h7er.oide.vrnncu1rrtu.s (Bloch and Schneider). Botete. Length 10 in. (25 cm). Distribution: Atlantic coast of the United States south to Guiana. Species: Tettwodorl lineatus Linnaeus. Puffer. Length 18 in. (46 cm). Distribution: Rivers of northern and western Africa.

Tetrorlorr (Puffer.) Poisoilirlg Method ofIntmication: Puffer poisoning or tetrodon poisoning is caused by ingesting the flesh, viscera, or skin of toxic tetraodontiform fishes (i.e., sharp-nosed puffers, puffers, porcupinefish, and other related fish). Certain goby fish, gastropod mollusks, and tropical reef crabs are also capable of transvectoring tetrodotoxin and causing a biotoxication. Puffer fish are more dangerous to eat imnlediately prior to and during the reproductive season, during which time the poison content in the body of the fish increases with the gonadal activity. The skin, liver, ovaries, and intestines arethe most toxic portions of the fish. The tnusculature of the fish usually is safer to eat than other parts of the fish, but at times it also may be toxic. The toxicityof the fish cannot be determinedby its appearance, freshness, or size since even small puffers may contain sufficient poison to be lethal. Puffer and fugu poisoning continuesto be the major causeof fatal food intoxications in Japan, where puffer meatis looked upon as a gourmet delicacy. The sale of toxic puffers is carefully regulated by public health authorities in Japan, butthis has not prevented periodic outbreaks of fatal food poisoning. TheU.S. Food and Drug Administration (FDA) recently has permitted the importation of Japanese puffers for sale in fugu restaurants in the United States (25,87), but this does not guarantee the safety of puffer products. All puffers are potentially toxic unlessthey have been cultivated artificially (123-125). There is evidence that several different strains of marine bacteria may play an important role in the biosynthesis of tetrodotoxin in the body of the fish (5,7). Clinicd CharLlcteri.7tic.s:The onset and symptomatology in puffer poisoning varies greatly depending upon the person and the amount of poison ingested. Initial symptoms usually consist of paresthesias of the lips and tongue, malaise, pallor, dizziness, and ataxia that develop within 10-45 minutes after ingestion of the fish, but may occur as much as 3 hours or niore after ingestion. The paresthesias are described as a tingling or a prickly to the fingers and toes, and gradually develops sensation that may subsequently spread into numbness.In severe cases,the numbness may involve the entire body, which has been described by victims as a floating sensation. Hypersalivation. profuse sweating, extreme weakness, precordial pain, headache, subnormal temperatures, hypotension, and a rapid, weak pulse usually appear early in the succession of symptoms. Nausea, vomiting, diarrhea, and epigastric pain frequently are present. The pupils are constricted during the early part of the intoxication, but later become dilated.As the disorder progresses,the pupillary and corneal reflexes are lost. Shortly after the development of the paresthesias, respiratory symptoms become a prominent part of the clinical picture. Respiratory distress, as noted by an increased rate of respiration, movements of the nostrils, and shallow respiration, generally is observed. Respiratory distress later becomes very pronounced, and the lips, extremities, and body become intensely cyanotic. Petechial hemorrhages involving extensive areas of the body, blistering, and subsequent desquamation may occur. Muscular twitching, tremor, andloss

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of motor coordination become progressively worse and finally terminate in extensive muscular paralysis. The first areas to be involved areusually the throat and larynx, resultingin aphonia, dysphagia, and, later, complete aphagia. The muscles of the extremities become of the patient paralyzed and the patient is unable to move.As the end approaches, the eyes become fixed and glassy, and convulsions may occur. The victim may become comatose, but in most instances the patient remains conscious and the mental faculties remain acute until shortly before death. The fatality rate of puffer poisoning is about 59% in untreated cases. There are no accurate mortality statistics worldwide. Treutmerlt: Early treatment seeks to remove the gastric stable toxin, which is partially inactivated by alkaline solutions. If the victim is seen within 3 hours of ingestion, 1 L 2%sodiumbicarbonate.Thisis gastriclavageshouldbeperformedwithatleast followed with activated charcoal in sorbitol solution. If the victim has difficulty swallowing or breathing, or is not alert, intragastric manipulation should be preceded by endotracheal intubation. Supplemental oxygen and ventilatory assistance should be promptly instituted as respiratory paralysis progresses. The physician should rememberthat the paralyzed victim may be fully conscious and should offer the victim frequent verbal reassurances. Hypotension induced by tetrodotoxin may require the intravenous (IV) administration of crystalloid fluid augmentation. Bradyarrhythmias generally respond to atropine (0.5 mg IV up to 2.0 mg). Severe heart block may require the placement of a temporary transvenous pacemaker. While a minor intoxication may be limited to paresthesias, all victims should be observed for at least 8 hours to detect deterioration, particularly respiratory failure. Under no circumstances should anyone with dysphagia be given liquids by mouth. The fruit of the nono tree (Morindcr citrijola Linnaeus) has been used for centuries by South Pacific islanders to treat the symptoms of ciguatera fish poisoning (2) and may be helpful in the treatment of tetrodon poisoning. The juice of the fruit is now sold in the United States and elsewhere throughout the world under the trade name “Noni.” The usual dosage is 3-4 ounces of the juice per day, or four capsulesof the concentrate. The product is nontoxic and should be tried. See Refs. 27-29. Preverltion: If one follows the old Mosaic sanitary laws in Deuteronomy 14:9- 10eliminate all scaleless fish from the diet-then puffer poisoning will never be a problem. If one is living in Japan and has a desire to eat fugu, he should purchase the fish from a first-class, authorized restaurant with a licensed puffer cook. However, even following this procedure will not absolutely guarantee food safety. Eating puffers, at best, is a game of Russian roulette. In any event, the skin and viscera of the fish should never be eaten. No cooking or drying procedure destroys the poison.

V.

ICHTHYOOTOXIC FISH

Ichthyootoxism is one of the lesser-known forms of fish poisoning. Ichthyootoxic fish constitute a group of fish that produce a poison generally restricted to the gonads. This group of toxic fish does not include the ichthyosarcotoxic puffers because the poison in puffers is distributed widely throughout the body. The musculature and other partsof the body in ichthyootoxic fish generally are safe to eat. Some of these fish are found only in freshwater. There does not appear to be any particular phylogenetic affinity other than the fact that the fish involved are all members of the class Osteichthyes, the true bony

Fish Toxins

41

fish (see Sec. 1v.C for a description of the class Osteichthyes). Most of the intoxications resulting from the ingestion of ichthyootoxic fish occur during the reproductive season, during which the gonadal activity of the fish is at its peak.

Repre.sewtative Species Family: Acipenseridae (sturgeons) Species: Huso huso (Linnaeus). Sturgeon. Length 6 ft (1.8 m). Distribution: Black Sea, Sea of Azov, Caspian Sea, Mediterranean Sea, and rivers that drain into these seas. Family: Lepisosteidae (gars) Species: Lepisosteus tristoechus (Bloch and Schneider). Alligator gar. Length 19 ft (6 m). Distribution: Rivers of Cuba, bays and coastal waters of the Gulf of Mexico. Family: Esocidae (pikes) Species: Esox lucius Linnaeus. Northern pike. Length 48 in. ( I .2 m). Distribution: Freshwaters of Europe, northern Asia, and North America. Family: Cyprinidae (minnows) Species: Brrrbus barbus (Linnaeus). Barbel. Length 35 in. (89 cm). Distribution: Freshwaters of northern and central Europe. Species: Schizothorctx irltermedius McClelland. Snow trout, marinka. Length 18 in. (46 cm). Distribution: Freshwaters of central Asia. Species: Tirlccrtinccz (Linnaeus). Tench. Length 24 in. (64 cm). Distribution: Freshwaters of Europe. Family: Stichaeidae (pricklebacks) Species: Stichaeus grigorjewi Herzenstein. Japanese prickleback. Length 20 in. (51 cm). Distribution: Freshwaters of Japan and Korea. Family: Cottidae (sculpins, cabezon) Species: S~.orl.’aenichtll?,smrrrmorrrtus (Ayres). Cabezon. Length 30 in. (76 cm). Distribution: Pacific coast of North America.

Ichthyootoxism (Fish Roe Poisoning) Mechanism of Intoxication: Ichthlyootoxism, or fish roe poisoning, results from ingestion of various salt- and freshwater fish of Europe, Asia, and North America, and to a lesser extent, the tropics. The of roemany freshwater and estuarine fish of eastern Europe and Asia are dangerous to eat during their reproductive period,usually March-June. Most ichthyootoxicfisharemembers of thefreshwaterCyprinidaeminnowgenera Burbus, Schizothorclx, and Tinca, found in Europe and Asia, and the genus Srichaeus of the family Stichaeidae, found in Japan and Korea. These fish have caused innumerable intoxications in Europe and Asia. A Pacific North American species of the Cottid genus, Scorpaenichthys, also has produced intoxications. Although cooking is said to destroy most ichthyootoxins, it cannot be relied upon as a completely safe procedure since the poison in some fish appears to be resistant to of ichthyootoxic fish generally are safe to cat during heat. The musculature and other parts the reproductive season. The chemical nature of ichthyootoxins is unknown. For a more comprehensive review of this topic, see Refs. 25 and 48. Cliniccrl Chctracteristics: Symptoms develop soon after ingestion oftheroeand

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consist of abdominal pain, nausea, vomiting, diarrhea, headache, fever, bitter taste in the mouth, dryness of the mouth, intense thirst, a sensation of constriction of the chest, cold sweats, irregular pulse, low blood pressure, cyanosis, pupillary dilation, syncope, chills, dysphagia, and tinnitus. In severe cases there may be muscle cramps, paralysis, coma, and death.Barbus roe usually does not cause death, but fatalities have resulted from eating Schizothom.r roe ( 5 3 ) . Treatment: Treatment is symptomatic. There are no known antidotes. Prevention: Avoid eating the roe of any fish during the reproductive season unless you have positive knowledge that the roe is safe to eat. This preventive advice is particularly pertinent to the freshwater and brackish water fish of Europe andAsia and all tropical marine species. Cooking fish roe cannot be relied upon to inactivate ichthyootoxins.

VI.

ICHTHYOHEMOTOXIC FISH

Ichthyohemotoxic fish consist of a variety of different species of eels. All of the fish of this group are membersof the order Anguilliformes (Apodes). The members of this group are characterized by an eel-like body, abdominal pelvic fins (when present), and an air bladder connected with the intestine by a duct. Gill openings are narrow or slitlike. The scales, if present, are cycloid. The dorsal and analfins are very long and usually confluent (1 26). The research on ichthyohemotoxicfish, or fish having toxic blood, reveals that there isverylittleclinical infomlation involving humans. Hemotoxins are largely parenteral poisonsandseldomtoxicwhentaken by mouth. Verylittle is known concerning the chemical nature of these posions.

Representcltive Species Family: Anguillidae (freshwater eels) Species: Anguilla anguilla (Linnaeus). Common European eel. Length 39 in. ( I m). Distribution: Europe, fresh- and saltwater rivers. Family: Congridae (conger eels) Species: Conger conger (Linnaeus). Conger eel. Length 79 in. (2 m). Distribution: Atlantic Ocean, Mediterranean Sea. Family: Muraenidae (moray eels) Species: Muraetza helena (Linnaeus). Moray eel. Length 59 in. (1.5 m). Distribution: Eastern Atlantic and Mediterranean Sea.

1chtk~ohemoto.rism~to.ris~n Mechanism of Intoxication: Ichthyohemotoxins are largely parenteral poisons, although there are a few instances on record in which individuals have become intoxicated due to ingestion of large quantities of the poison by mouth. Most of the ichthyohemotoxic or fish generally are recognized as good food fish. However, ingestion of fresh blood serum from these fish may cause food poisoning. This is a rare form of marine food poisoning. Clinical Characteristics: Very littleis known concerning the symptomatology of ichthyohemotoxism in humans. Fish serum intoxications may be of two types: syste1nic9 a form that results from drinking fresh, uncooked fish blood, and topical. The symptolns of the systemic form consist of diarrhea, bloody stools, nausea, vomiting, hypersalivation,

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skin eruptions, cyanosis, apathy, irregular pulse, weakness, paresthesias, paralysis, respiratorydistress,andpossiblydeath.Forthetopicalform,thereisasevereinflammatory response when raw eel serum accidentally comes in contact with the eye or the tongue. Oral symptoms consist of burning, redness of the mucosa, and hypersalivation. Ocular contact invokes a severe burning sensation and redness of the conjunctivae, lacrimination, and swelling of the eyelids. Eye irritation may persist for several days. Usually recovery is spontaneous. For a more comprehensive discussion of this subject, see Ref. 48. Treatment: Treatment is symptomatic. There are no known specific antidotes. Prevewtiow: Care should be takenin the handlingof eel blood. Raw eel blood should not be ingested. Cooking is said to destroy the toxic properties of eel blood.

VII. ICHTHYOHEPATOTOXIC FISH The livers of certain edible species of fish are sometimes found to be toxic to eat. Most of the outbreaks of ichthyohepatotoxism have occurred in Japan. Nothing is known concerning the chelnical nature of the poisons involved. It is believed that in some instances the intoxications are due to hypervitaminosis A. The fish involved are members of the class Osteichthyes.

Representdve Species Family: Scombridae (tunas, mackerels, albacore) Species: Scornberomorus niphonius (Cuvier and Valenciennes). Japanese mackerel. Length 39 in. ( 1 m). Distribution: Japan, Korea, China. Family: Serranidae (Sea bass, grouper) Species: Stereolepis ischinagi (Hilgendorf). Sea bass. Length 78 in. ( 2 m). Distribution: Japan, Korea.

I c h t h ~ o h e ~ ~ ~ t ~(Fish t ~ ~ Liver i s t r /Poisoning) Symptoms of ichthyohepatotoxism appear within 30 minutes- 12 hours after ingesting the fish liver. The initial symptoms consist of nausea, vomiting, fever, and headache. The headache may be very severe and is said to be intensely aggravated by the slightest movement of the body, head, or eyes. A mild diarrhea may be present, but abdominal pain generally is absent. The face of the victim usually becomes flushed and edematous, and a macular rash having large patchy erythematous areas develops. Within 3-6 days, desquamation appears. Large areas of skin may peel off around the nose, mouth, head, neck, and upper extremities, and gradually extends over the entire body. Epilation may 30 days. Vesicular formation of the oral result. Desquamation may continue for about mucosa and bleeding from the lips may occur. Orbital pain, joint aches, and cardiac palpitation with a rapid pulse may be present. Victims have complained of a slippery sensation on the tip of the tongue. Most of the more acute symptoms disappear in about 3-4 days. Residual symptoms consist of chapping of the lips, stomatitis, and mild hepatic dysfunction. Recovery usually is uneventful. No fatalities have been reported. The liver may be enlarged, but no jaundice has been observed. Treatment: Treatment is symptomatic. There are no specific antidotes. Prevention: Care should be taken in eating fish livers. In general, the liver is one of the most dangerous parts of a fish to eat. If a fish is poisonous, a greater concentration

Halstead

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of the poison is likelyto be foundin the liver than almost any other part of the fish. Cooking does not destroy the poison. Most outbreaks of ichthyohepatotoxism have resulted from eating fish livers that have been sauted or in soup. The toxicity of of fish liver cannot be determined by its appearance. It is recommended that fish livers be eliminated from the diet unless there is reliable information that it is safe to eat.

VIII. ICHTHYOALLYEINOTOXIC (HALLUCINOGENIC) FISH Ichthyoallyeinotoxism, or hallucinogenic fish poisoning, is caused by ingesting certain types of reef fish known to occur in the tropical Pacific and Indian Oceans. This biotoxication may result from eating either the head or flesh of the fish. The source and chemical nature of the poison is unknown. Most of the fish species incriminated in ichthyoallyeinotoxism also are involved in ciguatera fish poisoning. Whether there is a relationship between these two types of intoxications is not known. All of the hallucinogenic fish are members of the class Osteichthyes.

Representative Species Family: Kyphosidae (sea chubs) Species: Kyphosus citwrmcetIs (Forskil). Sea chub. Length 20 in. (50 cm). Distribution: Indo-Pacific. Family: Mugilidae (mullets) Species: Mugil cephalus (Linnaeus). Common mullet. Length 12 in. (30 cm). Distribution: Cosmopolitan. Family: Mullidae (Goatfish, surmullets) Species: Upetleus arge Jordan and Evermann. Goatfish. Length 12 in. (30 cm). Distribution: Indo-Pacific.

Ichtkgocrllyeinott,.risrn (Hdlucinogenic Fish Poisoning) Mech~rnistnqf Intoxication: Ichthyoallyeinotoxism, or hallucinogenic fish poisoning, is caused by eating the flesh or head of certain species of toxic reef fish, producing hallucinations. The poison reputedly is concentrated in the head of the fish, which is said to be the most dangerous part of the fish to eat. The nature of the poison is unknown. This type of biotoxication is sporadic, uncommon, and completely unpredictable. You cannot detect a hallucinogenic fish by its appearance. The poison is not destroyed by cooking. Clirlicrrl Chnrcrcteristics: The poison affects primarily the central nervous system. 2 hours after ingestion of the fish, continue The symptoms may develop within minutes to for about 24 hours, and then gradually subside. Symptoms consist of dizziness, loss of equilibrium, lack of motor coordination, hallucinations, and mental depression.A common complaint of the victim is that it feels as though “someone is sitting on my chest,” or there is a sensation of a tight constriction around the chest. The conviction that they are going to die or other frightening nightmares are characteristic aspects of the clinical picture. Other complaints consist of itching, burning of the throat, muscular weakness, and, rarely, abdominal distress. No fatalities have been reported. This form of poisoning is generally mild. Treatment: Treatment is symptomatic. No specific antidote is available. Prevention: Caution should be exercised in eating those species of reef fish that

Fish Toxins

45

have been incriminated in ichthyoallyeinotoxism. When possible, natives should be consulted before eating the fish in tropical areas. Hallucinogenic fish cannot be detected by their appearance.

ACKNOWLEDGMENT It is with deep appreciation that I acknowledge the technical assistance Medrano in the preparation of this chapter.

of Leonette C.

REFERENCES 1. ED Goldberg. The Health of the Oceans. Paris: Unesco Press, 1976. 2. BW Halstead. Poisonous and Venomous Marine Animals of the World. Washington, DC: U S . Government Printing Office, 1970. 3. M Aubert. Mediators of microbiological origin and eutrophication phenomenon. Mar Pollut Bull21:24-29,1990. of poisoning from the ingestion of red seaweed 4. BW Halstead, RL Haddock. A fatal outbreak Gmcilurin fsudoe in Guam-A review of the oral marine biotoxicity problem. J Natl Toxins l(l):87-Il5, 1992. of 5. T Yasumoto, A Endo, D Yasumura, H Nagai. M Murata, M Yotsu. Bacterial production tetrodotoxin and its derivatives. In: P. Gopalakrishnakone, CK Tan, eds. Progress in Venom and Toxin Research. Singapore: National University of Singapore, 1987, pp 310-313. 6. T Yasumoto. D Yasumura, M Yotsu, T Michishita, A Endo, Y Kotaki. Bacterial production of tetrodotoxin and anhydrotetrodotoxin. Agric Bioi Chem 50:793-795, 1986. 7. T Noguchi, D-F Hwang, 0 Arakawa, K Diago, S Sato, H Ozaki, N Kawai, M Ito. K Hashimoto. Palytoxin RS the causative agent in the parrotfish poisoning. In: P Gopalakrishnakone and CK Tan, eds. Progress in Venom and Toxin Research. Singapore: National University of Singapore, 1987, pp 325-335. 8. Y Kotaki, Y Oshima, T Yasumoto. Bacterial transformation of paralytic shellfish toxins in coral reef crabs and a marine snail. Bull Jpn SOCSci Fish 51:1009-1013, 1985. 9. T Noguchi, A Uzu. K Koyama, J Maruyama, Y Nagashima. K Hashimoto. Occurrence of tetrodotoxin as the major toxin in a xanthid crab Aterh.crtispo,-idirs. Bull Jpn Soc Sci Fish 4911887-1892,1983. IO. MNV Dimanlig, FJR Taylor. Extracellular bacteria and toxin production in protogonyaulax species. In: DM Anderson, AW White, DG Baden, eds. Toxic Dinoflagellates. New York: Elsevier,1985,pp103-108. ProtogonymI~~x 1 1 . T Ogata, M Kodama, T Ishimaru. Toxin production in the dinoflagellate mnorensis. Toxicon25:923-928,1987. S Sakamoto.UShimidu.Production of paralytic 12. TOgata,MKodama,KKomaru, shellfish toxins by bacteria isolated from toxic dinoflagellates. In: E Graneli, B Sundstrom, LEdler, DMAnderson,eds.ToxicMarincPhytoplankton.NewYork:Elsevier,1989, pp 311-315. 13. M Kodama, T Ogata. S Sato. Bacterial production of saxitoxin. Agric Bid Chem 52: 10751077,1988. 14. M Kodama, T Ogata. Toxification of bivalves by paralytic shellfish toxins [abstract]. Asia Pac J Pharm 3:99-109, 1988. B Sund15. M Kodama. Possible links between bacteria and toxin production. In: E Graneli, strom, L Edler,DM Anderson, eds. Toxic Marine Phytoplankton. New York: Elsevier, 1990, pp 52-61.

46

Halstead

16.TYasumoto.Production ofparalyticshellfishtoxinsbybacteriaisolatedfromtoxic dinoflagellates. In: E Graneli.B Sundstrom, L Edler, and DM Anderson. eds. Toxic Marine Phytoplankton. New York: Elsevier, 1989, pp 3-8. 17. EM Cospcr, C Lee, EJ Carpenter. Novel "brown tide" blooms i n Long Island elnbayments: asearchforthecauses.In:EGraneli.BSundstrom.LEdler, DMAnderson,eds.Toxic Marine Phytoplankton. New York: Elsevier. 1990, pp 17-28. 18. MKodama.TOgata. S Snkamoto.Possibleassociation ofnlarinebacteriawithparalytic shellfish toxicity of bivalves. Mar Ecol Prog Ser 61:203-206. 1990. ofparalytic 19. MKodama. T Ogata, S Sakamoto, S Sato, THonda,TMiwatani.Production shellfish toxins by a bacterium Momrelltr sp. isolated from PI-otogorl?nlrlcr.r fcrrrtcmt~.sis.Toxicon28:707-714,1990. 20. Y Shimizu, S Gupta, H-N Chou. Biosynthesis of red tide toxins. In: S Hall, G Strichartz. eds. Marine Toxins: Origin, Structure, and Molecular Pharmacology. Washington, DC: American Chemical Society. 1990, pp 21-28. 21. TJ Smayda. Novel and nuisance phytoplankton blooms in the sea: evidence for global epidemic. In: E Grancli, B Sundstrom, L Edler, DM Anderson, eds. Toxic Marine Phytoplankton. New York: Elsevier, 1990, pp 29-40. Ics enlpoisonnernents par les hiutres. les moules, 22. A Chevallier. EA Duchesne. Memoire sur les crabs, et par certains poissons de mer et de riviere [in French]. Ann Hyg Pub Paris 45: 387-45 1, 185 1. 73. HCouticre.Poissonsvenimeux et poissonsvcneneux[inFrench].TheseAgreg Carre et Naud,Paris,1899. 24.ENPawlowsky.Fische.Pisces.In:GifttiereUndlhreGiftigkeit [in German]. Jcna: Verlag von Gustav Fischer, 1927. pp 245-247. of theWorld.Washington,DC: 25.BW Halstcad.PoisonousandVcnolnousMarineAnimals U.S. Government Printing Office, 1967. 26.BW Halstead.Marinebiologicalhazards.JOceanTechno12(3):46-49.1968. 27. PS Auerbach. Clinical therapy of marine envenomation and poisoning. In: AT Tu, ed. Handbook of Natural Toxins. Vol. 3. Marine Toxins and Venoms. New York: Marcel Dekker, 1988, pp 493-565. 28. BW Halstead, PS Auerbach. Dangerous Aquatic Animals of the World. Princeton. NJ: Darwin Press, 1992, pp 241-255. 29.BW Halstead,PSAuerbach,DCampbell.AColourAtlas of DangerousMarineAnimals. Boca Raton. FL: CRC Press, 1990, pp182-186. 30. HEngelsen. 0111 Giftfiskoggiftigefisk[inNorwcgian].NordHygTidskriff 3316-325, 1922. 31, OV Linstow. Die Giftthiere und ihre Wirkungauf den Menschen [in German]. Berlin: August Hirschwald, 1894, pp 53-59. 32. U Anthoni, C Christopherson, L Gram. NH Nielsen, P Nielsen. Poisonings from flesh of the Greenland shark Sornirlos1r.s ,,lic,occ.l'ltlc/trs Inay be due to trimethylamine. Toxicon 29(10): 1205-1212,1991. 33. HB Bigelow, IP Farfante, WC Schroeder. Fishes of the Western North Atlantic. New Haven, CT: Sears Foundation for Marine Research, Yale University, 1948. Las masdel ralnomaritimo 34.AParra.Descriptiondediferenrespiezasdehistorianatural. representadas en setents ycinco laminas [in Spanish]. Havana:En la llnprenta de la Capitania General,1787.pp 1-135. 35. BW Halstead.PoisonousandVenolnousMarineAnimalsoftheWorld.Washington,DC: U.S. Government Printing Office. 1965. of toxiccrabs i n 36.YHashinloto, S Konosu,TYasumoto,AInoue,TNoguchi.Occurrence RyukyuandAlnami islands. Toxicon 5:85-90, 1967. 37. R Bagnis. Concerning a fatal case ofciguatera poisoning i n the Tuanlotu Islands. Clin Toxicol 31579-583. 1970.

Fish Toxins

38.

39. 40. 41.

42. 43.

44.

45. 46. 47. 4s

I

49 50 51 52.

47

KA Steidinger,DG Baden. Toxic marine dinoflagellates.In: DL Spector, ed. Dinoflagellates. Orlando, FL: Academic Press, 1984. pp 201 -261. JS Nelson. Fishes of the World. New York: John Wiley and Sons, 1984. M Murata, AM Legrand, Y Ishibashi, T Yasutnoto. Structures of ciguatoxin and its congener. J Am Chem Soc 11 1:8929-8931. 1989. M Murata, AM Legrand, Y Ishibashi, M Fukui, T Yasumoto. Structures and cOnfigUratiot1s of ciguatoxin from the moray eel G?.i,lttotlro~tcsjrr~,trnieus and its likely precursor from the dinoflagellate Garrrhidi.sclr.s toxicrrs. J Am Chetn SOCI12:4380-4386, 1990. T Yasurnoto, M Murata. Polyether toxins involved in seafood poisoning. In: S Hall and G Strichartz, eds. Marine Toxins: Origin, Structure, and Molecular Pharmacology. Washington. DC: American Chemical Society, 1990, pp 120- 132. c Frelin, M Durand-Clement, J-N Bidard, M Lazdunski. The molecular basis Of CigUatoXin action. In:S Hall and G Strichartz, eds. Marine Toxins. Washington, DC: American Chemical Society,1990, pp 192-199. A" Legrand, P Cruchet, R Bagnis, M Murata. Y Ishibashi. T Yasumoto. Chromatographic and spectral evidence for the presence of multiple ciguatera toxins. In: E Graneli. B Sundstrom. L Edler, and DM Anderson, eds. Toxic Marine Phytoplankton. New York: Elsevier, 1990, pp 374-378. CA Rakotoniaina, DM Miller. Assays for ciguatera-type toxins. In: DM Miller,ed. Ciguatera Seafood Toxins. Boca Raton, FL: CRC Press, 1991, pp 73-86. CW Venable, DM Miller. NMR of marine toxins. In: DM Miller.ed. Ciguatera and Seafood Toxins. Boca Raton, FL: CRC Press, 1991, pp 88-101. E Chungue, R Bagnis. N Fusetani, Y Hashimoto. Isolation of two toxins from a parrotfish Sccrnrs Rihbus. Toxicon15:89-93.1977. BW Halstead. Poisonous and Venomous Marine Animals o f the World. 2nd ed. Princeton. NJ:DarwinPress. 1988. MF Capra, J Cameron. Ciguatera i n Australia. In: DM Miller, ed. Ciguatera Seafood Toxins. Boca Raton. FL: CRC Press, 1991. K Terao, E Ito. TYasumoto.Lightandelectronmicroscopicstudies ofthemurineheart after repeated administrations of ciguatoxin or ciguatoxin-4c. Natural Toxins 1: 19-26, 1992. P Martyr.De Orbe Novo, the Eight Decadesof Peter Martyr d' Anghere. 1912 English translationbyFAMacNutt, 2 vols. New York: G. P. Putnam Sons, 1555. W Harmansen. An Account Taken From The Journal of the Voyages of Admiral Wolphart Harmansen to the East lndies [personal journal, in French]. Unpublished manuscript, 1601. pp 1-18.

53. G Knox. Poisonous fishes as means for prevention of cases of poisoning by them. VoennoMedZh 161399-464, 1888. 54. DR Steinbach. Aus dem Schutzgebiete der Marshall-Inseln [in German. with English translation]. In: F Danckelman, ed. Mittheilungen von Forschungsreisenden und Gelehrten aus den Deutschen Schutzgebieten. Berlin: Emst Siegfried Mittler und Sohn, 1895, pp 157-171. special reference to ciguatera in the 55 EW Gudger. Poisonous fishes and fish poisoning with West Indies. Am J Trop Med 10:43-55, 1930. 56. R Matsuo. Study of poisonous fishes at Jaluit Island [in Japanese]. Nanyo Gunto Chihobyo Chosa Ronbunshu 2309-326, 1934. 57. RL Gilman. Review of fish poisoning in the Puerto Rico-Virgin Islands area. US Navy Med Bull40:19-27,1942. 58. Y Hiyama. Poisonous Fish [in Japanese]. Odawara-shi, Juji, Japan: Japan Marine Product Research Laboratory, 1942, pp 1-24. 59. BW Halstead, WM Lively Jr. Poisonous fishes and ichthyosarcotoxism. US Armed Forces Med J 5:157-175, 1954. 60. Y Hashimoto. A note on the poison of a barracuda. Sphyrctenn picudn Bloch and Schneider. BullJpn Soc Sci Fish 21:1153-1157, 1956.

Halstead

48

61. JERandall.Areviewofclguatcra,tropical fish poisoning,with a tentativeexplanation of its cause. Bull Mar Sci Gulf Carib 8:236-267. 1958. 62. AH Banner, S Sasaki,PHelfrich. CB Alcnder.PJ Scheucr. Bioassay o f ciguateratoxin. Nature 189: 229-230,1961. 63. AH Bmncr, P Hclfrich. The Distribution o f Ciguatcra in the Tropical Pacific. An Epidemiological Survey of Fish Poisoning in the Tropical Pacilic. Technical report no. 3. Honolulu: HawaiiMarineLaboratory,1964. 64. MJ Cooper. Ciguatera and other marine poisonings i n the Gilbert Islands. Pac Sci 18:41 1 440, 1964. 65. PJ Schcuer, W Takahashi, J Tsutsumi, T Yoshida. Ciguatoxin: isolation and chemical nature. Science 155: 1267- 1268, 1967. 66. R Bagnis, F Berglund, PS Elias,GJ van Esch, BW Halstead, K Kojima. Problems o f toxicants i n marine food products. Bull World Health Organ 42:69-88, 1970. 67. DP De Sylva, AE Hine. Ciguatera-marine fish poisoning-a possible consequence of thcrmalpollutionintropical seas'? In:MRuivo, ed. MarinePollutionand Sea Life.Surrey. England: Fishing News Books, 1972, pp 594-597. 68.FERussell.Ciguaterapoisoning: ;I report of 35 cases.Toxicon 13:383. 1975. (grouper) tropical fish poisoning. 69. HR Jones, Jr.Acuteataxiaassociatedwithciguate1.a-typc Ann Neurol 7:49 I , 1980. J 281:948, 70. FM Tatnall, HG Smith, PD Welshy, PC Turnhull. Ciguatera poisoning. Br Med 1980.

71. HA Hanno.Ciguaterafishpoisoning in theVirginIsland[letter].JAMA 245:464, 1981. 72. JGMorrisJr.PLewin,NTHargrctt.CWSmith. PA Blake,RSchneider.Clinicalfeatures of ciguatera fish poisoning: a studyofthe disease i n the U.S. Virgin Islands. Arch Intern Med 142:1090. 1982. 73. NC Engleherg. JG Morris Jr. JN Lewis. JP McMillian, RA Polard, PA Blake. Ciguatera fish poisoning: a major cotnmonsource outbreak i n the U.S. Virgin Islands. Ann Intern Med 98: 336, 1983. 74. NW Withers.Ciguaterafishpoisoning.AnnuRevMed 3397-1 1 I , 1982. 75. AM Gelh, DMildran.Ciguaterafishpoisoning. NY State Med J79:1080,1979. 76.RPearce,CHinesJr.TWBurns, CE Clark.Ciguatera(fishpoisoning).SouthMedJ76: 560. 1983. 77. AM Lcgrand.RBagnis.Mode ofactionof ciguateratoxins. I n : EPRagelis, ed. Seafood Toxins. Washington,DC:AmericanChemicalSociety. 1984, pp 217-223. 78. DR Tindall, RW Dickey, RD Carlson, G Morey-Gaincs. Ciguatoxigenic dinoflagellates from the Caribbean Sea. In: EP Ragelis, ed. SeafoodToxins. Washington. DC: American Chemical Society. 1984. pp 225-240. 34:28-31. 79. Y Hokalna. Anupdate o f methodsforciguatoxindetection.SPCNewslett 1985. 80. JL Allsop, L Martin. H Lebris, J Pollard, J Walsh, S Hodgkinson. Les lnanifestations neurologiques de la ciguartera. RevNeurol 142:590, 1986. 8 I . NC Gillespic. RJ Lewis, JH Peam. ATC Bourke, MJ Hohnes, JB Bourke, WJ Shields. Ciguatera i n Australia: occurrence, clinical features, pathophysiology, and management. Med J Aust 145:584, 1986. 82. AMHHo,LM Fraser, EC Todd. Ciguatera poisoning: a reportofthree cases. Ann E m r g Med 15:1225. 1986. 83. RA Bagnis, AM Legrand. Clinical features on 12,890 cases of ciguatera (fish poiSOnillg) in French Polynesia. In: P Gopalakrishnakone and CK Tan, eds. Progress in Venom and Toxic pp 372-384. Research. Singapore: National University of Singapore, 1987. 84. ND Lewis. Ciguatera in the Pacific: incidence and ilnplications for marine resource development. In:EPRagelis. ed. SeafoodToxins.Washington,DC:AmericanChemicalSociety, 1984, pp 289-306.

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ND Lewis.Diseaseanddevelopment:ciguaterafishpoisoning.Soc SCi Med23:983. 1986. 86. p Gopalakrishnakone, CK Tan. Progress in Venom and Toxin Research. Singapore: National University of Singapore, 1987. 1991. 87. FE Ahmed, ed. Seafood Safety. Washington, DC: Natronal Academy Press, 88. GM Calvert. The recognition and management of ciguatera fish poisoning. In: DM Miller. ed. Ciguatera Seafood Toxins. Boca Raton, FL: CRC Press, 1991, pp 1- 1 1. 1991. 89. DM Miller, cd. Ciguatera Seafood Toxins. Boca Raton, FL: CRC Press, FE Russell, NB Egen. Ciguatcric fishes, ciguatoxin (CTX) and ciguatera poisoning. Toxicol 90. 1991. ToxinRev10:37-62, 91. J Laigret, G Bereziat, G Cuzon. J Polonovski. Etude comparative des acides gras extraits de s provenant de zones toxicogeneset non toxicogenes du lagon poissons C t e t l o d l ~ r m striurus de Tahiti. Bull Soc Pathol Exot 66:235-239, 1973. of ciguatoxin. 92. Y Hokama, AH Banner, D Boyland. A radioimmunoassay for the detection Toxicon15:317-325,1977. 93. PF Parc, R Ducousso, S Chanteau, E Chungue, R Bagnis. Problemes poscs par la detection ilnmunologique dc la ciguatoxine dam les tissus pisciaires [in Frcnch]. Med Oceanogr 14: 1-4,1980. 94. S Chanteau, I Lechat, F Parc, R Bagnis. Essai de detectionladeciguatoxine par une methodc immunoenzymatique [in French]. Bull Soc Pathol Exot 74:227-232, 1981. DC Baden,TYasumoto,MNukina, PJ 95. YHokama,LHKimura, MAAbad,LYokochi, Scheuer, Y Shimizu.An enzyme ilnlnunoassay for the detection of ciguatoxin. In: EP Ragelis, ed. Seafood Toxins. Washington, DC: American Chemical Society, 1984, pp 307-320. of ciguatoxin and related 96. Y Hokama. Simplified solid-phase immunobead assay for detection polyethers. J Clin Lab Anal 4:213-217, 1990. 97. Y Hokama. Immunological analysisof low molecular weight marine toxins. J Toxicol Toxin Rev 10:l-35, 1991. 98. Y Hokama, SAA Honda,MN Kohayashi, LK Nakagawa, AY Asahina, JT Miyahara. Monoclonal antibody (MAb) in detection of ciguatoxin (CTX) and related polyethers by the stickenzyme immunoassay (S-EIA) in fish tissues associated with ciguatera poisoning. In: S Natori.KHashimoto, Y Ueno. eds. MycotoxinsandPhycotoxins '88. Amsterdam:Elsevier Science, 1989, pp 303-310. 99. T Yasumoto, M Satake, Y Onuma, J Roux. Toxins involved in ciguatera, clupcotoxism and shark poisoning. Fifth Indo-Pacific Fish Conference, Noumea. New Caledonia, November 1997. 100. DS Jordan, BW Evermann. The Fishes of North and Middle America: A Descriptive Catalogue. Part 1. Washington,DC:SmithsonianInstitution.1896. 101. W Ferguson. On the poisonous fishes of the Caribbean Islands. Trans R SOC Edinb 9:6579, 1823. 102. RT Lowe. A History of the Fishes of Madeira. London: Bernard Quaritch, 1843. 103. A Kramer. Der purgierfisch der gilbcrtinsein [in German]. Globus 79( 12): 181-1 83, 1901, 104. Dl Macht, J Barba-Gose. Pharmacology of Rlr,Ietrlr.spreriosus, or "caster-oil fish." Proc Soc Exp Biol Med 28:772-774, 193 1. 105. Dl Macht, J Barba-Gose. Two new methods for pharmacological comparison of insoluble purgatives. J Am Pharm Assoc 20:556-564, l93 1, 106. SL Taylor, J Hui, DE Lyons. Toxicologyof scombroid poisoning. In: EP Ragelis, ed. Seafood Toxins. Washington, DC: American Chemical Society, 1984, pp 417-430. 107. HA Frank, DH Yoshinaga. Histamine formation in tuna. In: EP Ragelis, ed. Seafood Toxins. Washington, DC: American Chemical Society, 1984, pp 443-451. 1 08. SL Taylor. Histamine food poisoning: toxicology and clinical aspects. CRC Crit Rev Toxicol 17:92,1986. 109. T Kawbata, K Ishizaka, T Miura. Studies on the food poisoning associated with putrefaction 85.

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of marine products. I. Outbreaks of allergylike food poisoning caused by "samma sakuraboshi"(driedseasoned saury) andcannedseasonedmackerel.BullJpnSoc Sci Fish21: 335-340,1955. 1 IO. GM Dack. Food Poisoning, 2nd ed. Chicago: University of Chicago Press, 1956. 1 1 1. HBouder.ACavallo,JBouder.Poissonsveneneuxetichtyosarcotoxisme.BullInst Oceanogr 1-66, 1962. 112. JAWilliamson, PJ Fenner, JW Burnett, JF Filkin.cds.VenomousandPoisonousMarine Animals: Medical and Biological Handbook. Sydney: Universityof New South Wales Press. 1996. 113. JMeier, J, White.eds.ClinicalToxicology ofAnimalVenomsandPoisons.BocaRaton, FL:CRCPress,1995. 114. BW Halstead. Dangerous Marine Animals, 3rd ed. Ccntreville, MD: Cornell Maritime Press. 1995. 115. PSAuerbach, ed. WildernessMedicine.Management of WildernessandEnvironmental Emergencies, 3rd ed. NewYork:Mosby,1995. 116. E Geiger. Histamine content of unprocessed and canned fish. A tentative method of qualitative determination of spoilage. Food Res 9:293-297. 1944. 1 17. K Tsuda, R Tachikawa, K Sakai, C Tammura,S Ikuma, 0 Amakasu, M Kawamura, S Ikutna. O n the structure of tetrodotoxin. Chem Pharmcol Bull 12:642-645, 1964. 1 18. RBWoodward.Thestructureoftetrodotoxin.PureApplChem 9: 49-74,1964. 119. T Namhashi. Mode of aclion of dinoflagellate toxins on nerve membranes. In: VR LoCicero. ed. Proceedings of the First International Conference on Toxic Dinoflagellates. Wakefield, MA: Massachusettes Science and Technology Foundation, 1974, pp 395-402. I n : EP Ragclis, ed. Seafood 120. HS Mosher, FA Fuhrman. Occurrence and origin of tetrodotoxin. Toxins. Washington, DC: American Chemical Society, 1984, pp 333-341. 121. G Strichartz,NCastle.Pharmacologyofmarinetoxins.In: S Halland G Strichartz, eds. Marine Toxins. Washington. DC: American Chemical Society, 1990, pp 2-20. 122. JC Tyler. Osteology, Phylogeny, and Higher Classification of the Fishes o f the Order Plectognathi (Tetraodontiformes). Seattle: U.S. Department of Commerce. 1980. 123. TMatsui, S Hamada, S Konosu. [inJapanese].Bull Jpn Soc SciFish 47535, 1981. 124.TMatsui, S Hamada, K Yamamori.[inJapanese].BullJpnSocSciFish 48:l 179. 1982. 125. T Matsui, S Hamada,C Shimizu. [in Japanese]. Bull Jpn Soc SciFish 48:253, 1982. 126. LS Berg. Classification of Fishes Both Recent and Fossil. Ann Arbor, MI: J. W. Edwards, 1947.

3 Other Poisonous Marine Animals

1. 11.

Ill.

Introduction S I lnvertebratcs S 1 A. Poisonous marine Protista (Protozoa) S1 B. Poisonous cnidarians (Coelenterata) 53 C. Poisonous echinodcrms (sea cucumbers, sea urchins) D. Poisonous mollusks 56 63 E. Poisonousarthropods: crabs andlobsters Vertebrates 66 Poisonous marine turtles B. Poisonous marine tnammals Acknowledgtnent 72 References 72

A.

1.

54

67 68

INTRODUCTION

Although toxic marine fishes, paralytic shellfish poisoning, diarrhetic shellfish poisoning, brevitoxic (neurotoxic) shellfish poisoning, and amnesic shellfish poisoning probably account for the bulk of the foodborne intoxications caused by marine organisms, there are a variety of other toxic marine organisms capableof causing severe oral intoxications and even fatalities. This chapter deals with someof these lesser-known marine biotoxications. The topics discussed have been arranged phylogenetically.

II. INVERTEBRATES

A.

Poisonous Marine Protista (Protozoa)

The phylum Protista, or Protozoa, consists of single-celled microscopic organisms, most of which are free livingand inhabit an aquatic environment. Afew live i n the body fluids of other animals. Most protistans liveas independent cells, but some are grouped as colonies. Marine protistans poisonous to man are largely members of the class Mastigophora of the order Dinoflagellata. Since dinoflagellates exhibit both animal and plant characteris51

52

Halstead

tics (i.e., motility and chlorophyll), theyare claimed by bothzoologistsandbotanists. Hence they sometimes are referred to as plant-animals. They also are designated as phytoplankton. They abound in neritic waters and i n the high seas, ranging from the tropics to polar oceans. Dinoflagellates form an important part of the ocean plankton as synthetic producers of carbohydrates, proteins, and fats. During their periodic maxima, they may cause yellow, brown, green, black, red, or milky local discolorations of the sea. The “blooming” of thesetoxic plankton in excessive numbers frequently causes the mass mortality of the fish and other animals livingin the region. Phytoplankton blooms often areassociated with weatherdisturbancesorweatheralterationsthatbringabout changes in water masses or upwellings. Conditionsmost favorable for the growth of dinoflagellates are found more oftenin coastal waters than far offshore. Dinoflagellate blooms can causeseriouseconomiclosses i n a regionbecause of theirtoxicityandthemass mortalities of fish they cause. Toxic dinoflagellates play a major role as transvectors of poisons that are ingested by a variety of mollusks, causing paralytic, brevitoxic (neurotoxic), and diarrhetic shellfish to.ricus (Adachi and Fukuyo) serves as a poisoning.Thedinoflagellate Gtrmhie~~cli.scu.s transvector of theciguatoxincomplexinciguaterafishpoisoning. In addition,several other species of dinoflagellates are suspected as causative agents in ciguatera fish poisoning; they are A~nphidiniumcarterere (Hulbert), Ostreopsis ovuttr (Fukuyo), Prorocerltrurn conccrvum (Fukuyo), P. l i m r (Ehrenberg), and P. tuexicmrm (Tafall) (1).

Family: Pfiesteriaceae (dinoflagellate). Species: Pfiestericr piscicidn (Steidinger, Burkholder, et al.) Pfiesteria dinoflagellate. Distribution: Maryland, south along the southeastern coast of the United States.

Over the past 20 years there has been an increase in toxic dinoflagellate blooms worldwide which has resulted in mass mortalities of shellfish and finned fish capable of adversely affecting human health. One of the worst of these toxic blooms was caused by a new species of dinoflagellate known as Pfiesteritr pisciciclum, first reported by Burkholder et al. (2) in 1992. It was later determined by Steidinger et al. (3) that this dinoflagellate was a new species, a new genus, and a new family of the order Dinamoebales. This dinoflagellate is unique because of its polymorphic multiphasic life cycle. The dinoflagellate ranges in size from 5 to 250 pm. Moreover, the dinoflagellate produces potent toxin(s), the molecular structure of which has not been elucidated. It is estimated that this dinoflagellate has produced a mass mortality of more than one billion invertebrates and fish along the Atlantic coast involving the Pamlico and Neuse estuaries in North Carolina and the lower shores of Maryland. The cause of the toxic dinoflagellate bloom was due directly to excessive pollution by hog, chicken, and other untreated wastes that were being dumped into the rivers. The dinoflagellate requires fresh finfish or their excreta for excystment and the release of its potent neurotoxin(s). Mechnrrism of Intoxiccrtiorz: Human exposure to Pjiestericc may be in the form of an aerosol or water containing the toxic dinoflagellate or contact with the bodies of fish or shellfish that have been killed by Pfiesteria. Fish that have been killed by Pfiesteria have skin lesions over their bodies and should not be handled.

Other Poisonous Marine Animals

53

Cli~itwIC I ~ ~ ~ r ~ ~ ~ t eContact r i . s t i (with ~ ~ :Pjesteriu toxin may result in paresthesias of the extremities and circumoral area, joint and muscle aches, headaches, itching, nausea, vomiting,abdominalpain,memoryloss,disorientation,sweating,respiratorydistress, emotional changes, and skin lesions (4-6). Twtrtmetrt: The treatment is symptomatic. There is no known antidote. Pretvntiotl: Avoid handling any dead or dying fish having skin lesions in water contaminated by Pfiestcviel. Do not swim in estuaries or coastal waters contaminated by Pje.ster.it/.Persons working with cultures of Pjiesterin should be properly covered with protective clothing and masks to avoid contact with either water or air contaminated with Pje.steri(r toxin. Contaminated fish should not be eaten.

B. Poisonous Cnidarians (Coelenterata) Cnidarians or coelenterates are best known for their stinging abilities. However, i n this chapter, only their toxicity is discussed as it relates to biotoxications by ingestion. Cnidarians. or coelenterates, are simple metazoans having primary radial, biradial, or radiobilateral symmetry. They are composed essentially of two epithelial layers and an internal cavity, the gastrovascular cavity or coelenteron, which opens only throughthe mouth. Another dominant characteristicof the group is the presence of tentacles equipped with stinging nematocysts. The group is characterized further by showing a remarkable degree of polymorphism, having an alternation of generations with a sexual and asexual phase, as well as a specialization of individual polyps as in the siphonophores. A single species may present a variety of forms of either the sessile polyp or the free-swimming medusoid type. For at least a part of their life span, most coelenterates are attached or sedentary. The phylum Cnidaria (Coelenterata) includes three classes: Hydrozoa, the hydroids; Scyphozoa, the jellyfish; and Anthozoa, the sea anemones, corals, and alcyonarians. Apparently hydroids are not used as food. Jellyfish commonly are eaten in Japan and elsewhere, and there have been no reported cases of poisonings. The nematocysts of hydroids and jellyfish contain proteinaceous toxins that are inactivated by heating and gastric juices. However, oral biotoxications have resulted from the ingestion of sea anemones in the Philippines, New Guinea, and Samoa. Palytoxin, which is produced by anthozoan Ptrlyfhoa species. is not discussed in this section because palythoans generally are not considered to be a foodstuff. However, palytoxin has been found to be transvectored by fish and other organisms (7,8).

Represmttrtive Species Family: Actiniidae (sea anemones) Species: Phy.sobr~crchiacInu~~Itr.si(Kent). Lulnane (Samoa). Diameter 2 in. ( 5 cm). Distribution: Indo-Pacific. Family: Actinodiscidae (sea anemones) Species: Rhotfrrctis howc~si(Kent). Matalelei (Samoa). Diameter 4 in. (30 cm). Distribution:Indo-Pacific. Family: Stoichactiidae (sea anemones) Species: Rcrdicrr~tl~u.s p t r u r n o t e r r s i s (Dana). Matamala samasama (Samoa). Diameter 3 in. (8 cm). Distribution:Indo-Pacific.

Halstead

54

Sea Anemone Poisoning Intoxications resulting from the ingestion of poisonous sea anemones in Samoa and other parts of the tropical IndoPacific region have been reported by Farber and Lerke (9) and Martin (lo), and are discussed by Halstead ( 1 1.12) and Hashimoto (13). The nature of the poison is unknown. Mechanistn of Intoxication: Sea anemones commonly are eaten in Samoa and elsewhere in the tropical Indo-Pacific region,but they generally are cooked. Rhodncris howesi (Matalelei) andPhysobmchia douglasi(Lumane) generally are consideredto be poisonous when raw, but safe to eat when cooked. Rarlionthus pnurnotensis (matamala samasama) and some of the other members of this genus are considered to be poisonous to eat raw or cooked. Small children are frequent victims of sea anemone poisoning intheIndoPacific region. Clinical Chorcrcmiytics: The initial symptoms of sea anemone poisoning consist of acute gastritis with nausea, vomiting, abdominal pain, cyanosis, and prostration. Shortly after ingestion of the sea anemones, the victim may become comatose, which may last a period of 36 hours or more. During this comatose period, the superficial reflexes may be absent. The blood pressure and pulse remain normal. Pulmonary edema has been reported. The patient may go into profound shock, and death usually ensues. Trecrtment: The treatment is symptomatic. There are no specific antidotes. Prevention: The safest procedure for prevention is to avoid eating sea anemones.

C.

Poisonous Echinoderms (Sea Cucumbers, Sea Urchins)

Echinoderms include the starfish, sea urchins, and sea cucumbers. They are all members of the phylum Echinodermata. The members of this group are characterizedby their radial symmetry, a body with usually five radii around an oral-aboral axis, calcareous platesthat form a more or less rigid skeleton, or plates and spicules embedded in the body wall. Spines and pedicellariae are presentin the asteroids and echinoids,but are absent in some of the others. The coelom is complex and includes a water vascular system with tube feet. The digestive tract may or may not include an anus. The sexes usually are separate. Echinoderms, with the exception of a few planktonic holothurians, are all benthic and all are marine. The phylum Echinodermata is divided into four classes: Asteroidea, the starfish; Ophiuroidea, the brittle stars; Holothuroidea, the sea cucumbers; and Echinoidea, the sea urchins, heart urchins, and sand dollars. Some species of starfish are reported to be toxic, but little is known concerning the nature of the poisons or their effects on humans. Poisonings have resulted from eating sea cucumbers and the ovaries of sea urchins.

1. Poisonous Sea Cucumbers Sea cucumbers are free-living echinoderms having an elongate, wormlike or sausagelike body, without free arms but with a series of tentacles circling the mouth, located at the anterior end of the body. The intestinal tract is long and looped, terminating in an anus at the posterior end. The skeleton consists of variable-size, irregularly arranged plates embedded in the skin. Tube feet are present but are not situated in a furrow. I n some species of sea cucumber, a number of white,pink,orredtubulesareattached to the common stem of the respiratory trees. These are the so-called organs of Cuvier. or Cuvierian tubules. If the sea cucumber is irritated, the Cuvierian tubules are discharged through the anus. Upon contact with the water, they swell and elongate into sticky slender threads

Other Poisonous Marine Animals

55

that serve to entangle the predator. Only part of the tubules are emitted at any one time, and the expelled tubules are soon replaced by new ones. In some species, the organs of Cuvier are quite toxic, containing large concentrations of holothurin. Holothurians are sluggish creatures, generally moving over the bottom of the sea by means of rhythmic contractions of the body. The tube feet are used principally as organs of attachment. Sea cucumbers are not important i t e m of food for fish, but they are used as food by many Pacific islanders and Asians. Sea cucumbers are sold commercially under the names of “trepang” and “beche-de-mer.” They are prepared by boiling them, causing them to eviscerate, shorten, and thicken. After thorough drying, the trepang is ready for marketing. Trepang is used for flavoring soups and stews.

Represewt~rtiveSpccirs Family: Holothuridae (sea cucumbers) Species: Holotkuriu cwgus (Jaeger). Sea cucumber. Length 12 in. (30 cm). Distribution:Indo-Pacific. Species: Holothuria tl~b1do.w(Gmelin). Sea cucumber. Length 10 in. (25 cm). Distribution: Mediterranean and adjacent Atlantic Ocean.

Scrr C~vnr1x.rPoisorling Very little information is available on the clinical effects resulting from the ingestion of poisonous sea cucumbers. Mechnisrn of I~rto.vicrrtion:Intoxications have been reported from eating poisonous sea cucumbers (14,15). The causative toxins in seacucumbersarecalledholothurins, of sugars, steroid which involve a complex of saponins. Saponins are complex compounds moieties, or triterpenoid moieties and are characterized by forming a durable foam when their water solutions are shaken. See Refs. 12, 13. 16, and 17 for a more detailed discussion of the chemistry of holothurins. Clirlictrl Clltrmcteristics: Reported symptoms of dermal contact with sea cucumber poison are burning pain, redness, anda violent inflammatory reaction. Liquid ejected from the visceral cavityof some species, when contacting the eye, may cause blindness. Nothing appears to be known concerning the symptoms resulting from the ingestion of poisonous sea cucumbers, but fatalities have been reported ( 18-20). Treatrncnt: Treatment is symptomatic. Pharmacological studies suggestthat anticholinesterase agents may be effective in the event of ingestion of holothurin (21). Prevention: Check with the locals prior to eating sea cucumbers. This is particularly important in tropical regions.

2. Poisonous Sea Urchins Sea urchins are free-living echinoderms having globular, egg-shaped, ora flattened body. The viscera are enclosed within a hard shell, or test, formed by regularly arranged plates carrying spines articulating with tubercles onthetest.Betweenthespinesaresituated three-jawed pedicellariae, which are of interest to the venonlologist and have been described at great length elsewhere ( 1 l , 12). Tube feet are arranged in 10 meridian series rather than in furrows. A double porein the testcorresponds to each tube foot. The intestine is long and coiled, and an anus is present. The gonads are attached by mesenteries to the inner aboral surface of the test. The mouth, situatedon the lower surface, turns downward and is surrounded by five strong teeth incorporated i n a complex structure called “Aristotle’s lantern.” Sea urchins move by means of spines on the oral side of the test.

56

Halstead

Represerztc/tive Species Family: Echinidae (sea urchins) Species: P~rracet1trotu.slividus (Lamarck). European sea urchin. Diameter of test 3 in. (7 cm). Distribution: Atlantic coast of Europe, Azores, West Africa. Family: Toxopneustidae (sea urchins) Species: Tri/meusres g r d l l a (Linnaeus). Sea urchin. Diameter of test 4 in. (10 cm). Distribution: Indo-Pacific, Japan, East Africa, Australia. Secr Urchin Poisonitlg

Several species of European and Indo-Pacific echinoids serve as commercial food sources. Only the gonads are eaten, either raw or cooked. During the reproductive season, generally the spring and summer, the ovariesof certain species of sea urchins are reported to develop toxic products that are injurious to man (8,1 1 -13,22-25). Mecllcrnisrn o f Itltoxiccrtiorl: Sea urchin poisoning is the result of ingesting toxic sea urchin gonads. The chemical nature of the poison is unknown. Cliniccrl Characteristics: The symptoms of sea urchin poisoning consist of general epigastric distress, nausea, diarrhea, vomiting, severe migrainelike headaches, and swellto be an allergic type of reaction in some ing of the lips and mouth. There is believed cases. Ttwtmerzt: Treatment is symptomatic. There is no known antidote. Prevewtior1: Care should be taken when eating the ova of known toxic species of sea urchins during the reproductive season. It is especially important to contact the local inhabitants as to the edibility of sea urchin eggs, especially in tropical regions.

D. Poisonous Mollusks Mollusks have been incriminated in a number of types of oral food intoxications aside from paralytic shellfish poisoning, diarrhetic shellfish poisoning, and amnesic shellfish poisoning. Mollusks are members of the phylum Mollusca. Mollusks are unsegmented invertebrates with a soft body and usually secreting a calcareous shell. A muscular foot is present that may be modified to serve various functions. Covering at least a portion of the body is soft skin, the mantle, the outer surfaceof which secretes the shell. Respiration is by means of gills or a modified primitive pulmonary sac. Jaws are present in some species. In four of the five classes, food is obtained by the use of a rasplike device called a radula. In the cone shells and a few others, the radula ribbon is lost and the teeth are modified into hollow, harpoonlike structures that may contain venom. The phylum Molluscais generally divided into five classes: Amphineura, the chitins; Scaphopoda, the tooth shells; Gastropoda, the snails and slugs; Pelecypoda, the bivalves (scallops, oysters, clams); and Cephalopoda, the octopuses, squids, and cuttlefish. Most of the toxic species of mollusks are gastropods, pelecypods, and, rarely, cephalopods.

1. Poisonous Gastropods: Abalone Abalone are gastropod mollusks that have a rough, horny coating that quite frequently is hidden by a thick cover of algae and other growth. The spire of the shell is flattened and the epipoda is bordered with a fringe and tentacles that project around the margin of the shell. Along the margin of the shell in older specimens, there is a single row of holes through which feelers may project and from which water passing over the gills is discharged. The living mollusk projects its head out from under the edge of the shell in the

Other Poisonous Marine Animals

57

area where therow of holes terminate. The tip of the broad muscular foot is pointed backward from under the spiral. The lining of the shell is pearly iridescent. Abalone shell has been used extensively as a source of mother-of-pearl i n the manufacture of buttons. ornaments, and trinkets. The muscular foot is used i n the preparation of soups, chowders, and as steaks. The toxic substance foundin the viscera of abalone is believed to originate through their food web, namely, certain species of seaweeds belonging to the genus L)r~.srrraresticr (26).

Represmtrrtitv Species Family: Haliotidae (abalone) Species: Hrrliotis di.sc~u.s(Reeve). Abalone. Length 6 in. (15 cm). Distribution: Japan.

Abnlorle Viscer-lr Poisorrirrg Abalone poisoning has been reported fro111 eating Hrrliotis di.scw.s and H. siebolcli in Japan (27). Mechcrrrisrrr of 1rlto.riccrtiotr: Poisonings from abalone are theresult of eating the viscera of the mollusk. The custom of eating the entire mollusk is practiced in Oriental countries. Elsewhere, only the muscular foot (abalone steak) is eaten. The tnuscular foot generally is safe. Although only two species of Japanese abalone, H r r l i o t i s discus (Reeve) and H. sieboldi (Reeve), have been incriminated, the viscera of other species of abalone are suspect. Clirriccrl Clrrrmc.tr.ri.stics: The symptoms of abaloneviscerapoisoninghavebeen described as a sudden onset of a burning or stinging sensation over the entire body, followed by an urticarial rash, itching, erythema, pain in the face and extremities, edema. and subsequent development of skin ulceration. It has been observed that the skin lesions are limited to those parts of the body exposed to sunlight, and there is a distinct boundary between covered and exposed parts of the body (26). Tretrtmerrt: Treatment is symptomatic. Pre\wtion: Do not eat the viscera of the abalone. Only the muscular foot or steak should be eaten.

2. Poisonous Gastropods: Turban Shells The turban shells are members of the gastropod mollusk family Turbinidae. They

are related to the top,or trochus, shells of the Trochidae. As their name implies, they generally are conical or top-shaped, spiral, and have a pearly luster on the inner surface of the shell. The turban shells are largely algae feeders. Some of the turban shells have bee11 found to be toxic. Rel~r-e.~~~rltrrtitl~~ Species

Family: Turbinidae (turban shells) Species: Turho crrgyro.sfor,lu.s (Linnaeus). Turban shell. Dialneter 3 in. (8 ~ 1 1 1 ) . Distribution: Indo-Pacific. Species: Tur-hoIr1rrrwwrrrfus (Linnaeus). Turban shell. Dialneter 8 in. (20 cm). Distribution: Indo-Pacific.

T ~ r l ~Shell r t ~ Poisorrirlg Human outbreaks have beenreportedfromeatingturbanshellstaken at M;lrcus Island, the Marianas Islands, and the Western Pacific (12,13.28). Several toxic sLIbstallces

Halstead

58

have been isolated from 7'. trrggrostottlus (Linnaeus), butthe chemical nature of these compounds hasnot been fully determined.A poison has been isolated from T. rwI-tt1oI-utu,s (Linnaeus) thatisbelieved to beidentical to ciguatoxin (29). Saxitoxinhas also been found in the gut of this turban shell. Mechmism of Itltosicertion: The toxic substances apparently are foundin the midgut gland and the gut of theturbanshell and apparently are obtained by feeding on toxic algae. Ingestion of the whole mollusk may cause poisonings in humans. Ciguatoxin and saxitoxin are also believed to occur in these mollusks. See Chapter 2 for further information on ciguatoxin and saxitoxin. Clillicd ChNrcrL.teI-iSt1'c.s: The synlptoms of turban shell poisoning consist of gastrointestinal upset, nausea, vomiting, diarrhea, fatigue, temperature-reversal sensation, and pruritus. In general, the symptoms resemble those of ciguatera fish poisoning (28). TI-ecrttnerlt:Treatment is symptomatic. See Chapter 2, Sec. 1V.C on ciguatera fish. PI-evention: The turban shells are believedto be safe to eatif the viscera are removed. However, since ciguatoxin and saxitoxin Inay be present, to prevent poisoning one should be extremely cautious when eating these shellfish. It is advisable to check with the local inhabitants concerning their edibility.

3. Poisonous Gastropods: Whelks The whelks area large and aggressive familyof carnivorous gastropod mollusksthat range from tropical to polar seas. They have a vertical distribution that ranges from the littoral zone to great depths. Their shells come in a variety of shapes, sizes, and colors. Whelks commonly are observed clambering about on rocks or plowing their way through mud, sand, or gravel, with the muscular foot largely buried in the bottom. When at rest, the mollusk retracts the foot and thereby closes the aperture with a horny operculum. Most whelks are more scavengers than active predators, feeding on dead fish and other scraps, but they tend to shun anything in an advanced state of decay. Some of the whelks feed on live mollusks, and at times may cause considerable damage to oyster beds. Whelk poison is said to be concentrated i n their salivary glands.

Represeutdve Species Family: Buccinidae (whelks, ivory shells) Species: Bcrbylnrlirr jrrpotIiccr (Reeve). Japanese ivory shell. Length 3 in. (7 cm). Distribution: Japan. Species: Neptrrneer to~tiglrrr(Linnaeus). European whelk. Length 4 in. (10 cm). Distribution: Northern Europe. Species: Nepturlerr inteI-scwlpttr (Sowerby). Whelk. Length 6 in. ( I S cm). Distribution: Japan.

Whelk Poisotlitlg Several outbreaksof poisonings fromthe ingestion of the Japanese ivory shell (Btrbylotlicr jcrponi~tr)have been reported in Niigata, Japan (12,13,30,3 1). Human intoxications have been reported by Kanna and Hirai (32) for Neptunea itrtersarlptcr in Japan. Fange has reported finding the toxic substance tetrarnine i n the salivary glands of N. crllticqLdcr from Sweden (33-3). No clinical data were presented in the Swedish reports. M e c j ~ c r j l ic!fActiotl: .~~ Whelk poison is believed to be restricted to the salivary glands of the mollusk. Intoxication results when these glands are ingested in whole shellfish in the raw, cooked, or canned state.In B. jqmliccr, the poison was found to be concentrated in the lnidgllt gland. The poison found in B. jtrporlictr is called surugatoxin and has been

Other Poisonous Marine Animals

59

found to be a potent mydriatic, ganglion blocker, and hypotensive agent (36). Its molecular formula is C22Hl,,BrN,0,,, nlolecular weight 660 (13). Paralytic shellfish poison also has been detected in the digestive glands of N . decemcostlrta (Say) taken in eastern Canada (37). Clirlicrrl Cl~crmcterisfics: The toxic substance present in poisonous whelks istetramine. Tetramine is an autonomic ganglionic blocking agent. Intoxication from tetramine may result in nausea, vomiting, anorexia, weakness, fatigue, faintness, dizziness, thirst, dysuria, mydriasis, aphasia, numbness, photophobia, impaired visual accommodation, and dryness of the mouth. Trecrtrnent: Treatment is largely symptomatic. Preverltiorl: Poisonous whelks are said to be safe to eat if the salivary glands are removed.

4. Poisonous Bivalve Mollusks The pelecypods, or bivalves, have their bodies laterally compressed and are surrounded entirely by the lateral mantle folds, which are greatly enlarged and covered bya longitudinally divided shell. They lack a proboscis and a radula. The byssus gland, which is found at the base of the foot, secretes sticky threads that harden and serve as mooring lines to the substratum. The byssus threads of mussels are a good example of this attachment mechanism. The foot in bivalves is used as a burrowing organ. The mantle cavity lies on both sides of the foot and contains a pair of modified and enlarged gills, or ctenidia, also known as branchia. The stomach is associated with a crystalline style. The nervous system is simple and concentrated in the posterior portion of the body. Most bivalves are dioecious, and fertilization takes place in the water or in the mantle cavity. Pelecypods are largely filter feeders. In addition to paralytic, amnesic, brevitoxic (neurotoxic), and diarrhetic shellfish poisoning, there are other forms of bivalve shellfish intoxications, including callistin, venerupin, giant tridacna clam, and scallop poisoning.

5. Poisonous Bivalve Mollusks: Callistin Shellfish The genus Ctrllista belong to the family Veneridae, a group of bivalve mollusks of the class Pelecypoda. The members of this genus have an ovate shell that is acunlinate on the back and rounded at the front. The surface of the shell in Ccdlista brevi.si~~hotrrrtrr is marked by rough growth lines and is covered with a smooth and polished periostracum, under which light purplish-brown rays are seen on the yellowish background in young specimens. Members of the genus Cullisfa have been incriminated in human intoxications.

Repre.sewtative Species Family: Veneridae (Venus clams) Species: Ccrllisftr hrevisiphotlcrtrr (Carpenter). Japanese callista. Length in. 6 (15 cm). Distribution: Japan.

The history of callistin shellfish poisoning appears to be of recent origin. The first report of an outbreak of poisoning was by Asano et al. (38) and was caused by eating C. brevisil,hnntrttr taken in the vicinityof Mori, Hokkaido, Japanin 1950. There wasa second report of an occurrence in the same region in 1953. The Mori Health Ccnter subsequently banned the sale of this shellfish.

Halstead

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Mechcrnistn ofPoi.soning: Callistin shellfish poisoning results from eating the ovaries of the Ctrllistn bivalve, which contains a high concentration of choline. The shellfish are said to be toxic only during the spawning season, May-September. Clirricrrl Charcrcteristics: The onset of symptoms generally occurs within 1 hour after ingestion of the toxic shellfish. The chief symptoms are itching, flushing of the face, urticaria, a sensation of constriction in the chest, epigastric and abdominal pain, nausea, vomiting, dyspnea, cough, asthmatic manifestations, hoarseness, paralysis or numbness of the throat, mouth, and tongue, thirst, hypersalivation, drop in blood pressure, increase in pulse rate, leucocytosis, sweating, chills, and fever.In general, this biotoxication resembles a severe allergic reaction. No fatalities have been reported. Treatnrent: Treatment is symptomatic.. Antihistamines have been recommended. Pretrntiow:The callistin shellfish are saidto be safe to eat if the ovaries are removed. The ovaries can be identified by smearing a small amount of the whitish- or yellowishcolored gonads ona glass slide and observing the material undera microscope. The female ovary can be identified by the white, granular-appearing ova. The male gonadal substance appears as a milky paste. Cooking does not destroy the toxic principalof callistin shellfish poison. The freshness of the shellfish is not a factor in the occurrence of this disease.

6. Poisonous Bivalve Mollusks: Venerupin Shellfish Venerupin shellfish poisoning was named after the bivalves Dosinicl jcrporliccr and Tq’es serniclecusscrtr~,which are members of the pelecypod family Veneridae. In addition, the Japanese oyster (Crtmostrecr g i p s ) , a bivalve of thefamilyOstreidae,hasalsobeen incriminated as a causative shellfish species. The toxicity of the bivalves is believed to be due to the transvectoring of one or several toxic speciesof dinoflagellates of the genus Prorocerrtrum. One of the agents is thought to be P. rrltrric.le-lebouric~e(12).

RL~l~resenttrtitle Species Family: Ostreidae (oysters) Species: Crcrssostrecr gigas (Thunberg). Oyster. Length 25 in. (25 cm). Distribution: Japan, British Columbia, southern California. Family: Veneridae (Venus clams) Species: Dosinitr jcporriccr (Reeve). Japanese dosinia. Length 3 in. (7 cm). Distribution: Japan. Species: Ttrpes semidecussata (Reeve). Japanese littleneck. Length 2 in. ( 5 cm). Distribution: Japan, British Columbia south to California.

Vcrrerrrpir~Slrellfislr Poisorrirrg Outbreaks of venerupin shellfish poisoning were first reported i n 1957 near Niigata, Japan, and later in the Kanagawa and Shizuoka Prefectures, Japan, during the months of January-April ( 1 3,39). There is no record of this type of shellfish poisioning occurring elsewhere, despite the fact that the species of shellfish involved are found in other parts of Japan and have been introduced into the United States. There donot appear to be any recent outbreaks reported. M t 4 ~ a r r i s mc!fItrtc).~ictrtion:Venerupin poison appears to be transvectored by a toxic species of dinoflagellate of the genus Prorocentrur?~,which is ingested by the shellfish and concentrated in the digestive gland or “liver” of the mollusk. The chemical nature of the poison is unknown. Clinicer/ Cl?nmctPri,stic.s:The symptoms of venerupin, or asari, shellfish poisoning

Other Poisonous Marine Animals

61

usually develop within48 hours of ingesting the shellfish.The initial symptoms are nausea, gastricpain,vomiting,constipation,headache,andmalaise.Bodytemperatureremains normal. Within 36 hours, additional symptoms such as nervousness, hematemesis, and bleeding from the mucous membranes of the nose, mouth, and gums develop. Halitosis is a dominant part of the clinical picture. Jaundice, petechial hemorrhages, and ecchymoses of the skin generally are present, particularly about the chest, neck, and upper portion of the arms and legs. Leucocytosis, anemia, retardation of blood clotting time, and evidence of liver dysfunction have been noted. The liver generally is enlarged, but painless. In fatal cases, the victim becomes extremely excitable, delirious, then comatose. There is no evidence of paralysis or other neurotoxic effects as in paralytic shellfish poisoning. The low mortality rate has been credited to early diagnosis and prompt medical care.In severe cases, the victim usually dies within 1 week. Recovery is extremely slow and the victim remains in a weakened condition for an extended period of time. The fatality rate is about

33%. Trecltment: Treatment is symptomatic, buttheuse of IV glucose, vitamins B, C, and D, and insulin have been recommended. Prevetltim: ShellfishtakenintheShizuokaandKanagawaPrefectures,Japan, should not be eaten during the months of January-April. Ordinary cooking procedures do not destroy the poison. Toxic shellfish cannot be detected by their appearance. The only certain method to determine the safety of the shellfish is to prepare tissue extracts and test them on laboratory animals.

7. Poisonous Bivalve Mollusks: Tridacna Clams Some species of giant c l a m of the family Tridacnidae have shells nlore than 39 in. (1 m.) in length. Tridacna clams are recognized by their massive shells and large irregular teeth set on a broad hinge plate; they can be found burrowed or wedged into coralon the bottom of tropical seas. The shape and color of the shell often blends in with the coral rock, but the giant clam is detected quickly by the brilliant coloration of its siphon edges and mantle. Two species of the giant tridacna clams have been incriminated in French Polynesia in human intoxications, which clinically resemble ciguatera fish poisoning. Representntive Species Family: Tridacnidae (tridacna clams) Species: Triductra gigas (Linnaeus). Tridacna clam. Length 54 in. (137 cm). Distribution: Indo-Pacific. Species: Tridclctlo tmrima (Roding). Tridacna clam. Length 14 in. (35 cm). Distribution: Indo-Pacific.

TridLtcm Shelljsh Poisowing Tridacna shellfish poisoning clinically resembles ciguatera fish poisoning (see Chapter 2 . Sec. 1V.C). The symptoms consist of gastrointestinal, vasomotor, and various disturbances, including a loss of motor coordination. Bagnis (40) provides the most complete account of tridacna shellfish poisoning, which involved33 people and a numberof domestic animals that had eaten TridLroln nrnxirncr at Bora-Bora, Society Island, French Polynesia. The poison involved was believed to be of the ciguatoxin complex. Trecrtment: Treatment is symptomatic (see Chapter 2, Sec. 1V.C). Prrvention: It is advisable to check withthelocalinhabitantsprior toingesting tridacna clams.

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Halstead

8. Poisonous Bivalve Mollusks: Scallops Representative Species Family: Pectinidae (scallops) Species: Patinopectin yessoensis (Jay). Scallop. Length 6.3 in. (160 mm). Distribution: Northern Japan.

Sccrllop Poisoning Yasumoto et al. (41 ), Murata et al. (42), and Okada and Niwa (43) reported biotoxications in 1985 resulting from the eating of toxic scallops (Prrtinopectin yessoensi.s) harvested from northeast Japan. They isolated two toxins called pectenotoxin (PTXI and PTXI-5). PTXI was found to be a major component of dinophysotoxin (DTX) isolated from the mussel Mytilis eclulis taken from the northeast coast of Japan (41). Dinophysotoxin derives its name because of its association with the dinoflagellate Dinophysis forti;. Dinophysotoxin has also been found in toxic mussels found in Bantry Bay, Ireland (43). Another toxin, yessotoxin (YTX), has also been isolated from P. yessoensis by Murata et al. (42). Mechcrnism sf’ Action: Scallop poisoning is caused by eating poisonous scallops containing pectinotoxin or yessotoxin. Clinicnl Cl~trr.ncteri.stics:The symptoms generally consist of nausea, vomiting, and diarrhea. Neurological disturbances are generally not present. Trecrtnrent: The treatment is symptomatic. There is no known antidote. Prevention: Check with the local public health authorities if there is any question as to their edibility.

9. Poisonous Cephalopods Cephalopods are nlollusks of the class Cephalopoda that have bilateral symmetry and a well-developed head containinga circumoral crown of mobile appendages bearing suckers and/or hooks (exceptin Nrrutilus). The mouth has chitinous, beaklike jaws and a chitinous tonguelike radular band of teeth. The shell is variously modified, reduced, or absent and is enveloped by the mantle; an external shell occurs only in Nmrtilus, which is restricted to the Indo-Pacific area. They are soft-bodied animals, with their primary skeletal features consisting of a cranium-a mantle/fin support comprised of a cuttlebone or gladius. The central nervous system is highly developed and is associated with well-organized eyes. A funnel or siphon tube expels water from the mantle or body cavity. The coloration of cephalopods is variable, depending upon the species and habitat. Most cephalopods have numerous chromatophores and iridocytes i n the skin to accommodate rapid changes in colors and patterns. The size of cephalopods varies greatly, from less than 1 in. (2.5 cm) to 70 ft (20 m) in length and weighing over 1 ton. Locomotion is achieved by drawing water into the mantle cavityand expelling it i n a jetlike manner through the funnel. Octopi are sometimes observed crawling alongthe bottom of the sea on their a r m . Cephalopods are largely predatory and feed carnivorously on crustaceans, bivalves, and fish. Human intoxications resulting from the ingestion of octopus and squid have been reported. Repwsentrrtil>e Species Family: Ommastrephidae (squid) Species: Ommnstrephes slotrni p~rc$c~u,s(Steenstrup). Pacific squid. Length 12 in. (30 cm) Distribution: Pacific Ocean. Family: Octopodidae (octopi)

Other Poisonous Marine Animals

63

Species: Octopus vulgcrris (Lamarck). Octopus. Length 32 in. (80 cm). Distribution: Warm temperature and tropical waters, all oceans.

Cepkolnpod Poisorritrg Intoxications resulting from the ingestion of cephalopods generally are rare. However, a series of 779 outbreaks involving 2974 persons occurred in Japan from 1952 to 1955 caused by the ingestion of squid and octopus. Fatalities were reported (44). Apparently there have been no recent outbreaks reported. Meckrrrrisnr of 1rrto.ricrrtiorr:The nature of the poison involved in cephalopod poisonings is unknown. It is unknown whether bacteria may have playeda role in these cephalopod outbreaks. ClitriccrlCIr(1rcrcteristic.s: Symptoms did not develop in the Japanese outbreaks of cephalopod poisoning until about 10-20 hours after ingestion of the squid or octopus. The symptoms consisted of nausea, vomiting, abdominal pain, diarrhea, low-grade fever, headache, chills, weakness, dehydration, paralysis, and convulsions. Neurological sympa toms were not a dominant part of the clinical picture. Recovery was generally within period of 48 hours. In most instances, cephalopod poisoning resembles severe gastroenteritis, but death may occur. Trecrtrtrerrt: Treatment is symptomatic. There are no known specific antidotes. Prevetrtiorl: TheJapaneseoutbreaks of cephalopodposioningoccurredwithout warning and there was no evidence in the appearance or taste of the cephalopods. Both of the species of squid and octopus incriminatedin the Japanese outbreaks are eaten commonly throughout the world without any ill effects.

E. Poisonous Arthropods: Crabs and Lobsters The phylum Arthropodais the largest single group within the animal kingdom, containing more than 800,000 species. Arthropods are characterizedas having a body usually divided into a head, thorax, and abdomenof like os unlike somites, which are variously fusedand with each segment bearing a pair of jointed appendages. The exoskeleton is chitinous and is molted at intervals. The digestive tract is complete and divided into fore, mid-, and dorsally. The hindgut.The body spaces serve as ahemocoel,and theheartislocated phylum Arthropoda is divided intoa large numberof classes, but only twoof them, Merostomata, which includes the horseshoe crabs, and Crustacea, which includes the lobsters, crayfish, and crabs, are pertinent to toxicologists.

1. Poisonous Horseshoe Crabs The class Merostomata includes the horseshoe crabs or king crabs, which are characterized by an arched cephalothorax, a horseshoe-shaped carapace, a wide unsegmented abdomen possessing three-jointed chelicerae, pedipalpi, and six-jointed legs. They inhabit coastal waters with sandy or muddy bottoms at depths of less than 6 fathoms. Horseshoe crabs are scavengers and feedon polychaete worms, mollusks,a variety of small marine animals, and T d r y i e u s can be and algae. The Asiatic members of the genera Ctr~citroscorpir~s very toxic to eat.

Rcpreserrttltive Species Family: Xiphosuridae (horseshoe crabs, king crabs) Species: Crrrcirroscor1~ilr.srotrrrrrlictrudrr(Latreille). Asiatic horseshoe crab. Length 13 in. ( 3 3 cm).

Halstead

64

Distribution and habitat: Philippines, Indonesia, Malaysia. Species: Tdlypleu.7 gigas (Muller). Asiatic horseshoe crab. Length 19 in. (50 cm). Distribution: Torres Straits, Vietnam, east coast of Bay of Bengal.

Horseshoe Crcrh Poisoning Human intoxications from eating Asiatic horseshoe crabs have been reported by Smith ( 4 3 , Soegiri (46), Waterman (8,47,48), and Banner and Stephens (49). Mostof the reported outbreaks have occurredi n Thailand, but they probably occur elsewherein Southeast Asia wherever these horseshoe crabs are endemic. Horseshoe crab poisoning is referred to as mimi poisoning in Thailand. Mrchmism of Intmicrrtion: Asiatic horseshoe crab poisoning is caused by eating the unlaid green eggs, flesh, or viscera during the reproductive season. Despite their periodic toxicity, the large masses of unlaid green eggs are highly prized by Asiatic peoples (47,48). The poison in Asiatic horseshoe crabs is believed to be chemically identical to saxitoxin (50). Cliniccl/ ChLlrac.teristic,s:The onset of symptoms in Asiatic horseshoe crab poisoning usually occurs within 30 minutes of ingestion of the poison. The initial symptoms consist of nausea, vomiting, abdominal cramps, headache, dizziness, slow pulse rate, decreased body temperature, aphonia, cardiac palpitation, numbness of the lips, parethesias of the lower extremities, and generalized weakness. More-severe symptoms may occur in rapid succession: aphonia, sensation of heat in the mouth, throat, and stomach, inability to lift the arms and legs, generalized muscle paralysis, trismus, hypersalivation, drowsiness, and loss of consciousness. The mortality rate is unknown but is said to be very high. Death, when it occurs, takes place within a period of 16 hours. Trecrtmetzt: Treatment is symptomatic. Prevention: Although Asiatic horseshoe crabs commonly are eaten in many parts of Southeast Asia, they should be avoided during the reproductive season.

2. Poisonous Tropical Reef Crabs The class Crustacea includes the lobsters, crayfish, and crabs. Most of the known toxic reef crabs are members of the crustacean family Xanthidae. The xanthid crabs have a carapace that is transversely oval, hexagonal, or subquadrate, rarely subcircular, and almost always broader than long. The front of the carapace tends to be broad and is never produced in the form of a rostrum. The legs of the crab are of the ambulatory type rather than the swimming type. The family Xanthidae is found throughout the tropical littoral, inhabiting coral reefs. Most of the family feeds on a variety of foods, including algae. Some of the xanthid crabs have been foundto be extremely toxic and have caused human fatalities. Representative Species Family: Xanthidae (mud or reef crabs) Species: Atergutis yoridus (Linnaeus). Reef crab. Width of carapace 2 in. (5 cm). Distribution: Indo-Pacific. Species: Corpilius rntrculntlts (Linnaeus). Spottedreef crab. Width of carapace 4 in. (10 cm). Distribution:Indo-Pacific. Species: Demanin toxiccl (Garth). Poisonous reef crab. Width of carapace 2 in. ( 5 cm).

Other Poisonous Marine Animals

Distribution: Indo-Pacific. ?tl Reef crab. Width of carapace 2 in. Species: PlotypodiL/ ~ 1 y ( 1 1 1 ~ ~ 0 1 7 (Ruppell). cm). Distribution: Indo-Pacific. Species: Zozytmrs aeneus (Linnaeus). Width of carapace 3 in. (7 cm). Distribution: Indo-Pacific.

65

(5

Tropicul Reef Cmb Poisoning Reports of human biotoxications from tropical reef crabs have appeared at infrequent intervals. Most of these intoxications have occurred in the tropical Indo-Pacific region. References 2, 13, and 5 1-65 contain reports on poisonings by tropical reef crabs. Mechrrrlisrn of Itmxiccrtion: The poison contained in Zozymus aeneus, according to Refs. 8 and 66-68, appeared to be chemically identical to saxitoxin. Yasumoto et al. (697 I ) found saxitoxin, neosaxitoxin,and gonyautoxins to be present in various other species of tropical reef crabs including Nemanthias impressus (Lamarck), Actaeodes tornerltosus (H.MilneEdwards), Eriphicr scahricwltr (Dana), Pi1ur~111u.s vespertillo (Fabricius), Schi:.ophrys asperu (H. Milne Edwards), andThnlarnita species, and Halstead and Schantz (72) found themin Perctwn plani,s.sirnum(Herbst). Yasurnuraet al. (73) found tetrodotoxin in the reef crab Zozymus c m c u s . Clirlicnl Chcrrcrcteristics: The symptoms of tropical reef crab poisoning consist of paresthesias, muscular paralysis, aphasia, nausea, vomiting, and collapse. Death may occur within 2 hours to several days (66). The symptoms may resemble those of paralytic shellfish poisoning or tetrodotoxism (see Chapter 2, Sec. 1V.G.). Treatment: Treatment is symptomatic. Prevention: The eating of tropical reef crab is at best an extremely hazardous game of Russian roulette. Tropical reef crabs should not be eaten.

3. PoisonousCoconut Crabs Coconut or robber crabs (Birgus lntro) are land hennit crabsthat are equipped with powerful pincers capable of cutting coconuts from palm trees and opening them. Coconut crabs are capable of climbing palm trees and can do so with considerable agility. Under most circumstances, coconut crabs are eaten and looked upon as a great delicacy, but under certain conditions they may be toxic and may cause fatalities.

Representcltive Species Family: Coenobitidae (coconut crabs) Species: Birgus lntro (Linnaeus). Coconut crab. Length of carapace 5 in. (13 cm). Distribution: Indo-Pacific.

Coconut Crnb Poisonirlg Human biotoxications from coconut crabs have occurred from time to time in the Tuamotu and the Ryukyu Islands in the Indo-Pacific region (8,66,74). Mechanism ofI7?ro.rimrion: Coconut crabs commonly are eaten throughoutthe IndoPacific region, but under certain circumstances they may become extremely poisonous. It is believed that these crabs become toxic by feeding on the roots of certain toxic terrestrial plants. Native islanders in the Tuamotus have suggestedthat one of these toxic plants may be the “Piratea” [Ceoclosrmbrcrcul$ercr (Forster)], a member of the family Hernandiaceae, found in swampyareas on variousPacificatolls (74).Hashimoto(13)reported

Halstead

66

several other poisonous plants that the coconut crab may be feeding on in the Ryukyus, such as Diospyros tt~rrritimrr(Blume Bijdr) of the family Ebenaceae, Herncmdiu .so~~or(r (Linnaeus) of the family Hernandiaceae, and Aloccrsicr mtrcrorrhizc~(Linnaeus) Schott of the family Araceae. Clirlicrrl CI1omcteristics: The symptoms of coconut crab poisoning consistof a violent gastrointesintal upset, headache, chills, joint aches, extreme exhaustion, and muscular weakness. Deaths have been reported. Treatment: Treatment is symptomatic. Prevention: If there is the slightest question concerning the edibility of the coconut crab, avoid eating it. Unfortunately these crabs generally are considered edible anda great delicacy, and reliable local information is difficult to obtain to prevent poisoning. 4. Poisonous Lobsters The term lobster as used in the vernacular sense may include any member of the families Homaridae, the true lobsters; Palinuridae, the spiny lobsters; Scyllaridae, the slipper or shovel lobsters; and the deep-sea lobsters,all of which are members of the arthropod class Crustacea. The true and spiny lobsters are of commercial importance. Lobsters have a rigid, segmented exoskeleton, andfive pairs of legs, one or more pairsof which are modified into pincers or chelae, with the chela on one side usually larger than on the other. The eyes are on movable stalks, and there are two pairs of long antennae. Several pairs of swimmerets are on the elongated abdomen. A flipperlike tail is used for swimming; flexure of the tail and abdomen are used to propel the animal backward. All lobsters are marine and bottom dwelling. Lobsters are nocturnal in habit and scavenge for dead animals, fish, invertebrates, and seaweed. A number of poorly documented accounts have indicated that someof the spiny lobsters in French Polynesia have caused human intoxications. The exact species involved have not been documented (8.74).

Representrrtive Species Family: Palinuridae (spiny lobsters) Species: Paliwurus species. Spiny lobster. Length variable, about Distribution: Indo-Pacific.

12 in. (30 cm).

Lobster Poisorlirlg Lobster poisoning generally is considered to be a rare occurrence and apparently nothing is kllown concerning the nature of the poisons involved or the clinical characteristics of the intoxications they produce. It is assumed that the poisons involved originate through the food chain of the lobster, similar to the toxic tropical reef crabs which inhabit the same reef environment and have similar eating habits. Clirlictrl Cl7arcrcteristics: The clinical characteristics are unknown. Trmment: Treatment is symptomatic. prel~ptltiotl:Data on prevention of lobster poisoning are extremely meager. The best policy is to check with the local inhabitants.

111.

VERTEBRATES

Vertebrate fish poisonings are covered in Chapter 2, so this discussion is limited to poisonous marine vertebrates other than fish, that is, marine reptiles and mammals.

Other Poisonous Marine Animals

A.

67

PoisonousMarineTurtles

Marine turtles are reptiles of the order Chelonia (Testudinata) and are characterized by a broad body encased in a bony shell comprised of a rounded dorsal carapace and a flat ventral plastron that are joined at the sides and coveredby polygonal laminae (i.e., Scut% scales, or leathery skin). The jaws are edentulous and equipped with horny sheaths. The quadrate bone is united to the skull. The ribs are fused to the shell, and the sternum is absent. All turtles, tortoises, and terrapins are oviparous in their reproduction. There are five species of marine turtles that have been reported as poisonous to man.

Represetrtcrtive Species Family: Chelonidae (marine turtles). Species: Ccrretrcrcvrrettrr gigcrs (Deraniyagala). Loggerhead turtle. Length of carapace 47 in. (1.2 m). Distribution: Tropical Pacific and Indian Oceans. Species: Cl?e/orlinmyclcrs (Linnaeus). Green sea turtle. Length of carapace 47 in. (1.2 m). Distribution: All tropical and subtropical oceans. Species: Et-etmockelys irnhricntrr (Linnaeus). Hawksbill turtle. Length of carapace 35 in. (89 cm). Distribution: All tropical and subtropical oceans. Family: Derlnochelidae (leatherback turtles) Species: Dertnochelys coricrcea (Linnaeus). Leatherback turtle. Length of carapace up to 9 ft (2.7 m). Weight of about 1500 Ib (700 kg). This is the largest of all the turtles. Distribution: Largely circumtropical. Family: Trionychidae (soft-shelled turtles) Species: Peloche/ys Dibrotri (Owen). Soft-shelled turtle. Lengthof carapace 6 ft (1.8 m). Distribution: Rivers and coastal areas of Southeast Asia.

Moritre Turtle Poisonins (Cllelotlito~~icrrtio~~) Marine turtles have been reported as poisonous to eat at sporadic intervals over the years. Unfortunately very little specific information is available concerning manyof these outbreaks. References 8,12,52,66,75-82 report on turtle poisoning (chelonitoxications). Mechcrrrisnl o f htoxiccrtiotr : The origin of turtle poison (chelonitoxin) is unknown, of the opinion that the poison but most investigators who have studied the problem are originates in the food chain of the turtle. Most marine turtles appear to be omnivorous, and feed on marine algae, among other things. It is believed that they become poisonous in humans by feeding as a result of feeding on toxic algae. Turtle poisoning may be caused on the flesh, fat, viscera, or blood of various species of tropical sea turtles. The chemical nature of chelonitoxin is unknown. Clitlicd Clrnrcrcteristics: The symptoms of chelonitoxication appear to vary with the amount of turtle ingested and the individual. Symptom develop within a few hours to several days after ingestion. The initial symptoms consist of nausea, vomiting, diarrhea, of the exepigastric pain, tightness of the chest, pallor, tachycardia, sweating, coldness tremities, and vertigo. Frequently there is reported an acute stomatitis, consisting of a dry, burning sensation of the lips, tongue, and lining of the mouth, and thirst. Swallowing

68

Halstead

becomes very difficult, and hypersalivation may be pronounced. The tongue develops a white coating, and the breath becomes very foul. The tongue later may develop pinheadsize, reddened pustules. The oral symptoms may be slow to develop, buttheyusually become very severe after several days. The lingual pustules may persist for several months or may break down into ulcers. Some victims develop a severe hepatomegaly with right upper quadrant tenderness. The conjunctivae become icteric. Headaches and a feeling of “heaviness of the head” are reported. Deep reflexes may be diminished. Somnolence is one of the pronounced symptoms present in severe intoxications, and is usually indicative of an unfavorable prognosis. At first the victim is difficult to awaken, then becomes comatose, which is followed rapidly by death. The symptoms presented are typicalof hepatorenal disease. The clinical characteristics of marine turtle poisoning are discussed in Refs. 12,77,78,8I , and 83-85. Trentment: Treatment is symptomatic. There is no known antidote. Prevention: There are no reliable external characteristics indicativeof a toxic marine turtle to aid in prevention of poisoning. Natives frequently will test suspect turtle meat by feeding samples of it to dogs or cats. It is advisable to seek the advice of the local inhabitants before eating marine turtles.

B. PoisonousMarine Mammals Mamnals are characterized by bodies that are covered with hair and skin containing various types of glands. The skull possesses two occipital condyles. The jaws usually have differentiatedteeththatarecontained in sockets. The limbs are adapted variously for walking, climbing, burrowing, swimming, or flying. The feet have claws, nails, or hoofs. The heart is four-chambered, with only a left aortic arch. The lungs are large and elastic. There is a diaphragm between the thoracic and abdominal cavities. The male has a penis and fertilization is internal. The eggs are small or minute and usually are retained in a uterus for development. The females have mammary glands that secrete milk to nourish the young. Body temperature is regulated. Several orders of marine lnamnals have been found to be toxic to eat: the order Cetacea, which includes whales, dolphins, and porpoises; Pinnipedia, which includes walruses and seals; and Carnivora, which includes the polar bear.

1. Poisonous Cetaceans There are only four species of cetaceans (whales, dolphins, and porpoises) that have been reported as toxic to man: the sei whale (Brrlcrenoptercr borealis), white whale (Delphinq>terus leucrrs), sperm whale (Physeter crrtodorl), and Southeast Asiatic porpoise (Neophoerrem phocnenoide). Members of the family Bulnenopteridrre are the finback whales, which includes the sei whale. Some of the members of this family are among the largest of living animals. The largest member of the group, the blue or sulphur-bottom whale [Sibbddus 1nmculu.s (Linnaeus)], attains a length of 98 ft (30 m ) and a weight of 110 tons ( 1 12.500 kg). This is one of the two families of baleen whales i n which the embryonic teeth are replaced by baleen plates in the adult animals. Finback whales frequently are called rorquals, which refers to a whale having folds or pleats. The rorquals are equipped with longitudinal furrows, usually 10 to 100 in number, that are present on the throat and chest. These furrows increase the capacity of the mouth when it is opened. The members of this family are the

Other Poisonous Marine Animals

69

fastest swimmers of the baleen whales. Their food consists largely of euphausiid shrimp, copepods,amphipods,andotherzooplankton.Whalesareconsideredamongthemost healthy of all living mammals since evidence of pathology is seldom observed. The liver of the sei whale is considered to be toxic to eat. The family Monodontidae, or white whales, consists of only two species, the white whale (Dell,hirlcrl,ter-lrs leuccrs) and the beaked narwhal (Momdon monocerosj. They inhabit arctic seas and sometimes ascend rivers. Eating white whale has produced fatalities (86,87). There is no information available concerning the toxicity of the narwhal; apparently they are used for human food at times, but their meat is usually fed to sled dogs. White whales have a body shape similar to members of the Delphinidae. The snout is blunt and there is no beak. There are no external grooves on the throat. White whales usuallylive in schools, sometimes consisting of more than 100 individuals. They feed mainly on benthic organisms, cephalopods, crustaceans, and fish. White whales are of economic importance and are hunted mainly for their skins, which are sold as "porpoise leather." The family Physeteridae, or the sperm whales, inhabitsall oceans. The sperm whale (Physeter-cntodon) attains a large size of up to 65 ft (20 m ) and 55 tons (55,880 kg) in weight. The sperm whale is said to be the only cetacean with a gullet large enough to swallow a human. The characteristic featuresof the sperm whale areits tremendous barrelshaped head and the underslung jaw. The sperm whale feeds on squid, cuttlefish, fish, and elasmobranches. Theoil and fesh of the sperm whale have been reported to be toxic, but the documentation of this is poor (88). The family Delphinidae includes the dolphins and porpoises. They inhabit all oceans and the estuaries of many large rivers; some species may ascend rivers for great distances. Some species seem to prefer warm coastal waters; none of them are found in the polar regions. The term dolphin generally refersto small cetaceans having a beaklike snout and a slender streamlined body, whereas the term porpoise refers to small cetaceans having a blunt snout and a rather stout, stocky body. Their food consists of fish, crustaceans, and cuttlefish. The Asiatic porpoise( N e o l h ) c m n a phocaenoicle.~)ascends estuaries and rivers has been reported as toxic (89-91). for more than 1000 miles (1600 km). Neophocaer~~~

Family: Balaenopteridae (sei whales) Species: Bcrlcrenoptertr borecrlis (Lesson). Sei whale. Length 60 ft (1 8 m). Distribution: Atlantic Ocean, south to the Gulf of Mexico; Pacific Ocean, Bering Sea south to Baja California. Family: Monodontidae (white whales) Species: Delphitlnpter-us 1errccr.s (Pallas). White whale. Length l8 ft (5.5 m). Distribution: Arctic and subarctic seas. Family: Physeteridae (sperm whales) Species: Physeter- ctrtodon (Linnaeus). Sperm whale. Length 60 ft ( 1 8 m). Distribution: Polar, temperate, and tropical seas. Family: Delphinidae (dolphins, killer whales, and pilot whales) Species: Asiatic porpoise [Neophoc-amrrphocvenoides (Cuvierj]. Length 5 f t (1.5 m) for Asiatic porpoise. Distribution: Coastal areas, estuaries, rivers, and lakes of China.

Halstead

Some cetaceans havebeen reported as poisonous to eat including Baltrenoptercr borealis (92), Delphinrryterus 1eucrr.s (86,87), Physeter. catorlot1 (88), and Neophoccretm pkocmrtoides (89-91,93). The subject of poisonous cetaceans hasbeen reviewed by Halstead ( 12.8 1). Mechanism of Intoxication: Sei whale. The liver ofthesei whale (Bcrlcrettopter(t borealis) is reported to be toxic to cat. 1euccr.y) is White whale. The viscera and meat of the white whale (Del~hinrrl~terus in fatalities. reported to be toxic and may result Sperm whale. The oil and meat of the sperm whale (Physeter ccrrodon) may be poisonous in some localities. Asiatic porpoise. The liver, other viscera, and muscle of the Asiatic porpoise (Neophourettcr phocrrettoides) are reported to be poisonous and may cause death. The nature of the poisons has not been determined, but it is suspected that in some instances vitamin A may be the causative agent. Clitticcl1 CIlctracteristics: Seiwhalepoisoning.Theonset of symptomsbegins within 24 hours after ingesting the liver and consists of severe occipital headaches, neck pain, flushing of the face, nausea, vomiting, abdominal pain, diarrhea, fever, chills, photophobia, tearing, and erratic blood pressure. After several days, the victim's lips become dryanddesquamationdevelopsaroundthemouth,graduallyspreadingto the cheeks, forehead,andneck.Thedesquamationusuallydoes not involvetheentirebody.Although jaundice is not reported, there is evidence of liver impairment. Acute symptoms generally subside within 2 days, but the desquamation may continue for a longer period of time. White whale poisoning. Ingestion of the flesh of the white whale may cause death, but little appears to be known concerning the clinical characteristics. Sperm whale poisoning. No information is available on the clinical characteristics of sperm whale poisoning. Asiatic porpoise poisoning. The symptoms of Asiatic porpoise poisoning consist of abdominal pain, nausea, vomiting, bloating, swelling and numbness of the tongue, loss of vision, cyanosis, numbness of various areas of the body, hypersalivation, greenish tinge to the saliva, and muscle paralysis. Death may be rapid, and the fatality rate is said to be very high. Treatment: The treatment of cetacean poisoning is symptomatic. The nature of the poison is unknown. Prelwttion: T o prevent cetacean poisoning, the liver of the sei whale should never be eaten. The flesh of the white whale should never be eaten. The oil and flesh of the sperm whale reportedly are toxic in some areas and should be eaten only with extreme caution. The Asiatic porpoise viscera and flesh should not be eaten.

2. PoisonousWalrusesand Seals Walruses and seals are members of the mammalian order Pinnipedia. The pinnipeds are a group of marine mammals havinga spindle-shaped body and limbs modified into flippers for aquatic locomotion. The toes are included into webs, and the tail is very short. The males usually are larger than the females. The livers of walruses and certain species of seals may at times be poisonous.

Other Poisonous Marine Animals

71

Family: Odobenidae (walruses) Species: 0doI7en~1.srostncrrus (Linnaeus). Walrus. Length 1 1 f t (3.5 m). Distribution: Arctic Ocean, northeast coast of Siberia, northwest coast of Alaska, north to northwest of Greenland, Ellesmere Island. Family: Phocidae (seals) Species: EriSncrthus bnrbotus (Erxleben). Bearded seal. Length 9 ft (2.7 111). Distribution: Inhabits the edge of the ice along the coasts and islandsof arctic North Atnerica and northern Eurasia. Species: N(@10ccr ciwerctr (Peron). Australian sea lion. Length 8 ft (2.4 m). Distribution: South and southwest coasts of Australia. Species: Prrstr hi.s~,irr'tr(Schreber). Ringed seal. Length 4.5 ft (1.4 111). Distribution: Circumboreal, near the edge of the ice, to the North Pole.

Wdrm and S e d Poisot1ing Walruses (Od0berlrrs ro.smrru.s)have been reportedto have toxic livers(94). Several reports have also appeared on the toxicity of the liver of the bearded seal (Erignathus barbatus) (95-100). The Australian sea lion (Neophoca cinerea) hasbeen reported to have poisonous flesh and allegedly has caused produced deaths in humans and dogs (101,102). Mechmism of Itltoxicntiott: Walrus and seal poisoningis usually causedby ingesting the livers of the rogue bull walrus (Odobetlus) or the bearded seal (Erignr~thus),or the flesh of the Australian sea lion (Neophoca).Intoxications are believed to be caused by an excessive intake in vitamin A, which is present in the liver. However, in the case of the Australian sea lion, the nature of the poison is unknown. Clitliccrl Clr~rrcrcteristics:The symptoms of walrus and seal poisoning are said to be sirnilar to that of polar bear poisoning (see Sec. III.B.3). Tretrtnrent: The treatment of walrus and seal poisoning is similar to that for polar bear poisoning (see Sec. III.B.3). Preverltion: There is no reliable method of detecting a toxic walrus or seal by visual examination. Avoid eating walrus and seal livers and the flesh of Australian sea lions.

3. PoisonousPolar Bears Polar bears [Tllcllrlrctos tntrrifitnus (Phipps)] are marine carnivoresof the class Mammalia thatarecharacterizedbyhavingfourorfivetoes;claws;mobilelimbs;completeand separate radii and ulnas, tibias and fibulas; small incisors; and canines that are slender fangs. The only marine carnivore toxic to man is the polar bear. Most rnanmologists are of the opinion that there is only a single species. Relwesertttrtive Species Family: Ursidae (bears) Species: Tlln1arcto.s rnnritirnus (Phipps). Polar bear. Length 8 ft (2.5 m). Distribution: Arctic, circumpolar.

Polar Berrr Poisoning The biogenesis of the poison present in the polar bear has not been established, but it is believed that the toxin is due to the presence of excessive quantities of vitamin A.

72

Halstead

Meckcrnisnr cfb~toxic~rrion: Polar bear poisoning is caused by eating the liver or kidneys, which apparently concentrate large quantities of vitamin A. Clirzicd ChcrrLlc,reristics:The symptoms of polar bear poisoning usually begin about 2-5 hours after ingesting either the kidneys or the liver. The predominant symptoms are intense throbbing or dull frontal headaches, nausea, vomiting, abdominal pain, dizziness, drowsiness, irritability, weakness, muscle cramps, visual disturbances, and collapse. The headaches may become intense during the first 8 hours and may cause insomnia since they are aggravated by lying down. Gradually the headache lessens in severity and may disappear by the following day. Numerous cases have been cited i n which desquamation occurred on various parts of the body, particularly the face, arms, legs, and feet. Tonic a case in which photosensitiand clonic convulsions may be present. Sutton (103) reported zation was a prominent symptom. If fatalities do occur from these intoxications, they are rare. The amount of liver ingested appears to have a direct bearing on the severity of the symptoms. Eskimos believethat ingestion of polar bear liver may result in depigmentation of theskin.However,investigationfails to substantiatethisbelief (104). The clinical characteristics of polar bear poisoning are discussed in Refs. 12,96,99,103, and 105-1 IO. Trear~nent:Treatment of polar bear poisoningis symptomatic. Emetics and laxatives promptly administered sometimes are useful in relieving the severity of the symptoms. The clinical manifestations gradually disappear after ingestion of the toxic meat has been discontinued. Prevelltion: There is no reliable method of detecting toxic polar bear liveror kidneys by visual examination. The age of the bear seems to have no bearing on the edibility of the meat. The liver from cubs has been known to cause intoxication ( 1 1 I ) . In general, it is best to discard polar bear liver or kidneys. If these parts are eaten,they should be eaten in amounts of less than a 0.5 Ib (230 g).

ACKNOWLEDGMENT It is with deep appreciation that I acknowledge the technical assistance Medrano i n the preparation of this chapter.

of Leonette C.

REFERENCES 1.

2. 3.

4.

5. 6.

7.

KA Steidinger, DG Baden. Toxic marine dinoflagellates. I n : DL Spector, ed., Dinoflagellates. Orlando: Academic Press, 1984, pp 201-261. JMBurkholder.EJNoga.CJHobbs,HBGlasgow.New“phantom”dinoflagellateisthe causative agent of major estuarine fish kills. Nature 358:407-410, 1992. KA Steidinger, JM Burkholder, HB Glasgow, CW Hobbs, J K Garrett, EW Truby, EJ Noga. Gen. Et. Sp. Nov. (Pfiesteriaceae. Fam. Nov.), a newtoxic SA Smith. P’esreritr piscicid~~ dinoflagellatc with a complex life cycle. J Phycol 32:1.57-164, 1997. HB Glasgow, JM Burkholder. DE Schmecel, PA Tester. PA Rublee. Insiduous effects of a toxic dinoflagellate on fish survival and human health. J Toxicol Environ Health 46:SOl522, 199.5. DL Matuszak, JL Taylor, C Dickson, GC Benjamin. Toxic pfiesteria-surveillance for hunun disease in Maryland. Md Med J 47(3):144-147, 1998. BHilcman.Pfiesteriahealthconcernsrealized.ChcrnEngNews 75:14-15, 1997. BW Halstead, RL Haddock. A fatal outbreakof poisoning from the ingestion of red seaweed

Other Poisonous Marine Animals

8.

9.

IO. II.

12.

13.

14. 15.

16. 17. 18.

19.

20. 21.

22.

23. 24. 25.

26. 27. 28

29. 30. 31.

32.

73

~ r l ( c ; / ~ t.s[&e ( r ; ~ ~in Guan-areviewofthcoralmarinebiotoxicityproblem. J Natural Toxins l(l):87-l15, 1992. BW H&tcad. Dangerous marine animals, 3rd ed. Centrevilk, MD: Cornell Maritime Press, 1995. LFarbcr,PLerke.Studiesonthetoxicpropcrty of Rkodrrctis /rowe.sii (Matalnalu). In: I KeeganandWVMacfarlane,eds.VenomousandPoisonousAnimalsandNoxiousPlants o f the Pacific Region. Ncw York: Pergamon Press, 1963, pp 67-74. Rlrodactis kowe.sii (Coelenterata). Pac EJ Martin. Obscrvations on the toxic sea anemonc, Sci14(4):403-407,1960. BW Halstead. Poisonous and Venomous Marine Animals of the World. Washington. DC: U.S. Government Printing Office, 1965. BW Halstead. Poisonous and Venomous Marine Animals of the World, 2nd ed. Princeton, NJ:DarwinPress,1988. Y Hashimoto. Marine Toxins and Other Bioactive Marine Metabolites. Tokyo: Japan Scientific Socictics Press, 1979. T Yarnanouchi. On the poisonous substances containcd in holothurians. Pub1 Set0 Mar Biol Lab 4(2-3):183-203, 1955. RFNigrelli. S Jakowska. Effects of holothurin, a steroid saponin from the Bahamian sea cucumber (Actirro/Jygrro p s s i z i ) , on various biological systems. Ann NY Acad Sci 90:884892,1960. PJ Scheuer. Chemistry of Marine Natural Products. New York: Academic Press. 1973. PL Scheuer. Chemical and biological perspcctives contents. In:PJ Scheuer, ed. Marine Natural Products. NewYork:AcademicPress,1978,pp1-299. JB Clcland. Injurics and diseasesof man in Australia attributable to animals (cxcept insccts). J Trop Med Hyg 16:25-47, I91 3. ACastellaniand AJ Chalmers.Venomousanimals (continued): Piscesandamphibia.In: 203Manual of TropicalMedicine.3rded.NewYork:Wm.WochandCo.,1919,pp 241. S Frankel, C Jellinek. Ber essbare Holothurien [in German]. Biochem Z 185:389-391, 1927. SL Friess. Some pharmacological activities of the sea cucumber neurotoxin. Arner lnst Bio SciBull13(2):41,1963. M. Phisalix. Animal venilneux et venins (2 vols.) [in French]. Paris: Masson et Cie, 1922. EN Pawlowsky. Fische. Pisces [in German]. In: Gifttiere Und lhre Giftigkeit. Jena: Verlag von Gustav Fischer, 1927, pp 245-247. KV Earle. Pathological effects of two West Indian echinoderms. Trans R SOCTrop Med Hyg 33:447-452,1940. E Kaiser, H Michl. Die Biocheme der tierschen Gifte [in German]. Vienna: Franz Deuticke Wien,1958. Y Hashimoto, K Noito, J Tsutsurni. Photoscnsitization of animals by viscera of abalones, Hdiotis spp. Bull Jpn Soc Sci Fish 26: 12 16- 122 I , 1960. Y Hashirnoto, J Tsutsumi. Isolation of a photodynamic agent from the liver of abalone, Hdiotis discrrs /rccrlrrai. Bull Jpn Soc Sci Fish 272359-866, 1961. Y Hashimoto,TOkaichi,LD Dang, TNoguchi.Glenodinine,anichthyotoxicsubstance produced by a dinoflagellate, Peridini~mrpolonicurtr. Bull Jpn SOCSci Fish 34:524, 1968. T Yasumoto, Y Kotaki. Occurrence of saxitoxin in a green turban shcll. Bull Jpn Soc Sci Fish 43:207-21 1. 1977. C Sckiguchi,M Yokoyama, E Nishino. T Yonetani, M Shimpo. (NSA) [in Japanese]. Niigata Eiken Gyoho 95: 1, 1960. C Ji-Shcng. Marine toxins and toxicosis in China. In: A. Tu, ed. Tosin-Related D i s e ~ t , s c ~ . New Delhi: Oxford, 1993. pp 237-267. K Kanna, M Hirai. On the Toxicityof Nepturren irrterscdpta Sowerby. Lecture at the annual meting of the Japan Society of Fisheries, Tokyo.

74

Halstead

RFange. An acetylcholine-like salivary poison inthemarinegastropod Ne/7tlt1letl clrltiylta. Nature180(4578):196-197,1957. 34.RFange.Paperchromatographyandbiologicaleffectsofextracts of thesalivarygland of Neprrrnen nrrriqua (Gastropoda). Acta Zoo1 39:39-46, 1958. 35.RFange.Thesalivarygland of Nq7tur1ea cmtiyua. AnnNYAcad Sci90:689-694,1960. 36. S Hall, G Strichartz, E Moczydlowski, A Ravindran, PB Reichardt. The saxitoxins: sources, chemistry, and pharmacology. In:S Hall, G Strichartz, eds. MarineToxins: Origin. Structure, and Molecular Pharmacology, Washington, DC: American Chemical Society, 1990. pp 2965. in easternCanada.Fish 37. A Prakash, JC Medcof,ADTennant.Paralyticshellfishpoisoning Res Board Canada Bull 171:1-87. 197 1 . 38. M Asano, F Takayanagi, Y Furchuhara. Studies on the toxic substances in marine animals. 11. Shellfish poisoning from Ca//isttr hrer,isi/7/?or~~~r~~ Carpenter, occurred inthevicinity o f Mori, Kayabe County [in Japanese]. Sci Pap Hokkaido Fish Sci Inst (7):26-36. 1950. 39. T Akiba. Food poisoning due to oyster and baby clam in Japan, and toxicological effects of the toxic substance. Unpublished manuscript. 40. R Bagnis. A propos de quelques cas d’intoxications par des mollusques due genre “bnitier,” dans une ile de la socit [in French]. Bull Soc Pathol Exot 60:580-592, 1967. 41. T Yasumoto, M Murata. Y Oshima, M Sano, GK Matsumoto. J Clardy. Dimhetic shellfish toxins. Tetrahedron 41:1019, 1985. of yessotoxin. A novel 42. M Murata, M Kumagi, JS Lee, T Yasumoto. Isolation and structure polyether compound implicated in diarrhetic shellfish poisoning. Tetrahedron Lett 28:5869, 1986. in foodpoisoning.JToxicolToxicRev17: 43.K Okada, MNiwa.Marinetoxinsilnplicated 373-384,1998. 44. T Kawabata, BW Halstead, TF Judefind. A report of a series of recent outbreaks of unusual cephalopod and fish intoxications in Japan. Am J Trop Med Hyg 6:935-939, 1957. 45. HM Smith.Apoisonoushorseshoecrab.JSiamSocNatHist9:143-145,1933. 46. DR Soegiri. A case ofMimipoisoning[inDutch].In:CBonneandGMStreef.eds.Geneeskundig Tijdschrift voor Nederlandsch-Indie. Batavia: G. Kolff and Co., 1936, pp880-883. 47.THWaterman.PoisonousPacifichorseshoecrabs.Unpublished,1953. 48. TH Waterman.XiphosurafromXuong-Ha. AmSci41:301-302, 1953. 49. AH Banner, BJ Stephens. A note on the toxicity of the horseshoe crab in the Gulf of Thailand. Nat Hist Bull Siam SOC 21(3-4):197-203, 1966. 50. N Fusetani, H Endo. K Hashimoto, M Kodama. Occurrence and properties oftoxinsinthe horseshoe crab Curcinoscorpius rotundicauda. Toxicon Suppl 3: 165- 168, 1983. 51. AH Banner, JE Randall. Preliminary report on a marine survey of Onotoa, Gilbert Islands. AtollResBull13:43-62,1952. 52. MJ Cooper. Ciguatera and other marine poisonings i n the Gilbert Islands. Pac Sci 18:41 1440, 1964. 53. D Guinot. Les crabes comestibles de I’Indo-Pacifique [in French]. Paris: Editions de la Fondation Singer-Polignac, 1967. 54. A Inoue, T Noguchi, S Konosu, Y Hashimoto. A new toxic crab, Arerh.trti.s.porirllr.s.Toxicon 6.1 19-123, 1968. 55. S Konosu, Y Hashimoto. Occurrence of toxic crabs in the Ryukyu and Amami Islands and similarity of the crab toxin to saxitoxin. S Pac Comm Tech Rep Seminar: 1-6. 1968. 56. Y Hashimoto, S Knosu, A Inoue, T Saisho.S Miyake. Screening of toxic crabs in the Ryukyu andAmami Islands. Bull Jpn Soc Sci Fish 35:83, 1969. 57. S Konosu, A Inoue, T Noguchi, Y Hashimoto. A further examination of the toxicity of three species of xanthid crab. Bull Jpn Soc Sci Fish 35:88, 1969. 58. ACAlcala,BW Halstead.Humanfatalityduetoingestionofthe crab Dermrrria sp. inthe Philippines. Clin Toxicol 3:609-61 l , 1970.

33.

Other Poisonous Marine Animals

75

59. JP Erhardt, P Niaussat. De leventuelle toxicite du decapode brachyoure C~roililrs~ O ~ I L ' C ' . Y I I S Forskl, etude d'excmplaires provenant de I'atoll de Clipperton [in French]. Cah Pac (14): 114-1 15. 1970. 60. Y Hashimoto, S Konosu, M Shibota, K Watanabc. Toxicity of a turban-shell i n the Pacific. Bull Jpn Soc Sci Fish 36: I 163- 1 17 l . 61. S Konosu, T Noguchi, Y Hashimoto. Toxicity of a xanthidcrab Zosirtrfts oerrelrs and several other species i n the Pacific. Bull Jpn Soc Sci Fish 36:715-719, 1970. 62. GE Mote, BW Halstead, Y Hashimoto. Occurrence of toxic crabs in the Palau Islands. Clin Toxicol 3597-607. 1970. 63. YF Tch, JE Gardiner. Toxin from the coral reef crab Lop/toro,7!.rttlr.s pictor. Phartnacol Res Comm 2 2 5 1-256, 1970. 64. JS Garth. Derrrnrrin fo.uica. a new species of poisonous crab from the Philippines. Micronesia 7( 1-2): 179- 183. l97 1. 65. BW Halstead, KW Cox. A fatal caseof poisoning by the red-spotted crab Cmpi/i~tsr r r c t c h t l r . s (Linneaus) in Mauritius. R Soc Arts Sci Mauritius 4(2):27-30, 1973. in 66. Y Hashimoto, S Konosu, T Yasumoto, A Inoue, T Noguchi. Occurrence of toxic crabs Ryukyu and Amami islands. Toxicon 523-90, 1967. 67. S Konosu, A Inoue. T Noguchi, Y Hashimoto. Comparison of crab toxin with saxitoxin and tetrodotoxin. Toxicon 6.1 13-1 17, 1968. 68. T Noguchi, S Konosu, Y Hashimoto. Identity of the crab toxin with saxitoxin. Toxicon 7: 325-326,1969. 69. T Yasumoto, Y Oshima, T Konta. Analysis of paralytic shellfish toxins of xanthid crabs i n Okinawa. Bull Jpn SOCSci Fish 47:957-959, 1981. 70. T Yasutnoto, Y Oshima, M Tajiri, Y Kotari. Paralytic shellfish toxins in previously unrecorded species of coral reef crabs. Bull Jpn Soc Sci Fish 49:633-636. 1983. in 71. T Yasumoto, D Yasumura, Y Ohizutni, M Takahashi. AC Alcala, LC Alcala. Polytoxin two species of Xanthid crabs from the Philippines. Agric Biol Chem 50:163-167, 1986. 72. BW Halstead. ET Schantz. Paralytic Shellfish Poisoning. Rome: World Health Organization, 1984. 73. D Yasumura, Y Oshima. T Yasumoto. AC Alcala. LC Alcala. Tetrodotoxin and paralytic shellfish toxins in Philippine crabs. Agric Biol Chem 50593-598. 1986. 74. R Bagnis. F Berglund, PS Elias, GJ van Esch, BW Halstead, K Kojima. Problems of toxicants in marine food products. Bull World Health Organ 42:69-88, 1970. 75. EH Taylor. Amphibians and Turtles of the Philippine Islands [English edition]. Manila: Bureau of Printing, 1921, pp 162-163. 76 A Loveridge. Reptiles of the Pacific World. New York: Macmillan, 1945. 77 J Bierdrager. A caseof turtle poisoning in New Guinea [in Dutch]. Geneesk Tijdschr Nederlandsch-Indie 76( 18): 1933-1944, 1936. 78 P Deraniyagala. The Tetrapod Reptiles of Ceylon. London: Dula and Co., 1939. 79. A Carr. So Excellent A Fishe. Garden City, NY: Natural History Press, 1967. 80. BW Halstead, WM Lively Jr. Poisonous fishes and ichthyosarcotoxism. Pac Air Forces Conmand Surg Newslett l ( I): 1 -S, 1959. 81. BW Halstcad. Poisonous and Venomous Marine Animals of the World. Washington, DC: U S . Government Printing Office, 1970. 82. dcR Champetier, RN Rasolofonirivia, N Razafimahefa. JD Rakotoson, D Raboson. Intoxications by marine animal venoms in Madagascar (icthyosarcotoxism and chelonitoxism): recent epidetniological data. Bull Soc Pathol Exot 90286-290, 1997. 83. M Kinugasa, W Suzuki. An etiological investigation of a mass outbreak of food poisoning in the Shinchiku caused by eating a sea turtle caught along the coast at the town Koryu. district, Taiwan [English translation from Japanese]. Taiwan lgohkai Zassi 39(74):66-74. 1940. 84. T Romcyn, GT Haneveld. Vergiftigingcn door het eten van schildpadvless (Eretrtroc./te/y,s

76

Halstead irdwicccre) op Nedcrlands Nieuw-Guinea. Nederiandsch Tijschrift voor Genceskunde 1 156- I 159, 1956.

85

86. 87. 88.

89. 90. 91. 92. 93. 94. 95.

96. 97. 98.

99. 100.

101.

102. 103.

104. 105.

106. 107. 108.

109. 1 IO.

111.

100:

VK Pillai. MB Nair, K Ravindranathan, CS Pitchumoni. Food poisoning due to turtle flesh. J Assoc Phys India 10(4):181-187, 1962. V Stefansson. My Lifc with thc Eskimo. New York: Macmillan, 1924, pp 32-35. V Stefansson. Arctic Manual. New York: Macmillan, 1944, pp 98-283. Y Sahashi. Nurtritive value of sperm whale oil and finback whale oil. Sci Papers Inst Phy Chem Res 20(416):245-253, 1933. DJ Macgowan. Porpoise poison. Med Rcp Imp Marit Customs China (27):12. 1884. DJ Macgowan. Poisonous fish in China. Bull US Fish Comm 6: 130- 13 I , 1887. BE Read. Chinese Materia Medica: Fish Drugs. Peking: Peking Natural History Bulletin, 1939 M Mizuta, T Ito, T Murakami, M Mizobe. Mass poisoning from the liver of Sawara and Iwashikujira [English translation from Japanese]. Jpn Med J 1710:27-34, 1957. DJMacgowan.Poisonousfishandfish-poisoninginChina.ChineseRecMissionJ 17(2): 45-49,1886. FH Fay. Carnivorous walrus and some arctic zoonoses. Arctic 13: 111-122, 1960. J Lindhard. Sundhedsforholdene paa "Danmark-Expeditionen" [in Danish]. Hospitalstidcnde 325355347, 1910. A Krogh, M. Krogh. A study of the diet and metabolism of Eskimos[in Norwegian]. Meddel om Gronland 51:11-52, 1913. EO Jordan. Food Poisoning and Food-Borne Infection. Chicago: University of Chicago Press, 1931,pp55-64,1931. K Rodahl, T Moore. The vitamin A content and toxicity of bear and seal liver. Biochem J 37:166-168,1943. K Rodahl. Toxicity of polar bear liver. Nature 164(4169):530-531, 1949. R Rausch. The toxicity of polar bear liver. Unpublished, 1956. WH Leigh. Reconnoitering voyages and travels with adventuresin the new colonies of South Australia. In: Reconnoitering Voyages and Travels with Adventures in the New Colonies of South Australia, Cornhill, London: Smith, Elder, and Co. 1839, p 164. JB Cleland. Injuries from animals. Med J Aust 2(22):491-492, 1942. RL Sutton. Is polar bear liver poisonous? JAMA 118: 1026, 1942. V Stefansson. The Friendly Arctic: The Story of Five Years in Polar Regions. New York: Macmillan,1921. H Khl. Kann die Liber der Fleischfresser gifting sein'? In: R. Ostertag-Stuttgart, ed. Zeitschrift fur Fleisch-und Milchhygiene. Berlin: Verlagsbuchhandlung von Richard Schoetz, 1929; pp 45-49. 0 Boje.Toxin i n thefleshoftheGreenlandshark[inNorwegian]Mcddelom Granland 125(5):1-16,1939. JK Doutt. Toxicity of polar bear liver. J Mammal 21:356-357. 1940. RL Sutton. Is polar bear liver poisonous? JAMA 118: 1026. 1942. W Beckcr, C Klotzsche. Die hypervitaminose A [in German]. Arztl Woch 24545-550, 1955. H Jcghers, H Marraro. Hypervitaminosis A: its broadening spectrum. Am J Clin Nutr 6:335339,1958. EK Kane. Arctic Explorations: The Second Grinnell Expedition in Searchof Sir John Franklin, 1853, '54, '55. Philadelphia: Childs and Peterson, 1856.

4 Shellfish Chemical Poisoning

I. Introduction 78 11. Paralytic Shellfish Poisoning 79 A. Causativc toxin and its source 80 B. Molecular mechanism of action 81 C. Toxin uptake 82 D. Toxin metabolism, transport, and elimination 82 E. Treatment 83 111. Diarrhetic Shellfish Poisoning 84 A. DSP-causing agents and their sources 84 B. Known molecular pharmacology 85 C. Cellularimpactsofokadaicacidactions 85 D. Organandtissueeffects of DSTs 86 E. Toxinuptakemetabolism,transport,andelimination F. Treatment 86 IV. Neurotoxic Shellfish Poisoning 87 A.Thecausativetoxinsandtheirorigins 87 B. Molecular pharmacology of the toxin 87 C. Toxin uptake, metabolism, and distribution 89 D. Toxin elimination 89 E. Whole animal effects 90 F. Treatment for NSP 90 V. Amnesic Shellfish Poisoning 90 A. Causative toxin and its origin 91 B. Molecular target of domoic acid 92 C. Domoicaciduptakebythegut 92 D. Toxin distribution and elimination 92 E. Pathology 93 F. Treatments for ASP 93 VI.MinorShellfishToxins 94 A. Cyclized, peptidic hepatotoxins 94 B. Tetrodotoxin 95 of Toxins 95 VII.CO-Occurrence

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78

VIII.

ShellfishResponse t o Toxins 96

IX. Effect on Toxins by ShellfishPreparations07 X. Concluding Remarks 97 Acknowledgment 98

References 98

1.

INTRODUCTION

Molluscan shellfish have long been a gastronomic treat for humans, a delight that has tragically for some, ended in poisoning and even death. The cause of these poisoning’s can be twofold. First, poor food hygiene may lead to bacterial spoilage of the shellfish, resulting in human illness such as botulism( 1 ), a type of poisoning not unique to shellfish. The second cause, which rarely occurs i n other food sources and is the subject of this chapter, results from the shellfish sequestering toxic compounds. Many shellfish gain sustenance by sieving the water column and feeding upon the minute organisms therein. At times the organisms comprisingthe shellfish’s diet are toxic, so as well as nutrients, shellfish consume compounds that are seemingly harmless to them (2.3) but are toxic to those organisms further up the food chain. These toxic microorganisms are present in the environment as a matter of course, but are usually in numbers too low to present a problem. Their populations do on occasion bloom, intoxicating exposed shellfish to levels harmful tothe consumer. These toxins can remain withinthetissues of theshellfish for days, weeks, or months after exposure of the shellfish to the toxic microalgae. Therearefourmajor shellfish poisoningsyndromescaused by bioaccumulated toxins: paralytic shellfish poisoning (PSP), diarrhetic shellfish poisoning (DSP). amnesic shellfishpoisoning(ASP),andneurotoxic shellfish poisoning(NSP).Themicroalgal blooms thatgiverise to toxicshellfishareincreasing i n frequencyandimpactona globalscale (4). This has beenattributedtofactorssuchasglobalwarming (5,6) and increasedhumanimpactoncoastalwaters,includingagriculturalfertilizerrunoffand wastewater discharge (7,8). Globalization of the toxic shellfish problem may also be due, i n part, to the deposition of toxic microalgae from the ballast water of international shipping into areas where it lacks competitors and thus is able to flourish (9). Evidence of this global increase is the occurrence of toxic algal blooms and associated human intoxications in regions with no recorded history of such events. For instance, New Zealand experienced an NSP episode in 1993 (10) that was the first ever occurrence of this malady outside of the Americas. The public health impact of shellfish poisoning in naive regions is magnified because the local health authorities are inexperienced at recognizing the event unfolding before them, thus delaying implementation of suitable countermeasures. The shellfish that primarily cause human intoxication are the bivalve molluscs. This class of molluscs includes clams and oysters and is characterized by a two-piece hinged shell that protects the soft body. Some of the species reported i n shellfish poisonings are listedinTable 1. Thetoxinsaregenerallyaccumulated i n thedigestivegland,which counts among its functions excretion of digestive enzymes and some nutrient absorption, and in the siphon, the tubular organ that draws water into the animal’s body cavity and gut. Many adult bivalves attach to a solid surface or live in the sediment. This makes them ideal for farming as the main requirements for their growth are a suitable substrate

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Table 1 ShellfishSpeciesConfirmed to HaveCausedHuman Intoxications and the Syndromes Observed

Species

Shellfish poisoning syndrome

Example references

NSP PS P PSP ASP DSP DSP DSP DSP DSP PSP PSP PSP PSP PS P PSP ASP

10 11 12 13 14.15 16 17 15 15 18 19 11 20 21

22 23

and high-quality water from whichto feed. However, farmingcan also exacerbate shellfish toxicity as it concentrates the shellfish population, magnifying the toxic effectof an algal bloom. Rapid international freight and improved shipping technology mean that shellfish can be exported to many countries from a single region. Thus it is possible, that a shellfish poisoning event may occur far from where the product originated. As an example, the Galician Rios in Spain supplies approximately 40% of all European shellfish (24). Shellfish 200 people being hospitalized all over intoxication i n this one region led to more than Europe (25). For these reasons, monitoring shellfish toxicity has become a responsibility incumbent upon industries and government in virtually every region of the world. These programs ensure that shellfish bound for export and domestic consumption, and, at times, those imported. are either toxin free or contain toxin levels far below that which will cause human illness (26,27). These efforts have dramatically decreasedthe possibility of people being poisoned, but the safeguards arenot absolute. Public health authorities, other health professionals. government authorities, and shellfish farmers should therefore be aware of the various shellfish toxins, their effects, and etiology.

II. PARALYTIC SHELLFISH POISONING Paralytic shellfish poisoning (PSP) is the longest studied shellfish poisoning syndrome, with the toxic principle being recognized early this century (28,29). Human intoxications PSP is an are now global (Figure l ) , and i n some countries i n the Asia-Pacific region, almost annual concern (57). As the name suggests, paralysis is a prominent symptom of PSP. In theearlystages of theintoxication,victimstypicallyexperiencetinglingand numbness of the mouth, tongue, face, and extremities. Accompanying these effects may

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;9

\

V

Fig. 1 Regions of the world from where shellfish to havc caused human intoxication havc originated. (Amnesic shellfish poisoning A;diarrhetic shellfish poisoning m; neurotoxic shellfish poisoning V; paralytic shellfish poisoning ). (16,20.22,23,30-56).

be nausea and vomiting. Hypertension is usually evident in PSP victims (S8,59).In severe cases, the patient will exhibit advanced neurological dysfunction suchas ataxia, weakness, dizziness, a sense of dissociation, followedby complete paralysis. Thereis a compounding effect of central nervous system depression, rendering the diaphragm nonfunctional (60), and death mayresult from cardiorespiratory failure. Mortality rates from PSP have reached 40% (22).

A.

Causative Toxin and Its Source

PSP is caused by saxitoxin (STX; C,,,H17N704; molecular weight 299) and its chemical relatives (Figure 2); this family of toxins will be referred to here as the paralytic shellfish to guinea pigs at only S pg/kg when toxins (PSTs). STX is highly toxic, being lethal injected intramuscularly (61) and mice when injected intraperitoneally (29). PSTs are tricyclic molecules with the 1,2,3- and 7,8,9-guanidino groups of STX possessing ~ K , Jof 1 1.3 and 8.2, respectively (62-64). Thus, at physiological pH, the 1,2,3- guanidino carries a positive charge, whereas the 7,8,9-guanidino group is partially deprotonated (65). This polar nature of STX makes it readily soluble in water and lower alcohols but insoluble in organic solvents. It is extremely stable in solution at neutral and acidic pHs, even at high temperatures. However, alkaline exposure will oxidize the toxin to an inactive derivative (66). Both marine and freshwater microalgae produce PSTs. In freshwater, blue-green to freshalgae (Anlrbtrerltr circirdis) producePSTsandcantransfertheirtoxins water shellfish (Altrthyrilr conclollr) (69), although no reports exist of PST intoxication via thisroute.MarineshellfishsequesterPSTsfrommotilemarinemicroorganismscalled dinoflagellates, particularly Ale.randrium catenelllr, A. n~ir~lrtut?~, A. oster!feldii, A . tcrmcrreme, Gymnoclitliw?? ccrtencrturn, and Pyrodit~iuml~trharner~sew r . cor?IpressmI. At this

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STX neoSTX

B1 Gonyautoxin 2 Gonyautoxin 3 decarbamoylSTX

RI

R4 RZ

R3

H OH H H H

H H H H

H H H

H H

OSOS

OSOY

H H

H H H

H

soy

’ In this derivative, a proton replaces of all the structure beyond the wavy line, including R+ Fig. 2 Thc structure of saxitoxin and o f

naturally

occurring chemical variants (62,63,67,68).

time there is a debate concerning the possible production influence on PST production (70,7 I).

of PSTs by bacteria or their

B. Molecular Mechanism of Action PSTs prevent sodium ions from passing through the voltage-sensitive sodium channel (VSSC), binding to the channel with nanomolar affinity (72,73). This large protein, of approximately 260 kDa, spans cell membranes mainly in nerve and muscle. Depolarization of the cell membrane initiates a conformational change in the VSSC, opening a pore that selectively transports sodium ions into the cell. The VSSC is a multisubunit protein, with the largest subunit andthat which contains the pore, the a-subunit, containing four internal of these sequence repeats, it is hypothesized amino acid sequence repeats. Within each that six transmembrane a-helices assemble around the central ion transporting pore (74). Depending on the tissue and VSSC isoform, other smaller proteins, the P-subunits, attach to the a-subunit and affect channel properties such as the speed of ion conduction and channel activation and inactivation rates (75,76). To date, VSSCs have only been detected in animals, occurring in all vertebrates and in most invertebrate phyla, including molluscs (77), jellyfish (781, and flatworms (79). PSTs bind at the opening of the pore that allows the ions to pass through the VSSC (80,81). Whether ion passage is hindered by physical occlusion of the pore, or by an allosteric modulation of the pore’s structure making it unable to conduct ions, is still not fully resolved. Modified STX can havea dramatically altered ability to bind to the VSSC. For instance, the sulfated PST, B 1 (Figure 2), has an affinity 400 times less than STX for the VSSC rat skeletal muscle (73) and neoSTX (Figure 2) is four fold better than STX at binding to the same channel. Crucial also to the ability of STX to bind to the VSSC

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is the charge state of the 7,8,9-guanidino group, with its deprotonation preventing it from ligating to the channel (65). Mammals also express several VSSC isoforms to which STX can bind with only micromolar affinity. They exist in lnatnmalian sensory neurons (82), cardiac muscle(83). skeletal nluscle in the early stages of development (84), and experimentally denervated skeletal muscle (85). Differences in VSSC amino acid sequences underlie this diversity in toxin sensitivity, and mutation of a single amino acid can convert a toxin-insensitive VSSC to a channel that is easily blocked by STX (86).

C. ToxinUptake STX uptake from the gut appears to be quite efficient since no toxin is eliminated i n the feces of test animals (58,87,88). Absorption of drugs and toxins from the mammalian gut mainly occurs by passive processes and not via the specific active transporters for nutrients (89). Passive uptake across the gut epithelium favors nonionized and lipophilic molecules that readily penetrate cell membranes.It is curious thenthat the cationic STX is efficiently absorbed. Of importance for ionizable compounds the likePSTs, the pH of the gut environment affects toxin uptake by altering the charge state of the toxin. For example, since most absorption occurs in the alkaline intestine, in this part of the gut STX will lose a proton from the 7,8,9-guanidiniunl, making it less polar and therefore approaching a state more amenable to diffusion across the lipid bilayer. Also, the overall charge state in some PSTs can be modified by the presence of anionic sulfate groups, which can negate the positive charges provided by the guanidino groups. Passive absorption of charged, hydrophilic compounds occursin the gut by diffusion through the tight intercellular junctions of the epithelium (89). This process, called paracellular diffusion, is restricted to molecules of approximately 5 2 0 0 Da and is unaffected by pH. This exclusion limitisnotabsolute andmaybeaffected by agentsthatsequester calcium and magnesium. The complex biological matrix in which the toxin is ingested may contain natural ion chelating agents, making it easier for PSTs to enter the bloodstream via this route. Although paracellular diffusion is much less efficient than uptake through the epithelial cells, it may be a significant route of toxin absorption, as STX is not much greater than the size exclusion limit, even if not affected by ion chelators. Also, those PSTs significantly smaller than the parent STX molecule, for example, decarbamoylSTX (Figure 2; molecular weight 256), would be better able to pass through the tight junctions.

D. Toxin Metabolism,Transport,andElimination The first point where PSTs may be modified after ingestion is in the gut itself. Gastric acid can convert small amounts of the less toxic sulfated PSTs to more potent compounds (90). This may prove significant if a very large amount of these sulfonated toxins is ingested and critical amounts of the highly toxic STX or neoSTX are produced. Once in the blood, STX is returned to physiological pH, and thereforeto a predorninantly doubly charged and highly toxic state. With STX again being quite polar, there is little possibility of it crossing the blood-brain barrier, which is best traversedby lipophilic agents. This is borne out by a recent study where no toxin reached the brain of cats that received 2.7 pg/kg STX (87). Curiously though, at higher doses ( I O pg/kg) in this same study, STX did enter the brain. This dose dependence of STX’s ability to invade the

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brain may explain earlier reports of STX exerting central effects (60.91,92). One other explanation for this unexpected observation may be a process where charged drugs, to which STX can be equated, binds to plasma proteins (89), some of which may be actively taken across the blood-brainbarrier, carrying STX along with them. PST’s effects on central systems can also be achieved upon regions of the brain not encapsulated by the impermeant blood-brain barrier, such as the circumventricular region. Toxin is eliminated predominantly via the urine, with no elimination of PSTs via the feces (58,87,88). Excretion of PSTs occurs rapidly,no matter how the toxin is administered. For instance, the bulk of IV administered saxitoxinol, a chemically reduced STX, and STX was urinated by rats within hours of injection (66,88). In humans, the half-life after oral ingestion of toxic shellfish is approximately I O hours (58). Metabolic conversion of PSTs while passing through mammals has been little studied, especially in humans. Examination of the serum and urine of human PSP victims revealed a significant increase in Cl compared to its relative gonyautoxin-2, which differ only in an additional sulfate on the C type PST (58). This sulfation of gonyautoxin-2 in humans contrasts with rats, where there was no metabolism of STX or saxitoxinol during their-passage into the rat’s urine (66,88).

E. Treatment Victims should undergo immediate gastric evacuation to remove toxic contents unyet digested and absorbed. This can be achieved not only orally, but also by enemas to clear the intestines. This will also decrease the impact of any conversion of lesser toxic PSTs in the gut. Artificial ventilation for victims of severe PSP is the only recommended treatment to date. Symptomatic relief should accompany all other efforts. The dominant strategy for developing a medicinal treatment has been to use antibodies developed to STX, a carrier protein to make an antigenic epitope, that created against toxin conjugated to may sequester PSTs and out-compete the sodium channel to bind the STX (93-96). If the antibody has a significantly lower affinity than the sodium channel for STX, which was indeed the case with one antibody that possessed a micromolar affinity for the toxin (95). then it must compete for thetoxin by weight of numbers. This would requires administration of large amounts of the antibody. Another limitation of this approach is that the antibodies may be quite specific to single PSTs such as STX; other PSTs to which the victim was exposed maybe able to bypass the antibody and still act. A mixtureof antibodiesmay be necessary if this approach is to succeed. Despite these potential problems, there has been some successi n animal modelswhen treated with STX antibodies, although they have not been tested on PST mixtures (93-96). Since it seems that STX can cross the blood-brain barrier at high doses, antibodies will be ineffective in inhibiting this pool of toxin since they cannot cross the blood-brain barrier. Surprisingly, in guinea pigs, the potassium channel blocker 4-aminopyridine (4-AP) effectively counteracted the effectsof lethal IV doses of STX (61). This drug reverted the loss of blood pressure and enhanced neuromuscular transmission to allow the diaphragm to again function. Large dosesof 4-AP were necessary, however, and may cause serious side effects such as seizures and convulsions. If administered in a hospital, steps can be taken to ameliorate these side effects. A clue to an alternative drug strategy is present in a recent study on cats (87). Cats given lethal doses of STX were kept alive forthe duration of the experiments by continuous administration of the adrenergic agonist, dobutamine, in conjunction with mechanical

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ventilation. In these animals, because breathing was assisted, cardiac rather than respiratory arrest was the cause of death. Dobutanline increases cardiac contractile strength and volume, and providing it along with artificial ventilation, as was done with the cats, may be a suitable emergency medical strategy for PSP victims (87).

111.

DlARRHETlC SHELLFISH POISONING

The firstreport of what became known as diarrhetic shellfish poisoning (DSP) was in 1978 in the Tohoku district in Japan (IS). Since then reports of DSP have emerged from every continent except Africa and Australia (Figure1 ). DSP has never resultedin a human fatality, despite the causative toxins being lethal to mice when injected intraperitoneally (LDi0 of 192 pg/kg) (97). Diarrhea is the major symptom exhibited by victims as well as other gastrointestinal upsets such as vomiting, nausea, and abdominal cramps. These (IS). symptoms may become so severe as to incapacitate the patient

A.

DSP-Causing Agents and Their Sources

The major causative toxin for DSP is okadaic acid (OA) and its relatives, the dinophysistoxins (Figure 3). To be consistent throughout this chapter, the suite of DSP-causing toxinswillbereferredto asdiarrheticshellfishtoxins(DSTs).Okadaicacidderivesits name from the organism from which it was first isolated, the sponge Herlichondricr okcrh i , andwashypothesizedtobeproduced by microorganisms(97).Okadaicacid (C4,HtjXOl3; molecular weight 804) isa complex lipophilic polyether, readily soluble in many organic solvents (99) and sensitive to degradation by acid or base exposure (48). DSP isa somewhatconfusingsyndrome inthatseveralstructurallydistinctpolyether toxin families, the yessotoxins and pectenotoxins, often occur i n shellfish alongside OA and the DTXs and are also referred to as DSTs. Only dinophysistoxin and OA are truly diarrheagenic (IOO), and will be focused upon here, but the other toxins confuse toxicity monitoring results because of their effects on mice used i n the regulatory bioassay. Pectenotoxin is clearly an hepatotoxin (IOO), whereas how yessotoxin mediates its toxicity is still unclear (101); experimental animals injected with it die from cardiac failure (102).

H acid Okadaic Dinophysistoxin-l Dinophysistoxin-2

H H H

CH3 CH3

CH3 CH3 H

Fig. 3 Structure of' thepolyether diarrhetic shellfish toxins, okadaic acid and dino1Jhysistoxins (48,97.98).

Shellfish Chemical Poisoning

B.

85

Known Molecular Pharmacology

Okadaic acid is a very potent inhibitor of several classes of serinekhreonine phosphatase, acting at nanomolar and even picornolar concentrations (107-109). These enzymes are a variable subunit that then multisubunit proteins targeted to their substrate protein by forms a heterodimer with a structurally invariant subunit which catalyses the dephosphorylation of serine and threonine residues ( 1 IO). Serine-threonine phosphatases are subclassified depending upon their substrate specificity, their requirement for cofactors such as to several inhibitory peptides and divalent cations and calmodulin, and their sensitivity okadaic acid itself. OA-sensitive subclasses are PP-l and PP-2A, whereas PP-2C is unaffected by OA,andmicromolaramounts ofthetoxin arerequiredtoinhibitPP-2B ( l 0 9 , l I l , I 12). It is the catalytic subunits of PP-l and PP-2A that are affected by OA, mainly by noncompetitive inhibition (1 13,l 14). These two isoforms are highly homologous, explaining the similarity in their OA sensitivity. The homology is not complete and the differences i n amino acid sequence cause subtle differences in their affinity for OA. A sequence of four amino acids containing two acidic residues from rabbit PP-l can be a replaced by the corresponding amino acids from rabbit PP-2A, which contain instead cysteine and a basic amino acid, arginine. This increased the toxin sensitivity of the chimeric mutant so that it approached that of PP-2A from which the arginine-cysteine sequence was derived ( 1 15). The carboxylic acid of OA is essential to its activity, as demonstrated by methyl esterification, a modification which abolishes OA's abilityto inhibit its target phosphatases ( I 16,l 17). Also, oxidation of the 27-OH greatly reduced its abilityto inhibit PP-2A. From molecular modeling, this portion of molecule is not involved in bonding to the enzyme, but is more likely to be involved in binding the phosphate substrates (1 18). Apart from its acute toxicity, chronic exposure to OA may have a genotoxic effect. At low concentrations. OA forms DNA adducts in mammalian cell lines, between 2 and 100 adducts per 10' bases, a phenomenon not observed at higher concentrations of 2 10 nM ( 1 19). Regular dietary exposure to OA levels that may not elicit DSP may therefore have a teratogenic effect.

C. Cellular Impacts of Okadaic Acid Actions In those cells that take up DSTs, the toxins cause a buildup of phosphorylated proteins. Phosphatases are counterbalanced within the cell by protein kinases,which introduce phosphate groups onto proteins. Since the phosphorylation state of an enzyme, or receptor, often governs whether it is active or not, this interplay between kinases and phosphatases regulates cell function and manyof the cascades which underlie cellular processes. Further, of a kinases and phosphatases can act upon each other. For example, dephosphorylation kinase renders it inactive and phosphorylation of a phosphatase may switch the enzyme into action ( I 1 I ). In the presence of OA, protein kinases continueto function, but in those pathways containing PP- I and PP-2A, which are inhibited, phosphorylated proteins will become overabundant. Thus enzymic cascades and membrane receptors may continue to

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function long beyond their optimal time period, or may not switch on again until the OA is eliminated. Because OA has an effect on such a crucial enzyme, it may result in many effects within the same cell. This, of course, depends upon the efficiency of toxin uptake by the cell and the metabolic status of the cell when the toxin is absorbed, which would govern which subunit the PP-1 and PP-2A catalytic subunits would be dimerized to at the time. Multiple effects can be shown with vero cells, a monkey kidney cell line, where OA inhibited protein, DNA, and RNA synthesis, all at nanomolar concentrations ( 1 20). Certain cell types are also more sensitive to OA than others. For example, in culture, neuronal cells are affected at subnanomolar concentrations, whereas astrocytes and fibroblasts (121) require much more toxin for an effect to be realized. Another case in point would be rat hepatocytes, which require micromolar concentrations of OA for cytotoxicity to be observed ( 122). The role of celltype and cell susceptibility is also demonstrated by the differences between cellular and in vitro effects. Protein synthesis in a cell line was inhibited by SO nM OA, whereas in in vitro studies, generic protein synthesis using a rabbit reticulocyte lysate system was almost 10,000-fold more sensitive to OA, being inhibited by SO% at a concentration of 6.5 pM (120).

D. Organ and Tissue Effects of DSTs DSTsproducetwomajorphysiologicalresponses totheirmolecularactions: an acute effect-diarrhea-and a potentially chronic effect-tumor promotion. Intraperitoneal injection of DTX- 1 desquamates intestinal epithelia (100,123). This, in conjunction with induction of excessive fluid secretion in the intestine ( 124), underlies many of the gastrointestinal effects seen in DSP victims. DSTs alone do not induce tumor formation, but when applied to the skin of mice after a tumor-initiating substance, dimethylbenzanthracene, virtually all test animals developed skin tumors of varying malignancy ( 125,126).

E. Toxin Uptake Metabolism, Transport, and Elimination The sensitivity of these toxins to acid and base exposure would be expected to impact upon the stability of the DSTs while in the human gut before absorption, but the presence of DST degradation products in fecal material has never been reported. If not degraded to a form chemically different from the parent DST, the lipophilicity of the toxins would allow them to passively diffuse through the gut epithelium and toxin uptake would be expected to be quite efficient. Oral administration of tritiated OA to mice resulted in its In pregnant rats, 7% of distribution to liver (2%), kidney (2%), and blood (6%) (127). administered OA crossed the placenta and contaminated the fetal pups (127). Pregnant victims of DSP must receive special care because if DST canalso cross the human placenta it would be concentrated in a single fetus. OA was eliminated in urine, with almost 8% of the administered tritiated OA being detected in the urine within 24 hours (127). Curiously, OA has a secondary effect within the gut of increasing epithelial permeability via paracellular diffusion (128), which may increase uptakeof DSTs themselves or of hydrophilic compounds accompanying these toxins.

F. Treatment DSP is rarely life threatening and usual treatment is to make the patient as comfortable as possible for the durationof the intoxication. Symptomatic treatment for severe diarrhea,

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87

such as fluid replacement, should be employed. Because epithelial destruction by the DSTs contributes to 111uch of the diarrhetic effect, administration of classic antidiarrheals is unlikely to provide any relief.

IV. NEUROTOXIC SHELLFISH POISONING The Gulf of Mexico has a long history of toxic microalgae blooms that cause massive fish kills and respiratory irritation in humans. It was eventually recognized that the toxic to induce what agent from these blooms could also be transmitted to humans via shellfish NSP wasrestrictedfor becameknownasneurotoxicshellfishpoisoning(NSP)(129). manyyearstotheAmericasuntiltheearly1990s,whencaseswerereportedinNew Zealand and Australia (Figure l ) . Victims of this syndrome exhibit many of the same symptoms as people who suffer fromthe fish-derived seafood poisoning syndrome. ciguatera. Typical symptoms are tingling in the face, throat, and digits. dizziness, fever, chills, muscle pains, abdominal cramping, nausea, diarrhea, vomiting, headache, reduced heart rate, and pupil dilation (50). There have been no recorded deaths from NSP, although the causative toxin is fatal to test mammals ( 1 30- 132) when administered by various routes, including orally.

A. The Causative Toxins and Their Origins Like the PSTs and DSTs, the compounds that cause NSP originate from dinoflagellates. Inthis case, the culprit alga is Ptychodiscus hruvis (formerly known as Gyrrrnodiniunr b r e w ) ,which gives it name to the toxins, the brevetoxins. Many naming conventions have appeared over the years with regard to brevetoxins. but here we will refer to the toxins as PbTxs, an abbreviation derived from their taxonomic origin. PbTxs are lipophilic, 10to I I-ring polyether compounds (Figure 4) which can be divided into two classes. Type 1 brevetoxins contain l I hexameric rings except for two 7-membered and one 8-membered ring. Type 2 brevetoxins possess only IO rings with one more 8-membered ring than type l PbTxs and an unusual 9-membered ring. They also differ from type 1 PbTxs inthat they have a terminal pentameric ring ( 133). Decomposition of both types of brevetoxins is accelerated in aqueous solutions with pHs greater than 10 and less than 2 ( 135). When dry, however, PbTxs are extraordinarily stable with PbTx-2 and -3 being stable in this state to 300°C ( 1 3 5 ) . I n rats, PbTx-2 and -3 possess LDS,,s of 60 and 200 pg/kg via the intravenous route ( 130,131 ). Similar LD,,, values are obtained when administered intraperitoneally (131). but the toxin’s effectiveness is markedly reduced when ingested, with the respective oral LD,,,s being S20 and 6600 yg/kg ( 13 1 ).

B. Molecular Pharmacology of the Toxin PbTxs bind to the voltage-sensitive sodiunl channel described i n detail in Sec. 11. It binds, however, to a different region of the VSSC than that via which the PSTs act and elicits a very different effect. The VSSC binding site for PbTxs involves both the first and fourth of the four homologous domains within the a-subunit ( 136,137), and quite likely part of one of the extracellular loops of the channel ( 138). Theaffinity of the toxins for theVSSC is nanomolar ( 139,140).and unlike with the PSTs, toxin-insensitive channel isoforms have

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Me

H0 R

0

Fig. 4 Brevetoxins type 1 (A) and 2 (B) and some of the chemical variations that nature (133,134).

may occur in

yet to be reported and mutations of the channel which affect brevetoxin binding have not been reported. PbTxs shift the activation voltage of the VSSC to more negative potentials (141144). This puts the channel nearer the triggering threshold where the change in potential difference across the cell membrane initiates the seriesof conformational changesto open and allow conduction of sodium ions. Inactivation of ion conduction is also believed by some to be slowed by brevetoxins (132,141). The end result of either of these two effects, or their combined effect, is to lengthen the mean open time for the VSSC, allowing a higher overall number of sodium ions to enter the cell. This hyperexcitability of cells reliant upon action potentials can lead directly to cellular malfunction. The additional sodium loadmay also overwhelm theprocesses,such as the N a + / K ' transporter,that maintain the concentration gradient of sodium across the cell membrane, providing the

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electromotive force for action potentials. PbTx affected VSSCs also display subconductance states; that is, the rate of ion flow through the channel is reduced (141,145). The VSSC might not be the only receptor affected by the brevetoxins, because other smallpopulations of brevetoxinbindingsiteshavebeenobserved(138,139,146,147). These other binding sites were not identified, but the candidates include the nicotinic receptor-ionophore complexof the neuromuscular junction( 148)and the aryl hydrocarbon receptor, which is a ligand-activated transcription factor (149).

C. Toxin Uptake,Metabolism,andDistribution The lipophilic PbTxs easily pass through the gut epithelia into the bloodstream. Oral administration of PbTx-3 to rats resulted in almost all of the toxin being absorbed ( 1 SO). and within hours SO% of the toxin had accumulated equally in the liver and stomach. The ingested toxin was also readily distributed to the intestine (14%), heart (8%').and kidneys (695). Of the other tissues tested-spleen, lung, fat, muscle, plasma, testes, brain, and skin-no more than 4% of toxin appeared. Although IV administration of PbTx-3 results in a different pattern of tissue distribution in rats to that after ingestion, what is revealed is that uptake of brevetoxin is incredibly fast, with it disappearing from the blood within 1 minute of injection (IS1). Within 1 hour of the IV PbTx-3 injection, almost 70% of the 20% in the liver, and the remainder radiolabeled toxin appeared in the skeletal muscle, in the intestinal tract ( 1S 1 ). Orally ingested compounds first pass through the liver for detoxification, but in some cases, the compounds may attack the liver. In the case of the brevetoxins,thisisindeedthecase,wherePbTx-3affectedmouseliverefficiency by ( 152). enhancing sodium entry into liver cells, thereby inhibiting oxygen consumption Therefore the large anlount of PbTx that resides in the liver after its uptake could result in significant and possibly long-term liver dysfunction in NSP victims. The lipophilic nature of the brevetoxins would allow them to not only easily cross the gut epithelium, but also the brain barrier. This supposition is supported by a study with the chemically and pharmacologically related toxin ciguatoxin, which binds to the same sitc on the VSSC as the brevetoxins and elicits the same effects (153), which can induce many effects in the brains of mice (154). Curiously though, with PbTx-3, very little toxin reached the brain of rats, getting to no more than approximately 1% of h e toxin dose at any one time ( 150, IS l ) . PbTxs are vulnerable to metabolic conversion in mammals. This was demonstrated after rats were orally dosed with radioactive PbTx-3 and fecal extracts contained several radioactive compounds apart from the parent toxin, indicating some modification of the toxin had occurred during its movement through the body ( IS I ) .

D. Toxin Elimination PbTxs exit the lnamnalian body by both urination and defecation. In experimental m a n mals, the route of'toxin administration can affect which of the two routes will predominate. After IV administration of radiolabeled brevetoxin, 75% of the toxin was eliminated via the feces, whereas only 14% exited via the urine and the remainder was still in the rat's body after 6 days ( 15 I ) . After oral dosing with PbTx-3, however, some of the toxin is not absorbed, passing directly through the gut to be eliminated with the feces. Equivalent amounts of the toxin were eliminated in both the urine and feces, but because feces in-

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cludes unabsorbed toxin, it was suggested thaturinaryeliminationmight be dominant over the fecal route (150). The symptoms produced by the toxin may therefore accelerate in test mammals (135) and its elimination by increasing defecation and urination rates humans.

E. WholeAnimal Effects Intravenous injection of PbTx into anesthetized dogs caused apnea, bradycardia. uncontrolled spontaneous skeletal muscle twitching, and tonic contractions. Heart rate and blood pressure immediately dropped upon injection, but recovered quickly. Doses greater than 80 pg/kg causedrespiratory arrest in dogs ( 1 SS). This short-lasting bradycardia, hypotension, and respiratory inhibition is known as the Bezold-Jarish refex and also occurs in anesthetized cats given PbTx (156j.In awake rats, the respiratory rate is depressed, which the animal compensates for by increasing breath volume (157). A substantial decrease in core and peripheral body temperatures can occur in mammals given a brevetoxin (157). Thisobservation, in conjunction withtheneurologicaldysfunctionsuchasataxiaand simultaneousseizures of limbpairs,indicatesbrainandspinalchordinvolvement (156,157).

F. Treatmentfor NSP As with PSTs, antibodies to PbTxs have been investigated as a means of treating NSP victims. Pretreatment of rats with goat polyclonal antibodies directed toward PbTx-3 prevented any NSP symptoms i n rats after injection with PbTx-2 (130,157). Immediate injection of PbTx antibodies after injection with PbTxalsoprotectedthe animals from the toxin’s effects (130), except for some subtle emanations of toxicity such as mild ataxia. This was somewhat surprising since brevetoxin disappears from the blood so quickly and wouldnotbe exposed to the antibody. Brevetoxin is a reversible toxin ( I 5 8 ) , andan antibody may manage to sequester the toxin during the periods that it is dissociated from the receptor and before it reassociates. This is unlikely to be a suitable route for treating NSP victims, since quitea long time is likelyto occur between intoxication and realization by the victim that they may need medical attention.

V.

AMNESIC SHELLFISH POISONING

In 1987. on Prince Edward Island in Canada,an unusual shellfish poisoning event unfolded involving the blue mussel(Mytilus erlulis), a shellfish implicated in many different poisoning syndromes (TableI ) (46). Of the symptoms manifestedin the victims, themost curious was the effect on memory, an effect that lasted days or longer, which led to the syndrome being dubbed amnesic shellfish poisoning (ASP). Almost 200 people were affected and unfortunately, 3 of the victims died (159). Of interest, approximately 50,000 people are believed to have consumed mussels from the same batch that caused the poisoning (160). There have been few ASP events since (Figure l ) . Other symptoms experienced by ASP victims include nausea, vomiting, headache, diarrhea, and abdominal cramps. Neurological symptoms may follow, such as confusion, memory loss, and disorientation (161 ). I n severe cases, seizures, followed by coma and death may occur.

Shellfish

A.

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Poisoning

Causative Toxin and Its Origin

Because of the unusual nature of the s y m p t o m and the number of people affected in the first ASP event, Canadian authorities mobilized a substantial scientific team to ascertain whether the cause of the intoxication was anthropogenic or natural. They discovered that domoic acid (DA) (Figure3 , a known neuroexcitatory toxin, was the cause of the poisonings and had been accumulated in the shellfish from a diatom Pseurlo-t/it:schia (previously Nit:sc.hitr) ~nrrrget~s forma rtrultiseries ( 13,166). DA derives its name from the first organChotlclricr nrmtrttr, locally known ism in which it was found, the macroscopic red alga, as domoi (163). It was originally purified as the antihelminth in a long-used traditional medicine (163). This use, coupledwith the observation that approximately 50,000 people ingested DA-contaminated shellfish in Canada in 1987, giving a morbidity rateof approximately 0.4% and a mortality rate of 0.006%, indicates that DA is not very toxic compared to other shellfish toxins. Other DA-producing diatoms include Pseuno-rritcsc,hia seritrtcr, P. rtrultiseries, P. trustrnlis, P. psc.uek)cleliccrtis.sirtItr, P.clPlic.trti.ssitnci, and P. tur&ldcr (167-17 I). Of concern is an outbreak of DA poisoning in seabirds in California in 1991, where the vector was not shellfish but anchovies ( I7 1 ), a widely eaten organism not normally subjected to toxicity monitoring. Thus a different pathway exists that may lead to an ASP-like outbreak. Domoic acid (C,,H:,NO,,; molecular weight 31 I ) is a tricarboxylic acid that differs very little from the glutamate receptor agonist kainic acid, both being cyclized analogues

2 C O O H

'PCOOH

A

B

C

D

E

F

Fig. 5 Domoicacid ( A ) andthevariousanaloguesknown to date withregions o f isomerism depicted with the circular ;~rrows ( 162- 165). Note that not a l l of these compounds occur i n shellfish but arc specific to thc macroscopic red algae ( 164). Kainic acid, the ncurotoxin that defines the subset o f glutanmtc receptors activated by domoic acid and its analogues, is depicted in F.

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of L-glutamate (Figure 5 ) . DA is only weakly toxic to mammals, having an LDS,)in mice (intraperitoneally) of 3.6 mg/kg (172).It is a polar molecule, insolublein organic solvents, but soluble in water (-8 g/L) and slightly soluble in methanol (173). This polarity arises from three carboxylic acids and an amino group in the pentameric ring, which have pK,s of 2.1, 3.7, 5.0, and 9.8, respectively (163,174). The ionic state of DA affects its overall toxicity, with intraperitoneal DA injection into mice in an acidic vehicle significantly less toxic than at physiological pH( 175).

B. Molecular Target of Domoic Acid Domoic acid was recognized many years ago as a potent amino acid neuroexcitant (176), mainly through its structural similarity to kainic acid, a compound used to delineate a subclass of ionotropic glutamate receptors (IgluRs). IgluRs are ligand gated, neuronal ion channels, which respond to L-glutamate by opening and allowing the passage of cations. IgluRs trigger many intracellular cascades either directly by their introduction of cations or indirectly by the second messengers produced by these cascades, such as cyclic AMP or reactive oxygen species (177,178). IgluRs have been subdivided depending upon their sensitivity to three compounds: NMDA, AMPA, and kainate. It is primarily the kainate IgluRs that are sensitive to DA, although there is some evidence that AMPA IgluRs may also respond to the toxin (179). From the amino acid sequences of IgluRs cloned to date, hydrophobicity plots predict there are four transmembrane regions with both the carboxy and amino tails of the subunits being intracellular. The molecular diversity of the IgluRs arises not just from their differences in amino acid sequences, but also from the fact that they are made up of five, sometimes different, subunits which combine to surround the central ion-conducting pore (180,181). It is possible that domoic acid may affect other kainate-sensitive glutamate systems. For example, kainateis a nontransported competitive inhibitorof an excitatory amino acid transporter, EAAT2 ( 1 82). SinceDA can access the kainate binding site in kainate IgluRs, it is possible that it may also inhibit this enzyme.

C. Domoic Acid Uptake by the Gut The hydrophilicity and ionic properties of DA make it difficult for it to cross cell membranes in the gut epithelium. Evidence for the poor uptake efficiencyof DA by the human gut is its low morbidity rate. In fact, orally administered DA in both rats and mice was virtually completely eliminated in the feces and none of it made it to the urine (172). Since it possesses both basic and acidic pK,s, it can exist in charged states in both the stomach and intestines. For instance, in the stomach, a variety of charge forms from deprotonation of the various carboxylic acids can exist, whereas in the small intestine, all 3carboxylics will be charged while a pool of DA also bearing a positive charge on the imino will develop. The most likely route then for DA uptake is paracellular diffusion, an inefficient and size-selective method.

D. Toxin Distribution and Elimination Despite DA’s poor ability to cross the gut epithelium, it obviously can enter the human bloodstream and elicit drastic effects in ASP victims. It does not have much time to do this though, as it has been shown in monkeys that IV DA disappears from the blood very

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quickly, with 60-90% of the injected dose appearingin the urine within 6 hours of injection (183). Once DA has successfully entered the bloodstream it returns to physiological pH and all of the carboxylic acids will be charged, significantly hinderingit from crossing the blood-brain barrier, althoughnot absolutely (160). However, someof the human brain lacks the blood-brain barrier (184), exposing it to absorbed DA. After attacking these brain regions there may be a flow-on effect, whereby trauma in these exposed regions of the brain triggers excessive glutamate release within the brain and causes general excitotoxicity.Kainate-receptoractivationleads to activation of other IgluRs, with NMDAreceptor activation being the major cause of cellular death in cultured cerebellar granule neurons (185). DA that does enter the blood is primarily eliminated from mammals in their urine ( 1 86). Injection of radiolabeled DA into rats resulted in virtually the entire toxin dose being urinated within several hours of injection. Little metabolism of DA occurs during its time in the body because, in this same study, all of the radiolabel in the urine was associated with the parent domoic acid.

E. Pathology Kainate IgluRs are a prominent receptor family in the human brain, and since the bloodbrain barrier does not enclose all of the human brain,it is vulnerable to DA attack resulting in lesions and other neuropathologies (161,184,187). In mice, high doses were necessary to inflict observable damage upon these unprotected regions of the brain, the so-called circumventricular region. Lesions were generally localized but did extend a short way into neighboring regions of the brain ( 1 84), which may reflect the flow-on effect of DA neurotoxicity discussed above.Some animal models may be more susceptible to this effect. For instance, rats receiving an intraperitoneal injection of DA suffered significant lesions in most areas of the central nervous system (CNS), including cerebrum, cortex, hippocam( 1 88). As alluded to pus, hypothalamus, olfactory system and septum, and even the eye above, small amounts of DA can permeate the blood-brain barrier, albeit very slowly, and when it does, it can inflict additional neurological lesions apart from the circumventricular region (160).

F. Treatmentsfor ASP Symptomatic treatment and life support are the only recommended treatment. There are clues in some studies, however, of medicinal strategies that may be employed. The most direct strategy is the use of a kainate IgluR antagonist, CNQX, which ameliorated DA’s action on retinal tissue (1 89). And because kainate receptor activation by DA can elicit neuronal damage by their activation of the NMDA class of IgluRs, it is not surprising that the competitive NMDA receptor antagonist, D( -)-2-amino-5-phosphonopentanoic acid, and non-NMDA receptor antagonist, NBQX, greatly reduced domoate toxicity by almost 80% (185). Dextromethorphan is also another NMDA IgluR receptor antagonist that showed promise in mice as a DA antidote (190). Alternative pharmaceutical strategies may arise from inhibition of systems peripheral to the IgluRs. Pretreatment of rats with diazepam, a benzodiazepine sedative and anesthetic, can reduce DA-induced convulsions in rats at 5 mg/kg but achieved little else at this dose (191). Its efficacy if administered after DA intoxication was not explored. Kynurenic acid, a tryptophan metabolite found in the mammalian brain, protected mice

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from DA toxicity for several hours, even when administered some time after the onset of motor seizures (192). Gastric lesions, which appear after DA administration in mice, were also significantly reduced by kynurenic acid (193). Administration of tryptophan and the organic acid transport blocker, probenecid, augmented the protective effects of kynurenic acid (190). Activation of the 5-HT1A subclass of serotonin receptors in rat hippocampi by the drug 8-hydroxy-dipropylaninotetralinnegated most of the effects of DA that was injected directly into the hippocampus (194).

VI. MINOR SHELLFISH TOXINS Several peptidic hepatotoxins, previously considered to occur only in freshwater microalgae. have now been found in marine shellfish and phytoplankton. They have not, as yet, been implicated in any shellfish poisoning, but we should be wary of their potential to causehumanintoxication.Also, the long-knownmarine toxintetrodotoxin,which has caused human intoxications from gastropod nlollusc ingestion rather than bivalve molluscs, has been found in a dinoflagellate known to be a source of PSTs. This raises the possibility that i t may toxify bivalve shellfish. In this section, these two toxins will be briefly summrtl-ized.

A.

Cyclized, Peptidic Hepatotoxins

Microcystins are cyclic heptapeptides from species

of thefreshwaterblue-grcenalgae

Microcystis (Figure 6), long known as hepatotoxins (197). The amino acid composition of the microcystin may vary, but the presence of the novel hydrophobic amino acid, 3amino-9-methoxy- IO-phenyl-2,6,8 trimethyl deca, 4,6 dienoic acid (ADDA)is essential to its bioactivity. Like okadaic acid, microcystins are potent inhibitors of the serine/threonine protein phosphatases, binding to the same site on the enzymes as OA (198j, and also act as tunlorpromoters (199). Althoughacutehumanintoxication bythesepeptidesafter eating marine shellfish has not been reported, they have been detected in the blue mussel

Me 0

Fig. 6 The structure of the cyclic heptapcptide toxin. microcystin (195,196). Thc side chain of the essential and unique amino acid. ADDA is to thc left of the molecule.

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Shellfish Chemical Poisoning

(Mytilus edulis) (200), a shellfish responsible to date for ASP, DSP, and PSP (Table l), and other commercial marine bivalves (201). We must be wary of their presence in the marine environment because chronic exposure to subacute doses of microcystins may impact upon human health, especially their potential role in cancer development.

B. Tetrodotoxin Tetrodotoxin (TTX) (Figure 7), is the famous pufferfish poison, highly prized in the fugu tradition in Japan. Human intoxication and death has occurred after consuming mollusks as in all of infested with this toxin. although the mollusks involved were not bivalves, the shellfish poisoning syndromes described above, but were gastropods. Seventeen Taiwanese in 1994 were poisoned after eating samples of Nrrs.sari~r.scustus and N. conoiddis, with one elderly victim dying, although most were probably from complicationsthat arose after the initial effects of the poisoning had abated (205). TTX competes with a comparable affinity for the same binding site on the VSSC as the PSTs, acting in the same manner as PSTs. Despite these similarities in their molecular pharmto elicit the same effects acology, TTX is structurally dissimilar to the PSTs (Figure 7), but like the PSTs, they possess a guanidino group (PK,~8.8) essential to its toxicity (202-204). The possibility of TTX intoxication from bivalve shellfish is of concern because of a recent report that thedinoflagellate Alexmdriurn n l i m t w r l , a dinoflagellateknown to havecausedPSP intoxicationviabivalveshellfish,mayalsoproduce TTX(206).Thisdiscoverywas made due to the fact that toxic individuals of the commercial edible scallop, Patirzopecten yessoerlsis, contained TTX along with PSTs. Thus TTX may abecause of PSP and analysis of culprit samples is necessary to confirm that PSTs and not TTX were the causative toxin.

VII.

CO-OCCURRENCE OF TOXINS

From Table 1, it can be seen that some shellfish, such as Mytilus edulis, are responsible for more than one type of shellfish poisoning. The conditions that initiate and maintain a bloom for one toxic microalgae may be just as suitable for other toxic species and so a bloom may contain several speciesof toxin-producing organisms. Also, slow detoxification of one toxin from a shellfish may overlap with a bloom of another toxic species, adding to the shellfish’s toxin load. Thus it is possible that people may experience more than one type of intoxication, making the diagnosis of such an event far more difficult

pK, = 8.8 Fig. 7 The neurotoxin,tetrodotoxin (202-204)

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and the treatment even more problematic. In fact, both diarrhetic and paralytic shellfish toxins have co-occurred in mussels from Spanish waters (24). A potential complication of the co-occurrence of toxins is the effect by okadaic acid on paracellular diffusion. As described above, this process is the means by which molecules traverse the gut epithelia through the tight junction barrier, allowing small hydrophilic molecules not amenable to passing through cellular barriers to enter the human system. Okadaic acid has been found to increase this permeability ( 1 28) which may not only affect its own uptake, but also of hydrophilic toxins such as the PSTs or domoic acid with which it may co-occur.

VIII. SHELLFISH RESPONSE TO THE TOXINS Many of the factors that govern the ability of a toxin to be taken up across the human gut also influence the ability of shellfish to accumulate the toxin. Domoic acid, which is poorly taken up across the gut epithelium, is also very poorly accumulated by shellfish (207). Lipophilic compounds like the brevetoxins, however, which are retained in the fat tissue of mammals for long periods (151), may be expectedto be accumulated effectively and retained by shellfish for long periods after a toxic algal bloom. This is indeed the case, with detectible levels of brevetoxin remaining in shellfish some months after the toxic dinoflagellate Prychodiscus brevis had disappeared (208). Poisonous shellfish do detoxify when no longer exposed to the intoxicating organism, with the rateof detoxification being species dependent and influenced by salinity, water temperature, and the size of the shellfish (207,209-21 1). Different life stages of toxifying microalgae can produce differentamounts oftoxin.Gametes,zygotes,and thefirstfew stages of vegetatively growing cellsof the DA-producing diatomP.seud~-tzit,-.schie/ purrpws f. rwltiser'ies do not produce domoic acid (212). The age structure o f a toxic algae bloom influences the amount of toxin a shellfish is exposed to. What effect then. do these toxins have on the shellfish themselves. By definition, shellfish that accumulate these toxins must be resistant to some degree, as the compounds often reside in the animals' tissue for long periods of time. Shellfish do possess the receptors that are inhibited by some of these toxins. The mollusk Aplysicr is known to have kainate-sensitive glutamate receptors (213) and a VSSC (77). Resistance may arise from the possession of insensitive isoforms of the enzymes and receptors targetedby the toxins. As has been discussed, simple mutations of the VSSC can result in much reduced PST sensitivity, and a naturally occurring mutation may underlie the apparent resistance of shellfish nerves to PSTs (2,3). Similarly, a single point mutation in a serine-threonine protein phosphatase can cause a 50-fold decrease in the sensitivity of this enzyme to OA (214). The toxin sensitivity of these receptors i n toxin-accumulating species of shellfish has yet to be elucidated. Alternatively, physical separation of the toxin away from its active site, such as in the digestive gland, may confer resistance. Depuration rates of particular toxins are complicated by their metabolic conversion within the shellfish which may modify their chemistry. Incubation of sulfated PSTs with homogenates of different tissues of the scallop (Plmqm~retrnrrrRe1lcrnicv.s) and littleneck clam (Prororhcrca stcrmitwa) desulfated the toxins, converting them to the most potent PST, saxitoxin (215,216). This transformation would remove a negative charge from the molecule, changing its overall charge state and therefore its pharmacokinetics in the shellfish.

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IX. EFFECT ON TOXINS BY SHELLFISH PREPARATIONS The means of preparing shellfish may impact on the amount and state of the toxins that are ingested by potential victims. Raw shellfish, which are commonly consumed, would obviously bethe most dangerous way to eat any toxic shellfish. Some of the shellfish toxins are heat stable and water soluble, such as the PSTs and domoic acid. One would expect therefore that boiling of shellfish in water, as in the making of soup, would extract the toxin. Although this possibility has not been studied with shellfish, anecdotal evidence comesfromJapanesecaseswhereparticularcrabswhichcanaccumulatePSTswere cooked as part of miso soup and consumersof the broth died from PSP (217). In contrast, research with the hepatopancreas of the lobster (Hotntrrus arner-iccrnus), which can accumulate PSPs, showed that boiling removed very little of the PSTs present in the samples (218). Steaming, however, which caused a loss of tissue water, did significantly reduce the toxicity. It may be that the toxin is biochemically anchored in the tissue and not easily extracted by cooking, withthis anchorbeingpresentonly in somespecies. A similar scenario can be raised with lipophilic shellfish toxins and their possible extraction from shellfish tissues into cooking oils.

X.

CONCLUDING REMARKS

It must be remembered that the toxins described herin are naturally occuring compounds, unlikely to have evolved for the purpose of killing humans. Rather,they have some natural function, or are the physiological resultof the organism’s metabolism,not yet fully understood. Their occurrencc in nature is probably underestimated, only being detected after our attention has been attracted by some event such as a human intoxication. efficacy Individual variation in the healthof shellfish consumers can impact upon the of shellfish toxins in humans. For instance, people who suffer stomach and intestinal ailments that affect gut pH may experience different pharmacokinetics with regard to the toxins, possibly improving the efficiency of toxin uptake. Medication given to counteract these ailments, such as drugs to inhibit acid production, could have the opposite effect. Also, the existenceof an ulcer may provide a direct method for toxin entry into the bloodstream for toxins such as domoic acid which are taken up poorly across the gut wall. The occurrence of shellfish poisoning may also be lower than the reality. Mild poias sonings may notbe recognized by victims as poisonings, but rather self-diagnosed generic food poisoning or as an allergic response. The general community is often more aware of these two ailments than shellfish poisoning, and unless they present themselves to a shellfish toxin-aware health professional. the intoxication may never be identified. The adage thatthe world is getting smaller due to modern technology is as true with the food we eat as with anything else. Shellfish have increasingly become a staple of the global diet. Toxins transmitted from the environmentto the consumer are a danger faced today by many people in many countries, not just those which traditionally have a seafood diet. A shellfish poisoning eventnot only affects the victims and their loved ones, but can have an economic impact due to closure of the shellfish industry and a flow-on effect to the whole seafood industry due to reduced consumer confidence in general seafood safety. Steps have been taken in many countries to ensure that their shellfish are safe. These include monitoring of water supplies for harmful microorganisms and direct

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toxicity testing of selected shellfish. These monitoring studies have upper limits that are usually far below the real acute toxic dose, but little is known about the effects of chronic consumption of some of thesetoxins.Increasedsensitivity of analyticalmethodsand expansion of surveys to other members of the marine and aquatic environment may extend our knowledge of the actual taxonomic distribution of these toxins and fully measure how exposed humans are to these compounds. In due course, it will also reveal the role the toxins play in the physiology and ecology of the organisms.

ACKNOWLEDGMENTS This is contribution number 987 of the Australian Institute of Marine Science.

REFERENCES 1.

2.

3.

4.

5.

6. 7.

X.

9.

10.

I I. 12.

13

IN Ashie, JP Smith. BK Simpson. Spoilage and shelf-life extensionof fresh fish and shellfish. Crit Rev Food Sci Nutr 36537-121, 1996. innerves BMTwnrog. T Hidaka, H Yalnaguchi. Resistance to tetrodotoxin and saxitoxin 10: of bivalve molluscs. A possible correlation with paralytic shellfish poisoning. Toxicon 273-278,1972. RC Kvitek, MK Beitler. Relative insensitivity of butter clam neurons to saxitoxin: a preadaptation for sequestering paralytic shellfish poisoning toxins as a chemicnl defense. Mar Ecol Prog Ser 69:47-54, 1991. GM Hallegraeff. A review of harmful algal blooms and their apparent global increase. Phycologia 32:79-99, 1993. JA Patz, PR Epstein, TA Burke. JM Balhus. Global climatechange and emerging infectious diseases. JAMA 275:217-223, 1996. PA Tester. Harmful lnarinc phytoplankton and shellfish toxicity. Potential consequences of climate change. Ann N Y Acad Sci 740:69-76, 1994. CWY Lam, KC Ho. Red tides in Tolo Harbour. Hong Kong. In: DM Anderson, T Ncn1oto, eds. Red Tides: Biology, Environmental Science and Toxicology. New York:Elsevier. 1989, pp 49-52. TJ Smnyda. Novel and nuisance phytoplankton blooms i n the sexevidence for a global epidemic. In: E Graneli, B Sundstrom, L Edlcr. DM Anderson. eds. Toxic Marine Phytoplankton. New York:Elsevier, 1990. pp 29-40. GM Hallegraeff, CJ Bolch. Transport of diatom and dinoflagellate resting spores via ship’s ballast water: implications for plankton biogeography and aquaculture. J Plankton Res 14: 1067-1084, 1992. H Ishida, N Muramatsu, H Nukaya. T Kosuge. K Tsuji. Study on neurotoxic shellfish poisoning involving the oyster, Crmwstren gigcts. in New Zealand. Toxicon 34:1050- 1053, 1996. I Karunasagar, Y Oshimo, T Yasumoto. A toxiu profile for shellfish involved in an outbreak of paralytic shellfish poisoning in India. Toxicon 28:868-870, 1990. 0 Arakawa, T Noguchi, D Hwang,D Chang. J Jeon, K Hashimoto. Paralytic shellfish poisoning bythemussel MTrihrs eddis i n Korea. In: T Okaichi, DM Anderson, T Ncmoto, e d ~ . Red Tides: Biology, Environmental Science and Toxicology. New York: Elsevier. 1989. pp 407-4 10. JLC Wright. RK Boyd, ASW de Freitas, M Falk, RA Foxall. WD Janlicson, MV Laycock. AW McCulloch, AG McInnes. P Odense, VP Pathak, MA Quilliam, MA Ragan. PG Sink

Shellfish Chemical Poisoning

14.

15.

16.

17.

18. 19.

20.

99

P Thibault, JA Walter, M Gilgan, DJ Richard, D Dewar. Identification of domoic acid, a neuroexcitatory amino acid,in toxic mussels from eastern Prince Edward Island. Can J Chem 67:48 1-489, 1989. M Kumagai, T Yanagi, M Murata. T Yasumoto, M Kat, P Lassus, JA Rodriguez-Vazquez. Okadaic acid a s the causative toxin of diarrhetic shellfish poisoning i n Europe. Agric Biol Chem50:2853-2857,1986. T Yasumoto, Y Oshima, M Yamaguchi. Occurrence of a new type of shellfish poisoning in the Tohoku district. Nippon Suisan Gakkaishi 44: 1249-1255, 1978. G Lelnbeyc,TYasumoto,JZhao,RFernandez.DSPoutbreak in ChileanFiords.In:TJ Smayda, Y Shimizu,eds. Toxic Phytoplankton Blooms in the Sea. New York: Elsevier, 1993, pp 525-529. L Boni, A Milandri, R Polctti, M Pompe. DSP cases along the coast of Emilia-Rornagna (northwestern Adriatic Sea). In: TJ Smayda, Y Shimizu, eds. Toxic Phytoplankton Blooms. New York: Elsevier, 1993. pp 475-481. RE Long,JCSargent,KHammer.Paralyticshellfishpoisoning: a case reportandserial clcctrophysiologic observations. Neurology 401:3 10-3 12, 1990. I Karunasagar, B Joseph, KK Philipose, I Karunasagar. Another outbreak o f PSP in India. HarmfulAlgaeNews l7:l, 19Y7. RQ Gacutan, MY Tabbu, EJ Aujero, F Icatlo Jr. Paralytic shellfish poisoning due to Pyrodirti w r l hoIImItvI.se var. c w r r y m s s o in Mati, Davao Oriental. Philippines. Mar Biol 87:223-227, 1985.

21.

22.

23.

24.

25.

26. 27. 28. 29. 30. 31.

32. 33.

D Hwang, T Noguchi. Y Nagashima.1 Liao, K Hashimoto. Occurrence of paralytic shellfish poisons in the purple clam Solrctellino tliphos (bivalve). Nippon Suisan Gakkaishi 53:623626.1987. F Rosalcs-Loessener, E De Porras, MW Dix. Toxic shcllfish poisoning in Guatemala. In: T Okaichi. DMAnderson,TNemoto,eds.Red Tides: Biology. Environlncntal Science and Toxicology. New York: Elsevier, 1989. pp 113- I 16. KW Kizer. Domoic acid poisoning. West J Med 16159-60. 1994. A Gag-Martinez, JA Rodriguez-Vazquez, P Thibault, MA Quillianl. Simultaneous occurrence of diarrhctic and paralytic shellfish poisoning toxins in Spanish mussels in 1993. Nat Toxins 4:72-79, 1996. J Luthy. Epidemic paralytic poisoning in western Europe, 1976. In: DL Taylor, HH Seliger. eds. Toxic Dinoflagellate Blooms. Amsterdam: Elsevier. 1979, pp 321-326. HH Huss. Development and use of the HACCP concept i n fish processing.Int J Food Microbiol15:33-44,1992. DL Park. Surveillance programmes for Inanaging risks from naturally occurring toxicants. FoodAddit Contan 12:361-371, 1995. in the common sandcrab. Science H Sonuncr.The Occurrence of the paralytic shellfish poison 76574-575. 1932. H Sonuner. KF Mcyer. Paralytic shellfish poisoning. Arch Pathol 24:560-598, 1937. CT Tan, EJ Lee. Paralytic shellfish poisoning i n Singapore. Ann Acad Med Singapore 15: 77-79.1986. D Montebruno. Paralytic shellfish poisoning in Chile. Mcd Sci Law 33:243-246, 1993. MEE Popkiss, DA Horstman. D Harpur. Paralytic shellfish poisoning. a report of 17 cases in Capetown. S AfrMcd J 55:1017-1023, 1Y7Y. BC Rodrigue, RA Etzel, S Hall. E De Porras, OH Velasquez, RV Tauxe, E Kilbournc. PA Blake. Lethal paralytic shellfish poisoning i n Guatemala. Am J Trop Med Hyg 42:267-27 1, 1990.

34. E Todd. G Avery, CA Grant. An outbreak of severe paralytic shellfish poisoning in British Columbia. Can Conunun Dis Rep 19:99- 102, 1993. 35 EWC Todd, T Kuiper-Goodman. W Watson-Wright, MW Gilgan, S Stephen. J M:llT, S pleas:incc.MA Quilliam. H Klix, HALuu. CFB Holmes. Rece11t illnesses from seafood toxins

7 00

36. 37 38. 39.

40. 41. 42.

43. 44. 45. 46. 47.

48. 49. 50. 51.

52. 53.

54.

55.

56. 57. 58.

Lle wellyn in Canada: paralytic, amnesic and diarrhetic shellfish poisoning. In: TJ Smayda, Y Shimizu, eds. Toxic Phytoplankton Blooms i n the Sea. New York: Elsevier, 1993, pp 335-339. BD Gcssner, JP Middaugh. Paralytic shellfish poisoning in Alaska: a 20-year retrospective analysis. Am JEpidemiol141:766-770,1995. DM Anderson, JJ Sullivan, B Reguera. Paralytic shellfish poisoning in northwest Spain: the toxicity of the dinoflagelatte Gyr?~r~orlinium c m w m m . Toxicon 27:665-674, 1989. LA Proenca, L Rorig. Mussel production and toxic algal blooms in Santa Catarina State, southern Brazil. Harmful Algae News 12/13:5. 1995. TJ Laila. First report of Gyrmodirriurn caterltrturlr from Atlantic Morocco. Harmful Algae News12/13:7,1995. E Macdonald. Dirwphysis bloom in West Scotland. Harmful Algae News 93, 1994. N Lundholm, J Skov. Psr~rdorzif,.sc.hinpse~cdor/c~/ic~ari.ssir,ltr i n Scandinavian coastal waters. HarmfulAlgaeNews5:4,1993. G Lcmbeye. Major PSP outbreak in Chile. 1991-1992. Harmful Algae News 2: 1-2, 1992. FH Chang, DM Anderson, DM Kulis, DC Till. Toxin production of A/csodriur,r tniuufuur (Dinophyceae) from the Bay of Plenty, New Zealand. Toxicon 35393-409. 1997. Anonymous. Shellfish poisoning: boy dies and 13 treated. Harmful Algae News 8:6, 1994. Anonymous. Paralytic shellfish poisoning: New Brunswick. Canada. Morbid Mortal Wkly Rep 25347-348, 1976. MA Quilliam, JL Wright. The amnesic shellfish poisoning myslery. Anal Chem 61:1053AI060A, 1989. JM Hughes, MA Horwitz, MH Merson, WH Barker, EJ Gangarosa. Foodborne disease outbreaks of chemicaletiologyintheUnitedStates,1970-1974.Am J Epidenliol 105233244,1977. T Yasumoto, M Murata, Y Oshima, M Sano, GK Matsumoto, J Clardy. Diarrhetic shellfish toxins. Tetrahedron 41:1019-1025, 1985. M Kat. Diarrhetic shellfish poisoning in the Netherlands related to the dinoflagellate D i m p/y.si.s otwrtrirrtrfo. Antonie Van Leeuwenhoek 49:417-427, 1983. PD Morris. DS Campbell, TJ Taylor, JI Freeman. Clinlcal and epidemiological features of neurotoxic shellfish poisoning in North Carolina. Am J Public Health 81:471-474, 1991. Q Yuzao, Z Ziaping, H Ying, L Songhui, Z Congju. Occurrence of red tides on the coasts Sea. New of China. In: TJ Smayda, Y Shimizu, eds. Toxic Phytoplankton Blooms in the York: Elsevier, 1993, pp 43-46. Q Adnan. PSP and red tide status in Indonesia. In: TJ Smayda,Y Shimizu, eds. Toxic Phytoplankton Blooms in the Sea. New York: Elsevier. 1993, pp 199-202. t Gyrfmr/i~liun~ c'crtcJrltrtur?f ALBarbera-Sanchez, S Hall,EFcrraz-Reyes. A / e . ~ r ~ m / r i r wsp.. and PSP in Venezuela. In: TJ Smayda, Y Shimizu. cds. Toxic Phytoplankton Blooms in the Sea. NewYork: Elsevier, 1993. pp 281-285. D Mcdina, G Inocentc. C Lopez. PSP in bivalve molluscs along the Uraguayall co21st. In: TJ Smayda, Y Shimizu, eds. Toxic Phytoplankton Bloonls i n thc Sea. New York: Elsevier, 1993, pp 425-428. Y Fukuyo, K Yoshida, T Ogata, T Ishimaru, M Kodama. P Pholpunthin, S Wisessang, V Phanichyakam, T Piyakarnchana. Suspected causative dinoflagellates of paralytic shellfish poisoning in the Gulf of Thailand. In: T Okaichi. DM Anderson, T Nemoto, eds. Red Tides: Biology, Environnxntal Science and Toxicology. New York: Elsevier, 1989, pp 403-406. GM Hallegraeff, C Sumner. Toxic planktonblooms affect shellfish f a r m . Aust Fish 45(December):15-18,1986. RA Corrales. JL Maclean. Impacts of harmfuld g on~seafarming in the Asia-Pacific areas. J Appl Phytol 7:151-162, 1995. BD Gcssner. P Bell, GJ Doucette, E Moczydlowski, MA Poli, FVan Dolah, S Hall. Hypertcnand identification of toxin in human urine and serum following a cluster of musselassociated paralytic shellfish poisoning outbreaks. Toxicon 35:7 1 1-722, 1997.

Shellfish Chemical Poisoning

101

59. JPK McCollum, RCM Pearson, HR Ingham, PC Wood, HA Dewar. An cpidcmic of mussel poisoning in north-castEngland.Lancet1968:767-770,1968.

60. HL Borison, WJ Culp, SF Gonsalves, LE McCarthy. Central respiratory and circulatory depression caused by intravascular saxitoxin. Br J Pharmacol 68301-309, 1980. 61. FT Chang. RM Bauer,BJBenton, SA Keller, BR Capacio.4-aminopyridineantagonizes saxitoxin-andtetrodotoxin-induccdcardiorespiratorydepression.Toxicon34:671-690. 1996. 62. EJ Schantz, VE Ghazarossian, HK Schnoes, FM Strong,JP Springer, JO Pczzanite, J Clardy. The structurc of saxitoxin. J Am Chem Soc 97:1238-1239, 1975. 63. Y Shimizu, C Hsu, WE Fallon, Y Oshima, I Miura, K Nakanishi, K. Structure of neosaxitoxin. J Am Chcm Soc 100:6791-6793, 1978. 64. RS Rogers, H Rapaport. The pKa's of saxitoxin. J Am Chem SOC102:7335-7339, 1980. 65. G Strichartz. Structural determinants of the affinity of saxitoxin for neuronal sodium channels. Elcctrophysiological studies011frog peripheral nervc. J GenPhysiol84:281-305. 1984. 66. RC Stafford, HB Hines. Urinary elimination of saxitoxin after intravenous injection. Toxicon 33:1501-1510, 1995. 67. T Harada. Y Oshima, T Yasumoto. Structures of two paralytic shellfish toxins, gonyautoxins V andVI,isolatedfrom a tropical dinoflagellate, Pyrodirlium bakamer~sevar. cwnprcwa. Agric Biol Chem 46: 1861- 1864, 1982. 68. T Noguchi, Y Ueda, K Hashimoto, H Seto. Isolation and characterization of gonyautoxin1 from the toxic digestive gland of scallop Pntinopecten yessoensis. Nippon Suisan Gakkaishi 47:1227-1231, 1981. 69. AP Negri, GJ Jones. Bioaccumulation of paralytic shellfish poisoning (PSP) toxins from the cyanohacterium Ambaerlcc rircimlis by the freshwater mussel Ahthyria c m r l o k c . Toxicon 33:667-678.1995. 70. S Gallacher, KJ Flynn, JM Franco, EE Brueggemann, HB Hines. Evidence for production o f paralyticshellfishtoxins bybacteria associatedwith A / ~ . V ~ I ~ spp. I ~ U(Dinophyta) III in culture. Appl Environ Microbiol 63239-245, 1997. 71. ES Silva. Intracellular bacteria: the origin of dinoflagellate toxicity. J Environ Pathol Toxicol Oncol 10: 124- 128, 1990. 72. JB Wcigele, RL Barchi. Analysis of saxitoxin binding in isolated rat synaptosomes using a rapid filtration assay. FEBS Lett 91310-314. 1978. 73. X Guo, AUehara, A Ravindran,SHBryant. S Hall,EMoczydlowski.Kineticbasisfor i n sodium channels of canine heart and denervated insensitlvity to tetrodotoxin and saxitoxin rat skeletal muscle. Biochemistry 26:7546-7556, 1987. of voltage-gatedionchannels.Natunvis74. HTcrlau, W Stuhmer.Structureandfunction senschaftcn85:437-444,1998. 75. SC Cannon, AI McClatchey, JF Gusclla. Modification of the Na' current conducted by the rat skeletal muscle alpha subunit by coexpression with a human brain heta subunit. Pflugers Arch 423: 155- 157, 1993. 76. N Makita, PB Bennett, AL Gcorge. Molecular determinants of beta1 subunit-induced gating modulation in voltage-dependent Na' channels. J Neurosci 16:71 17-7127, 1996. of the 77. JRDyer,WLJohnston. VF Castellucci, RJDunn.Cloningandtissuedistribution Ap!\sia Na' channel alpha-subunit cDNA. DNA Cell Biol 16:347-356, 1997. 78. JD Spafford, AN Spencer, WJ Gallin.A putative voltage-gatcd sodium channel alpha subunit (PpSCNI ) from the hydrozoan jellyfish, Po/yorchi.s penicilllltus: structural comparisons and evolutionary considerations. Biochem Biophys Res Commun 244:772-780, 1998. 79. MC Jeziorski, RM Grcenberg, PA Anderson. Cloning of a putative voltage-gated sodium channel from the turbellarian flatworm Bdellolrru cnndirirr.Parasitology 115:289-296, 1997. 80. J Satin, JT Limbcris, JW Kyle, RB Rogart, HA Fozzard. The saxitoxin/tetrodotoxin binding site on cloned rat brain IIa Na channels is in the transmembrane electric field. Biophys J 67: 1007-1014.1994.

102

Llewellyn

GM Lipkind. HA Fozzard. A structural model of the tetrodotoxin and saxitoxin binding site of the Na' channel. Biophys J 66: 1- 13, 1994. 82. AN Akopian,LSivilotti,JNWood.Atetrodotoxin-resistantvoltage-gatedsodiumchannel expressed by sensory neurons. Nature 379257-262, 1996. 83. LL Cribbs, J Satin, HA Fozzard, RB Rogart. Functional expression of the rat heart I Na' channel isoform. Demonstration of properties characteristic of native cardiac Na channels. FEBS Lett 275: 195-200, 1990. 84. MMWhite,LQ Chen, RKleinfield,RGKallen,RL Barchi. SkM2, a Na' channel cDNA clone from denervated skeletal muscle, encodes a tetrodotoxin-insensitive Na' channel. Mol Pharlnacol39:604-608,1991. 8 5 . RC Kallen,ZHSheng,JYang,LQ Chen, RB Rogart,RLBarchi.Primarystructureand expression of a sodium channel characteristicof denervated and immature rat skeletal muscle. Neuron 4:233-242, 1990. 86. J Satin, JW Kyle, M Chen, P Bell, LL Cribbs, HA Fozzard, RB Rogart. A mutant of TTXresistant cardiac sodium channels with TTX-sensitive properties. Science 256: 1202-1 205, 1992. 87. D Andrinolo, LF Michea, N Lagos. Toxic effects, pharmacokinetics and clearance of saxitoxin, a component of paralylic shellfish poison (PSP), in cats. Toxicon 37:447-464, 1999. 88. HB Hines, SM Naseern, RW Wannemacher. ['H]-saxitoxinol nletabolis~nand elimination in the rat. Toxicon 3 1 :905-908, 1993. 89. AG Gilman, TW Rall, AS Nies, P Taylor. The Pharmacological Basis of Therapeutics, 8th ed. NewYork:PergamonPress,1990. 90. T Harada, Y Oshima, T Yasumoto. Assessment of potential activation of gonyautoxin V in the stomach of mice and rats. Toxicon 22476-478, 1984. 91. FT Chang, BJ Benton, RA Lenz, BR Capacio. Central and peripheral cardio-respiratory effects of saxitoxin (STX) in urethane-anaesthetized guinea-pigs. Toxicon3 1:645-664, 1993. of saxitoxin in the cerebrospi92. HL Borison,LE McCarthy. Respiratory and circulatory effects nal fluid. Br J Pharmacol 61:679-689. 1977. 93. BJ Benton, VR Rivera, JF Hewetson, FCChang. Reversal of saxitoxin-induced cardiorespiratory failure by a burro-raised alpha-STX antibody and oxygen therapy. Toxicol Appl Pharmacol 124139-5 I , 1994. 94. SR Davio. Neutralization of saxitoxin by anti-saxitoxin rabbit serum. Toxicon 23:669-675, 1985. 95. R1 Huot, DL Armstrong, TC Chanh. In vitro and in situ inhibition of the sodium channel blocker saxitoxin by monoclonal antibodies. J Toxicol Environ Health 27:381-393, 1989. 96. B Kaufman, DC Wright, WR Ballou, D Monheit. Protection against tetrodotoxin and saxitoxin intoxication by a cross-protective rabbit anti-tetrodotoxin antiserutn. Toxicon 29:58l 587, 1991. V Endcn,JClardy,YGopichand, F 97. KTachibana,PScheuer.YTsukitani,HKikuchi, Schmitz. Okadaic acid, a cytotoxic polyether from two marine sponges of the genus Hrrlic.hondricr. J Am Chem Soc 103:2469-2471, 1981. 98. M Murata, M Shimitami, H Sugitani, Y Oshima, T Yasumoto. Isolation and structural ehcidation of the causative toxin of the diarrhetic shellfish poisoning. Nippon Suisan Gakkaishi 48:549-552,1982. 99. MA Quilliam, JLC Wright. Methods fordianhetic shellfish poisons. In GM Hallegraeff, DM Anderson,ADCernbella. eds. Manual on HarmfulMarineMicroalgae.Paris:UNESCO, 1995, pp 95-1 1 I . on experimental marine 100. K Terao, E 110, T Yanagi, T Yasumoto. Histopathological studies toxin poisoning. I. Ultrastructural changes in the small intestine and liver of suckling mice induced by dinophysistoxin-l and pectenotoxin-l. Toxicon 24: I141 - I 151, 1986. of yessotoxin. Nat Toxins 5: 101. H Ogino, M Kumagai, T Yasumoto. Toxicologic evaluation 255-259,1997.

81.

Shellfish

Poisoning

103

102. K Terao, E110, M Oarad:\, M Murata. T Yasumoto. Histopathological studies on experimental marine toxin poisonit1g-5. The effects i n mice of yessotoxin isolated from Ptrtitl~)/~e~Tetl yes,soc~t~.si.sand of a dcsulfated derivative. Toxicon 28: 1095-1 104, 1990. 103. T Y:lsumoto. y Oshima, W Sugawara, Y Fukuyo, H Oguri, T Igarishi, N Fujita. Identification

104. 105.

106.

107. 108.

109.

1 IO.

I II. 112.

113.

114. 115.

116. 117.

118.

119.

120

121

122.

of I);no/,lly.s;.s,fi,r.tii as the causative organismof diarrhetic shellfish poisoning. Nippon Suisan Gakkaishi 46: 1405- 13 1 1, 1980. y Murakatni. Y Oshima, T Yasulnoto. Idcntificatlon of okadaic acid as a toxic component of :l marine dinoflagellate P r o r ~ ~ t w ~ t rlinrtr. t m Nippon Suisan Gakkaishi 48:69-72, 1982. T Hu, J Marr, ASW Defreitas, MA Quilliam, JA Walter, JLC Wright. S Pleasance. New diol esters (of okadaic acid) isolated from cultures of the dinoflagellates froroccnfrum h r r r and frorocerltrlrrtr cotwnww. J Nat Prod 55: 1631 - 1637, 1992. JS Lee, T Igarashi, S Fraga, E Dahl, P Hovgaard, T Yasumoto. Determination of diarrhetic shellfish toxins in various dinoflagellate species. J Appl Phycol 1: 147-152, 1989. A Tnkai, M Murata. K Torigoe, M Isobe, G Mieskes, T Yasumoto. Inhibitory effect of okadaic acid derivatives on protein phosphatases. Biochetn J 284:539-544, 1992. YM Li, JE Casida. Cantharidin-binding protein: identification as protein phosphatase 2A. Proc Natl Acad Sci USA 89: 1 1867- I 1870, 1992. TA Haystead, AT Sim, D Carling, RC Honnor, Y Tsukitani, P Cohen, DG Hardie. Effects of the tumour promoter okadaic acid on intracellular protein phosphorylation and metabolism. Nature337:78-81. 1989. J Goldberg, HB Huang. YG Kwon, P Grcengard. AC Nairn, J Kuriyan. Three-dimensional structure of the catalytic subunit of protein serine/threonine phosphatase-I . Nature 376:745753.1995. P Cohen. The structure and regulation of protein phosphatases. Ann Rev Biochetn 58:453508.1989. CW Laidley. E Cohen, JE Casida. Protein phosphatase in neuroblastoma cells: ['Hlcantharidin binding site in relation to cytotoxicity. J Phartndcol Exp Ther 280:1152-1158, 1997. C Bialojan. A Takai. Inhibitory effect of a marine-sponge toxin, okadaic acid, on protein phosphatases. Specificity and kinetics. Biochem J 256:283-290, 1988. A Takai. G Mieskes. Inhibitory effect of okadaic acid on the p-nitrophenyl phosphate phos1991. phatase activity of protein phosphatases. Biochem J 275:233-239, Z Zhang, S Zhao, F Long, L Zhang. G Bai,H S h i m , M Nagao, EYC Lee. A mutant of protein phosphatase-l that exhibits altered toxin sensitivity. J Biol Chem 269:16997- 17000, 1994. CFB Holmes, HA Luu, F Carrier. FJ Schmitz. Inhibition of protein phosphatases- 1 and-2A with acanthifolicin. Comparison with diarrhetic shellfish toxins and identification of a region on okadaic acid important for phosphatase inhibition. FEBS Lett 270:216-218. 1990. S Nishiwaki.HFujiki,MSuganuma,HFuruya-Suguri, R Matsushima,Ylida,MOjika, Yamada. D Uemura, T Yasumoto. Structure-activity relationship within a series of okadaic acid derivatives. Carcinogenesis 1 1 : 1837- 1841, 1990. K Sasaki, M Murata, T Yasumoto, G Mieskes, A Takai. Affinity of okadaic acid to type-l and type-2A protein phosphatases is nlarkedly reduced by oxidation of its 27-hydroxyl group. Biochem J 298259-262, 1994. V Fessard, Y Grosse. A Pfohl-Leszkowicz, S Puiseux-Dao. Okadaic acid treatment induces DNA adduct formation in BHK2 1 C 13 fibroblasts and HESV keratinocytes. Mutat Res 361 : 133-141.1996. WG Matias, M Bonini, EE Creppy. Inhihition of protein synthesis in a cell-free system and vero cells by okadaic acid. a diarrhetic shellfish toxin. J Toxicol Environ Health 48:309317, 1996. a potent MT Fernandez, V Zitko, S Gascon, A Novelli. The marine toxin okadaic acid is neurotoxin for cultured cerebellar neurons. Life Sci 49:PL157-PL162, 1991. T Aune, T Yasumoto, E Engeland. Light and scanning electron microscopic studies on effects

104

Llewellyn

ofmarinealgaltoxinstowardfreshlyprepared hepatocytes. J Toxicol Environ Health 34: 1-9,1991. 123. E Ito, K Tcrao. Injury and recovery process of intestine caused by okadaic acid and related compounds. Nat Toxins 2 3 7 1-377, 1994. 124. L Edebo, S Lange, XP Li, S Allenmark. Toxic mussels and okadaic acid induce rapid hypersecretion in the rat small intestine. APMIS 96:1029-1035, 1988. 125. M Suganuma, H Fujiki, H Suguri, S Yoshizawa, M Hirota, M Nakayasu, M Ojika, K Wakamatsu, K Yamada, T Sugimura. Okadaic acid: an additional non-phorbol-l2-tetradecanoate13-acetate-type tumour promoter. Proc Natl Acad Sci USA 85: 1768-1771, 1988. 126. H Fujiki, M Suganuma, H Suguri, S Yoshizawa, K Takagi, N Uda, K Wakamatsu,K Yamada, M Murata, T Yasumoto, T Sugimura. Diarrhetic shellfish toxin, dinophysistoxin-I , is a potcnt tumor promoter on mouse skin. Jpn J Cancer Res 79: 1089-1093, 1988. 127. WC Matias, EE Creppy. Transplacental passage of [‘HI-okadaic acid in pregnant mice measured by radioactivity and high-performance liquid chromatography. Hum Exp Toxicol 15: 226-230,1996. 128.JTripuraneni,AKoutsouris,LPestic,PDeLanerolle,GHecht.Thetoxinofdiarrhetic shcllfishpoisoning,okadaicacid,increasesintestinalepithelialparacellularpermeability. Gastroenterology 1 12:100- 1 IO. 1997. 129. SMRay,DVAldrich. Gyrnnodiriiunr breve: inductionofshellfishpoisoninginchicks. Science148:1748-17.50,1965. 130. CB Templeton, MA Poli. R Solow. Prophylactic and therapeutic use ofan anti-brevctoxin (PbTx-2) antibody in conscious rats. Toxicon 27: 1389-1395, 1989. 13 1. DC Baden, TJ Mende. Toxicity of two toxins from the Florida red tide marine dinoflagellate, Phckodiscus brevis. Toxicon 20:457-461, 1982. 132.REGawley,KSRein,MKinoshita, DC Baden.Binding of brevetoxinsandciguatoxinto the voltage-sensitive sodium channel and conformational analysis of brcvetoxin B. Toxicon 301780-78.5,1992. 133. Y Lin, M Risk, SM Ray, D van Engen, J Clardy, J Golik, JC James, K Nakanishi. Isolation and structure of brevetoxin B from “red tide” dinoflagellate Phchodiscus hrevis (Gyrnrtodinitmr breve). J Am Chem Soc 103:6773-6776, 1981. 134.KNakanishi.Thechcmistryofbrevetoxins: a review.Toxicon23:473-479,1985. 135. DC Baden.Brevetoxins:uniquepolyetherdinoflagellatetoxins.FASEB J 3:1807-1817, 1989. 136. MA Poli, TJ Mende, DC Baden. Brevetoxins, unique activators of voltage-sensitive sodium channels,bindtospecificsitesinratbrainsynaptosomes.MolPharmacol30:129-135, 1986. 137. VL Trainer, DC Baden, WA Catterall. Identificationof peptide components of the brevetoxin receptor site of rat brain sodium channels. J Biol Chem 269: 19904-19909, 1994. 138. VL Trainer, WJ Thomsen, WA Catterall, DC Baden. Photoaffinity labelling of the brevetoxin receptor on sodium channels in rat brain synaptosomes. Mol Pharmacol 40:988-994, 1991. 139. RA Edwards,VLTrainer, DC Baden.Brevetoxinsbindtomultipleclassesofsitesinrat brain synaptosomes. Brain Res Mol Brain Res 14:64-70. 1992. 140. VL Trainer, E Moreau, D Guedin, DC Baden, WA Catterall. Neurotoxin binding and allosteric modulation at receptor sites 2 and 5 on purified and reconstituted rat brain sodium channels. J Biol Chem 268:17114-17119, 1993. 141. G Jeglitsch, K Rein, DC Baden, DJ Adams. Brevetoxin-3 (PbTx-3) and its derivatives modulate single tetrodotoxin-scnsitive sodium channels i n rat sensory neurons. J Pharmacol Exp Ther 284516-525, 1998. 142. WD Atchison, VS Luke, T Narahashi, SM Vogel. Nerve membrane sodium channels as the target site of brevetoxins at neuromuscular junctions. Br J Pharmacol 189:731-738, 1986. 143. JM Huang, CH Wu, DC Baden. Depolarizing action of a red-tide dinoflagellate brevetoxin on axonal membranes. J Pharmacol Exp Ther 229:615-621, 1984.

Shellfish Chemical Poisoning

105

147. PL Whitney, J A Delgado, DG Baden. Complex behavior of marine animal tissue extracts ill thc competitive binding assay of brevctoxins with rat brain synaptosomes. Nat Toxins 5 : 193-200, 1997. 148. JP Gallagher, P Shinnick-Gallagher. Effectsof crude brevetoxin on membranc potential and spontaneous or evoked end-platcpotentials in rathemidiaphragm.Toxicon23:489-496, 1985. Rein, DGBaden,PJWalsh, DE Hinton, K Tullis,MSDenison.Bre149. BSW&burll,KS vetoxin-6 (PbTx-6). a nonaromatic marine neurotoxin, is a ligand of the aryl hydrocarbon receptor. Arch Biochem Biophys 343: 149- 156, 1997. 150. M Cattet, JR Geraci. Distribution and elimination of ingested brevetoxin (PbTx-3) in rats. Toxicon31:1483-1486,1993. 151. MA poli, CB Templeton. WL Thompson, JF Hewetson. Distribution and elimination of brevetoxin PbTx-3 in rats. Toxicon 28:903-910, 1990. (PbTx-3) inhibits oxygen consump152. FA Rodriguez, N Escobales, C Maldonado. Brevetoxin-3 tion and increasesNa' content in mouse liver slices througha tetrodotoxin-sensitive pathway. Toxicon 32:1385- 1395, 1994. 153. A Lombet, JN Bidard, M Lazdunski. Ciguatoxin and brevetoxins share a common receptor site on the neuronal voltage-dependent Na' channel. FEBS Lett 219:355-359, 1987. of 154. YG Peng, TB Taylor, RE Finch,PDMoeller.JSRamsdell.Neuroexcitatoryactions ciguatoxinonbrainregionsassociatedwiththermoregulation.Neuroreport6:305-309. 1995. 155. GL Johnson, JJ Spikes, S Ellis. Cardiovascular effects of brevetoxins i n dogs. Toxicon 23: 505-515. 1985. 156. HL Borison, LE McCarthy, S Ellis. Neurological analysis of rcspiratory, cardiovascular and neuromuscular effects of brcvetoxin in cats. Toxicon 23517-524, 1985. 157. CB Templeton, MA Poli, RD LcClaire. Cardiorespiratory effects of brevetoxin (PbTx-2) in conscious, tethered rats. Toxicon 27: 1043- 1049, 1989. 158. T Yuhi, A Wada, R Yamamoto, M Urabc, H Niina, F Izumi, T Yanagita. Characterization of ['Hlbrevetoxin binding to voltage-dependent sodium channels in adrenal medullary cells. ArchPharmacol 350:209-212, 1994. 159. TM Perl, L Bedard, T Kosatsky, JC Hockin, ECD Todd, RS Remis. An outbreak of toxic encephalopathy caused by eating mussels contaminated with domoic acid. Eng J Med 322: 1775- 1780, 1990. 160. E Preston, I Hynie. Transfer constants for blood-brain barrier permeation of the ncuroexcitatory shellfish toxin, domoic acid. Can J Neurol Sci 18:39-44, 1991. 161. MJ Teitelbaum, R Zatorre, S Carpenter, D Gendron, AC Evans, A Gjedde, NR Cashman. Neurologic scquclac of domoic acid intoxication due to the ingestion of contaminated musscls. N Eng J Med 322:1781-1787, 1990. 162. JLC Wright, M Falk, AG McInnes, JA Walter. Identification of isodomoic acid D and two new geometrical isomers of domoic acid in toxic mussels. Can J Chem 68:22-25, 1990. 163. TTakemoto, K Daigo.Constituents of C/wtrrlrirr nrrtrnta. Chem PharmBull 6578-580, 1958. 164. M Macda, T Kodama,T Tanaka, H Yoshizumi, T Takemoto,K Nomoto. T Fujita. Structures of isodomoic acids A, B and C, novel insecticidal amino acids from the red alga Chondrin nr'nrrrfn.Chem Pharm Bull 34:4892-4895, 1986.

106

L le wellyn

165.LZaman, 0 Arakawa,A Shimosu, Y Onoue, S Nishio, Y Shida, TNoguchi. Two new isomers of domoic acid from a red alga, Clzmdrier arrncctcc. Toxicon 35:205-212, 1997. 166. SS Bates,JCBird, ASWDeFreitas,RFoxall,MGilgan, LA Hanic,GRJohnson, AW McCulloch, P Odense, R Pocklington, MA Quilliam, PG Sim, JC Smith. DV Subba Rao, Nir:.sc/ricr purlgerls as the primary source ECD Todd, JA Walter, JLC Wright. Pennate diatom of domoic acid, a toxin in shellfish from eastern Prince Edward Island, Canada. Can J Fish Aqaut Sci 46:1203-1221, 1989. 167. DV Subba-Rao, MA Quilliam, R Pocklington. Dornoic acid-a neurotoxic amino acid produced by the marine diatom Nit3chirr p~cngc’nsin culture. Can J Fish Aquat Sci 45:20762077.1988. 168. DL Garrison, SM Conrad, PP Eilers, EM Waldron. Confirmation of domoic acid production by Pseudonitz.shia ctusfr.cr/is (Bacillariophyceae) cultures. J Phycol 28:604-607, 1992. 169.MCVillac, DL Roelke, FP Chavez, LA Cifuentes. CA Fryxell. P.srrr~lor~ir~sc~hin trrrstrcrlis Frenguelli and related species from the west coast of the USA: occurrence and domoic acid production. J Shellfish Res 12:457-465, 1993. Nitzschicr psercr/oc/e~/ic~rrtissir,,N-asource of 170. JL Martin, K Haya, LE Burridge, DJ Wildish. domoic acid in the Bay of Fundy, eastern Canada. Mar Ecol Prog Scr 67:177-182, 1990. 171. L Fritz, MA Quilliam, JLC Wright. An outbreak of domoic acid poisoning attributed to the uustr.cr1i.s. J Phycol 28:439-442, 1992. pennate diatom P.seLrd~)~lir~.s~/liu 172.FIverson.JTruelove,ENera,LTryphonas,JCampbell,ELok.Domoicacidpoisoning and mussel-associated intoxication: preliminary investigations into the response of mice and rats to toxic mussel extract. Food Chem Toxicol 27:377-384. 1989. 173. M Falk, PF Seto, JA Walter. Solubility of domoic acid in water and in non-aqueous solvents. Can J Chem 69: 1740- 1744, 1991. of domoicacid.Can J Chem67: 174.MFalk,JAWalter,PWWiseman.Ultravioletspectrum 1421-1425,1989. 175. MS Nijjar, MS Madhyastha. Effect of pH on domoic acid toxicity in mice. Mol Cell Biochem 167:179-185,1997. 176.TJBiscoe,RHEvans, PM Headley,MMartin.JCWatkins.Domoicandquisqualicacids 255: 166-167, aspotentaminoacidexcitantsoffrogandratspinalneurones.Nature 1975. 177. EK Michaelis. Molecular biology of glutamate receptors inthe central nervous systeln and their role in excitotoxicity, oxidative stress and aging. Prog Neurobiol 54:369-415. 1998. 178. S Ozawa, H Kamiya, K Tsuzunki.Glutamatereceptorsinthemammalianccntralnervous sytem. Prog Neurobiol 54581-618, 1998. 179. JA Larm, PM Beart, NS Cheung. Neurotoxin domoic acid produces cytotoxicity via kainateand AMPA-sensitive receptors in cultured cortical neurones. NeurochemIn1 3 1 :677-682, 1997. 180. WWisden,PH Seeburg. Mammalian ionotropic glutamate receptors. Curr Opin Neurobiol 3:291-298,1993. 18 1. S Nakanishi. Molecular diversity of glutamate receptors and impkations for brain function. Science258:597-603,1992. 182. RJ Vandenberg, JL Arriza, SG Amwa, MP Kavanaugh. Constitutive ion fuxes and subslrate 1. 1995. binding domains of human glutamate transporters. J Biol Chem 270:17668-1777 183. J Truelove, F Ivcrson. Serum domoic acid clearance and clinical observations in the CynOmOlgus monkey and Sprague-Dawley rat following a single i.v. dose. Bull Environ Contaln Taxicol 52:479-486, 1994. 184. JE Bruni,RBose,CPinsky.GBGlavin.Circumventricularorgalloriginofdomoicacidinduced neuropathology and toxicology. Brain Res Bull 26:419-424, 1091. 185. FW Berman, TF Murray. Domoic acid neurotoxicity in cultured cerebellar granule neurons is mediated predominantly by NMDA receptors that are activated as a consequence of exitatory amino acid release. J Neurochem 69:693-703, 1997.

Shellfish

Poisoning

107

~ ,Hierlihy. Renal clearance of domoic acid in the rat. Food Chcm Toxicol 10: 186. CA S U Z USL 701-706. 1993. 187. L Tryp[lollas, F [verson. Neuropkithologyof excitatory neurotoxins: the domoic acid model. Toxicol Pathol 18: 165- 169. 1990. 188. L Tryphonas. J Truelove, F Iverson. Acute parenteral neurotoxicity of domoic acid i n c p o 1990. lnolgus monkeys. Toxicol Pathol 18:297-303. to excitatory amino acidsand excite189. CD Zcevalk, WJ Nicklas. Nitric oxide in retina: relation toxicity. Exp Eye Res 58:343-350, 1994. 190. R Base, C Pinsky, GB Glavin.Sensitive murine model and putative antidotes for behavioural toxicosis from contaminated mussel extracts. Can Dis Weekly Rep 16:91-98. 1990. 191. S NakajiIna, JL Potvin. Neural and behavioural effects of domoic acid, an amnesic shellfish toxin, i n the rat. Can J Psycho1 46:569-581, 1992. 192. C Pinsky, GB Glavin, R Bosc. Kynurenic acid protects against neurotoxicity and lethality of toxicextractsfromcontaminatedAtlanticcoastmussels.ProgNeuropsychophannacol BiolPsychiatry 13595-598, 1989. 193. GB Glavin, R Base, C Pinsky. Kynurenic acid protects against gastroduodenal ulcerationi n mice injected with extracts from poisonous Atlantic shellfish. Prog Neut.opsychopharmaco1 BiolPsychiatry 13569-572, 1989. 194. SK Shartna, K Dakshinamurti.Suppressionofdomoicacidinducedseizuresby%(OH)DPAT. J Neural Transm 93:87-98, 1993. 195. DP Boles, AA Tuinman, PL Wessels, CC Viljoen, H Kruger, DH Williams, S Santikarn, RJ Smith, SJ Hammond. The structure of cyanoginosin-LA, a cyclic heptapeptide toxin from the cyanobacterium Micrmysris nerugirtosa. J Chcm Soc Perkin Trans 1:231 1-2318, 1984. S Santikarn, RJ Smith. JCJ Bru-na, DH 196. DP Botes, PL Wesscls, H Kruger, MTC Runnegar. Williams. Structural studies on cyanoginosins-LR,-YR, -YA, and -YM, peptide toxins from cyanobacterium M i c w c w i s ~ e r t c g i ? ~J .Chem ~ ~ . Soc Perkin Trans 1 :2747-2748, 1985. 197. WW Carmichael. Thc toxins of cyanobacteria. Sci Am 270:78-86, 1994. 198. C MacKintosh, KA Bcattie, S Klumpp, P Cohen, CA Codd. Cyanobacterial rnicrocystirl-LR is a potent and specific inhibitor of protein phosphatases 1 and 2A from both manmnls and higher plants. FEBS Lett 264: 187-192, 1990. 199. R Nishiwaki-Mntsushima, S Nishiwaki, T Ohta, S Yoshizawn, M Suganuma, K Harada, MF Watanabe, H Fujiki. Structure-function relationships of microcystins, liver tumor promoters. in interaction with protein phosphatase. Jpn J Cancer Res 82:993-996, 1991. 200. DE Williams, SC Dawe. ML Kent, RJ Andersen, M Craig, CFB Holmes. Bioaccumulation and clearance of microcystins from salt water mussels, Mytilus ed~tlis,and in vivo evidence for covalently bound microcystins in mussel tissues. Toxicon 35: 1617-1625, 1997. RJ Andersen, CF Holmes. Identification 201. DZX Chen, MP Boland, MA Smillie, H Klix, C Ptak, of protein phosphatase inhibitorsofthe microcystin class in the marine environment. Toxicon 31:1407-1414, 1993. 202. K Tsuda, S Ikutna, M Kawamura, R Tachikawa, K Sakai. Tetrodotoxin. VII. On the structure of tetrodotoxin and its derivatives. Chem Pharrn Bull 12: 1357-1 374, 1964. 203 T Goto, Y Kishi, S Takahashi. Y Hiratn. Tetrodotoxin. Tetrahedron 21:2059-2088, 1965. 204 RB Woodward. The structure of tetrodotoxin. Pure Appl Chem 9:49-74, 1964. 205. C Yang, K Han, T Lin, W Tsai, J Dcng. An outbreak of tetrodotoxin poisoning following gastropod mollusc consumption. Hum Exp Toxicol 14446-450, 1995. 206. M Kodama,S Sato, S Sakamoto, T Ogata. Occurrenceof tetrodotoxin in Alesnndrilrnr fctr)tNrerrse, a causativedinoflagellateofparalyticshellfishpoisoning.Toxicon34:1101-1105, 1996. 207. GD Wohlgeschaffen, KH Mann, DV Subba Rao, R Pocklington. Dynamics of the phycotoxin dolnoic acid: accumulation and excretion i n two comnercially important bivalves, J Appl Phycol 4297-310, 1992. 208. H Ishida, N Muralnatsu, T Kosuge, K Tsuji. Studyof neurotoxic shellfish poisoning involving

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209.

210. 21 1.

212.

213. 214. 215. 216. 217. 218.

Llewellyn

New Zealand shellfish,CrcrssosrrecI gigrts. In: T Yasumoto, Y Oshima,Y Fukuyo, eds. Harmful and Toxic Algal Blooms. Paris: UNESCO, 1996, pp 491-494. J Blanco, A Morono, J Franco, MI Reyero. PSP detoxification kinetics in the mussel Mytilus gefllul”nvirlcicr1is. One- and two-compartment models and the effect of some environmental variables. Mar Ecol Prog Ser 158:165-175, 1997. W Silvert, DV Subba-Rao. Dynamic model of the flux of domoicacid, a neurotoxin, through a Mxtilus edulis population. Can J Fish Aquat Sci 49:400-405. 1992. W Silvert, AD Cembella. Dynamic modelling of phycotoxin kinetics in the blue mussel, Mytilus rdulis, with implications for other marine invertebrates. Can J Fish Aquat Sci 52: 521-531,1995. DV Subba Rao, ASW De Freitas, MA Quilliam, R Pocklington, SS Bates. Rates of production of domoic acid, a neurotoxin amino acid in the pennate marine diatom Nir:.schier purtgerls. In: E Graneli, B Sundstrom.L Edler, DM Anderson, eds. Toxic Marine Phytoplankton. New York: Elsevier, 1990, pp 413-417. LE Trudeau, VF Castellucci. Excitatory amino acid neurotransmission at sensory-motor and interneuronal synapses of AplysicI ccrlifornica. J Neurophysiol 70: 1221- 1230, 1993. L Zhang, Z Zhang, F Long, EYC Lee. Tyrosine-272 is involved in the inhibition of protein 1, 1996. phosphatase-l by multiple toxins. Biochemistry 35: 1606-161 in YShimizu,MYoshioka.Transformationofparalyticshellfishtoxinsasdemonstrated scallop homogenates. Science 21 2547-549, 1981. JJ Sullivan, WT Iwaoka, J Liston. Enzymatic transformation of PSP toxins in the littleneck Biochem Biophys Res Commun 114:465-472, 1983. clam (Prorothncrr srcu~rir~ecr). Y Hashimoto. Marine toxins and other bioactive marine metabolites. Tokyo: Japan Scientific Societies Press, 1979, pp 53-55. JF Lawrence, M Maher, W Watson-Wright. Effectof cooking on the concentrationof toxins associated with paralytic shellfish poisonin lobster hepatopancreas. Toxicon 32:57-64, 1994.

5 Pathogens Transmitted by Seafood"

I. Introduction 109 11. BacterialandViralPathogensAssociatedwithRawandUnderprocessedShellfish

121

Bacterial and Viral Pathogens Primarily Associated with Improper Processing or Handling of Seafood 159 of Seafood-BornePathogens 164 IV. Development ofRapidMethodsforDetcction 165 V. ConclusionsandRecommendations VI. Additional Sources of Information: World Wide Web Sites for Pathogens Associated with Seafoods 17 1 References 172 111.

1. A.

INTRODUCTION Overview of Seafood-Borne Disease

Risks and hazards are associated with all forms of human activities. While humans may choose to minimize or eliminate some risks by changing or eliminating a certain type of behavior or activity, all humans must consume food and water to live. In some regions of the world, the consumptionof food and watercan be, unfortunately, routinely associated with exposure to pathogenic organisms. Inhabitants of developed countries generally assume that their supply of food and water is safe, and government regulatory and public health agencies are charged with the task of ensuring the safety of these items. In many regions, however, infectious disease is a wayof life, responsible for the deathsof millions of people each year in developing countries. Part of this infectious disease is associated with the transmission of pathogenic microorganisms in the food and water that is consumed. The World Health Organization( I ) reported that in 1997, of a global total of 52.2 million deaths, 17.3 million or one-third were due to infectious and parasitic diseases.

* This chapter is dedicated to the educational and scientific contributions of John Liston and Jack R. Matches, two former faculty members and microbiologists in the School of Fisheries, University of Washington. 109

110

Hetwig Other and unknown causes 9%

Infectious and parasitic diseases 43%

9%

Perinatal and maternal causes 10%

Fig. 1 Major causes of death in developing countries (1).

The leading causes of death from infectious diseases were acute lower respiratory infections (3.7 million), tuberculosis (2.9 million), diarrhea (2.5 million), H I V / A T D S (2.3 million), and malaria (1.5-2.7 million). Diarrheal and parasitic diseases may be associated with the consumption of contaminated water and food. Major differences in the causes of human death exist between developing and developed countries (Figs. 1 and 2). Infectious and parasitic diseases cause43% of the deaths in developing countries and approximately 1% of the deaths in developed countries. An excellent choice of food for protein and other essential nutrients is seafood. In many regions of the world seafood and other aquatic products are a major portion of the diet, while in other regions of the world seafood is viewed as a delicacy or something

Diseases of the circulatory system 46% Fig. 2 Major causes of deathin developed countries (1).

Pathogens Transmitted by Seafood

111

unusual. Besides supporting the healthy livesof millions of people, seafood is also capable of pathogenic of supporting the growth of or acting as a vehicle for the transmission organisms. The principalbiologicalagentsthat cause seafood-borne disease are bacteria, viruses, and parasites. The major human diseases caused by fish-borne parasites are trematodiasis, cestodiasis, and nematodiasis. Worldwide, but primarily in developing countries, trematodes are the most important food safety hazard linked to fish and fishery products. The trematodes infecting the greatest number of people are species of the genera Clonorchis, Opisthorchis, and Parvrgonirnus (2). The World Health Organization (2) estimates that tens of millions of people are affectedby fish-borne trematodes. Most of these people live in developing countries. Parasites area minor problem for seafood consumersin most developed countries. The focus of this chapter is on the bacteria and viruses associated with aquatic foods that are pathogenic to humans, although the reader should understand that parasitic infections are themajor problem associated with the consumption of seafood in many regions of the world. Bivalve mollusks present a much greater risk of infecting humans with bacterial and viral pathogens than do crustaceansandfinfish.Thegreatestnumberofseafoodassociated disease outbreaks and cases in developed countries are causedby the consumption of raw or insufficiently cooked bivalve mollusks that were harvested from waters contaminated with human sewage or waters wherea fraction of the indigenous microflora are pathogenic to humans. These two types of contamination are associated with enteric bacteria and viruses (Escherichiu coli, Salmonella, hepatitis A, Norwalk-like viruses) and bacteria naturally found in surface water and sediments (Vibrio vuln$cu.s, V. pcrrclhaetnolyticu.7, V. cholerae, Aercmo11c1.s hydrophila, Clostridiutn Dotdinurn). Another group of pathogenic microorganisms (suchas Stclphvlococcus aureus) are primarily introduced into fishery products by humans who handle or process seafoods after harvesting.

B. Consumption of Seafood and the Rise in Aquaculture 1. Increasing Consumption of Seafood In recent years the supply of fish has continued to increase steadily and in 1995 the total world production of finfish, crustaceans, and mollusks from capture fisheries and aquaculture reached 1 12.9 million metric tons. Much of the increase in annual aquatic production is attributable to aquaculture. For cultured finfish and shellfish, the annual contribution to total finfish and shellfish rose from 11.7% in 1989 to 18.5% i n 1995. For food fish, more than one-quarter of the total world supply was derived from aquaculture. Aquaculture is one of the fastest growing food-producing sectors, providing an acceptable supplement and substitute for wild fish and plants. The relative importance and role of aquaculture in different countries varies widely. I n 1995, for example, more than 60% of the total aquatic production in China came from aquaculture. This was nearly twice that seen in France, India, Republic of Korea, and the Philippines. A considerably lower contribution was reported in Thailand (13%), Norway (9%). and the United States (7%) (3).

2. The Safety of Aquaculture Products There are increasing concerns related to the safetyof food products from aquaculture and studies are now focusing on these concerns. Are there greater hazards associated with aquaculture products versus wild-caught products? Are different pathogens associated with

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Table 1 Prevalence of Snlrrrorlrllrr i n AquaculturalSpecies,’

Country Product Tilapia Catfish Catfish Eel

Prawns

Reference % Positive Africa South U.S.

us. Japan Philippines

55 3 21 26

187 188 1x9 190

16

191

aquacultural products? The greatest amountof aquaculture is done i n developing countries i n tropical and subtropical regions of the world, yet most of the scientific literature and research related to seafood safety has been performed i n “Western” countries with seafood captured or raised in temperate waters. Historically, cultured fish have not been considered important vectors of human pathogens. This situation may be changing, partly due to increasing animal densities as a consequence of a rapidly growing industry and partly due to increasing awareness by health care providers of pathogens in aquatic species that may result in human illness (4). Not surprisingly, bacteria that are known to be pathogenic to humaw have been isolated from locations where aquaculture products are produced and from products that have been sent to the marketplace. Table 1 lists the results from several studiesas summarized by D’ Aoust (5) that indicate the prevalence of Srrlnlowello i n different aquacultural species, including tilapia, catfish, eel, and prawns. A more recent review (6) summarized the association of pathogenic bacteria with cultured fish (Table 2). Although evidence has been published showing the presence of a variety of pathogenic organisms in culturedfishandshellfish,whetherthere is a proportionately

Striped bass hybrid

Pathogens Transmitted by Seafood

113

larger number of cases or outbreaks of seafood-borne disease associated with cultured animals has not been established. More research is required in this emerging sector of seafood production.

C. Cases and Outbreaks: Statistics and Government Agencies to Oversee Seafood Safety 1. United States-Developingan Interest in Shellfish Sanitation and the Food Safety Initiative Many i n the public may perceive food safety issues as something completely new, but several federal, state, and local agencies have been concerned with these issues for many years. Regarding seafood safety, there is a lengthy history of government involvement i n the safety of shellfish. Most of the pathogenic bacteria and viruses associated with seafood-borne disease are associated with the consumption of bivalve mollusks. The early history of shellfish-borne disease and the establishment of shellfish sanitation regulations in the United States is summarized i n the manual of operations for the National Shellfish Sanitation Program (7). While the exact cause or microbiology of disease was not known at the time, public health controls on shellfish became a national concern in the United States in the late 19th and early 20th centuries. At that time, public health officials noted a large number of illnesses associated with the consumption of raw oysters, clams, and mussels. Shellfish-associated disease outbreaks were also recorded in Europe. During the winter of 1924-1925, widespread outbreaks of typhoid fever occurred in New York, Chicago, and Washington, D.C. These outbreaks were traced to oysters that had been contaminated with sewage. Local and state public health officials and the shellfish industry became so alarmed that they requested that the surgeon general of the United States Public Health Service develop the controls necessary to ensure a safe supply of shellfish. The surgeon general called a conference in 1925 of representatives from state and municipal health authorities, state conservation commissions, the Bureau of Chemistry [later to become the Food and Drug Administration (FDA)], the Bureau of Commercial Fisheries [now called the National Marine Fisheries Service (NMFS)], and the shellfish industry. TheNationalShellfishSanitationProgram(NSSP)developedfromtheprinciplesand shellfish controls formulated at this conference. To strengthen the mission of the NSSP. in 1982officialsfrom22statesformedtheInterstateShellfishSanitationConference (ISSC). The ISSC allows state regulatory officials to establish uniform guidelines and to exchange information about the sources of safe shellfish. The ISSC adopted the NSSP manual of operations and has established procedures that enable it to adopt changes i n the manual (7). The Centers for Disease Control and Prevention (CDC) is largely responsible for collecting and tabulating data related to prevention and control of disease, injury, and disability in the United States. Alerts and information about current foodborne disease outbreaks and cases are disseminated in a publicationcalled Mnrbidify and Morralip Weekly. The most recent publications that summarize the numbers and causes for foodborne and seafood-related disease outbreaks are the “Summary of Notifiable Diseases, United States 1997” (8) and “Surveillance for Foodborne-Disease Outbreaks-United States, 1988-1992” (9). Data related to shellfish-borne disease was previously collected by the FDA’s Northeast Technical Services Unit (NETSU). The NETSU data were used

114

HeMg

and cited in previous reviews (10,ll). While the NETSU considers reports of outbreaks (two or more persons who become ill after consumption of a common food) and individu cases, the CDC database usually includes only outbreaks. In addition, the CDC data are collected exclusively through the voluntary submission of outbreak forms from state public health departments. The NETSU data are considered more inclusive and precise than the CDC data for shellfish-borne disease (10). In 1994, Rippey(1 1) published a review that summarized the infectious diseases associated with molluscan shellfish consumption. The data presented were related to reports presented through 1990, and as thorough as he could be, Rippey admitted that his data represented only a small portion of the actual number of cases that occur annually because of the weakness of the reporting system and the nature of most foodborne disease. Unfortunately, data collection related to shellfish-born disease is no longer being performed by NETSU, and no other agency in the United States has formally begun to collect this data in a similar manner. The result of these weaknesses is that the data for the number of cases of seafood-borne illness in the United States are most likely poorer and less representative than that collected 10 years ago. With these caveats in mind, data reported by the CDC suggest that among the dif ent kinds of food that are tabulated, there are a significant number of foodborne-disease outbreaks (25%) and a smaller fractionof cases that are associated with seafood (Figs.3 and 4). For the purposes of this review, seafood is considered “shellfish” and “other fish” in the categories that are listed in the CDC data. The data suggest that seafood has fewer cases associated with each outbreak compared to other typesof foods. From 1988 to 1992, 6095 foodborne disease outbreaks representing 77,373 cases were reported to the CDC. Seventy-five deaths were reported to be caused by foodborne disease (9). The outbreaks reported include only a fraction of the cases of foodborne disease that occur each year. For the average of 15,500 cases and 14 deaths reported each year by the CDC’s surveillance system, there are an estimated 6 million cases that actually occur each year (12). The etiology was not determined for 59% of the reported outbreaks. Most of the outbreaks that had an unknown etiology had an incubation period of 15 hours or more, suggesting an infectious agent. Manyof these outbreaks may have been caused by viruses (9). The capability of testing serum for antibodies to foodborne viruses is not widely available.

6,095 outblreah

Fig. 3 Distribution of foodborne disease outbreaks in the United States, 1988-1992 (9).

115

Pathogens Transmitted by Seafood

Shellfish

Other Fish

1Yo

2%

Other Foods 97%

Fig. 4 Distribution of foodborne disease cases in the United

States, 1988-1992 (9).

Shellfish-borne and “otherfish” diseases represented 1% and 2%of the total number of cases, but 2% and 23% of the outbreaks, respectively. In general, seafood-borne disease outbreaks are characterized by a small number of cases. On the other hand, an “outbreak” represented by only a single case is not reported in the CDC data. The responsible pathogen is not identified in more than half of the foodborne disease outbreaks that are reported to the CDC. During this 5-year period there were no cases associated with “other fish” that were attributable to Vibrio cholerae, V. parahuemolyticus, and V. vulniJcus. Examining outbreaks that were associated with shellfish during this same period, these three pathogens together caused one to four outbreaks (9). In 1988, a multistate hepatitis A outbreak with 61 cases was caused by eating raw oysters (13). An outbreak of V. cholerae occurred in Guam, an island in the U.S. Pacific territory, was caused by eating contaminated reef fish. In this single outbreak, 26 people became ill and 1 died. In 1991, there were two outbreaks of V. chokrae caused by tainted food imported to the United States. One outbreak with two cases was attributed to crabs that were imported illegally from Ecuador (14). Most of the disease outbreaks and cases associated with seafood, as reported by the CDC (9), are caused by chemical agents such as scombrotoxin, ciguatoxin, and paralytic shellfish poisoning. These seafood-borne disease agents are discussed elsewhere in this book. During the past 3 years there has been growing public concern about the safety of the food supply in the United States. Much of this concern was ignited by outbreaks caused by the emerging pathogen E. coli 0157:H7. In May 1997, a report called “Food Safety from Farm to Table: National Food-Safety Initiative” was presented to President Clinton. This report presented an intergovernmental agency strategy to prevent foodborne disease and included plans to develop elements of an improved foodborne disease surveillance system. The first component of the new system is the Active Foodborne Disease Surveillance System, known as FoodNet, a collaborative effort between the CDC, FDA, and the U.S. Department of Agriculture (USDA), and selected counties in seven states (California, York, and Oregon)participating in Connecticut, Georgia, Maryland, Minnesota, New CDC’s Emerging Infections Program. FoodNet is designed to conduct population-based

116

Herwig

active surveillance of seven bacterial foodborne pathogens (Saln~onella, ShipAlcr, C m 7 p ~ lobacter, E. coli 0157:H7, Listeria, Yersirlia, and Vibrio),and to determinethe magnitude of diarrheal illnesses and the proportion of these illnesses that are attributable to foods. The population of these seven FoodNet sites i n 1997 was 20.3 million people (7.7% of the U.S. population). The objectives of FoodNet are to (a) describe the epidemiology of new and emerging bacterial, parasitic, and viral foodborne diseasesof national importance, (b)morepreciselydeterminethefrequency andseverity of foodbornediseases in the United States, and (c) determine the proportion of foodborne disease caused by eating specific foods. To addresstheseobjectives,FoodNetconductsactivesurveillanceand related studies: a population survey, a physician survey, and a case-control study of E. coli 0157:H7 infections ( 1 516). A positive outcome from the initiation of FoodNet is perhaps a more realistic estimate about the incidence of foodborne diarrheal disease in the United States. Data from FoodNet suggest that the incidence of diarrheal illness in the United States is about 1.4 episodes/person/year, or some 370 million episodes each year. If only 25% of diarrheal disease is food related, the burden of foodborne diarrheal disease in the United States far exceeds current estimates (16). In theUnited States, four federal agencies play major roles in carrying out food safety regulatory activities: the FDA, the Food Safety and Inspection Service (FSIS) of the USDA, the Environmental Protection Agency (EPA), and the NMFS. Seafood safety is under the jurisdiction of the FDA, EPA, and NMFS. The FDA has jurisdiction over domestic and imported seafoods thatare marketed in interstate commerce. The FDA’s Center for Food Safety and Applied Nutrition (CFSAN) seeks to ensure that seafoods are safe, sanitary, nutritious, wholesome, and honestly labeled. The CFSAN also has control over seafood processing plants. The EPA establishes tolerances for pesticide residues in seafoods, and is responsible for protecting against other environmental chemical and microbial contaminants in water that might threaten the safety of seafoods. The NMFS conducts a voluntary seafood inspection and grading program that is primarily a food quality activity (16). The seafood industry is in the early stages of transitioning to hazard analysis critical control point (HACCP) programs. It is generally accepted in the food science community that the use of HACCP programs i n all aspects of food production, processing, and distribution is proactive and an excellent approach toward food safety. In 1995, the FDA issued itsfinalruleon HACCP programs for seafood. Written HACCP plans for seafood are now required, and must be specific for each processor and type of seafood. In response to the need to train members of the seafood industry i n HACCP procedures, the National Seafood HACCP Alliance for Training and Education was created. Thisand other organizations provide HACCP training courses and model plans that can be used as templates for developing specific plans ( I 6).

2. Asia: The Importance of Seafood in Their Diets The people of Asian countries consume a larger portion of fish and fishery products in theirdietsthancitizensinnlostWesterncountries,thereforeseafood-bornediseaseis generally a much larger percentage of the overall problems associated with foodborne disease. I n 1996, Lee et al. (17) published an epidemiological study of food poisoning in Korea and Japan. From 1981 to 1990, the most commonly incriminated vehicles of foodborne illness in Korea were fish and other seafood (31.8%). meat and animal products (25.0%), grainsandvegetablesincludingmushrooms(17.5%),andcompoundfoods

Pathogens Transmitted by Seafood

117

( 1 8.3%). During the same time,in Japan the most comnlon vehicles were seafood ( 2 I .7%), meat and animal products (3.6%),and graimand vegetables (14.6%). Amongthe seafoods, the major causes of food poisoning in Korea were shellfish (9.3% of total) and puffer fish (5.0%).In Japan, shellfish and puffcr fish accounted for 6.9% and 2.8%, respectively, of the total number of disease outbreaks. In Korea, of the 115 deaths from food poisoning from 1981 to 1990, 42.6% were caused by seafoods, 13.1%J by meat and animal products, and 33.1% by grains, vegetables, and mushrooms. In Japan, of the 106 deaths from food poisoning during the same period, 62.3% were caused by seafoods and 22.5% by grains, vegetables, and mushrooms. No fatalities were attributed to meat and animal products. In Korea, 58.6% of the outbreaks of food poisoning from 1981 to 1990 were due to bacteria, 18.6% were due to toxic compounds, and 23.3% were due to unknown causes. In Japan, 61.3% of food poisoning cases were due to bacteria, 23.5% were due to toxic compounds,and 15.2% weredue to unknowncauses.Thefollowing bacterialspecies were incriminated in Korea: Vibrio spp. (37.6%), Strlrrrotrellcr spp. (23. I %), other species (17. I %), S t t r ~ ~ l r ~ l o ~ ~ (14.9%), o c c u s and E. coli (6.8%). In Japan, the bacterial foodborne problemswere Vibrio spp.(47.3%). Strrl,l,ylococclrs spp.(24.8%), Salmowellcr spp. (14.8%), and other species(9.6%).With data such as these,it is understandable why there needs to be research related to seafood safety in these countries (17).

D. Microbial Classification and Identification of Pathogens 1, Prokaryotes (Bacteria and Archaea) A revolution in describing the taxonomy of microorganisms has occurred over the past 20 years, largely initiated by the contributions by Carl Woese and his coworkers (182 1 ). The taxonomy of microorganisms is now based on the phylogenetics. or evolution, of organisms rather than on interpretation of the results of phenotypic properties. The phylogeny of microorganisms can be inferred by finding the changes that have occurred i n molecules that act as chronometers of evolutionary history over thousands of years. The 16s rRNA (ribosomal RNA) and 30s rRNA are the molecules of choice today. Since the fossil record for microorganismsis extremely poor and virtually nonexistent, the relationship between microorganismsis now determinedby sequencing representative molecules. After the sequences are obtained, the phylogenetic relationship is suggested by performingintensivecalculations withtheaid of personalormainframe computers. A variety of algorithms have been developed to create phylogenetic trees, also known as "trees of life." The work by Woese and others clearly illustrate that there are three major groups or domains of organisms, called the Btrcteritr, Arclraea, and Errkatptr. The natnes A ~ ~ J ~and I CErrkrrryr YI were formerly known as Archaebacteriaand Eukaryotes, in use and found in the literature today. Besides respectively, and these older names are still observing the three major trunks that can be determined by sequencing the representative molecular chronometers, these three domains of life have major differences in their cellular biochemistry and genetic organization. The 16s rRNA sequences and phylogenetics for many of the bacterial food pathogens have been determined and are available fromvariety a of electronic database servers. One of the best resources for phylogenetic analysisand 16s rRNA sequences is the Ribosomal Database Project (RDP) (22) located at Michigan State University. The URL for their web site is www.cllle.lllsu.edt1/RDP/.

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118

Of interest, most of the seafood-borne bacterial pathogens cluster into two phylogenetic groups and arenot widely distributed throughout the "tree of life." No human microbial pathogens are found in the Archaea. Bacterial pathogens associated with seafood are members of the y (gamma) subdivision of the Proteobnctericr and the gram-positive bacteria (Fig. 5). Besides the basic scientific curiosity about the phylogenetic position of seafood-borne pathogenic bacteria, this information can also be used to develop molecular probes and polymerase chain reaction (PCR) primers. Protocols developed for the phylogenetic analysis of microorganisms are used to examine the composition or identity of organi s m present on a food or environmental sample without havingto culture or grow organisms on media. y SubdivisionProteobucteriu Vibrio group Vibrio cholerue subgroup Vibriocholerae Vibrio vulnificus Vibriofisheri assemblage Vibrioparahaemolyticus Aerornonus group Aerornonas hydrophilu subgroup Aeromonas hydrophila

Enterics and relatives Eschericl~iu-Salmor~ell~~ group Escherichiacoli

Salmonellaspecies Yersiniu group Yersiniaenterocolitica Plesiomonas shigelloides Catnpylobacter and relatives Canlpylobacter fetus subgroup Campylobacter jejuni

Gram positive phylum Clostrium and relatives Clostridium botulinum subgroup Clostridiumbotulinum Bacillus-Lactobacillus-Streptococcu~. subdivision

group

StUphY~OCOCCUS

Staphylococcus aureus Listeriu-Bruc/lot}fri~~ Group Listeria monocytogenes

Streptococci Streptococcusiniae

Fig. 5 Phylogeneticposition of pathogenicbacteriaassociatedwith sedood. No111ellclalure for the lnajor groups and subgroups are thc t e r m used by the Ribosomal Database Project ( 2 2 ) .

Pathogens Transmitted by Seafood

119

The discovery of foodborne bacterial pathogens was made possible by the ability to culturetheorganismsonbacteriologicalmedia.Today, therapididentificationand differentiation of many foodborne pathogens is possible because of molecular genetic and immunologic methods. Nevertheless, for the development of theserapid tools, a pure culture of the pathogenic organism was required, meaningthat the culture was most likely initially isolated, characterized, and grown on bacteriological media in the laboratory. In other words, if the pathogenic organism has not been cultured, it has not been identified or implicated as causing seafood-borne illness.

2. Viruses In addition to the bacteria that may cause disease, animal viruses that can cause human illness are associated with seafood. Compared to bacteria, viruses are small, ranging from 25 to 75 nm, and are therefore not observable under the light microscope. While animal viruses come in a variety of shapes, mostof those associated with food are spherical. The of DNA or RNA, but most of the genetic material of animal viruses may be composed food viruses contain RNA, usually single stranded. Virus particles do not have any metabolism of their own. but require living animal host cells for replication. to host by direct or indirect transmission. Direct transmission, Viruses pass from host also known as contact transmission,is probably the most common way that a virus particle passes from one host to another. For example, direct transmission of viruses that cause gastrointestinal disease occurs through an anal-oral route, usually by hands that are contaminated with fecal material containing the infectious virus. Indirect transmission of viruses may occur by (a) vectors, which are intermediate animals within which the viruses are transported; and Inay multiply; (b) fomites, inanimate objects on which the viruses (c) vehicles, foods and water which may transport the viruses. Viruses that have indirect modes of transmission are required to be more stable and durable compared to those that can only be transmitted directly. Viruses are classified using a different scheme compared to other microorganisms and other higher forms of life. A “tree of life” that consolidates and organizes all of the different viruses does not exist.The primary viruses associated with seafoods are members of two viral families, the Picortuviridue and the Crdiciviriche. The Picortmviridcre are snlall single-stranded RNA viruses comprising some of the important pathogens of humans and animals. These viruses are small, having an icosahedral structure, and their nucleic acid consists of positive sense, linear, single-stranded RNA. The Cdiciviridw is also a positive sense, linear, single-stranded family of viruses. Fig. 6 shows the taxonomic groups of viruses associated with foods using the taxonomic nomenclature as described by the International Committee on the Taxonomy of Viruses (23). Six kindsof viruses causing infectionin humans are associatedwith the consumption of seafood: hepatitis A, human caliciviruses (Norwalk virus and Norwalk-like virus), hepatitis E, astrovirus, group A rotavirus,and human adenovirus. Human poliovirus in seafoods is primarily of interest for historic reasons and since this virus is routinely used in laboratory experiments with seafood. The morphologies of human viruses and the detection of these viruses is often performed with theaid of an electron microscope. Samples containing virus particles are stained with a solution containing an electron-dense compound that creates a “negativestain” image that can be observed under an electron microscope. Fig. 7 displays the electron micrographs of representative viruses that may cause seafood-borne disease.

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Positive Stranded Single StrandedRNA Viruses Family Picornaviridae Genus Enterovirus human poliovirus Genus Hepatovirus hepatitis A (HAV) Family Caliciviridae Genus Calicivirus human caliciviruses, alsoknownas small round structured viruses (SRSV) Norwalk virus Norwalk-like viruses, including Hawaii strain, Snow Mountain strain, Taunton strain, Southampton strain Hepatitis E Family Astroviridae Genus Astrovirus Small Round Viruses (SRV) (probable classification) Double Stranded RNA Viruses Family Reoviridae Genus Rotavirus group A rotavirus Double Stranded DNA Viruses Family Adenoviridae Genus Mastadenovirus human adenovirus Fig. 6 Viruses and virus families associated with food. Viruses that are most frequently associated with seafood are in boldface print. Taxonomic nomenclature as described by the Sixth Report by the International Conunittee on Taxonomy of Viruses (23).

While hepatitis A, the Norwalk viruses, and other viral pathogens are thought to cause many outbreaks and cases of gastroenteritis and hepatitis in humans, the good news for food lnicrobiologists and the seafood industry is that there are many human infectious viruses that are not known to be transmitted by seafoods to humans. These include human immunodeficiency virus (HIV), herpesvirus, Hantavirus, rabies virus, rhinoviruses causing the common cold, and the agent of bovine spongiforrn encephalopathy. The last disease agent is not a virus, but a prion.

E. Summary of Clinical Presentations of the Diseases Table 3 summarizes the symptoms,infectivedose,incubationperiod,andduration of seafood-borne diseases. The time intervals listed for the incubation period and the duration of disease are approximations and vary between individuals, episodes, pathogenic strains,

ransmitted Pathogens

h

by Seafood

727

Fig. 7 Electronmicrographs of representative viruses associated wlth seafood (A) calicvirus, (B) astrovirus, (C) human rotavirus, (D) human adenovirus. Micrographs were electronically retrieved from the Universal Virus Database approved by the International Committee on Taxonomy of Viruses. This site is maintained at the Australian National Universlty.

and the numberof bacteria or virus particles consumed. In this table, pathogenic organisms are separated from each other based on the types of symptoms that are commonly observed. For some of the pathogens the infective dose is based on feeding trials with human volunteers and/or the number of organisms that were enumerated on the incriminating or may item of food. Most of the foodborne pathogens cause a diarrheal disease that may not be accompanied by vomiting and nausea, but some seafood-borne pathogens may cause septicemia or neurologic effects that may lead to death. Additional details about the individual seafood-borne pathogens are provided below.

II. BACTERIAL AND VIRAL PATHOGENS ASSOCIATED WITH RAW AND UNDERPROCESSED SHELLFISH A.

Bacteria

In developed countries, most of the problems associated with seafood are caused by the consumption of raw, underprocessed, or mishandled shellfish, particularly bivalve mollusks. These invertebrates concentrate microorganisms that arein surrounding waters by a filter-feeding process. Someof the pathogenic organisms that are concentrated are indigenous to the aquatic environment, while other pathogens are introduced from terrestrial sources or by the fecal pollution of humans or other warm-blooded animals. After fish

Table 3 Symptoms, Infective Dose, Incubation Period. and Duration of Seafood-Borne Diseasesa Organism

Symptoms

Infective dose

Upper ~~a.stroiritestirin1 tract syniptoins (riatisea, \vmiirirrg) occur Jirsr or predomiriute Sraph~lococci~.s nurect.s Nausea. vomiting, diarrhea. Less than I mg of toxin will abdominal pain produce symptoms.

Incubation period or onset time to symptoms 1-6 hour, mean 2-4 hours

Loic,er ,qa.rrstroiritesfinof rmcr symprotns (abdominal cramps, diarrhea) occiir first or predominate 1- 10 cells 8-72 hours Snlmoriella spp. Abdominal pain, diarrhea, vomiting, nausea Some serotypes are highly in- Variable. depending on seroEscherickia coli Abdominal pain. diarrhea. fective for infants and nausea; some strains may type young children. E. coli is a cause watery or bloody dinormal inhabitant in the arrhea. gut of humans and other mammals. 18-36 hours Cunipylohucter jejLoii Small, perhaps less than 200 Abdominal pain. diarrhea. vomiting. nausea cells 24-48 hours Yersiiiia enterocolitico Fever and abdominal pain are Unknown. but thought to be the primary symptoms; fregreater than 10' cells quently have diarrhea and/ or vomiting; may imitate flu and acute appendicitis. Vihrio cholerue (01 8-96 hours 10" cells with production of Abdominal pain. diarrhea, and 0 1 3 9 ) cholera toxin (CT). Much vomiting, nausea. "rice walower number if stomach ter" stool acid is neutralized 18-36 hours Suspected to be 10"-10' cells Vihrio cholerue (non1. Abdominal pain. diarrhea. vomiting. nausea 01 and non-0139) 2. Extraintestinal infection such as septicemia. wound infections. ear infections 3-76 hours Diarrhea, abdominal pain. Vihrio parahuenrolyti10-10- Kanagawa positive cells CllS vomiting, nausea

Duration of disease

6

2-3 days

5 days Variable. depending on serotype

7-10 days 3-21 days

Varies between individuals, mild, watery diarrhea to acute diarrhea Diarrhea may be quite severe lasting 1 week

X Illness usually mild, with duration of 3-5 days

2

aF

L

v.

e,

U2

0

Pathogens Transmitted by Seafood

-d 0 0

d

c

a

I

r-

-

e,

0

D

124

3

S .M

Henvig

3

S .-

Pathogens Transmitted by Seafood

Hewig

126

and shellfish are harvested, pathogenic microorganisms may also be introduced when the food is processed and transported. y-Proteobacteria and gram-positive A variety of bacteria, including members of the organisms, are associated with shellfish-borne disease in humans. Table 4 lists the Gram’s stain reaction, cellular morphology, major phenotypic characteristics, and the natural hab tats where these pathogenic bacteria are normally found. For many of these organisms, more thorough and detailed reviews about the individual genera and species have been published. In the following sections I will summarize the major features of each of the pathogenic organisms and briefly outline protocols that are routinely used to detect and enumerate these pathogens. Additional details can be found in the citations listed.

1. Vibrio Species Of the several species that comprise the genus Vibrio, three species areof primary concern in seafood-borne disease:V. cholerae, V. parahaemolyticus,and V. vulnijicus. To a much lesser extent, some additional speciesof Vibrio have been suggested to cause human disease such as wound infections and gastroenteritis, includingV. alginolyticus, V. carchariae, V . cincinnatiensis, V. damsela, V. jluvialis, V. fumissii, V. hollisne, V. metschnikovii, and V. mimicus (24). Fig. 8 shows the distribution of cases caused by Vibrio species in the United States from 1989 to 1998 (M. Glatzer, personal communication). During this Vibrio species were attributed same period89%of the deaths caused by seafood-associated to V. vuln$cus (Fig. 9) (M. Glatzer, personal communication). (Since the database was established primarily to follow the number of cases associatedV.with vulnijicus, the number of deaths and cases associated with the other Vibrio species should be examined with caution.) All species of the genus Vibrio are gram-negative rods that are often motile with either peritrichous or single polar flagella. They are facultative anaerobes with both fermentative and respiratory metabolisms. In cultural studies, members of the genus Vibrio are frequently isolated from estuarine water, sediments, and biota. Many strains are chitinolytic, capable of hydrolyzing chitin, a biopolymer composed of monomers of N-acetyld-glucosamine (25). Chitin is found in the exoskeleton of arthropods and in certain fungi. In aquatic environments, strains of Vibrio have been foundin close association with crusta-

V. flwralis V V. mrmrcus

-

3%

alginolyticus 1%

5%

V. vulnificus

55%

parahaemolytlcus 16%

Fig. 8 Cases of Vibrio species causing seafood-borne illness in the United States, 1989-1998 m. Glatzer, personal communication).

127

Pathogens Transmitted by Seafood V. parahaernolyticus -

V. cholerae 6%

!io/.

V. vulnificus

89%

Fig. 9 Deaths caused by seafood-associated Vibrio species in the United States, 1989-1998 (M. Glatzer, personal communication).

ceans. These include crabs, shrimp, and lobsters, seafoods that are of great importance for commercial and recreational harvesters. The reported incidence of potential pathogenic species of Vibrio in fish and shellfish is relatively high. Unlike many of the other pathogenic bacteria listed in this chapter, pathogenic and nonpathogenic strains of the genus Vibrio are autochthonous microorganisms in freshwater, estuarine, and the marine environment. U.

Vibrio cholerue

Disease. Vibrio cholerue is the causative agent responsible for the disease called cholera and in the most severe form is sometimes called cholera gravis. Cholera has killed many people and it continues to be a problem in many developing countries. Outbreaks, epidemics, and pandemics of cholera have been recorded throughout history. Since 1817, the world has been affected by seven pandemics of cholera (26). Specific distinctions in the genus are made based on the production of cholera enterotoxin, also known as cholera toxin (CT), serogroup, and the potential for epidemic spread. The basis of serotyping V. cholerue is the lipopolysaccharide somatic (0)antigen. Until recently, the distinction was simple, V. cholerue 0 1 that produced CT were associated with the epidemics and all other strains were nonpathogenic or occasional pathogens. Now, two serogroups-01 and 0139-are associated with epidemic disease, but not all strains of these serogroups produce CT. These strains do not produce cholera, and are not pathogenic (24). V. cholerue strains of the 0 1 serogroup that produceCT have long been associated with pandemic cholera. CT-negative V. cholerae strains have been occasionally isolated in cases of diarrhea or extraintestinal infections. This serogroup can be further divided into two serogroups called Ogawa and Inaba. V. cholerue 0 1 can also be divided into two biotypes, classical and El Tor. The causative agents of the first four pandemics are not known, but those of the fifth and sixth pandemics are due to the classical biotype of V. cholerue 0 1 . For the past 20 years the classical biotype has only been isolated in Bangladesh. The causative agent of the present, seventh pandemic cholera, the El Tor biotype of V. cholerue, began in 1961 in Sulavesi, Indonesia. The seventh pandemic extended across Asia into the Middle East during the 1960s and through Africa, southern Europe, and the Pacific Islands during the 1970s.This pandemic reached South America

128

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in January 199 I . Within a year a majority of countries in South and Central America were affected (26).Until the emergence of the 0139 serogroup, all isolates that were identified as V. cholerere on the basis of biochemical tests, but were negative for 01 serology were referred to as “non-01.” By late 1992,a new cholera epidemic was caused by the emergence of a new serotype, V. cholerrre 0139,also known as V. cholerrre Bengel (24,26). V. cholercw 0139, which emerged in theBay of Bengalarea i n 1992 hassince been detected in 10 countries. This new strain continues to be confined to southeast Asia (Bangladesh, China, India, Indonesia, Malaysia, Myanmar, Nepal, Pakistan, Sri Lanka, Thailand). Upuntil 1994,imported cases were reported in Estonia, Germany. Hong Kong, Japan, Korea, Singapore, Switzerland, Thailand, and the United States (27).V. cholerrre 0139 appears to be a hybrid of the 01 and non-Ol strains. The clinical presentation and modes of transmission of V. cholercre 0139 are the same as V. cholerere 0 I . In important virulence characteristics, V. cholerne 0 1 39 is indistinguishable from V. cholercrc 01 El Tor strains (28). However, this organism does not produce the 0 1 lipopolysaccharide and has a polysaccharide capsule that is similar to non-Ol strains (24). Cholera remains a global threatand one of the key indicators of social development. While the diseaseno longer poses a threat to countries with a minimum standardof healthy living, it remains a challenge in countries where accessto safe drinking water and adequate sanitationcannotbeassured.Almosteverydevelopingcountryisnowfacingeither a cholera outbreak or the threat of an epidemic (27). In India, cholera is estimated to be responsible for the deaths of 20 million people during this century. Since1991,the cholera pandemicin South America hasbeen responsible for the deaths of 10,000people. Death from cholera is caused by extreme dehydration and loss of electrolytes. An individual with cholera may lose up to 20 L of liquidlday and the tnortality rate may be 30-50% if an infected person is untreated (26). Three major types of V. cholerrre are recognized and are differentiated based 011 serology: 01, 0139,and non-OI/non-0139. The 0 1 serotypeisdivided into classical (nonhemolytic) or El Tor (hemolytic) types. V. c h o l e r m 01 causes cholera, which may be a mild case of diarrhea or a life-threatening disorder. The more recently discovered serotype, 0139,is very similar to 01. Non-Ol V. cholerere causes a less severe disease, usually a gastroenteritis or soft-tissue infection and septicemia. Most strains of non-01/ non-0 139 V. cholertre are non pathogenic (24,29). The symptoms associated with V. cholerne 01 range from a mild, watery diarrhea to an acute diarrhea with rice water stools. The mechanisms for pathogenesis have been well described and include two important aspects, colonization of the small intestine and of intracellular cyclic adenoproduction of cholera toxin. The toxin causes increased levels sine monophosphate (CAMP) and the secretion of water and electrolytes (Na‘, K - , Cl-, bicarbonate) into the lumen of the small intestine (26).

Ecology. Cholera is a disease that is primarily waterborne, but food that has contacted contaminated water may also carry organisms of V. c~l~olcrrre 01. The food that has been most frequently implicated in outbreaks is seafood, both molluscan shellfish and crustaceans. Estrada-Garcia and Mintz (30)showed that seafood was the most commonly implicated vehicle in foodborne cholera outbreaks around the world, with more than 12 outbreaks associated with seafood since 1961 (Table 5 ) . The primary reservoir for V. (Aolerrre is infected humans. Short-tertn carriage of this pathogen by humans is important in transmission of the disease. Persons with acute cholera excrete 107-10s V. cholerrre cells per gram of stool. People who are excreting

nd

Pathogens Transmitted by Seafood

129

Table 5 SeafoodsImplicatedinCholeraTransmissiond

Location Scverlrh pcrrlderrlic

1973 1974 I974 1977 1978 1982 1994

Raw shellfish Raw and undercooked shellfish Salted raw fish Raw salted fish and clams Steamed prawns Cooked squid "Samba1 sotong" Rawfish

198 199 200 20 1 35 36 202

1991 1991 1991 1992

Raw seafood ceviche Cooked crab Cooked crab Shrimp and fish

203 14 204 205

Louisiana Louisiana

1978 1986

34 206

Colorado

1988

Cooked crab Cooked crab and shrimp, raw shrimp and crab Raw oysters

Italy Portugal Guam Gilbert Islands Singapore Singaporc Italy L n r i r ~Arrlericrr epirlenlic

Ecuador Ncw Jersey (Ecuador) New York (Ecuador) California (Pcru) U.S. Gldf Cotr.st

207

large volumes of diarrhea may easily release a trillion viable and pathogenic cells in I O L of diarrhea. Asymptomatic carriers may live in the same household as an individual suffering from acute illness. In various studies 4-22% of individuals may be asymptomatic carriers. Although there is evidence that V. cholerne can survive and multiply in the environment (31), the rates of isolation of CT-producing V. cholera 0 1 from the environment correlate primarily with the degree of sewage contamination (29). Several reports have illustrated the close association between Vibrio species and crustacean zooplankton. While most research related to public health has focused on the shellfish that humans purchase and consume, smaller planktonic animals may be causing very significant problems for humans. Huq et al. (32) described a simple filtration method to remove plankton-associated V. cholercre in raw water supplies in developing countries. In laboratory experiments, the bulk of the plankton was removed with a filter constructed from either nylon net or sari material, the latter being inexpensive and readily available in Bangladeshi villages. Seafood may be contaminated if harvested from water polluted by sewage or from environments where V. cholerne 0 1 occurs naturally independentof human fecal contamination. In artificially contaminated oysters and clams, V. ckolerne 0 1 survived for more than 3 weeks when refrigerated (33). Crabs boiled for less than 10 minutes or steamed for less than 30 minutes may still harbor viable V. choler-ne 0 1 organisms (34). In the United States, the first cholera outbreak associated with seafood occurred in Louisiana. Eleven cases of cholera were associated with the consumptionof home-cooked crabs that were either boiled or steamed. Secondary contamination of seafood has been reported. Steamed prawns (35) and cooked squid left for at least 4 hours at room temperature (36) were implicated in two cholera outbreaks in Singapore. In businesses that maintain fish live in aquaria for human consumption, the aquaria

130

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water may contain pathogenic organisms such as V. choler.cre. Kam et al. (37) described the investigation of 12 cholera cases in Hong Kong. Microbiological investigations demonstrated that contaminated seawater in fish tanks that were used for keeping seafood alive was the most likely vehicleof transmission. The source of seawaterin some facilities Inay be contaminated. Restaurant owners in Asia who serve seafood often receive seawater for their holding tanks from “water vehicles.” The operators of these vehicles Inay draw seawater from polluted harbors or typhoon shelters, protected anchoring areas that provide shelter for fishing junks and other small vessels during monsoons (38). Estrada-Garcia and Mintz (30) listed several factors that make seafood a possible also important for the other Vibrio vehicle for cholera transmission. These factors are species that are pathogenic to humans: In many parts of the world seafood is consumed raw or undercooked. Seafood may be contaminated with V. cholcrcre in the aquatic environment. V. cholercre persists for many weeks in seafood, even if refrigerated (39) and multiplies rapidly if introduced onto cooked seafood (40). Therefore secondary contamination may be a problem. V. cholernc adheres to chitin, and chitin-absorbed bacteria seem to be more resistant to variations in pH and heat than free-living bacteria (41). V. cholercle colonize the surfaceof copepods and may concentratein the gastrointestinal tracts of animals that ingest copepods (42). MicrobiologicalProcedures.Traditionalmicrobiologicalproceduresfor

V. chol-

erne and other pathogenic Vibrio strains are well established and have changed little in

recent years. Most species of Vibrio grow well at alkaline pH and a key step in the protocols istheinitial enrichment in alkaline peptone water. Enrichments are streaked onto thiosulfate citrate bile salt (TCBS) agar and typical colonies are tested with a series of biochemical and physiological tests. Fig. 10 summarizes the protocol for V. cholercre (43). The key confirmation for the identificationof V. cholercre 0 1 is agglutination in polyvalent antisera. Antiserum against 0 1 3 9 is now available.

b.

Vibrio vulniJcus

Disease. Vibrio vultlificus isthe mostseriouspathogenic Vibrio species in the United States, responsible for 95% of the seafood-related deaths. A disease caused by V. vultzificus was first described by Roland in 1970 (44) who detailed the case of endotoxic syndrome and leg gangrene acquired in New England coast water. The etiologic agent was misclassified as V. parnhaernolvticus (44). Investigators later examined their collections of lactose-positive vibrio strains and a new species of Vibrio, V. [email protected], was declared in 1979 because the pathogen could produce cutaneous lesions (vulnificus, meaning wounds in Latin) (45,46). In the state of Florida, this organism is the leading cause of foodborne deaths. While most of the cases that have been documented are from the southeast region of the United States,V. vulngcus was recently found in the waters and shellfishin Europe. In 1996, Dalsgaard et al. (47) reported the first clinical and epidemiological data about a series of V. drzificus infections in northern Europe. Arias et al. (48) presented the first report of the detection of V. vult$ficus naturally present in seawater and edible shellfish along the Spanish Mediterranean coast. This pathogenic bacterium may be Inore widespread in temperate and tropical waters than previously thought. v . \)uln.$cu.y manifests itself in three fornls of human disease, with two forms of seafood-borne disease having a very high rate of mortality. First, v. v u i t l i f i c w lnay cause

131

Pathogens Transmitted by Seafood

Vibrio cholerae Oysters

Oysters

Weigh 25 g sample and add 225 ml of APW Homogenize for 2 min

Weigh 50 g sample and add450 ml Alkaline Peptone Water (APW) Homogenize for 2 min

Split homogenatein half

250 m1 anddilutions

Than Foods Other

Prepare serial dilutionsof homogenate 250 ml anddilutions

t

Incubate homogenate and APW dilutions 6-8 h, 16-24 h at 35-37°C

Incubate homogenateand APW dilutions 6-8 h, 16-24 h at 42°C

Streak all enrichments onto Thiosulfate Citrate Bile Salts (TCBS) agar (and, optionally, Modified Cellobiose PolymyxinB Colistin (mCPC) agar) TCBS is incubated 18-24h at 3537°C; mCPC is incubated 18-24 h at 39-40°C

t

Pick typical colonies onto 1%Tryptone + 1% NaCl (T,N,) or Trypticase Soy agar (TSA) + 1.5% NaCl Incubate 12-24h at 35-37°C

I

t

Perform preliminary biochemicaland physiological tests Triple Sugar Iron (TSI) Kligler Iron Agar (KIA) Arginine Glucose Slant (AGS) 1% Tryptone + 3% NaCl TIN^), 1% Tryptone + 0% NaCl (TINo) Gelatin Agar (GA), Gelatin Salt (GS) Hugh-Leifson glucose broth Oxidase Gram stain

t

Confirm V. cholerae 01, V. cholerae 0139, V. cholerae non-01, V. cholerae non-0139, and V. mimicus using serological and biochemical tests

Fig. 10

Enrichmcnt.isolation,andidentificationprotocol

for Vihrio cholerrre (43).

Henvig

132 Table 6 Characteristics Among Patients with

Vibrio vulnificus Infections in the United States,

1988-1996O

Gastroenteritis septicemia Wound Characteristic Primary Median age, years (range) Males (9%) Fever Diarrhea Abdominal cramps Nausea Vomiting Shock Localized cellulitis Bullous lesions Hosuitalized

infection 54 (24-92) 89 91 58 53 59 54 64

-

49 97

35 (0-84) 57 57 100 84 71 68 0

0 65

59 (4-91) 88 88

-b

30 91

89

' Data presented by Shapm et al. (49). Characteristlc not associated with syndrome.

a primary septicemia often resulting from the consumption of raw oysters. The mortality rate for this form is about 60%. Second,V. vulnijicus may cause wound infections on the skin. This disease may be causedby organisms present in seawater andlor shellfish, and has a mortality rate of 20-25%. The third form of the disease is a gastroenteritis that rarely causes death. Table 6 summarizes the characteristics among patients with V. vulnacus infections in the United States from 1988 to 1996 (49). V. vuZn$cus septicemia is nearly always associated with the consumption of raw oysters. Previous reports have shown the prevalence of raw oyster consumption in the general United States population to be around 17%, with a prevalence reaching as high as 32% in coastal states such as Florida(50). Data clearly indicate the hazards associated with the consumption of raw shellfish, particularly oysters, harvested from the Gulf Coast of the United States. Oysters causingV. vulnijkus disease have been harvestedin Louisiana, Florida, and Texas (Fig. 11). Although many of the shellfish-associated cases of V. Unknown

Fig. 11 States of harvest for shellfish associated withVibrio vulnificuscases (M. Glatzer,Personal communication).

Pathogens Transmlfted by Seafood Unknown

133

9 states

Anzcna

2%

New York 2%

Florida

38%

Alabama

Texas

9%

11%

Fig. 12 States of consumption of shellfish for cases associated with Vibrio vuln$cus personal communication).

(M.Glatzer,

vulnijicus occurred in these harvest states, there were also a significant number of cases as California, and states that in states where there are major population centers, such receive shipmentsof shellfish from the Gulf (Fig.12). These data also illustrate the complexity of the problems that are faced by federal and state public health officials in dealing with the interstate transport of raw shellfish distributedto retail and food service establishments. Nearly three-quarters of the cases of V. vulnifcus disease are associated with the consumption of shellfish in restaurants (Fig. 13). The common symptoms are fever, chills, nausea, and hypotension (low blood pressure). Symptoms associated with gastroenteritis are often present, but are less common. An unusual symptom that also occurs is the development of secondary lesions that are often found on the extremities. These lesions may develop into necrotizing fasciitis or vasculitis that may require the amputation of limbs or surgery. Fortunately infection with V. vulnijicus is not a serious problem for healthy people; nearly all serious and deadly Unknown Miscellaneous

Party

4%

4%

5%

qestaurant 72% Fig. 13 Location of shellfish consumption for cases associated with Vibrio vuln$cus (M. Glatzer, personal communication).

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Herwig

infections occur in people with an underlying disease that causes an elevated level of iron of patients in the blood or in those who are immunosuppressed. About three-quarters with raw oyster-associated V. vulnificus primary septicemia have preexisting liver disease ( 5 1S 2 ) . In Florida, the reported rateof raw oyster-associated V. vuln$cu.s primary septicemia is 60 times greater among personswith liver disease thanin those without liver disease (53). Liver or blood-related diseases may predispose an individual to serious V. vulrl$cr~ septicemia. In recentyears,additionalwork has indicated that patients who are immunosuppressed are also susceptible to V. vuln$cus and V. ckolerue infections. Raw oyster-associated V. vulnificus septicemia has been reported in at least one patient with HIV infection (54) and a fatal case of V. cholerae non-01 sepsis was described i n a 51-year-old patient who was undergoing chemotherapy for leukemia and reported consuming raw oysters 48 hours before he became ill ( 5 5 ) . A report (56) describing the acquisition of V. vulr~ijcus septicemia from wounds in two patients with solid organ transplants supports the notion that immunosuppressed persons are at increased risk for severe Vibrio infections. Some patients with underlying liver diseases continue to consume raw oysters even after they are informed about the risks. Tayloral. et (57) studied the raw shellfish consumption practices of patients with liver disease being seen in the outpatient gastroenterology clinic at Walter Reed Army Medical Center. One-fifth of patients with known liver disease, even when previously informed about the risks, reported eating raw shellfish. In 199 I , California was the first state to require restaurants and other establishments that serve or sell Gulf Coast oysters to warn prospective customers about the possible risks. Florida and Louisiana also use these warning regulations. In October 1995, the ISSC rejected an FDA proposal to ban the sale of raw oysters obtained from the Gulf of Mexico during warm-weather months. Instead, oyster harvesters are now required to refrigerate oysters within 6 hours after harvesting from this region. The U.S. government has received pressure from national consumer groups to set a standard requiring the Gulf Coast shellfish industry to eliminate pathogenic bacteria, such as V. ~ ~ u l n ~ j i cfrom r ~ s ,oysters. The consumer groups notethat technologies are available that can eliminate this organism. A mild heat pasteurization technology is offered by AmeriPure Oyster Companies of Empire Louisiana. Another technology that appears to be promising, a high-pressure treatment, is also being developed (58).

Ecology. V. vulnificw is a normal inhabitant of the estuarine environment, having of the United States, the Gulf of Mexico, and in estuarine been isolated on both coasts environments around the world. The ecology of V. \~uh$cu.sis very similar to V. pcrrcrhrremolvticus. As filter-feeding shellfish, oysters concentrate the number of microorganisms found in the water column in their gut. For example, one study found a nearly 10,000-fold increase in concentration, from 7 colony forming units (cfu)of V. vuln[jicu.s in seawater to 10" cfu/g of oyster. As found with other marine vibrios, there is no correlation between the presence of V. vulrlificus and fecal coliforms. Therefore, measuring thelevel of E. coli or fecal coliforms is not a good monitoring method for V. vulrlificus. Some of the highest concentrations of V. vulniJcus have been found in the intestines of finfish (59). V. vulnificus also undergoes seasonal population variations correlated with temperature. The data of Motes et al. (60) clearly show the relationship between temperature and the levels of V. vulr1ificu.s in oysters. During warm-weather months,when more than 85% of the shellfish-associated V. vuln$cus occur, most probable number (MPN) counts were usually 103-10'/g of oyster meat. During cold-weather months,when infections have not

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135

been reported, MPN counts were less than IO/g. Gulf Coast data suggest that the number of V. wtln[ficus organisms i n oysters is strongly correlated with water temperature until the temperature reaches 26”C, above which there appears to be no additional increase in the number of bacteria. Seasonal temperature change explains most of the variability in the Vibrio levels in the Gulf. Salinity explains an additional 10% of the variability in these levels and also explains the differences between sites. Shapiro et al. (49) summarized data that was collected by the Gulf Coast Vibrio Surveillance System, which includes public health agencies in the states of Florida, Alabama, Louisiana, and Texas. Using data collected by the CDC, they performed oyster trace-backs and examined the temperature of the place of harvest. For primary septicemia infections caused by V. vulrz(ficus, they found that all of the implicated oysters were harvested in the Gulf of Mexico when water temperatures were greater than 22°C. Among the 72 traced cases between 1988 and 1995, only 3 infections occurred in persons who consumed oysters harvested in waters with temperatures less than 20°C. While water temperature can serve to predict infections by V. vuh~[ficus,Shapiro et al. (49) did not prove that higher temperature is the cause of the increased incidence of V. vuln$cus infections. Unfortunately for the oyster industry in the Gulf, their data support the strong association between “summer harvesting” and illness, but summer harvesting represents 6 months of the year (May-October). Since 1972, oyster harvesting during this period has increased from 15% to 40% of the total annual production. A simple response to this problem, such as a closure of the harvest from May to October, would resultin a severe economic impact to the Gulf Coast oyster industry. Methods to reduceV. vuln$cus in live oysters after harvest include depuration(also called relaying), irradiation, and heat treatment. Gamma irradiation, although effective in reducing V. vuln$clrs in oysters in doses of 1.O- 1.5 kGy, also increases oyster mortality during storage. Also, this process is not approved by the FDA. Conventional depuration of oysters with an indigenous microflora of V. vulw$cus has been unsuccessful (61,62) and if conducted at temperatures higher than 21°C may actually increase the number of V. vuln$cus in oysters. Motes and DePaola (63) evaluated suspension relaying of Gulf Coast oysters to offshore waters that are normally free of V. vuln$cus. They observed a decline of V. v~rlr~$cus in relayed oystersthat was suggested tobe associated with exposure to high-salinity environments essentially devoid of V. v u l ~ z $ c ~ ~ s . Besides using methods to reduce the populations of potentially pathogenic v. vu/r~$cus cells in oysters, procedures can be used to tninilnize the increase in v. vuln(fifi~us numbers after harvest. Reducing the time oysters remain outside of refrigeration can decrease consumer exposure to high numbers of V. vulrz$cus; the oysters must be cooled immediately after harvest to eliminate postharvest growth of V. vuln$cus. Under typical Gulf Coast industry practices, oysters are held on the deck of harvest vessels without refrigeration or icing until the vessel docks. The NSSP does not require shellstock to be refrigerated on harvest vessels. By the summer of 1995 most Gulf Coast states had regulations on the time shellstock can remain outside refrigeration that are more restrictive than those set forth in the NSSP manual (64).

MicrobiologicalProcedures. Theenrichment,enumeration,andisolation procedures for V. vuln$c~u are very similar to those used for V. parcrhaemolyticus. Many studies attempt to perform enumerations for both Vibrio species using the same samples. Mostprotocolsbeginwith an enrichment step in alkalinepeptonewater,followedby plating onto a selective and differential medium called TCBS agar. Similarto V. cholerae,

Herwig

136

V. vu1rlijicu.s can grow under alkaline conditions. Inrecentyears, however, a modified cellobiose polymyxin B colistin (mCPC) agar has replaced the use of TCBS for isolating V. vulnificus. Suspected colonies are examined in a variety of biochemical tests. Fig. 14 outlines the enrichment and identification procedures for V. vulrzijicus and V. purcrhnetno-

lyricus (43). Some V. vultz@xs researchers believe that the environment contains a very diverse population of V. vultlificw strains and that only a certain subset appears to be associated with human disease(65,66).To examinethe diversity that exists within the species, molecular methods have been used including restriction fragment length polymorphism (RFLP) and ribotyping. Tamplin etal. (65) described RFLP and biotype profiles for environmental

Vibrio vuinificus

Vibrio parahaemolyticus

Weigh 50 g of sample and add 200 ml of Phosphate Buffer Saline(PBS)

Weigh 50 g of sample and add 200 ml of 2-3% NaCl solutionor Phosphate Buffer Saline (PES)

Prepare serial hutions in PBS

PreDare serial dilutions in saline or PBS

Inoculate Alkaline PeptoneWater (APW) MPN Incubate 12-16 h at 3537°C

Inoculate APW or Alkaline Peptone Salt broth (APS) MPN Incubate 16-18 hat 3537°C

t

Streak enrichments onto Modified Cellobiose Polymyxin B Colistin (mCPC)agar Incubate 18-24 hat 39-40°C

+

Streak enrichments onto Thiosulfate Citrate Bile Salts (TCBS) agar Incubate 18-24 hat 35-37°C

Pick typical colonies onto l%Tryptone1% NaCl (TINl agar) or Trypticase Soy agar + 1% NaCI, and Gelatin agar (GA) or Gelatin Salt agar (GS) Incubate 12-24 hat 35-37°C

+

Perform prelimmary biochemical testing to differentiate species Gram stain Oxidase Motility Arginine Gelatin Salt (AGS) Triple Sugar Iron (TSI) 0/129 vibriostat sensitivity ONPG test

Perform additional physiological, biochemical, serological, and pathogenicity tests

Fig. 14 f?ruiyticlls

Pathogens Transmitted by Seafood

137

and clinical V. vlrlw$c.u.s strains. They found high levels of variation in RFLP profiles among 53 clinical and 78 environmental isolates as described by pulsed-field gel electrophoresis. In contrast, ribotype profiles showed greater similarity. Jackson et al. (66) presented evidence that V. vuln;fic~rsinfections result in the proliferation of a single pathogenic strain, not a mixture of strains, and that high-risk individuals may be susceptible torelativelylowconcentrations of V. r~uln~ficus, as low as 10' cells/g of oyster if the pathogenic strain is present. Characterization of V . 1~1rl11$crrs strains has led to subdivision of the species into two biotypes which are now suggested to be basedon serological properties and host range, rather than the more variable biochemical properties. Biotype 1 strains are pathogenic for humans, exhibit several immunologically distinct lipopolysaccharide(LPS) types, and are indole positive (67). Biotype 2 strains appear to be virulent for both humans and eels and express a conmon LPS type, and the majority of biotype 2 strains are indole negative (67,68).

c'. Vihrio ~~nr~r11tremolytic'rl.s Disease. The firstreport of a V. pnr~rhaernolyticusfoodborneoutbreakwas in 1950. This organism was first isolated in 1953 as the causative agent for food poisoning i n Osaka, Japan (69). In some regions of the world V. parnkaernolyticw is the leading cause of gastroenteritis associated with food. In 1994, 102 outbreaks of foodborne disease involving 4726 cases were reported to the Taiwan Department of Health. Of these outbreaks, 72.5% were caused by bacterial pathogens with V. partrknemolyticus responsible for 56.7%, Stcll'h?.lococc.lrs mre1r.s 20.3'76, Bcrcilllrs cereu.s 14.9%, and Salmonella spp. other than S. typhi and S. pcrrcrtyphi 8.1% (70). V. ~'c~r~~hrrenrol~ticus has been the leading high proportion (54%) of the cause of foodborne illness i n Taiwan for many years. A large outbreaks, defined as having more than 50 cases, was associated with commercial lunch-boxes suppliedto elementary and juniorhigh schools. In Japan, V. pczr~rkezernolyticus is the leading cause of foodborne disease, with as much as 70% of the bacterial foodborne disease i n the 1960s being caused by this organism. Most of these outbreaks were associated with the consumption of seafood. Whereas most Japanese outbreaks involve fish, in the United States outbreaks primarily involve crab, shrimp, lobster, and oyster. The largest outbreak caused by V. p r o hrlenlo1yticu.s occurred in the summer of 1978 and affected 1133 of 1700 people attending a dinner in Port Allen, Louisiana, in which boiled shrimp was served. The shrimp were boiled in the morning then repacked i n the wooden crates in which they had been stored before they were cooked. The boiled shrimp were stored in an unrefrigerated truck until they were served approximately 8 hours later. In the last 2 years, two major outbreaks caused by V. pcrrtrhrrenro1~ticu.shave occurred in the United States. Thereis some concern among regulatory officials and within the industry about whether the recent outbreaks are an anomaly or the start of a new trend. During July-August 1997, the largest outbreak i n North America in recent years of confirmed V. ~~nrcrhaemo1~~ticu.s infections occurred (7 1). Illness in 209 people was associatedwith eating raw oysters harvested from California, Oregon, and Washington in the United States and British Columbia in Canada. One person died from the outbreak. During this outbreak, most ill people had no underlying illness. There was a suggestion that a slightly higher than normal water temperature contributed to this outbreak. Mean Pacific coast sea surface temperatures recorded by the U.S. Navy ranged from 12°C to 19°C from May through September. These temperatures were I"C-5"Chigherthanthe temperaturesfor the sameperiod in1996.Thelargest

138

Henvig

previous outbreak of V. PLrrah~lerno1yticu.sinfections reported in North America occurred in 1982 and resulted in 10 culture-confirmed cases (71). In 1998, an even larger V. purerIlnernolyticus foodborne outbreak occurred along the Gulf Coast. This outbreak caused illness in more than 400 people (M. B. Glatzer, personal communication). The symptoms associated with V. ~~arrrhtret~~ol~ticr~,s are primarily diarrhea and abdominal cramps with fever, nausea, and vomitingto a lesser extent. The infectious doseis thought to be large, somewhere between lo5and IO’ cells. Pathogenic V. ~~~rrahnemolyticus strains, which cause acute gastroenteritis after consumption of raw or partially cooked seafoods, have been known to produce thermostable direct hemolysin (TDH), also known as the Kanagawa phenomenon, or TDH-related hemolysin (TRH), orboth TDH and TRH (72,73). Most clinical isolates (96.5% of 2720 strains) have been Kanagawa phenomenon positive, whereas 99% of 650 environmental and food cultures have been found to be Kanagawa phenomenon negative (74,75). Suthienkul et al. (76) used PCR to analyze genes encoding TDH (tdl1) and TRH (tr.11) in 489 clinical strains and found that 81 % of the 489 strains were TDH’TRH-, 5.5% were TDH-TRH’, 2% were TDH-TRH’, and 11.5% were TDH-TRH-. Hence the investigators thought that identification of V. parahnemol~ti~us, irrespective of its virulence factors, is necessary for public health (77). V. ~~a~~rhnemolyticus is not a reportable disease in all states. During the 1997 outbreak,publichealthofficials in Washington,California,andBritishColumbiabecame aware of the outbreak through routine reporting. An editorial note in Morbidity mcl Mortality Weekly (71) suggested that all states should consider making V. ~~trr~rh~retrrolyticus and other vibrioses reportable.

Ecology. V. ~~arc~haerrrolyticus is a naturallyoccurringbacteriuminestuaries around the world. It is found in sediments, plankton, and in a variety of fish and shellfish. Pioneering work performed in the 1970s in Rita Colwell’s laboratory clearly documented the ecology of this human pathogen in the Chesapeake Bay (78). V. ~~arahaemolyticus was found to have a strong association with zooplankton during the warm summer months, and appeared to “overwinter” in the sediments during the cooler periods of the year. Most Vibrio species are chitinolytic, and thus are involved with the biodegradation of crustacean exoskeletons and other forms of chitin (79). Several studies have not found a correlation between the number of V. pLrrCrhClern[)lyti(.u,~ and fecal coliforms in environmental samples (61$0). V. yrrrcrhc~emolyticus has a remarkably rapid growth time, as short as 8 or 9 minutes at 37°C in bacteriological media. Even in seafoods, generation times of 12-18 minutes havebeenreported(24).Withsuch a shortgenerationtime,thisspeciescangrowto remarkably high concentrations i n a relatively short time. Recently another type of shellfish has been implicated as a vehicle in seafood-borne V. f,crr~rhnemolyticusdisease. Bean et al. (81 ) demonstrated that the consumption of cooked crayfish was associated with V. pcrrcrhaemolyticus infection. Crayfish are commercially harvested along the Gulf Coast of the United States. Of interest, no crayfish consumption was reported in V. vuln$cus infections. Crayfish have been described as “nouvelle cuisine” and are becoming an increasingly popular seafood item in many areas outside the Gulf Coast region. MicrobiologicalProcedures. Procedures verysimilartothoseusedfor V. vulr1ificu.s are used for V. parahaemolyticus (Fig. 14) (43). Sometimes additional amounts of NaCl are included in the diluent or enrichment broths used for V. ~~~rrrhaemolyticus. Isolates of V. pcrrahcretnolyticus can be serotyped according to their somatic (0)and capsular

Pathogens Transmitted by Seafood

139

(K) antigens, based on a scheme developed by Sakazaki et al. (82) who exanlined 2720 strains. Presently there are 120 antigens and 59 K antigens. Although many environmental and some clinical isolates cannot be typed bythe K antigen, the majority of clinical stains have recognized 0 types. While the antigenic characteristic is helpful in identifying and monitoring different strains, there appearsto be no correlation between serotype and virulence. Not all strains of V. / ~ r ~ ~ r r l ~ r ~ e t ~cause r ~ ) / yhuman t i c ~ ( . ~disease.I n many environmental studies pathogenic strainsof V. pcr~rrhcrenrolytit~us are never orrarely found. To enumerate or identify pathogenic strains, special attention is paid to finding strains that produce a hemolysin, called TDH (thermostable direct hemolysin) or Kanagawa hemolysin. A special blood agar called Wagatsuma agar (83) can be used to detect the production of this special hemolysin. but i n recent years a gene probe has been used to detect tdh genes. Primer sets for specific amplification of the TDH gene fragment have been described, and by using a preenrichment step i n alkaline peptone water for 8 hours, I O cells can be detected (84). Kaysner et al. (85) concluded that the urease-positive biotype is a useful marker for identifying potentially pathogenic strainsof V. ptrrnhtrernolyticlrs isolated from mollusks grown in the Pacific Northwest. This phenotype may only be useful for a preliminaryscreen,since all TDH-positivestrainswereureasepositive,however, notall urease-positivestrainsproducedTDH.Theassociation of positiveureaseactivityand TDH. has not been observed, however,in strains isolated from other regionsof the world (86). Chen and Chang (86a) described a rapid method for detecting V. /,rr~rrhaemo/yticrrs in oysters by immunofluorescencemicroscopy.Antibodieswerepreparedagainsttwo outer membrane proteins of V. pcrmhrretrrolyticus and an indirect staining method was employed. It was suggestedthat this could beused as a rapid screen ofV. ~~tr~crhLlerr~o!\‘ricu.s in oysters, with a presumptive positive result obtainable within 24 hours. Some problems remained, however, with cross-reactions between closely related but generally nonpathogenic organisms, such as V. crlgir~olyticrrs.

2 . Salmonella (1. Diserrse. Species of Soltwtrelltr are gram-negative, facultative anaerobic, nonspore-forming, peritrichously flagellated rods that fernlent glucose and utilize citrate as a sole sourceof carbon. Some species arenot motile. S c h r o t w l l t r spp. are very closely related ~ ~ ~ r rspecies e. of Srrlrrronellcr are to E. coli and are lnenlbers of the E l l t e ~ o b t r c ~ t e r - i ~ ~Several recognized and hundreds of different serotypes have been identified. The nomenclature of the Salmonella group has changed over the years and is based on a taxonomic scheme that uses biochemical and serologic infomiation and on the principles of numerical taxonomy and DNA/DNA homology (87). Manywould argue that all of the different “species” and strains are actually a single species with numerous subspecies. The three species that are generally recognized on a biochemical basis include S. typhi, S. cholertresui.~,and S. erlteritidis (88). Theserotypes are identified according to the Kauffnlann-White antigenic scheme and are segregated based on their somatic (O), capsular (Vi), and flagellar (H) profiles. Serologic tests are complex and labor-intensive techniques involving the agglutination of surface antigens with Salmonella-specific antibodies. Before 1950 typhoid fever, the disease caused byS. phi, was the primary infection caused by Salttrorwlltr in Western countries. This disease was the most frequent form of water-borne disease, and thousands of people died of typhoid fever during the early part

140

Hewig

of this century. Most of the outbreaks occurring earlier this century were related to S. hphi in the consumption of raw mollusks. At present, nontyphoid Srrltnonella spp. are one of the leading causes of bacterial foodborne disease in the United States and in many other non-Asian countries. Scrlrnorrell~ is associated with shellfish that is harvested from contaminated water or with seafoods that may have come in contact with other meats, poultry, or dairy products during preparation. The acute symptoms of the disease, called salmonellosis, are nausea, vomiting, ab1-2 days. dominalcramps,diarrhea,fever,andheadaches.Acutesymptomsmaylast Some people may suffer more chronic symptoms including arthritis-like symptoms that may follow 3-4 weeks after the onset of the acute symptoms. The infectious dose may be low, withas few as 15 cells causing the disease. As with other gastrointestinal diseases, the severity of the infection depends on the health and age of the person and strain differences within the genus.All age groups are susceptible, but symptoms are most severe in the elderly, infants, and the infirm. AIDS patients frequently suffer salmonellosis (estimated at 20-fold more than the general population) and suffer from recurrent episodes. The disease is caused by penetration and passage of Srrlrnonella organisms into the epithelium of the small intestine where inflammation occurs. There is evidence that an enterotoxin may be produced (87). Strlnwnella is among the leading causes of foodborne illness in the United States. It has been estimated that 2-4 million cases of salmonellosis occur in the United States each year. The CDC estimates that 75% of S. enterifidis outbreaks are associated with the consumption of raw or inadequately cooked Grade A eggs (87).

h. Ecology. Salmonellu arewidelydistributed amonganimals,particularlydomesticatedpoultryandswine,and in the environment.Environmentalsourcesinclude water,soil,insects,factorysurfaces,kitchensurfaces,animalfeces,andwildanimals. Scdrnor~ellaare primarily a problem associated with raw meats, poultry, and eggs, but they are also associated with milk and dairy products, fish, shrimp, frog legs, sauces and salad dressing, and manufactured foods that have ingredients that include the above.SLrlmonella species can be readily isolated from fish from aquaculture farms. Table 1 lists the prevalence of S d r n o r w l l c r in aquaculture farms in South Africa, the United States, Japan, and the Philippines ( 5 ) . There is evidence that Scrlrnonelkr is frequently isolated from these operations. Many aquaculture farms may be using antibiotics to safeguard the health of farmed fish and shellfish. c. Microhiologictrl Procedures. Traditionalmethodsfortheenrichment,isolation, and identification of Sdrnonella often include several steps or transfers and may take more than a week to perform. Usually, a preenrichment step in lactose broth precedes enrichment steps in tetrathionate broth and Rappaport-Vassiliadis (RV) broth. These steps are followed by plating on selective media that may include bismuth sulfite agar, xylose lysine deoxycholate agar, and hektoen enteric agar, biochemical screening, and serotyping. Fig. 15 describestheprotocolrecommended by the FDA.Recentstudiesindicate that modified semisolid Rappaport-Vassiliadis (MSRV) broth is very effective at yielding results that are better than or equal to those obtained with RV broth (89).In the last 10 years several research laboratories and commercial firms have described more rapid screening methods (90). Several of these methods have been recognized and approved by international agencies.

147

Pathogens Transmitted by Seafood Weigh 25 g of tissue Add 225 ml Lactose broth and blendfor 2 minutes

+

i

Transfer to wide-mouth, screw cap jar (500 ml and let stand for 60 min at room temperature

*

Mix well, adjust pH, if necessary to 6.8 0.2 Add up to 2.25 ml of surfactant (Tergitol Anionic 7 or Triton X - l 00)

+

Loosen jar lids and incbbate 24 k 2 h at 35°C

Transfer 0.1 ml of mixture

Rappaport-Vassiliadis (RV) broth Incubate 245 2 h at 42 rt 0.2%

Tetithionate (TT) broth Incubate 24 rt 2 h at 43f 0.2%

+

Transfer loopful from TT and RV broths onto: Bismuth Sulfite (6s) agar Xylose Lysine Deoxycholate (XLD) agar Hektoen Enteric (HE) agar Incubate 24 f 2 h at 35°C

/

Pick 2 or more colonies from each selective agar Pick typical colonies onBS agar at 24 rt 2 h, 48 f 2 h Transfer to Triple Sugar Iron (TSI) agar, Lysine Ironagar Incubate 24 k 2 h at 35°C

+

Apply biochemical and serological tests to presumptive Salmonella from TSI agar Urease test Polyvalent flagellar(H) test Lysine decarboxylase broth Phenol red dulcitol broth KCN broth Malonate broth Indole test Polyvalent somatic (0) test Phenol red lactose broth Phenol red sucrose broth Methyl Red-Voges Prokauer tests Simmons citrate

Fig. 15

Enrichmentandisolationprocedurefor

Salnronella

species

(90).

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142

3.

Escherichia coli

(1. Disecrse. The bacterial species E. coli has been described and known for many years. The organism is a common inhabitant of the facultative anaerobic microflora found in the intestinal tracts of humans and other warm-blooded animals. E. coli is a gramnegative, motile rod, capable of fermenting lactose and other sugars at 35°C. This organism is a member of the family of bacteria known as the Enterobacteriaceae. Isolates can be serologically differentiated on the basis of three major surface antigens, the somatic (O), flagellar (H), and capsule (K) antigens. Most of the strains found i n humans and in contaminated water samples are avirulent. Only certain strains of E. coli are pathogenic to humans and these strains are referred to as theenterovirulent E. coli (EEC) group. Four classes of E. coli are generally recognized: (a) enterotoxigenic E. coli (ETEC), (b) enteropathogenic E. coli (EPEC), (c) enterohemorrhagic E. coli (EHEC). primarily of the serotype 0157:H7, and (d) enteroinvasive E. coli (EIEC) (91). In addition to these four classes, diffuse-adhering E. coli (DAEC) and enteroaggregative E. c d i (EaggEC) strains have been recently recognized and associated with diarrhea in children in certain parts of the world (92). ETEC causes gastroenteritis andis sometimes called travelers’ disease. The infective dose is IO’ or more cells, possibly less for infants. This E. coli class is not a problem in countries with high sanitary standards. Humans are the principal reservoir of ETEC that cause human disease. EPEC causes watery or bloody diarrhea primarily i n infants, with few cases reported for adults. The most common foods associated with this disease are raw beef and chicken, and infant formula prepared with contaminated water. Humans are an important reservoir for EPEC. EHEC, primarily associated with serotype 0157:H7, causes hemorrhagic colitis, a disease that can be particularly severe for infants and young children. The vehicle generally associatedwith this disease is raw or undercooked ground beef. Cattle appear to be the natural reservoir for E. coli 0157:H7. EIEC causes bacillary dysentery, a mild form of dysentery, where there is the appearance of blood and mucous i n stools. The infective dose for EIEC is thought to be low and it is associated with food contaminated with human feces, since humans are a major reservoir.

h. Ecology. E. coli andothercloselyrelatedentericbacteriaarenonnalinhabitants of the guts of humans and other warm-blooded animals. Pathogenic strains of E. c d i may be a problem in shellfish and fish that are harvested from water containing human and other fecal pollution. Even though there are few reported cases of E. coli causing shellfish-borne disease, E.coli is a useful organism for monitoring the environment and shellfish for fecal contamination.

c. Mic.rohiolo~~iic.LI1 Procedures. Proceduresfor the enumeration of E. coli and fecal coliforms have beenusedformanyyears. The bacteriological quality of surface water, drinking water, and shellfish growing water is generally determined and regulated based on the presence and levels of fecal coliform and E. coli. The term “fecal coliform” is described by an operational definition, that is, by the ability to ferment and produce gas in particular media, at an elevated temperature, within a specific time. Classical enumeration procedures for fecal coliforms include the inoculation of a series of broth tubes for initial enrichnlent, followedby the transfer of positive tubes into more selective media. Using a series of dilutions of broth tubes results in the calculation of an MPN based on the number of positive tubes overa series of three dilutions. Conventional MPN procedures for total and fecal coliforms and E. coli require several days for incubation of the inoculated

Pathogens Transmitted by Seafood

143

media and aliquots from positive tubes from one type of medium to another at very specific times. Fig. 16 describes the conventional MPN procedure beginning with a presumptive MPNs (91). To test using lauryl sulfate tryptose broth and brilliant green lactose broth perform the fecal coliform test, small aliquots from positive lauryl sulfate tryptose tubes are transferred toEC broth tubes. Positive tubes from this step are streaked onto a differential medium and later presumptive colonies of E. coli are examined in a series of biochemical tests (Fig. 16). For the enumeration of fecal coliforms in shellfish growing waters A-

Weigh 50 g tissue, add450 ml phosphate-buffered dilution water Blend for 2 minutes

t

Prepare declmal dilutionsof blended material

Inoculate Lauryl Sulfate Tryptose(LST) MPN tubes Incubate for 48 ? 2 h at 35°C Examine after 24 rt 2 h and again at 48 k 2 h

+

Tubes are scored positiveif gas is present Transfer loopful from positive tubesto Brilliant Green Lactose Bile (BGLB) tubes

t

Calculate coliform MPN based on positve BGLB tubes, those with gas

EC Broth Method for Fecal Coliforms and Confirmed Test for E. coli Transfer loopful from positive LST tubes from the PresumptiveTest to EC tubes Incubate 48 rt 2 h at 45.5"C Examine after24 rt 2 h and again at 48 rt 2 h

+ +

Tubes are scored positive if gas is present

Streak loopful from positivetube to Levine's Eosin Methylene Blue (L-EMB) agal Incubate 18-24 hat 35°C

Transfer suspected E. coli colonies to Plate Count agar slants Perform biochemicaltests Gram stam Indole production Voges-Proskauer (VP)-reactive compounds Methyl red-reactwe compounds Citrate utilization Gas lrom lactose

Calculate E. coli MPN based on propbrtionof EC tubes that contain E. coli

144

Herwig

+ + +

Collect water sample

Prepare decimal dilutions

Inoculate A - l MPN tubes Incubate 3 h at 35"C, followed by 21 h at 44.5%

Tubes are scored positiveif gas is present

Fig. 17 MPN protocolusing A - l medium for shellfish growing waters (91).

1 brothcanbeused. Water samples are incubated at 35°C for 3 hours before theyare moved into a 44.5"C water bath incubator (Fig. 17). A newer variation of the conventional protocol incorporates the use of a fluorogenic substrate, 4-methylumbelliferyl-~-D-glucuronide (MUG), thatwillfluoresce under UV light when the enzyme P-glucuronidase cleaveslnethylutnbelliferonefromthesubstrate(Fig. 18). Achromogenicsubstrate 5 bron~o-4-chloro-3-indoyl-~-D-glucuronide (S-GLUC or BCIG) can also be used. These substrates have been incorporated into several selective media for the Enterobacteriaceae for rapid detection of E. coli (93). Chromogenic and fluorogenic substrates are used in several commercially available systems, such as those with the trade name Petrifilm, for the enumeration of coliforms and E. coli (94). At the present time there are no simple procedures available for the direct cultivation of EPEC, ETEC, and EIEC from foods. Weigh 25 - 50 g shellfish meat and add phosphate-buffered dilution water Blend for 2 minutes

PreDare serial dilutionsof blended material

Incubate Lauryl Sulfate Tryptose(LST) tubes as in Conventional MPN Procedure

I

t

Transfer loopful from positive LST tubes to EC-MUG broth tubes Incubate 24 h at 44.5 f 0.2%

t

Determine fluorescencein tubes All tubes that fluoresceare positive Calculate E. coli MPN

Fig. 18 EC-MUG method for dctermining E. coli MPN i n shellfishnle:l(s (91 ).

Pathogens Transmitted by Seafood

145

Since Shiga toxin-producing E. coli (STEC), especially serotype 0157:H7, arewell recognized as in1portallt food pathogens, several selective media have been described for these in detail in regulatory manuals organisnls (89). The conventional procedures are described such as the Bacteriological Analytical Manutrl (9 l), Oflcial Metl1od.s of Anc4wi.s (?fAoAC IilterilatiollLl[(95), and Recornmended Procedures for the Ex-nminarion of Sea W m r and Shellfish (96).

4.

Campylobacter jejuni

(1. Diseclse. Ctlinpglobacter jejuni is a gram-negative,curved,andmotilerod.It is a nlicroaerophilic organism requiring reduced concentrations of atmospheric oxygen for growth. Because of its microaerophilic requirements, optimum conditions for growth are achieved in the laboratory when 3-5% oxygen and 2-10% carbon dioxide are Provided. Before 1972 and after techniques were developed to culture this Organism from i primarily associated with abortion and enteritis feces, it was thought that C. j e j ~ n was in sheep and cattle. In recent years, however, C. jejuni is believed by some to bethe leading cause of bacterial diarrheal disease in the United States, causing more cases than S c h n o n e l k ~and Slligellrr combined (97,98). C. jejuni andothercloselyrelatedspecies,forexample, C. lari, C. coli, and C. }~?'ointestir~e~lis, cause diarrhea that may be watery or sticky and may contain blood and fecal leukocytes. It is believed that 99% of cases are caused byC. jejuni. Other symptoms that are often present include fever, abdominal pain, nausea, headaches, and muscle pain. The illness usually occurs 2-5 days after ingestion of contaminated food or water and than usually lasts 7-10 days. The infective dose is considered to be small, perhaps less 200 organisms (99). The pathogenic mechanisms for C. jejuni are still not completely understood. Death from C. jejuni is extremely rare, but there are reports of abortion induced by the organism. Some investigators suspect that C. jejuni is the leading cause of gastroenteritis in the world (100).

b. E c o l ~ ~ q y .C. jejuni isnotusually found in healthyhumans, but it is found in healthy farm animals, birds, and flies. In foods, C. jejuni is frequently associated with raw chicken; raw milk is also a source. C. jejuni is sometimes found in surface waters, such as streams and ponds. Shellfish have been implicated as a vehicle in human cases of C. jejunienteritidis (101,102).In 1993,69% of investigated mussels and 27%of oysters traded in the Netherlands were found to be contaminated with Ccrtnpylobacterspp. (1 03). Initial and later testing suggestedthat most of the isolates were C. lari. C. lari is a thermophilic species that was first isolated from gulls and subsequently from other avian species, dogs, cats, and chickens. Throughout the world, C. jejuni is the most significant known agent of human gastroenteritis. C. lari is isolated much less frequently from clinical samples, but is also recognized as a causative agent of gastroenteritis in humans (104). Studying the distribution of Cnrnpylobacter along the West Coast of the United States over an 8-year period, Abeyta et al. ( I 02,105) indicated that Camnpylobacter are well distributed in shellfish growing waters. In temperate aquatic environments, Campglobacter may exhibit a seasonality in its population numbers. Wilson and Moore (106) reported that few Cmlpylobacter were recovered between May and August; most were foundin the cooler winter months (November-March). Less than 6% of the shellfish examined between May and August contained C~rmpylobacter.Thermophilic Campylobocter spp. were detected in 42% of the shellfish. The greatest percentageof these (57%) were urease-positive thermophilic campylobacters

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146

(UPTC), atypical types that do not appear to be closely associated with domestic or farm animals or man. Their significance in foodborne human diseaseis probably minimal. These atypical Ccrmpvlobncters have not been commonly reported elsewhere. This may be the result of misidentification as C. coli.

c. Microbiologicc~lProcedures. Cc~mpylobcrc~ter areusuallypresentin highnumbers in the diarrheal stools of individuals, but isolation requires antibiotic media and a special microaerophilic atmosphere. Crrmpylobcrcter was difficult to culture until protocols for using the appropriate gas atmospheres were developed for enrichment and isolation of this organism. Ccrmpylobc~cterwill grow only under microaerophilic (microaerobic) conditions which can be supplied using special gas mixtures purchased from gas vendors or by using special “gas-paks” that generate the proper atmosphere within an anaerobe jar. More recently Abeyta et al. (105) reported that selected enrichment broths supplemented with the commercially available enzyme Oxyrase were evaluated for recovery of Ce~mpylobercterunder nornlal atmospheric conditions from shellfish. Results indicated that the Oxyrase enzyme was useful. Growth of the organism is not very fast, and isolation of C. jejurzi from food may take several days to a week. Water samples can be tested for the presence of Ce~mpylobc~ctrrby filtering samples through a 0.45 pm positively charged filter and adding the filter to an enrichment broth, a Bolton broth withantibiotics.Afterincubation in the enrichment broth, aliquots are streaked onto selective medium and incubated under microaerophilic conditions. Presumptive Cclmpylobacter colonies are tested with a variety of physiological and biochemical tests. Shellfish and other fishery products are examined in a similar fashion, except that the samples are placed in a stomacher or blendedto prepare a homogenate. Fig. 19 shows the suggested protocols for testing for Ccrmpylobcrcrer in water and shellfish samples as suggested by Hunt et al. (97). 5. Listeria monocytogenes cl. Disease. Listericl monocytogerws is a gram-positiverod,motile by means of peritrichousflagella.Theorganism is not a spore former. Some studies suggest thata significant number of humans Inay be intestinal carriersof this organism. It has been found in a variety of mammals, both domesticated and wild, a number of different bird species, and in some species of fish and shellfish (107,108). Lennon et al.(109) described a perinatal outbreak in Auckland, New Zealand, and hypothesizedbut did not prove an association with raw fish or shellfish. Schlech (1 IO) provided an extensive review on listeriosis and suggested that there has been a significant increase in the incidence of the disease. Listeriosis is an atypical foodborne pathogenof public health concern because of the severity and the nongastroenteric nature of the disease. While a relatively unco1nn1on disease, the high mortality rate suggests the need for continued vigilance and study. Listeriosis is clinically defined when the organism is isolated from blood, cerebrospinal fluid, or from otherwise sterile sites. The symptoms and outcome of listeriosis can be very serious and include septicemia, meningitis, encephalitis, and intrauterine or cervical infections in pregnant women which may result in spontaneous abortion or stillbirth. The onset of these disorders is usually preceded by influenza-like symptoms such as nausea, vomiting, and diarrhea. The onset time of serious forms of listeriosis is unknown but may range from a few days to 3 weeks

(107,108). The infective dose for

L. mo~zoc~ytogenes is unknown but is believed to vary with

147

Pathogens Transmitted by Seafood Water Samples Filter 2 - 4 liters through 0.45 pm positively-charged filters

t

Add filter to 100 m1 of Bolton broth with antibiotics Incubate 3 h at 30"C, followed by 2 h at 37°C under microaerobic conditions

+ +

Incubate, with shaking28 h at 42 C, without shaking 48 h at 42°C

I

t

Streak enrichmentsonto Abeyta-Hunt-Bark agar or Modified Campylobacter Blood-Free Selective agar base (CCDA) using anerobic jars under microaerobic conditions at 37-42°C

Test presumptive Campylobacter colonies with physiological and blochemical tests Hippurate hydrolysis Antibiotic sensitivities Nitrate reduction H2S from cysteine

Triple Sugar Iron (TSI) Temperature range of growth 3.5% NaCl

Glucose utilization MacConkey agar Glycine

Shellfish Samples Measure 100-200 g shell liquor and meat Blend at low speedor stomach for 60 seconds

Remove 25 ghomogeGate for sample analysis Add to 225 Bolton broth(1:l 0 homogenate)

t

+

Remove 25 g of i:l 0 homogenate Add to 225 Bolton broth (1:lOO homogenate)

Incubate 4 h at 37°C - for samples producedor processed c10 days previously OR Incubate 3 h at 30°C, followed by 2 h at 37°C - for frozen samplesor samples produced or processed 2 10 days previously

t

Incubate, with shaking28 h at 42°C: without shaking 48 h at 42°C

Streak enrichments onto Abeyta-Hunt-Bark agar'or Modified Campylobacter Blood-Free Selective agar base (CCDA) using anerobic jars under microaerobic conditions at 37-42°C

Test presumptive Campylobacter colonies with physiological and biochemical tests

Fig. 19 Protocols for examining water and shellfish salnples for Cunrpylohactcr species (97)

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148

the strain and the susceptibility of the human. Fewer than 1000 organisms can cause the disease.Ingested L. tnonocytogenes invades thegastrointestinalepithelium,entering monocytes, macrophages, or polymorphic leukocytes. The presence of L. rrrorrocytoSerlc’s in phagocytic cells permits access to the human brain and probably allows for migration tothe fetus in pregnant women. The pathogenesis of L. rrror~ocyrogerrescenters on its ability to survive and multiply in phagocytic host cells (107,108).

b. Ecology. L. monoc.ytogerles differs in many aspects from most other foodborne pathogens because it is ubiquitous and is resistant to diverse environmental (and food processing) conditions such as low pH and high NaCl concentrations. This pathogen is microaerobic and psychrotrophic. Two properties that contribute to its widespread distriit bution in the environment are its ability to survive for long periods of time, although is not a spore former, and its ability to grow at low temperatures. L. ttrorlocyroserlc’.s is widely distributed in a number of mammalian, avian, fish, and shellfish species (107,108). The organism appears to be widely distributed in freshwater and less so in bottom sediments. Colburnet al. (1 1 1) conducted a study to determine the incidence of Listeritr species in freshwater tributaries draining into Jumboldt-Arcta Bay in California. Lister-icr species were detected in 8 1% of freshwater samples and30% of sediment samples. Whether these organisms are indigenous to freshwater or are the result of runoff from livestock found in the vicinity of the rivers is uncertain. The organism is very hardy and resists the effects of freezing, drying, and heat. L. tnowocytogetzes has been associated with raw milk, cheeses (primarily soft varieties), ice cream, raw vegetables, fermented raw-meat sausages, poultry, raw meats, and raw and smoked fish. Seafoods, particularly ready-to-eat forms, have been frequently reported to be contaminated with L. rtrotloc~togewes( 1 12-1 14). The organism has the abilityto grow in temperatures as low as 3OC, permitting growth i n refrigerated foods. This characteristic may be a particular problem in ready-to-eat foods, such as cold smoked salmon and cooked fish products, since these products are not further heat processed before they are consumed. A relativelyhigh incidence of the organism (6-369’0) has been found in these products ( 1 13).

c. Microbiological Procedures. The traditional methods of analyzing food for L. monocytogenes are complex and time consuming, requiring a total time of 5-10 days. Protocols ( 1 15) are outlined in Fig. 20. Sample processing begins withan enrichment step using trypticase soy broth which is supplemented with yeast extract, phosphate salts, and pyruvic acid. Selective agents (acriflavin, nalidixic acid, and cyclohexamide) are added to the enrichment. Aliquots from the enrichment are streaked onto selective media and then presumptive L. rr7orlocytogerle.s colonies are examined in several biochemical and physiologicaltests.Finally,serologic,mousepathogenicity,andtheChristie,Atkins, Munch-Peterson (CAMP) testare performed. In recentyears,specificnonradiolabeled DNA probes have been developed which allow for simpler and faster confirmation of suspect isolates. Agersborg et al. (1 16) detected L. nrot1ocytoRene.s in seafood products using PCR and primers for fragments of the lysteriolysin 0 (/dy) gene and forthe invasionassociated protein gene (icrp). They were able to detect one to five L. trrorlocytogerlescells in S g of product in 55 hours.

6.

Yersinia enterocolitica

N. Disease. Thegenus Yersinicr hasthreepathogenicspeciesforhumans: Y. ellterocolitic.cr, a common intestinal pathogen in humans; Y. pseuclotuberculnsis, an intestinal pathogen in rodents which occasionally infects humans; and Y. pestis, the infectious agent

149

Pathogens Transmitted by Seafood Weigh 25 g of sample and add 225 ml of Enrichment Broth (EB) [Trypticase Soy Broth with Yeast Extract (TSBYE) supplemented with monopotassium phosphate, disodium phosphate, pyruvic acid]

I

+

Blend or stomach. Transfer to 500 ml flask Incubate 4 h at 30°C

+

Add selective agents acriflavin, nalidixic acid,and cycloheximide, and incubate additional44 h for a total of 2 days at 30°C

At 24 and 48 h, streak EB culture ontoboth Oxford Medium (OXA) and Lithium Chloride Phenylethanol Moxalactum (LPM)agar or LPM + esculin/Fe3+ agars. PALCAM agar may be substituted for LPM agars Incubate OXA andPALCAM media for 24-48 h at 35°C Incubate LPM mediafor 24-48 h at 30°C

Streak typical Listeria colonies from OXA and LPM (or PALCAM) onto TrypticaseSoy Agar with Yeast Extract (TSAYE)

+ +

Perform biochemical and physiological tests Cellular morphology and motility Catalase Gram stain Carbohydrate testing Hemolytic activity

Perform serological, mouse pathogenicity,Christie-Atkins-Munch-Peterson (CAMP) test

of bubonic and black plague. As members of the Enterobacteriaceae, Yersini(l are small, rod-shaped, gramncgative. oxidase-negative, facultative anaerobes that ferment glucose. Y. cwter’ocdificlr is often isolated from clinical specimens such as wounds, feces, sputum, and lymph nodes. It is not considered part of the normal human microflora. Y. entewcolirictr is a heterogeneous species, being separated into several subgroups according to biochemical activity and somatic (0)lipopolysaccharide antigens ( 1 17- 119). Yersiniosis in humans is characterized by synlptoms of gastroenteritis with diarrhea and/or vomiting. Most systematic infections occur in children, especially those less than 5 years old. Fever and abdominal pains are the hallmarks of this disease, with the organism causing physicians to misdiagnose appendicitis. Crohn’s disease, and mesenteric lymphadenitis. Most symptomatic infections result in self-limiting diarrhea. Yersiniosis may also give rise to a variety of autoimmune complications including reactive arthritis, erythema nodosum, iridocylitis, Homerulonephritis, carditis, and thyroiditis. These diseases have been reported to follow acute infection ( 1 19). This outcome is infrequent before the age of I O years and

750

Herwig

occurs most often in Scandinavian countries. Yersiniosis is a rare disease in the United States, but appears to be a more common disease in northern Europe, Scandinavia, and Japan. b. Ecology. Y. etlterocoliticcl hasbeenfound in a variety of environmentsand has been isolated from the intestinal tracts of many different mammalian species, as well as from birds, frogs, fish, flies, fleas, crabs, and oysters ( 1 19). Y. etlterocoliticn can be found in meats, raw milk, fish, and oysters. This organism is also commonly found in a variety of terrestrial and freshwater habitats, including soil, vegetation, lakes, rivers, wells, and streams. Pigs are the only animal species from which Y. enterocoliticcr of biovar 4 serovar 0 3 , the variety most commonly associated with human disease, has been isolated with any degree of frequency. Individual isolates of Y. erlterocditicxr from pigs and humans appear identical with each other in terms of serovar, biovar. RFLP of chromosomal and plasmid DNA, and virulence determinants ( 1 17). Y. enterocoliticrr, including pathogenic strains, can survive for extended periodsin the environment, such as in soil, vegetain rivers than tion, streams, lakes, wells, and spring water (120). Its survival was lower i n soils; Chao et al. ( 120) suggest that the presence of protozoans in the aquatic environment are responsible for their enhanced decline. The relatively low optimum temperature for growth may account for the higher incidence of yersiniosis in temperate regions of the world and the tendency for infections to peak during late autumn and winter where Y. crlterocoliticrr is endemic (121 ). c. Mic.robiologicer1 Procedures. If highconcentrations of Y. erlteroc-oliticcr are 36-48 hours;however, suspectedthen thisspeciescanbepresumptivelyidentifiedin confirmation or enrichment of samples for Y. enterorditiea may add another 2-3 weeks to the procedure. Fig. 2 1 summarizes the protocol ( 1 18) for the enrichment and isolation of Y. enterocoliticcr. Samples for enrichment are placedin peptone sorbitol bile broth and incubated for 10 days at 10°C. This step takes advantageof the ability of Y. erlterocoliticcr to grow at lower temperatures. Following the enrichment step, samples are streaked onto selective agars and presumptive colonies are tested through a series of differential media, physiological, and biochemical tests. Some investigators have recommended the use of two enrichment media, suggesting thatnosingle procedure will recover all pathogenic serotypes. The second enrichment method uses irgasan ticarcillin chlorate broth followed by plating on Snlrnot~ellrr-Shi~~e/ler agar (89).If high countsof this organism are suspected, aliquots of the homogenized sample can be directly plated on selective media and later colonies can be probed with a virulence gene probe. Determination of pathogenic strains requires additional steps and may include an autoagglutination test, the low calcium response Congo red agarose virulence test, and the crystal violet binding test. Other tests include DNA colony hybridization with probesto detect virulence factor genes. and intraperitoneal infection of adult mice pretreated with iron dextran and desferrioxamine B. Screening isolates for invasive potential usingan in vitro HeLa cell assay is alsoan effective procedure for determining pathogenic strains.

7 . Plesiomonas shigelloides cr. Disetrse. P1esionIoncr.s.skigelloitle.s is a motile,oxidase-positive,catalase-positive, facultative anaerobic, gram-negative rod that has been isolated from freshwater, freshwater fish, and shellfish and from many types of domesticated and wild animals. The genus are consists ofa single species. Manyof the phenotypic properties found in P. .shi,~e/loidr.s shared with the genera Vibrio and Aerornonas. For example, P. .shic~e//oicfe.es is Silnilar to

751

Pathogens Transmitted by Seafood Weigh 25 g of sample, add 225 ml Peptone SorbitolBile Broth (PSBB) Homogenize 30 seconds

t

Incubate 250 ml of homogenate for 10 days at 10°C OR

1

If high counts of Yersiniasuspected, spread-plate0.1 r n l of homogenized sample onto Macconkey agar or Celfsulodin-lrgasan-Novobiocin(CIN) agar, and transfer 1.O ml of homogenate to 9.0 ml solution of 0.5% KOH in 0.5% saline and spread-plate onto MacconkeyAgar and CIN Agar Incubate for 24 h at 30°C

Perform colony hybridization withYersinia virulence gene probe Transfer 0.1 PSBB of enrichment to 1.0 ml solution of 0.5% KOH in 0.5% saline and mix Transfer 0.1 ml enrichment to 1.O ml to 0.5% saline Streak from each saline tube onto Macconkey agar and CIN agar Incubate 24 h at 30°C

4-J

Pick presumptive Yersinia colonies and inohlate Lysine Arginine Iron (LAI)agar slants, Christensen's Urea agar,and Bile EsculinAgar Incubate 48 h at room temperature

t

Cultures yielding typical reactions onLA1 agar are streaked onto Anaerobic Egg Yolk (AEY) agar Incubate at room temperature

Perform gram stain, biochemical,and other physiological tests Lysine, arginme. ornithine decarboxylase Phenylalanine deaminase Motility at 2226°C and 3537°C Acids from: mannitol, sorbitol, cellobiose, adonitol. Inositol, sucrose, rhamnose, raflinose, meliblose. salicin, trehalose, xylose Simmons citrate Indole Voges-Proskauer test Llpase beta-D-glucosldase test Pyrazinamidase

152

Herwig

Vibrio spp. in its susceptibility to the vibriostatic agent O/ 129. Like Aerornontrs species, P. shigelloides does not have a requirement for sodium and is unable to grow in 6% NaC1. Humans and other animals may be carriersof the organism, showing no overt sylnptoms of disease. Much of the earlier information about this pathogen came from outside of the United States, but in recent years studies from the United States are supporting the role of this organism as a pathogen that causes diarrhea. Many of these cases are associated with the consumption of raw bivalve mollusks or with foreign travel (122,123). p . shigel1oide.s may cause a mild self-limiting gastroenteritis with fever, chills, abdominal pain, nausea, diarrhea, and vomiting with symptoms beginning 20-24 hours after consumption of the contaminated food or water. The infection may cause diarrhea for 12 days in healthy adults. There may be high fever and chills, and protracted dysenteric symptoms in infants and children under 15 years of age. The infectious dose is presumed to be quite high and is thought to be more than one million organisms. In healthy people, gastrointestinal illness caused by P. skigelloides infection may be so mild that infected individuals do not seek medical treatment. consequently the number of cases associated with this organism is assumed to be vastly underreported (124,125). A cluster of cases occurred in North Carolina in November 1980 following an oyster roast. Thirty-six of 150 people who had eaten roasted oysters experienced nausea, chills, fever, vomiting, diarrhea, and abdominal pain beginning 2 days after the roast. The averag duration of the symptoms was 2 days. P. skigelloides was recovered from oyster samples and patient stools (126). b. Ecology. P. shigelloides is a gram-negativerod thathas beenisolatedfrom freshwater, freshwater fish, and shellfish and from many types of domesticated and wild aninlals ( 123,127-130). Most human infections caused by this organism are suspected to be waterborne. The organism may be found in unsanitary water that is used for drinking water, recreational water, or water used in food processing. Studies indicate a seasonal effect from environmental sources, with an increase i n the reported casesof diarrhea during the warmer months ( 127,128).

c.MicrobiologicalProcedures. P. s1~igelloide.scan beculturedonInany of the media used for isolation and enrichment ofthe enteric bacteria. This speciesis tolerant of bile salts and brilliant green. Because lactose is generally slowly fermented, P. shigelloides appears as lactose negative on solid media. Because of a lack of competitiveness, enrichment techniques may be of limited usefulness (13 l). Procedures for the enrichment and isolation of P. shigelloides are not standardized and have not been published by the FDA ( 132). Koburger and Wei (124) suggesteda protocol where samples are diluted and plated onto Inositol brilliant green bile salt agar and PL agar. One to 10 g of sample are added to 90 m 1 of (etrathionate broth. Following a 24-hour incubation period, suspected colonies are examined in differential media and the enrichment broth is streaked onto the isolation media. Fig. 22 outlines the procedure (124). P. sl~igelloidescan be differentiated from Aeromonas spp. using a series of biochemical tests.

8. Aeromonas hyrophila and Other Aeromonas Species

N. Disease. Aerotnonu h y h p k i l n andother Aerornonas speciescanoften be isolated from food and the environment. CertainAerolnonus species are human pathogens that may cause gastroenteritis. Among the suspected foods are prefrozen or inadequately cooked seafood and oysters (133). Based on hybridization studies, the taxonomy and speci-

Pathogens Transmitted by Seafood

153

Add 10 g of sample to 90 ml Tetrathionite Broth Incubate at 40°C for 24 h

+

Streak onto duplicate platesof two of the following selective media Incubate at 35°C for 24 h Macconkey agar Billiant Green Lactose Bileagar PL agar Salmonella shigella agar Inositol Brilliant GreenBile Salts agar

+ +

Pick three typical coloniesfrom each of the selective media into Triple Sugar Iron agar and Inositol Gelatin deeps Incubate at 35°C for 24 h

Perfom oxidase test and gram stain from TSI slant

ation of the genus A P I Y ) I ) I Ounderwent II~ revision in 1980s. The taxonomy of the genus is still evolving and still confusing. Since DNA-DNA hybridization is not routinely employed in most clinical laboratories. most still rely on a series of phenotypic tests to differentiate Aeronlo/m species. The species name A. hydrophilrr has been broadly used i n the older literature to refer to the whole group of mesophilic aeromonads, but it should he more narrowly definedby the phenotypic characterizationof Popoff or DNA-DNA hybridization. If the speciation of a strain is in question, or if an isolate has not been thoroughly Aer.otnmm sp. or aeromocharacterized, then investigators should refer to the isolate as nads. Based on phenotypic testing and definitions, the generally accepted speciesof Aeromo)uI.s associated with diarrhea in humans are A. hydrophiltr, A. ( ~ r v i wA., \ ~ c ~ l n t r ibiovar i sobricl and biovar lvrorrii ( 134,135). Two types of gastroenteritis have been associated with A. hydrophila, a cholera-like illness with “rice water” diarrhea, and a dysenteric of illness characterized by loose stools containing blood and tnucus. The infective dose the organism is unknown, but SCUBA divers who have ingested small amounts of watcr have become ill and A. hydrophilu has been isolated from their stools ( 1 36).

b. E ~ o l o ~ y y .AcJromo1russpp. are commonly associated withfish and seafood and arewidelydistributed in the aquatic environment (137). Most of the published studies have dealt with A. /ryhophi/u, a bacterial species that is present in all freshwater environments and in estuaries. Some strains are capable of causing disease in fish and amphibians as well as humans. who may acquire infections through open wounds or by ingestion of organisms in food orwater. A. /rydrop/riI~rhas beenfrequentlyisolatedfromfishand shellfish. It has also been found in market samples of red meats and poultry. There are

154

Herwig Prepare dilutionsor use 25 gof original samplefor enrichments For dilutions, weigh 25 g and blend with 225 ml of 0.1% peptone water

+

For enrichments and MPN procedures, inoculate sample into Alkaline PhosphateWater or Tryptic Soy Broth Containing30 mg/L ampicillin Incubate at 28°C for 24 h

Streak enrichmentsor MPN tubes onto Starch Ampicillin (SA) agar or Bile Salts Brilliant Green Starch (BBGS) agar Incubate at 28°C for 18 to 24 h

+ + +

Flood the surface of the agar plates with Lugol's iodine solution Count typical colonies that have surrounding zones of starch hydrolysis

Pick typical colonies and streak onto nutrient agar that does not contain carbohydrate, such as Nutrient agar, TrypticSoy agar Streak DNase testagar

Perform gram stain, catalase test, resistance to 0/129 vibriostatic agent Inoculate Kaper's medium and observe reactions

relatively few published cases in which foodborne gastroenteritis.

Aerotnolm species have been associated with

c. Mictnbiological Procedures. Protocolsfor the enrichmentandisolation of pathogenic species of Aerovronm have not been officially accepted by the FDA i n the United States. Media for isolation of aeromonads usually exploit a resistance to ampicillin. Palumbo et al.(138) described a protocol for the isolation and identificationof Aerorwrltrs species from food (Fig. 23). Food samples can be blended and diluted i n peptone water and spread onto a surfaceof starch ampicillin agar or bile salts brilliant green starch agar. The latter medium is suggested by investigators who formulated it for samples i n which a large number of Proteus spp. might be encountered. Typical colonies on these media are identified after incubating by flooding the surface with an iodine solution. If low numbers of A e r o ~ n ~spp. ~ ~are m expected, food samples can be inoculated into enrichment broths or a seriesof MPN tubes with alkaline peptone water or tryptic soy broth containing ampicillin. After 24 hours, aliquots from the enrichments are streaked on the plating media. Suspected colonies from the plating media canbe tested for additional phenotypic proper-

Pathogens Transmitted by Seafood

155

ties to identify whether A. hyiro~philaor other Aewtw11m species were isolated. Species identification is confirmed by performing a series of biochemical tests (125,138).

B. Viruses There are four major categories of viruses that cause gastroenteritis in humans: rotavirus, enteric adenovirus, calicivirus (that includes Norwalk virus and its relatives). and astrovirus. Hepatitis virus is a foodborne virus whose symptoms and signs are associated with liver disorders. Raw shellfish havebeen implicated in outbreaks of foodborne viral gastroenteritis in the United States, Europe, and Australia (139-143). Outbreaks have occurred following the consulnption of shellfish harvested from waters contaminated with human sewage (143,144). Mostof the viruses associated with seafood-borne disease are calicivirus and hepatitis A. Table 7 lists the structural features and diseases caused by viruses associated with foodborne disease. Unlike most bacteria, viruses are not easily inactivated by normal cooking procedures. Shellfish tissue may "protect" the virus and individuals may become ill after consuming cooked shellfish that are contaminated with enteric viruses. To study the extent of the hazard presented by oysters contaminated with virus, DiGirolamo et al. (145) contaminated samples of whole and shucked Pacific oysters with IO' plaque-fonning units of poliovirus/ml and heat processed the oysters in four ways: stewing, frying, baking, and steaming. Results indicated that a significant portion of virus in oysters withstood these methods of processing. The survival rate varied from 7% to 10%. Cooking experiments performed with contaminated mussels revealed that S minutes after the opening of the mussels' valves, rotaviruses and hepatitis A virus could still be recovered in the steamed shellfish (146). While depuration or relaying shellfish may be a satisfactory method for removing pathogenic bacteria from these animals, such methods must be used with caution for the removal of human virus. Power and Collins (147) studied the elimination of poliovirus, E. coli, and a coliphage from the common mussel (Myrillrs cdulis). In their experiments. the relative rates of elimination during depuration were E. c w l i > coliphage > poliovirus. It was concluded that E. coli cannot beareliableindicator of viruseliminationfrom mussels. A dramatic experiment that showed the potential danger of relying on indicator bacteria and depuration was performed in Australia. Following the widespread outbreaks of oyster-associated gastroenteritis in Australia in 1978. several programs were introduced to minimize the occurrence of further outbreaks. One program included the depuration of oysters and the use of human volunteers, as an interim measure, to test samples before sale to the public. After eating Georges River oysters that were thought to be safe. S2 volunteers became ill and Norwalk virus was foundto be the cause of the infection. Depuration, as carried out in pollution-free water for 7 days, was therefore concluded to be an unsatisfactory method ( 148).

1. Hepatitis A Virus (1. Disctrw. HepatitisAvirus (HAV) is also referred toas Heptovirus A. and i n older literature as infectious hepatitis. Five kinds of hepatitis are recognized: A, B. C, D. and E. Hepatitis A and E are of concern with food, but type E is not found inNorth America. HAV is classified with the enterovirus group of the Picornaviridae family. Like other enteric viruses, HAV is transmitted by the fecal-oral route. The most common form

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156

Table 7 Structural Features and Symptoms of Human Viruses Associated with Seafoods,’

Virus Virus most ,frequently trssociaterl with seafood:

Hepatitis A

Calicivirus [includes Nonvalk virus and other small round structured viruses (SRSV)]

diameter, 27virus, round nm icosahedral symmetry, not enveloped, single stranded RNA, genome length of 7500 nth 35-39 nm diameter. round virus, icosahedral symmetry, not enveloped, one or two structural proteins, single stranded RNA, genome length of 7700 nt

Virus less ,frequently associated with seufood: 80 diameter, nm double-

Human adenovirus

Human poliovirus

shelled wheel-like capsid, icosahedralsymmetry.11 segments of doublestranded RNA, genome length of 18,500 nt 27-30 nm diameter, round surface and pointed star with 6 points, polyhedral symmetry, several structural proteins, single stranded RNA, genome length of 6800-7900 nt 75 nm diameter, not enveloped, fastidious growth in culture, double-stranded DNA, helper or satellite virions may be present, genome length is 30000360000 nt 28-30 nm diameter, round virus, icosahedral symmetry, not enveloped, single stranded RNA, genome length of 7400 nt

Structure and disease symptoms from sunlrnarles present (208). ” n t = nucleotides.

Epidemics of acute diarrhea and vomiting in older children and adults, often food or water borne

Major cause of severe dehy-

Rotavirus (group A)

Human astrovirus

Fever, malaise, nausea, anorexia, abdominal discomfort, followed by jaundice

111

drating diarrhea in infants and young children

Watery endemic diarrhea of children, in day care ccnters, some disease outbreaks, role in HIV-related diarrhea

Endemic diarrhea of infants and young children

Cause of polio in humans

Murphey et al. (23) and Blacklow and Herrmann

Pathogens Transmitted by Seafood

157

of transmission is person to person by fecal contamination. The incubation period for HAV ranges from 10 to 50 days with a mean of approximately 30 days, depending upon the number of virus particles consumed. The infectious dose is thought to be 10-100 particles. The virus infects the liver producing a debilitating, low-mortality disease that sometimes includes jaundice. The infected person is characterized by a sudden onset of fever, malaise, nausea, anorexia, and abdominal discomfort, followed by several days of jaundice. Death from HAV isveryrare. Most adultshaveanimmunity to HAV that provides lifelong protection against reinfection (23,149,150).

b. Ecology. HAV is an entericvirusthatiscloselyassociatedwithinfectedhumans and fecal contamination. Less than 10% of HAV cases are food associated or water associated. With foods, it is most frequently associated with bivalve mollusks, but it may also be found in foods associated with a large amount of handling, such as salads. In the later association, contamination may occur during preparation by a food handler who is infected with HAV. In the United States, between 5000 and 35,000 cases of food poisoning caused by HAV are estimated to occur each year. One of the more noteworthy outbreaks associated with seafood includes an outbreak in Sweden in 1955. Oysters held in a harbor awaiting sale around Christmas were contaminated with fecal pollution from a toilet that was over theharbor.Oystersfromtheharborwereeatenrawand629peoplebecame ill. This outbreak was the first report of HAV transmission by shellfish. In 1988 an HAV outbreak occurred in Shanghai, China. Clams taken from water that was contaminated with human sewage were eaten raw, resulting in nearly 300,000 cases of HAV infection (126). c. Microbiologiccrl Procedures m c l Detectiorl. Thediagnosis of an HAVinfection can be performed by detecting antibodies to HAV in human serum (126).

2. Human Calicivirus (Norwalk and Norwalk-Like Viruses) (1. Disetrse. Thehumancaliciviruses,whichincludeNorwalk-likevirusesorthe Norwalk family of viruses, are called small, round, structured viruses (SRSV) i n Europe. These viruses are 35-39 nm in diameter and are a serologically related group of viruses. Norwalk viruses are classified in the Caliciviridae family, and are positive sense, linear, single-stranded RNA viruses. Norwalk viruses cause gastroenteritis, typically with diarrhea and vomiting, with an incubation period of 24-48 hours and a duration of infection lasting from 24 to 60 hours. The disease caused by Norwalk viruses is often referred to as viral gastroenteritis. The infectious dose is thought to be low. The attack rate for Norwalk viruses is particularly high; often more than 50% of individuals who consulne contaminated food become ill. Humans appear to be susceptible to repeated infectionwith the same strain since the virus does not elicit a strong immunological response (23,149.150).

b. Ecology. Some investigatorssuspectthatNorwalkvirusesmaybeamongthe leading agents causing foodborne disease. Water is the most common source of outbreaks, but raw or insufficiently cooked bivalve mollusks have also been recognized as vehicles for transmission. Food outbreaks are often associated with the consumption rawofshellfish. In 1978 morethan 2000 people who ate oysters were infected in New South Wales, Australia. Some groups who consumed oysters showed attack rates of 85% (140). In December 1994 and January 1995, Florida experienced the largest outbreak of oyster-associated gastroenteritis ever reported. The largest shellfish and trout harvest for the Apalachicola Bay fishing industry occurs for the New Year’s holiday, when oyster

158

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roasts are a tradition. Of 223 oyster eaters, 58% became ill, compared with 3% of nonoyster eaters. Most oyster eaters ate only cooked (grilled, stewed, or fried) oysters. Oyster eaters who reported eating thoroughly cooked oysters were as likely to become ill cornpared to those who ate raw oysters. The outbreak demonstratedthat oysters cooked to the point where consumers consider them done or overdone werestill capable of transmitting enough virus to cause disease in a substantial proportion of people. The outbreak may have resulted from overboard dumpingof feces during a cornmunity outbreak of diarrheal illness. McDonnell et al. (15 1) suggested that using fecal coliform monitoring to define water quality is inadequate. Contamination of oysters with SRSV occurred during a time when routine water quality monitoring indicators were in acceptable ranges both in Florida and Texas. c. Mic~rohiologicdProceclrrres N I I ~Detectiorl. Norwalkvirusescanbedetected using serologic methods, but in recent years gene probes and PCR amplification methods have become increasingly more popular. While the DNA molecular procedures are theoretically very sensitive and rapid, nluch of the research effort has been directed toward developingmore efficientextractionproceduresandreducingtheproblemsassociated with inhibitory compounds that are present i n complex food matrices. I n 1996. Atmar et al. (152) described a multicenter, collaborative trial that was performed to evaluate the reliability and reproducibility of a reverse transcription PCR method ( 153) for the detection of Norwalk virus in shellfish tissues. The sensitivity and specificity of the assays were 85% and 9170, respectively, when results were detcrmined by visual inspection of ethidium bromide-stained agarose gels. The test sensitivity and specificity improved to 87% and 100%afterconfirmation by hybridizationwith a ctioxigenin-labeled,virusspecific probe.

3. Group A Rotavirus CL Disctrsc.. Rotaviruses are members of the Reoviridae family. They have a genome consisting of l l double-stranded RNA segments surrounded by a distinctive twolayered protein capsid. Particles are 80 nm in diameter. Six serologic groups have been identified, three of which (A, B, and C ) infect humans. Rotaviruses cause acute gastroenteritis. The most widesprcad group A rotavirus is also known as infantile diarrhea, winter diarrhea. acute nonbacterial infectious gastroenteritis, and acute viral gastroenteritis. Rotavirus infection is the most common cause of dehydrating diarrhea in children in the United States. Rotavirus, the most important cause of pediatric gastroenteritis in the United States, is responsible for an estimated one-third of all hospitalizations for diarrhea in children less than 5 years of age (154). Rotavirus gastroenteritis is a mild to severe disease characterized by vomiting. watery diarrhea, and low-grade fever. The infective dose is presumed to be low, from 10 to 100 virus particles. Since a person with a rotavirus diarrhea excretes large numbers of virus particles ( IOs-lO"' particles/ml of feces), infectious doses can easily be acquired from contaminated hands or utensils ( 154,155).

h. Ecology. Rotavirusesarctransmitted by thefecal-oralroute.Person-to-person contact is thought to be the most common means of transmission. Rotaviruses are very stable in the environment ( 154,155). These viruses do not appear to be the most frequent virus type associated with seafood.

Pathogens Transmitted by Seafood

159

c. Microbiolo,yiccrl Procedures ~rrrdDetection. Specificdiagnosis of the disease is made by identification of the virus in the patient's stool. Enzyme immunoassay is the test most widely used to screen clinical specimens, and several commercial kits are availableforgroupArotavirus.Electronmicroscopyandpolyacrylamidegelsareusedin some laboratories. More recently a reverse transcription PCR (RT-PCR) method has been developed to detect all three groups of human rotavirus (156-158).

4. Other Viruses:HumanAstrovirus,HumanAdenovirus, and Poliovirus Other viruses may also be found associated with shellfish, including human astrovirus, human adenovirus, and poliovirus. These viruses have notbeenreported as being frequently associated with seafood.

111.

BACTERIAL AND VIRAL PATHOGENS PRIMARILY ASSOCIATED WITH IMPROPER PROCESSING OR HANDLING OF SEAFOOD

A.

Bacteria

1.

Clostridium botulinum

(7. Diserrsc~. Foodbornebotulism, as distinguishedfromwoundbotulism andinfant botulism, is an extremely severe form of food poisoning caused by the ingestion of foods containing a potent neurotoxin that is formed during growth of Clostridium botulir u m . The toxin is heat sensitive and can be destroyed if food is heated to 80°C for 10 minutes or longer. The incidence of the disease is low, but the mortality rate is very high if the infected patient is not treated quickly. Approximately 10-30 outbreaks are reported in the United States each year, and these are mostly associated with inadequately processed home-canned foods. Some cases of botulism may go undiagnosed because symptoms are transient or mild or are misdiagnosedas Guillain-Barr6 syndrome. Seafood products have been involved in botulism outbreaks ( 126,159,160). C. b o t r r l i r r u r r r is a gram-positive, obligately anaerobic, spore-forming rod that may produce a neurotoxin. Seven types of C. botulirzrrrn (A, B, C, D, E, F, and G) are recognized, based on the antigenic nature of the toxin produced. Types A, B, E, and F cause human botulism, and typeE is mostly associated with seafoods in the United States. Different types of C. botrrlitrum are found in different areas of the world. Overall, type E is infrequcntly found around the world, but predominates in northern regions and in most temperate aquatic environments. This type is found in high numbers around the Great Lakes and in the coastal areas of Washington and Alaska. Along the Pacific coast of the United States, however, the prevalent type shifts from E to A and B below latitude 36"N. In Europe, type B predominates in the aquatic environments around the United Kingdom, while in other aquatic environments, type E is most frequently found. Surveys of Asia report high levels of type E spores around the Caspian Sea and in the northern areas of Japan. I n tropicalareas of Asia,typesCandDarefoundathigherlevelsinaquatic environments thantype E. Nanogram quantities of botulinum toxin can cause illness. Onset of symptoms in foodborne botulinum usually occur 18-36 hours after ingestion of the toxic food, although this can vary from 4 hours to 8 days. Early signs of intoxication

160

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include marked lassitude, weakness, and vertigo, usually followed by double vision and progressive difficulty in speaking and swallowing. Difficulty in breathing, muscle weakness, abdominal distention, and constipation may also occur ( 126,159,160). Storage of fresh and processed fish using modified atmospheric packaging (MAP) has gained widespread application in Europe. The technology has been approached more cautiously in North America because of the risk associated with C. borulinum E. The conditions often encountered in MAP fish products are conducive to the growth andtoxin production of C. borulinurrr E evenat temperatures as low as 33°C (161 ). Mild temperature abuse conditions of MAP fish products are of additional concern. Furthermore, the restricted growth of normal spoilage organisms may enhance the growth of C. horulitrum. These possibilities were shown by Eklund ( 162) in sallnon inoculated with C. b ~ t u l i t ~ ~ t t ~ spores and stored under MAP conditions. While spoilage was delayed for 10 days, toxin production occurred earlier. Whether toxin production precedes or follows spoilage is debated by some, but there is little question that with mild temperature abuse, botulinum toxin can be detected prior to organoleptic deterioration. To ensure the safety of MAP fish, strict temperature control at low temperatures ( 100 mg/ 100 g) are nuts, soybeans, whole grains, and dried beans. Good sources of Mg (50-100 mg/ 100 g) include crustaceans and spinach. Poor sources of Mg ( 15 mg/dl) dueto vitamin D intoxification are listed in Table 3. It has been known for some time that vitamin D may represent a potential concern in cardiovascular health (344,345). Epidemiology studies have reported a lower incidence of atherosclerosis in underdeveloped countries, where food is not fortified with vitamin D (337). These workers indicated that dark-skinned individuals were less susceptible to the toxic effects of vitamin D compared to their fair-skinned counterparts, as evidenced by the existence of large differences i n the average incidence of atherosclerosis among Caucasian and Negro populations. The rate of decline in age-adjusted heart disease deaths recorded from 1968 to 1975 among women and blacks was half that of Caucasian men, which is consistent with the attenuated effect of vitamin D in blacks and premenopausal women. In a community-based study, Scragg et al. (346) showed that the increased exposure to sunlight was protective against CHD. A photometabolic interaction between cholesterol and vitamin D is based on the fact that both vitamin D and cholesterol are derived from squalene, andthe conversion of squalene to 7-dehydrocholesterol could be enhanced bysunlight,whereasintheabsence of sunlight,cholesterolisformed(347).Supplementing the dietof 19 1 volunteers, ages 63-76 years, with vitaminD to evaluate a relationship between cardiovascular health and vitamin D indicated that serum 25-hydroxyvitamin D waspositivelycorrelatedwithserumLDLandnegativelycorrelatedwithHDL (348,349). Other workers have shown a strong relationship between artery calcification and an increase in total serumcholesterol(350).Aorticcalcification is asrelevant in predisposing animals to atherosclerosis as is lipid infiltration and plaque formation (351). There are case studies describing vitamin D toxicity that were attributed to both supplementation and fortification practices. Gross hypercalcemia attributed to vitamin D toxicitywasreported ina 6-month-old boy who had beenadministered ancxcessive amount of a vitamin mixture composedof vitamins A, C , and D since the ageof 4 months. The child exhibited symptomsof vomiting, constipation, and increasing apathy, as well as an undetectable parathyroid hormone plasma concentration (352). Milk and infant formula preparations can be either underfortified or overfortified as well. In 1990 a reported eight cases of hypervitaminosis D resulting in hypercalcemia, anorexia, and other symptoms in weredetectedcaused by amassivefortification of milkwithvitamin D atadairy Massachusetts (353). More than 500 times the amount of vitamin D listed on the label was present i n the product, occurring as a result of equipment malfunction. A follow-up study conducted to determine the variance in vitamin D levels i n milk and infant formula

Table 3 AdverseEffects of Vitamin D Toxicity

Organ Skeletal Cardiovascular

reduced bone nuss Hypcrcalcifcation of bone, Ahnormal contraction of vnscular smooth muscle (hypcrtension), calcification of arteries

Calcium-phosphateprecipitation within renal tubules. urinary tract stones in skin, artery, and gastric mucosa Soft tissue Calcification Renal

414

Kitts

from several dairies i n five states produced results that showed only 62% of the 42 milk samples contained less than 80% of the amount claimed on the label (354). No vitamin D was detected i n 3 of the 14 samples of skim milk tested, i n which one milk sample labeled as containing vitamin D (ergocalciferol) actually contained vitamin D (cholecalciferol). Seven of the I O infant formulas contained tnore than ZOO%, of the amount stated on the label; one containing more than 400%)of the stated amount. A similar study conducted in the United States reported 80% of milk samples contained either 20% less or 20% more vitamin D than the amount stated on the label (355). Of particular concern was of whole milk thereportedfindingthat one sample of chocolate milk and one sample contained more than 900% and 300% vitamin D. respectively, of the amount stated on the label. These studies identified the need for monitoring fortified products for vitamin D content and possibly improving the technologies used to fortify milk with vitamin D. There is concern that abolishing the current directive on vitamin D supplementation would cause a resurgence of rickets. which was a health issue i n parts of Canada even in the late 19SOs, prior to when vitamin D was added to milk (356). Despite the potential risks of vitamin D toxicity due to inaccuraciesin fortification and supplementation technologies or practices, it may be unrealistic to expect that oral vitamin D supplements could reach all populations atrisk (e.g., pregnant women, infants, disabled persons) who are presentlyprotected by vitamin D fortifiedfootls. Thesituations of chronically ill and elderly persons warrant additional attention dueto the fact that they tend toconsume lower levels of vitamin D-containing foods and beverages and have less access to the outdoors and sunlight.

B. Vitamin E Vilamin E, which appears to be the least toxic of the f a t soluble vitamins, includes more than 30 different tocopherols (a-.p-, r-, and 6-tocopherols) witha common homolog. Excellent sources o f tocopherols are soybean. cottonseed, canola, and wheat germ oils. The accepted daily intakeof a-tocopherol is 0.15-2 mg/kg of body weight. and generally a clinical deficiency of vitamin E is rare. Although tocopherols are found naturally in all high plants, fortification is still required where food products arc easily exposed to thermal oxidation or decomposed by exposure of light. The limit of uses in olive oil is 200 mg/ kg. in coconut oil, palm kernel oil, and tallow is S00 mg/kg, and i n infant food it is 300 mg/kg fat (Codex Alimentarius). The antioxidant activity of tocopherols is i n the following descending order: a- > p- > z- > F- at 37°C and 6- > r- > p- > a- at 5O0C-l00"C. It is importanttonote thatwhenpresent i n highconcentrations in foodsystems.prooxidant effects of vitamin E may also result (357,358). Vitamin E deficiency i n humans is rare, but it canoccur in prematurcneonates and i n people suffering from cystic librosis anti biliary atresia. I n most cases, vitamin E deficiencies occur becauseof lipid malabsorption syndromesand the dependence of enteric absorption of vitamin E on adequate lipid absorption. Low dietary intake of vitamin E, coupled with high physiological demands can result in oxidative stress symptoms such as erythrocyte hemolysis and hemolytic anemia. For example. an increase i n the requirement for vitamin E has been associated with oxidative stress generated by excessive fishoil consumption (359). Moreover, a low body content of tocopherols has been shown to result i n an increased tissue (360) and HDL (361) cholestcrol content. This observation o f vitamin E in protecting may be attributed to the important and often overlooked role against depletion of important membl.ane PUFAs which are required for preserving both

415

Nutritional Toxicology

physical properties and enzymatic activities involved in cholesterol metabolism. Further support for this idea comes from the importance of vitamin E in preventing a cholesterolinduced increase in aortic catalase and glutathione peroxidase activity (362). There has been strong support for a positive role for vitamin E in the prevention of ischemic heart disease by induction of antiatherogenic properties (363). Inhibition of lipid oxidation by vitamin E occurs by donating a hydrogen atom from one of the hydroxyl groups i n the vitamin peroxyl (LOO) and alkoxyl (LO') radicals or tocopheryl free radical [Toc'; Eq. ( S ) ] . The inhibition reactions are as follows:

+ TocH H LOOH + Toc' + Tot' + LOO TOC + TocH + LOH + Toc' + TOC'-+ LO TOC

LOO'

LOO' LO' LO'

-

-

Inhibition of peroxyl and alkoxyl free radicals by tocopherol (TocH) can prevent lipid oxidation and decomposition reactions. The rates of reaction Eqs. (S)-@) are important in determining the anti- and pro-oxidant activities of tocopherols. The faster the forward reaction (e.g., the larger the k value). and the slower the backward reaction (e.g., the smaller the - k value), the greater the antioxidant activitiesof tocopherols. The antioxidant activity of vitamin E has alsobeen shown to enhance antioxidant defense mechanisms and prevention of chronic disease. In vitro studies have demonstratedan efficacy for vitamin E to delay the oxidation of low density lipoproteins (363,364). Studies conducted in vitro with rabbits fed D,L a-tocopherol reported a resistance to in vitro forced peroxidation reactions (36S), but no effect i n preventing atherosclerosis. Tocopherol (TocH) can also be converted to a tocopheryl radical (Toc'), which in turn can promote lipid oxidation by abstracting PUFA (LH) to produce an alkyl free radical (L'). In addition, a tocoquinoperoxy1 radical (QOO') can be produced by the oxidation of tocopherol radical (Q'), which also abstracts hydrogen from lipids, thereby promoting lipid oxidation. The pro-oxidant reactions are as follows: TOCH+ 0: + HOO' Toc'

+ TOC'

+ LH + TocH + L'

Toc' -+ Q'

+ 0 2 + QOO. QOO' + LH + QOOH + L' QOOH + LH -+ QOH + L' + OH

Q'

The rates of these reactions dictate the anti- or pro-oxidant activity of tocopherols under different conditions. For example, the reaction rate constant in Eq. ( S ) is many-fold relatively higher than the rate constants in Eqs.(5') and ( I O ) at high tocopherol concentrations, resulting in the large quantities of tocopheryl radicals produced i n Eq. (10). The accumulation of excessive tocopheryl radicals results i n the production of more peroxyl radicals, which promotes Eq.( 1 l ) . These reactions generate free radicals for further propagation reactions i n lipid oxidation. A pro-oxidant effect of vitamin E attributed to photosensitization has also been shown with an intravenous lipid emulsion product usedto treat neonatal jaundice in premature infants (366). Following a 24-hour exposure to phototherapy, thelevel of hydroperoxides increased almost sixfold. The potential physiological significance of these reactions, or the relation between the concentrations of anti- and pro-

Kitfs

416

oxidant activities of tocopherols is unclear (367). However. high concentrations of vitamin E can reduce transition metals and produce radical species from redox reactions (368). The radical center in the aqueous phase is transferred to the lipid phase and a tocopheryl radical (Toc’) is generated when an aqueous peroxyl radical (LOO’) comes into contact with the surface of the LDL, where endogenous a-tocopherol is present to donate a hydrogen to the peroxyl radical according to

The a-tocopherol radical formedin LDL, being water-soluble, is trapped in the lipid phase of LDL, and a chain reaction propagating additional tocopheryl radicals (Toc’) occurs according to the following scheme:

The exposure of LDL to pro-oxidant depletes a-tocopherol prior to generation of lipid peroxidation products, which is consistent with the free radical scavenging property of vitamin E (369). Halliwell(370) demonstrated that the reduction of Cu” to Cu‘ by vitamin E also promotes lipid oxidation. The pro-oxidant activity of vitamin E in the presence of Cu” prevented a characteristic antioxidant effect when tested in a detergent dispersion model micellar solution containing Cu” and a-tocopherol (37 I). The tocopheryl radical (Toc’) generatedby abstraction of hydrogen from tocopherol can be efficiently regenerated in the presence of ascorbic acid (AscoH) according to the scheme

+ TocH + Toc’ + LOOH L‘ + TocH(slowreaction,inefficient) Toc’ + LH Toc’ + AscoH + Asco’ + TocH (fast reaction, efficient) LOO’

Asco’

+ ROO’ -+

Asco-OOR (stable, and inactive product)

The synergistic antioxidant activity between ascorbic acid and a-tocopherol (372) is characterized by the return of tocopherol to antioxidant status and the generation of ascorbic radical that can further stabilize the peroxyl radical by forming a stable and inactive product. In addition to regeneration of tocopheryl radicals, the peroxyl radical stabilizing effect makes ascorbic acid an effective synergistic antioxidant.

C. Vitamin C Vitamin C (ascorbic acid) is a water-soluble reducing agent that has important roles in the regulation of antioxidant and pro-oxidant functions, xenobiotic detoxification, and iron metabolism. Ascorbic acidis found in both fruits (e.g., papaya, oranges, cantaloupe, strawberry) and vegetables (e.g., broccoli, green peppers, cauliflower, kale); however,it is generally accepted that extreme conditions in food processing and storage will reduce the ascorbic acid content dramatically. Exposure of food to extremes of heat, light, oxygen, and pH result in losses associated with thermal destruction, photo- and enzymatic oxidation, and leaching of ascorbic acid. A deficiency of vitamin C is well documented to result in hemorrhage, hyperkeratosis, hypochondriasis, and blood abnormalities, the symptoms

Nutritional Toxicology

41 7

of the condition scurvy. Epidemiological evidence indicatesthat ascorbic acid is a significant variable in modulating the incidence of gastric cancer (373). Other workers have reported a significant inverse relationship between vitamin C status and both diastolic and systolic blood pressure (374,375). Plasma vitamin C concentrations have also been found to be significantly lower in individuals with CHD; however, unlike vitamins A and E, plasma vitamin C levels are not related to a risk of coronary artery disease (376). These findings are potentially related to the affinity of vitamin C to protect against oxidative stress, as evidenced by a strong reducing power both in vitro (377) and in vivo (378), and an affinity to delay LDL lipid peroxidation (379). Vitamin C has been referred to as thefirst defense andmost importantantioxidantinplasma(380).Asisthecasewith vitamin E, ascorbic acid can delay LDL lipid peroxidation and may provide longer protection by inhibiting aqueous peroxyl radicals(38 1) and sequestering transition metals (382). The significance of vitanlin C antioxidant activity may also be the underlying cause for the observed prolonged survival of patients with terminal cancers (383). Reactions specificto enhanced intestinal iron absorption attributable to ascorbic acid and associated transition metal-induced redox cycling or mixed-function cosubstrate activity has resultedin a potential pro-oxidant toxicityrisk associated with vitamin C (384,385). Both mutagenic and genotoxic effects of ascorbic acid have been demonstrated in vitro (386,387). Although these studies are often conducted in the absence of catalase, they denlonstrate the importance of reducing transition metal ions (e.g.,Fe‘+, Cu”) in generating Fenton reaction-induced hydroxyl radicals from the Habcr-Weiss cycle in vitro. The interpretation of these findings to an in vivo condition requires caution, however, since it should not be overlooked that the interactions between antioxidant enzymes and other nonenzymatic antioxidants provide both a diverse and synergistic function in protecting cells from oxidative stress.

D. Folic Acid Folate represents an important vitamin for coenzyme activity with single-carbon transfer in biochemical reactions and has important roles in nucleic acid synthesis, erythrocyte function, and hair health. Fortification of cereal grain food systems with folic acid has been accepted asa necessary strategy for preventingboth lnacrocytic anetnia and debilitating neural tube birth defects (388). This practice is based on the understanding that the folate requirement of 400 pg/day is not obtainable from an average diet consumed by women of childbearing age.In general, folate deficiency is conmot1 in the North American diet and has been regarded as a risk factor for many chronic diseases, including anemia, cancer, and cardiovascular disease. Current fortification levels for folate are listed at 140 pg folate/100 g cereal grain (389). This notwithstanding, concern hasbeen expressed that high folate levels could also mask vitamin B deficiency in some populations, including the elderly (390).

IX. NATURALLYOCCURRING TOXINS A.

Aflatoxins

Aflatoxins are secondary metabolites produced by the molds A.spr~yillusj f m u s and A. prrrmiticus i n agricultural commodities such as corn, peanuts, figs, tree nuts. rice, dried fruits, cassava, and various seeds. Moreover, aflatoxin residues can also occur in egg or

418 Table 4

KittS

ExposureRoutesfor Aflatoxin

Routc DirectconsumptionDietary

intake of AFB, from cereal, grain. and nut products IndirectconsumptionMetabolites of allatoxin (AFM, in milk) Inhalation Occupational risk: aflatoxincontaining dust

milk products as a result of ingestion of aflatoxin-contaminated feed by poultry or lactating dairy cows. Toxic components isolated from peanut meal used as an ingredient in poultry feed were resolved into four fluorescent spots when applied to silica gel and eluted using chloroform-methanol as the tnobile phasei n a thin layer chromatographic procedure (391 ). The two fluorescent blue and two fluorescent green spots identified under a UV light were named aflatoxins B1, B2, G I , and G2, respectively (392). The structures of aflatoxin are derived from a condensed bisfuradcoumarin ring, with isomers B? and B I representing dehydro- derivatives of aflatoxins B1 and G I . respectively. Aflatoxins M1 and M2 are typicallytnammalianmetabolites of B1 andB2.respectively,althoughtheymayalso occur in fungal cultures. Special research intcrest has been placedon aflatoxin B I (AFB 11, since it is the most prevalent and the most toxic secondary metabolite. Food and feeds are susceptible to invasion by Aspergillus at all stages of production (production, preharvest, harvest, processing, transportation, and storage). Relative humidities of 88 to 95% and storage temperatures in the 25"C-3OoC range are optimal for growth of Aspet.gi//rts species from air, soil, or insect vectors (393). Essential of oilscloves, cinnamon, and onion and garlic extracts, and eugenol all have inhibitory effects on aflatoxin production and growth of A. ,flmws. In addition, caffeine is effective in inhibiting growth and mycotoxin production of A.spct;qillus and Pmicilliwn species (394); a finding which appears to be specific to caffeine since other mcthylxanthines such as theobromine are less effective (395). The possible routes of human exposure (Table 4) and subsequent health risks are presented in Table S . Direct consumption of aflatoxins is related to the intake of principle aflatoxins from dietary sources, which are distinct from the indirect consumption of aflatoxin metabolites from edible animal tissues. Inhalation of aflatoxins poses an occupational risk of allatoxicosis for workers in grain mills, oil presses, and employees of livestock Table 5 TargetOrgansforAllatoxin

Organ systctn Respiratory Gastrointestinal Hepatic Reproductive

B,

Characterization of discasc Lung, trachea, and bronchus tumors Colon carcinotna, small intestine tumors Hcpatocellular carcinotna, xute hcpatitis, preneoplastic lesion Etnbryotoxicity. reduced fertility. inhibition of lymphocyte function, reduced cell mediated immunity

Nutritional Toxicology

419

feed-processing facilities. Although acute toxicity of AFB 1 results in hepatitis and death in countries such as India and Kenya (396). it is the chronic exposure to low levels of aflatoxins that represents themost c o m m n risk associated with carcinogenesis. Aflatoxins are relatively low molecular weight. lipophilic compounds, suggesting efficient absorption from the small intestine into the mesenteric circulation prior to entering the liver through the portal circulation. Allatoxins act predominantly at the liver and biliary tract. AFB 1 is a procarcinogen that once metabolically activated to the 8,9-epoxide intermediate, a putative and ultimatecarcinogen,formsadducts(N7-guaninederivative2,3-dihydro-2-(N7puanyl)-3-dehdroxya~atoxinB 1) that are primarily found i n GC-rich regions of DNA (Fig. IO). Thcse adducts result in a GD-TA nucleic acid transversion and DNA mutation (397). Biliary excretion of AFB 1 has been reported to involve an AFB l-glutathione complex as a major metabolite excreted at 6 to 8% of administered dose in I O minutes, with the peak rate of excretion occurring at 30 minutes (39X). Elimination of AFB 1 is relatively slow, with an apparent plasma half-life of 92 hours for cumulative AFB 1 fecal excretion com-

Procarcmogen (without polar groups) Aflatoxin B1 Phase I Enzyme (Cytochrome P450)

Polar groups introduced Electrophilic Intermediate (AFBI 8.9 oxide)

Conjugate

Reaction

I

Phae II Enzyme (Glutathione S-transferase) Aflatoxin Glutathlone

Carclnogenic Adducts

I

Excretion 8,9dihydro-B,9dihydroxy Aflatoxin B1

9-Hydroxyaflatoxin B1

420

Kitts

pared to IS% urinary excretion 23 daysafter dosing (399). The three major urinary metabolites recovered by radiotracer methods are AFM 1 (41-SO%), AFPl ( MeIQx > DiMeIQx > IQ (431). PhlP fied in the diet in decreasing order were PhIP is present in the greatest amounts in fried beef and represents the greatest exposure to HCA i n the North American diet. It is noteworthy that the carcinogenic potencies of these HCAarealmost in reverse order (IQ > DiMeIQx > MeIQx > PhlP), whichmaybe explained by the fact that PhIP, while undergoing phase I activation, is cleared efficiently by extrahepatic tissues. From dietary records collected from more than 3500 individuals on intakes of HCA, an upper bound estimate of incremental cancer risk was estimated at 1.1 X 10", when using cancer potencies based on body surface area. Nearly half (46%) of the incremental risk was due to the ingestion of PhIP, with the consumption of meat and fish products contributing most (80%) of the total risk.

XI. CONCLUSIONS Epidemiological, experimental, and metabolic studies have provided convincing evidence that the human diet contains both nutrients and bioactive nonnutrients which provide protection against degenerative diseases including atherosclerosis and cancer (Table 7). Xenobiotic compounds are metabolized by enzyme systems that have evolved in response to selection pressure and are dependent on the nutritional status and the macro- and micronutrient composition of the diet. The role of dietary constituents in influencing the toxicity of xenobiotic agents needs to be considered along with nutrient-nutrient interactions in evaluating risks to dietary-induced environmental carcinogenesis, mutagenesis, teratogenesis, or other chronic diseases such as CHD. Phase I reactions that are necessary for the detoxification of xenobiotic materials will be suppressed by fasting or induced by highprotein diets or the availability of key nutrients such as niacin or riboflavin. Phase I1 enzymes, located in the cytoplasm and endoplasmic reticulum, are active in conjugation reactions that produce more polar metabolites from GSH conjugates, sulfates, and glucuronides in removal of electrophilic xenobiotics derived by endogenous phaseI enzyme activity, or from exogenous sources. Phase I1 enzymes are also regulated by nutritional status concerning various specific nutrients, as well as other food constituents that include iso-

425

Nutritional Toxicology

Table 7 NonnutritiveChctnoprevcntiveAgents

in Foods

Agent Resveratrol

Grape.mulberry

I3C.I Phenyl isothiocyanate Sulphoraphone Crambene

Cabbage, broccoli Cabbage, broccoli Broccoli Cabbage, broccoli, mustard Spice (Cucltttrrr longcl)

Turmeric

Eugenol

CLA-conjugated linoleic acid

Oil extracts from clove, cinnamon. basil, nutmeg Beef, cheese

Inhibition of cyclooxygenase and hydroperoxidase activity, anti-inflammatory activity, induction of phase I1 NADPHquinone reductase activity Inhibition of flavin tnonooxygcnase Inhibition of P-450 cnzytncs Induction of phase 11 enzymes Induction of GST activity Antioxidant activity, inhibition o f cyclolipoxygenase, scavenging of diolepoxides Anti-inflammatory activity, induction of Phase I1 enzymes, modulation of immune system Antioxidant activity, modulation of cyclolipoxygenase activity

thiocyanate and that are present in many cruciferous plants (broccoli, cabbage, kale, and cauliflower) (3,432). The result is dietary nutrients as well as nonnutrients that work in concert with the general nutritional status of the organism to minimize the toxicity of xenobiotic agents. Attempts to relate the chenloprotective properties of various dietary components to the development of chronic disease states will continue to be an active area of study in nutritional toxicology.

REFERENCES I.

2.

3.

4. 5.

6. 7.

8.

A Boveris, B Chance. The mitochondrial generation of hydrogen peroxide: general properties and the effect of hyperbaric oxygen. Biochem J 134:707-716, 1973. YVYuan,DDKitts.Endogenousantioxidants:roleofantioxidantenzytnes in biological systems. In: F Shahidi,ed. Natural Antioxidants: Chemistry, Health Effects and Applications. Champaign. IL: AOCS Press, 1997, pp. 258-270. DD Kitts. Bioactive substances in food: identification and potential uses. Can J Physiol Phartnacol72:423-434,1997. MA Moorc, CD Stoner. Cancer chemoprevention: principles and prospects. Carcinogenesis 14~1737-1746, 1993. ID Capel. Factors affecting antioxidant defense potential. In: CK Chow, ed. Cellular Antioxidant Defense Mechanisms, vol. 11. Boca Raton, FL: CRC Press, 1988, pp. 191-215. R Weidruch, RL Walford. Dietary restriction in mice beginning at 1 year of age: effect on life-span and spontaneous cancer incidence. Science 215:1415-1418, 1982. KE Cheney. RK Liu, CS Smith, PJ Meredit, MR Mickey, RL Walford. The effect of dietary restriction of varying duration on survival, tumour patterns, immune function and body temperature in BIOC3FI female mice. J Gerontol38:420-430,1983. H Nohl, D Hegner. Responses of mitochondrial superoxide dismutase, catalase and glutathione peroxidase activities to aging. Mech Ageing Dev l 1 : 145-151, 1979.

Kitts

426

9. GA Hnzelton, CA Lang. Glutathione contents of tissues in the aging tnouse. Biochctn J 188: 25-70, 1980. 10. EWKellog. I Fridovich.Superoxidedismutascintheratandmouse as afunctionofage and longevity. J Gerontol 3 1 :405-408. 1976. 1 I . E Xia, G Rao, H Van Remmen,AR Hcydari, A Richardson. Activitiesof antioxidant enzymes in various tissues of male Fischer 344 rats are altered by food restriction. J Nutr 125:195-

201,1995. 12. LJ Davis, B Tadolini, PL Biagi, RL Walford, F Licastro. Effect of age and extent of dietary restriction on hepaticmicrosomallipidperoxidationpotential i n mice.MechAgeingDev

72:155-163.1993. 13. LD Younglnan. Protein restriction, and caloric restriction compared: effectson DNA damage. carcinogenesis, and oxidative damage. Mutat. Res 295: 165-179. 1993.

14. LD Youngtnan, JK Park, BN Ames. Protein oxidation associated with aging is reduced by dietary restriction of protein or calories. Proc Natl Acad Sci USA 8 9 9 1 12-91 16. 1992. 15. R McCarther, EJ Masoro, BP Yu. Does food restriction retard aging by reducing metabolic rate? Am J Physiol 248:E488-E490, 1985. 32: 16. CK Chow. Nutritional influence on cellular antioxidant defense systems. Am J Clin Nutr 1066- 1OX l , 1979. 17. DV Godin. SA Wohaieb. Nutritional deficiency. starvation and tissue antioxidant status. Free 1088. Radic Biol Med 5:165-176, 18. RASunde, GE Gutzke,WGHoekstra.Effect of dietarymethionineonthebiopotency of selenite and selenornethioninc i n the rat. J Nutr 1 I 1:76-86, 198 I . 19. CJ Huang,MLFwu.Proteininsufficiencyaggravatestheenhancedlipidperoxidationand reduced activities of antioxidative enzymes i n rats fed diets high i n polyunsaturated fat. J Nutr 122: 1 182- 1 189, 1992. 20. K Mouri, Y Hayafune. 0 Igarashi. Effectof dietary protein on vitamin E levels in erythrocyte and tissues of rats. J Nutr Sci Vitaniinol 32: 145- 155, 1986. 21. JF VanFleet, GD Boon,VJ Ferrans. Induction of lesions of selenium-vitamiti E deficiency in weanling swine fed silver, cobalt, tellurium. zinc, cadmium and vanadium. Am J Vet Res 42:789-800.1976. 22. MHN Golden, D Ramdath. Free radicals in the pathogenesis of kwashiorkor. Proc Nutr Soc 46~53-68, 1987. 23. AEA McLean, EK McLcan. Nutrition and toxicant liver injury. I n : J Maseh, K Osaneova, DP Cuthbertson, eds. Nutrition Proceedings of the 8th International Congress, Prague. Glasgow. Exccrpta medica, 1969, pp. 457-460. 24.EARogers,PHNewbcrne.Dietandaflatoxin B, toxicity i n rats. ToxicolAppl Pharmacal 20:113-121.1971. 25. RA Shakman. Nutritional influences on thc toxicity of environmcntal pollutants. Arch EnvironHealth 28:105-113, 1974. 26. GHAnderson.Sugarsandhealth:areview.NutrRes17:1485-1498,1997. 27. E Shafrir. Fructosdsucrose metabolism, its physiological and pathological implications. In: N Kretchtncr, CB Hollcnbeck,eds. Sugar andSweetners.NewYork:CRCPress, 1991, pp. 63-98. 28.DVZyznk,JMRichardson,SRThorpe,JWBaynes.Formationofreactiveintermediates 3 16547from Amadori compounds under physiological conditions. Arch Biochem Biophys 554, 1995.

29.JDMcPherson, BH Shilton,DJWalton.Role of fructose i n glycationandcrosslinking o f proteins. Biochemistry 27: 1901- 1907, 1988. 30. DG Dyer, JA Blackledge, SR Thotpe, JW Baynes. Formation ofpentosidine during nonenzymatic browning of proteins by glucose. Identification of glucosc and other carbohydrates as possible precursors of pentosidine i n \ * i w .J Biol Chem 266: I 1654-1 1660, 1991. 31. MX Fu, KJ Wells-Knecht,JABlackledge,TJLyons,SRThorpe,JWBaynes.Glycation,

Nutritional Toxicology

32.

33. 34.

35. 36.

37. 38. 39.

40. 41.

427

glycoxidation and crosslinking o f collagen by glucose. Kinetics. mechanisms and inhibition o f late stages of Maillard reaction. Diabetes 43:676-683. 1994. RC Khalifoh, P Todd, AA Booth. SX Yang, JD Mott. BC Hudson. Kinetics o f nonenzymatic glycation o f ribonuclease A leading to advanced glycation end products. Paradoxical inhibition by ribose leading t o facile isolation o f protein intermediate for rapid post Atnadori studies.Biochemistry 35:4645-4654. 1996. JA Dunn, JS Patrick. SR Thorpe. JW Baynes. Age-dcpcndcnt accumulation of Ne-(carhoxymethyl) lysine in lens proteins. Biochemistry 2839464-9468, 1989. N Araki, N Lleno. B Chakr;tbarti. Y Morino, S Horuchi. Itnmunochemical evidence for the presence o f advanced glycationend products i n human lens proteins and its positive correlationwith aging. J Biol Chem 266:7329-7332. 1992. R Bucala, A Cerami. Advunced glycosylation: chemistry, biologyand implications for diahetes and aging. Adv Phartnacol 23: 1-34, 1993. T Hayashi. M Namiki. Role o f sugar fragmentation i n the Maillard reaction. In: M Fujimaki. M Namiki, H Kato.cds. Amino-Carbonyl Reactions i n Food and Biological Systems. Amsterdam: Elsevier. 1986. pp. 29-38. MA Cilornh. VM Monnicr. Mechanism o f protein modification by glyoxal and glyxoslaldehyde. reactive intcrmcdiatcs ol' the Maillard reaction. J Biol Chetn 270: 10017-10026, 1995. SP Wolff. ZY Jiang, JV Hunt. Protein glycotion and oxidative stress in diabetes mellitus and ageing.FreeRadBiolMed 10:339-352, 1991. SP Wolff. RT Dean. Glucose autoxidation m d protein oxidation: the role of autoxidative glycosylation in diabetes mellitus and ageing. Biochem J 245:243-250. 1987. SP Wolff. RT Dean. Aldehydes and ketoaldchydes i n the non-enzymaticglycosylation of proteins.Biochetn J 249:617-619. 1988. M Brownlee. Advanced protein glycosylation in diabetes and ageing. Annu Rev Mcd 46:

223-234. 199s. AS Eblc. SR Thotyc. JW Bayncs. Nonenzymatic glycosylationand glucosc dependent crosslinking o f proteins. J Biol Chem 259:9406-9412, 1983. 43. F Hayasc. RH Nagaraj, S Miyata. FG Njorogc. VM Monnier. Ageing proteins: immunological dctcction of a glucose-derived pyrrole formed during the Maillard reaction irt l i l y ) . J Biol

42.

44.

4s.

46.

47. 48.

Chetn2643758-3764,1989. S Teichberg. HFull.YMLi.M Stcflcs. Advancedglycation end HVlassnra.LJStriker. productsinduceglotnerularsclerosis and albuminurin i n nortnalrats.Proc Natl Ac;tti Sci USA 9 I I 1204- I 1708. 1994. DR Sell. VM Monnicr.End-stage renal disease and diabetescatalyze theformation of :I pentose-derived crosslink from aging human collagen. J ClinInvest 85:380-384. 1990. R Bucala, A Makita. T Koschinsky, A Cerami. HVlassara.Lipidadvancedglycosyl;ltio11: pathway for lipidoxidation i r l ,i\w. Proc Natl Acud Sci 90:6434-6438. 1993. WC John. EJLtttmb. The Mnillard o r browning reaction i n tliabetes. Eye 7:230-237, 1993, M Brownlee. The pathological implications of 1"'otein-glycatiotl. Clin Invest Mcd 18:275281. 199s.

49.

RS Sohal, R Wcindruch. Oxidative stress, caloric restriction and aging. Science 273:59-63, 1996.

so.

JA Dunn, DR McCance, SR Thorpe. TJ Lyons,JWBnyncs.Age-tlependentaccuI11ulation o f N'-(carhoxymethyl) lysine and N'-(carboxymethyl) hydroxylysine in human skincollagen. Biochemistry 30:120S-1210, 1991. SI. VM Monnier. DR Sell, RH Nagarji, S Miyata. S Grandhee, P Odetti, SA Ibrahim. Maillard reaction-mediated molecular datnaget o extraccllular matrix and other tissue proteins i n diabetes. aging and uremia. Diabetes 41:S36-S41, 1992. 52. MC Wells-Knecht. SRThotpe, JW Baynes. Pathway o f formation of glycoxi&ation products during glycation of collagen. Biochemistry 34: 1.5 134- 15 141, 1995. 53. Y Aoki. K Yazaki, K Shirotori, Y Yanagisawa. H Oguchi, K Kiyosawa, S Furuta. Stiffclling

428

Kitts

of connective tissue i n elderly diabetic patients: relevance to diabetic nephropathy and oxidative stress. Diabetologia 36:79-83. 1993. 54. H Valssara, H Fuh, Z Makita, S Krungkai, A Cerami, R Bucala. Exogenous advanced glycosylation end products induce complex vascular dysfunction in normal rats: a model for dia89: 12043- 12047, 1992. betic and aging complications. Proc Natl Acad Sci USA 55. JVHunt, CCT Smith, SP Wolff.Autoxidativeglycosylationandpossibleinvolvement of peroxides and free radicals i n LDL modification by glucose. Diabetes 39: 1420-1424, 1990. 56.TLyons, MF Lopes-Virella.Glycosylation-relaledmechanisms. In: BDraznin,REckel. eds. Diabetes and atherosclerosis. Molecular basis and clinical aspects. New York: Elscvier Science,1993, pp. 169-189. 57. PB Duell, JF Oram, EL Bicrman. Non enzymatic glycosylation of HDL and impaired HDLreceptor-mediated cholesterol efflux. Diabetes 40:377-384, 1991. 58. R Bucala, Z Makita, G Vega, S Grundy. T Koschinsky, A Ccrami, H Vlassara. Modification of low-density lipoprotein by advanced glycation end products contributes to thc dyslipidctnia of diabetes and renal insufficiency. Proc Natl Acad Sci USA 91:944-9445. 1994. on 59.MPHabib, F Dicherson.ADMooradian.Effects of diabetes,insulinandglucoseload lipid oxidation in the rat. Metabolism 43:1442-1445, 1994. 60. S Picard.Lipoproteinglycooxidation.DiabctcsMetab (Paris) 2139-94, 1995. 61. ADMooradian, CC Lung, JL Pinnas.Glycosylationenhancestnalondialdchydebindingto proteins. Free Radic Biol Med 21:699-701, 1996. 62. MSSwany,ECAbraham.lnhibltion of lenscystallinglycationandhighmolecularweight aggregate formation by aspirin in virro and i r ~I ~ I Y I Invest . Opthalmol Vis Sci 30:I 120- I 126. 1989. 63. D Edclstein, M Brownlce. Mechanistic studies of advanced glycosylation and product inhibition by aminoguanidinc. Diabetes 41 :26-29, 1992. 64. JT Skamarauskas, AG McKay,JVHunt.Aminoquanidienanditsprooxidanteffects on an 21:801-802. 1996. experimental model of protein glycation. Free Radic Biol Med 65. AA Booth. RC Khalifah, BC Hudson. Thiamine pyrophosphate and pyridoxamine inhibit thc formation of antigenic advanced glycation end products: comparison with aminoguanidinc. Biochem Biophys Res Commun 220: 1 13- 1 19, 1996. of fonnation of 66. AABooth,AA Khalifah, RC Todd, BC Hudson.Invitrokineticstudies antigenic advanced glycation end products(AGES):novel inhibition of post-Amadori glycation pathways. J Biol Chetn 272:5430-5437, 1997. 67. SK Jain, R McVie, JJ Jaramillo, M Palmer, Smith. T The effect of modest vitamin E reduction on protein glycosylation in diabetes. Diabetes Care 14:68-72, 1996. 68. SK Jain, M Palmer. The effect of oxygen radicals metabolites and vitamin E on glycosylation of proteins. Free Radic Biol Mcd 22:593-596, 1997. N'-(carbocylmethyl)lysine 69. S Reddy, J Bichler,KJWells-Knecht,SRThorpc,JWBayncs. is a dominant advanced glycation end product(AGE) antigen in tissue proteins. Biochemistry 34:10872-10878,1995. 70. RT Holman. George 0 . Burr and the discovery of essential fatty acids. J Nutr 1 18535-540. 1988. or 71. DDKitts,PJHJones.Dietaryfats:discriminativepartioningforenergyandsynthesis triacylglycerols. Food Res Int 2957-69. 1996. 72. RT Holman. SB Johnson, TF Hatch.Acaseof humanlinolenicaciddeficiencyinvolving neurological abnormalities. Am J Clin Nutr 35:617-623. 1982. 73. H Tanaka, C Date, H Chen, T Nakayatna, T Yokoyama, N Yoshiike, H Iwaoka, M Iwaya, MM Zaman. M Yamaguchi, Y Matsumura, M Sugiyama, W Kushiro, T Ichimura, A Noji, A Chowdhury, IS Kim. TB Kwan, BM Cho. A brief review of epidemiological studies on ischemic heart disease in Japan. J Epidemiol 6:S49-S59, 1996. 74. HCSpenser.Perspectivesofpublichealthandprevention. Am J MedSci 310:S83-S85, 1995.

Nutritional Toxicology

429

75. A Zanchetti. Hyperlipidemia in the hypertensive patient. Am J Med 96:3S-8S, 1994. 76. SM Grundy, MA Denke. Dietary influences on serum lipids and lipoproteins. J Lipid Res 31:1l49-ll72. 1990. 77. K Sanders, L Johnson, K O’Dea, A Sinclair. The effect of dietary fat level and quality on in nortnocholesterolemic subjects. Lipids plasmalipoproteinlipidsandplasmafattyacids 29: 129- 138. 1994. 78. KC Hayes, A Pronczuk, S Lindsey, D Dierscn-Schadc. Dietary saturated fatty acids (12:0, 14:0, 16:O) differ in their impact on plasma cholesterol and lipoprotcins in non human primates. Am J Clin Nutr 53:49 1-498, I Y Y 1. 79. YV Yuan, DD Kitts, DV Godin. Influence of dietary cholesterol and fat source on atherosclerosis in the Japanese quail (Corurnixjnporlicw). Br J Nutr 78:993-1014, 1997. 80. LA Woollct, DK Spady, JM Dietschy. Saturated and unsaturated fatty acids independently regulate low density lipoprotein receptor activity and production rate. J Lipid Res 33:7788, 1992. 81. ML Fcrnandez, ECK Lin, DJ McNamara. Regulation of guinea pig plasma low density lipoprotein kinetics by dietary fat saturation. J Lipid Res 33:97-109, 1992. 82. ML Fernandez, ECK Lin, DJ McNamara. Differential effects of saturated fatty acids on low density lipoprotein metabolism in the guinea pig. J Lipid Res 33:1833-1842, 1992. 83. ML Garg, AA Wierzbicki, ABR Thornson, MT Clandinin. Dietary saturated fat level alters the competition between a-linolenic and linoleic acid. Liptds 24:334-339, 1989. 84. AHirai, T Terano,THamazaki, J Sajiki, S Kondo.AOzawa,TFujita,TMiyamoto,Y Tamura, AKumagai.The effects of the oral administration of fish oil concentrate on the releaseandthemetabolismof [“C] arachidonicacidand [“C] eicosapentaenoicacidby human platelets. Thromb Res 28:285-290, 1982. 85. SH Goodnight, WS Harris, WE Connor. The effectsof dietary omega-.? fatty acidson platelet composition and function in man-A prospective, controlled study. Blood 58:880-881. 1981. 86. l Ikeda, K Wakamatsu, A Inayoshi. K Imaizumi. M Sugano,K Yazawa. Alfa-linolenic, cicosapentaenoic and docosahexaenoic acids affect lipid metabolism differently in rats. J Nutr 124:1898-1906,1994. in dietaryfatandcholesterolintakesmodify 87. YV Yuan,DDKitts,DVGodin.Variations antioxidant status of SHR and WKY rats. J Nutr 128:1620-1630, 1998. 88. TV Fungwe, JE Fox, LM Cagen, HG Wilcox. M Heimberg. Stimulation of fatty acid biosynthesis by dietary cholesterol and of cholesterol synthesis by dietary fatty acid. J Lipid Res 35:31 1-318, 1994. 89. MH Lin, SC Lu, JW Hsieh, PC Huang. Lipoprotein responses to fish, coconut and soybean oil diets with and without cholesterol in the Syrian hamster. J Formos Med Assoc 94:724731, 1995. 90. A Pronczuk, P Khosla, KC Hayes. Dietary myristic, palmitic and linoleic acids modulate cholesterolemia in gerbils. FASEB J 8: 1l91-l200, 1994. 91. WH Ling, PJH Jones. Enhanced efficacy of sitostanol-containing versus sitostanol-free phytosterol mixtures in altering lipoprotein cholesterol levels and synthesis inrats. Atherosclerosis 118:319-331. 1995. 92. JA Weststrate, GW Meijer. Plant sterol-enriched margarines and reduction of plasma total andLDL-cholesterolconcentrationsin normocholesterolaen~icandhypercholesterolaernic subjects. Eur J Clin Nutr 52:334-343, 1998. 93. JM Hodgson, ML Wahlquist, JA Boxall, ND Balnzs. Can linoleic acid contribute to coronary artery disease? Am J Clin Nutr 58:228-234, 1993. 94. J Regnstroem, J Nilsson. Lipid oxidation and inflammation-induced intimal fibrosis in coronary heart disease. J Lab Clin Med 124: 162- 168, 1994. 95. MI Uusitupa, J Sarkkinen, J Torpstroem, P Pietinen, A Aro. Long term effects of four fatmodified diets on blood pressure, J Hum Hypertens 8:209-218. 1994. 96. MC Morris. Dietary fats and blood pressure. J Cardiovasc Risk 1:21-30. 1994.

430

Kitts

97. S Sacks, M Stampfer, A Munoz, K McManus, M Canessa, E Kass. Effect of linoleic and oleic acids on blood pressure, blood viscosity and erythrocyte cation transport. J AmCol1 NuW 6:179-185, 1987. 98. AM Gotto. Lipid lowering, regression and coronary events-a review of the interdisciplinary council on lipids and cardiovascular risk intervention. Seventh council meeting. Circulation 921646-656,1995. 99. M Modan, H Halkin, S Almog, A Lusky, A Eshkol, M Shefi, Z Shitrit-Fuchs. Hyperinsulinemia-a link between obesity and glucose intolerance. J Clin Invest 75:809-817, 1985. 100. 0 Samuelsson, T Hedner, B Persson, 0 Anderson, G Berglund, L Wilhelmsen. The role of diabetes mellitus and hypertriglyceridemia as coronary risk factors in treated hypertension: 15 years of follow-up of antihypertensive treatment in middle-aged men in the primary prevention trial in Goteborg, Sweden. J Intern Med 235:217-227, 1994. 101. MR L’Abbe, KD Trick, JL Beare-Rogers. Dietary (n-3) fatty acids affect rat heart, liver and aorta protective enzyme activities and lipid peroxidation. J Nutr 12 1 : 1331- 1340, 1991. 102. 0 Tirosh, R Kohen, J Katahendler, A Alon, Y Barenholz. Oxidative effect on the integrity of lipid bilayers is modulated by cholesterol level of bilayers. Chem Phys Lipids 87: 17-22, 1997. 103. M Naruszewicz, E Wozny, E Mirkiewiz, G Nowicka, WB Szostak. The effect of thermally oxidized soya bean oil on metabolism of chylomicrons. Increased uptake and degradation of oxidized chylomicrons in cultured mouse macrophages. Atherosclerosis 66:45-54, 1987. 104. T Kaneko, K Kaji, M Matsuo. Cytotoxicities of a linoleic acid hydroperoxide and its related aliphatic aldehydes towards cultured human umbilical vein endothelial cells. Chem Biol Interact 67:295-304, 1988. 105. KK Carroll, HT Khor. Dietary fat in relation to tumorigenesis. Prog Biochem PharmacolIO: 308-353,1975. 106. LACohen,JYChen-Backlund,DW Sepkovic, S Sugie. Effect of varying proportions of dietary menhaden and corn oil on experimental rat mammary tumour promotion. Lipids 28: 449-456,1993. 107. LU Thompson, SE Rickard, LJ Orcheson, MM Seidl. Flaxseed and its lignan and oil components reduce mammary tumour growth at a late stage of carcinogenesis. Carcinogenesis 17: 1373-1376,1996. 108. CPJCaygill,ACharlett, MJ Hill. Fat fish, fish oilandcancer.Br J Cancer74:159-164, 1996. JK Huttunen, JF Navajas,BC 109. N Simonsen,Pvan’tVeer,JJStrain,JMMartin-Moreno, Martin, M Thamm, AF Kardinall, FJ Kok, L Kohlmeier. Adipose tissue omega-3 and omega6 fattyacidcontentandbreastcancer intheEURAMICstudy.EuropeanCommunity multicenter study on antioxidants, myocardial infarction and breast cancer. Am J Epidemiol 1471342-352.1998. 1 IO. DF Horrobin. The role of essential fatty acids and prostaglandins in breast cancer. 1n:BS Reddy, LA Cohen, eds. Diet, Nutrition and Cancer: A Critical Evaluation. Boca Raton, FL: CRCPress,1986,pp.101-124. 111. A El-Sohemy, MC Archer. Regulation of mevalonate synthesis in rat mammary glands by dietary n-3 and n-6 polyunsaturated fatty acids. Cancer Res 573685-3687. 1997. 112 DD Kitts. Toxicology and safety of fats and oils. In: YH Hui, ed. Bailey’s Industrial Oil and Fat Products, vol. I , 5th ed. New York: John Wiley & Sons, 1996, pp. 215-280. 113. MA Saleh, KA Ahmed, AN Sharaf, LM Abdel. Mutagenicity of heated cottonseed frying oil. J Food Safety 7:203-213. 1986. 114. AV Gastel, R Mathur, VV Roy, C Rukmini. Ames mutagenicity test of repeatedly heated edible oils. Food Chem Toxicol 22:403-405, 1984. of thermally-oxidized sunflower oil by 115. PJA Sheehy, PA Morrisey, A Flynn. Consumption chicks reduces a-tocopherol status and increases susceptibility of tissues to lipid oxidation. Br J Nutr 7153-65, 1994

Nutritional Toxicology

431

116. V Ruiz-Guien-ez, FJG Muriana. Effect of ingestion of thermally oxidized frying oil on desaturase activities and fluidity i n rat-liver microsomes. J Nutr Biochem 3:75-79. 1992. 117. CM Yang. CWC Kendall, DStamp, A Medlinc, MC Archer, WR Bruce. Thermally oxidized dietary fat and colon carcinogenesis in rodents. Nutr Cancer 30:69-73, 1998. r r u m fatty acids. An1 J Clin Nutr 66S:1006S118. A Ascherio, WC Willet. Health effects of IOIOS, 1997. 119. FB Hu, MJ Stampfer. JE Manson, E Rimm, CA Colditz, BA Rosner, SH Hcnnekens, WC Willet. Dietary fat intake and the risk of coronary heart disease i n women. N Engl J Med 337:1491-1499,1997. 120. T Byers. Hardened fats, hardened arteries. N Engl J Med 337:1544-1545, 1997. 121. A Tavani, E Negri, B D'Avanzo, C La Vecchia. Margarine intake and risk of nonfatal acute myocardial infarction i n Italian women. Eur J Clin Nutr 51:30-32, 1997. 122. S Shapiro. Do trm.s fatty acids increase the risk of coronary artery disease? A critique of the epidemiologic evidence. Am J Clin Nutr 66S:lOl IS-1017S. 1997. 123. A Nordoy, SH Goodnight. Dietary lipids and thrombosis, relationships to atherosclerosis. Arteriosclerosis 10:149-163, 1990. 124. D Steinberg, S Parthasarathy. TE Carew, JC Khoo, JL Witztum. Beyond cholesterol: modifications of low-density lipoprotein that increase its atherogenicity. N Engl J Mcd 320:915924, 1989. 125. SK Peng, CB Taylor, JC Hill, RJ Morin. Cholesterol oxidation derivatives and arterial endothelial damage. Atherosclerosis 54: 121- 133. 126. P Addis. SW Park. Role of lipid oxidation products in atherosclerosis. In: SL Taylor, RA Scanlan,cds.FoodToxicology:APerspectiveontheRelativeRisks.NewYork:Marcel Dekkcr, 1989, pp. 297-330. 127. MS Jacobson, MC Price, AE Shamoo, FP Hcald. Atherosclerosis in white carncau pigeons: effects of low-level cholestane-triol feeding. Atherosclerosis 57:209-217. 1985. 128. GAS Ansari, RD Walker, VB Smart, LL Smith. Further investigations of lnutagaenic cholesterol preparations. Food Chem Toxicol 20:35-41. 1982. SD Bergmann, JH Zavoral. Plasma cholesterol oxida129. HA Emmanuael, CA Hassel. PB Addis, tion products (oxysterols) in human subjects fed a meal rich in oxysterols. J Food Sci 56: 843-847. 199 1. 130. T Parasassi,AM Giusti, M Raimondi, G Ravagnan,0 Sapora, E Gratton. Cholesterol protects the phospholipid bilayer from oxidative damage. Free Radic Biol Med 1951 1-516, 1995. 131. A El-Sohemy, WR Bruce. MC Archer. Inhibition of rat mammary tumorigenesis by dietary cholesterol.Carcinogencsis17:159-162,1996. 132. A El-Sohemy, CWC Kendall, AV Rao, MC Archer. WR Bruce. Dietary cholesterol inhibits the development of aberrant crypt foci in the colon. Nutr Cancer 25: 11 1-1 17, 1996. 133. CWKendull,SAJanezic.DFriday, AVRao.Dietary cholesterolenhancespreneoplastic aberrant crypt formation and alterscell proliferation in the murine colon treated with azoxymethane. Nutr Cancer 17: 107- 1 14, 1992. 134. MLKruger,DFHorrobin.Calciummetabolism,osteoporosisandessentialfattyacids:a review.ProgLipidRes36:131-151,1997. 135. S Garcia-Lopez, CD Miller. Bioavailabilityof calcium from four different sources. Nutr Res 11:1187-1196.1991. 136. LA Keane, NN Potter, JW Shcrbon. Estimation of calcium status in selected food systems. J Food Sci 53:l I 1 1-1 112, 1988. 137. RR Recker, A Bammi. MJ Barger-Lux, RP Heancy. Calcium absorbability from milk products, an imitation milk, and calcium carhonate. Am J Clin Nutr 47:93-95, 1988. 138. TM Smith, JC Kohlars, DASavaiano,MDLevitt.Absorption of calciumfromInilkand yogurt. Am J Clin Nutr 42:1197-1200, 1985. 139. MS Buchowski. DD Miller. Calcium bioavailnbility from ripening cheddar cheese. J Food Sci55:1293-1295,1990.

Kitts

432

140. MS Buchouski, DD Millcr. Lactosc, calcium source and age affect calcium bioavailability i n rats.JNutr121:1746-1754, 1991. 141. YV Yuan. DD Kitts, T Nagasawa. The effect of lactose and fermentation products on paracellular calcium absorption and femur biomechanics. Can Inst Food Sci Techno1 J 23:74-80. 1991.

142. RH Wassertnan, F W Lcngemann. Further observations of lactose stimulationof the gastrointestinal absorption of calcium and strontium i n rat. J Nutr 70377-844, 1960. 143. AG Poneros, JW Erdman. Bioavailabilityof calcium from tofu, tortillas, nonfat dry milk and mozzarella cheese in rats: effectof supplemental ascorbic acid.J Food Sci 53:208-210, 1988. 1 4 4 . YS Lee, T Noguchi, H Naito. Intestinal absorption of calcium in rats given diets containing casein or amino acid mixture: the roleof casein phosphopcptides. Br J Nutr 49:67-76, 1983. 145. DD Kitts, YV Yuan. T Moriyama. T Nagasawa. Effect of casein, casein phosphopeptides and calcium Intake on ileal "Ca disappearance and temporal systolic blood pressurei n spontaneously hypertensive rats. Br J Nutr 68:765-781, 1992. 146. YV Yuan, DD Kitts. Confirmation of calcium absorption and femoral utilizationin spontaneously hypertensive rats fed casein phosphopeptide supplemcnted diets. Nutr Res 1 1 : 12571272, 1991. 147. YV Yuan, DD Kitts. Effectof dietary calcium intake and protein source on calcium utilization and bone mechanicsin the spontaneously hypertensive rat.J Nutr Biochem 3:452-460, 1992. 148. RP Heaney, CM Weaver. Calcium absorbability from spinach. Am J Clin Nutr 47:707-709. 1990.

149. RP Heaney, RR Rccker, CM Weaver. Absorbability of calcium sources: thc limited role of 1990. solubility. Calcif Tissue lnt 46:300-304, 150. PR Saha, CM Weaver, AC Mason. Mineral bioavailability in rats from intrinsically labeled whole wheat flour if various phytate levels. J Agric Food Chem 42:2531-2535. 1994. 151. HAndersson,BNavcrt,SABingham. HNEnglyst,JHCummings.Theeffectofbreads containing similar amounts of phytate but different amounts of wheat bran on calcium, zinc and iron balance in man. J Nutr 50:503-510, 1993. 152. CM Weaver. Effect of phytate on mineral bioavailability. Symposium Proceedings: Protein and Co-Products Division. Champaign, IL: American Oil Chemists' Society. 153. CM Weaver, RP Heaney. RM Berdine. ML Fitzsimmons. Human calcium absorption from whole wheat products. J Nutr 121 : 1769- 1775, I99 1. 154. A Pointillart, L Gucgucn. Meal feeding and phosphate ingestion influencc calcium bioavailability evaluated bycalciumbalanceandboncbreakingstrength in pigs. Bone Miner 21: 75-8 I , 1993. in persons with mild 155. DA McCarron. CD Morris. Blood pressure rcsponsc to oral calcium to moderate hypertcnsion. A randomised double-blind, placebo controlled, crossover trial. 1985. AnnInternMed103:825-831, i n human hypertension. Science 217: 156. DA McCarron, CD Morris, C Cole. Dietary calcium 267-269. 1982. 157. D Reed, D McGee, K Yano, J Hankin. Diet. blood pressure and multicollinearity. Hypertcnsion7:405-410, 1985. Ugoretz, S Krutzik. Disturbances of calcium metabolism in 158. DA McCarron, NN Yung, BA the spontaneously hypertensive rat. Hypertcnsion 3(suppl I):l162-I 167. 1981. 159. HL Newmark, MJ Wargovich, WR Bruce. Colon cancer and dietary fat, phosphorous and calcium--a hypothesis. J Natl Cancer Inst 72: 1323-1325, 1984. 160. C Garland, RB Shekelle, E Barctt-Comer, M Criqui, AH Rossof, 0 Paul. Dietary vitamin D and calcium and risk of colorectal cancer: a 19-year prospective study i n man. Lancet i: 307-309, 1985. 161. MJ Wargovich, LC Stephens, KN Gray. Effect of two human nutrient density levels of calcium on the promotional phaseof colon tumorigenesis in the F344 rat. Proc Am Assoc Cancer Res 30: 196, 1989.

Nutritional Toxicology

433

of dietary fat promoted colon carcinogenesis in KltS by 162. BC Pence, F Buddingh. Inhibition supplemental calcium or vitamin D3. Carcinogenesis 9: 187- 190, 1988. 163. MT Weis, c Saunders. Magnesium and arachidonic acid metabolism. Magnes Res 6: 179190,1993. 164. BT Altura, M Brust. S Bloom, RL Barbour, JG Stempak, BM Altura. Magnesium dietary intake modulates blood lipid levels and atherogenesis. Proc Natl Acad Sci USA 87:18401844, 1990.

165.

166.

167. 168.

169.

170.

171.

172. 173.

174. 17.5. 176.

177.

178.

179. 180.

181.

182.

BF Dickens,WBWcglicki, YS Li, I Mak.Magnesiumdeficiency in vitroenhancesfree s. Lett 31 1: radical-induced intracellular oxidation and cytotoxicity in endothelial ~ ~ 1 1 FEBS 187-191,1992. RM Forbes, KE Weingartner. HM Parker, J Erdman. Bioavailability to rats of zinc, magnesium and calcium i n casein-, egg-, and soy protein- containing diets.J Nutr 109:1652-1660, 1979. KB Prakash. K Shivakutnar, CC Kartha, K Rathinam.Magnesiumdeficiencyandcerium promote fibrogenesis in rat heart. Bull Environ Contatn Toxicol 57517-524. 1966. J Freubis, TE Carew, W Palinski. Effect of vitamin E on atherogenesis in LDL rcceptordeficient rabbits. Athcrosclerosis 1 17:2 17-224, 1995. A Zhang, TPO Cheng, BM Altura. Magnesium regulates intracellular free ionized calcium concentration and cell geometry in vascular smooth muscle cells. Biochim Biophys Acta 1134125-29.1992. JAM Maiaer. C Malpuech-Btugere, E Rock, Y Rayssiguier, A Mazur. Serum from magncsium-deficient rats affects vascular endothelial cells i n culture: role of hyperlipidemia and inflammation. J Nutr Biochem 9:17-22, 1998. C Luthringer. Y Rayssinguier, E Gueux, A Berthelot. Effect of moderate magnesium deficiency on serum lipids, blood pressure and cardiovascular reactivity in normotensive rats. Br J Nutr 59:243-250, 1988. Y Rayssiguier, E Gueux, D Weiscr. Effect of magnesium deficiency on lipid metabolism in rats fed a high carbohydrate diet. J Nutr 1 1 1: 1876-1883, 1981. MM Mahfouz, TL Smith, FAKumerow.Decreaseduptakeoflowdensitylipoproteinby LLC-PK cells cultured at low magnesium concentration. Magnes Res 5:249-254, 1992. J Freubis, TE Carew, W Palinski. Effect of vitamin E on atherogenesis in LDL receptordeficient rabbits. Atherosclerosis 1 17:217-224, 1995. Y Rayssinguier, E Gucux. Magnesium and lipids in cardiovascular disease. J Am Col1 Nutr 5:507-519.1986. S Tongyai, Y Rayssinguier, C Motta, E Gueux, P Maurois, FW Heaton. Mechanistn of increased erythrocyte membrane fluidity during magnesium deficiency in weanling rats. Am J Physiol257:C270-276,1989. FA Kummcrow, E Wosowicz, T Smith, NL Yoss, J Thiel. Plasma lipid physical properties in swine fed margarine or butter in relation to dietary magnesium intake. J AmCol1Nutr 12:125-132.1993. Y Rayssiguier. E Gueux. The reductionof plasma triglyceride clearance by magnesium deficient rats. Magnes Res 2:132-138, 1983. K Shivakumar. Magnesium deficiency and the cardiovascular system. Curr Sci 68:12211226, 199.5. BF Dickens, WB Weglicki, YS Li, I Mak.Magnesiumdeficiency in vitroenhancesfree radical-induced intracelllular oxidation and cytotoxicityin endothelial cells. FEBS Lett31 1: 187-191,1992. KB Prakash, K Shivakumar, CC Kartha. Magnesium deficiency-related changes in lipid peroxidation and collagen metabolism in vivo in rat heart. Int J Biochem Cell Biol 29:129134, 1997. KB Prakash. K Shivakumar,CCKartha, K Rathinam.Magnesiumdeficiencyandcerium promote fibrogenesis in rat heart. Bull Environ Contarn Toxicol 57:517-524, 1996.

434 183.

184. 185.

186. 187. 188.

189. 190. 191. 192.

193. 194. 195. 196. 197. 198.

199. 200. 201. 202. 203. 204.

205. 206.

207. 208.

Kitts p Laurant, RM Touyz, EL Schiffrin. Effect of magnesium on vascular tone and reactivity in pressurizedmesentericresistancearteriesfromspontaneouslyhypertensiverats.CanJ Physiol Pharmacol 75:293-300, 1997. CF Mills. Dietary interactions involving the trace elements. Annu Rev Nutr 5: 173-193, 1985. WH Crosby. The safety of iron fortified food. JAMA 239:2026-2027, 1978. RBLauffer.Ironstoresandtheinternationalvariationinmortalityfromcoronaryartery disease. MedHypotheses35:96-102,1990. MJ Burt, JW Halliday, LW Powell. Iron and coronary heart disease: iron’s role is undecided. BMT 307575-576, 1993. H McFarlane, S Reddy, KJ Adcock, H Adeshina, AR Cooke, J Akcne. Immunity, transferrin and survival in kwashiokor. Br Med J 4:268-270, 1970. E Graf, JR Mahoney, RG Bryant, JW Eaton. lron catalyzed hydroxyl radical formation. Stringent requirement for free iron coordination site. J Biol Chem 2593620-3626, 1984. A Poppi, B Halliwell. Formation of hydroxyl radicals from hydrogen peroxide in the presence of iron. Biochem J 249:185-190, 1986. J Stocks,M Kemp, TL Donnandy. Increased susceptibility of red blood-cell lipids to autooxidation in haemolytic states. Lancet i:266-269, 1971. DA Adelekan, Dl Thurnham. Plasma ferritin concentrations in anemic children: the relative importance of malaria, riboflavin deficiency and other infections. Am J Clin Nutr 51:453456,1990. A Ascherio. Dietary iron intake and risk of coronary disease among men. Circulation 89: 969-974,1994. CT Semptos, AC Looker, RTF Gillum. Iron and heart disease: the epidemiologic data. Nutr Rev54:73-84,1996. HE Bristow, JJ Strain, RW Welch. lron status and blood lipids in male and female rats. lsr J MedSci 160:140,1991. JL Sullivan. The iron paradigm of ischemic heart disease. Am Heart 1J17:1177- I 187, 1989. JL Sullivan. Stored iron and ischemic heart disease: empirical support for a new paradigm. Circulation 86: 1036-1037, 1992. RA Bradbear, C Bain, V Siskind. et al. Cohort study of internal malignancy in genetic hemochromatosisandchronicnonalcoholicliverdiseases.JNatlCancerInst75:81-84, 1985. A Bomford, R William. Long term results of venesection therapy in idiopathic hemochromatosis. Q J Med 45:611-613, 1976. RG Stevens. Body iron stores and the risk of cancer. N Engl J Med 319:1047-1052, 1988. OHMuth.Symposium:SeleniuminBiomedicine.FirstInternationalSymposiumOregon State University. Wesport, CT: Avi Publishing, 1967. AS Majaj, LL Hopkins. Selenium and kwashiorkor. Lancet 2592, 1966. RF Burk, WN Pearson, RP Wood,F Viteri. Blood Se levels and in vitro red blood cell uptake of Se in Kwashiorkor. Am J Clin Nutr 20:723-727, 1967. E Kauf, H Dawczynski, G Jahreis, E Janitzky, K Winncfcld. Sodium sclenitc therapy and thyroid-hormone status in cystic fibrosis and congenital hypothyroidism. Biol Trace Elem Res 40:247-253, 1994. B Pence, F Buddingh. Effects of dietary selenium deficiency on incidence and size of 1.2 dimethylhydrazine-induced colon tumors in rats. J Nutr 1 15: 1 196-1202, 1985. PL Selvais, C Labuche, NX Ninh, JM Ketelslegers, JF Denef, DM Maiter. Cyclic feeding behavior and changcs in hypothalamic galanin and neuropeptide Y gene expression induced by zinc deficiency in the rat. J Neuroendocrinol 9 5 - 6 2 . 1997. HG Schutz, M Read, R Bendel, VS Bhalla, l Harrill, JE Monagle, ET Sheehan, BR Standal. Food supplement usage in seven western states. Am J Clin Nutr 362397-901, 1982. EJMurray,BLanghaus,HHMesscr.Theeffects of zincandcalciumdeficiencesonthc bone resorption in the rat. Nutr Res 1: 107- 1 15, 1981.

Nutritional Toxicology

435

209. TM Bray, WJ Bettinger. The physiological role of zinc as an antioxidant. Free Radic Bid Med 8:28 1 -29 1, 1990. 210. J Szebeni. CD Eskelson, M Chvapil. The effect of zinc on iron-induced lipid peroxidation in different lipid systems including liposomes and micelles. Physiol Chem PhysMed NMR. 20:205-211,1988. 211. PB Patel, RA Chung, JY Lu. Effect of zinc deficiency on serum and liver cholesterol in the female rat. Nutr Rep Int 12:205-209, 1975. 212. MA Fernandez, BL O’Dell. Effect of zinc deficiency on plasma glutathione in the rat. Proc Soc Exp Biol Med 173564-567. 1983. 213. AA Shaheen, AA Aod El-Fattah. Effect of dietary zinc on lipid peroxidation, glutathione, protein thiol levels and superoxide dismutase activity in rat tissues. Int J Biochem Cell Biol 27:89-95,1995. P Faure, M Roussel, A Favier. Superoxide dismutase 2 14. C Coudray, MJ Richard, F Laprote, activity and zinc status: a study in animals and man. J Nutr Med 3:13-26, 1992. 215. JC Philcox, MH Tilley, P Coyle, AMRofe. Metallothionein and zinc homeostasis during tumor progression. Biol Trace Elem Res 40:295-305, 1994. 216. I Yucel, F Arpaci, A Ozet, B Doner, T Karaylanoglu, A Sayar, 0 Berk. Serum copper and zinc levels and copperlzinc ratio in patients with breast cancer. Biol Trace Elem Res 40: 3 1-38, 1994. 217. MJ Richard, P Guiraud, MT Leccia, JC Beani, A Favier. Effect of zinc supplementation on resistance of cultured human skin fibroblasts towards oxidant stress. Biol Trace Elem Res 371189-199,1993. 218. LS Chubatsu, R Meneghini. Metallothionein protects DNA from oxidative damage. Biochem J2911193-198.1993. increase in the ratio of zinc to 219. LM Klevay. Hypercholesterolemia in ratsproducedbyan copper ingested. Am J Clin Nutr 26:1060-1068, 1973. 220. MK Yadrick, MA Kenney, EA Winterfeldt. Iron, copper and zinc status: responseto supplementation with zinc or zinc and iron i n adult females. Am J Clin Nutr 49:145-150, 1989. 221. MK Song, NF Adham, ME Ament. Levels and distribution of zinc, copper, magnesium and calcium in rats fed different levels of dietary zinc. Biol Trace Elem Res 11:75-88, 1986. 222. JD Bogden, JM Oleske, MA Lavenhar, EM Munves, FW Kemp, KS Bruening, KJ Holding. Effects of one year of supplementation with zinc and other micronutrients on cellular immunity in the elderly. J Am Col1 Nutr 9:214-225, 1990. 223. JR Prohaska. Changes in Cu, Zn-superoxide dismutase, cytochrome c oxidase, glutathione peroxidase and glutathione transferase activities in copper-deficient mice and rats. J Nutr 121:355-363,1990. 224. K Eder. M Kirchgessner. Concentrations of lipids in plasma and lipoproteins and oxidative stability of low-density lipoproteins in zinc-deficient rats fed linseed oil or olive oil. Nutr Biochem8:461-468,1997. 225. T Atsumi, F Numano. Blood zinc levels in patients with atherosclerosis obliterans, thromboangitis obliterans and Takayasn’s disease. Jpn Heart J 16:644-669, 1975. 226. SJ Stohs, D Bagchi. Oxidative mechanisms in the toxicity ofmetal ions. Free Radic Biol Med 18321-336, 1995. 227. JB Nielson. Toxicokinetics of mercuric chloride and methyl chloride in mice. J Toxicol EnvironHealth 3735-122, 1992. 228. HI Popescu, L Negru,I Lancranjan. Chromosomal aberrations induced by occupational exposure to mercury. Arch Environ Health 34:461-463, 1979. 229. SF Ah, CP LeBel, SC Bondy. Reactive oxygen species formation as a biomarker of methyl mercury and trimethytin neurotoxicity. Neurotoxicology 13:637-648, 1992. H 2 0 2formation and oxidative 230. BO Lund, DM Miller. JS Woods. Studies on HgIl induced stress in vivo and in vitro in rat kidney mitochondria. Biochem Pharmacol 45:2017-2024, 1993.

436

Kitts

231. J Albrecht, G Szumauska. R Gadamski, B Gagkowska. Changes in activity and ultrastructural localization of alkaline phosphatase i n cerebral cortical microvessels of rat after singlc intra-

peritoneal administration of mercuric chloride. Neurotoxicology 15:897-902, 1994. 232. G Girardi, MM Elias. Mercuric chloride cffects on rat rcnal redox enzymes activities: SOD

protection. Free Rad Biol Med 18:61-66, 1995. 233. M Yasui, K Ora, RM Garmto. Aluminum decreases the zinc concentration of soft tissues andbonesofratsfed a low calcium-tnagnesiutll diet. Biol Trace ElcmRes 31293-304, 1991. 234. ACAlfrey.Aluminum.AdvClinChem23:69-91.1983. 235. J Fleming, JG Josh. Ferritin isolation of alutninum-ferritin cotnplcx from brain. Proc Natl AcadSeiUSA84:7866-7870,1987. 236. JMC Gutteridge, GJ Quinlan, I Clark, B Halliwell. Aluminium salts accelerate peroxidation of membrane lipids stimulated byiron salts. Biochetn Biophys Acta 835:441-447, 1985. 237. RKatyal.BDesigan,CPalSodhi, S Ojha.Oralaluminumadministrationandoxidative injury. Biol Trace Elem Res 57:125-130, 1997. 238. ES Luis, AH Balagot, AC Villaflor, FC Sanchez, EN Develles. Pb. Cd, and Hg contents of bivalves collected during the different seasons of thc year. Food Chetn 32:239-255, 1989. 239. PW Washko, RJ Counsins. Role of dietary calcium and calcium binding protein in cadmium toxicity in rats. J Nutr107:920-928,1977. 240. K Ravi, VK Paliwal, R Nath. Induction of Cd-tnctallothioneini n cadmium exposed monkeys under different nutritional stresses. Toxicol Lett 22:21-26, 1984. 241. MRS Fox, SHTao, CL Stone,BE Fry. Effect of zinc, iron, and copper dcficicncy on cadmium in tissues of Japanese quails. Environ Health Perspect 5457-65, 1984. 242. DM Dhavalc, VB Masurchar, BA Gridhar. Cadmium induced inhibition of Na-K ATPasc reactivity in tissues of crab Scyllu .serrcm (Forskal). Bull Environ Contam Toxicol 40:759763, 1988. 243. KP Das. PC Das, S Dasgupta, CD Dey. Serotonergic-cholitlergic neurotransmitters function i n brain during cadmium exposure in protein restricted rat. Biol Trace Elem Res 36:119127,1993. 244. N Abd Elghany, MC Schumachcr, ML Slattery. DW West, JS Lee. Occupation. cadmium exposure and prostate cancer. Epidemiology 1: 107-1 15, 1990. 245. GRimback.JPallauf.Cadmiumaccumulation,zincstatusandmineralbioavailabilityof growing rats fed diets htgh in zinc with increasing amount of phytic acid. Biol Trace Elem Res 5759-70, 1997. 246. ML O’Riordan, EG Hughes. HJ Evans, Chromosome studies on blood lymphocytes of men occupationally exposed to cadmium. Mutat Res 58:305-3 I I , 1978. 247. RR Bell, JL Early, VK Nonavinakere, Z Mallory. Effect of cadmium on blood glucose level in the rat. Toxicol Lett 54: 199-205. 1990. in mice in relationto 248. NSugawara. Effect of dietarycadmiumonscrumandliverlipids interaction of csscntial metals, zinc, copper, and iron. Toxicol Sci 9:20-36, 1084. 249. AMV Crespo. MA Reis, MJ Lanca. Effect of selenium supplementation on polyunsaturated fatty acids in rats. Biol Trace Elem Res 47:335-341. 1995. Effect of cadmium on 250. S Gumuslu. P Yargicoglu, A Agar, M Edremitlioglu, Y Aliciguzcl. antioxidant status in alloxanc-induced diabetic rats. Biol Trace Eletn Res 57: 10.5-1 14, 1997. In: Hardness of 251. R Masironi. Cardiovascular diseases in relation to trace element balance. Drinking Water and Public Health. Commission of the European Communities. New York: Pergatnon Press, 1976, pp. 41 1-440. 252. SE Larsson, M Piscator. Effectof cadlniutn on skeletal tissue i n normal and calcium-deficient rats. Isr J Med Sci 7:495-498, 1071. Am J Clin Nutr 45: 1277-1288, 253. JV Joosens, LJ Geboers. Dietary salt and risks to health. 1987. 254. JG Sebranek. DG Olson, RC Whiting, RC Benedict, RC Rust,AA Kraft, JH Woychik. Physi-

Nutritional Toxicology

255. 256. 257. 258.

259. 260.

261.

262.

263.

437

ological role o f dietary sodium inhumanhealthand in~plicationsof sodiumreductionin nlusclc foods. Food Techno1 37:s 1-59, 1983. KA Reddy, EH Marth. Reducing the sodium content of foods: a review. J Food Protect 54: 138-150, 1991. KT Khaw, E Buret[-Connor. Dietary potassium and stroke associated morta~ity.N Engl J Med 3161235-240. 1987. JD Swales. Salt substitutes and potassium intake. Br Med J 303:1084-1085, 1991. RE Hoyt. Hyperkalemia due to salt substitutes. JAMA 256:1726, 19x6. D McCaughan. Hazardsof non-prescription potassium supplements. Lancet 13-5 i:5 14. 1984. B Ardendt, LH Skibsted, HJ Andcrsen. Antioxidant activity of nitrite in metmyoglobin induced lipid peroxidation. Food Res Techno1 204:7-12, 1997. CF Wang. RG Casscns. WC Hoekstra. Fate of ingested "N-labelled nitrate and nitrite in the rat. J Food Sci 46:745-748. 1981. S Odashima. Co-operative progranune on long-term assays for carcinogcnicity in Japan. In: Molecular and Cellular Aspects of Carcinogen Screening Tests. IARC Scientific Publication 27. Lyon: International Agency for Research o n Cancer, 1980, pp. 315-326. M Grecnblatt, S Mirvish. BT So. Nitrosamine studies: induction of lung adenomas by concurrent adnlinistration of sodium nitrite and secondary amines in Swiss mice.J Natl Cancer Inst 46:1029-1034,1971.

264.

A Mackawn, T Ogiu, H Onodera, K Furuta, C Matsuoka,Y Ohno, S Odashima. Carcinogenicity studies of sodium nitrite and sodium nitrate in F-344 rats. Food Chem Toxicol 20:25-

33. 1982. 265. RD Kimbrough. Pathological changes i n human beings acutely poisoned by dimcthylnitrosamine. In: PN Magcc, ed. Banbury Report 12: Nitrosamines and Human Cancer. Cold Spring 1982, pp. 25-36. Harbor, NY: Cold Spring Laboratory Press, of endogenousnitrosation i n humans by 266. HOhshima.HBartsch.Quantitativeestimation monitoring N-nitrosoproline excreted in the urine. Cancer Res 41:3658-3662, 1981. 267. SS Mirvish. A Cardcsa, L Wallcave, P Shubik. Induction of nmuse lung adenomas by anlines or ureas plus nitrite and by N-nitroso compounds: cffect of ascorbate, gallic acid, thiocyanate and cafleinc. J Natl Cancer lnst 55:633-636, 1975. 268. ME Moller. R Dahl, OC Bockman. A possible role of the dietary fibre product wheat bran, as a nitrite scavenger. Food Chcm Toxicol 26:841-845, 1988. 269. HF Stich, H Ohshima. B Pignatclli. J Michelon. H Bartsch. Inhibitory effect ofbetel nut extracts on endogenous nitrosation i n humans. J Natl Cancer lnst 70:1047-1051, 1983. 270. IR Rowlnnd, AK Mallctt. A Wise. E Bailey. Effect of dietary carrageenan and pectin on the reduction of nitro-compounds by the rate caecal microllora. Xenobiotica 1325 1-256, 1983. 271. SJ Whiting.HLWhitney.Effectofdietarycaffeine and theophylline on urinarycalcium excretion i n the adult rat. J Nutr 11731224-1228, 19x7. 272. LK Massey,MSOpryszek.Nocffectofdietarycaffeine on calciumexcretion inyoung women.NutrRes 10:741-747, 1990. 273. P Avogaro, C Capri. M Pais, G Cazzolato. Plasma and urine cortisol behavior and fat mobilization in man after coffee consumption. Isr J Med Sci 9: 114-1 19, 1973. 274. J Blanchard, SJA Sawers, J Jonkman. DS Tang-Lin. Comparison of urinary metabolite profiles in young and cldcrly males. Br J Clin Pharlnacol 19:225-232, 1985. 275. M Bonati, R Latini, F Galetti, JF Young, G Tognoni, S Garrattini. Caffeine disposition after oral doses. Clin Pharmcol Thcr 32:98-106, 1982. 276. MJ Arnaud. The pharmacology of caffeine. Prog Drug Res 31:273-3 13, 1987. 277. DM Grant, BK Tang, ME Campbell. W Kalow. Effect of allopurinol on caffeine disposition in man. Br J Clin Phunnacol 21:454-458, 1986. 278. MJ Amaud. Metabolism of caffeine and other components of coffee. In: S Garratini, ed. Caffeine. Coffee and Health. NewYork:RavenPress, 1993. 279. JW Daly, P Butts-Lamb, W Pagctt. Subclasses of adenosine receptors in the central nervous

438

280.

281. 282. 283. 284.

285.

286. 287. 288. 289. 290. 291. 292.

293.

294. 295. 296. 297. 298. 299. 300.

301. 302.

Kifts system: interaction with caffeine and related methylxanthines. Cell Mol Neurobiol 1:67-80, 1983. J Moraidis, D Bingmann, A Lehmenkuehler, EJ Soeckmann. Caffeine-induced epileptic discharges in CA3 neurons of hippocampal slices of the guinea pig. Neurosci Lett 12951-54, 1991. Y Tanakea, M Sakurai, M Goto, S Hayashi. Effect of xanthine derivatives on hippocampal long-term potentiation. Brain Res 522:63-68, 1990. SP Tofovic, KR Branch. RD Oliver, WD Magee, EK Jackson. Caffeine potentiates vasodilator-induced renin release. J Pharmacol Exp Ther 256:850-860, 1991. L Whisett, CV Manion, HD Christensen. Cardiovascular effects of coffee and caffeine. Am J Cardiol 53:918-922, 1984. JL IZZO,A Ghosal, T Kwong, RB Freeman, JR Jaenike. Age and prior caffeine use alter the cardiovascular and adrenomedullary responses to oral caffeine. Am J Cardiol 52:769-773, 1983. HR Superko, W Bortz, PT Williams, JJ Albers, PD Wood. Caffeinated and decaffeinated a concoffee effects on plasma lipoprotein cholesterol, apolipoproteins and lipase activity: trolled, randomized trial. Am J Clin Nutr 54599-605, 1991. RE Freid, DM Levine, PO Kwiterovich, EL Diamond, LB Wilder, TF Moy, TA Pearson. The effect of filtered-coffee consumption on plasma lipid levels. JAMA 267231 1-815, 1992. DS Thelle, E Arnesen, OH Forde. The Tromso heart study. Does coffeee raise serum cholesterol’? N Engl J Med 308: 1454-1457, 1983. D Robertson, D Wade, R Workman, RL Woosley. Tolerance to the humoral and hemodynamic effects of caffeine in man. J Clin Invest 67: 111 1-1 1 17, 1981. M van Dusseldorp, P Smits, T Thien, MB Katan. Effect of decaffeinated versus regular coffee on blood pressure. A 12 week, double-blind trial. Hypertension 14563-569, 1989. AL Klasky, DB Petitti, MA Armstrong, CD Friedman. Coffee, tea and cholesterol. Am J Cardiol 55577-578, 1985. SM Haffner, JA Knapp, MP Stem, HP Hazuda, M Rosenthal, LJ Franco. Coffee consumption, diet and lipids. Am J Epidemiol 122: 1- 12, 1985. DJ Naismith,PAAkinyanju, S Szanto, J Yudkin.Theeffectinvolunteersof coffee and decaffeinated coffee on blood glucose, insulin, plasma lipids and some factors involved in blood clotting. Nutr Metab 12:144, 1970. S Heyden, HA Tyroler, G Heiss, CC Hames, A Bartel. Coffee consumption and mortality (total mortality, stroke mortality and coronary heart disease mortality). Arch Intern Med 138: 1472-1475,1978. J1 Mann, M Thorogood. Coffee-drinking and myocardial infarction. Lancet ii:1215, 1975. B Stavric. An update on research with coffeelcaffeine. Food Chem Toxicol 30533-555. 1992. R Fears. The hypercholesterolaemic effect of caffeine in rats fed on diets with and without supplementary cholesterol. Br J Nutr 39:363-374, 1978. HU Aeschbacher. Mutagenesis of coffee. Trends Pharmacol Res 5512-513, 1984. CA Blair, T. Shibamoto. Ames mutagenicity tests of overheated brewed coffee. Food Chem Toxicol 22:971-975, 1984. AN Wijcwickreme, DD Kitts. Modulationof metal-induced cytotoxicity by Maillard reaction products isolated from coffee brew. J Toxicol Environ Health 55:15 1-168, 1998. JA Little, HM Shanoff, A Csima, R Yano. Coffee and serum lipids in coronary heart disease. Lancet i:732-734, 1966. M van Dusseldorp, MB Katan, PNM Demacker. Effect of decaffeinated versus regular coffee on serum lipoproteins. A 12 week double blind trial. Am J Epidemiol 132:33-40, 1990. AN Wijewickreme, DD Kitts, TD Durance. Reaction conditions influence the elementary composition and mctal chelating affinity of nondialyzabel model Maillard rcaction products. J Agric Food Chem 45:4577-4583, 1997.

Nutritional Toxicology

439

coffee beans and coffee 303. JM McKenzie. Content of phytate and minerals in instant coffee, beverage. Nutr Rep Int 29387-395, 304. PW Harvey, KGD Allen. Decreased plasma 1ecithin:cholestcrol acyltransferase activity in copper-deficient rats. J Nutr 11*1:1855-1858, 1981. 305. PL Zock, MB Katan, MP Mekus, M van Dusscldrop, JL Harryvan. Effect of alipid-rich fraction from boiled coffee on serum cholesterol. Lancet 335:1235-1237, 1990. 306. MPME Weustcn-Van der Wouw, MB Katan, R Viani, AC Huggett, R Liardon, PG LundLarsen, DS Thelle, I Ahola, A Aro, S Meyhoom, AC Beynen. Identity of the cholesterolraising factor from boiled coffee and its effects on liver function enzymes. J Lipid Res 35: 721-733,1994. 307. WMN Ratnayake, R Hollywood, E O’Grady, B Stavric. Lipid content and composition of coffee hrews prcpared by different methods. Food Chem Toxicol 31:263-269, 1993. 308. A Aro, P Pietincn, U Uusitalo, J Tuomilehto. Coffee and tea consumption, dietary fat intake and scrum cholesterol conccntrations in Finnish men and women. J Intern Med 226:127132,1989. of a coffee extract. J Toxicol Environ Health 309. DD Kitts. Studies on the estrogenic activity 20:37-49,1986. 3 IO. HU Aeschbachcr, C Chappuis. Non-mutagenicity of urine from coffee drinkers compared with that from cigarette smokers. Mutat Res 89:161-168, 1981. 311. HUAeschbacher,HPWurzner.AnevaluationofinstantandregularcoffeeintheAmes mutagenicity test. Toxicol Lett 5 : 139-145, 1980. 312. BS Shane. AM Troxclair, DJ McMillin,CB Henry. Comparative mutagenicityof nine brands of coffee to Sdrrwnello typhirrrur.iurn TA 100, TA 102 and TA 104. Environ Mol Mutagen 1 1: 195-206, 1992. 313. Y Sasaki, T Shibamoto, C1 Wei, S Fernando. Biological and chemical studies on overheated brewed coffee. Food Chem Toxicol 25:225-228, 1987. 3 14. R Stalder, A Bexter, WP Wurzner, H Luginhuhl. A carcinogenicity study of instant coffee in swiss mice. Food Chem Toxicol 285329437, 1990. 315. X Shi, NS Dalal, AC Jain. Antioxidant behaviour of caffeine: efficient scavenging of hydroxyl radicals. Food Chern Toxicol 29: 1-6, 1991. 316. LA Rosenburg, AA Mitchell, S Shapiro. D Slone. Selected birth defects in relation to caffeine-containing beverages. JAMA 247: 1429- 1432, 1982. effects 317. B Watkinson, PA Fried. Maternal caffeine use before, during and after pregnancy and upon offspring. Neurobehav Toxicol Teratol 7:9-17, 1985. 318. DD Kitts, K Shckhtman. Chronic dose dependent effects of caffeine in pregnant rats. Can Instit Food Sci Techno1 20:273-278, 1987. 319. A Aldridge, J Bailcy, AH Ncims. The disposition of caffeine during and after pregnancy. SeminPerinatol 5310-314, 1981. 320. SP Govindwar, MS Kachole, SS Pawar. In vivo and in utcro effects of caffeine on hepatic mixed-function oxidases in rodents and chickens. Food Chem Toxicol 22371-375, 1984. 321. TR Tephly, GJ Mannering. Inhibition of drug metabolism. V. Inhibition of drug metabolism by steroids. Mol Pharmacol 4:lO-14, 1968. 322. H Nishimura, K Nakai. Congenital malformations in offspring of mice treated with caffeine. ProcSocExpBiolMed104:140-142,1960. 323. T Fujii, H Nishimura. Reduction in frequency of fetopathic effects of caffeine in mice by pretreatment with propranolol. Teratology 10: 149- 152, 1974. 324. MMoriguchi,WJ Scott. Preventionofcaffeine-inducedlimbmalformations bymaternal adrenalectomy. Teratology 3 3 3 19-322, 1986. 325. PE Schneider, HI Miller, T Nakamoto. Effectsof caffeine intake during gestation and lactation on bones of young growing rats. Res Exp Med 190:131-136, 1990. 326. MMA Elmazar, P McElhatton. F Sullivan. Studics on the teratogenic effects of different oral preparations of caffeine in mice. Toxicology 2357-71, 1982.

Kifts

440

327. I Fortier, S Marcoux, L Beaulac-Baillargeon. Relation of caffeine intake during pregnancy to intrauterine growth retardation and premature birth.Am J Epiderniol 137:93 1-940, 1993. 328. B Larroque, M Karninski, L Lelong, D Subtil, P Dehaenc. Effects on hirthweight of alcohol and caffeine consumption. Am J Epidemiol 137:941-950, 1993. 329.DPurves, FM Sullivan.Reproductiveeffects of caffeinc.Experimentalstudiesinanimals. In: S Garratini, ed. Caffeine, Coffee and Health. NewYork:RavenPress.1993. pp. 317342. 330. A Nehlig, G Derbry. Potential teratogenic and neurodevelopmental consequences of coffce and caffeine exposure. A review on human and animal data. Neurotoxicol Teratol 16531543. 1994. 331. SM Tarka, RA Applebaum, JF Borzclleca. Evaluation ofthe teratogenic potential of cocoa powder and thobromine in New Zealand white rabbits. Food Chcm Toxicol 24:363-374, 1986. 332. SM Tarka, RS Applebaum, JF Borzelleca. Evaluation of the perinatal, postnatal and tcratoin Sprague-DawleyKD rats. Food Chem genic effects of cocoa powder and theobromine Toxicol 24375-382. 1986. 333. H Tanaka, K Nakazawa. Maternal caffeine ingestion increases the tyrosine level in neonatal cerebrum. Biol Neonate 57:133-139, 1990. 334. R Guillet. C Kellogg. Neonatal exposure to therapeutic caffeine alters the ontogenyof adenosine AI receptors in brain of rats. Neuropharmacology 30:489-496, 1990. 335. DS Groissier, P Rosso, M Winick. Coffee consumption during pregnancy: subsequent behavioral abnormalities of the offspring. J Nutr 112:829-832, 1983. 336. JG Haddad. Vitamin D-solar rays and the milk way, or both. N Engl J Med 18: 1213-1215, 1992.

337.JMoon,BBandy, AJ Davidson.Hypothesis:etiology of atherosclerosisandosteoporosis: Are imbalances in the calciferol endocrine system implicated'? J Am Col1 Nutr 11:567-583, 1992. 338. JJB Anderson, SU Tovcrund.DietandvitaminD: a reviewwith an emphasisonhuman function. J Nutr Biochem 5 3 - 6 5 . 1994. 339. C Ryan, P Eleazer, J Egbert. Vitamin D in the elderly. Nutr Today 30:228-232, 1995. 340. MJ Beclunan, RL Reinhardt, DC Beitz. Up regulation of the intestinal 1.25 dihydroxyvitamin D receptor during hypervitaminosis D: a comparison between vitamin D? and vitamin D l . Biochem Biophys Res Commun 169:9 10-9 14, 1990. 341. RW Chesney. Vitamin D: Can an upper limit be defined? J Nutr 1 19: 1825-1828, 1989. T Klorume. Hypervitaminosis D after 342. Y Nako, N Fukushima, T Tomomasa, K Nagashima, prolonged feeding with a premature formula. Pediatrics 928624364, 1993. 343. RB Mazes, HS Barden, C Christiansen, AB Harper, WS Laughlin. Bone mineral and vitamin D in Aleutian Islanders. Am J Clin Nutr 42: 143-146, 1985. 344. HB Taussig.Possibleinjury to the cardiovascularsystemforvitamin D. Ann J Mcd 65: 1195-1200,1966. 345. WF Friedman. Vitamin D as a cause of the supervascular aortic stenosis syndrome. Am Heart J73:718-726,1967. 346. R Scragg, R Jackson, IM Holdaway, T Lim, R Bcagleholc. Myocardial infarction is inversely associated with plasma 25-hydroxyvitamin D, levels: a community-based study. I n t J Epidemiol19559-563.1990. Q J Med 347.DSGrimes,EHindle,TDyer.Sunlight,cholesterolandcoronaryheartdisease. 89:579-589,1996. 348. RScragg, K-T Khaw, S Murphy.Lifestylefactorsassociatedwithwinterscrum 25hydroxyvitamin D levcls in elderly adults. Age Ageing 24:271-275, 1995. 349. R Scragg, K-T Khaw, S Murphy. Effect of winter oral vitamin D , supplementation on cardiovascular risk factors in elderly adults. Eur J Clin Nutr 49:640-646, 1995. 350. R Danielsen, H Sigvalason, G Thorgeirsson, N Sigfussion. Predominance of aortic calcifica-

Nutritional Toxicology

44 1

tion a s an atherosclerotic manifestation i n women: the Reykjavik study. J Clin Epidenliol 49~383-387, 1996. 351. TG Hines. NL Jackobson, DC Beitz, T Littledike. Dietary calcium and vitamin D: risk factors in the development of atherosclerosis in young goats. J Nutr 115: 167-178, 1985. a renlintlcr.Arch 352. MLBKO,MMLiberman,MSalzmann.ChronicvitaminDoverdosage: Dis Child 66: 100, 1991. EW 353. CH Jacobus. MF Holick, Q Shao, TC Chcn. IA Holm, JM Kolodny. GEH Fuleihan, Seely. Hypervitaminosis D associated with drinking milk. N Engl J Med 326: 1 137-1 177, 1990. 354. MF Holick, Q Shao, WW Liu, TC Chen. The vitamin D content of fortified milk and infant formula. N Engl J Mcd 326: I 178- 1 18 1. 1992. 355. TC Chen. Q Shao, H Health, MF Holick. An update on the vitamin D content of fortified 1993. milk from the United States and Canada. N Engl J Med 329: 1507, 356. R Victh. SW Kooh. T Murray, J Houpt. Vitamin D supplementation. Can Med Assoc J 149: 396-397,1993. a- and P-tocopherols in bulk 357. SW Huang. EN Frankcl. JB German. Antioxidant activity of and oils and in oil-in water emulsions. J Agric Food Chcln 42:2108-2114, 1994. 358. AN Wijewlckreme, DD Kitts. Oxidative reactions of model Maillard reaction products and a-tocopherol in a flour-lipid mixture. J Food Sci 63:466-47 l , 1998. i n enhancement of cholesterol-induced athcroscle359. J Thiery. D Seidel. Fish oil feeding results rosis i n rabbits. Atherosclerosis 63:53-56, 1987. 360. N Chupukcharocn, R Komaratat, P Wilalrat. Effects of vitamin E deficiency on the distribution of cholesterol in plasma cholesterol in plasma lipoproteins and the activity of cholesterol 7a-hydroxylase i n rabbit liver. J Nutr 115:468-472, 1985. E have differential efl'ccts 361. HW Chen, CK Lii, CC Ou, ML Wang. Dietary fat and vitamin on serum lipid Icvcls. Nutr Res 15:1367-1376. 1995. 362. SV Subrahnxmyam. M Prasad. J Kalra. K Prasad. Antioxidant enzymes in hypcrcholcstcrolernia and effects of vitamin E i n rabbits. Atherosclerosis 101:135-144. 1993. 363. HMG Princcn. G van Poppcl. C Vogelezang, R Buytanhek. FJ Kok. Supplementation with vitamin E but not beta carotene in vivo protects low density lipoprotein for lipid peroxidation in vitro. Arterioscler Thromb Vasc Biol 12554-562, 1993. 364. 1 Jialnl, SM Grundy. Effectof dietary supplementation with alpha-tocopherol on the oxidative 33:899-906, 1992. modification of low density lipoproteins, J Lipid Res 365. DW Morel,MDe La Llcra-Moya, KE Friday.Treatment of cholesterol-fedrabbitswith dietary vitanlin E andC inhibits lipoprotein oxidation but not developmentofatherosclerosis. JNutr 1242123-2130. 1984. 366. J Ncuzil, BA Dnrlow. TE Indcr. KB Sluis. Oxidation o f pxenteral lipid cnwlsion by ambient and phototherapy lights: potential toxicity of routine parenteral feeding. J Pediatr 126:785790.1995. 367. PK Whitting. VW Bowry, R Stocker. lnvcrsc deuterium kinetic isotope effect for peroxidation in human low-density lipoprotein(LDL):a simple test for tocophcrol-mediated peroxidation in LDL lipids. FEBS Lett 375:45-49. 1995. 368. M Iwatsuki, E Niki, D Stone,VM Darley-Usmar. a-tocopherol rnediatcd peroxidation in the copper (11) and mctmyoglobin induced oxidation of humanlow density lipoprotein: the inlluencc of lipid hydroperoxides. FEBS Lett 360:271-276, 1995. 369. B Holliwell. Oxidation of low-density lipoproteins: questions of initiation. propagation, and the effect of antioxidants. Am J Clin Nutr 61:6703-677S. 1995. 370. B Halliwell. How to characterize a biological antioxidant. Free Rad Res Commun 9: 1-32. 1990. 371. MMaiorino,AZamburlini,ARoveri, F Ursini.Prooxidant role of vitaminE in copper induced lipid peroxidation. FEBS Lett 330: 174-176. 1993. 372. ENiki.Antioxidantconqxxmds. In: KJA Davics,cd.OxidativeDamageandRepair:

442

Kitts

Chemical,BiologicalandMedicalAspects. NewYork:PergamonPress,1991,pp.5764. 373. M Cohen, HN Bhagavan. Ascorbic acid and gastrointestinal cancer. J Am Col1 Nutr 14:565578, 1995. 374. DA McCarron, CD Morris, HJ Henry, JL Stanton. Blood pressure and nutrient intake in the United States. Science 224:1392-1398, 1984. 375. JP Moran, L Cohen, JM Greene, X Guifa, EBFeldman, CC Hamcs, DS Feldman. Plasma ascorbic acid concentrations relate inversely to blood pressurei n human subjects. Am J Clin Nutr 57:213-217, 1993. 376. R Delport, JB Ubbink, JA Human, PJ Beckcr, DP Myburgh, WJ Hayward Vcrmaak. Antioxidant vitamins and coronary heart disease risk in South African males. Clin Chim Acta 278: 55-60,1998. 377. PA Glascott, MTsyganskaya,EGilfor,MAZern, JL Farber.Theantioxidantfunctionof the physiological content of vitamin C. Mol Pharmacol 50:994-999, 1996. 378. I Jialal, SM Grundy. Preservation of the endogenous antioxidants in low density lipoprotein by ascorbate but not probucol during oxidative modification. J Clin Invest 87597-601, 1991. 379. K Kunert, AL Tappel. The effect of vitamin C on in vivo lipid peroxidation in guinea pigs as measured by pentane and ethane production. Lipids 18:271-274, 1983. 380. BFrei,LEngland, BN Ames.Ascorbateis an outstandingantioxidant in humanblood plasma. Proc Natl Acad Sci USA 86:6377-6381, 1989. 381. KL Retsky,MWFreeman, B Frci.Ascorbicacidoxidationproduct(s)protecthumanlow density lipoprotein against atherogenic modification. Anti- rather than prooxidant activity of vitamin C in the presence of transition ions. J Biol Chem 268:1304-1309, 1993. of 382.KLRetsky,BFrei.VitaminCpreventsmetalion-depcndentinitiationandpropagation lipid peroxidation in human low density lipoprotein. Biochim Biophys Acta 1257:279-287, 1995. 383. E Cameron, L Pauling. Supplemental ascorbatei n the supportive treatment of cancer: reevaluation of prolongation of survival times in terminal cancer patients. Proc Natl Acad Sci USA 75:4538-4542,1978. 384. A Samumi, J Aronovitch, D Godinger, M Chevion, G Czapski. On thc cytotoxicity of vitamin D and metal ions. A site-specific Fenton mechanism. Eur J Biochem 137: 119-124, 1983. 385. V Herber. Viewpoint: Does mega-C do more good than harm or more harm than good? Nutr Today28:28-30,1993. 386. HFStich,JKarim, J Koropatnick,LLo.Mutagenicaction of ascorbicacid.Nature260: 722-724,1976. 387. MP Rosen, RHC San. HF Stich. Mutagenic activity of ascorbate in mammalian cell cultures. Cancer Lett 8:299-305, 1080. 388. GS Ranhotra, PM Keagy. Adding folic acid to cereal-grain products. Cereal Food World 40: 73-76.1995. 389. S Daly, JL Mills, AM Molloy, M Conley, YJ Lee, PN Kirke, DC Weir. JM Scott. Minimum cffective dose of folic acid for food fortification to prevent neural-tube defects. Lancet 350: 1666-1669,1997. 390. LB Bailey.Folateintakerecommendationsfrom a nutritionalscienccperspective.Cereal Food World 40:63-66, 1995. Nature 195:1062391. BF Ncsbitt, J O’Kelly, A Sheridan. Toxic metabolite ofA.s/~e,.Xi/lfts~~ffkl,ft.s. 1063,1962. 392. LL Zaika, RL Buchanan. Review of compounds affecting the biosynthesis and bioregulation of aflatoxins. J Food Protect 50:691-708, 1978. 393. DL Park, B Liang. Perspectives on aflatoxin control of human food and animal feed. Trends Food Sci Techno1 4365-381, 1993. 394. RL Buchanan, DC Hoover, DB Jones. Caffeine inhibition of aflatoxin production: mode of action. Appl Environ Microbiol 46: I 193-1 200, 1983.

Nutritional Toxicology

443

395. RL Buchanan, RS Applebaum, P Conway. Effect of theobromine on growth and aflatoxin production by Aspergillus porrrsiticus. J Food Safety 1:211-216, 1978. 396. JM Cullen, PM Newbeme. Acute hepatotoxicity of aflatoxins. In: DL Eaton, JG Groopman, pp. 3-26. eds. The Toxicology of Aflatoxins. New York: Academic Press, 1994, in the content of the multistage nature of 397. YP Dragan, HC Pitot. Aflatoxin carcinogenesis cancer. In: DL Eaton, JG Groopman, eds. The Toxicology of Aflatoxins. New York: AcademicPress,1994,pp.179-206. 398. CJ Holeski, DL Eaton, DH Monroe, GM Bellamy. Effect of phenobarbitol on the biliary excretion of aflatoxin PI-glucorunide and aflatoxin Bl-S-glutathione in the rat. Xenobiotica 17:139-153,1987. 399. RA Coulombe, RP Sharma. Clearance and excretion of intratracheal and oral administration of aflatoxin B1 in the rat. Food Chem Toxicol 23:827-830, 1985. 400. CD Groopman, LG Cain, TW Kensler. Aflatoxin exposure in human populations: measurements and relationship to cancer. CRC Clin Rev Toxicol 19:113-145, 1988. 401. JP Whitty, LF Bjeldanes. The effectsof dietary cabbage on xenobiotic metabolizing enzymes and the binding of aflatoxin B1 to hepatic DNA in rats. Food Chem Toxicol 25:581-587, 1987. 402. DH Phillips. Fifty years of benzo(a)pyrene. Nature 303:468-472, 1983. 403. AW Wood, RL Chang, W Levin, RE Lehr, M Schaeffer-Ridder, JM Karle, DM Jerina, AH Conney. Mutagenicity and cytotoxicity of benzo(a)anthracene diol epoxides and tetrahydro epoxides: exceptional activity of the bay region 1,2 epoxides. Proc Natl Acad Sci USA 74: 2746-2750,1977. 404. 0 Plekonen, DW Nebert. Metabolism of polycyclic aromatic hydrocarbons: etiologic role in carcinogenesis. Pharmacol Rev 34: 189-222, 1982. 405. BR Smith, WR Brian. The role of metabolism in chemical-induced pulmonary toxicity. Toxicol Pathol 19:470-48 I , 199l. 406. DA Wiersma, RA Roth. Clearance of benzo(a)pyrene by isolated rat liver and lung: alterations in perfusion and metabolic capacity. J Pharmacol Exp Ther 225: 121-125, 1983. 407. RA Roth, A Vinegar. Actionby the lungs on circulating xenobiotic agents, witha case study of physiologically based pharmacokinetic modeling of benzo(a)pyrene disposition. Pharmacol Ther 48:143-155, 1990. 408. SG Bowes, AG Renwick. The intestinal metabolism and DNA binding ofbenzo(a)pyrenein guinea-pigs fed normal, high-fat and high cholesterol diets. Xenobiotica 16:543-552, 1986. 409. P Lesca. Protective effects of ellagic acid and other plant phenols on benzo(a)pyrene-induced neoplasia in mice. Carcinogenesis 4: 1651-1653, 1983. 410. SK Katiyar,RAganval,MTZaim, H Mukhtar.ProtectionagainstN-nitrosodiethylamine and benzo(a)pyrene induced forestomach and lung tumorigenesis in A/J mice by green tea. Carcinogenesis14:849-855,1993. 41 1. T Sigimura. Carcinogenicity of mutagenic heterocyclic amines formed during the cooking process.MutatRes 15033-41, 1980. 412. JS Felton, MC Knize. Occurrence, identification and bacterial mutagenicity of heterocyclic amines in cooked food. Mutagen Res 259205-218, 1991. 413. T Sigimura, S Sato. Mutagen-carcinogen in food. Cancer Res 43:S2415-S2421, 1983. 414. T Sigimura, K Wakabayashi. Mutagens and carcinogens in the diet. In: MW Pariza, JS Felton. HU Aeschbacaher, S Sato, ed. Mutagens and Carcinogens in the Diet. New York: WileyLiss,1990,pp. 1-18. 415. H Ohgaki, K Kusama, N Matsukura, K Morino, H Hasegawa, S Sato, S Takayama, T Sugimura. Carcinogenicity in mice of a mutagenic compound, 2-amin0-3-methylimidazo[4,5-f] quinoline from broiled sardine, cooked beef and beef extract. Carcinogenesis 5921-924, 1984. 416. S Takayama, Y Nakatsuru, M Masuda, S Sato, T Sugimura. Demonstration of carcinogenicity in F344 ratsof IQ from broiled sardine, fried beef and beef extract. Gann 75:460-470, 1984.

Kitts

444

417. RS Adamson. UP Thorgeirsson. EG Snyderwine, SS Thorgeirrson. J Reeves. DW Dalgard. S Takayama. T Sugimura. Carcinogenicity of 3-amino-3-n~ethyli1nida~o[4,5-f]quinoline in nonhuman primates: induction of tunwurs in three macques. Jpn J Cancer Res 81:10-14, 1990. 418. JK Lin, JT Cheng. SY Lin-Shiau. Enhancement ofthemutagenicityofIQandMeIQby nitrite in Salmonella system. Mutat Res 278977-287, 1992. 419. C Sasagawa,M Muramatsu. T Matsushima. Formationof direct mutagens from amino-imidazoazaarcnes by nitrite treatment. Mutat Res 203386, 1988. 420. T Matsushima, T Sugimura. Mutagen-carcinogens i n amino acid and protein pyrolysates and in cooked food. Prog Mutat Res 2:49-56. 198 1. 421. RH Adamson, UP Thorgeirsson. Carcinogens in foods: heterocyclic amines and cancer and 369:211-220, 1995. heart disease. Adv Exp Mcd Biol 422. JW Gaubatz, S Rooks.Dietary-linked DNAdamagein cardiaccells.FASEBJ10:A967. 1995.

423.

424.

425.

426.

427. 428. 429.

430.

431.

432.

433. 434.

435. 436.

437.

EOvcrvik,MOchiani.MHirose,TSugimura,MNagao.Theformation of heartDNA adducts in F344 rats following dietary administrationof heterocyclic amines. Mutat Res256: 37-43, 1991. CD Davis, HAJ Schut, EG Snyderwine. Enzymatic phase I1 activation of the N-hydroxyl14: amines of IQ, MeIQx and PhIP by various organs of monkeys and rats. Carcinogenesis 207 1-2096, 1993. AR Boobis, J Segura, NJ Gooderham, DS Davies. CYPIA2-catalyzed conversion of dietary heterocyclic amines to their proximal carcinogens is their major route of metabolism in humans. Cancer Res 54:89-94. 1994. HX Zu, HAJ Shutt. Inhibition of 2-a~nino-31nethylin~idazo[4,5-f]quinoline-DNA adduct formation in CDF, mice by heat altered derivatives of linoleic acid. Food Chem Toxicol 30: 9-16, 1992. AJ Alldrick, J Flynn,IR Rowland. Effects of plant-derived flavonoids and polyphenolic acids on the activity of mutagens from cooked food. Mutat Res 163:225-232. 1986. L Busk, UG Ahlborg. L Albanus. Inhibition of protein hydrolysate mutagenicity by retinol (vitamin A). Food Chern Toxicol 20:535-539. 1982. P Lindeskog, E Overnik, L Nilsson. C-E Nord, J-A Gustafsson. Influence of fried meat and fibre on cytochrome P-450 mediated activity and excretmn of mutagens in rats. Mutat Res 202:553-563, 1988. DW Layton. KT Bogen. MC Knize. FT Hatch, VM Johnson, JS Fclton. Cancer risk of heterocyclic amines in cooked foods: an analysis and implications for research. Carcinogenesis 16: 39-52. 1995. Y Zhang. TW Kenslcr,CC Cho, CH Posner, P Talalay. Anticarcinogenic activities of sulforaphane and structurally related synthetic norbornyl isothiocyanatcs. Proc Natl Acad Sci USA 91:3147-3150. 1994. M Jang, L Cai.GO Udeani, KV Slowing, CF Thomas, CWW Beechcr. HH Fong.NR Farnsworth, AD Kinghorn, RG Mehta, RC Moon, JM Pczzuto. Cancer chemopreventive activity of resveratrol, a natural product derived from grapes. Science 275218-220, 1997. EN Frcnkel, AL Waterhous. JE IOnsella. Inhibition of human LDL oxidatlon by resvcratrol. Lancet 34111 10.3-1 104, 1993. MTHuang,TLysz. T Ferraro, AF Abitli, JD Laskin, AH Conney.Inhibitoryeffects of curcumin on in vitro lipooxygcnase and cyclooxygenase activities in mouse epidermis. Cancer Res 51:813-819, 1991. K Polasa, B Sesikaran,TP Krishna. K Krishnaswamy. Turmeric-induced reduction in urinary 1991. mutagens. Food Chem Toxicol 29699-706, AC Reddy, BR Lokesh. Effect of dietary turmeric on iron-induced lipid peroxidation in the rat liver. Food Chcm Toxicol 32279-283, 1994. CJM Rompelberg. SJC Evertz, GCDM BruijnlJesrozier. PD Van Den Heuvel, H Vcrhagen.

Nutritional Toxicology

438.

439.

440.

441.

442.

443.

445

Effect of eugenol on the genotoxicity of established mutagens in the liver. Food Chem Toxicol34:33-42,1996. CJM Rompelberg. M-J ST Steenwinkel, JG Van Asten, JHM Van Dalft, RABaan, H Verhagen. Effect of eugenol on the mutagenicityof benzo(a)pyrene and the formationof benzo(a)pyrene-DNA adducts i n the -1acZ-transgenic mouse. MutatRes369:89-96, 1996. TH March, EH Jeffery, MA Wallig. The cruciferous nitrile. crambene, induces rat hepatic and pancreatic glutathione-S-transferase. Toxicol Sci 42:82-90, 1998. Y Li, EJ Wang, L Chen, AP Stein. KR Rcuhl, CS Yang. Effects of phenethyl isothiocyanate on acetaminophen metabolism and hepatotoxicity. Toxicol Appl Pharnmol 144:306-314, 1997. S Larsen-Su, DE Williams. Dietary indole-3-carbinol inhibits F M 0 activity and the expression of flavin-containing nmnooxygenase form 1 i n rat liver and intestine. Drug Metab Dispos 24:927-93 l , 1996. YL Ha, NK Grimm, MW Pariza. Newly recognised anticarcinogenic fatty acids: identification and quantification i n natural and processed cheese. J Agric Food Chem 73:75-81, 1989. C Ip. JA Scinleca. Conjugated linoleic acid and linoleic acid arc distinctive modulators of manunary carcinogenesis. Nutr Cancer 27:13 1-1 35, 1997.

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15 Food Additives

I. Introduction 448 11. Functions of FoodAdditives 448 Preservation A. 448 448 Processing B. C. Appeal and convenience 449 Nutrition D. 449 111. Food Additive Categories 451 N . FoodAdditiveSupplyIndustry454 V.ResearchandDevelopment455

VI. Manufacturing 458 VII. Government Regulations 459 United A.States 459 B. European Union (EU) 463 C. Japan 466 VIII.Trends and Issues 466 1X. Description of MajorFoodAdditives 468 Sweeteners A. 468 B. Thickeners and stabilizers 474 C. Colors 480 substitutes Fat D. 485 Enzymes E. 489 Vitamins 490 F. G . Antioxidants 495 H. Preservatives 499 I. Emulsifiers 502 J. Flavors 504 X. AdverseEffectsofFoodAdditives508 A. Food additives banned from use 509 Industrial B. chemicals 510 C. Foodallergiesandotheradversefoodreactionstofoodadditives D. Food additives derived from allergenic food 515 Bibliography 5 15

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1.

INTRODUCTION

The broadest definition of a food additive is any substance that becomes part of a food product, either directly or indirectly, during some phase of processing, storage, or packaging. The universe of food additives encompasses Direct food additives, those that are intentionally added to food for a functional purpose, in controlled amounts, usually at low levels (from parts per million to 1-2%, by weight), and Indirect or incidental food additives, those entering into food products in small quantities as a result of growing, processing, or packaging. The difference between food ingredients and additives is mainly in the quantity used in as food (e.g., sucrose), any given formulation. Food ingredients can be consumed alone while food additives areused in small quantities (usually less than 2%) relative to the total of desirable and food compositionbut which nonetheless playa large part in the production safe food products. as minor ingredients incorporated into foods Food additives may be looked upon to affect their properties in some desired way. Most commonly, the effects desired relate to color, flavor, texture, nutritive value, or stability in storage. There is no rigorous definition that meets all needs. The Codex Alime~mrius,which dominates actions in international circles, considers as a food by itself and normally an additive as an ingredient “not normally consumed by the used as a typical ingredient.” This obviously leaves great latitude for judgment committee. The U.S. Food, Drug, and Cosmetic Act has a complex definition of food additives that comes closeto any component of food introduced into U.S. commerce after 1957 and it will be addressed in the section dealing with government regulations.

11.

FUNCTIONS OF FOOD ADDITIVES

Direct food additives serve several major functions. Many additives, in fact, are multifunctional (Table 1). The basic functions of direct food additives include the following:

A.

Preservation

Food preservation techniques have advanced in the past 100 years and now include thermal processing,concentrationanddrying,refrigerationandfreezing,modifiedatmosphere, and irradiation. However, the use of chemical preservatives frequently augments these basic preservation techniques and represents the most economical way for food manufacturers to ensure a reasonable shelf life for their product. Antioxidants and antimicrobial agents perform some of these functions as well.

B. Processing Food processors are increasingly using food additives to ensure the integrity and appeal of their finished products. Emulsifiers maintain mixtures and improve texture in breads, dressings, and other foods. They are used in ice cream when snloothness is desired, in breads to increase shelf life and volume and to distribute the shortening, and in cake mixes

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Table 1 FoodAdditivesandTheirFunctions

Food additive Anticalilng agents Antioxidants

Appeal Preservation improvement modification Nutrition

Process X

X

Colors

Emulsifiers Enzymes Fat substitutes Flavors Humectants Leavening agents agents pH control Preservativcs Processing aids Sweeteners (sugars only) Sweeteners. high intensity Thickeners stabilizers and Vitamins and minerals

X X

X X X X X

X X X

X X X X

X X X X

X X X

X X

X X X

to achieve batter consistency. Stabilizers and thickeners assist in presenting an appealing product with consistent texture. Sorbitol, a humectant and sweetener, is used to retain moisture and enhance flavor. With the removal of sugar from many foods for dietetic reasons, a substitute bulking agent is needed.

C. Appeal and Convenience The changing eating habits of consumers, partly brought about by the large increase in the number of women who work outside the home, is creating a growing need for convenience foods. In many of these types of foods, it is essential that a variety of additives be used to provide the taste, color, texture, body, and general acceptability that are required. This need for convenience, while maintaining aesthetic appeal and taste, is becoming extremely important. Most food additives such as gums, flavoring agents, colorants, and sweeteners are includedby food processors because consumers demandthat food look and taste good as well as be easy to serve.

D. Nutrition There have been tremendous advancesin the knowledge of human nutrition, and consurners are increasingly awareof the value of good nutrition. Vitamins, antioxidants, proteins, and minerals are added to foods and beverages as supplements in an attempt to ensure proper nutrition for those whodo not eat a well-balanced diet.In addition, additives suchas antioxidants are often used to prevent deterioration of natural nutrients during processing. Recently more importance has been attributed to disease prevention through proper nutrition, as well as to increasing performance through sport nutrition products. On the other hand, the desire for good nutrition througha balanced diet may adversely affect consumer demand for some food additives such as fat substitutes.

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Adopted from the National Academy of Sciences/National Research Council national survey of food industries, the following ternls describe the physical and technical effects of various food additives: Anticaking agent orfree-flow agent: substance added to finely powdered or crystalline food products to prevent caking, lumping, or agglomeration. Antimicrobial agent: substance used to preserve food that prevents the growth of microorganisms and subsequent spoilage, including fungistats, mold and rope inhibitors, antimicrobial agents, antimyotic agents, preservatives, and mold preventing agents (indirect additives). Antioxidant: substance used to preserve food by retarding deterioration, rancidity, or discoloration due to oxidation. Boiler water additive: substance used in a steam or boiler water system as an anticorrosion agent to prevent scale or to effect steam purity. to preserve or enhance the color or Color or coloring adjunct: substance used shading of a food including color fixatives and color-retention agents. a unique flavor and/or color to Curing or pickling agent: substance imparting food, usually producing an increase in shelf-life. Dough strengthener: substance usedto modify starch and gluten, thereby producing more stable dough. Dryingagent:substancewithmoisture-absorbingabilityusedtomaintainan environment of low moisture. Emulsifier or emulsifier salt: substance which modifies surface tension in the component phase of an emulsion to establish a uniform dispersion or emulsion. Enzyme: Used to improve food processing and the quality of finished food. Firming agent: substance addedto precipitate residual pectin, thus strengthening the supporting tissue and preventing its collapse during processing. Flavor enhancer: substance added to supplement, enhance, or modify the taste and/or aroma of a food without imparting a characteristic taste or aroma of its own. Flavoring agent or adjuvant: substance added to impart or help impart a taste or aroma in food. Flour treating agent: substance added to milled flour to improve its color and/ or baking qualities, including bleaching and maturing agents. Formulation aid: substance used to promote or to produce a desired physical state or texture in food, including carriers, binders, fillers, plasticizers, film-formers, and tableting aids, etc. Freezing or cooling agent: substance that reduces the temperatureof food materials through direct contact. Fumigant: volatile substance used for controlling insects and pests. Humectant: hygroscopic substance incorporated in food to promote retention of moisture. Leavening agent: substance used to produce or stimulate production of carbon dioxide in baked goods in order to impart a light texture, including yeast, yeast foods, and calcium salts. Lubricant or release agent: substance added to food contact surfaces to prevent ingredients and finished products from sticking to them (direct additives), including release agents, lubricants, surface lubricants, waxes, and antiblocking agents (indirect additives).

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Malting or fermenting aid: substance used to control the rate or nature of the malting or fermenting process, including microbial nutrients and suppressants and excluding acids and alkalis. Masticatory substance: substance that is responsible for the long-lasting and pliable property of chewing gum. Nonnutritive sweetener: substance having less than 2% of the caloric value of sucrose per equivalent unit of sweetener. Nutrient supplement: substance necessary for the body’s nutritional and metabolic processes. Nutritive sweetener: substance having greater than 2% sucrose per equivalent unit of sweetening capacity. Oxidizing or reducing agent: substance which chemically oxidizes or reduces another food ingredient, thereby producing a more stable product. pH control agent: substance addedto change or maintain active acidity or basicity, including buffers, acids, alkalis, and neutralizing agents. to enhance the appeal Processing aid: substances used as a manufacturing aid or utility of a food or component, including clarifiers, clouding agents, catalysts, flocculents, filter aids, crystallization inhibitors, etc. Propellant: gas used to supply force to expel a product or to reduce the amount of oxygen in contact with the food in packaging. Sequesterant: substance which combines with polyvalent metal ions to form a soluble metal complex to improve the quality and stability of products. Solvent or vehicle: substance used to extract or dissolve another substance. Stabilizer or thickener: substance used to produce viscous solutions or dispersions, impart body, improve consistency, or stabilize emulsions, including suspending and bodying agents, setting agents, and bulking agents. Surface-active agent: substance used to modify surface properties of liquid food components for a variety of effects, other than emulsifiers. Includes solubilizing agents, dispersants, detergents, wetting agents, rehydrating enhancers, foaming agents, defoaming agents, etc. Surface finishing agents: substance used to increase palatability, preserve gloss, and inhibit discoloration of foods, including glazes, polishes, waxes, and protective coatings. Synergist: substance used to act or react with another food ingredientto produce a total effect different from or greater than the sum of the effects produced by the individual ingredients. Texturizer: substance which affects the appearance or feel of the food. Tracer: substance added as a food constituent (as requiredby regulation) SO that levels of this constituent can be detected after subsequent processing and/or combination with other food materials. Washing or surface removal agent: substance used to wash or assist in the removal of unwanted surface layers from plant or animal tissues.

111.

FOODADDITIVECATEGORIES

Substances that come under the general definition of direct food additive number in the thousands and include

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Inorganic chemicals (e.g., phosphates, sulfites, calcium chloride, etc.) Syntheticorganicchemicals(e.g.,dyes,benzoates,aromachemicals,vitamin A, etc.) Extraction products from and derivatives of natural sources (e.g., pectin, essential oils, vitamin E, etc.) Fermentation-derived products (e.g., enzymes, citric acid, xanthan gum, etc.) Most food additives have a long history of use; others are the result of recent research and development to fill particularrequirements of modcrnfoodprocessing.Someare common chemicals of industry that are upgraded in t e r m of purity to allow their use i n food. Major categories of foodadditivesincludepreservatives,colorants,antioxidants, flavors, thickeners and stabilizers, emulsifiers, acidifiers and buffers, enzymes, and sweeteners. Examples of major products in each category are shown in Table 2. Within this same category, products may belong to several chemical classes and offer specialized functionality (e.g., water- and oil-soluble antioxidants that include ascorbic acid and hindered phenols, respectively, and water-soluble azo dyes and water-dispersible carotenoids as food colors). Basic foodstuffs are excluded from the definition, although ingredients added to foods (e.g., high fructose corn syrup, MSG, and protein concentrates) are often included among food additives. Certain food additives, such as colors, flavors, gums, emulsifiers, and preservatives may find use also in pharmaceutical products and in toiletries and cosmetics (e.g., toothpaste, lipstick, etc). The same Food Chemical Codex (FCC) grade as in food is typically used in these applications, however, the combined value of the additive for these other applications does not exceed 10% of food use. Indirect food additives have no purposeful function in food and may be divided into the following categories: Components of adhesives (e.g., calcium ethyl acetoacetate 1,4-butanediol modified with adipic acid) Components of coatings (e.g., acrylate ester copolymer coatings and polyvinyl fluoride resins) Components of paper and paperboard (e.g., slimicides, sodium nitratehrea complex, and alkyl ketone dimers) Basic components of single- and repeated-use food contact surfaces (e.g., cellophane,ethylene-acrylicacidcopolymers,isobutylenecopolymers,andnylon resins) Components of articles intended for repeated use (e.g., ultrafiltration menlbrancs and textiles and textile fibers) Compounds controlling growth of microorganisms (e.g., sanitizing solutions) Antioxidants and stabilizers (e.g., octyltin stabilizers in vinyl chloride plastics) Certain adjuvants and production aids (e.g., animal glue, hydrogenated castor oil, synthetic fatty alcohols, and petrolatum)

as contaminants. In the United In many countries, these materials are defined and regulated States, these materials are food additives under the law. They are commonly classed as indirect food additives, but the FDA handles them in the same way as direct additives. Just as with direct additives, they may be generally recognized as safe (GRAS) substances and thereby escape explicit regulation because that status makes them, in fact, not food

453

Food Additives Table 2 SelectedMajorFoodAdditives

Thickeners and stabilizers Agar Alginates Carageenan Carboxymethyl ccllulose (CMC) Casein Gelatin Gellan gum Guar gum Gum Arabic Locust bean gum Modified starches Pectin Xanthan gum

Sweeteners Acesulfame-K Aspartame Dextrose Lactitol Mannitol Sorbitol Saccharin Xylitol Colors Certified food colors Dyes Lakes Noncertified colors Caramel Plant extracts Synthetic carotenoids Fat substitutes Partially or nonmetabolizable Sucrose polyester (Olestra) Caprenin Fat tnimetics Carbohydrate based products Protein based products Emulsifiers Flavors Aroma chemicals Vanillin Essential oils/natural extracts Menthol Flavor compositions Strawberry flavor Enzymes Amylases (alpha-amylase, etc.) Glucose isomerase Pectinases Proteases Rennin

Vitamins VitaminA Vitamin A acetate Vitamin B I Thiamin hydrochloride Vitamin B. Thiamin mononitrate Vitamin B,, Pyridoxtne hydrochloride VitaminB Cyanocobalamin Vitamin C Ascorbic acid VitaminD Ergocalciferol, cholecalciferol VitaminK Menadione Antioxidants Ascorbic acidlsodium ascorbate Erythorbic acidlsodium erythorbate BHA (butylated hydroxyquinone) BHT (butylated hydroxytoluene) PG (propyl gallate) TBHQ (tert-butyl hydroquinone) Tocopherols Sulfur dioxide/sulfite salts Preservatives Benzoic acidlbenzoates Propionic acidlpropionates Parabenes Sorbic acidlsorbates Sulfites Emulsifiers Mono- and diglycerides Lactylated esters Lecithin Polysorbates Propylene glycol esters Sorbitan esters Sucrose csters Anticaking agents Aluminum calcium silicate Calcium silicate Salts o f fatty acids (stearates) Silicon dioxide Tricalcium silicate Yellow prussiate of soda pH control agents Citric acid Malic acid Phosphoric acidlphosphatcs Sodium citrate Sodium hydroxide

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additives. In general, however, regulation of these materials is more extensive and more rigorous in the United States than in other counties. As might be expected, packaging materials which have been used for a long time, such as glass, receive less close scrutiny than more newly introduced materials and those materials just being proposed for introduction.

W. FOOD ADDITIVESUPPLY INDUSTRY Food additive suppliers are an important part of the food manufacturing system, supplying products to both commodity processors and food processors (Figure 1). Practically every food manufacturing operation depends to some degree on the use of food additives, but the range of additives necessary for the formulation varies (Table 3). Overall, the food additive industry appears to be highly fragmented, consisting of more than 500 companies supplying a variety of chemically and functionally different products that serve a common end-use market-the food industry. However, suppliers tend to beeitherhighlyspecializedparticipantsinthemajorproductcategories(e.g., Novo with enzymes, Warner-Jenkinson Company with certified food colors, etc.) or large chemical companiesthat offer food-grade versionsof a few industrial products (e.g., Lonza's emulsifiers, FMC Corp.'s cellulose derivatives). Manufacturers are typically involved in supplying additives in a limited number of product categories (e.g., colors, vitamins, or enzymes) or servicing selected food sectors (e.g., processed meats, dairy-based products, or bakery products). While a company or group of companies may tend to dominate sales in each of the specific categories (e.g., Novo with enzymes, Rhodia with vanillin, or NutraSweet/KelcoCo. with aspartame and biogums), no single company or small group of companies dominates the entire food additive industry. Forty years ago it was relatively easy and lucrative for chemical companies to stumble into the role of food additive supplier and reap profits by upgrading the purity and quality of chemicals originally developed for other industrial markets. Today, however, the long time and high costs associated with gaining regulatory approval (estimated 510 years and $1.5-$40 million) have taken away the incentive to commercialize products

Fig. 1 Integrated view of the U.S. food manufacturing system.

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from basic research. As a result, food additives represent only a minor portion of large chemical companies’ overall business. Most large chemical companies that supply food additives, such as FMC, Monsanto, Hercules, Lonza, Hoffman-La Roche, Huls, Rh6nePoulenc, and Eastman Chemicals, have diversified chemical operations, with perhaps only 5% or less of total sales generated by food additives. Figure 2 depicts the food additive industry structure and the flow of its products. Some 60-70% of food additives are used in the manufacture of food; about 20-30% are usedin commodity processing operations such as flourmilling,meatpacking,oilseed crushing and refining, vegetable packaging, animal feeds, and fruit juice processing; and the remaining 5-10% are used for things such as pharmaceuticals and cosmetics.In addition to basic additive producers, the food additives industry includes companies that specialize in compounding of specialty product mixes, and national and local distributors (Figure 2). Specialty compounders formulate mixed products for the food industry such as dairy ingredients, baker’s mixes, curing blends, thickener and emulsifier blends, cheese aids, ethnic flavors, total seasoning packages, and spice blends. They are generally very knowledgeable about additive and ingredient properties and are experiencedin food technology overall. Compounding companies are often relatively small, sell directly to a food processor, are highly service oriented, and market product lines that have a high level of perceived differentiation. Their formulations offer convenience and enjoy higher gross profit margins than single food additive sales. Distributors also playan important rolein the distribution of food additives. Additive producers typically use distributors to service their smaller accounts or for warehousing and servicing of accounts that geographically the producers cannot cover effectively or economically.

V.

RESEARCH AND DEVELOPMENT

Because there are so many dissimilar and unconnected segments of the food additives industry, the participating companies exhibit different approaches to research and development (R&D). Many stress applications research to uncover new niches for existing additives or modifications of currently FDA-approved additives. Some emphasize innovative research or new, high-value products, but these are very few because of the cost and time for basic research, development, regulatory approval, and market acceptance of a new food additive product. For example, NutraSweet’s aspartame product took more than 11 years to gain FDA approval; acesulfame K took 6 years for FDA approval and a total of 21 years since development. The total costs of research, development, and approvals for aspartame were close to $25 million. Procter & Gamble’s OlestraTM was in research and development for 20 years, yet in developwasn’t submittedto the FDA until June 1987. After spending over $200 million ment costs and waiting more than 8 years for FDA approval, the fat substitute received approval in January 1996. Approval of thefoodadditive islimited to snacks such as potato chips and tortilla chips. Moreover, it has a special regulatory constraint: OlestraTMcontaining products require fortification with vitamins A, D, E, and K to compensate for the limited absorption of these fat-soluble vitamins, and the products must be labeled with the statement, “This product contains Olestra. Olestra may cause abdominal cramping and loose stools. Olestra inhibits the absorption of some vitamins and other nutrients. Vitamins A, D, E, and K have been added.”

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COMMODITY ADDITIVES PROCESSORS Sugar refining Grain milling 9 Oil seed processing Fruit and vegetables processing

4

FOOD MANUFACTURERS Acidulanls Emulsiliers Flavors, colors, etc. High-Intensity sweeteners

510%

OTHER USES * Pharmaceutical

Arumal feed, etc.

60-7m DISTRIBUTORS BLENDERS

REFINED INGREDIENTS

A

FOOD MANUFACTURERS

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(hmedhzen food *sMckfood

Daily products Bread. c m k i e s , etc.

I

FaSt-lood meals Hospital f w d etc. meals, Airiine

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L

Fig. 2 Food additives, pattern of use in food, and other applications.

In general, only large, well-financed companies can afford the

R&D efforts neces-

sary to bring a new food additive product to market. Small companies simply are unable

to deal with the complexity, costs, and required time. Personnel staffing requirements for R&D of food additives vary significantly. Because statistics for the food additives business of most producing companies are not reported separately, only estimates can be made. R&D expenditures as a percentage of sales typically range from 1% or less for products such as preservatives, to 5-6% for more technically oriented products such as fat substitutes and certain natural colors, and 5-1076 for flavors.

VI.

MANUFACTURING

Manufacturing processes for food additives vary widely in their nature and technological sophistication. Some of the specific processes for the more important food additives are described in later sections of this report. A common characteristic of all food additives manufacturing, however, is that the products must be made to a high degree of purity and under sanitary conditions similarto those of food processing plants. Production equipment must be dedicated to food additive products and cannot be used forother industrial production. Plants producing food additives are subject to periodic inspection by the regulatory agencies. Typically chemical additives made by synthesis (e.g., BHT, saccharin) or by fermentation(e.g.,aspartame,microbialenzymes,xanthanandgellangums)require a high level of capital investment. The former additives have industrial uses and are likely to share their basic production costs with the industrial-grade material; a small portion of

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the total production is then upgraded and purified to food-grade quality in separate dedicated plant units. Accurate long-term market forecasts for products are essential in order to minimize the risk associated with capital investment decisions in single-purpose plants. Other participants in the food additives business that are not involved in chemical production on a large scale for the extraction and purification of natural products, small-scale synthesis of aromatic chemicals, and for flavor and ingredient compounding have much lower capital requirements.

VII. A.

GOVERNMENT REGULATIONS

United States

The application of food additives is highly regulated worldwide, although regulatory philosophy, the approval of specific products, and the level of enforcement differ from country to country. Basic regulations in the United States, Western Europe, and Japan are described below. These three major industrial regions are the largest consumers of food additives. With only 13% of the world’s population, these countries account for more than twothirds of the food additive market. The U.S. Food and DrugAdministration(FDA) istheprincipal U.S. regulatory body controlling the use of food additives. It does so through the 1958 Food Additives Amendment to the Food, Drug & Cosmetic (FD&C) Act of 1938. The amendment was enacted with the threefold purpose of 1.

2. 3.

Protecting public health by requiring proof of safety before a substance can be added to food. Advancingfoodtechnology. Improving the food supply by permitting the use of substances in food that are safe at the levels of intended use.

According to the legal definition, food additives that are subject to the amendment include “any substance the intended useof which results or may reasonably be expected to result directly or indirectly in its becoming a component or otherwise affecting the characteristics of any food.” This definition includes any substance used in the production, processing, treatment, packaging, transportation, or storage of food. If a substance is added to a food for a specific purpose it is referred to as a direct additive. For example, the low-calorie sweetener aspartame, which is used in beverages, puddings, yogurt, chewing gum, and other foods, is considered a direct additive. Indirect food additives are those that become part of the food in trace amounts due to its packaging, storage, or other handling. This class includes all materials that would not usually become part of food if man could completely control food production. In practice, indirect food additives are found in agricultural produce in quantities well within acceptable and legal tolerances. For example, minute amounts of packaging substances may find their way into foods during storage. A variety of chemicals, including plastic monomers,plasticizers,stabilizers,printingink,andothersubstances,migrate at extremely low levels into foods. Lead andtin are perhaps the main concerns associated with packaging materials. The storage of acidic foods in inappropriate containers can result in the leaching of toxic heavy metals, such as zinc and copper, into the food. Food packaging manufacturers therefore must prove to the FDA that all materials coming in contact with food are safe before they are permitted for use in such a manner.

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A wide variety of chemicals are used in modern agricultural practice. Residues of these chemicals can linger in raw and processed foods, although federal regulatory agencies evaluate the safety of such chemicals, and regulate and monitor their use on food products. The major categories of agricultural chemicals include insecticides, herbicides, fungicides. fertilizers, and veterinary drugs, including antibiotics. Industrial and/or environmental pollutants may migrate into foods i n small amounts. On rare occasions, hazardous levelsof polychlorinated biphenyls (PBCs) and polybrominated biphenyls (PBBs) have been found in foods. For regulatory purposes, all food additives fall into one of three categories: Generally recognized as safe (GRAS) substances Prior sanctioned substances Regulated direct/indirect additives. GRAS substances (approximately 700 compounds) are a group of additives regarded by qualified experts as “generally recognized as safe.” These substances are considered safe because their past extensive use has not shown any harmful effects. Prior sanctioned substances (approximately 1400 compounds) are products that were already in use in foods prior to the 1958 Food Additives Amendment and are therefore considered exempt from the approval process. Some prior sanctioned substances also appear on the GRAS list. This is the grandfather clause of the amendment. The FDA is involved in an ongoing review of the GRAS and prior sanctioned substance lists to ensure thatthesesubstances are tested by means of thelatestscientific methods. Likewise, the FDA also reviews substances that are not currently included on the GRAS list to determine whether they should be added. All other additives are regulated-that is, a specific food additive petition must be filedwiththe FDA requesting approval for use of theadditive in anyapplicationnot previously approved. A food or color additive petition must provide convincing evidence that the proposed additives perform as intended. Animal studies using large doses of the to show that the substance will not cause additive for long periods are often necessary harmful effects at expected levels of human consumption. In deciding whether an additive should be approved, the agency considers the composition and properties of the substance, the amount likely to be consumed, its probable long-term effects, and various other safety factors. Absolute safety of any substance can never be proven. Therefore the FDA must determine if theadditiveissafeunderthe proposed conditions of use, based on the best scientific knowledge available. I n addition, the FDA operates an Adverse Reaction Monitoring System (ARMS) to help serve as an ongoing safety checkof all additives. The system monitors and investigatesall complaints by individuals or their physicians that are believed to be related to specific foods, food additives, or nutrient supplements. Color additives for food represent a unique and special category of food additives. They have historicallybeen so considered in legislation and regulation. The current legislation governing the regulation and use of color additives in the United States is the Food, Drug & Cosmetic Act of 1938, as a mended by the Color Additive Amendment of 1960. Colors permitted foruse in foods are classified eitheras certified or exempt from certification.Certifiedcolorsare man-made, with each batch being tested bythe manufacturer and the FDA (certified) to ensure that they meet strict specifications for purity. Color additives that are exempt from certification include pigments derived from natural sources. However, color additives exempt from certification also must meet certain

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legalcriteriaforspecificationsandpurity.One of thefeatures of theColorAdditive Amendment of 1960 was the equal treatmentof synthetic colors and the so-called natural colors in respect to pretesting requirements. Moreover, currently all color additives, certified and uncertified (“natural”), are designated on the label as artificial color. Flavor substances are regulated somewhat differently, and the rules are less restrictive. However, the use of aroma chemicals as flavor ingredients is regulated under laws that may differ from country to country. Following the lead of the United States, inclusion on a positive list that spells out which chemicals are permitted for food use has become the prevalent legislation for regulating flavor chemicals worldwide. The United States has a list of flavor substances that are deemed GRAS based on the history of use, review of of experts. These GRAS lists (through GRAS 18) available toxicology, and the opinion have been compiled since 1977 by the expert panel of the Flavor Extracts Manufacturers Association of the United States (FEMA). Over the years, more than 1800 materials appeared on FEMA lists. Formed in 1909. FEMA is an industry association that originally started pursuing voluntary self-regulation and later was granted quasi-official status on regulatory matters regarding flavor chemicals by the FDA. The FEMA expert panel was formed in1960. Thisindependentpanel,composed of eminentlyqualifiedexpertsrecruitedfromoutsidetheflavorindustry,hasexpertiseinhumannutrition,physiology, metabolism,toxicology,andchemicalstructure-activityrelationships.Mostindustrial countries more or less follow the U.S. system. Although the FDA has primary jurisdiction over food additives, clearance for use of additives in certain products must be obtained from other government agencies as well. For example, the U.S. Department of Agriculture (USDA) through the Meat Inspection Division (MID) exercises jurisdiction over additives and ingredients for meat and poultry; the Bureau of Alcohol, Tobacco, and Firearms (BATF) of the U.S. Department of the Treasury controls the ingredients used in alcoholic beverages. The standards of identity specify in detail what can and cannot be packaged under a given product name. Standards of identity exist for milk, cream, cheese, frozen dessert. bologna products, cereal products, cereal flours, pasta, canned and frozen fruits and vegetables, juices, eggs, fish,nuts, nonalcoholic beverages, margarine, sweeteners, dressings, and flavorings. An approved food additivein the United States may be precluded from use in certain foods characterized by the standards of identity unless the additive is specifically required by or is listed as an optional ingredient in the standards. The standardsof identity establish the ingredient composition of a given food, which can then be labeled by its common name. If the manufacturer does not adhere to the standard composition, the food must be labeled “imitation.” The Federal Insecticide, Fungicide and Rodenticide Act (FIFRA), which was issued in 1972 and amended in 1988, covers pesticides used on raw agricultural products. The FDA, however, is responsible for enforcing tolerances for pesticide residues that end up in food products. In the United States, label disclosure of food additives is mandated with few exceptions. Under FDA, USDA, and BATF regulations, the ingredients of a food or beverage must be stated on the product label i n decreasing order of predominance. For many direct additive categories, chemical constituents must be identified by their common names and the purpose for which they were added. One of the recent regulations involving the food industry, as well as food additive manufacturers, came with the passing of the Nutrition Labeling and Education Act of 1990 (NLEA), which anlends the Federal Food, Drug & Cosmetic Act, to make nutrition

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labeling mandatory for most FDA-regulated foods. The nutrition labeling regulations issued by the FDA and the USDA Food Safety and Inspection Service (FSIS) required compliance by August 8, 1994. The FDA’s nutrition labeling regulations focus on nutrients currently accepted as significantly affecting consumer health. The serving size is the basis for reporting each food’s nutrient content. Serving sizes are defined for most foods reflecting the amount people actually eat and are shown in both common household and metric measures. The amount per serving of the following nutrients are required to be included on labels: total calories, calories from total fat, total fat, saturated fat, cholesterol, total carbohydrates, complex carbohydrates, sugars, dietary fiber, protein, sodium, vitamin A, vitamin C, calcium, and iron. Listing other essential vitamins and minerals such as thiamin, riboflavin, and niacin, among other nutrients, is optional. A simplified nutrition label format is allowed for foods containinginsignificant amounts of more than half the required nutrients. The minimum label includes total calories, total fat, total carbohydrates, protein, and sodium. The FDAregulation requires the nutrition content be based on amounts of the product customarily consumed, and expressedin both common household and metric measures (e.g., 1 cup and 240 ml). Serving size reference amounts are based on food consumption survey data on amounts of food commonly consumed per eating occasion by persons 4 years of age and older. Manufacturers must follow the procedures to convert the reference amounts to serving sizes appropriate for their specific products. Any package containing less than two servings is considered a single-serving container. Nine terms are presently allowed by the FDA to describe a food, including free, low, high, source of, reduced, light/lite, less (or, for calories, fewer), more, and fresh. Claims for cholesterol are tied to levels of saturated fat in the food. Meal-type products are not allowed to use the term reduced. Health claims are allowed for only the following nutrient/disease relationships: a a a a

a a

a a a a

a

Calcium and osteoporosis Sodium and hypertension Unsaturated fats, low cholesterol intake, and cardiovascular disease Dietary lipids and cancer Fiber-containing grain products, fruits and vegetables, and cancer Fruits, vegetables, and grain products that contain fiber and risk of coronary heart disease Dietary fiber from fruits and vegetables and cancer Folic acid and neural tube defect Sugar alcohols and tooth decay Psyllium-containing foods and the riskof heart disease (when consumed as part of a diet low in saturated fat and cholesterol Soy protein and reduced risk of coronary heart disease (FDA proposal as of January 1999, not finalized yet)

Changing dietary recommendations and labeling requirements impact food additive producers both positively and negatively. Products used for fat-sparing/substitution (e.g., hydrocolloids, starches, other fat substitutes) and low-calorie sweeteners fare well, as food manufacturers striveto lower the caloric and saturated fat content of their products. Natural colors (provided they can be substituted), as well as other natural or seemingly natural

Food Additives

463

products will also be in higher demand so as to provide consumers with a more healthy and nutritious product (or at least one with a more healthy-sounding label). The Food Additives Amendmentalso contains what is knownas the Delaney clause, which mandatesthe FDA toban any food additive found to cause cancer in manor animals, regardless of dose level or intended use. The clause applies not only to new food additives but also to those in use prior to 1958. The Delaney clause is totally inflexible in that it does not recognize any threshold level below which the additive might not present a health hazard. Thus it has caused a number of problems for the food industry and for food additives. Certain additives (e.g., the sweetener cyclamate, etc.) have been banned after they were found to be potential carcinogens-even though feeding tests in animals at massive dose levels may not bear any correlation to the potential risk to man of chronic ingestion at very low levels. Were it not for a moratorium mandated by Congress, saccharin would also have been banned in the United States several years ago by the FDA in compliance with current U.S. food laws. Although congressional sentiment has been running for some time in favor of repealing the Delaney clause, to date, attempts to replace it with a more practical and realistic law have been unsuccessful.

1. Approval Process A new substance gains approval for fooduse through the successful submission of a food additive petition that must document the following: Safety, including chronic feeding studies in two species of animals. Intended use. Efficiency data at specific levels in the specified food system. Manufacturingdetailsandproductspecifications. Methods for analysis of the substance in food. Environmentalimpactstatement. Quite frequently, this process can be lengthy-up to I O years in the case of aspartame and OlestraTM-and costly in terms of man-hours and dollars. There is little doubt that every level of the U.S. food additives business is affected by regulations, and operates with a constant awarenessof the importanceof FDA decisions.Not only is the introduction of a new food additive impossible without FDA approval, but the additives in use are under constant scrutiny by the regulatory agency and remain vulnerable to new unfavorable toxicology findings. While the barring of an additive may create opportunities for suppliers to develop new or substitute materials, the potential market is often too small loss of the ingredient may cause havoc within afto create sufficient incentive, and the fected sectors of the food industry. For example, the ban on cyclamates, followed by the close call on saccharin, almost caused the demise of the diet soft-drink industry. The wellrecognized need for alternative safe sweeteners undoubtedly was a stimulus for G . D. to engage in a IO-year effort to have Searle (now Monsanto’s NutraSweet Kelco division) aspartame cleared for food use.

B.

European Union (EU)

Food additives intended for human consumption are regulated by the member states as described in Directive 89/107/EEC of December 21, 1988. The EU food additive law recognizes 106 food additives. Later, several amendments and adaptationsof the directive were introduced or proposed including

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A list of additives the use of which is authorized to the exclusion of all others. The list of foodstuffstowhichtheseadditivesmaybeadded,theconditions under which they may be added and, where appropriate, a limit on the purpose of their use. The rules on additives used as carrier substances and solvents, including their purity criteria. In 1990 the commission proposed a first specific directive relating to sweeteners and food additives other than colors and sweeteners. The Sweetener Directive took effect on July 30, 1994. Member countries were asked to adopt the directive by December 3 1, 1995, which did not occur. The sweetener guidelines are expected to open up new markets for low-calorie food products and will simplify logistical matters. Efforts have been toward a uniform registration process so that a registration obtained in one country would be valid in all EU member countries. The new EU food additive law, however, will not prevent individual countries from asking for additional or country-specific requirements for new product registrations. At the EU level, several institutions and groups are involved in the development of food additives law, illcluding of the institutions of the European Commisthe Scientific Committee for Food (SCF), one sion which deals with safety issues, representatives from different national professional organizations, representatives from the food industry, retailers, etc. The Standing Committee on Foodstuffs ensures close cooperation between the commission and the member states. and labeling of novel food such as The EU rules for theevaluation,marketing, genetically modified foods are also being developed. The new nlarketing rules wouldalso oblige manufacturers to obtain permission before placing new foods or ingredientson the market, with the exception of products that are substantially equivalent to existing foods. The new rules have still to be cleared by the European Parliament, which has the power to veto under the new co-decision procedure introduced in 1995. In many countries, additives must be declared in the labeling. Within the EU, some additive groups have been uniformly codified with “E” numbers for the orientation of consumers. Some countries, suchas Germany, have gone further, adopting regulations on an acceptable daily intake (ADI) basis that build on the latest toxicological knowledge. Some examples of “E” numbers are presented in Table 4. Under EU food law, any claim that a food has the property of preventing, treating, or curing a human disease or condition, or any implication of such properties, is prohibited. This aspect of the law has been strictly enforced in all member states of the EU. As early as 1980, the European Commission recognized that the area of food claims required harmonization and circulated the first proposal for a directive. By the end of 1998, this approach had not succeeded. Recently the introduction of genetically modified (GM) corn and soy into Europe has caused considerable activity within governments and consumer organizations. European Parliament and Council Regulation no. 258/97 on novel foods and novel food ingredients requires prior approval of foods and food ingredients containing or consisting of a GM organism, and food and food ingredients produced from, but not containing GM organisms. More recently, Council Regulation1 139/98 came into force, requiring that any product containing GM soy or corn, or derivatives of GM soy or corn containing protein or DNA, must be labeled with the statement “produced from genetically modified soy” or

465

Food Additives Table 4

Selected EUFoodAdditivcsandCodes

Colorants El00 El01 El02 El 10 E120 E150 El60 E 160a El 60c E162 E163 Preservatives

-

Curcumin Riboflavin Tartrazin Yellow no. 6 Carmin Caralnels Annatto Beta-carotene Paprika Beetroot red (betanin, betanidin) Enocyanin (grape-skin cxtract)

Sorbic acid Sodium sorbate E20 1 Potassium sorbate E202 Calcium sorbate E203 Benzoic acid E210 Sodium benzoate E21 1 Potassium benzoate E212 Sodium propionate E28 1 Potassium propionate E282 Calcium propionate E283 Antioxidants L-ascorbic acid (vitamin C) E300 Synthetic alpha-tocopherol (vitamin E) E307 Propyl gallate (PG) E31 1 Butylhydroxyanisole (BHA) E320 Thickcners and stabilizers alginate Sodium E40 l E415 E420 E440a Emulsifiers E322 E47 1 Mono- diglycerides and of fatty acids Polyglycerol E475 acids fatty esters of Sodium steroyl-2 lactilate E48 I E200

"produced from genetically modified maize." However, refined oils or lecithin that are very unlikely to contain GM protein or DNA are exempt from such labeling statement requirements. A further labeling change that comes into force in February 2000 is the quantitative declaration of ingredients (QUID). This applies to foods and beverages with more than oneingredient,withvery fewexceptions. The quantity of ingredients,expressed as a percentage of the food or drink, must appear in or immediately next to the name of the food or i n the list of ingredients next to the ingredient concerned.

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C. Japan In Japan, the Food Chemistry Division of the Ministry of Health and Welfare (MHW) has jurisdiction over food additives through the Food Sanitation Law. It was in 1948 that the term “food additive” appeared in the law (in the Food Sanitation Law) and a positive list of food additives was created in Japan.It was the first positive list created in the world, and it did not distinguish between syntheticor natural additives. Several amendments were adopted later. Amendments to the regulations, as well as additions or deletions to Kohetish0 (the Japanese Codexof Food Additives), were mostlyinfluenced by two major objectives: protection of food sanitation and customer safety, and harmonization with international regulatory requirements. In the Food Sanitation Law, the term “additive” means anything added to, mixed into, permeating, or otherwise put in or upon food forthe purpose of processing or preservto defining ing it. Most discussions on regulating food additives in Japan have been related what food additives should be under legal restriction and on labeling requirements. Very often in these discussions, differentiating “synthetic” and “natural” food additives had been at issue. In Japan, those two generally used terms have often misled customers into a blind belief in natural food additives. However, regulatory bodies, as well as the food additive industry, no longer distinguish additives with these terms. The latest amendment of the law (May 24, 1995) includes deletion of the term “chemically synthesized substances.”Thus“natural”foodadditivesareregulatedunder the amended law (being enacted from May 24, 1996), unless theyarelisted as “existingfoodadditives.” The MHW then disclosed the list of “existing food additives” on August IO, 1995. Today, when new natural food additives are used in Japan, suppliers also need to report them to the MHW. However, in general, data requirements for natural additives are still not as strict as those for chemical substances. Natural food additives reported to the MHW are listedin a table separate from the conventional positive list for chemical food additives. There are about 1200 items in the natural additive list, while the conventional list contained 349 compounds as of 1992. In 1983 the MHW addeda new regulation that requires labeling by the name of the compound used as a food additiveas well as by the purpose of its use. Today both synthetic and natural food additives need to be labeled.

VIII. TRENDS AND ISSUES While there are many differences in food tastes and preferences among consumers, the major trends driving the food additives industry appear to be very similar: Concern for health and nutrition. Food safety/health consciousness. Desire for convenience. The concept of value added. High costs associated with R&D and product commercialization. Growing awareness of the connection between diet and diseases suchas cancer and heart disease has caused consumersto reexamine their diets and lifestyles and seek healthier alternatives. Consumer desire for healthier, more nutritious foods favors natural addi-

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467

tives and ingredients (and those that are perceived as natural), as well as those that reduce calories, sodium, cholesterol, and the overall fat in foods. Fortification withthe “right level” of vitamins, amino acids, and trace minerals is important, and additives that sound natural(e.g.,gelatin,pectin,vitamins,etc.)versuschemical(e.g.,potassiumbenzoate, butylated hydroxyanisole, etc.) have a more favorable consumer image. The shiftawayfromcommodity to moreprocessed,higher-valuefoodproducts favors an increased use of additives in processing. Additives that are perceived favorably by consumers as healthy or natural foods are likely to grow faster. Finally, demands are high for fat replacers, high-intensity sweeteners, low-calorie bulking agents, certain gums, freezehhaw stabilizers, and natural flavors. Sales of ingredient and additive blends will dominate in the future. The synergistic effects that enhancethefunctionality of thesematerials,whilereducingthequantity needed, will play an ever-more significant role in formulated foods. Information on these blends will be scarce, because they will be developed in house by food additive suppliers and food manufacturers wishingto maintain confidentiality in order to optimize exclusive commercial benefit. Other issues affecting the growth and broadening of the food additives industryincludeincreasinggovernmentregulatoryactivity;increasing R&D andlegal expenses; and the great length of time needed to perfect, gain approval for, and market a new food additive product. In addition to traditional processed food products, a variety of health-related products known as “functional foods” and “nutraceuticals” have appeared on the market. Functional foods are food products that improve performance or provide a health benefit beyond meeting the basic nutritional needs of humans. Although functional foods are consumed for their taste, aroma, or nutritional value, they are also consumed by health conscious adults for their perceived benefits in preventing the onset of degenerative diseases such as arthritis, cancer, or heart disease. Nutraceuticals are specific vitamins, minerals, amino acids, herbs and other botanicals, or constituent parts thereof that are taken in oral form to promote natural ways of preventing or treating various degenerative disease conditions. Nutraceuticals differ from functional foods in that they are only consumed for their health benefits rather than for taste, aroma, or nutritive value. In the United States nutraceuticals will have to overcome regulatoryconstrainedbeforetheycangainalargemarket. In constrast,inJapanand several countries in Europe the conceptof nutraceuticals is well established,both in terms of regulations and consumer acceptance. The market for fat replacers is the number one concern of customers in the United States, and following the recent approval of Olestra, the first truly heat-stable fat substitute will increase new product activity and interest in reduced or no-fat products. The fastest growth for fat substitute-containing products is expected to be in the United States, where diets have historically been higher in fat and sugar and consumers appear to have more problems with obesity/weight control and associated diseases. Europe may be the next window of marketing opportunity for light products. Especially in the UK, France, and Germany, the popularity of foods and beverages with less fat, sugar, and calories appears to be moving toward US.levels. In Japan the market for fat substitutes is currently very small, because the problem of excessive consumption of fat is not as serious as in the United States or Western Europe. However, with the growing influx of Western culture and along with it “fast food,” demand for low-calorie/low-fat foods is likely to grow. The success of these products will depend on the success/acceptance of low-calorie/lowfat products in the United States and Western Europe.

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The safety of the food supply continues to receive a great deal of attention from the press, the public, and the governments. In 1993, an outbreak of food poisoning i n the United States, eventually traced to undercooked beef, caused fundamental changes in regulatory policies and demonstrated to food processors the need for increased caution against food pathogens. The recent European outbreaksof bovine spongiform encephalopathy (BSE), known as “mad cow disease,” have created more serious and worldwide concerns about cattle-derived food products, including some dietary supplements (e.g.. gelatin capsules) and personal care products. Fast-paced lifestyles will continue to drive the demand for savory, high-quality convenience foods. Microwaveable and shelf-stable products that are tasty and healthy require additives such as specialized flavors, colors, and stabilizers to enhancelmaintain quality and will result i n continuing growth of the market for these additives. The concept of value-addedproducts is also ofgreatinterest tofoodprocessors as foods with added value, or at least perceived added value (e.g., low-fat, low-calorie, vitamin fortified, more convenient form/package, perceived prevention against particular diseases), garner higher margins. Therefore consumption of additives that can aid in adding value to processed foods will continue to increase. Following trends in the United States,the European market shows increased interest in ethnic foods and vegetarianism. The motivation in both cases is to promote health. Also, more products are being introduced which are aimed at very specific groups. These include children, teenagers, women, and in particular the growing population of elderly persons.

IX.

DESCRIPTION OF MAJOR FOOD ADDITIVES

Direct food additives comprise more than 30 types. With about 3000 food additives, including more than 1800 flavoring substances currently approved for use i n the United States (and more petitioning for approval), it would be difficult in a chapter such as this to discuss each and every substance. Ten major food additives were selected for discussion in this chapter.

A.

Sweeteners

Sweeteners are used in formulated foods to impart sweetness and to perform several other functions. They render certain foods palatableand mask bitterness; add flavor, body, bulk, and texture; change the freezing point and control crystallization; control viscosity, which contributes to body and texture; and prevent spoilage. Certain sweeteners act as preservatives by binding moisture in food that is required by detrimental microorganisms. Alternatively, some sweeteners can serve as food for fermenting organisms that produce acids that preserve the food, thus extending shelf life by retaining moisture. These auxiliary flmctions must be kept in mind when considering applications for artificial sweeteners. Sweeteners can be classified in a variety of ways: 0

0

Nutritive or nonnutritive. Materials either are metabolized and provide calories, or are not metabolized and thus are noncaloric. Natural or synthetic.Commercialproductsthataremodifications of anatural product, for example, honey or crystalline fructose, are considered natural.

Food Additives 0

0

469

Regularor low-calorie/dietetic/high-intensity. Althoughtwosweetenersmay have the same number of calories per gram, one may be considered low-calorie or high-intensity if less material is used for equivalent sweetness. As foods. For example, fruit juice concentrates can impart substantial sweetness.

Sweetness is measured via sensory methodsby taste panels. It is a subjective perception influenced by a multitude of variables including the temperature of the food being tasted, pH, other flavors and ingredients in the food, physical characteristics of the food sweetener, concentration, rate of sweetness development, and permanence of sweetness and flavor. Also, results can vary depending on the foods consumed priorto testing (even several hours before testing), the flavors to which the taster is accustomed, tasting experience of the panelist, time of day, and the physical surroundings in the test room. Sucrose, commonly known as table sugar (orrefined sugar), is the standard against which all sweetenersaremeasured in t e r m of quality of tasteandtaste profile.It is consumed in the greatest volume of all sweeteners. Sucrose, high-fructose corn syrup (HFCS), and other natural sweeteners (e.g., molasses, honey, maple syrup, and lactose) are food commodities and are not considered as food additives, therefore they will not be covered here. The discussion that follows is limited to the polyol alternative sweeteners and the high-intensity sweeteners.

1. Polyols Polyols(sugaralcoholsorpolyalcohols)arechemicallyreducedcarbohydrates.These compounds are important sugar substitutes that are utilized where their different sensory, special dietary, and functional properties make them desirable. Also polyols are utilized in low-calorie food formulations. The sweetness of polyols relative to sucrose and their caloric values are shown in Table 5. Moreover, because polyols are absorbed more slowly from the digestive tractthanis sucrose, they are useful in certain special diets. When consumed in large quantities (in excess of 25-50 g/day), however, they have a laxative effect, apparently becauseof the comparatively slow intestinal absorption.In the EU countries, if a food product contains morethan 10% by weight of a polyol, a warning statement must be added to the label stating that excessiveconsumption may induce a laxative effect. I n the United Statesfood products sweetenedwith polyols and containingno sucrose can be labeled as “sugarless.” “sugar free,” or “no sugar” but must also bear the statement “Not a reduced calorie food,” “Not a low calorie food,” or “Useful only for not promoting tooth decay.” Table 5 RclativcSwcctncss and Caloric Value of Polyols

Relative swectncss Caloric valuc (sucrosc = 100)

(U.S. allowance: kcal/g)

Erythritol Hydrogenated starch Hydrolysates

60-70 25-50

0.4 3.0

lsolllalt

45-65 40 90 70 50-70 100

2.0 2.0 3.0

Polyol

Lactitol Maltitol Mannitol Sorbitol Xylitol

I .6 2.6

2.4

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Sorbitol occurs naturally in many edible fruits and berries including pears, apples, 1974 the cherries, prunes, and peaches. Its nontoxic nature has long been recognized. In FDA included sorbitol as one of the first four chemicals in its revised list of GRAS substances. Sorbitol is only70% as sweet as sucrose. However, it has many functional properties to body desirable in a sweetener, such as bulking agent ability, high viscosity (contributing and texture), hygroscopicity (resulting in its humectant as well as its softening nature), cool taste, sequestering ability, and crystallization modification (retardation). Because sorbitol can be digested without insulin and is also noncarinogenic, it is used as a sugar substitute in diabetic and sugarless foods and candies. In general, sorbitol is used in foods to aid retention of product quality during aging, or to provide texture or other product characteristicsto the formulation. In its major applications-sugarless chewing gum, candies, and mints-liquid sorbitol is used primarily as a bulking agent and not for its sweetness. Sorbitol’s noncariogenic nature and the fact that it does not promote tooth decay may account for its wide use in these applications. Mannitol is only about 70% as sweet as sucrose and is also noncariogenic. Because of its nonhygroscopic nature, mannitol is used as a dusting powder and anticaking agent, besides its special dietary food application. The highest demand for mannitol is in sugarless chewing gum and sugar-free chocolates. However, mannitol has a more serious laxative effect than sorbitol and a warning label is required when consumption is likely to exceed 20 gtday. Xylitol is a five-carbon poly01 with sweetness similarto sucrose. It is found in small amounts in a variety of fruits and vegetables, and is formed as a normal intermediate in the human body during glucose metabolism. Xylitol has good solubility, blends well with foods, and has a lower melting point than sucrose, an advantage in the manufacture of confectionery products. There is also evidence that xylitol is not only noncariogenic but reduces tooth decay when used as a replacement for sucrose.It is mainly used in compressed candies, chewing gum. and overthe-counter pharmaceutical products. Xylitol is expensive, therefore it is usually used in small amounts in combination with other sweeteners. In a blend with aspartame, the two compounds havean excellent synergistic effect. Also, xylitol is blended with other polyols to minimize undesirable properties, such as hygroscopicity or the laxative effect of sorbitol, or to improve the solubility of mannitol. Lactitolmonohydrate,asugaralcohol,hasphysicochemicalpropertiesdifferent from those of sugars. It has a sweetness value approximately one-third that of sucrose and is therefore suitable where bulking with low sweetness is required. To increase the sweetness it can be blended with high-intensity sweeteners. It is derived from milk sugar and used as a sweetener in Japan, Israel, and Switzerland. In the United States a selfaffirmation GRAS statement petition has been submitted to the FDA for its use in chocolate, confections, and baked goods.

2. High-Intensity Sweeteners High-intensity sweeteners, once used mainly for dietetic purposes, are now used as food additives in a wide variety of products. They are termed high-intensity because they are many times sweeter than sucrose. But because of their very low use levels, high-intensity sweeteners cannot perform other key auxiliary functions in food and often must be used in conjunction with other additives such as low-calorie bulking agents. High-intensity sweeteners are also used in pharmaceuticals, cosmetics, animal feed, and biocides. The

471

Food Additives Table 6

Rcgulatory Status andSweetnessRelative to Sugar" Sweetness (sucrose = 1)

Cyclamate, Na salt Aspartam Accsulfarne K Saccharin Sucralose" Thaumatin (talin) Alitarne' Neohesperidin DC Stevioside Glycyrrhizin

30 200 200 300 600 3000 2000 2000 300 50

U.S.

Canada

Europc

Japan

P A A A A N P N N

A A A N' A N

A

P

P

N N

N"

N

A N N

N A N A N A P N A A

A A A P N

" A = approved: P = petitiontiled; N = not approved. "Sucralose is approved in U.S.A. Australia. Russia. Brazil. New Zealand. Quasar. Romania. and Mcxico. ' Alitam IS approved 111 Australia. New Zcsland. Pcoplc's Republican o l Chlna. Indonesia, Colombia. and Mexico. "Glycyrrhizin is approved as a llavoring. but not as a sweetener i n thc United States. "Sacchurm i n Canada is limited for use In personal care products and pharmaceutical. but it is banned In Ibods and beverages.

regulatory status and sweetness relative to sugar of high-intensity sweeteners are showr! in Table 6. Aspartame was approved in the United States i n 1981 for use in prepared foods, dry beverage mixes, and as a tabletop sweetener, and in 1983 for use in liquid soft drinks. It gained instant popularity and has become the sweetener in virtually all diet soft drinks in the United States. Aspartame has impacted not just the dietetic soft drink market but also many other sweetener markets. Its success has encouraged R&D, and FDA approval is currently being sought for its use in baked products, since aspartame can now be made heat-stable through an encapsulation process. Aspartame first appeared inthe U.S. dietsoft drink market in combination with saccharin (30% aspartam and 70% saccharin). Presently about 98-99% of canned or bottled diet sodas contain 100% aspartame. However, aspartame may be replaced in many products because i n 1998 other high-intensity sweeteners were approved for beverages. Aspartame can be used in many diverse applications. It is approved for use in more than 100 countries worldwide, and more than 5000 products cor.tain aspartame. In the United States, FDA approval is being given to more and more applications. In 1981 it was approved for use in prepared foods, dry beverage mixes, and as a tabletop sweetener and for carbonated liquid products in 1983. More recently, in 1993, FDA approval was extended to many other products, and thelist of approved products now includes the following categories:

0

Nonalcoholicbeveragesandready-to-servenonrefrigerated,pasteurized,aseptically packaged fruit juice beverages, including sport drinks Frozendesserts(dairyandnondairy) Refrigerated,flavoredmilkbeverages Fruit and wine beverages containing less than 7% alcohol

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Yogurt-type products in which aspartame is added after pasteurization and culturing Refrigerated,ready-to-servegelatindesserts Confectionaries(hardandsoftcandies) Bakedgoods,includingbakingmixes Low-alcoholbeer(containing less than 3% alcohol) Aspartame is about 200 times sweeter than sucrose. Unlike many other low-calorie sweeteners, aspartame isdigestedbythebody to amino acids, which are metabolized normally.However,because of itsintensesweetness,theamountsingestedaresmall enough that aspartame is generally considered noncaloric. Aspartame has a sugarlike taste, and enhances some flavors. Before aspartame was approvedby the FDA, it underwent the most rigorous review the agency ever gave a food additive. The process took approximately 10 years to complete. In early 1984. aspartame’s safety in beverages was again brought into question by researchers at the University of Arizona and the Community Nutrition Institute. The FDA, however, rejected a request for further hearings, saying it was satisfied that aspartame is safe in soft drinks. More recently, some research reports show that artificial sweeteners have had no effect on lowering weight levels and suggest that artificial sweeteners may actually increase appetite and thirst. To date, these findings have not appeared to affect the American consumer’s perceived benefit of low-calorie sweeteners. A few cases relating aspartame consumption to severe medical reactions have been reported in medical journals. About 4000 consumer health complaints of headaches and other reactions havebeen received by the FDA, allegedly due to the consumption of aspartame. The clinical validity and resultant outcome of these claims are not known at the present time. Saccharin was discovered in 1879 and has been used as a food additive since the early 1900s. Saccharin is approximately 300 times as sweet as sucrose. Because it is acidic and not very soluble in water, it is used primarily as its sodium salt. Saccharin combines well with other sweeteners and has an excellent shelf life. Its main disadvantages are a bitter, metallic aftertaste and concern over its safety. In the United States, a warning label regarding its safety must be attached to all food products containing saccharin. Saccharin is the most widely used nonnutritive sweetencr worldwide and is the least expensive on a swcetness basis. The FDA took saccharin off the GRAS list in the early 1970s as a result of a study in the United States was suggesting it caused cancer in rats. A ban on saccharin used proposed by the FDA but was stayed by Congress i n 1977 because of the ensuing public uproar fueled by the fact that there was then no noncaloric sweetener to replace it. However, saccharin has now been cleared of the possibility of causing bladder cancer by n number of studies. It is banned in Canada. Saccharin has been used primarily i n soft drinks, but also as a tabletop sweetener and in a wide range of other beverages and foods. A drop in the demand for saccharin for use in soft drinks occurred in early 1985 after Coca-Cola and Pepsi-Cola substituted a major portion of their saccharin use with aspartame. However, it is still used in other products in the rapidly growing dietetic soft drink market. In July 1988, the FDA approved the use of Hoechst AG’s acesulfameK (SunetteTM) for use in chewing gum, dry beverage mixes, instant coffee and tea, gelatins, puddings, and nondairy creamers. In 1998, the FDA approved its use i n nonalcoholic beverages. 200 times that of sucrose. It has a Acesulfame K has a rapidly perceptible sweet taste

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good shelf life and is relatively stable across temperature and pH ranges associated with the preparation and processing of foods. Baked goods, candies, and dry mixes are believed to be the most viable markets for this low-calorie sweetener. A limitation is an unusual taste detected at levels required for adequate sweetness, which will no doubt prevent its widespread use in diet soft drinks. No toxicity problems have been reported in a multitude of studies to date. Sucralose is the onlylow-caloriesweetenermadefromsugar.Since 1991 it has been authorized for use in foods and beverages in more than 30 countries worldwide, including Canada, Mexico, Brazil, Australia, New Zealand, Argentina, Lebanon, Russia, and Romania, and it received FDA approval in April 1998. Developed by Tate & Lyle (UK), sucralose is a chloroderivative of sucrose, 600 times sweeter than sucrose, made by altering the sucrose molecule. Unlike sugar, sucralose is not converted into energy by the body, and therefore containsno calories. In addition, sucralose does not promote tooth decay, and is stable in a wide range of pH and thermal process conditions. Its uses include soft drinks, dairy products, baked and extruded products, puddings, breakfast cereals, jams and jellies, canned fruit, and chewing gum. Other high-potency sweeteners not approved for use in the United States but used elsewhere include the following compounds: Cyclamate is 30 times sweeter than sucrose. It has a sugarlike taste, a good shelf life, and a synergistic effect when combined with saccharin or aspartame. Cyclamate was introduced as a food sweetener in the 1950s, but was banned in 1970 because of its suspected carcinogenic potential. Since then, Abbott Laboratories, the developer and main producer of cyclamate, has undertaken further studies and submitted petitions to the FDA that demonstrate its safety. In June 1985, the National Academy of Sciences concluded that cyclamate was not a carcinogen. The FDA, however, has not reapproved use of the sweetener. Cyclatnate use is currently permitted in more than 40 countries, including Canada and the EU (excluding the United Kingdom). Cyclamate is used as a tabletop sweetener, in beverages, and in low-calorie foods, particularly in combination with saccharin. The use of cyclamate with saccharin givesa better taste to beverages than saccharin alone. Thus saccharin producers would welcome reintroduction of cyclamate inview of competition from aspartame. Developed by Pfizer in 1979 (prior to selling its food business), alitame isa dipeptide made of two amino acids, L-aspartic and D-alanine. It is 2000 times as sweet as sugar, with the same taste as sugar; thus its use level would be 25-400 ppm. Composition and use patents had been issued in 32 countries. The U.S. patent expires in 2000. Alitame is approved in Australia, New Zealand, the People’s Republic of China, Indonesia, and Mexico for use in food, beverage, and tabletop applications. Approval is still pending in the United States, Japan, Canada, and the EU. Potential market applications for alitatne include bakery products, snack foods, candies and confectionaries, ice cream, and frozen dairy products. A reported advantage of alitame over aspartame is lower loss during cooking and heating, since it is heat-stable. Thaumatin, a mixture of sweet-tasting proteins from the seeds of T l ~ ~ u t m cotro cmceus dcrniellii, a West African fruit, is about 2000-2500 times sweeter than sucrose. Its taste develops slowly and leaves a licorice aftertaste. Thaumatin acts synergistically with saccharin, acesulfame K, and stevioside. Potential applications include beverages and desserts; it cannot be used in baked products. Thaumatin is generally recognized in the United States as safe for chewing gum, and the supplier, Tate & Lyle, is seeking GRAS extensions for other foods. Thaumatin has been permittedin Japan as a natural food addi-

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tive since 1979. Although it is approved for use as a sweetener in the United Kingdom and Australia, it is used primarily as a flavor enhancer. Stevia rebaudinnrr, a plant native to South America, is the source of the stevia extract, which is a natural sweetener. Stevia can beused in food products that require baking or cooking because of its stability in high temperatures. This product is approved for use as a sweetener in Japan,but is not approved for use in the United States. Thereis currently a proposal in Brazil that the sweetener be included in any sugar-free soft drink in that country. Brazil is believed to be the third-largest soft drink market in the world, after the United States and Mexico. Dihydrochalcones (DHCs) are derived from bioflavonoids of citrus fruits and are 300-2000 times sweeter than sucrose. They leave a licorice aftertaste and give a delayed 2000 times sweeter than sucrose is properception of sweetness. Currently DHC that is duced from bitter Seville oranges by hydrogenation of natural neohesperidin (the main flavone of some oranges). In low concentration in combination with other sweeteners, it has potential usesin chewing gum, candies, some fruit juices, mouthwash, toothpaste, and pharnlaceuticals. It is approved for use in Spain, the Netherlands, Germany, Belgium, and Zimbabwe. Several other high-potency plant constituents (in addition to stevia and thaumatin) that have been considered as food sweeteners include monellin from the African “serendipity berry”; glycyrrhizin, also discussed as a flavor enhancer and extracted from the licorice root; and hernandulcin, an oil extracted from a Mexican plant. Such sweeteners could potentially be used in addition to or as substitutes for synthetic sweeteners that are now used to sweeten low-calorie or dietetic foods and beverages.

B. Thickeners and Stabilizers Thickeners and stabilizers (also called hydrocolloids, gums, or water-soluble polymers) provide a number of useful effects in food products. The technical base for these effects results from the ability of these materials to modify the physical properties of water. Most food and beverage products largely consist of water. Water-soluble materials function as rheology modifiers, affecting the flow and feel (mouth) of food and beverage products; act as suspensionagentsforfoodproductscontainingparticulatematter;stabilizeoil/ water mixtures; act as binders in dry and semidry food products; and create both hard and soft gels in food products that require this physical form. During the 1990s, fat replacement (discussed in detail in a later section) became a major application for modified starches and gumsas these additives provide unique texturizing, bulking, and emulsifying properties. Moreover, natural gums have been preposed as good sources of dietary fiber. Thickeners and stabilizers are generally used in very small anlounts in most food products (e.g., 0.15% in jam, 0.35% in ice cream, and 1-2% in salad dressings). Table 7 indicates the primary functions of many food thickeners. Two principal classes of these materials are recognized: natural materials obtained from plants and animals, and semisynthetic nlaterials that are manufactured by chemical derivatization of natural organic materials, generally based on a polysaccharide on microbial fermentation-based substances. A third class known as “synthetic polymers,” obtained from petroleum or natural gas precursors, is not used as a food additive. Figure 3 shows the sources and the various hydrocolloids used by the food processing industries. Unmodified or natural corn starch, produced by the wet millingof tield corn. supplies the majority of thickening material for the American food and beverage market. Other

Table 7 Major Food Thickeners and Stabilizers and Their Functions Emulsion Thickening stabilization Unmodified starches Modified starches Casein Gelatin Carboxymethylcellulose Methylcellulose Guar gum Alginatcs Xanthan gum Pectin Locust hean gum Gum arabic Carageenan Agar

X X X X X X X X X X X

X X X X X X X X

Suspending properties Gclation

X X

Crystallization control

X X

Water binding

Mouth feel

X X

X

X

X

X X

X X

X

X

X X

X X X

Flavor fixation

Protective film forming

Synergistic Fat cffect substitution X

X X

X X

Foam stabilization

X

X X X X

X

X

X X

X X X X X X X

X

X X X

X X

X X

X X

X X

X

X

X

X

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P 1)

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Food Additives

4 77

natural starches of significance include potato, tapioca, arrowroot, and sago starches. Unmodified cornstarch, commonly called pearl starch, is used in the food processing industry in the preparation of sauces, gravies, and fillings. It is the choice thickening ingredient in many formulated food products because of its modest price. It is modestly priced in comparison to modified starch, but even more so compared with additives such as cellulose ethers, guar, xanthan, and alginates. Major use categories for unmodified starch include 0 0

0

0 0

Meat gravies Cookedpuddings Cream-stylesauces Pie fillings Barbecue sauces Saladdressings Bakedgood fillings

Modified starches used in food products or food processing have been modified to Extend the bodying or gelling effect of norlnal starches Improvc resistance to acid or heat degradability and to low temperature and frecze/ thaw (eliminating aggregation) Improve texture Modify gelling tendencies as desired Increase viscosities at high temperature without gelling on cooling Provide instant solubility and gelling i n cold water Modified or derivatized starches are generally designed for more selective food applications than unmodified starch. Modified starches are used in a wide variety of products, including baby foods, purees, candy (e.g., bonbons and butter creams), jellies, cake mixes, dough, various soup powdcrs and liquids, instant noodles, puddings, pie fillings, batter mixes, sauces, salad dressings, dairy desserts, snack foods, and canned foods. In meat products such as sausage, ham, and luncheon meat. modified starches serve as a binder as well as a thickener. Recently, modified starchcs have been used as fat substitutes in margarine-like spreads, salad dressings, cookies, and baked products. Casein is a protein occurring naturally in, and obtained from, milk: it is thc main ingredient in cheese. Casein is marketed as sodium, calcium, potassium, or magnesium caseinatc and is uscd in confections, puddings, bakery fillings and frostings, coffee whiteners, and whipped toppings. I n 1970 only five protein products were available from milk. Since then, nunlerous advances have been made used in the methods to isolate and modify proteins. As a result, most suppliers now offer multitudes of specialty casein products. Gelatin is obtained from pork skin and bones (type A), or beef skin and bones (type B). Type A is mostly used for confectionery products and type B for dairy applications. Gelatinisabout97%protein,but it has no beneficialvalueto humannutrition.Food applications for gelatin includes dairy products such as yogurts, confectionary products such as g u n m y animal chcwables, meats suchas canned hams, and gelatin desserts. Gelatin is hygroscopic, capable of absorbing up to 10 times its weight in water. Under refrigeration i t forms a thermally reversible gel of high strength. Gelatin seems to exhibit little synergy with other thickeners and stabilizers. Therefore it appears to be of little benefit to blend gelatin with other gums to produce custom

formulations.

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Carboxymethylcellulose (CMC) is the primary cellulose ether consumed in food and beverage applications, mainly in pet foods, frozen dairy products, beverages, bakers’ goods, dry drink mixes, syrups, glazes, icings, and toppings. The current search for microwave-compatible food additives makes CMC a candidate for this rapidly growing formulated food market. CMC, a nonnutritive substance, is also popularin diet food formulations requiring thickeners and stabilizers. Methylcellulose (MC) and hydroxypropylcellulose (HPC) are also used in specialized food and beverage applications, but their relatively high market prices preclude them from large volume applications. Guar gum is the galactomannan derived from the endospenn of guar seeds (Cynrnoposis tetmgonolobus) grown in India and Pakistan since ancient times. It is one of the most economical and widely used gums, with extensive use in a variety of food applications since the 1950s. In spite of several attempts by major domestic guar concerns to encourage domestic production of guar in Texas and other arid agricultural areas of the American Southwest, mostof the guar gum consumedin the United States is derived from imported degermed guar beans (splits), mostly from India. Becauseof changing supplies due to weather and harvest conditions, as well as the demand for guar gum for industrial applications, the price of guar gum tends to shift dramatically. However, guar is expected to remain one of the most cost-effective thickeners on the market. Major uses for guar gum include ice cream, dessert toppings (e.g., Cool Whip@), frozen and refrigerated prepared meals, cheese, imitation bakery jellies and dry-mix bakery formulations, fruit drinks, soups, gravy and sauce mixes, water-based frozen desserts, salad dressings, and instant hot cereals. Alginates are extracted from different types of seaweeds, mainly from brown seaweed, Mcrcrocytis pyrifern and Lroninuria sp. The alginates include the various salts of alginic acid and propylene glycol alginate (PGA). Sodium alginate is used primarily as a binder in frozen desserts, reconstituted onion rings, crab and shrimp analogs, instant pudding mixes, fabricated puddings, sauces and gravies (particularly those containing milk or requiring the low “weep” property of alginates), and re-formed meats. Propylene glycol alginate (PGA) is used in the United States as a foam stabilizer in beers and ales. In addition, PGA is used by major food manufacturers in salad dressing formulations. The nonsodium light metal salts of alginic acid are used as sodium alginate alternatives in low-sodium and dietary food specialties. Xanthan gum, a fermentation product, is used in salad dressings, relishes, syrups, sauces, bakery fillings, prepared puddings, glazes and toppings, processed cheese products, dry cake and beverage mixes, and fruit and carbonated beverages. A significant use is in dairy products, where it prevents the separation of the contained whey from the rest of the food product. Xanthan doesnot exhibit any reactivity with milk proteins and therefore is often used in combination with other hydrocolloids, particularly carrageenan. Moreover the stability of xanthan gum to acid and high salt content makes it very useful for many types of foods. Gellan gum isthe latest hydrocolloid approved for food use, produced with a fermentation process likethat used for the fermentationof xanthan gumby the organismAur-omonas elodecr. The FDA approved gellan gum for use in icings, frostings, bakery fillings, and low-solids jams and jellies and confections.It is also approved for food usein Japan. Gellan gum can be used at levels substantially below those required by conventional hydrocolloids. There are two forms of gellan gum. The first is a high-acetyl gum, which is partlyacetylatedandprovidesthermoreversiblegels.Thesecondisalow-acetyl gum forming a firmer and more brittle gel.

Food Additives

479

Pectin is a fruit extract from the peel of citrus fruits and apple pomace. The main commercial types used in foods are pectin itself and potassium pectinate, sodium pectinate, of esterification and amidated pectin. Commercial products include high ester [degree (DE) of 501 or low ester (DE of less than 50) pectins. Traditional food uses of pectins are in jellies and jams. Newer applications include gummy candies and fruit-flavored juices and carbonated drinks.In gummy candies and jellies, it is replacing starch for improvement of fruity flavor.In fruit-flavored drinks,it stabilizes the constituents and makes the product more appealing. A constraint on the supply of pectin is the approval required by the EPA to start up a new plant. Because of the high costs of compliance to dispose of the large volume of waste generated during pectin production, the last North American pectin production plant was relocated from Florida to Mexico. Locust bean gum is obtained from the carob tree. The major source of locust bean gum is the Mediterranean countries. The size and quality of the crop is directly related to climatic conditions, producing periodic shortages of supply and great fluctuations of price. Chemically locust bean gumis similar to guar gum. Anionic, cationic, and hydroxyalkyl derivatives are also produced commercially. Locust bean gum swells in cold water, but heating is necessary for maximum solubility. Locust bean gum is widely used in frozen dairy products, in conjunction with guar gum and carrageenan, and is used for preventing syneresis in cream cheese. In addition, locust bean gum is used in many nonemulsified sauces and dressings as a thickener, in prepared meals, and in bakery products as a moisture retention aid. Much of the locust bean gum is supplied in a blended form to the dairy industry. Gum arabic is obtained from various trees of the genus Acacia, primarily from A. senegd. It is highly soluble in water (up to 50%), and its solutions are of relatively low viscosity. Other advantages of gum arabic as a food additive are its nontoxicity and lack of odor, color, and taste. These properties are especially useful in systems requiring emulsifying properties, such as high solid suspensions. It is used as an emulsifier in beverages for citrus oil and flavors,a foam stabilizer in beer, as a crystallization retarder and emulsifier in confectionaries, and as a stabilizer in dairy and bakery products. Since the source of supply has sometimes been unreliable because of political and social events in the Middle East, many U.S. users have turned to substitutes, including starch derivatives. Carrageenan is extracted from Irish moss (Chondrusand Gignrfincr species) that is harvested off the Atlantic shores of New England, the Canadian Maritime Provinces, and several European countries. Carrageenan is readily soluble i n water to form an inelastic gel and is commonly used with other gums. Its most unique property is a high degree of reactivity with certain proteins, such as casein. The largest application of carrageenan in food use is in dairy products (e.g., frozen desserts,flavored milk powder, nondairy creamers). For example, cocoa can be suspended in milk with the use of about 0.025% carrageenan. One of the most significant recent developments for carrageenan suppliers has been its widespread use in poultry applications for moisture retention. The product Serves to retain moisture before and during cooking and allows the poultry to be pumped with large amounts of water. In 1990, the FDA decidedto allow an unrefined seaweed extract knownas Philippine Natural Grade (PNG) to be sold under the carrageenan heading. Traditionally, refined carrageenan is made in a 10-step process in which carrageenan is extracted from the seaweed and then filtered to remove the cell walls, or cellulose, and other substances from the seaweed. PNG carrageenanis prepared in a five-step process that extracts the unwanted

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substances from the seaweed, leaving both carrageenan and about 10-14% cellulose, as opposed to the less than 1% found in traditionally refined product. PNG is currently being used in the meat, cheese, and pharmaceutical markets. Agar (also called agar-agar) is obtained from various red-purple seaweeds of the class Rhodophycae. Agar is used primarily in baked foods (icings, toppings, meringues) and in confectionary products. Because agar is the most expensive of the seaweed extracts, there have been efforts to substitute with other gums such as carrageenan. A number of other thickening agents are used by the food industry, but these represent a very minor portion of the food additives market. Most are higher priced, in erratic supply, and face increasing competition from the principally used thickeners. Such other thickening and stabilizing agents and their principal uses include Ghatti gum. Obtained from India and Sri Lanka; no other functional properties are known than thickening and emulsion stabilization. Tragacanthgum.ObtainedfromtheMiddleEastandusedinsaladdressings and sauces. Karaya gum. Obtained from India and used for extreme thickening to pastelike gels.

C. Colors Colors are additives used to improve the overall appearance of foods and influence the perception of texture and taste. Products are derived from either natural origins or producedsynthetically. IntheUnitedStates,colorsaredividedintotwotypes:certified (FD&C) and natural (exempt from certification) colors. Food colors are listedi n the Code of Federnl Regultrtions (CFR) Title 21 parts 70-82. If an additive is not specifically included in these sections, it may not be used for coloring food, drug, or cosmetic products that will be sold in the United States. There is some confusion about the term “natural” colors. The definition of “natural” varies between the United States, Europe, and Japan. This section will concentrate on the U.S. regulations, with occasional reference to others. In the United States, from a regulatory point of view, there is no definition for natural colors, only “certified dyes” and “color additives exempt from certification.” Certified colors are synthetic materials whose purityis checked by theFDA.Colorsobtainedfromanimal,plant, or mineral origins are not certified because they often contain complex mixtures of many components. The exact composition of natural color varies from plant variety to plant variety, from region to region, and from season to season. Users depend on the integrity of their suppliers to ensure product quality. The certification process concerns only batch purity, it does not guarantee the safety of the color molecule. There is no inherent reason why certified colors are either more safe or less safe than natural colors (colors exempt from certification).In order to market their products, U.S. producers must submit product samples from each batch of material in an FDA laboratory to ensure and pay a certification fee. The materials are analyzed that they meet specific purity specifications. In other parts of the world, only self-certification exists, except in Japan where certification of synthetic colors has been required since 1994. Certified food colors, both primary and blends, are produced in a variety of forms including powder, liquid, granules, plating blends, nonflashing blends, pastes, and dispersion; the least expensive form is powder.

Food Additives

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A number of formerly certified FD&C colors have been banned under the provisions of the Delaney clause of the Food, Drug & Cosmetic Act, either because they were found to be carcinogenic or because there was no assurance that they could be made free from carcinogenic impurities. These actions have steadily reduced the number of certified dye in 1950 to 7 in 1998. (An colors available to the U.S. food industry from more than 22 additional color, FD&C citrus red no. 2, is permitted for coloring the skins of oranges that are not intended or used for processing, but it has not been produced in the United States in recent years.) Certified food colorants can be divided into dyes and lakes. Dyes are chemical compoundsthat exhibit their coloring power or tinctorial strength when dissolved in a solvent. Lakes are insoluble colored materials that color by dispersion. Table 8 shows the physical properties of these seven certified food colorants. Color regulations specify a legal minimum of 85% pure dye for primary colors,but most dye lots contain from 90-93% pure dye. Certified dyes fall into several chemical classes: azo-dyes (yellowno. 5 , yellow no. 6, red no. 40, citrusred no. 2),triphenylmethane dyes (blue no. 1, green no. 3), xanthine type (red no.3), and sulfonated indigo (blue no. 2). FD&C dyes are also used in the production of lakes, which are pigments prepared by combining a certified dye with an insoluble alumina hydrate substratum. Lakes are both water and oil insoluble and impart color through dispersion in food. Thus they are suitable for coloring foods that cannot tolerate water and products in which the presence of water is undesirable.Examplesinclude bakery products(icings,fillings,cakeand doughnutmixes),confections,dairyproducts(hardfatcoatingsforicecreamnovelties, wax coatings for cheese, yogurtwith fruit syrups), drypet foods, dry beverage bases, and dessert powders. The FD&C lakes do not have a legally specified minimum dye content; manufacturers use formulations of from 1 1 % (standard) to 42% pure dye (concentrated). Noncertified colors can be from either natural origins (primary sources), such as vegetables and fruits, or produced synthetically. Traditional markets for noncertified food as butter, margarine, shortening, colors include lipid-based, high-fat food systems such popcorn oil, processed cheeses and spreads, salad dressing, and snack foods. Water-soluble forms are also available and are used in beverages, baked goods, confections, and dairy products. Food color additives exempt from certification, their colors and sources listed are in Table 9 and described in more detail below. Annatto extract (Bixin, Norbixin, etc.) is an extract of a seed from a shrub called Bixa or-ellrrrw L. that grows in South America, East Africa, and the Caribbean. Oil- and water-soluble fonns exist depending on the method of extraction. Annatto extracts exhibit various yellow shades, and are commonly used in cheddar cheese and bakery products, often in combination with turmeric or paprika oleoresin. Beet juice/powder (betanin, beet-root red, etc.) is a water-soluble color found as the predominant pigment in red beets (Betcl v ~ d g m i ~Several ). forms exist, including dried ground beets, or dehydrated beet powder; beet juice concentrate, the liquid obtained by concentrating the expressed juice from mature beets; and beet powder, made by spray drying beet juice concentrate onto a carrier of maltodextrin. Canthaxanthin (Roxanthin) is a synthetically prepared carotenoid that is conm1ercially available as a water-dispersible powder. It exhibits reddish orange to dull violet shades. Caramel (burnt sugar) color results from the controlled heat treatment of food-grade carbohydrates. Often catalysts are added to drive the reaction to the desired color end point. Caramel colors exhibita colloidal charge anda variety of shades from yellow brown

Table 8 Physical and Chemical Properties of Certified U.S. Food Colorants Stability to FD&C name

Common name

Light

Red no. 3 Red no. 40 Yellow no. 6 Yellow no. 5 Blue no. 1

Erythrosine Allura red AC Sunset yellow FCF Tartrazine Brilliant blue FCF

Fair Very good Fair Good Fair

Fair Fair Fair Fair Poor

Blue no. 2

Sodium indigo disulfonates Fast green FCF

Very poor Fair

Green no. 3

Oxidation

pH change

Compatibility with food components

Tinctorial strength

Hue

Poor Very good Good Good Good

Very good Very good Good Good Excellent

Blue Yellow Red Lemon yellow Green- blue

Poor

Poor Good Good Good Good (unstable in alkali) Poor

Very poor

poor

Deep blue

Poor

Good

Good

Excellent

Blue

Water solubility 9 25 19 20 20 1.6

20

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Food Additives

Table 9 Food Color Additives Exempt from Certification"

Additive

Color

Source

Annatto extract Beet juice Dehydrated beets Canthaxanthin" Caramel Apocarotenal' Beta-carotene Carrot oil Cochineal extract (carmine) Fruit juice (grape and cranberry) Grape skin extract" (enocianina) Paprika Paprika oleoresin Riboflavin Saffron Titanium dioxide" Turmeric Turmeric oleoresin Vegetable juice

Yellow Red Purple Red Brown Orange Yellow Yellow Red

Vegetable Vegetable Vegetable Synthetic Semi-synthetic Synthetic Synthetic Vegetable Insect

Red

Fruit

Red

Fruit

Red Red Yellow Yellow White Yellow Yellow Red

Vegetable Vegetable Synthetic Vegetable Synthetic Vegetable Vegetable Beet and red cabbage juice

"Under the Code of Federal Regulations, Title 21. No color additive may be used in foods for which standardsof idcntity have been promulgated under Section401 of the Federal Food.Drug & Cosmetic Act, unless the use of added color I S authorized by such standards. "May not exceed 66 mg/kg of solid. o r pint of liquld, food. ' May not exceed 33 mg/kg of solid, or pint of liquid, food. "Used only in beverages. "May not exceed l % by weight of the food.

to reddish brown, and is available in powder and liquid forms. Caramel has a very large market in cola beverages. It is also used in bakery products and confectionaries. Apocarotenal (beta-apo-8'-carotenal) is a red-orange synthetically prepared carotenoid that is oil soluble. The pigment is found in oranges and tangerines and is commonly used in products such as cheese spreads and snack foods. In the United States, a usage restriction of 15 mg/lb of semisolid or solid food exists. Commercial products of natural beta-carotene exist from several sources, including the alga Dunaliella salina and palm oil. Beta-carotene can be also synthesized. It is oil soluble and exhibits a characteristic butter to egg-yolk shade. It is commonly used in baked goods, beverages, and confections. Cochineal extract, or carmine, the lake pigment of cochineal extract, is an extract of a female cochineal insect Dacrylopius coccus, or Coccus cacti. It is a stable colorant used since antiquity. At pH 4 and below it is orange, at pH 4-6 it is magenta-red color, and at pH greater than 6, it has a blue-red shade. The insect is commonly cultivated in

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Peru, Ecuador, and the Canary Islands. Approximately 70,000 insects are requircd to produce one lb of SO% carminic acid lake. It is cotnmonly used in beverages, sausage products, aperitifs, and confections. Cochineal extract is not kosher. Fruit juicesthat typically contain carotenoid- or anthocyanin-type pigments are often usedin concentrated or single-strength forms as coloring agents. In the United States, fruit juices mustbe expressed from mature varietiesof edible fruits or a water infusionof the dried fruit. Fruit juices that are used for coloring include cranberry, cherry, raspberry, elderberry, grape, orange, and tomato. Grapeskinextract(enocianina) is obtained by an aqueousextraction of fresh, deseeded marc remaining after grapes have been pressed to produce grape juice or wine. It contains the common componentsof grape juicebut not in the same proportion. During the steeping process, sulfur dioxide is added, and most of the extracted sugars are fermented to alcohol. The extract is concentratedby vacuum evaporation, during which practically all of the alcohols are removed. A small amount of sulfur dioxide may be present. I n the United States, grape skin extract is permitted only for use in coloring beverages. Paprika is the ground form of sweet red peppers (Cqxicurw ~ I I U ~ HPaprika ) . oleoresin is a solvent extract of the coloring principles of sweet red peppers. Extraction of the peppers is cauied out withseveralpermittedsolvents,includinghexane,ethylene dichloride, and various alcohols. Oil is commonly added to the extract to standardize the strength, with typical designations in American Spice Trade Association (ASTA) units and color value units (CVU). Paprika oleoresins are oil soluble, reddish orange shades. Typical applications include coloring salad dressings, snack foods, cheese product, baked goods, breading, and crackers. lo as lactoflavin and vitamin B'. Riboflavin, a bright yellow color, is also referred It is a naturally occurring yellow pigment isolated from milk, and it can also be synthesized. It has limited solubility, a bitter taste, and is light sensitive, therefore it has limited USC.

Saffron is the dried stigmas or extractof Crocus .wtivu.s. The predominant pigments arecrocinandcrocetin.Saffronislimitedinitsapplicationduetoitsveryhighcost; approximately 165,000blossoms are required to produce 1 kg of colorant. Saffron is commonly used as a spice and colorant i n rice products. Its bright lemon-yellow color is also used in applications such as soups, baked goods, and certain dairy products. Titanium dioxide is a white pigment that is reacted product from a mineral oxide called ilmenite, a type of iron ore. The crystal form, anatase, is the form of choice as a colorant for food. In the United States, purity of 99% or greater is required. Titanium oxide isthe onlywhitepigmentcurrentlypermittedasacoloradditive intheUnited States. It is often used to opacify systems such as low-fat/no-fat salad dressings and dairy products, pet foods, baked goods, sugar-coated candies, and other confections. It colors by dispersion, as it is not water or oil soluble. Turmeric and turmeric oleoresin is a bright yellow pigment from the rhizome CurL ' I I I I I ~ ~lotzgcr, which is grown predominantly in India. The principle coloring agent is curcumin. The oleoresin form is extracted by solvents, such as alcohol and acetone. It is available with or without flavor components. Some vegetable juices, typicallyin a concentrated form, are used as coloring agents. In the United States, vegetable juices must meet the criteria of the federal regulation, which describes juice expressed from mature varieties of ediblc vegetables. An example of a commercially available vegetable juice colorant isred cabbage juice, which contains anthocyanins. Most other vegetable juice concentrates contain chlorophyll pigments and

Food Additives

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are often not of sufficient color concentration nor stable enough to be used commercially. In addition, the flavor impact is often undesirable. Eight synthetic dyes are permitted for use in the EU countries. Twelve water-soluble dyes and eight of their lake colors are allowedi n Japan for foods. Compared to American consumers, European and Japanese consumers are more prone to demand that their food products contain natural colorants. There is a growing body of evidence that many natural colorants perform additional functions. They also act as vitamins, antioxidants, and antimicrobial and antiviral agents. Natural colorants also may have anticancer properties and can be used to treat vascular diseases and improve night vision. This information was discovered in recent years.

D.

Fat Substitutes

Although fats are essential for a healthy diet, excessive consumptionof fat has been related to health problems. Fat replacers are those ingredients that can help to reduce a food’s fat and calorie levels while maintaining some of the desirable qualities fat brings to food, such as “mouth feel,” texture, and flavor. Fat replacers can be carbohydrate, protein, or fat based. Three alternative approaches are being pursued in this area: Fatsubstitutes(Table IO). Thesearepartiallyor fully nonmetabolizablecompounds that possess fatlike properties and can replace fats on a one-for-one basis. Most fat substitutes are synthetic compounds that possess fatlike properties and can replace fat in a broad range of applications. Fat mimetics (Table 1 l ) . These are nonfat ingredients that mimic the mouth feel and other functional properties providedby fat, but have fewer caloriesthan fat. I n recent years, numerous approaches have been undertaken to partially replace or to eliminate fatsin food by using FDA-approved traditional nonfat food ingredients suchas novel carbohydrates and gums,as well as other innovative ingredients, including microparticulated milk and egg proteins, and modified oat fibers. These products are capable of duplicating many of the functional properties of fats, such as lubricity, tenderization, opacity, flavor release, slipperiness, melt. and plasticity. These products cannot substitute for fatson a one-to-one replacement basis. Moreover, these ingredients are suitable as fat replacements only in foods that do not require extensive heat processing (e.g., salad dressings, frozen desserts, margarine-like spreads, etc.). Emulsifiers are fat- or fatty acid-derived compounds that have the ability to modify the surface properties of solids or liquids and possess many of the properties Table 10 FatSubstituteApproved

in theUnitedStatcs

Composition

Name

Olcstra (OleanTM) Caprenin“ Salatrim (BenefatTM) Medium-chain triglycerides (MCT)”

Sucrose hexa-, hepta-, and octaesters Monobehenin esterified with C, ,, and C,,,l, acids Monostcarin esterified with C2 C , ,I, and C, I, acids Esters of fractionatcd coconut oil fatty acids

“Application is limited t o soft candy and confectionary coatings ”Because o f ~ t shigh prlce, ~t IS used in medical foods only.

(,,

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486 Table 11 SelectedProducersandDevelopers

Producer Carbohydrate-based products AVEBE America Inc. Cerestar International Conagra Inc. Crompton & Knowles, Inc. Cultor Food Science, Inc. FMC Corporation

Grain Processing Corporation Hercules, Incorporated National Strach and Chemical Co. The Quaker Oats Company Remy Industries N.V. Rhodia Food Ingredients A. E. Staley Manufacturing Co.

Zumbro, Inc. Protein-based substitutes Cultor Food Science, Inc. Nutrasweet/Kelco Opta Food Ingredients Emulsifiers American Lecithin Co. Central Soya Co., Inc. Cultor Food Science, Inc. Lonza Inc. Lucas Meyer Inc. Gumslhydrocolloids Alko Ltd. (Finland) Asahi Kasei Kogyo K.K. (Japan) Dow Chemical Co. FMC Corporation Hercules Incorporated Nutrasweet/Kelco

of FatMimetics

Compound

Trade name

Potato maltodextrine Modified high-amylose corn Oat maltodextrine Cellulose gel Polydextrose Fatlcarbohydrate emulsion Microcrystalline cellulose Mixtures of cellulose, maltodextrine, etc. Corn maltodextrine Pectin Modified waxy corn, tapioca starches

Paselli SA2 Amalean I and I1 oatrim Miracle Middles Litesse Very-low Novagel 200 Avicel

Oat maltodextrine Rice starch Oat maltodextrine Modified corn starch Modified potato, tapioca or waxy corn starch Hydrolized oat flour Rice maltodextrine

Maltrin Spledid N-Oil, N-Lite, Slenderlean Oatrim Remygel Oatrim Instant Stellar Sta-Slim Trimchoice Ricetrin 3

Whey protein Concentrate Microparticulated egg white and milk protein Zein, a corn gluten derivative

Dairy-Low Simplesse

Phospholipid fraction derivative of soy lecithin Lecithin Emulsion of Soybean Oil or milk fat Emulsifier Modified soy lecithin

N.A.

Enzyme modified cellulose Cellulose (>l pm) Methylcellulose, hydroxypropyl methylcellulose Cellulose Pectin Xanthan gum Gellan gum

N.A. Cellucream Methocel

Lita

Centrolex VeryLo N.A. M-C-Thin HL66

Avicel Splenda Dricoid 280

food Additives

487

of a fat or an oil. The caloric value of most emulsifiers is similar to that of triglycerides. However, depending on the degree of esterification and polymerization, some emulsifiers such as polyglycerol esters may have a lower calorie content. Polyglycerol esters contribute only 6 kcal/g. Typically 2% fat in a formula can be replaced with1% emulsifier with no loss of functionality. However, due to regulatory constraints and flavor considerations, emulsifiers are usually used at 1% concentration or less in formulated foods.

1. Low andNoncaloricLipids Currentlyonlyfournewlystructuredlipidsareapprovedforuse as fatsubstitutesin theUnitedStates(Table 10). Thesearemade bytheinteresterification of natural oils. Olestra (brand name OleanTM), developed by Procter & Gamble Co. (P&G), was approved by the FDA in January 1996 for use in preparing potato chips, tortilla chips, and other savory snacks. Olestra is a sucrose polyester made from sucrose backbone and six to eight fatty acids. The number and type of fatty acids vary depending on the performance characteristics desired. The fatty acids are derived from vegetable oils found in soy, corn, and cottonseed oils. Olestra molecules are much larger than those of ordinary fats, so the body’s digestive enzymes cannot break it down. Thus Olestra is neither digested nor absorbed, passing straight through the body. It is noncaloric and nonsweet. Olestra was discovered about 25 years ago. Its submission to the FDA was withdrawn numerous times as the information on it was refined. At the time of its approval, more than 300 volumes covering more than 100 laboratory studies on seven species and 98 clinical investigations involving 2500 humans comprised the body of knowledge on this compound. P&G spent more than $200 million for the development and regulatory approval process of olestra. There are some concerns about olestra because it blocks the absorptionof fat-soluble vitamins consumed with it. Therefore the FDA requires that fat-soluble vitamins A, D, E, and K be added to foods made with olestra. Also it is reported to cause abdominal discomfort and may act as a laxative in some cases. Therefore products with olestra have to carry a warning label that these effects are possible. Approval of olestra brought a range of responses from scientists and consumer advocates who disapprove of its use, as well as endorsements by groups such as the American Dietetic Association, which identifies olestra as “one more choice for consumers in the war against fat.” Caprenin, or caprocaprylobehenin, is a reduced-calorie alternative to cocoa butter and other confectionary fats. Caprenin is a fat, a triglyceride composed of naturally occurring fatty acids-caprylic (C8,(,),capric (C,,,(,), and behenic (C??(,) acids. The mediumchain fatty acids are derived from coconut and palm kernel oils. The long-chain behenic acid, which comes from hydrogenated rapeseed oil, is mostly unmetabolized in the gastrointestinal track. Therefore caprenin hasa caloric density of 5 kcal/g, instead of the 9 kcal/ g in conventional fats. According to P&G’s patent, this material can substitute for about 70% of the fat in confectionary products, which contain usually 25-45% fat. Instead of the traditional FDA clearance processthat requires several years to complete, Caprenin has been cleared by a “self-affirmation process.” That process was based on the recommendationof an expert panel (convened by the Life Sciences Research Office of the Federation of American Societies for Experimental Biology), which reviewed published data and P&G’s research and concluded that caprenin is safe as a confectionary fat.

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Salatrim (BenefatTM),an esterified monostearin, is a family of reduced-calorie fat products consisting of short- and long-chain fatty acids. These fats are derived from ingredients found in nature and provide only 5 kcal/g instead of the 9 kcal/g provided by traditional fats. This calorie reductionis achieved because the short-chain fatty acids (acetic, propionic, or butyric) used are inherently low in calories, and the long-chainfatty acid (stearic acid) used is not fully metabolized. Because BenefatTMis fat, it delivers the taste and performance of conventional fats. Medium-chain triglycerides (MCT) are estersof fractionated coconut oil fatty acids. These compounds provide 8.3 kcal/g, only slightly less than conventional fats. However, recent physiological studies suggest that MCTs are burned readily for energy and have little tendency to be incorporated into tissue lipids that are not deposited as fat. MCTs are GRAS compounds, have been used in medical and infant feeding products for more than 30 years, but have not been used in consumer food products because of their high cost. More recently, their use has been expanded into sport/nutrition foods.

2. Fat Mimetics Table 1 1 lists some conventional food ingredients that have been used as fat replacers. Many new ingredients are being recommended, therefore this list should not be considered exhaustive. However, it provides an overview of the various types of replacers and fat extenders presently available.

N. Cnrboh~dmte-BasedSubstitutes. Nearly 40 different products based on starch have been recommended for fat replacement. Some of these exist as products with other uses, although several havebeen developed specifically as fat mimetics. Mostof the materials in this category are used to form a gel containing modified starch and water. The gel is then substituted for fat in the formula on an equal-weight basis. Starch-based fat mimetics have many different properties dependingon the parent starch and on the degree of cross-linking,substitution,andacidmodification. In manyinstances,two ormore starches must be used together to give the desired effect, or they can be combined with other polymers and emulsifiers. Maltodextrins are productsof the acid hydrolysisof starch, and actas bulking agents, giving the mouth-feel qualities of fat. One of the first on the market, in 1984, was N-Oil, a hydrolyzed tapioca maltodextrin. The substance forms a thermoreversible gel in aqueous foods, and therefore creates the mouth feel of fat. N-oil and several similar products are used in frozen desserts, salad dressings, margarine-type spreads, dips, baked products, and snacks. Several other starch-based maltodextrin fat replacers are available. One of the most widely used fat mimetics is AvicelTM,a cellulose derivative used in frozen desserts, salad dressings, and baked goods. Methocel, a food gum, is made of cellulose ethers for use in bakery products, fried foods, and salad dressings. Pectin is a gum that forms a gel. It has a large water-holding capacity and therefore helps to overcome some of the dry impression of fat-free foods. A gum is a soluble fiber, so it must be countedas a carbohydrate (4 kcal/g) in the calculationof calories for labeling purposes. In 1991 Splenda, a specialty pectin, was introduced as a fat replacer. Another gum fat replacer, carrageenan, that was used in low-fat hamburger, failed to achieve wide consumer acceptance. Other hydrocolloids and gumsthat are not listed in Table 1 1 are frequently promoted as fat-sparing agents. Xanthan, gelatin, carrageenan, algin, guar konjak, locust bean gums, etc., can be utilized as well for their fat-sparing function.

Food Additives

489

Polydextrose-“LitesseTM” and “Veri-LoTM”-is recommended for replacement of fats. Polydextrose is a water-soluble, reduced-calorie polymer of dextrose that contains small amounts of sorbitol and citric acid. It provides 1 cal/g as it is only partially metabolized by the human body. Oatrim, developed in the USDA laboratory in Peoria, Illinois, is an amylodextrin with 5% P-glucan extracted from oat flour. It is used as a fat replacer in baked products.

b. Protein-Bused Substitutes. SimplesseTM is a mixtureof eggwhite and milk proteins, water, sugar, pectin, and citric acid subjected to high shear (e.g., microparticulation) that f o r m a gel of protein spheroids. The small spheres produced by microparticulation provide the mouth feel of fat. This product is used in frozen desserts. A similar version, Simplesse 100, is made from whey protein and is approved for use in baked products. With its 1-2 kcal/g, 1 g of microencapsulated protein can replace I g of fat. Several other protein-based fat replacers are on the market and are used in both cooked and uncooked products. Lita is based on zein, a microencapsulated protein from corn. Like other proteinbased fat replacers, it contributes less than 2 kcal/g. It is used in frozen desserts, whipped toppings, and mayonnaise. c. En~u/s~)?er.~. Emulsifiersarefat-basedsubstances thatareused withwater to replace all or part of the shortening content in cake mixes, cookies, icings, and vegetabledairy substitute. Most emulsifiers provide the same calories as fat, but less is usell, resulting in fat and calorie reductions. Many emulsifiers simply play a “fat-sparing’’ role. However, polyglycerol esters may have a lower caloric content than triglycerides, depending on the degree of esterification and polymerization. The commonly used food emulsifiers that have applications as fat replacers include lecithin, mono- and diglycerides, and derivatives such as acetylated, succinylated, and diacetyl tartaric esters of distilled monoglycerides, polyglycerol esters, polysorbates, and sucrose esters. More information on specific compounds can be found in the emulsifier section of this chapter.

E.

Enzymes

Enzymes are catalysts used during food processing to make chemical changes to the food. They are biological catalysts that make possible or greatly speed up chemical reactions by combining with the reacting chemicals, bringing them into the proper configuration for thereaction to take place. They are notaffectedbythereaction. All enzymes are proteins and become inactive at temperatures greater than about 40°C or in unfavorable conditions of acidity or alkalinity. Some of the specific functions food enzymes perform include Speed up reactions Reduce viscosity Improve extractions Carry out bioconversions Enhance separations Develop functionality

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Create/intensify flavor Synthesize chemicals Food enzymes are usually classified into the following categories: Carbohydrasesandamylasesarecommercially the mostimportantsubgroup, hydrolyzing 1,4-glycosidic bonds in carbohydrates Proteases,hydrolyzepeptidebondsinproteins Lipases,splithydrocarbonsfromlipids Pectic enzymes and cellulases, hydrolyze plant cellwallmaterial Specialtyenzymes These enzyme categories can be divided further into 15-20 subgroups. The traditional roles of enzymes in the food industry have been in the processing of bakery goods, alcoholic beverages, and starch conversion. But interest is now focused on newer and more varied applications, such as hydrolysis of lactose, the preparation of modified fats and oils, the processing of fruit juices, and other processes where newer enzymes are being identified. Today many food processes utilize enzymes. Food-grade enzymes encompass a wide variety of commercial products that are employed in the production, conversion, and modification of foods because of their highly efficient and selective catalytic functions. Table 12 lists many of the major food enzymes and gives some applications in foods and food processing. The largest application of enzymes in the food industry is the use of alphaamylase, glucoamylase, and glucose isomerase for starch conversion and production of high fructose corn syrup. Rennin, a protease enzyme used in cheese making, is also of significant value, followed by a host of other enzymes, including pectinases, invertase, lactase, and maltase (used for the modification of starches and sugars), catalase, pepsin, glucose oxidase (an antioxidant for canned foods), and bromelin, ficin, and papain (plant proteases used for tenderizing meat and producing easily digestible foods). Enzymes are highly specific and can act only on a single classof chemicals, such as proteins, carbohydrates, or fats. These same enzymes are also used in nonfood applications such as pharmaceuticals, textiles, detergents, and waste treatment. Enzymes are produced from animal tissues (e.g., pancreatin, tripsin, lipase), plant tissues (e.g., ficin, bromelin), and most frequently by microorganisms (e.g., pectic, starch enzymes). Microbial production from a variety of species of molds, yeasts, and bacteria is increasingly becoming the predominant source of enzymes. Application of genetic engineeringto the developmentof enzymes has already made a significant impact. The first food additive produced by genetic engineering was chymosin, “Chy-MaxTM”, a microbial rennet that has been approved by regulatory agencies intheUnitedStates,Canada, theUnited Kingdom,Australia,Italy,andseveralother countries. Advantages of the bioengineering product are increased yields, relative ease of manufacture, lower price, and the ability to label the product as kosher.

F. Vitamins Vitamins are nutritive substances required for norrnal growth and maintenance of life. They play an essential role in regulating metabolism, converting fat and carbohydrates into energy, and forming tissues and bones. Vitamins can used be as functional ingredients

49 1

food Additives Table 12 Applications for Enzymes intheFoodIndustry

Food Alcohol production Baking

Amylases Fungal proteases

Brewing

Microbial proteases, papain, pectinase Invertase Pectinases, cellulase

Confections Coffee

Dairy

Rennins, lactase, lipase

Fats and oils Flavors

Lipase, phospholipase Protease, lipase

Fruits and vegetables Fruit juice and wine

Cellulase Pectinases

Protein

Bromelin, papain pepsin, pancreatin

Sugar processing

Amylases, cellulase

Starch conversion

Glucose isomerase

Other

Proteases

Starch liquefaction Dough conditioning, flour bleaching, malting, and antistaling Low-calorie beer, chill proofing, barley, altemative adjunct liquefaction, and saccharification Cream candy centers Removal of burnt flavor in UHT (ultra-heattreated) milk Separation of bean, viscosity control of extracts Cheese making, accelerated cheese ripening, natural cheese flavor concentrates, whey utilization, lactase intolerance Coca-butter substitutes, flavor-ester synthesis Synthesis of savory flavors, natural flavor esters Breakdown of cellulose structure Mash treatment, depectinization, starch/araban haze removal, citrus pulp wash viscosity reduction, natural cloud production Rendering, soy milk production, egg white replacement emulsifier production, functional hydrolysates Removal of undesirable starches and polysaccharides in the processing of cane sugar High fructose corn syrup, maltose, and dextrin syrups Meat tenderizing, coffee soluble-extract viscosity reduction

in foods. Vitamin E (tocopherol) and vitamin C (ascorbic acid) protect foods by serving as antioxidants to inhibit the destructive effects of oxygen. This helps protect the nutritive value, flavor, and color of food products. In addition, ascorbic acid enhances the baking quality of breads, increases the clarity of wine and beer, and aids color development and inhibition of nitrosamine formation in cured meat products. Beta-carotene and beta-apo8”carotenal are vitamin A precursors, which are brightly pigmented and may be added to foods such as margarine and cheese to enhance their appearance. The roles of these substances outside their nutritional functions are discussed elsewhere in this chapter (see Antioxidants, Preservatives, and Color). Thirteen vitamins are recognized as essential for human health, and deficiency diseases occur if any one is lacking. Because the human body cannot synthesize most vitamins, they must be addedto the diet. Most vitamins are currently consumed as pharmaceutical preparations or over-the-counter vitamin supplements. Some, like vitamins B, C, D, and E are added directlyto food products. Ready-to-eat breakfast cereals are a good example of fortification. Because the primary use of these cereals is as a complete breakfast entree, they are commonly formulatedto provide 25% or more of the daily value (% DV) per serving of the 10-12 important vitamins and minerals common to cereals. Another

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important example is the fortification of fruit drinks with vitamin C. Other foods that typically have added vitamins include margarine. infant formula, meal replacements, and breakfast bars (Table 13). Vitamins are added to processed foods for several reasons: T o restorevitalnutrientslostduringprocessing-thisisimportantwithdried milk, dehydrated vegetables, canned foods, and refined and processed foods. To standardize nutrient levels in foods when these fluctuate because of seasonal variations, soil differences, and methods of preparation. To fortify fabricated foods that are low in nutrients and promoted as substitutes for traditional products; this includes complete breakfasts, breakfast drinks, meat extenders, and imitation products such as eggs, milk, cheese, and ice cream. To fortify a major staple, such as bread, with a nutrient known to be i n short supply. For the preparation of designer foods (nutraceuticals) containing vitamins that are shown to be useful in preventing chronic diseases. Vitamins are typically divided into two groups: fat soluble and water soluble. Fatsoluble vitamins are usually measured i n international units (IUS) and consist of vitamins A, D, E, and K. The water-soluble group, usually measured in units of weight, consists of vitamin C (ascorbic acid) as well as the B vitamins. Humans need eight nutritive Bcomplexvitamins:niacin,riboflavin,panthotenicacid,pyridoxine,folicacid,thiamin, biotin, and vitamin B ,?.Table 14 outlines synonyms for andthe most commonly marketed forms of the major vitamins consumed in the United States as food additives. I n 1993 the FDA introduced reference daily intakes (RDI) [formerly recommended daily allowance (RDA)] for the labeling of foods and vitamin supplements. The RDI was designed to spell out the nutritional requirements of an average American. Those with greater than average needs (young woman, the elderly, and cigarette smokers, for example) are responsible for knowing their additional requirements and supplementing their diet. RDI values for vitamins established by FDA regulations are listed in Table 14. Table 13 FortifiedFoodGroups

Vitamin

Food Milk

Vitamin D

420 IUA

Beverages (noncarbonated) Cereals

Vitamin C

15-100% of U.S. RDI

Most essential vitamins Thiamin, Riboflavin, Niacin Vitamin A

25-100% of U.S. RDI

Flout Margarine Miscellaneous foods (e.g.. instant breakfast, energy bars. etc.)

per serving

Most essential vitamins

per serving S - W k of U.S. RDI per 2 oz. serving 33,100 lU/kg

Optional, but generally added Optional; also added as an antioxidant Optional; added to 90% of cold cereals Mandatory Optional, hut gcncrally added Added to position food as complete meal replacement

Food Additives

493

Table 14 VitaminsConsumcd as FoodAdditivesand U.S. RDIValues

U.S. RDI

ajor synonyms Principal Vitamin Vitamin A A, Az Vitamin B Niacin Thiamin Riboflavin Pantothenic acid Pyridoxine Cyanocobalamin Folic acid Biotin Vitamin C

Vitamin D D1 D, Vitamin E

Vitamin K K, K,

5000 IU

Retinol

Vitamin A acetate Vitamin A palmitate

Dchydroretinol Vitamin B? Vitamin B , Vitamin B2 VitaminB, Vitamin B,) Vitamin B Vitamin B,

Nicotinic acid Thiamin hydrochloride Riboflavin Calcium pantothenate Pyridoxine hydrochloride

Ascorbic acid

Ascorbic acid Sodium ascorbate Calcium ascorbate

Folate

20 mg 1.5 mg 1.7 mg 10 mg

2 mg 6 Pg 0.4 mg 0.3 mg 60 mg

400 IU

Ergocalciferol Cholecalciferol Tocopherols

Phytonadione Menadione

DL-alpha tocopherol acetate D-alpha tocopherol D-alpha tocopheryl acid succinate

30 IU

Phylloquinone

65 Pg

Vitamin A is generally added to margarine and milk. Muchof the vitamin A content of milk is obtained by feeding cows supplements of the vitamin. In addition, vitamin A is frequently added to instant breakfast foods, granola bars, and quick preparation or energy bar food products to better position those foods as complete meal replacements. Most natural vitamin A is derived from fish oils or carotenoid pigments found in chlorophyll-containing plants. These carotenoid pigments are the source of several provitamins, of which alpha- and beta-carotene are the most important. Important commercial forms include beta-carotene, retinol, retinol acetate, and retinol palmitate. Practically all the vitamin A used today is obtained by synthesis from the chemical intermediate, betaionone. Thiamin (vitamin B , ) is found in all plants, but cereal grains, milk, legumes, nuts, eggs, and pork contain large amounts. Thiamin is essential for the proper functioning of the central nervous system. Important commercial products include thiamin hydrochloride and thiamin mononitrate. Thiamin is obtained synthetically by several different routes, including linking chlorate-thylpyrimidine with 4-methyl-5-(hydroxy-ethyl)thiazole. Another method is the conversion of 4-amino-5-cyano-pyrimidine into a thioformylaminomethyl derivative via catalytic hydrogenation and reaction with sodium dithioformate.

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Riboflavin (vitamin B:) occurs in plant and animal cells. Important dietary sources include organ meats, yeast, and dairy products. Riboflavin is produced synthetically from D-ribose and fermentation processes. Pantothenic acid (vitamin B,) occurs i n all animals and plants and i n some microorganisms. Natural sources of pantothenic acid include liver, eggs, broccoli, cauliflower, tomatoes, and molasses. Commercially available forms include the liquid D-pantothenyl alcohol (panthenol), as well as calcium D-pantothenate and racemic calcium pantothenate. It is produced commercially by condensation of D-pantolactone with beta-alanine. Niacin (vitamin B,) is a generic term that includes both niacin (nicotinic acid) and niacinamide (nicotinamide). Poultry, meats, and fish are the most important sources of niacin. Both niacin and niacinamide are important commercial forms. Niacin is produced synthetically by the oxidation of quinoline, or 2-methyl-5-ethyl-pyridine. Niacinamide is produced by amidation of niacin. Pyridoxine (vitamin B,) refers to naturally occurring pyridine derivatives that have vitamin B, activity. Most forms of the vitamin occur in plants and animals, but chemical synthesis is a far more efficient and economical method of production than natural isolation. Pyridoxine hydrochloride is produced by the condensation of ethoxyacetylacetone with cyanoacetamide. Cyanocobalamin (vitamin B,:) is found in dairy and meat products. Cyanocobalamin and hydroxocobalamin are the important commercial forms, produced by fermentation using either Streptomycetes griseus or S. crureofkciens. Vitamin B is essential for bone as well as fornormal marrowcells,thenervoussystem,andthegastrointestinaltract, blood function. Folic acid, a member of the vitamin B complex, is a yellow-orange crystalline powder found in brewer’s yeast, wheat, nuts, legumes, and liver tissues. Folic acid and the calcium salt of folic acid can be obtained synthetically by a number of routes from triaminohydroxypyridine and para-aminobenzoylglutamic acid. Folic acid functions as a coenzyme in the synthesis of nucleic acid, purine-pyrimidine metabolism, and other systems. Recently folic acid gained importance because of its role in reducing the chances of neural tube birth defects and its role of controlling homocysteine, a risk factor in atherosclerosis. Medical studies indicate that folic acid and pyridoxine (vitamin B,) can reduce high levels of homocysteine, an amino acid,in the blood. A high levelof blood homocysteine was found to be an independent factor from cholesterol leading to increased risk of heart attack and stroke. Therefore folic acid and possiblyvitamin B , use in nutraceuticals and other fortified food products (e.g., breakfast cereals, cereal bars, calorie control and fitness food products, etc.) will increase substantially in the next few years. Vitamin C (ascorbic acid) is the most important vitamin used as a food additive in terms of volume. Primary applications include fruit juices, still beverages, juice-added sodas, and dry cocktail or beverage powder mixes. Ascorbic acid is also used as a food to preserve and preservative. Fruit juice makers, in particular, are applying the vitamin protect against color change in fruit ingredients. By doing so, they can also promote the high vitamin content of juices. Vitamin D: (ergocalciferol) and vitaminD l (cholecalciferol) are produced synthetically by the irradiation of the provitamins ergosterol and 7-dehydrocholesterol. respectively. Vitamin D! can also be isolated from fish liver oils. Although vitamins D: and Dl areboth important commercial products, most of thevitamin D issynthesized by the photochemical conversion of 7-dehydrocholesterol to D,.

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Vitamin E is found widely throughout nature, but the main dietary sources include vegetable fats and oils, dairy products and meat, eggs, cereals, nuts, and leafy green and yellow vegetables. The role of tocopherol as an antioxidant that can reverse the damaging effects of free radicals and therefore prevent certain chronic diseases has received increasingly wide attention in recent years. Naturally occurring vitamin E can be obtained from vegetable oil sources by distillation, and recently two major vegetable oil processors initiated the commercial production of natural tocopherols. However, large quantities of tocopherols are synthetically derived. Vitamin K, found i n green leafy vegetables, tomatoes, cauliflower, egg yolks, soybean oil, and liver, is essential for the formation of prothrombin and other blood-clotting factors in the liver. Menadione and its sodium bisulfite and diphosphoric acid ester derivatives are the lnost common commercial forms of the vitamin K group of compounds. Menadione (vitamin K \ ) is produced synthetically by treating 2-methylnaphthalene with chromic acid in the presence of sulfuric acid.

G. Antioxidants Antioxidants are food additives that retard atmospheric oxidation and its degrading effects, thus extending the shelf life of foods. Examples of food oxidative degradation include products that contain fats and oils in which the oxidation would produce objectionable also rancid odors and flavors, some of which might even be harmful. Antioxidants are used to scavenge oxygen and prevent color, flavor, and nutrient deterioration of cut or bruised fruits and vegetables. Recently, definitive studies have shown and been widely publicized in the news media that antioxidant nutrients suchas ascorbic acid (vitaminC) and tocopherols (vitamin E) can protect against harmful cell damage and thus prevent certain human diseases. Foods formulated with antioxidants and other vitamins are now recommended to prevent and cure cancer, cardiovascular diseases, and cataracts. The same antioxidants that are used to prevent oxidative deterioration of food may be used in functional foods (nutraceuticals, designer foods, etc.)to create products that prevent or cure certain chronic diseases.In this section, however, only the food preservation function of antioxidants will be discussed. To improve the performance of antioxidants,twoothertypes of foodadditives, sequestrants (e.g., EDTA, citric acid) and synergists (e.g., mixtures of antioxidants and lecithin), are frequentlyused with them. Antioxidants may also be presentin food packaging as indirect food additives, but such use is not covered in this chapter. Food antioxidants can be divided into water-soluble and oil-soluble compounds and also classified as natural or synthetic, as shown in Table 15. The most frequently used natural antioxidants are ascorbic acid (vitamin C), its stereo isomer erythorbic acid, and their sodium salts, plus the mixed delta and gamma tocopherols. While ascorbic acid finds its major use as a nutritive supplement orin pharmaceutical preparations, smaller amounts are intentionally used for antioxidant purposes. Erythorbic acid (iso-ascorbic acid) is virtually devoid of vitamin C activity (only 5% that of ascorbic acid). Citric acid and tartaric acid are also natural antioxidants (and antioxidant synergists), but are predominantly added to foods as acidulants. Synthetic antioxidants used as direct food antioxidants include butylated hydroxyanisole(BHA),butylatedhydroxytoluene(BHT),tert-butylhydroquinone(TBHQ), and propyl gallate (PG). These antioxidants are effective in very low concentrations (0.01%

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Table 15 FoodAntioxidantsandTheirManufacturingProcesses

Manufactured compound Antioxidant Oil-soluble products Butylated hydroxyanisole (BHA) Butylated hydroxytoluene (BHT) Tert-butyl-hydroquinone (TBHQ) Propyl gallate (PG) Tocopherols Thiodipropionic acid Dilauryl thiodipropionate Ascorbyl palmitate Ethoxyquin Water-soluble products Ascorbic acid Sodium ascorbate Erythorbic acid Sodium erythorbate Glucose oxidase/catalasc enzymes Gum guaiac Sulfites

Rosemary extract

by Synthesis Synthesis Synthesis Synthesis Extraction or synthesis Synthesis Synthesis Synthesis Synthesis Fermentation or synthesis Fermentation or synthesis Fermentation or synthesis Fermentation or synthesis Fermentation Extraction Synthesis Extraction

or less in animal fat) and not only retard rancidity but also protect the nutritional value of the food by minimizing the breakdown of vitamins and essential fatty acids. At one time, safety questions were raised about several synthetic antioxidants. These have largely been resolved, although the impact on the market for synthetic antioxidants still exists. The major applications of antioxidants in foods are listed in Table 16. The fats and oils industry and the snack/fast food/convenience food industries are the major users of food antioxidants. While the growth of fat-containing foods is declining because of consumer concerns related to the adverse effectsof fat and high caloric intake to health, the increasing preference for “healthier” unsaturated fats will increase demands for oil-soluble antioxidants because these fats require more protection against rancidity. Butylated Hydroxyanisole (BHA) was introduced commercially in foods in 1948. The major applications for BHA are in frying fats and oils, salad oils, and shortenings. BHA can beused in blends with BHT, TBHQ, or PGto optimize performance. For example, blends of BHA and propyl PG are used as stabilizers in edible lard and tallow. BHA is approved by the FDA as a GRAS substance, but the use is limited to a maximum 0.02% of the total fat and oil content of the product. Past developments have had a detrimental effect on the demand for BHA. In 1982, the Japanese government reported that, according to feeding studies done in Japan with BHA at 2% of the entire diet, BHA was found to be carcinogenic. Consequently BHA would not be allowed in food products sold in Japan after July 1982. Various governments, including the United States, Canada, and the UnitedKingdom,requestedadelay intheimplementationdate until further studies could be done. The date was then deferred to February 1, 1983, and the ban was never implemented. Subsequently the World Health Organization’s Food and Agriculture Organization studies showed that BHA dosage levels would have to be high

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Food Additives Table 16 FoodApplications for Antioxidants

Oil-soluble antioxidant applications (to retard onset the rancidity) of

Water-soluble antioxidant applications (to prevent oxidative deterioration of color, flavor, and nutrients)

Edible fats vegetables and fruits Fresh Vegetable oils Dried fruits uit Nuts ruits Frozen Shortenings Margarine Salad oils productsConfectionery frying foodFast oils Flavoring compositions products Bakery Processed 111eat spreads poultryand Meat Cheeses fruits processed Thermally chicken Processed Snack foods, nuts Canned meat and poultry Pancnke/cake mixes Breakfast cereals Dehydrated potatoes Chewing gums

(e.g., about2% of the oil or fat content of the food) beforeany carcinogenic effects would become apparent. (The normal BHA content level is 200 ppm of the fat or oil content of the food.) However, because of Japan’s announcement of its initial study, BHA was removed from some of the food and food packaging sold in the United States and Japan. The findings of the Japanese study relative to BHA were surprising since several other studies conducted worldwide had found BHA to be noncarcinogenic. The original Japanese researcher has now agreed that BHA is not carcinogenic: however, irreparable damage to BHA as a food antioxidant has occurred, and the product’s unhealthy image is unlikely to be reversed in the future. Butylated hydroxytoluene (BHT) was approved for use as a food antioxidant in 1954. BHT is often used in blends with BHA or BHA/PG in vegetable oils and in edible animal fats to take advantage of the synergism obtained. Although BHT was never removed from the FDA’s GRAS list, demand for BHT as a direct food additive dropped significantly because of an FDA proposal to restrict the use of BHT as a food additive throughout the 1980s. That proposal, as well as the general trend toward the use of allnatural ingredients in foods, has negatively impacted BHT use in foods. Producers of both BHA and BHT have petitions filed with the FDA to recognize the existence of “prior sanctions” for the use of the chemicals as food antioxidants at levels not to exceed 0.02%. Such recognition would eliminate the necessityof classifying the chemicals as food additives. Tertiary-butyl hydroquinone (TBHQ) is relatedto BHA and has good heat stability. It was first introduced for food applications in 1972. TBHQ shows exceptional ability i n protecting unsaturated vegetable oils and animal fats from rancidity. One of its largest applications is in soybean oils. Although mostly used by itself, TBHQ canbeused in combination with BHT and BHA. TBHQ is often used as a replacement product for PG.

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Propyl gallate (PG) has been used as a food antioxidant since the 1950s. Its current primary use is more as a synergist in combination with BHA and BHT. The active part gallic acid, can be extracted from natural sources and can be synthesized. Propyl gallate is effective in vegetable oils as well as animal fats, but it is not heat stable, even at cooking temperatures. Total consumption of PG is very small because of its relatively high price and competition from TBHQ. Ascorbic acid (vitamin C) and sodium ascorbate are widely used as natural antioxidants and vitamin supplements. As an antioxidant, ascorbic acid is used primarily in prepared foods (canned fruits and vegetables, juice drinks, applesauce, potatoes) and in processed meats (sausages). Manufacturers use it if for its protective function in soft drinks, for example, but declare it as “added vitamin C.’’ Ascorbic acid is insoluble in fats and oils, and its almitoyl ester is synthesized to impart some lipid solubility. Used alone, the ester is not very effective in fats and oils, and it is normally used in combination with tocopherol. Erythorbic acid (iso-ascorbic acid) and sodium erythorbate are used primarily as antioxidants in cured meats (e.g., bacon, sausages) and by salad bars as an oxygen scavenger. They arealso used in frozen fruits, vegetablefats and oils, and frozen fish and seafood. Erythorbic acid (and its salts) has benefited significantly from the FDA’s ban on the use of sulfites for fresh or uncooked vegetables i n salads. Approximately 80% of the total U.S. consumption is estimated to be in the form of sodium erythorbate. The greatest use is i n cured meat to minimize the formation of nitrosamines during the curing or cooking process. USDA regulations governing the maximum level of nitrite permitted for curing uses of erythorbacon require the useof 500 ppln of ascorbates or erythorbates. Other food bates are in fresh cut meat, frozen fruits and vegetables, and raw fresh cut vegetables. In Europe, erythorbic acid use has been permitted since 1995. Tocopherols are the fastest growing antioxidants used in the United States. Because the United States trades heavily with Japan, where synthetic antioxidants are banned,U.S. food exporters are reformulating products using natural antioxidants. Mixed tocopherols appear to be the product of choice. Although all isomeric forms (alpha, gamma. and delta) of tocopherol show antioxidant activity, the 80% garnma/20% delta mixture of natural tocopherols has the best antioxidant activity. Mixed natural tocopherol products can be used to protect a variety of food products, including dehydrated and processed vegetables, pasta and noodles, animal fats, salad dressings and oils, snacks, meats, and baked foods. Residues from vegetablerefining contain a small but significant level of tocopherols. Using techniques such as molecular distillation, these can be concentrated to give a brown oily product with goodantioxidantproperties. Thecompositionvaries withtheorigin (type of vegetable oil), and both gamma-rich and delta-rich versions are used. Sulfites serve multiple functions in foods: ( 1 ) inhibition of enzymatic and nonenzymatic browning, and (2) control of microbial growth. For years, sulfur dioxide and sulfite to help preserve the color of dried fruits and vegetables. salts have been widely used Sulfites areused in wine making and the wet milling of corn to prevent undesirable microbial growth.Restaurantsandotherfoodserviceoutletspreventthebrowningoffresh produce with the use of sulfites. But because of the allergic reactions of some consumers (especially asthmatics) to sulfites, regulations were issued and alternatives sought. In July 1986, six sulfiting agents-sulfur dioxide, sodium sulfite, sodium bisulfite, potassium bisulfite, sodium metabisulfite, and potassiunl metabisulfite-were banned by the FDA for use i n raw vegetables and fruits on salad bars. In July 1987, the FDA ruled that all pack-

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aged foods containing I O ppm or more of sulfur dioxide equivalents must disclose on the label that sulfiting agents are present. In 1990. the use of sulfites on fresh potatoes was banned. Treatment of fruits and vegetables with sulfitesis the most effective means available today to control browning. However, because sulfites have been banned in certain food categories and their regulatory status i n other categories is in question, alternative treatments to retard enzymatic browning and other oxidative reactions havebeen investigated. To date, however, alternatives to sulfites are not equivalentto sulfites in their effectiveness, cost, or functionality. The promising antioxidantlpreservativealternatives generally contain ascorbic acid or erythorbic acid in combination with one or more adjuncts, such as citric acid or some other acidulant, a calcium salt. a phosphate, sodium chloride, cysteine, or a preservative such as potassium benzoate or sorbate. The ascorbic acid derivatives, ascorbic acid 2phosphate and ascorbic acid-6-fatty acid esters, are also reportedly effective. Another suggested substitute (which functions in water but not with fats and oils) is the sequesterant and chelating agent ethylenediaminetetraacetic acid (EDTA), which hasbeen widely used i n processed potatoes, salad dressings, sauces, and beverages. Cyclodextrin is another sulfitealternativethatcanbeusedtopreventbrowning. Finding a good substitute for sulfites, however, has not yet been realized. This is because sulfites not only act as antioxidants to prevent browning, but also perform preservative functions i n preventing unwanted microbial spoilage. The above chemicals are ineffective against microbes. Ethoxyquin is included in the FDA regulation but limited to specific applications only. It is cleared for retarding oxidation of carotene, xanthophylls, and vitamins A and E in animal feed and fish food, and as an aid in preventing the development of organic peroxides i n canned pet food. Gum guaiac is an approved antioxidant for natural flavoring substances and other natural substances used in conjunction withflavors. It is also approved for addition to aninlal feed and food-packaging materials.

H. Preservatives Preservatives (antimicrobial agents) are capable of retarding or preventing the growth of microorganisms such as yeast, bacteria, molds, or fungi and subsequent spoilage of food. The principal mechanisms are reduced water availability and increased acidity. Sometimes these additives also preserve other important food characteristics, such as flavor, color, texture, and nutritional value. Important food preservatives used include sorbic acid and its potassium salt, calcium and sodium propionates, sodiumand potassium benzoates, and parabens. Sulfur dioxide and sulfites are also used extensively for controlling undesirable microorganisms in soft drinks, juices, wine, beer, and other products. Salt, organic acids, sugar, alcohol, spices, essential oils, and herbsalso inhibit the growth of microorganisms, but usually their primary function is different when added to food. Chemical preservatives play a very important role i n the food industry, from manufacture through distribution to the ultimate consumer. The choice of a preservative takes into consideration the product to be preserved, the type of spoilage organism endemic to it, the pH of the product, the shelf life, and the ease of application. No one preservative all organisms, and therefore combinations are canbe used in every producttocontrol

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oftenused. In certainfoods,specificpreservativeshaveverylittlecompetition.Inthe concentrations used in practice, noneof the preservatives discussed here is lethal to microorganisms in foods. Rather, their actionis inhibitory. Major uses for preservativesby food industry sector are listed in Table 17. In general, increased demand by consumers for lightly processed, lightly prepared foods (as people tend to do less cooking at home but at the same time are looking for products that are fresh, suchas prepared salads) has stimulated use of antimicrobial preservatives over the past several years. At the same time, however, media and consumer reacof several preservatives in tion to chemical preservatives has stymied or limited the growth favor of “all natural” and “no preservatives added” food products. However, significant displacement of traditional preservatives with naturally derived new products is not expected in the near future. Important areas for preservatives are in fruit beverages and convenience foods. For example, low fat/low calorie salad dressings require a preservative, while the traditional high oil-containing products had lower water activity and therefore an acceptable shelf life without chemical preservatives. Potassium sorbate and sorbic acid are used as preservatives in a great variety of foods and can be used as directadditives, as spraysordipbaths,and as coatingson wrapping materials, inhibiting yeasts, molds, and bacteria. Potassium sorbate is used where high water solubility is desired. Because sorbates have no effect on the microorganisms that produce lactic acid, they can be utilized to prevent yeast and mold spoilage of foods, such as mostcultureddairyproductsandpickles,withoutinterferingwiththedesired bacterial cure. Potassium sorbate solutions may also be used for spray and dip bath applicat i m s on cheese, dried fruits, smoked fish, and similar products. The effectiveness of potassium sorbate is based on its ability to depress fatty acid metabolism in the microorganisms. Useof sorbic acid is limited because of its low solubility in water. Therefore potassium sorbate is the primary form used in foods. It is effective against microbes at pH 6.5 or less. As the pH decreases, the antimicrobial activity of this preservative increases. Onan equal weight basis, potassium sorbate has 74% of the activity of sorbic acid. Sorbic acid and potassium sorbate are GRAS additives. Normal use levels are in the range of 0.05-0.10/0. Sorbates are used in cheeses, baked goods, spreads, margarine, dried fruits, jams, and jellies. Because of its corrosive nature, propionic acid, a liquid, is rarely used in the food industry. Its sodium and calcium salts are used in its place, yielding the free acid within

Table 17 MajorUSCSofPreservatives by Use Sector

Preservative SorbatesMoldandyeastinhibition in proccssedcheeseandspreads, other low-acid foods, and dried fruits. Effective in the acidic pH range up to pH 6.5 BenzoatesBeverages,fruit juice, pickles.Effective in pHrange 2.5-4.0 PropionatesMoldandropeinhibitors inbreadandbakedgoods ParabensAntibacterialforuseinlow-acidfoods(pHgreaterthan 5.0) such as meat and poultry products

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the food at low pH. They are highly effective mold inhibitors. but have essentially no effect against yeast. They have negligible activity against bacteria, except for their effectiveness against the rope-causing Bacillus rtleset1tericu.s. Propionic acid occurs naturally in Swiss cheeses at levels as high as 1 %. Its calcium salt, and to a lesser extent its sodium salt, have been used for more than 30 years as an inhibitor of mold growth in bread. The main market for propionate salts isinbakery products, chiefly because these salts do not inhibit yeast action (they also have almost no activity against bacteria). The propionates have GRAS status for use i n foods and have no upper limits imposed except for breads, rolls, and cheese, which come under the Standards of Identity. They can be used up to 0.3% in cheese products and to 0.32% by weight of the flour in white bread and rolls. Benzoic acid is one of the oldest chemical preservatives used in foods, having been described as a preservative in the 1800s. It has been used in foods since the early 1900s. Benzoic acid occurs naturallyin some fruits and spices, suchas cranberries, prunes, cinnamon, and cloves. Sodium or potassiunl benzoates are most effective in the pH range of 2.5-4.0. Benzoates have activity against yeasts, molds, and bacteria. However, benzoates are not recommendedfor bacterialcontrol becausetheirantimicrobialactivity is poor in above pH 4, where bacteria are the greatest problem. As benzoates are very efficient controlling yeasts, they cannot be used i n dough or in other yeast-raised bakery products. The most important uses for benzoates are in fruit juices and carbonated beverages, jams and jellies, and condiments. In carbonated drinks, 0.03-0.05% is used; in noncarbonated drinks, up to0.1 % is used. Benzoates arealso used for fats and oils, gravies, frostings, puddings, and gelatins. Potassium benzoate became commercially available in 1984 and can be used as a substitute for sodium benzoate in many of the above food products. It is also useful in margarine, potato salad, fresh fruit cocktails, and pickles. Although the amount of sodium added with the benzoate salt is nutritionally insignificant, the potassium salt was developed specifically for use in reduced or low-sodium food products to avoid sodium declaration on the label. The potassiunl or sodium salts of benzoic acid are more soluble in water than is benzoic acid and consequently are preferred for use in many food products. They do not destroy yeasts or molds but instead retard further growth of organisms already present, provided the degree of contamination is not too high. Benzoic acid and benzoates are GRAS substances and are pernlitted for usei n foods up to a maximum of 0.1% concentration. Parabens are esters of para-hydroxybenzoic acid. A combinationof methyl and propyl esters and sodium benzoate ismostoftenused,buttheethyl and butyl esters also have utility. Parabens are the only phenols approved for microbiological preservation of foods. Parabens are effective against molds and yeasts and are relatively ineffective against bacteria, especially the gram-negative bacteria. Their antimicrobial activity extends up to pH 7.0, making the parabens the only antimicrobial agents effective at higher pH values. The methyl and propyl parabens are GRAS ingredients, but their use is limited to 0.1% (combined). tz-Heptyl paraben is permitted in beer at a maximum concentration of 12 ppm. Parabens are used in baked goods, beverages, fruits, jams and jellies, and olives and pickles, but not in dairy products. Because parabens are the most expensive of the availablepreservativesandhavesometechnicalproblemsassociatedwiththeirusein foods, use by the food industry remains limited.

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About I O years ago, sodium nitrite and sodium nitrate were used i n curing bacon and other meats to prevent the growth of bacteria that cause botulism. The nitrate and nitrite were linked to the formation of nitrosamines in the meat, which were considered carcinogenic in experimental animals. Though the public outcry has largely subsided and nitrites continue to be used in smaller amounts, the continued use of these preservatives probably s t e m from the absence of suitable alternatives. Sodium ascorbate and sodium erythorbate are effective catalysts in the curing process, and the addition of one of these antioxidants to bacon makes it possible to reduce the quantity of sodium nitrite used. Most chemical preservativesin use today have specialized uses and established niche markets in the food industry. A great amountof interchangeability does not exist because of specific inhibitory actions toward bacteria, molds, or yeasts. Blendsof antioxidants and preservatives (some natural and some synthetic chemicals) can provide multiple functions for multiple food products. One such combination of ingredients is a blend of erythorbic acid, citric acid, and potassium sorbate as an antioxidant and antimicrobial substitute for sulfites on fresh vegetables.

1.

Emulsifiers

Emulsifiers are additives that allow normally immiscible liquids, such as oil and water, to form a stable mixture. They are widely used in foods in order to achieve the texture, taste, appearance, fat reduction,and shelf life desired in foods. Bread and bakery products is the largest food segment utilizing emulsifiers. In this application, they soften the bread and strengthen the dough by distributing the fat within the product so less fat (shortening) needs to be added. Emulsifiers are utilized as fat-sparing agents i n salad dressings and bakery and dairy products. Visible fats and oils routinely need emulsifiers for food-product processing, appearance, maintenance of shelf life, texture, and taste uniformity. They are also included i n low-fat formulations (e.g., frozen desserts, bakery products), often more so than in formulations with normal fat levels. In addition, food emulsifiers are widely used in convenience, snack, and microwaveable food products. The multiple applications and functions of food emulsifiers are shown in Table18, and several of the more prominent food uses of emulsifiers are shown in the following listing:

Breads Frozen desserts Icings Cream fillings Chocolate milk Whipped toppings Coffee creamers Instant breakfasts Infant formula Dessert mixes Rolls

Cakemixes Fresh cakes Donuts Cereals

Food coatings Instant potatoes Pastas Snack foods Ice cream Dips

Shortenings Margarine and diet spreads Peanut butter Candy Caramels Chewing gum base Chocolate Toffees

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Table 18 Functions of Emulsifiers

Function

Antistaling

Margarine, creamy salad dressing. coffee whiteners, frozen desserts Most baked goods

Modifying texture

Bread, cakes, macaroni

Wetting

Coffee whiteners. drink mixes, instant breakfasts Color and flavor systems Shortenings, margarine, peanut butter Ice cream, frozen desserts

Emulsifying

Solubilizing Crystal modification Preventing agglomeration Foaming

Defoaming Reducing tackiness Fat sparing

Improving palatability

Whipped toppings, icings. cakes. convenience desserts, ice cream Processing of syrups, yeast Candies, chewing gums Baked products, frozen desserts, whipped toppings, margarine, spreads, imitation sour cream Icings, confectionary coatings

Disperses small droplets of immiscible substances Complexing action on starch reduces firming of crumbs Complexing action 011 starch reduces clumping, improves consistency and uniformity Reduces interfacial tension between liquid and solid Improves solubility Modifies mode and ratc of crystal formation Controls coagulation of fat particles Controls dispersion of a gas in a liquid Breaks emulsions Assures texture Reduces size of fat globules, resulting a wider dispersion and reduced fat levels Improves mouth feel

The most common and commercially important emulsifiers are monoglyceridesand diglycerides of fatty acids and their esters (e.g., glyceryl monostearate), lactylated esters (e.g.,sodiumstearoyllactylate),propyleneglycolmono-anddiesters(e.g.,propylene glycol monostexate), lecithin, sorbitan esters (e.g., sorbitan monostearate), and polysorbates (e.g., polyoxyethylene 80 sorbitan monolaurate). With the exception of lecithin, few emulsifiersareused as a single additive. Most food emulsifiers are used as blends of emulsifiers, water, fats, and other classes of food additives such as gums. These products are formulated for specific applications (or specific customers) so that the combination provides both enhanced performance and ease of use. Emulsifiers are regulated as food additives in most countries. The FDA classifies lecithin, monoglycerides and diglycerides, diacetyl tartaric acid ester (DATEM), and triethyl citrate as GRAS substances. The other emulsifiers have specific regulations that permit their use in specific products at set levels. Monoglycerides and diglycerides are used in the largest amounts (more than 50% of the total volume for emulsifiers), mainly because of their low cost. Important applications are in the preparation of shortenings, in bread and other bakery products, and in ice cream. Lecithin. Commercial lecithin is usually a by-product from the refining of crude soybean oil. The term lecithin describesa complex mixtureof phospholipids, triglycerides, fatty acids, and other componentsthat occur naturally in soybean oil. The major phospholipids of lecithin are phosphatidylcholine(PC), phosphatidylinositol (PI), phosphatidylethanolamine (PE), and phosphatidic acid (PA). The unique structureof these phospholipids

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and other minor constituents gives lecithin its emulsification properties. Lecithin is often modified to improve its effectiveness as an emulsifier. The common modified lecithins thatare commercially available are the hydroxylated, acetylated, and enzyme-modified lecithins. The result is more hydrophilic, water-dispersible lecithin with enhanced oil-inwater emulsion properties. The primary applications for lecithin are in baked products, dairy blends, baby foods, nutritional drinks, margarines, chocolates, chewing gunis, and confectionaries. Polysorbates are a group of emulsifiers that contain sorbitans, various types and amounts of fatty acids, and polyoxyethylene chains. Heating sorbitol with stearic acid in the presence of a catalyst cyclizes sorbitol andf o r m an ester to produce sorbitan monostearate and tristearate. Other sorbitan esters of importance are monooleate and tristearate. Any of the three esters maybe reacted with ethylene oxide to give polyoxyethylene derivatives, which are much more hydrophilic than sorbitan esters. The monostearate derivative is known as polysorbate 60, the tristearate is polysorbate 65, and the monooleate is polysorbate 80. Polysorbates and sorbitol esters are used chiefly in ice cream. imitation dairy products, and in baking applications. Polyglycerol esters contain polymerized glycerol and various types and amounts of fatty acids. The polyglycerol portion is synthesized by heating glycerol in the presence of an alkaline catalyst. The polyglycerol backbone is then esterified either by direct reaction with a fatty acid or by interesterification with triglyceride fat. Sucrose esters are manufacturedby adding fatty acids to a sucrose molecule. Sucrose has eight free hydroxyl groups that are potential sites for esterification with fatty acids. Derivatives containing one to three fatty acid esters are emulsifiers and are approved for food use. There are a large number of other emulsifiers used in the food industry. but their volumes are negligible. Examples include lactylated esters, used in direct baking (notthe shortening) and in imitation dairy products, and propylene glycol esters, used i n various prepared mixes, shortening. and baking.

J.

Flavors

Flavors consist of concentrated preparations, with or without solvents and carriers, used to impart a specific taste to food. Flavor ingredients are the largest single group of direct food additives utilized by the food industry. They also represent the highest value among the food additives segments. Flavoring substances are classified as Naturalflavoringsubstance-obtained by physical separation, enzymatic processes, or microbial processes from vegetable or animal sources, either in the raw state or after processing (including drying, torrefaction, and fennentation). Nature-identicalflavoringsubstance-obtained by synthesisorisolated by chemical processes and chemically identical to substances naturally present in the vegetable or animal sources (this classification is used in Europe butnot allowed in the United States). Artificial flavoring substance-obtained by chemical synthesis and not found in nature. Flavoring preparation-products other than natural substances, whether concentrated or not, with flavoring properties, obtained by physical separation or enzymatic or microbial processes from material of vegetable or animal origin, either

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i n the raw state or after processing (including drying, torrefaction, and fernlentation). Process flavorings-products obtained by heating to a temperature not exceeding 180°C for a period not exceeding 15 minutes using a mixture of ingredients, not necessarily having flavoring properties themselves,of which at least one contains nitrogen (amino) and another is a reducing sugar. Smoke flavorings-smokeextractsusedintraditionalfoodstuff smokingprocesses. Flavor enhancers-some amino acids and nucleotides, as well as sodium salts (such as monosodium glutamate, sodium inositate, and sodium guanylate), have only a weak taste by themselves but have the power to considerably enhance the taste sensation caused by other ingredients in savory flavors.

The flavor industry is not a single homogeneous entity, but a composite of closely interrelated and somewhat overlapping sectors including essential oils and natural extracts, aroma chemicals, and compounded flavors. The first two sectors providethe raw materials used for compounding flavors. Essential oils are usually defined as the volatile aromatic material obtained from botanical or animal sources by the process of distillation, expression, solvent extraction, or maceration. The most common physical process used for removal of essential oils is steam or water distillation. to a material that has been removed from a plant by a The term “extract” refers solvent, after which the solvent is evaporated to concentrate the oil. Absolutes, which are alcohol-soluble liquids, and concentrates, which are usually waxy solids, are both extracts. Oleoresins are thick, viscous products obtained by extraction of plant material with a nonaqueous solvent (e.g., hydrocarbon)that is subsequently removed. Extractsof vanilla beans and other fruit extracts are the most important product examples of this class. Essential oils and natural extracts represent complex aroma mixtures containing hunto condreds of chemical constituents. They may be used for imparting scent or aroma sumer products or lnay be used as raw materials for compounding flavor and fragrance compositions, or they may be the source of isolated aroma chemicals, also used in compounding. Essential oils can be classified into three chemical groups: straight hydrocarbons, oxygenated compounds, and benzene derivatives. Aroma chemicals comprise organic compounds with a defined chemical structure thatareisolated from microbial fermentation, plant or animal sources, or produced by of organic synthesis. Isolation consists of the physical removal of the flavor compound interest from a natural source that contains it (e.g., L-menthol isolated from cornmintoil). Isolates may be further chemically modified. Aroma chemicals used to compound flavors are of two types: ( l ) isolates, which have been physically removed from natural sources that contain them and which may be further chemically modified; and(2) synthetic aroma chemicalsthat duplicate the structure and aroma characteristicsof their counterparts found in nature. Synthetic aroma chemicals that duplicate the structure and aroma characteristics of their counterparts found in nature are known as “nature identical.” Those that are not known to occur in nature but display an aroma reminiscent of known natural products with unrelated chemical structure are defined as “artificial.” However. the legal definition of natural and artificial varies, depending on each country’s legislation. Aroma chemicals are used as raw materials for flavor compositions. While technical merits are not at issue, naturally occurring aroma

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chemicals may enjoy preferential status for theiruse i n certain countries because of labeling regulations. More than 80% of aroma chemicals in use contain only carbon, hydrogen, and oxygen in their structure, the large majority being esters, ketones, aldehydes, and alcohols. A few contain nitrogen (nitro and nitrile groups, pyrazines) and/or sulfur (mercaptane, thiazoles). About 4% of the chemicals are unsaturated hydrocarbons, primarily with cyclic and acyclic terpene structures (e.g., limonene, pinenes, etc.). Mostof the aroma chemicals are oil-soluble, water-insoluble liquids. Aroma chemicalsof commerce can be broadly classified according to their chemical structure and are grouped into three categories as follows: Benzenoids (including naphtalenoids): chemicals containing a benzene or naphthalene ring, including alcohols, acids. esters, aldehydes, ketones, phenols, phenol esters, and lactones. Terpene and terpenoids: chemicals with (or closely related to) characteristic terpene structures, both acyclic and cyclic, having two or more isoprene (CiH,) moieties and oxygenated derivatives of the terpene hydrocarbons, including alcohols, aldehydes, ketones, and esters. Other aroma chemicals: includes aliphatic, alicyclic, and heterocyclic compounds and esters of lower fatty acids. Of the thousands of aroma chemicals includedi n compounded flavors. the following compounds are used in very large quantities: 3-phenetyl alcohol and esters, vanillin, ethyl vanillin, esters of lower fatty acids, benzyl acetate. alpha-hexyl-cinnamaldehyde, 1m e n thol (synthetic), geranioUnerol and esters, and anethol. The universally applicable definition of flavor compositions is that of mixtures of aromatic materials that are added to foods and beverages in order to improve palatability. Flavor compositions consist of complex mixtures of various aromatic materials from few to 100 or more constituents. Compounded flavors may contain aroma chemicals, natural extracts, essential oils, solvents, andin some cases other functional additives (e.g.. antioxidants,acidulants.emulsifiers,etc.).Certainrawmaterialsthatcanbeuseddirectly as flavors without compounding (e.g., vanilla, peppermint) and those products with a taste of theirown,such as sweeteners,acidulants,andsalts,arenotincluded i n theabove definition. Flavors serve all sectors of the food processing industry, including carbonated and still beverages, processed foods, confectionary, and dairy foods, and are added to foods and beverages for the following reasons: T o create a totally new taste To enhance, extend, round out, or increase the potency of flavors already present To supplement or replace flavors to compensate for losses during processing To simulate other more expensive flavors or replace unavailable flavors To mask less desirable flavors-to cover harsh or undesirable tastes naturally present in some foods Thetypes of flavor compositions,theirmanufacturingprocess andthestarting materialsformanufacturingthem,andtheircommonproductformaresummarized in Table 19.

Food Additives

507

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508

X.

ADVERSE EFFECTS OF FOOD ADDITIVES

The practice of adding chemicals (e.g., salt, spices, herbs, vinegar, and smoke) to food dates back many centuries. In recent years, however, the ubiquitous presence of chemical additives in processed foods has attracted much attention and public concern over the long-term safety of additives to man. Although the safety issue is far from subsiding, there is scientific consensus that food additives are indispensable in the production, processing, and marketing of many food products. Moreover, the judicious use of chemical additives-typically in the range of a few parts per million (ppm)to less than 1 % by weight of the finished food-contributes to the abundance, variety, stability, microbiological safety, flavor, and appearance of food. While food additives offer a major contribution to the palatability and appeal of a wide variety of foods, their level of use is relatively insignificantinthetotalhumandiet.Forthemostpart,thepermittedfoodadditivesaresafe, highly effective, and have been in continuous use for a long time. There is much discussion about whether a food additive or food product is natural or synthetic. The fact is that this classification, in many instances, has become somewhat arbitrary. Many food additives synthesized in chemical laboratories are also naturally occurring in normal food. Monosodium glutamate, a flavor-enhancing food additive, is the sodium salt of glutamic acid, an amino acid found i n many foods such as mushrooms and tomatoes and metabolized by the human body using the same biochemical pathways of digestion. Synthetic vitamin C (ascorbic acid) and its isomer, erythorbic acid, are the same chemicals that are found in citrus fruits. Similarly, citric acid, which is today produced commercially by enzymatic fermentation of sugars, is the same chemicallyas the naturally occurring chemical that has been found to make lemons and limes tart. Much of the worldwide public concern about the use of food additives relates to fearsaboutsafetyandhasgeneratedsomesort of regulatorystructure in every major country, as well as in international bodies, to monitor this aspect of the field. There is a Joint Experts Committeeon Food Additives, set up by the Food and Agriculture Organization and the World Health Organization, to consider the safety of additives and set specifications and limits for them. These limits take the form of an acceptable daily intake (ADI). The Codex Committee on Food Additives is required to follow the safety guidelines of the Joint Experts Committee. Its safety criteria are generallynot very different from those used in the United States, although they are not codified. In the United States, criteria for food additives are stated in 21 CFR 9170.22, “Safety factors considered,” and 21 CFR 9 170.20, “General principles for evaluating the safety of food additives.” The key sentence, which also runs through the decisions in other countries, says, “A food additive for use by man will not be granted a tolerance that will exceed I / 100‘” of the maximum amount demonstrated to be without harm to experimental animals.” apply It should be remembered, however, that in the United States, these criteria only to substances that are legally food or color additives and, by interpreting regulation, to thosesubstancesthatare GRAS on thebasis of “scientific procedures.” For those substances that are GRAS because of “experience based on common use in food,” there are no rules. Decisions of safety depend on the knowledge and judgment of the “experts qualified by scientific training and experience to evaluate its safety.” One other aspect of the safety question deserves discussion. In the United States only, there is a special provision, known as the Delaney clause, that says “no additive shall be deemed to be safe if it is found to induce cancer when ingestedby man or animal.”

Food Additives

509

This ~neansthat an additive is not to be permitted at any level, no matter how low, if it induces cancer at any level, no matter how high. The risks posed to the consumer by the food supply are rated in decreasing order of severity as follows:

1.

Microbiological hazards (food poisoning from bacteria or bacterial toxins salmonellosis, botulism, etc.). 2. Nutritional hazards (excessive consumption of sodium, saturated fat, etc.). in fish,lead from car exhaust, etc.). 3. Environmental pollutants (mercury 4. Natural toxicants (mushroom poisoning, solanine in potatoes and other solanaceous plants, shellfish toxins, etc.). 5. Pesticide residues (maximum residue levels are enforced in the United States but may exceed federal limits i n imported produce). 6. Food additives (documented cases of poisoning due to food additives are rare and were due to noncompliance with federal regulations). Although the risk to human health from food additives ranks the lowest anlong food hazards, some potential risks from food additives do exist.

A.

FoodAdditivesBanned from Use

In the United States, the FDA prohibited the use of a number of chemicals in foods for human consumption because they either present a risk to public health or have not been shown tobesafe by adequate scientific data. Table 20 lists the food additives that are presently prohibited from addition to food. Use of any of these substances causes the food to be in violation of FDA regulations. In the years since 1970, food colors, especially the synthetic dyes, have received tremendous publicity-nearly all of it bad. Color additives for food represent a unique and special category of food additives. They have historically been so considered in legislation and regulation. The current legislation governing the regulation and use of color additives i n the United States is the Food, Drug & Cosmetic Act of 1938, as amended by the Color Additives Amendment of 1960. This amendment allowed forthe provisional of scientific studies determining or temporarylisting of food colorants, pending completion the suitability of these colorants for permanent listing. Pharmacological testing of synthetic “certified” colors was initiated in 1957. Many of the synthetic colors that had been approved for use at some time i n the past have been removed from the approved list as a result of new toxicological test results. This has steadily reduced the number of certified dye colors available to the U.S. food industry from more than 22 in 1950 to 7 in 1999 (an additional color, FD&C citrus red no. 2, is permitted for coloring the skins of oranges that are not intended or used for processing, but it has not been produced in the United States i n recent years). Table 21 provides a history of the status of synthetic colorants in the United States. Another eight dyes are permitted in the EU, but are not permitted in foods in the United States. The EU works on a positive list system using EU Directive no. 95/2/EC, which is the general directive on food additives (other than colors and sweeteners that are covered in separate directives). Thislaw recognizes 106 food additives. If the additive is mentioned in the doctrine then it is allowed, if not it is forbidden. However, the directive includes a list of substances that cannot be used i n flavorings (Table 22).

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Table 20 FoodAdditivesProhibitedfromUseinHumanFood

Food additive

21CFR section Date ruling of

Calamus and its derivatives Cinnamyl anthranilate Cobaltous salts and its derivatives Coumarin Cyclamate and its derivatives Diethylpyrocarbonate (DEPC)

189.110 189.113 189.120 189.130 189.135 189.140

May 9, 1968 Oct.23,1985 Aug.12,1966 March 5 , 1954 Oct.21,1969 Aug. 2, 1972

Dulcin Monochloroacetic acid" Nordihydroguaiaretic acid (NDGA) P-4000 (5-nitro-2-n-propoxyaniline) Safrole Thiourea (thiocarbamide) Chlorofluorocarbon Flectol H ( I ,2-dihydro-2,2,4-trimethylquinoline Lead solders Mercaptoimidazoline and 2-mercaptoimidazoline 4,4'-methylenebis (2-chloroanaline)

189.145 189.155 189.165 189.175 189.180 189.190 189.191 189.220

Jan.19, 19.50 Dec.29,1941 Apr.11,1968 Jan.19,1950 Dec.3,1960 Mar. 17, 1978 Mar. 15, 1977

Flavoring compound Flavoring compound Foam stabilizer Flavoring compound High-intensity sweetener Ferment inhibitor in beverages High-intensity sweetener Preservative in beverages Antioxidant High-intensity sweetener Flavoring compound Antimycotic preservative Propellant Food packaging adhesive

189.240 189.250

June 27, 1995 Nov.30,1969

Can solder Packaging material

189.280

Dec,2,1969

Hydrogenated 4,4'-isopropylidenediphenolphosphite ester resins Tin-coated lead foil capsules for wine bottles

189.300

Feb.17,1989

189.301

Feb. 8, 1996

Packaging adhesive and polyurethane resin Antioxidant and stabilizer in vinyl chloride resins Capsule for wine cork

Functionality

-

"Pernutted i n food package adheslves wlth an accepted m~grat~on levcl up to 1 0 ppb under

4

175.105.

B. Industrial Chemicals Polychlorinated biphenyls (PCBs) and polybrominated biphenyls (PBBs) are toxic industhey trialchemicals.Because of their widespread, uncontrolled industrial applications, have become a persistent and ubiquitous contaminant in the environment. As a result, certain foods, principally those of animal and marine origin, contain PBCs and PBBs as environmental contaminants. PCBs are transmitted to the food portion (meat, milk, and eggs) of food-producing animals ingesting PCB-contaminated animal feed. In addition, a significant percentage of paper food-packaging materials contain PCBs, which may migrate to the packaged food. Therefore temporary tolerances for residues of PCBs as unavoidable contaminants are established by the FDA (21 CFR 3 109.15 and $109.30). The temporary tolerances for residues of PCBs are as follows: 1.5 ppm in milk (fat basis) 1.5 ppm in manufactured dairy products (fat basis) 3 ppm in poultry (fat basis) 0.3 ppm in eggs 0.3 ppnl in finished animal feed for food-producing animals

51 1

Food Additives Table 21 ChronologicalHistory of Certified Food Colors i n the United States Year listed for food

additive 1907 1907 1907 I907 I907 1907 I907

Nanw of certified food color

Red no. 1 Redno.2 Red no. 3

Orange no. 1 Yellow no. I Green no. 2 Blue no. 2

I959

Yellow 110. 5 Yellow no. 3 Yellow no. 4 Green 110. 1 Green no. 3 Red 110. 4 Yellow no. 6 Blue no. 1 Yellow no. 2 Orange no. 2 Red no. 32 Violct no. I Citrus red no. 2

1966 l97 I

Orange B Recl no. 40

1916 1918 1918

1922

1927 l929 I929

1929 1939

1929 1939 1950

Y car delistcd

1961 1976 .4-

I956

I959 I966 b I

1959 1959

1966 E

1976 E E

1969 1956 1956

I973

*

:%

:v :E E

2 ppm in animal-feed conlponents of animal origin, including fish meal and other by-products of marine origin and i n finished animal feed concentrates, supplements. and premixes intcnded for food-producing animals 2 ppm i n fish and shellfish (edible portion) 0.3 ppm in infant and junior foods 10 ppm in paper food-packaging material intended for use with human food or finished animal feed

C.

Food Allergies and Other Adverse Reactions to Food Additives

Although food allergy rarely constitutes a serious, life-threatening concern, it may result in chronic illness. As complete avoidance of the incriminated food is the best defense against adverse reactions. informationis of foremost importance. Food allergensidentified to date are prcdominantly proteins, although some may be polysaccharides. Despite the n d t i t u d e of additives used in foods, only a small number have been associated with advcrse reactions. Table 1.3 provides a list of the food additives that have been associated with adverse reactions.

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Table 22 EuropeanUnionList of Food Additives Prohibited for Usc in Flavorings

(Directive 95/2/EC) EU number E230 E23 1 E232 E233 E234 E235 E239 E242 E249 E250 E25 1 E252 E280 E28 I E282 E283 E284 E31 I E312 E315 E316 E579 E620 E62 1 E622 E623 E624 E625 E626 E627 E628 E629 E630 E63 1 E632 E63 3 E634 E635 E912 E9 14 E927b E950 E957 El 105

Compound Biphenyl, diphcnyl Orthophenyl phenol Sodium orthophenyl phenol Thiabendazole Nisin Nntamycin Hexamethylene tctramine Dimethyl dicarbonate Potassium nitrite Sodium nitrlte Sodium nitrate Potassium nitrate Propionic acid Sodium proprionatc Calcium proprionate Potassium proprionate Sodium tetraborate (Borax) Octyl gallate Dodecyl gallate Erythorbic acid Sodium erythorbate Ferrous gluconate Glutamic acid Monosodium glutamate Monopotassiurn glutamate Calcium diglutarnate Monoammonium glutamate Magnesium diglutalnate Guanylic acid Disodium guanylate Dipotassium guanyhte Calcium guanylate Inosinic acid Disodium inosinate Dipotassium inosinate Calcium inosinate Calcium 5’-ribonucleotides Disodium 5’-ribonucleotides Montan acid esters Oxidized polyethylene wax Caramide Acesulfame K Thaumatin Lysozy1ne

Food Additives

513

Burning of sulfur-containing coal has been used for centuries to preserve food. In addition, sulfite salts (sodium and potassium sulfite, bisulfite, or metabisulfite) are used as a sanitizing agent for fermentation containers and are added to a wide variety of food products, including dried fruits and vegetables, wine, shrimp and other seafood, and citrus beverages. Because of complaints about severe allergic reactions from asthmatic consuniers, in 1986 the FDA banned the use of sulfites in fresh cut fruits and vegetables, and sulfites must be listed on the label if a food product contains i n excess of I O ppm sulfite. The FDA estimates that about 1% of the U.S. population may be sulfite sensitive. However, among the asthmatic patient population, the sensitivity to sulfites is more prevalent, ranging from 2% to 5%. The antimicrobial food preservatives benzoate and paraben are believed to cause adverse reactions, such as asthmatic reactions i n some individuals. Benzoates occur naturally in certain berries and are usedin beverages. and their use is limited 0.1% to concentra-

Somogyi

514 Table 24 SelectedFoodAdditives Derived from Allergenic Food Staples Milk proteirl rlc,ri\wti\vs

Casein Caseinates Lactose Lactitol Whey Egg protcirl &riw~fi\v.s Albumin Globulin Livetin Lysozyme Ovalbumin Soyhem deriIdw.7

Hydrolyzed soy protein Hydrolyzed vegetable protein Natural flavoring Meat flavoring (natural) Lecithin Soy protein Soy concentrate Soy isolates WllcYrf &;\~rlti\~es

Gluten Vital gluten Starch Vital gluten Vegetable gum Vital gluten Cor11 dc.ri,uti\~es

Caramel coloring Corn sweetener Citric acid Dextrin Dextran Erythritol Food starch Gellan gum Lactic acid Maltodextrine Mannitol Modified food starch, vegetable gum Sorbitol

Xanthan gum

Food Additives

515

tion. Parabens are effective antioxidantsi n low-acid products. However, they are primarily used in cosmetic and pharmaceutical products and rarely in food. They have been implicated as a cause of eczematous or contact dermatitis reactions. Butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) have been linked to adverse reactions in a small number of individuals. These antioxidants are frequently used in fats and oils and in cereal products to retard rancidity. Aspartame (L-aspartyl-L-phenylalanine methyl ester), a widely used artificial sweetener, is a dipeptide. Humans metabolizeit. Aspartame has been citedas the most frequently complained-about product. Soft drinks havebeen mentioned most often as the aspartamecontaining product, and headaches are the common reaction reported.In the United States. the FDA requires that aspartame-containing products include the following label declaration: “Phenylketonurics:containsphenylalinine.”Also, in the EU countries, thelabel declaration “contains a source of phenylaniline” is required. Monosodium glutamate (MSG) isused as a foodadditivebecause of itsflavorenhancing properties. The most commonly reported adverse reaction associated with MSG consumption is Chinese restaurant syndrome. Symptoms of the Chinese restaurant syndromeincludenausea,headache,sweating,thirst,facialflushing, and abdominalpain. These symptoms typically occur 15-30 minutes after consuming food containing a large amount of MSG. A Chinese food meal may contain from S to I O g of MSG. Among the coloring agents used in thefood industry, tartrazine(FD&C yellowno. S ) has most often been implicated as a cause of allergies, especially urticaria and asthma. Respiratory problems subsequent to tartrazine ingestion have been reported by several sources. Tartrazine produces a bright yellow color and it is used in a variety of beverages, bakedproducts,confectionaries,dessertmixes,etc.Tartrazineisalsoused to produce other food colors such as green, maroon, and rust. Lack of yellow color, therefore, is not a guarantee of tartrazine’s absence. Thus the FDA requires that FD&C yellow no. 5 be specifically stated by name on food ingredient labels. Food colorings other than tartrazine (listed in Table 23) have alsobeen implicated as causing adverse reactions in some individuals.

D. Food Additives Derived from Allergenic Food of allergic reactions. In adults, these foods include nuts, peanuts, fish, and shellfish. In children, the main culprits include eggs, milk, peanuts, soy, wheat, and fish. Elimination of these foods from the allergic individual’s diet is essential. However, many food additives are derived from these basic food items, and the allergen compound may be carried over even into highly refined derivatives. Recognition of the presence of such potentially allergenic compounds is sometimes difficult. In Table 24, selected food additives derived from allergenic natural food sources are listed. Recognition of these additives is crucial to avoid potential health hazards to consumers sensitive to certain foods.

A few foods are responsible for the majority

BIBLIOGRAPHY AT Brannen. Food Additives. New York: Marcel Dekker, 1980. Code of Federal Regulations, “Title 21“Food and Drugs. Subchapter B: Food for Human Consumption. Parts 100-199. Washington. DC: U.S. Government Printing Office, 1998.

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MDrozenandTHarrison.Structure/Function Claim forFunctionalFoodsandNutraceuticals. Nutraceut World 1 : 18-20, 1998. FJFrancis. Colorants. St. Paul, MN: American Association of Cereal Chemists, 1998. TE Furia. Handbook of Food Additives, 2nd cd. Boca Raton, FL: CRC Press. 1980. M Clicksman. Food Hydrocolloids. Boca Raton. FL: CRC Press. 1982. YH Hui. Principles nndIssuesin Nutrition. Monterey, CA: Wadworth Health Scienccs Division, 1985. Institute of Medicine. Food Chemicals Codex, 4th cd. Lancaster, PA: Tcchnomic Publishing. 1996. 1989. RJ Lewis, Sr. Food Additives Handbook. New York: Van Nostrand Rcinhold, DD M e t ~ ~ l fHA e . Sampson, RA Simon. Food Allcrgy: Adverse Reactions to Foods and Food Additives, 2nd cd. Cambridge, MA: Blackwell Science, 1996. TNagodawithanaandGReed.Enzymes i n FoodProcessing.SanDiego,CA:AcademicPress, 1993. P Newberne. GRAS flavoring substances, 18th list. Food Techno1 52(9):65-92, 1998. L O’Brien Nabors. RC Geraldi. Alternative Sweeteners. 2nd cd. New York: Marccl Dekkcr, 1991. JE Perkin. Food Allergies and Adverse Reactions. Gnithcrsburg, MD: Aspen Publishers. 1990. G Reineccious. Source Book of Flavors, 2nd ed. New York: Chapman & Hall, 1994. LD Roscnberg, LP Somogyi. The U.S. FoodIndustry in thc1990s.BIPreportD96-2033.Menlo Park, CA: SRI Consulting, 1996. LP Somogyi. Direct food additives in fruit processing. In: LP Sotnogyi. HS Rarnaswamy, YH Hui, eds. Processing Fruits, Biology, Principles. and Applications. Lanccster, PA: Tcchnornic Publishing,1996. LP Somogyi. The flavour and Fragrance industry. Chetn Ind March 4:170-173, 1996. LP Somogyi, H Janshekar. Y Ishikawa, S Bizzari. Food additives.In: Spccialty Chemicals forStratcgies and Success. Specialty Chemicals Handbook. Mcnlo Park. CA: SRI Consulting. 1996.

16 Analysis of Aquatic Contaminants

Introduction S 17 General Consitlelations 5 1 X A. Sampling 518 B. S m p l e storage 518 C. Quality :wurancc 5 18 Ill. Organics 519 A. Introduction 5 19 B. Extraction 520 C. Cleanup 522 D. Analysis of particular organics 524 E. Determination of tainting 526 IV. Inorganics and Organomctallics S26 I.

11.

Introduction 526 B. Matrix dcstruction 527 C. Analysis 527 Rel'erenccs S29

A.

1.

INTRODUCTION

A single chapter cannot provide a complete guide to the analysis of all of the comnlon contaminants of seafood; this chapter is therefore intended as an introduction to the enornlously complex matter of analyzing contaminants in food and to provide some direction for performing these analyses. The considerationsof contaminants in seafood derive from real orperceivedpublichealthconcerns.Knownpublichealthconcernsaregenerally regulated by government agencies whoset allowable limits. Such regulations are generally based on the toxicity and the safety margin of the contaminant and on the expected daily consumption of the particular food. Analysis of contaminants in seafoods is done for two reasons: (a) routine monitoring for compliance purposes (food safety), and (h) for research to determine the effects on various organisms and the expected daily dietary intakeof contaminants by humans. Routine monitoring of seafood safety is meant to identify excessive amounts of contaminants 51 7

Kiceniuk

518

i n batches of seafood and requires analytical methods capable of quantitation at the regulated levels. The analytical methods used for research need to have lower detection limits than those used for monitoring of food safety, since it is necessary to quantitate contaminants not only near the regulated levels but also to quantitate existing levels of contaminants in samples in order to do statistical analysis of the data or for the calculation of expected daily intakes of contaminants.

II. GENERAL CONSIDERATIONS A.

Sampling

One of the first considerations after deciding to analyze a seafood for contaminants is to in individual organisms can vary take the samples for analysis. The contaminant levels considerably (1). Season, food availability, temperature, and other conditions contribute to the variability. It is essential to consider the purpose of the undertaking. If it is desirable to understand sources of individual variability, it is necessary to take many (20-30) Samples of individuals; if onthe other hand thedrivingreasonfortheanalysisisone of consumer health, pooled samples will provide the required answers. However, since seafoods are either from individual organisms or from groups of individuals, precautions must be taken to ensure that the samples are representative of the particular food in question. This can be done by taking pieces of individuals selected at random from a batch of the seafood (in the case of large organisms) or whole organisms (if small), grinding or otherwise homogenizing the resulting pooled sample, and retnoving subsamples of the homogenate for analysis.

B. Sample Storage Having decided on an appropriate sampling strategy, the samples must be stored so as to prevent deterioration of the food or the contaminant, or contamination of the sample during storage. Freezing is the preferred method to prevent spoilage of samples and is generally the most common storage method, but it is best to check with the analyst for conditions for the specific contaminants of interest and for suitable storage containers to prevent contamination of samples during storage (2).

C. Quality Assurance Assuring the quality of measurements is a critical concern, as it is central in determining the validity of conclusions drawn fromthe data. All aspects of analysis must be evaluated, not only the measurement of the analyte. This chapter is not concerned with the levels of contaminants in living organisms, but rather the levelsof contaminants in the organism as it is consumed or presentedto the consumer. The distinction may be trivial for persistent contaminants, but can be very important for any contaminants that are sensitive to pH or redox changes, or subject to enzymatic alteration or to redistribution postmortem. The number of samples, the method of sampling, and the handling of the samples can have considerable impact on the resulting data quality. Many laboratories are now acredited, meaning that they have proved their ability to controlthe analytical processes within their laboratory. The processes outside their facility may, however, compromise the data quality if due consideration is not given to all aspects of the analysis. A detailed account of all

Analysis of Aquatic Contaminants

519

aspects of quality assurance can be foundin the text of Taylor (3) and many works dealing with detailed application of the principles (2,4-6). There are two aspects of analysis that are often confused; these are accuracy and precision. Consider, for a moment, an individual shooting at a target. The individual in question attempts to hit the bulls-eye repeatedly.If after say 10 shots the target isexamined and all of the shots fall in a small area, the shooter can be said to be precise. Notice that no mention has been made as to what part of the target was hit. If the (small) pattern of shots matches the intended mark, the bulls-eye i n this case, the shooter can be said to be accurate as well as precise. The two aspects are quite different, as are solutions to problems with either. In the case of a lack of precision, it is necessary to find the source of the variability. A problem with accuracy can be corrected by appropriate calibration of the method. Accuracy can be assured by the analysis of appropriate certified reference materials.

111. ORGANICS A.

Introduction

This is a very diverse group of compounds. Those compounds considered in this chapter include: highly volatile compounds that can impart off-flavors or taint seafoods, aromatic hydrocarbons, a number of groups of persistent industrial chemicals, of which there are thousands of individual congeners, as well as pesticides and their persistent metabolites. Some examples of groups of compounds of interest include: aromatic hydrocarbons, also referredto as polycyclicaromatichydrocarbons(PAHs),polychlorinatedbiphenyls (PCBs), polychlorinated terphenyls (PCTs), polychlorinated naphthalenes (PCNs), chlorinated dibenzo-p-dioxins (PCDDs or “dioxins”), chlorinated dibenzofurans (PCDFs or “furans”), chlorinated pesticides (e.g., DDT, mirex, toxaphene, hexachlorocyclohexane, As is evident from cyclodienes), chlorinated cthers, chlorophenols, and n-nitrosamines. this list, chlorinated compounds are the largest group; this is due to the tendencyof highly chlorinated compounds to persist in the environment and to accumulate in organisms. Brominated analogues of many persistent chlorinated compounds werealso manufactured andarefoundintheenvironmentand i n marine organisms (7-16), although inlesser amounts than the chlorinated counterparts. Those organic compounds that tend to persist in the environment, althoughthey are a very diverse group, share some common properties. They have low polarity and thus tend to be much more soluble in the lipidsof organisms than in water; as a result they tend to bioaccumulate. Although these compounds are not highly volatile, they are sufficiently volatile to vaporize from soils, particularly in warm climates, and be transported by air currents over very long distances (17-20). For example, toxaphene was reportedly the most abundant organochlorine in lake trout in the Great Lakes (21) and in Arctic fish (22), even though it was never used in the Arctic or the Great Lakes watershed. Organisms have considerable capabilityto metabolize foreign compounds and either recyclethecarbonorexcretethemetabolites.Many of thechlorinatedenvironmental contaminants are metabolizedvery slowly due to the presence of halogens or other groups that interfere with the binding of degrading enzymes with the particular contaminant. In the environment, abiotic processes such as photodegradation also play an important role in the degradation of many compounds. The compoundsthat tend to persist in the environment do so because of their chemical structure, which enables them to resist degradation

520

Kiceniuk

by biological and environmental processes. As a result they accumulate in organisms and circulate through the food chain and the environment. The total complement of halogenated compounds that are extractable with organic solvent are called extractable organohalogens (EOX). These compounds canbe those containing chlorine (EOCI), bromine (EOBr), or iodine (EOI). The quantitatively predominant ones are the EOC1. Although EOCl is the group about which the most is known, 85-95% of the chlorine that is extractable is on compounds that are as yet unidentified (23,24). The 5-15% of the EOCl that is known constitutes the pesticide and industrial chemicals commonly referredto as organochlorines. Some pesticides and industrial chemicals are known to be metabolized to an oxygenated form (hydroxyl or carboxylic acid). These, being more polar than the original compound, are mostly excreted by organisms. Some of these compounds can. however, enter lipid synthesis pathways andcan be conjugated to lipids. Such compounds are more lipophilic than the parent compound; they are of larger size and are likely to be retained in body fat (25-29). only The reported accumulation of such a conjugate in humans is that of pentachlorophenol-palmitate in human body fat (30). However, since these larger compounds are of the same size as lipids (and are, in fact, lipids) theywouldberoutinelydiscarded with other troublesome lipids during sample clean-up, and it is therefore likely that they are undocumented for lack of looking rather than because of lack of presence. As a group, EOCl is no doubt the largest group of organic contaminants in marine organisms, as well as being the least understood. Although there are about 2500 documented naturally produced halogenated compounds (3l ) , there are no naturally produced organohalogens that bioacculnulated or are knownto be persistent environmental contaminants. Some fractions of lipids containing high levels of EOCl are known to be toxic in animal assays (32), and as a result it seems prudent that these materials should be investigated further. N-nitroso compounds are a major group of chemical carcinogens (33). Although they are not found in living fish or shellfish, they can be formed during processing, storage, or cooking and may therefore be present in seafood as it is consumed.

B. Extraction Most seafood samples contain organic contaminants, at microgram per gram or less levels, in a complex matrixof proteins, lipids, water, and minerals. Modern instruments generally havesufficientsensitivitytodetectevenlowerlevelsofcontaminants,butthematrix components cannotbe introduced into analytical instruments and as a result it is necessary to extract the organic contaminants from the matrix and to further remove any undesirable components (cleanup) prior to analysis in order to prevent interference with the analysis. Highly volatile contaminants, such as the odoriferous compounds that can cause tainting, can be removed from seafood by steam distillation (34,35), or by purging a macerated sample with aninertgasandtrapping the contaminantsforanalysis (36,37). Another approach is that of headspace analysis (38) in which a macerated sample is sealed in a vial, heated to volatilize the contaminants of interest, and vapor in the headspacc gas is analyzed by gas chromatography or other means. For all other types of organic contaminants, an extraction with solvent (generally a nonpolar one) must be used. Many different solvent systems havebeen reported, ranging from supercritical carbon dioxide to the more common petroleum ether, hexane and hexane-acetone, and hexane dichloromethane mixtures (39-41). Different solvent systems are favored for different groups of contaminants

Analysis of Aquatic Contaminants

52 1

due not only to the need to extract the particular contaminant reliably but also to prevent breakdown of the contaminants (41-43). Whatever the solvent, extraction can be accomplished only if the sample is suitably macerated. This is generally accomplishedby grindingthe sample first or by blending the sampledirectly in theextractionsolvent.The blending not only macerates the samplebut also provides energyto speed up the extraction process. An alternative procedure is cold column extraction in which a macerated sample is mixed with anhydrous sodium sulfate (to remove water), packedin a glass column, and extracted with solvent passed down the column. Methods of solvent extraction of aromatic hydrocarbons havebeen reviewed by Dunn (44), Griest and Caton (45), Kiceniuk and Holoubek (41), Vassilaros et al. (46), and Zitko (47). Themost commonly used procedure for the extraction of these compounds from seafood is alkaline hydrolysis of the sample followed by extraction with a nonpolar solvent such as hexane, pentane, isooctane, cyclohexane, diethyl ether, benzene, or a mixture of solvent (41). Dichloromethane is a good solvent for aromatic hydrocarbons and has the added advantage that it is less prone to the formation of stable emulsions than solvents such as diethyl ether (41). Emulsion formation causes analytes to be lost during subsequent steps, thus increasing variability in the analysis. The formation of emulsions can be reduced by acidification (48) and phase separation can be improved by addition of salt (46). The sample can be ground with anhydrous sodium sulfate and column extractedwithmethyl-t-butylether/dichloromethane(49)orwithdichloromethane in a blender or tumbler (50,51). Other methods such as Soxhlet extraction and various combinations of methods are also used (41). Phenols and halogenated phenols are found in tissues in the free form as well as conjugates of sulfate, glucuronide, and fatty acids (30). The sulfate esters and glucuronides are water soluble, whereas the fatty acid conjugates are lipophilic. Therefore in order to extract all of the phenols it is first necessary to hydrolyze the conjugates prior to solvent extraction or steam distillation. The procedures for the removal and analysis of phenols are reviewed by Benvenue and Beckman (52), and Dougherty (53). The most conmon methods for hydrolysis of phenolic conjugates reported in recent literature are those using enzymes (53-55). Acid hydrolysis using mineral acids, such as 10% sulfuric acid, has been found to be equally successful (56.57). After deconjugation the phenolic compounds are removed equally effectively by either steam distillation or solvent extraction (53,56). Numerous solvent systems have been used for the extraction of chlorinated ethers (58). The methods used are similar to those for the extraction of polychlorinated dibenzop-dioxins and polychlorinated dibenzofurans (58). The tissue is first homogenized with sodium sulfate followed by Soxhlet extraction with a mixture of hexane/acetone/diethyl ether/petroleum ether (59-62) or, alternatively, the homogenate is packed in a glass column and extracted with dichloromethane (63-65). Alkaline hydrolysis and other methods are also used (58). There are about 750 pesticides, metabolites, and organic impurities that are commonly monitored in food (40). Dueto the similar (lipophilic) natureof these residues there has been a trend to thedevelopment of so-called multiresidue methods for the extractionof groups of similar compounds. Residues can be extracted from tissue by any of the methods previouslydiscussedforremoval of other lipophilic materials. Allextractionmethods coextract lipids which must be removed prior to analysis of most residues (see cleanup). One recently developed method, called matrix solid-phase dispersion (MSPD), combines extraction and clean-up into one procedure. In the MSPD procedure the tissue sample is blended with octadecyl derivatized silica (C,J, transferred to a column prepackcd with

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Florid, and the residues are eluted with methanol or acetonitrile (66-68). There are presently about five methods in use for the isolation and subsequent detection of 200 residues (40). Dibenzofurans are unstable at high pH, therefore alkaline hydrolysis of samples to break down the matrix is of limited use in the extraction of chlorinated furans and dioxins (43). Acid digestion with hydrochloric or sulfuric acid is preferred for matrix breakdown. Often the acid digestion is combined with a solvent such as hexane, pentane, or toluene to extract the dioxins and furans at the same time as doing the digestion (69). Seafood by mixing with having a low fat content can be freeze dried and subsequently extracted ether/acetone/hexane/diethyl dichloromethane(70).Soxhletextractionwithpetroleum ether has been used to extract dehydrated fish holnogenate as well (7 1 ). Homogenate mixed with sodium sulfate can also be packed in a column on top of material used for cleanup, thus combining theextractionandfirststage of cleanup (72). For a detailed review of procedures for the extraction of dioxins and furans see Clement et al. (69). Polychlorinated terphenyls and naphthalenes can be extracted from homogenized tissue with a variety of cold solvents (73-75). Steam distillation and extraction of condensate with hexane (76,77), Soxhlet extraction(78,791, and acid (80) or base (80-82) digestion followed by solvent extraction are also used (75). The extraction of polychlorinated biphenyls (PCBs), has been reviewed by Metcalfe (83). Cold column extraction methods have lower recoveries of PCBs than the more popular Soxhlet methods (84). Saponification followed by extraction with isooctane was used for isolation of PCBs from lobster tissue (85). Extraction by blending with a solvent is also used (83). Supercritical fluid extraction methods have been developed (86,871 and will probably become more prevalent. Volatile nitrosamines are extracted from homogenized food by a multistage extraction using ammonium sulfamate in water, followed by dichlorolnethane (33). For extraction of nonvolatilenitrosaminesthehomogenizedfoodsarnpleismixedwithCelite, packed in a glass column, and extracted with pentane followed by ethyl acetate. The first 225 ml of ethyl acetate eluate is discarded and the last 300 nd is used for analysis ( 3 3 ) . Extractable organohalogens are extracted from tissue by blending with combinations of solvent suchas hexane/isopropanol(50/50)(32,88), acetonelhexane (2/1) (24) acetone/ cyclohexane (2/1) (89). The extraction is repeated, the solvent layers pooled and evaporated, and the solvent exchangedto hexane (24) or cyclohexane(89). The resulting extract is then washed with distilled water (24,88,89) acid, or salt solutions (32,90) to remove halides prior to neutron activation analysis of the organic phase for chlorine, bromine, and iodine.

C. Cleanup Methods for the cleanup of extracts prior to analysis of organic residues can be grouped according to the following mechanisms: Separation by molecular size Gel permeation Dialysis Absorption Liquid-liquid partition Chemical degradation of lipids

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Often a number of these lnechanisrns are required to remove all of the interfering materials. Since lipids form the bulk of the material extracted from tissue, they are usually removed first. Most lipids have molecular weights of 500-1500, whereas the contaminants con.1Inonly analyzed are smaller. Gel permeation chromatography and dialysis methods separate moleculesby size. Bothof these methods can handle the large amount of lipids present in Some seafood and provide nearly complete recovery of the contaminants. Gel permeation can by automated readily and is a common method in analytical laboratories. Dialysis, using polyethylene membrane in organic solvent, is slower than gel permeation chromatography but uses much less solventto fractionate the same amount of material. Dialysis has been used for the preparation of large amounts of COlltaminant fraction for tOXiCOlOgical work (91) as well as for analysis (92-94). Sterols present in many seafood samples will pass through the membrane and mustbe removed prior to most analytical procedures (91). Adsorption chromatography is a commonly used method for cleanup of more than 400 contaminants in food (95), including organochlorine compounds (75) and aromatic hydrocarbons (41). Chemical degradation of lipids by the addition of concentrated sulfuric acid to extracts in hexane is a simple and time-proven method of cleanup of extracts for the analysis of organochlorine compounds such as PCBs and DDTs. The recoveryof compounds after sulfuric acid cleanup has been examined in detail for 39 organochlorine contaminants a Florisil cleanup to that of the sulfuric (96). The same study compared recoveries for acid method fora variety of contaminantsin certified reference materials, including lyophilized fish, and reported comparable results for organochlorines (96). Dioxins and furans are amongthe most toxic contaminants and are foundin seafood at several orders of magnitude lower levels than other organic contaminants. The very low levels of these contaminants, together with the fact that many compounds interfere with the detection and quantitation of dioxins and furans, requires that not only lipids but also the interferents be removed priorto analysis. Lipids constitute the bulkof the material in the extract and are removed by gel permeation chromatography, adsorption on silica gel, or by washing the extract with concentrated sulfuric acid (69). The rigorous removal of interferents prior to analysis is a time-consuming but essential process. Such a cleanup requires from three to five columns (69), depending on the procedure used. For a detailed comparison of methods and a complete review of dioxin and furan analysis see Clement et al. (69). After lipid removal by gel permeation or other means, polychlorinated naphthalenes can be further cleanedup on charcoal (78) or charcoal on polyurethane foam (75) columns to remove compounds such as nonplanar PCBs and other compounds that interfere with the analysis of polychlorinated naphthalenes. Extracts for the analysis of polychlorinated terphenyls can be cleaned up on a silica cartridge with n-hexane (97). Methods for removal of lipids and organochlorine interferents from extracts prior to analysis of PCBs have been reviewed by Erickson (73) and Metcalfe (83). Gel permeation is perhaps the most widely used initial cleanup procedure. Sulfuric acid treatment following GPC has been reported for congener-specific analysis of PCBs in lobster hepatopancreas tissue (85). Methods for the cleanupof samples for analysisof chlorinated pesticides have been reviewed (40,98). Because of the very large number of compounds in this category there are a wide variety of methods for cleanup of samples prior to analysis. Since, i n most cases, a number of pesticides are of interest (multiresidue methods), sample cleanup and initial fractionation are combined to provide fractions such as PCBs and DDE, toxaphene

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and DDT, etc. The initial fractionation is done on columns such as silicic acid, alumina. or Florisil (40,98). Depending on the particular application, the initial fractions may require further cleanup stepsby different columns or other separation methods. For example, nitration has been used to remove compounds other than toxaphene in the analysis of samples for the toxaphene group of compounds (98-100).

D. Analysis of Particular Organics Nitrosamine compounds were first determined (in the 1960s) by thin-layer chromatography (101,102), and by gas chromatography (GC) usingan electrolytic collductivity detector (103). Alkali flame ionization and the more selective Coulson electrolytic conductivity detectors were used in the early to mid-1970s (104). The thennal energy analyzer (TEA), which was developed specifically forthe analysis of N-nitroso compounds, provides high sensitivityandselectivityforthedeterminationofnitrosaminesand is thedetector of choice. In the TEA breaks, N-NO bonds to produce a nitrosyl radical, which is then reacted with ozone to produce exited nitrogen dioxide. Nitrogen dioxide emits therlnal energy (33) which can be detected and measured. This detector canbeused for GC or high performance liquid chromatography (33). Extractableorganohalogensareanalyzed by neutronactivationanalysis.This method detects all chlorine, bromine, and iodine regardless of the chemical bonding state of these elements and, sincethe analysis is done on organic extracts from which all halides have been removed, it measures all of the organohalogen present i n the extract. The sanv ples, typically about 750 p1 of extract or comparator standards, are irradiatedin a neutron fluxto produceradionuclides of the halogens (activation). The irradiated samples are opened in a fume hood and a 500 p1 portion of the contents is transferred to a fresh 1.2 1111 polyethylene vial to eliminate theneed for a vial blank. The new sample vial and contents are then decayed for 5 min and counted for 30 min. The 443 keV, 617 keV, and 1642 keVpeaks of ‘?‘I, ‘“Br, and 3xC1areusedtoquantitatetheiodine, bromine, and chlorine content of the samples. The absolute detection limits are typically 3 ng for iodine, 6 ng for bromine, and 30 ng for chlorine (89). In order to determine individual organic contaminant compounds it is necessary to either use a detection and quantitation method that is highly specific and not subject to interference from other materials or to first separate the individual compounds and then quantitate them. The only detection methods that can be used to identify and quantitate individualcompounds in mixturesareShpol’skiifluorometryfor the determination of some of the aromatic hydrocarbons (41,105,106) and immunoassay. Immunoassay uses antibodies that are specific for a particular compound to quantitate the compound in question. Methods of immunoassay are available for a variety of contaminants, and in some cases provide sensitivity comparableto mass spectrometric methods (107). This methodology has considerable potential for automation and more widespread use particularly for initial screening of large numbers of samples. For all other detection methods, the components must first be separated. The separation process is generally started during cleanup with silica, Florisil, or various combinations of columns. Each fraction from such a column or combination of columns contains a large number of contaminants of similar properties. The chromatographic method that provides the highest resolution of organic compounds is gas-liquid chromatography using wall-coated open tubular columns. The “capillary” columns used for separation of chlorinated pesticides and chemically similar industrial chemicals are

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typically 10-60 In long and 0.2-0.32 mm inside diameter coated with a moderately polar material to a film thickness of about 0.25 pm. The specific column types used for PCBs have been reviewedby Metcalfe (83). Clementet al. (69) have reviewed the gas chromato(58) reviewed the analygraphic separation of dioxins and furans. Paasivirta and Koistinen sis of chlorinated ethers. Columns for the separation of aromatic hydrocarbons have also been reviewed (41), as have those for the analysis of phenols and chlorophenols (53), n-nitrosamines (33), polychlorinated terphenyls and chlorinated naphthalenes (75), and chlorinated pesticides (40). The detector types used for the detection and quantitation of chlorinated pesticides and other compounds are electron capture detectors, electrolytic conductivity detectors, atomic emission detectors, mass spectrometry, and tandem mass spectrometry (mshns). Electron capture and electrolytic conductivity detectors are sensitive to contamination and fouling. They have linearity and overload problems as well, and are unable to discriminate among the halogens (40). These detectors are, however, cheaper than mass spectrometers and remain the methods of choice for routine monitoringof chlorinated pesticides i n large numbers of samples. Atomic emission detectors can detect various elements of interest, but largely due to their expense, havenot gained wide use.Mass spectrometry has become the method of choice for analysis of organic contaminants. This is due to the ability of mass spectrometry to not only quantitate the individual compounds after separation by high-resolution gas chromatography but also to confirm the identification of the compound. When usedinthenegativeion chemical ionization mode, mass spectrometry provides extremely high sensitivity for polychlorinated aromatic compounds while being insensitive to other potentially interfering compounds (74,78). The useof mass spectrometry for the determination of dioxins and furans has been reviewed (108,109). Low-resolution mass spectrometers are often used for screening of samples for high levels of dioxins and furans. When more critical measurements have to be made, however, the more expensive highto resolution machines must be used to obtain the lower detection limits necessary, and provide more freedom from interferences(75). Tandem mass spectrometry provides about I O times higher detection limits than high-resolution mass spectrometry but is capable of quantitation of dioxins and furansin samples with less stringent cleanup than that required forhigh-resolutionmachines (75). Iontrap mass spectrometryhas also beenused for analysis of chlorinated pesticides ( 1 IO, 1 1 1) and polycyclic aromatic hydrocarbons (1 12). High-performance liquid chromatography (HPLC) as well as high-resolution gasliquid chromatography are used for the separation and analysis of aromatic hydrocarbons (41).The detectors used for determination of aromatics by HPLC are ultraviolet absorption detectors or fluorescence detectors. With gas chromatography, flame ionization detectors may be used for routine screening of samples. Most determinations of aromatics are now done by gas chromatography (GC) with some type of mass spectrometric detection (41). Single-ion monitoring (SIM) can be used to filter out interfering spectra and focus on the determination of specific compounds, thus increasing the peak response ( 1 13). The GCSIM parameters for a large number of aromatic hydrocarbons and heterocyclic aromatic hydrocarbons have been published ( 1 13,114) and together with retention indices can be used for the routine identification of these compounds (41). Toxaphene is the trade name for an insecticide that was produced starting in the mid-1940s by the chlorination of camphene. This process results in a mixture consisting of some 200 congeners, many of which have enantiomers (1 15). About half a million metric tons of toxaphene were produced ( 1 16) and presumably used and released to the environment. Although its use was banned in the United States in 1982 (98). its use has

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continued in some countries. Many of the compounds in toxaphene are highly persistent, and even in remote parts of the northern hemisphere, such as the Arctic and the Atlantic, toxaphene is the most or second most common organochlorine contaminant (22,116). The of the analysis of individual compounds in toxaphene is so complex thattheanalysis enantiomers of some of the chiral forms was first reported in 1994 ( 1 1 S).

E. Determination of Tainting Since tainting is by definition “the presence of an objectionable odor or flavor in a food,” it is based on human perception (of what is objectionable). Tainting in the strict sense must therefore be determined by human senses. This is doneby presenting known (clean) samples as well as suspected samples to groups of individuals in one of a number of presentation formats and asking the individuals to evaluate them (taste, smell, or both). The results are then tallied, evaluated, and compared to statistical tables. For a detailed examination of the determination of tainting see Botta ( 1 17). Tainting substances canbe accumulated by organisms and remain in subsequent products or they may originate from packaging materials or other process-related sources ( 1 18). Many types of compounds have been shown to impart undesirable odors or flavors to seafood ( 1 18- 12 1 ). In those cases where a particular compound is identified as a cause of tainting it can be analyzed by instrumental methods. Since these are rather volatile compounds, they can be isolated by steam distillation, purging, or direct headspace analysis. Separation is generally by gas chromatography, with detection by flame ionization or sniffing of the components as they come off the column. For a detailed account of the determination of oderiferous materials, see the review by Karahadian and Lindsay (35). A semiconductor sensor array instrument is now on the market for the characterization of volatile molecules (122,123). Although this instrument is primarily sensitive to polar compounds like the spoilage products of seafood, other sensor arrays maybe developed that will be capable of quantitating other materials, such as tainting compounds.

IV.

INORGANICS AND ORGANOMETALLICS

A.

Introduction

The elements of general interest as contaminants are cadmium, selenium, lead, mercury, and arsenic. Selenium is an essential trace element, but is toxic at high doses. The margin between nutritional levels and toxicity is small( 1 24,125). In seafood, selenium is of interest not only for its potential toxicity but also for its protective role against arsenic, cadmium, and mercury toxicity ( 124,126). Four oxidation statesof selenium have been documented in nature, as well as dimethyl and diethyl organic forms (124). The species of selenium differ in their absorptive ability ( 127) and in their ability to protect against mercury toxicity ( 1 24). It is therefore necessary to analyze not only the total selenium but to also measure the individual species in some cases. All aspects of the analysis of selenium are reviewed by Cappon (124). Mercury is a relatively rare element on earth, but is ubiquitous in the environment due to the volatility of elemental mercury and to pollution. Since mercury is present in seafood mostly in organic form, and mostly as methylmercury (the most toxic form), many investigations of mercury in seafood determine total mercury and assume that all mercury is methylmercury (the worst-case scenario). Theidentification of the specific form (specia-

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B. Matrix Destruction Most analytical methods for the determination of metals require that the sample be in liquid form; therefore the matrix of seafood must be destroyed or otherwise solubilized prior to analysis. Methods such as neutron activation analysis of freeze-dried ( I 34,135) or homogenized samples are done routinely, and energy dispersive X-ray fluorescence spectrometry can be done on freeze-dried samples ( 1 36). Some high-temperature analytical methods such as inductively coupled plasma or flame atomic absorbance spectrometry Hocan tolerate incomplete sample digestion (as long as the material is homogeneous). mogenized slurries of food material can be analyzed by atomic absorbance spectrometry ( 137). For the other methods of analysis the matrix is destroyed by acid digestion or ashing to provide a soluble material. Voltometry or spectrophotometry methods require more complete digestion of samples ( 1 38). Dry ashing consists of heating the sample in the presence of air to first dry it out and then to oxidize the dried material. The remaining ash is then dissolved and analyzed. This method cannot be used for the preparation of samples for mercury analysis, as most forms of mercury are volatile enough to be lost. Cadmium, aluminum, selenium, and lead chloride are also volatile and require precautions to prevent the formation and loss of halides (2). For a review of digestion procedures see Taylor et al. ( 137) and Novozamsky et al. (138).

C.

Analysis

All aspects of methods for the analysis of mercury ( 1 39), cadmium ( 1 28), lead (140), arsenic (141 ), selenium ( I 24), and tin (142) have been reviewed (2). Recently, atomic spectrometry methods for the analysis of all metals and elements of interest have been reviewed ( 137). 1. Total Content Most of the samples analyzed for metals are analyzed for the total metal content of the particular metal. The analytical methods for total metal content can be grouped into two catagories: direct analysis and analysis of a digested sample. The predominant method of direct analysis (homogenized freeze-dried sample)of elements is neutron activation analysis (NAA). NAA has a number of advantages over other methods: (a) analysis can be done without physical destruction of the sample; (b) reagent blanks are not needed; (c) the potential for contamination can be reduced by minimal preirradiation handling; (d) the process is virtually free from matrix interferences; (e) solids, liquids, or gasses can be analyzed; ( f ) qualitative information is obtained as well as quantitative analysis; (g)

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there is multielement specificity; (h) almost all of the elements can be determined; (i) excellentsensitivityanddetectionlimitsareattainable; ( j ) thereishighprecisionand accuracy, with (k) extensive linear range;(l) the cost is reasonable; (m)total analysis time is short, and (n) fully automated systems are available. The most important advantage is that the chemical state of the element does not influence the analysis (143; Chatt, personal communication). Because of these advantages, NAA has been used for the analysis of foods ( 1 34,144) and for the certification of trace elements in reference materials ( 1 34,135). If the element of interest is present at less than the detection limits or there is interference fromotherelements,theelement of interestcanbeseparatedoutbyradiochemical ( 134,145) or preconcentration methods. There are a variety of methods for the analysis of contaminant elements that normally require digested samples. Mostof the methods involve atomic spectrometry, electrochemical methods, or a combination of gas or HPLC with a detector for metals such as atomicabsorbancespectrometry,inductivelycoupledplasma,ormicrowave-induced plasma emission spectrometry.It has been argued that among the presently available methods for the analysis of trace elements (e.g., contaminants) in biological material, the most sensitive, fast, selective, and precise multielement method is inductively coupled plasma mass spectrometry (ICP-MS) (146). However, the special consideration required during sample preparation for the analysis for a particular element can effectively reduce the capability to analyze multiple elements using the same sample preparation. This may account for the few articles using ICP-MS for the analysisof seafoods ( 137). The less expensivegraphitefurnace(GFAAS)orelectrothermalatomicabsorptionspectrometers (ETAAS) are among the most popular methods for analyzing total metal contaminants in seafood, particularly selenium ( 124), cadmium ( 1281, and lead ( 140).

2. Speciation Organometallic compounds tend topartitionintolipids of organisms andthustendto accumulate in organisms i n a manner similar to other organic contaminants. Analytically speaking, organometallics are often detected and analyzed by the metals they contain. Some knowledge of speciation of metals and arsenic is necessary in order to interpret the toxicologic risk of levels of these elements in a particular seafoc-id (141). For example, mercury is often present mostly as the most toxic form (methylmercury) ( 1 39). while arsenic is often i n the form of arsenobetaine, which is relatively nontoxic (141). In addition, although selenium counteracts the toxicity of mercury, the species of selenium have different absorption and protective abilities (124). Speciation analyses are done by extraction, separation, or a combination of both, followed by detection with an element-specific detector. The most prevalent detection methods for contaminant elements are atomic absorbance spectrometry (AAS) and inductively coupled plasma (ICP). Selenium species have been separated by both ion exchange chromatography and ion pairing chromatography followed by ICP-MS ( 137) or flame atomic absorption spectrometry ( 125). Selective hydride generation has also been used to determine selenium species i n biological material ( I 24). For detailed reviews of methods for the analysis of selenium species, see Cappon ( 124) and Taylor et al. ( 137). Arsenic compounds rangei n polarity from water soluble to lipid soluble; as a result, a number of solvent combinations must be used to recover the various arsenic species. Separation of arsenic compounds can be done usingthinlayer chromatography, highperformance ion exchange chromatography, or a combination of methods (141). Detection

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of the species can be by atomic absorption spectrometry, atonic emission spectrometry, 01' ICP-MS (141). Methods for extraction of organomercurials have been reviewed (139,147-150). The earlier procedures generally used dilute acidto liberate protein-bound organic mercury compounds, a halide to promote the formation of halide derivatives, and extraction with an organic solvent such as benzene or toluene. The organomercurials werethen converted to water-soluble thiosulfate complexes and back-extracted from the organic phase with water,leavingnonmecurialorganicsbehind i n theorganicphase.Thecoextraction of lipids tends to form emulsions which are difficult to break and lead to losses of analyte. Extraction of lipids with acetone before the extraction of organomercurials was reported by Hight and Cocoran (15 I ) . A simplified method for the extraction of methyl- and ethylnlercury from tissues has been reported (152) and Inay be applicable to seafood samples. This method uses thiosulfate complexationwith no prior acidification, bromide treatment, and solvent extraction. Another method that has been used for the analysis of methylmercury i n seafood uses sulfuric acid and iodoacetic acid to release the mercury and convert it to a volatile form which can be determined by gas chromatography ( 153). This method is carried out with the sanlples contained in headspace vials and the procedure can be semiautomated to reduce labor costs. A supercritical fluid extraction method hasalso been reported ( 1 54).

REFERENCES DJH Phillips. Use o f biological indicator organisms to quantitate organochlorine pollutants i n aquatic environments--a review. Environ Pollut 16:167, 1978. New York: 2. JW Kiceniuk,S Ray. eds. Analysis of Contaminants in Edible Aquatic Resources. VCH Publishers,Inc..1994. 3. JK Taylor. Quality Assurance of Chemical Measurements. Chelsea, MI: Lewis Publishers, Inc.,1987. 4. RE Clement, GA Eiceman. and CJ Kocster. Environmental analysis. Anal Chem 67:271R, 1.

1995.

S. 6.

7. 8.

9. 10. 11.

12.

J Lawrence. K1 Aspila. Quality assurance for environmental monitoring. Water Qual Res J Can 30:1, 1995. W Horwitz, R Albert. Reliability of the determinations of polychlorinated contaminants (biphenyls, dioxins, furans). J AOAC Int 79:589, 1996. LB Willelt, HA Irving. Distribution and clearanceof polybrominated biphenyls in cows and calves. J Dairy Sci 59: 1429, 1976. GWSovocool. RK Mitchum,YTondeur, WD Munslow,TLVonnahme, JR Donnelly. Bromo- and brotnochloro-polynuclenr aromatic hydrocarbons, dioxins and dibenzofurans i n municipal incinerator fly ash. BiomedEnvironMassSpectrom15:669,1988. JWS Jamieson. Polybrominated biphenyls in the environment. Economic and Technical Review Report EPS-3-EC-77- 18, 1977. B Jansson, L Asplund, M Olsson. Brominated flame retardcnts-ubiquitous environmental pollutants'?Chemosphere 162343, 1987. I Watanabe, T Kashimoto, M Kawano, R Tatsukawa. A study of organic bound halogens in humanadipose.marineorganismsandsedimentbyneutronactivationandgaschromatographicanalysis.Chemosphere16:849,1987. U Sellstrom, U-B Uvemo, C WahlB Jansson, R Andersson, L Asplund, K Lit&, K Nylund, berg, U Wideqvist, T Odsjo. M Olsson. Chlorinated and brominated persistent organic compounds in biological samples from the environment. Environ Toxicol Chem 12:1163, 1993.

Kiceniuk

530

13. A Pettigrew. Flame retardants (halogenated).In: J Kroschwitz, M Howe-Grant, eds. Encyclo-

pedia of Chemical Technology, 4th ed. New York: John Wiley &L Sons, 1991, p. 958. 14. V Zitko.Thefateofhighlybrotninatedaromatichydrocarbonsinfish.In:MAQKhan,JJ Lcch, JJ Menn, eds. Pesticide and Xenobiotic Metabolism i n Aquatic Organisms. Washington,DC:AmericanChemicalSociety.1979.p.177. 15. DW Kuehl,RHaebler.Organochlorine,organobromine,metal.andseleniumresidues in bottlenose dolphins (Tursiops ttuncatus) collected during an unusual mortality event i n the Gulf of Mexico, 1990. Arch Environ Contam Toxicol 28:494, 1995. 16. PS Haglund, DR Zook, H-R Buser. J Hu. Identification and quantification of polybrominated diphenyl ethers and tnethyoxy-polybroluitlated diphenyl ethers i n Baltic biota. Environ Sci Technol 313281, 1997. 17. CP Rice,SMChernyak.Marinearctic fog: an accumulator o f currentlyusedpesticide. Chetnospherc35:867,1997. 18. F Wania, J-E Haugen, YD Lei. D Mackay. Temperature dependence o f atmospheric concen32:1013, 1998. trations of semivolatile organic compounds. Environ Sci Technol 19. TF Bidleman.EJChristensen. WNBillings.RLeonard.Atmospherictransportoforganochlorines in the North Atlantic gyre. J M a Res 39:443, 1981. 20. TF Bidletnan,GWPatton,MDWalla, BT Hargravc, WP Vass, PErickson,BFowler, V Scott, DJ Gregor. Toxaphene and other organochlorines i n the Arctic Ocean fauna: evidence for atmospheric delivety. Arctic 42307. 1989. 2 1. WH Newsome, P Andrews. Organochlorine pesticides and polychlorinated biphenyl congeners in cotntnercial fish from the Great Lakes. J AOAC lnt 76:707, 1993. 22. DCGMuir,RJNorstrom,MSimon.Organochlorinecontaminants in arcticmarinefood chains:accunmlation ofspecific polychlorinatedbiphenylsandchlorodane-relatedcompounds. Environ Sci Tcchnol 22:1071, 1988. 23. RK Boyd. Extractable Organically Bound Chlorine (EOCI) in Fish and Sediments; the Unno. 69. Halifax: NationalResearchCouncil, identified 85%). IMB Technical Report 1993.

24.WHNewsome,PAndrews,BSConachcr.RRRao,AChatt.Totalorganochlorinecontent of fish from the Great Lakes. J Assoc Anal Chetn 76:703, 1993. 25. RM Hollingworth, N Kurihara, J Miyatnoto. S Otto, CD Paulson. Detection and significance of active metabolites of agrochemicals and related xenobiotics in animals. Pure Appl Chcnl 67: 1487, 1995. 26. EG Lcighty, AF Fentitnan Jr., RM Thompson. Conjugation of fatty acids to DDT i n the rat: possible mechanism for retention. Toxicology 15:77, 1980. I n : CD Paulson, J Caldwell. DH 27. GB Quistad, DH Hutson. Lipophilic xenobiotic conjugates. Hutson,JJMenn,eds.XcnobioticConjugationChemistryACSSymposiumSeries299. Washington, DC: American Chemical Society, 1986, p. 204. 28. APryde,RPHanni.Outdoordissipation oftheexperimentalacaricide2-naphthylmcthyl cyclopanecarboxylate on apple trees: formation of lipophilic conjugates. J Agric Food Chem 31:564, 1983. 29. DH Hutson. PF Dodds, CJ Logan. The significance of xcnobiotic-lipid conjugation. Biochem Soc Trans 13:854, 1985. 30. GAS Ansari, SG Britt. ES Reynolds. Isolation and characterization o f paltnitoylpentachlorophenol from human fat. Bull Environ Contam Toxicol 34:661. 1985. 31. GW Gribble.NaturallyOccurringOrganohalogenCompounds-ACornprchcnsiveSurvey. NewYork:Springer-Verlag/Wien.1996. 32. H Hikansson, PSundin,TAndersson.BBrunstriim, L Dencker,MEngwall, G Ewald,M Gilck, G Holm, S Honkasalo, J Idestam-Almquist, P Jonsson, N Kautsky, G Lundberg, A Lund-Kvernheim. K Martinsen, L Norrgren. M Pcrsoncn,M Rundgren. M Stilberg, M Tarkpea, C Wesin. In vivo and i n vitro toxicity of fractionated fish lipids, with particular regard to their content of chlorinated organic cotnpounds. Pharnlacol Toxicol 69:459, 1991.

Analysis of Aquatic Contaminants

53 1

33. T Kawabata, K Nakamura. N-nitrosamines. In: JW Kiceniuk, S Ray, eds. Analysis of Con-

taminants i n Edible Aquatic Resources. New York: VCH Publishers, Inc.. 1994, p. 467. i n the detcrtnination of petrotcum 34. P Donkin,SVEvans.Applicationofsteamdistillatiotl hydrocarbons in water and mussels (Myrihts ethtlis) from dosing experiments with crude oil. AnalChimActa:156:207.1984. 35. C Karahadian, RC Lindsay. Chemical isolationof oderiferous componcnts. In: JW Kiceniuk, S Ray, eds. Analysis oPContaminantsin Edible Aquatic Resources. New York: VCH Publishers. Inc., 1994, p. 275. 36. DAJ Murray, WL Lockhart. Microextraction and gas chromatographic analysis of selected petroleum hydrocarbons in watcr and fish tissuc. J Chromatogr 212:305, 1981. in fish tissues 37. DAJ Murray, WL Lockhart. Determination of trace volatile organic compounds by gas chromatography. J Assoc Anal Chem 71: 1086. 1988. 38. T Sato, N Matsuoka, H Sugihara. H Aknzawa, and T Motohiro. Petroleum-like off-flavour i n seasoned herring roc. Water Sci Tcchnol 20:49. 1988. 39. J Paasivirta. J Koistincn, S Ray. Extraction, cleanup. and separation of organochlorincs. In: JW Kiceniuk, S Ray, eds. Analysisof Contaminants in Edible Aquatic Resources. New York: VCH Publishers, Inc., 1994, p. 287. 40. HM Lott, SA Barker. Chlorinated pesticides. In: JW Kiceniuk, S Ray, cds. Analysis of Contaminants inEdibleAquaticResources.NcwYork:VCHPublishers,Inc.,1994,p.379. 41. JWKiceniuk. I Holoubck. Polycyclic aromatic compounds. In: JW Kiceniuk, S Ray, eds. Analysis of Contaminants in Edible Aquatic Resources. New York: VCH Publishers, Inc., 1994, p. 429. 42. GKC Low, GE Batley, Cl Brockbank. Solvent-induced photodegradation asa source of error i n the analysis of polycyclic aromatic hydrocarbons. J Chromatogr 392:199, 1987. 43. JS Jasinski. Multiresidue procedures for the determination of chlorinated dibenzodioxins and dibenzofurans in a variety of foods using capillary gas chromatography-electron capture detection. J Chromatogr 478349, 1989. 44. BP Dum. Determination of polycyclic aromatic hydrocarbons in sediments and marine organisms. In: A Bjorseth, ed. Handbook of Polycyclic Aromatic Hydrocarbons, Vol. 1. New York: Marcel Dekker,. 35. WH Gricst, JE Caton. Extraction of polycyclic aromatic hydrocarbons for quantitative analysis. In: A Bjorseth,ed. Handbook of Polycyclic Aromatic Hydrocarbons.New York: Marcel Dekker, 1983, p. 95. 46. DL Vassilaros, PW Stoker, GM Booth, ML Lee. Capillary gas chromatographic determination of polycyclic aromatic compounds in vertebrate fish tissue. Anal Chem 54:106. 1982. 47, V Zitko. Organic contaminants in fish and shellfish. In: IF Lawrence, ed. Food Constituents and Food Residues. New York: Marcel Dekkcr, 1984, p. 533. 48 JA Lebo. JL Zajiork, TR Schwartz. LM Smith, MP Beasley. Distribution of monocyclic and polycyclic aromatic hydrocarbons in fish tissue. J Assoc Anal Chetn 74:538, 1991. 49 JA Lcbo, LM Smith. Determination of fluorene in fish, sediment. and plants. J Assoc Anal Chcm 69944, 1986. 50. WD MacLeod, DW Brown, AJ Friedman, DC Burrows, 0 Maynes, RW Pearce, CA Wigren, RC Bogar.StandardAnalyticalProcedures oftheNOAA AnalyticalFacility1985-1986. Extractable Toxic Organic Compounds. NOAA Technical Memo NMFS FINWC-92, 2nd ed. Washington, DC: U.S. Department of Commerce. 51. MM Krahn, LKMoore. RC Bogar, CA Wigren, S Chan, DW Brown.High-performance liquid chromatographic method for isolating organic contaminants from tissue and sediment extracts. J Chromatogr 437: 161, 1988. 52. A Bevenue, H Beckman. Pentachlorophenol: a discussion of its properties and its occurencc as a rcsidue in humanandanimaltissue.ResidueRev19:83,1967. 53. RC Dougherty. Phenols and chlorophenols. In: JW Kiceniuk, S Ray, eds. Analysis of Contaminants in Edible Aquatic Resources. New York: VCH Publishers, Inc., 1994, p. 453.

Kiceniuk

532

54. MMKrahn,DGBurrows,GMYlltalo,DWBrown, CW Wigren, TK Collier, S-L Chan, U Varanasi. Mass spectrometric determination of metabolites of aromatic compounds i n bile of fish capturedfrom Prince WilliamSound, Alaska, after theExxon Valdez oil spill. Environ SciTechnol26:116, 1992. 55. U Varanasi, M Nishimoto, WL Reichert, BTL Eberhart. Cotnparative Inetaholism of benzo (a)pyrcne andcovalentbindingtohcpaticDNA i n Englishsole,starryflounder,andrat. Cancer Res 463817, 1986. 56. DW Kuehl, MJ Whitaker. RC Dougherty. Micromethods for toxic residue screening by negative chemical ionization mass spectrometry. Anal Chern 52935, 1980. 57. Y Tondcur, RC Dougherty. New screening methods for acidic toxic substances using negative chemicalionizationmassspectromctry.Tetrachloroteq>hthalatc in human urines.Environ SciTcchnol15:216,1981. 58. JPaasivirta, JKoistinen. Chlorinatedethers.In: JWKiceniukand S Ray,eds.Analysis of Contaminants i n Edible Aquatic Resources. New York: VCH Publishers, Inc.. 1994. p. 41 I . 59. GA Dhopcshwarkar. Naturally occurring food toxicants: toxic lipids. Prog Lipid Rcs 19: 107, 1981.

60.JKoistinen.Residues of planarpolychloroaromaticcompounds i n Balticfishandseal. Chemosphere 20:1043, 1990. 61. J Paasivirtn, P Klien, M Knuutila, J Knuutincn, M Lahtiperl, R Paukku. A Veijanen. L Welling. MVuorinen, PJ Vuorincn.Chlorinatedanisolcsandveratroles in fish.Modcl conpounds. Instrumental and sensory determination. Chemosphere 16: 123I , 1987. 62. J Soikkeli. J Tarhancn, J Paasivirta. A Witick. Multicotnponcnt analysis for dimeric chlorophenol ethers (PCDEs, PCPAs, and PCBAs) i n biological samples. Chemospherc 15:2103, 1986. 63. JP Sherry, HA Tee. A procedure for the determination of polychlorinated dibcnzo-p-dioxins i n [ish.Chemosphere20:865, 1990. 64. LM Smith, DL Stalling, JL Johnson. Determination of part per trillion levels of polychlorinated dibenzofurans and dioxins in environmental samples. Anal Chem 56: 1830. 1984. 65. RJ Norstrom,MSimon, MJ Mulvihill.Agel-pertneationlcolumnchrotnatographyclean-up in animal tissue. I n t J Environ Anal Chem 23:267, method for the determination of CDDs 1986. 66. HMLott. SA Barker.Matrixsolidphascdispersion (MSPD) extractionandgaschromatographic screcning of 14 chlorinated pesticides in oysters (Crcl.sso.sfr-cx/vir,qirIico).J Assoc Anal Chem 76:67, 1993. 67. AR Long, MD Crouch, SA Barker. Multiresidue matrix solid phase dispersion (MSPD) extraction and gas chromatographic screening of nine chlorinated pesticides in catfish ( k f d w i s p ~ t r ~ c t c c f ~muscle rs) tissue. J Assoc Anal Chcm 74:667, 1 99 1. 68. HM Lott, SA Barker. Extraction and gas chromatographic screening of14 chlorinated pesti1993. cides I n crayfish hepatopancreas. J Assoc Anal Chem 76:663, 69. RE Clement, CJ Koester, L Grey. Dioxins and furans. In: JW Kiccniuk, S Ray, eds. Analysis ofContaminants i n EdibleAquaticResources.NcwYork:VCHPublishCrs,Inc..1994, p. 347. K Dillon,RDonnelly.EHorn, 70. PO’Kcefe,CMeyer,DHilker,KAldous.BJelus-Tyrol-, R Sloan. Analysis of 2, 3. 7, 8-tcrtachlor-p-dioxin i n Great Lakes fish. Chctnosphere 12:325, 1983.

71. J Koistinen. Alkyl polychlorobiphcnyls and planar aromatic chlorocompounds in pulpmill products, effluents, sludges and exposed biota. Chemosphere 24559, 1992. 72. LM Smith. DL Stalling. JL Johnson. Determination of parts-per-trillion levels of polychlorinated dibenzofurans and dioxins in environmental samples. Anal Chem 56: 183 1 , 1984. 73. MD Erickson.AnalyticalChemistry o f PCBs.Boston:Butterworths.1986. 74. RC Hale. J Greaves, K Gallagher, GG Vadas. Novel chlorinated terphenyls in sediments and shellfish o f an estuarine environment. Environ Sci Technol 24:1727. 1990.

Analysis of Aquatic Contaminants

533

75. S Ray. Polychlorinated terphenyls and naphthalenes. I n : JW Kiccniuk, S Ray. eds. Analysis of Contaminants in EdibleAquaticResources.NewYork:VCHPublishcrs.Inc.,1994. p. 339. 76. CD Veith, IM Kiwus. An exhaustive steam-distillation and solvent-extraction unit for pesticides and industrial chemicals. Bull Environ Contam Toxicol 17:631, 1977. 77. M Cooke, DJ Roberts, ME Tillctt. Polychlorinated naphthalenes. polychlorinated biphenyls and sDDT residues i n British birds of prey. Sci Total Environ 15:237. 1980. 78. RC Dougherty, MJ Whitaker, LM Smith. DL Stalling.DW Kuehl. Negative chemical ionizationstudiesofhumanandfoodchaincontaminationwithxenobioticchemicals.Environ Health Perspect 36: 103, 1980. 79. JTarhanen, J Koistinen,JPaasivirta, PJ Vuorincn,JKoivusaari, I Nuuja,NKannan. R Taksukawa.Toxicsignificance ofplanararomaticcompounds in Baltic ecosystcn-new studies on extremely toxic coplanar PCBs. Chemosphere 18: 1067, 1989. 80. J Jan, S Malnersic. Dctermination of PCB and PCT residues in fish by tissue acid hydrolysis and destructive clean-up of extract. Bull Environ Contam Toxicol 19:772, 1978. 81. I Watanabe, T Yakushiji, N Kunita. Distribution differences between polychlorinated terphenyls and polychlorinated biphenyls in human tissues. Bull Environ Contam Toxicol 25:XlO. 1980.

de Vries. UAT Brinkman. An evaluation of chromatographic 82. A de Kok, RB Geerdink. G methods for the analysis of polychlorinated terphenylsin environmental samples. Int J EnvironAnalChem 1299, 1982. S Ray, cds. Analysis of Contami83. CD Metcalfe. Polychlorinated biphenyls. In: JW Kiceniuk, p. 305. nants in Edible Aquatic Resources. New York: VCH Publishers, Inc., 1994. 84. ML Hattula. Some aspects of the recovery of chlorinated residues (DDT-type compounds and PCB) from fish tissue using different extraction methods. Bull Environ Contam Toxicol 12:301,1974. 85. TL King,BK Haines, JF Uthe. Non-, mono-, and di-o-chlorobiphenyl concentrations and 2,3,7,8,-tetrachlorodibenzo[p]dioxin in aroclorsanddigestive theirtoxicequivalentsto glandsfromAmericanlobsters (Hortrtrrlrs arrlericctnlrs) captured in AtlanticCanada.Bull Environ Contarn Toxicol 57:465, 1996. 86. C Bcrger, M Pc~rut.Preparative supercritical fluid chromatography. J Chromatogr 50537, 1990. 87. KS Nam, S Kapila, AF Ynnders, RK Puri. A multiple sample extraction and on-line systcm for the analysis of chlorinated compounds. Chemosphere 23: 1 109, 1990. in marine oils. Envi88. G Lunde, E Steinncs. Presenceof lipid-soluble chlorinatcd hydrocarbons ron Sci Technol 9:155, 1975. 89. JW Kiceniuk, J Holzbccher, A Chatt. Extractable organohalogens in tissues of beluga whales from the Canadian arctic and the St. Lawrence River. Environ Pollut 97:205, 1997. 90. K Martinsen, A Kringstad, GE Carlberg. Mcthods for determination of sum parameters and characterization of organochlorine compounds in spent bleach liquors from pulp mills and water,sedimentandbiologicalsamplesfromreceivingwaters.WaterSciTechno120:13, 1988.

91. JC Meadows, DE Tillit. TR Schwartz, DJ Schroedcr. KR Echols, RW Gale, DC Powell, SJ Bursian. Organochlorine contaminants in double-crested cormorants from Green Bay, WI: I. Large-scale extraction and isolation from eggs using semi-permeable membrane dialysis. Arch Environ Contam Toxicol 3 1:218, 1996. 92. JN Huckins, MW Tubergen, JA Lebo, RW Gale. TR Schwartz. Polymeric film dialysis in

organic solvent media for cleanup of organiccontaminants.JAssocAnalChem73:290, 1990. 93. CA Stern, DCG Muir, CA Ford, NP Grift, E Dewailly. TF Bidleman, MD Walla. Isolation and identification of two major recalcitrant toxaphene congeners in aquatic biota. Environ Sci Technol 26: 1838. 1992.

Kiceniuk

534

94. J Falandysz, L Strandberg.P-A Bcrgqvist. SE Kulp, B Strandberg. C Rappe. Polychlorinated naphthalenes i n sediment and biota from the Gadafisk Basin. Baltic Sea. Environ Sci Tcchnol

303266. 1996. 95. W Specht, M Tillkes. Gas-chromatographkche bestimmung von Ruckstanden an pflanzen-

96. 97. 98.

99. 100.

101

102. 103.

104.

105. 106.

107. 108.

109.

1 IO. 11I.

112.

113.

114.

I IS.

hehandlugsmitteln nach clean-up ubcr gel-chromatographie und mini-kicselgel-saulcn-chromatographic. Fresenius Z Anal Chcm 322:443. 1985. JL Bernal. MJ del Nozal, JJ Jiminez. Some observations on clean-up procedures using sulphuric acid and floisil. J Chromatogr 607303, 1992. STC Gonzalez-Barros, MEA Piiieiro. JS Lozano. and MAL Yusty. PCBs and PCTs in wolves (Curtis / u p s ) in Galicia (N.W. Spain). Chemosphere 35:1243. 1997. MA Saleh. Toxaphene: chemistry, biochemistry. toxicity and environmental fate. Rev Environ Contam Toxicol 1 l8:1, 1998. in Canadianeast CJMusial, JF Uthc.Widespreadoccurrence ofthepesticidetoxaphene coast marine fish. Int J Environ Anal Chem 14: 117, 1983. DCG Muir, CA Ford, NP Grift, DA Metner. WL Lockhart. Geographic variation of chlorinated hydrocarhons in burbot (Lota Iota) from remote lakes and riversin Canada. Arch EnvironContamToxicol 19530, 1990. R Preussman, G Nerath. G Wulf-Lorcntzen, D Diaber, H Hengy. Methods of colour formationandthinlayerchromatographyfororganicN-nitrosocompounds.FreseniusZAnal Chem 202:188, 1964. NP Sen, DC Smith, L Schwinghamer. JJ Marleauy. Diethylnitrosamine and other N-nitrosamines i n foods. J Assoc Anal Chem 52:47. 1969. DM Coulson. Electrolytic conductivity detector for pas chromatography. J Gas Chromatogr 3: 134, 1965. JF Palfratnan, J Macnab, NT Croshy. An evaluation of the alkali flame ionisation detector and the Coulson electrolytic conductivity detectori n the analysis of N-nitrosaminesin foods. J Chromatogr 76:307, 1973. F Ariesc, C Gooijer, NH Velthorst, JW Hofstraat. Shpol’skii spcctrofluorimetric dctermination of polycyclic aromatic hydrocarbons i n biota. Anal Chim Acta 232:245, 1990. CC DeLima.TheShpol’skiieffect as ananalyticaltool.In:WLZielinskiJr.. ed. CRC Critical Reviews in Analytical Chemistry. Boca Raton. FL: CRC Press, 1992, p. 177. L Stanker. Residue detection by immunoassay. I n : JW Kiceniuk, S Ray, eds. Analysis of Contaminants in Edible Aquatic Resources. New York: VCH Publishers. Inc., 1994. p. 523. REClement,HM Tosinc.The gas chromatography/mass spectrometrydetermination of chlorodibenzo-p-dioxins and dibcnzofurans. Mass Spectrom Rev 7593 1988. GRB Wcbster. K Olie, 0 Hutzinger. Polychlorodibenzo-p-dioxitlsand polychlorodihenzofurans. In: FW Karasek, 0 Hutzinger, S Safe, eds. Mass spectrometry i n Environmental Sciences. NewYork:PlenumPress, 1985. p. 257. GC Mattern, CH Liu, JB Louis, JD Rosen. GC/MS and LC/MS determination of 20 pesticides for which dietary oncogenic risk has been estimated. J Agric Food Chem 39:700, 1991. JDRosen.Pesticideanalysisbymassspectrometry.In:RGrecnhalgh,TRRoberts. eds. Pesticide Science and Biotechnology. Proceedings of the 6th International Congress of Pesticide Chemistry (IUPAC). Oxford:BlackwellScientific.1987. p. 301. AA Mosi, WR Cullen, GK Eigendorf. Ion-molecule reactions of halocarbons with polycyclic aromatic hydrocarbons in a quadrupole ion trap: part 11-applications to environmental analysis. J Mass Spcctrom 33:250. 1998. TL King, JF Uthe, CJ Musial. Polycyclic aromatic hydrocarbons in the digestive glands of the American lobster, Hotnarus amcricanus, capturedi n the proximity of a coal-coking plant. BullEnvironContamToxicol50:907,1993. I Tuominen, K Wickstrnm, H Pyysalo. Determination of PAHs by GLC-selected ion monitoring (SIM) technique. J High Resolut Chromatogr Commun 9:469. 1986. H-R Buser, MD Muller. Isomer- and enantiomer-selective analyses of toxaphene components

Analysis of Aquatic Contaminants

116.

117.

118.

119.

120. 121.

122.

123.

124. 125.

535

using high resolution gas chromatography and detection by mass spectrometry/mass spectrometry. Environ Sci Technol 28: 119, 1994. J de Boer, PG Wester. Determination o f toxaphene in human milk from Nicaragua and fish and marine tnatntnals from the northeastern Atlantic and North Sea. Chetnosphere 27: l 879, 1993. S Ray. cds. JRBotta.Sensoryevaluation oftainted aquaticresources.In:JWKiceniuk, Allalysis of Contaminants in Edible Aquatic Resources. New York: VCH Publishers, Inc., 1994, p. 257. A Veijanen, M Lahtiperae, J Paasivirta. Analytical methods of off-flavour compounds. Lake Paeijacnne Symposium 1987. pp. 66-67. A Veijanen, J Paasivirta, M Lahtipcrii. Structure and sensory analyses of tainting substances in Finnish freshwater environments. Water Sci Technol 20:43, 1988. TH Heil. RC Lindsay. Volatilc compounds in flavor-tainted fish from the uppcr Wisconsin River. J Environ Sci Health B23:489. 1988. M Ogata, Y Miyake. Identitication of substances in petroleum causing objectionable odour in fish. Water Res 7:1493, 1973. TAGill.Advancedanalyticaltools in seafoodscience.In:JBLuten, T Borrescn,JOchlenschliiger, eds. Seafood from Producer to Consumer. Integrated Approach to Quality. Amsterdam: Elsevier. 1095, p. 479. K Persaud. Multieletnent arrays for sensing volatile chemicals. Intcll Inst Comp 9: 147. 1991. CJ Cappon. Selenium and organic selenides. In: JW Kiceniuk, S Ray, eds. Analysis of Contaminants in Edible Aquatic Resources. New York: VCH, 1994, p. 205. G Kiilbl, J Lintschinger. K Kalcher, KJ Irgolic. Reversed-phase ion-pair chromatographic separation of selenite and selenate with selenium-specific detection. Mikrochitn Acta I 19: 113, 1995.

126. ARuiter.Contaminants in fish.In: A Ruiter, ed. FishandFisheryProducts.Wallingford: CAB International, 1995. p. 26 1. ;ind in vitro bioavailability of selenium 127. LH Shen, H van Nieuwenhuizen. JB Luten. Speciation in fishery products. In: JB Luten, T B~rresen,J Oehlenschliiger. eds. Seafood from Producer to Consumer, Integrated Approach to Quality. Atnsterdam: Elsevier. 1995, p. 653. 128. S Ray. Cadmium. In: JW Kicenium,S Ray, eds. Analysis of Contaminants in Edible Aquatic 1994, p. 91. Resources. New York: VCH Publishers, Inc.. 129. W Salomons, U Fiirstner. Metals i n the Hydrocycle. Berlin: Springer-Verlag. 1984. pp. 212287. 130. JF Uthe, DP Scott. CL Chou. Cadmiutn contaminationin American lobster, Hotnarus americanus, near a coastal lend smelter: use of multiple linear regression for management. Bull Environ Contam Toxicol 38:687, 1987. 131. JF Uthe, CL Chou. Cadmium in sea scallop (Placopecten magcllanicus) tissues from clean I , 1987. and contaminated areas. Can J Fish Aquat Sci 44:9 132. JF Uthe, CL Chou. DH Loring, RTT Rantala, JM Bewers, J Dalxiel, PA Yeats. R Levaque Charron. Effect of waste treatment at a lead smelter on cadmium levels i n American lobster (Hotnarus americanus). sediments and seawater in the adjacent coastal zone. Mar Pollut Bull 17:l 18, 1986. 133. DA Wright, PM Welbourn. Cadmium in the aquatic environment: a review of ecological, physiological, and toxicological effects on biota. Environ Rev 2: 187. 1994. 134. SJ Parry. Biomedical applications. In: Activation Spectrometry in Chemical Analysis. New York: John Wiley & Sons. 1991, p. 163. 135. RM Parr. On the role of neutron activation analysis in the certification of a new reference material for trace-elenlent studies, mixedhuman diet, H-9. J Radioanal Nucl Chetn 123:259, 1988. 136. TH Nguyen, J Boman, M Leermakers, W Bacyens. Mercury analysis in environmental samJ Anal Chern 360:199, 1998. ples by EDXRF and CV-AAS. Frcsenius

536

Kiceniuk

137. A Taylor, S Branch, H Crews, DJ Halls, LMW Owen, M White. Atomic spectrometry update-clinical and biological materials, food and beverages. J Anal Atom Spectrom 12:1 19R. 1997. 138. I Novozamsky. HJ van der Lee, VJG Houba. Sample digestion procedures for trace element determination. Mikrochim Acta 1 19: 183, 1995. 139. CJ Cappon. Mercury and organomercurials. In: JW Kiceniuk, S Ray, eds. Analysis of Conp . 175. taminants in Edible Aquatic Resources. New York: VCH Publishers. Inc.. 1994, 140.RLobinski,FCAdams.Leadandorganoleadcompounds.In: JW Kiccniuk, S Ray. eds. Analysis of Contaminants in Edible Aquatic Resources. NewYork:VCH Publishers. Inc., 1994, p. 1 15. 141. Y Shibata. M Morita. Arsenic and organoarsenicals. In: JW Kiccniuk, S Ray, eds. Analysis of Contaminants in Edible Aquatic Resources. New York: VCH Publishers. Inc.. 1993, p. 159. 142. RE Sturgeon. KWM Siu. Tin and organotin. In: JW Kiccniuk, S Ray. eds. Analysis of Contaminants in Aquatic Resources. New York: VCH Publishers, Inc.. 1994. p. 225. 143. WD Ehtnann, DE Vance.Nuclearactivationanalysis.In:WDEhmann,DEVance,cds. & Sons. 1991. Radiochemistry and Nuclear Methods of Analysis. New York: John Wiley p. 253. 144. WC Cunningham, WB Stroube Jr. Application of an instrumental neutron activation analysis procedure to analysis of food. Sci Total Environ 63:29. 1987. 145. I Bayat, ND Raufi. M Nejat. Determination of mercury and other toxic elements i n fish and of foodstuffs using destructive neutron activation analysis. In: Health-Related Monitoring Trace Element Pollutants Using Nuclear Techniques. Vienna: International Atomic Energy Agency. 1985. p.141. 146. CK Mathews Trace toxic elements in nutritional health. In: Proceedings of the International Conference of Health and Diseases: Effects of Essential and Toxic Trace Elements, 4th ed. New Delhi: Wiley Eastern, 1995. p. 43 I . 147.JARodriguez-Vasqucz.Agaschromatographicdctcrtnination of organomercury (11) compounds. A mini review. Talanta 25:299, 1978. 148. AK Shirvastran, SG Tandon. The determination of mercury: a mini-review. Toxicol Environ Chem 5 3 1 I . 1982. In: DFSNatusch, PK Hopke.eds.AnalyticalAspectsof 149.RSBraman.Chemicalspeciation. & Sons. 1983, p. I . AnalyticalChemistry.NewYork:JohnWiley 150. CJCappon.GLCspeciation of selectedtraceelements.LC-GC5:400.1987. 151. S Hight.MCocoran.Rapiddetermination of methylmercury infishandshellfish:method development. J Assoc Anal Chetn 70:24. 1987. 152. AGFBrooks,EBailey,RT Snowden. Determinationofmethyl-andethylmercury in rat blood and tissue samples by capillary gas chromatography with electron-capture detection. J Chromatogr 374:289, 1993. 153. P Larsen, W Baeyens. Improvement of the semiautotnatated headspacc analysis method for the determination of methylmercury i n biological samples. Anal Chitn Acta 228:93, 1990. 154. W Holak. Determination of methylmercury after supercritical fluid extraction. J AOAC Int 78:l 124. 1995.

17 Agricultural Chemicals Debra L. Browning and Carl K. Winter U t ~ i w r s i hof CtrlIfi)r-r~itr-Da\~is, Davis. Califontin

Introduction 537 History A. 538 TI. PesticideTypes539 A. Insecticides 539 B. Herbicides 540 C. Fungicides 541 111. PesticideUsageLevels541 Regulations 543 IV. Pesticide 544 V. PesticideResidueLimits of 1996546 VI. FoodQualityProtectionAct VII. International Regulations 547 VIII. MonitoringandEnforcement547 A. U.S. Department of Agrlculture 549 B. State programs 550 Risks 551 IX. Dietary X. NondietaryRisks 553 XI. Conclusions 554 References 554 I.

1.

INTRODUCTION

“Pesticide” is a generic term for a variety of agents that control pests. Insecticides, herbicides, and fungicides are all commonly known pesticides, but less well known types includemolluscicides,bacteriocides,nematicides,scabicides,pheromones(attractants), plant growth regulators, acaricides, and repellants. Traditionally, pesticides have been used i n agriculture to maximize yield and minimize crop loss to pests. Pesticides can be applied before planting a crop to protect seeds/ seedlings, sterilize soil, protect roots, and kill competing weeds; during growth to mini-

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mize unwanted pests; and after harvesting to minimize damage from rodent, insect, and fungal populations (1 ). It has been estimated that the average time i t takes a farmer to tend and harvest I acre of cropland has decreased from 56 hoursin I830 to 2 hours today ( 1 ). Lower maintenance and increased crop yields have encouraged farmers to apply nlore pesticides. It has been estimated that 40% of the world's food supply would be at risk without pesticides. In developed countries, economic benefits may range from $3.50to $5.00 for every $ 1 .OO spent on pesticides (2). The United States accounts for nearly one-third of the pesticide user expenditures worldwide and one-fifth of the pesticide usage worldwide. The United States uses 4.5 billion pounds of pesticides i n a typical year (of which I .2 billion pounds are used in agriculture) at a user cost of $1 1.3 billion ($7.9 billion on agricultural pesticides) (3).

A.

History

Humans have used pesticides to control invertebrate, vertebrate, and microorganism infestations since as early as 1000 B.c., when the Chinese used sulfur as a fungicide against powdery mildew on fruit ( l ) . Sulfur remains an important pesticide today (79-89 million pounds used in the United States in 1995) (3).Arsenical compounds were popular insecticides in the 16th century, while nicotine, rotenone, and chrysanthemum extract have all been used as insecticides since the 17th century and are still used today ( l). AstheUnitedStatestransitionedfromhorsepower to mechanizedpower i n the 1920s, farm production increased and the use of farm labor decreased. The small and medium-sized farms of the Midwest could not typically afford nor get capital to exploit the advantagesof size; subsequently onlythe large farms survived. The large farms became moreheavilydependent on pesticidesandmechanizedpower to maintainproduction. Heavy use of arsenic pesticides led to the beginnings of consumer concern in this country regarding the use of pesticides on agricultural crops (4). The 1938 creationof the Federal Food, Drug and Cosmetic Act (FFDCA) and subsequent amendments established the requirement for pesticide tolerances to be established when pesticide use could result in residues on food or feed crops. In 1947 the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) was passed by Congress. I t grouped all pesticide products under one law and mandated labeling and registration requirements. The law was amended in 1975, 1978, 1980, 1984, 1988, and 1996. Additional requirements under these amendments included Appropriate chemical, toxicological. and environmental i n p c t studies Label specification Use restrictions Responsibility to monitor pesticide residue levels i n foods Pesticide reevaluation and reregistration Special consideration for the sensitivity and exposure of infants and children Consideration of aggregate risk from food, water, and domestic exposure Cumulative risks from pesticides possessing a common mechanism of toxicity Despite government intervention and regulation of pesticide use, pesticide residues

in food continue to generate significant societal concern. I n a national consunler attitude survey conducted annually over the past decade, between 72% and 82% of U.S. consumers considered pesticide residuesto be a serious hazard ( S ) . Contributing to the public's aware-

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ness and concern over pesticide residues are several widely publicized incidents and reports. These include an incident in which the insecticide aldicarb was used illegally on watermelons in California in 1985 and resulted in morethan 1000 suspected cases of human illness (6). Other notable events include areport released by the Natural Resources Defense Council and widely covered by the national media alleging that U.S. children faced “intolerable” risks from exposure to a number of pesticides, including the plant growth regulator daminozide (Alar) (7,8), and a 1993 report from the National Research Council that recommended changes in the risk assessment and regulation practices to more appropriately consider the differences in susceptibility and exposure of infants and children to pesticide residues relative to adults (9).

II. PESTICIDE TYPES It is important to appreciate that all pesticides possess some degree of toxicity to some living organisms, otherwise they would have no practical use. It is equally important to recognize that pesticides are usually developed for a target species but physiological and biochemical system similarities in nontarget organisms can lead to undesired responses. Adherence to labelinginstructionsandsafeusepracticescanminimizehumanhealth hazards (1). There are several types of pesticides:

Pest

Type of pesticide

Insect Weeds Fungi Nematodes Mites Leaves Bacteria Rodents Snails Algae

Insecticide Herbicide Fungicide Nematicide Acaracide Defoliant Bacteriocide Rodenticide Molluscicide Algacide

Primarydamage to agriculturalcommodities isattributedtoweeds,insects,and fungi. The U.S. Environmental Protection Agency (EPA) reported that of the estimated 1 billion pounds of conventional pesticides (measured on the basis of active ingredients) used in the United States in 1995, 55% were herbicides, 32% insecticides, 7% fungicides, and 6% other (3).

A.

Insecticides

Insecticides are pesticides designed to control crop-damaging insects. They can be nerve poisons, muscle poisons, dessicants, or sterilants. Chlorinated hydrocarbons, organophosphates, and carbamates are the most common chemical classes of insecticides. Chlorinated hydrocarbons such as DDT, aldrin, dieldrin, and chlordane were developed during the

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1930s and 1940s and ledto rapid improvements in insect pest control throughout the world because of their high insect toxicity and their significant environmental persistence. This same persistence led to problems of environmental build-up and biological magnification, and was manifested in significant ecological and environmental upset that ultimately resulted in the elimination of the majority of the uses of chlorinated hydrocarbon insecticides in the United States. Chlorinated hydrocarbons have more recently been associated with estrogenic and enzyme-inducing properties that could possibly interfere with fertility and reproduction i n human and other nontarget organisms ( l ) . Organophosphate and carbamate insecticides have replacedmost chlorinated hydrocarbons. From two very different chemical classes (esters of phosphoric or phosphorothioicacidandcarbamicacid,respectively), they share a conmlonmechanism of action involving inhibition of cholinesterase enzymes in both insects and in mammals. Organophosphate and carbamate insecticides are less persistentthan the chlorinated hydrocarbon to mammals insecticides that they largely replaced, but are generally more acutely toxic and other nontarget organisms ( I , 10). There are as many as 200 different organophosphate insecticides, of which 39 cornpounds are currently registered for U.S. food use. Examples of organophosphate insecticides include parathion, malathion, diazinon, chlorpyrifos, and azinphos-methyl. About 25 carbamate insecticides are in the marketplace and 14 are registered for U.S. food use. Carbaryl, aldicarb, and carbofuran are examples of carbamate insecticides. One of the newer classes of synthetic insecticides is the pyrethroids. Derived from pyrethrins (the natural extracts of chrysanthemum flowers), pyrethroids are more stable inlightthantheirnatural predecessors and are therefore nlore effective as agricultural insecticides. Pyrethroids are considered excellent broad-spectrum insecticides, cause rapid “knockdown” and mortality in insects at low doses, are of lower toxicity to mamn1als by oral, dermal, and inhalation routes of exposure than the organophosphates and carbamates, and break down fairly rapidly i n the environment. They are in significant demand worldwide and synthesisof new analogs continuesto represent an important research area. Though more selective to target species, the pyrethroids suffer from significant environmental lability that reduces their effectiveness and are more costly than other classes of insecticides ( 1 l ) .

B. Herbicides As indicated previously, herbicides contributeto the majority of U.S. agricultural pesticide use in terms of pounds applied. There are a large number of different types of herbicides availableontheU.S.andworldwidemarkets;examplesincludetriazine,sulfonylurea, phenoxy, and quaternary ammonium herbicides. The wide variety of herbicides also results i n a wide variety of toxicological actions on plant material. Preplant herbicides are applied before crop seeding has begun, preemergents are applied (to the soil) before the appearance of unwanted weeds, and postemergents are used after germination of the crop and/or weeds. Some herbicides, such as glyphosate, are toxic to virtually all types of plant material (although glyphosate resistance is being incorporated into genetically modified plant varieties such as soybeans), while others may be more selective, such as phenoxy herbicides that are toxic to broadleaf plants but do not harm narrow-leaf plants such as grasses. Some herbicides exert their toxicity through direct plant contact, while others are applied to the soil or foliage and are translocated throughout the plant following absorption into the plant (I).

Agricu/fura/ Chemicals

C.

541

Fungicides

Fungicides are chemicals derived from compounds such as inorganic metals and sulfur, aryl, and alkyl-mercurial compounds and chlorinated phenols (1). They are used to control molds and other plant diseases by inhibiting the metabolic processes of growing fungal organisms. Many storage conditions (increaseddecreases in heat, moisture) can lead to massive microbial growth, especially on grain products. Many types of fungi can stress or damage crops and stimulate plant toxin development, and some fungi Aspergilsuch as lus jlavus and Fusarium monilifomze produce aflatoxins and fumonisins, respectively, which represent mycotoxins of significant human and animal health concern (12,13).

111.

PESTICIDE USAGE LEVELS

The EPA provides estimates of pesticide use and expenditures for the United States and the world. It bases estimates on surveys, U.S. Department of Agriculture (USDA) reports, other published reports, and proprietary data when it is available. Not all proprietary data is made available, therefore the EPA can only estimate use data. Pesticides have both agricultural and nonagricultural uses, including household and garden pest control, mosquito abatement, sanitation, and wood preservation. Fifty-one percent (Fig. 1)of the pesticide use in the United States in 1995 involved chlorine and sodium hypochlorite. This is not surprising when one considers the large quantities required for disinfecting potable and wastewater. Large volumes are also used as bleaching disinfectants and in swimming pools (3). Of the 4.5 billion pounds of pesticides used in 1995, only 27%or 1.2 billion pounds were devoted to conventional pesticide usage, which excludes biocides, wood preservatives, and disinfectants. Of that 1.2 billion pounds, 939 million pounds were used in the production of food and fiber products (Fig. 2) and the remainder went to home, govern(3). The average amount ment, industrial, and commercial facility, site, and land uses spent on pesticides is$4200 per farm and$20 per homeowner, though it is estimated that only three-fourths of farms and homes use pesticides (3). The types and amounts of pesticides used in food and fiber production are shown in Fig. 3. Herbicides and plant growth regulators were responsible for nearly halfof the pounds of pesticides applied, followed by fumigantshematicides, insecticideshiticides, and fungicides.

(720 Million Pounds)

6%

Conventtonal Pestlclder (1.22 Billion Pounds) Includes *othef pesticide chemicals not developedlproduced

use as oesticides. Thlsincludes: -sulfur -petroleurn -chlorine

pecialty Blocidea 60 Million Pounds)

Pounds)

Chlorlne#ypochlorltea (2.32Billion

Usage Total U.S. (4.5 Billion Pounds)

Fig. 1 U.S.pesticide use, 1995. (Adapted from EPA (3).)

Browning and Winter

542 IndustrlaUGowmmenWHom~er

I

Production of Food and Fiber Products

Fig. 2 Conventional pesticide use in the United States, 1995. (Adapted from EPA (3).)

Fig. 4 suggests an overall decrease in pesticide use since 1979,though agricultural use of pesticides fluctuates yearly depending on many factors such as floods/unseasonable weather, pest outbreaks, and increases in planted acreage of pesticide intensive crops. Flooding conditions in 1993 led to higher weed infestation problems in 1994,resulting in greater pesticide use(3). Care should be exercised when developing definitive conclusions of pesticide use trends based on comparisons of total pounds applied. The effectiveness of various pesticides may differ dramatically; some may be active at rates of pounds of an ounce per acre.A more effective per acre, while others may be effective at fractions method to determine pesticide use trends is to compare the number of pesticide applicationsratherthantheamountsapplied.Unfortunatelythistypeofdataisfrequently lacking. California has the most comprehensive pesticide use reporting system in the nation, having established the nation’s first full-use reporting system in 1990 (14). Growers are required to report to the county agricultural commissioners all site-specific information related to use of each pesticide, including the total amount of product applied and the specific number of acres treated, as well as the precise location of application. The Calif

Fig. 3 Types of pesticides used (by volume) in the United States in 1995. (Adapted (3)J

from EPA

Agricultural Chemicals

543

O I0 0

800 600

400

200 0 1979 1981 1983 1985 1987 1989 1991 1993 1995

Fig. 4 Annual volume of pesticides used in food and fiber production in the UnitedStates, 19791995. (Adapted from EPA (3.)

nia pesticide reporting system generates realistic pesticide use data which can be used to more accurately guide dietary risk assessment (15).

IV. PESTICIDE REGULATIONS The EPA, the U.S. Food and Drug Administration (FDA), and the USDA are responsible for regulating and monitoring pesticide use in the United States. The EPA registers pesticides for use and establishes allowable levels of pesticides (tolerances) on food and feed crops. The FDA monitors domestic and imported foods, and the USDA monitors meat and poultry. The USDA also has responsibility for the Pesticide Data Program, which, in contrast to the FDA's tolerance enforcement focus, more randomly samples primarily fruits and vegetables in ready-to-eat form to aid EPA's risk assessment activities (16). The USDA was given the initial responsibility in 1947 to administer FIFRA. The original statute was intended to group all pest control products under one law and provide one agency with jurisdiction to deny, suspend, or cancel registrationsof pesticide products. The newly created EPA assumed responsibility forFIFRA administration in 1972, while the FDA and USDA retained basic responsibility for both monitoring residue levels and seizure of foods not in compliance with the regulations. Since 1972 the EPA has been authorized to grant pesticide registration and to establish pesticide tolerances and regulate pesticide residues in food and feed under FIFRA. FIFRA is primarily a risk/balancing statute;if the benefits of the useof a pesticide (such as increased productivity, lower food cost, or public health protection) are deemed by EPA to outweigh competing risks (such as adverse effects to humans, including consumers and agricultural workers) or environmental damage, the EPA has the authority to register the pesticide for specified uses (17). For food use pesticides, the EPA requires a full battery of toxicological tests typically performed on rats, rabbits, and nonhuman primates. Such studies include, but are not limited to, acute, subchronic, and chronic exposure, carcinogenicity, metabolic fate, teratogenicity, and mutagenicity studies. The EPA also requires that the manufacturer provide studies on pesticide effects on nontarget organisms, the environmental offate the

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pesticide and its breakdown products, and residue studies. It is estimated that it may take 10 years to fulfill the EPA’s testing requirements for a new pesticide at a cost of approximately $30 million (1). Once collected, the EPA reviews this data to estimate the risks associated with the specified uses of the pesticide and to determine if all appropriate tests have been completed.If such risks are consideredto be lower than the benefits anticipated from the pesticide’s use, the EPA will grant a registration and will specify the pest/comrnodity combinations for which the pesticide may be used and appropriate conditions for use anddisposal,whichincludeconsumer,occupational,andenvironmentalconsiderations. Failure to obey such legal requirements, which are printed on the pesticide label, constitutes a federaloffenseandmayresultinfinancialpenaltiesand/orimprisonment. After registration with the EPA, individual states can restrict or denyofuse a particular pesticide within that state. California is one such state with stringent use restrictions.

V.

PESTICIDE RESIDUE LIMITS

As described previously, when the use of a pesticide on a food or feed crop may have the potential to leavea residue, the EPA typically requires thata “tolerance” or maximum allowable level be established for that pesticide/commodity combination. Residues deto be illegal and may result in tected in excess of the established tolerance are deemed seizure and possibly removal of the commodity from the market andfines to the producer. Illegal residues also result when residues are detected, regardlessof level, on commodities for which the pesticide is not registered for use. The process EPA uses to establish tolerances is rather confusing and often misunderstood; this process is described in much more detail by Winter (17). Briefly, tolerances are established to represent the maximum expected residues of a pesticide on a particular commodity resulting from legal applications of the pesticide under conditions specified for use. As such, tolerances are usefulas enforcement tools to indicate whether application conditions were followed, since it is highly unlikely that residues in excess of tolerances will be encountered under the specified use conditions. In addition, the presence of a pesticide on a commodity for which the pesticide does not have an established tolerance could indicate that the pesticide was used on the wrong commodity or could illustratethat care was not exercised to prevent contamination of other commodities through factors such as drift or residual soil uptake. Tolerances, unfortunately, are often considered as “safety standards,” which is a misnomer, since illegal residues rarely meet toxicological criteria as “unsafe” residues (17). Before granting a tolerance. EPA makes assessments of potential human exposure resulting from all registered (and proposed) uses of the pesticide. Typically the EPA initially calculates the theoretical maximum residue contribution (TMRC), which represents the maximum legal exposure to the pesticide and assumes that (a) the pesticide is always used on all commodities for which it is registered, (b) residues are always present at thc tolerance level, and (c) there is no reduction in residue levels resulting from postharvest factorssuch as transportation,washing,peeling,cooking,processing,etc.The TMRC value is compared with toxicological criteria such as the reference dose (RfD), which represents, based upon the results of animal toxicology studies and extrapolations to humans, the typical daily exposure level not considered to represent any appreciable level of risk. If the TMRC is below the RfD, the risk from the use of the pesticide is deemed

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negligible and the tolerance petition is typically approved, provided that the oncogenic (cancer) risks posed at the TMRC are below the “negligible risk” of level 1 excess cancer per million. The calculation of oncogenic risks uses conservative (risk magnifying) assumptions concerning low-dose extrapolations from the results of animal cancer studies performed at moderate and high doses (18). In cases where the TMRC exceeds the RfD or the oncogenic risk exceeds the negligible risk level, the EPA may use refinements in its risk assessment practices to more accurately assess “anticipated” levels of exposure by considering more realistic pesticide use, residue, and/or postharvest data. Some studies have indicated that the TMRC may often exaggerate exposure levels by offactors 10,000100,000 times (10). Since the tolerance values represent, by definition, the maximum residues anticipated from the proper use of a pesticide, is it clear that assuming all residues As an example, Fig.5 compares tolerance are present at the tolerance level is not realistic. levels, anticipated residues using controlled field trials, and actual regulatory monitoring findings for the fungicide captan on apricots, cherries, and peaches. Both the highest observed regulatory monitoring levels and the anticipated residues from legal worst-case field trials represent only a small fraction of the established tolerance level. In many cases, the artificially high exposures estimated using the TMRC cause the RfD to be exceeded or the oncogenic to exceed a negligible risk level, necessitating the need for refinements in the exposure calculations. Such refinements may include adjustments of actual pesticide use (in contrast to the assumption that 100% of the acreage is treated), the useof more realistic residue data, and/or considerationof potential postharvest effects such as processing that could significantly reduce actual consumer exposure to the pesticide. These refined exposure estimates commonly represent the anticipated residue contribution (ARC) (18). If the ARC is below the RfD, and the oncogenic risk at such an exposure is less than 1 excess cancer per million, the tolerance is usually established.

Apricots

m

level

m

Peaches Cherries Anticipated

observed Maximum

residue’

Fig. 5 Comparison of captan tolerances and residues from controlled field studies and monitoring programs. (Adapted from Winter (17).) “Average residue from controlled field studies using most severe legal applicahon of pesticide. hCalifomia Department of Food and Agriculture Monitoring Programs, 1981-1984.

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I n summary, while dietary health risks from exposure to residues are considered before a tolerance is approved, the actual tolerance level established is not based on safety, but rather represents the maximum residue anticipated from the legal use of the pesticide.

VI.

FOOD QUALITY PROTECTION ACT OF 1996

In August 1996, the Food Quality Protection Act (FQPA) was signed into law and promised to significantly alter the way pesticides would be regulated in the United States. The act was intended to end a paradox in the statutes governing pesticide residues found in FFDCA and FIFRA. The Delaney clause, a 1958 FFDCA amendment. stipulatedthat any food additive shown to cause cancer in humans or laboratory animals could not be used. By definition, pesticide residues were considered to be food additives only in cases where a pesticide concentrated to greater levels i n the processed food than in the raw commodity a processed food form. Inconsistencies werc or when a pesticide was added directly to developed in the regulation of raw versus processed foods, and additional inconsistencies surrounded the two major federal laws; the Delaney clause, under FFDCA, was a “risk only” statute, while FIFRA was a “risk balancing” law that allowed consideration of bothrisksandbenefits.Afteralengthystudy,theNationalResearchCouncilin1987 recommended that the EPA adopt a uniform “negligible risk” policy for the regulation of potentially carcinogenic pesticides in all foods as a reasonable substitute for the zerorisk Delaney clause that applied to processed food forms. The EPA adopted this policy in 1988 but wasimmediatelychallenged in court.After the U S . 9thCircuitCourt of Appeals ruled that EPA’s “negligible risk” policy didnot comply with the Delaney clause ( 19) and subsequent consent decree agreements were developed that specified a time table forwhichpesticideregistrationssubject tothe Delaneyclause wouldbe revoked,the FQPA emerged as a legislative tool to eliminate the Delaney clause from pesticide regulation. on processed The new act establishes one law for all pesticide residue tolerances food and raw agricultural commodities, aswell as standards that applyto all risks, whether oncogenic or nononcogenic. The EPAmust determine that tolerances are “safe,” defined as “a reasonable certainty that no harm will result from aggregate exposure” IO the pesticide (20). “A reasonable certainty of no harm” has historically been interpreted as a one i n one million additional risk of cancer over a lifetime and exposure below the reference dose. TheFQPAalsoadoptsseveral oftherecommendationsfrom the 1993National Research Council report on pesticides in the diets of infants and children. Important new provisions of the FQPA include thepotentialuse of anadditional IO-fold uncertainty factor when extrapolating the results of animal toxicology data of possible human effects to provide additional protection for infants and children, consideration of “aggregate” risk from water and domestic exposure to pesticides in addition to dietary exposure, and consideration of “cumulative“ exposure to familiesof pesticides (suchas the organophosphate insecticides) that possess common mechanisms of toxicological action. The FQPA also expedites approval of safer pesticides, creates incentives for the development and maintenancc of effective crop protection tools for American farmers, requires periodic reevaluation of pesticide registrations and tolerances, requires the EPA to account for possible endocrine disruption, and provides consumer right-to-know provisions (20).

Agricultural Chemicals

VII.

547

INTERNATIONAL REGULATIONS

Pesticide residues on foods entering the United States from other countries must comply with U S . tolerances. Throughout the world, however, pesticide residue standards may vary from country to country. Some pesticides used on commodities in foreign countries may not be allowed on the same commodities in the United States, while other pesticides permitted in the United States may not be allowed in other countries ( 18). The Codex Committee on Pesticide Residues (CCPR) was created to provide international guidance on pesticide residue issues. The CCPR reconmends international pesticide tolerance standards to facilitate international food trade. The Food and Agriculture Organization (FAO) and the World Health Organization (WHO) established the CCPR i n 1961-1962 (21 ). Membership is open to all members and associate members of FAO and/or WHO. Scientific experts from the Joint Expert Committee on Food Additives and the Joint Meeting on Pesticide Residues make independent recommendations to WHO and FAO on pesticide residue limits known as maximum residue limits (MRLs) (22). These MRLs are not recommended unless an acceptable daily intake (ADI), analogousto the U S . reference dose, has been established from relevant data. Any question of the pesticide’s safety will result in reevaluation, and all nations participating may comment on the limits set by the Codex (21). Thcre are currently several pesticides subject to Codex MRLs for one or more food conmodities common to international trade. The United States is one of Inany countries participating in Codex thathasnot accepted theseinternationalpesticidelimitsunless they match those already established by the EPA. When comparing Codex Alimentarius equivalent MRLs to U.S. tolerances, the standards were equal47% of the time, the Codex MRLs were lower 34% of the time, and U.S. standards were lower 19% of the time (23). Because differing data sets are used, pesticide metabolites are regulated differently, and agricultural production/pest control practices differ, it is difficult to reach a consensus on the best international regulatory numbers (23).

VIII. MONITORING AND ENFORCEMENT Although the EPA, USDA, and FDA all have roles in the regulation of pesticides, the FDA is charged withenforcingtolcrances in domestic and imported foods shipped in interstate commerce (24). The objective of the FDA monitoring program is to monitor foods and feed for illegal pesticide residues andto take regulatory action when tolerances are exceeded or when residues are detected on commodities for which the pesticide is not registered. The FDA primarily performs commodity monitoring from which fruits. vegetables, and other commodities are sampled and analyzed for residues of more than 200 possible pesticides using multiresidue screening techniques (24). Commodity monitoring programs encompass both surveillance monitoring and compliance monitoring. I n the surveillance monitoring program, the typesof conmodities chosen for sampling and the origins of the samples are targeted to improve FDA’s ability to identify violative residues ( 2 5 ) . While targeted, samples collected in this program are far more random than those analyzed in the compliance monitoring program i n which samples are usually drawn as a follow-up to illegalresiduedetection or similar problems (typically related to n spccific shipper,

Browning and Winter

548 1.6%

1,2% 8%

66.0

88.1

Domestic

Import

4429 samples

5223 samples

Residue Found Residue Found 0No Residue Found Violative Violative Not

Fig. 6 Summary of FDA's surveillance monitoring program, 1997. (Adapted from FDA (24).)

grower, geographic area, or country). Most samples gathered by the FDA are from surveillance sampling. Domestic samples are usually collected near the source of production or at the wholesale level, whereas imported foods are sampled at the pointof entry into the United States. Raw agricultural commodities are preferable for commodity samples, whic are then analyzed before washing or peeling. The FDA also tests some processed foods (24). In 1997 the FDA analyzed 9652 surveillance and 191 compliance samples. Fig. 6 compares results from domestic and import monitoring and Table 1 provides a more detailed breakdown by commodity. The FDA notes that the majority of the illegal residues from foodof imported origin occurred when residues were detected for which no tolerance was established on the commodity, not from residues in excess of legal tolerances (24). In the vast majority of these cases, the violative pesticide is registered for use (by the

Table 1 FDASurveillanceMonitoring Program1997 byCommodity Number samples violative limits found within legal of Domestic surveillance Samples (by commodity) Fruits Vegetables Graindgrain products Milk/dairy productdeggs Fish/shellfish/other Baby foods/formula Import surveillance Samples (by commodity) Fruits Vegetables Graindgrain products Milk/dairy products/eggs Fish/shellfish/other Baby foods/formula Source: Adapted from FDA (24).

No residue Residue found Residue found

1171 1707 397 628 369 51

44.1% 69.1% 59.5% 97.0% 68.0% 82.8%

54.7% 28.5% 40.6% 3.0% 32.0% 17.2%

1.2% 2.4% 0.0% 0.0% 0.0%

2034 2356 322 85 158 268

60.6% 63.0% 86.0% 89.4% 93.7% 86.6%

38.2% 34.9% 13.0% 10.6% 6.3% 10.8%

1.2% 2.1% 0.9% 0.0% 0.0% 2.6%

0.0%

549

Agricultural

EPA) on other commodities. Results clearly indicate that residue levels rarely approach the tolerance levels and that illegal residues are infrequently encountered. The FDA also performs its Total Diet Study annually to acquire incidence/level data on particular commodity/pesticide combinations. This study uses a market basket approach, with 261 foods comprising each market basket which are gathered once a year in each of the four geographical regions of the United States from three different cities in each region. Each collectionof foods is prepared for table-ready consumption and then analyzed for pesticide residues. As an example, FDA inspectors may purchase apples, flour, eggs, and sugar to use in the baking of an apple pie and the pie is analyzed for pesticide residues at the time it is ready to be consumed (24). By combining analytical results with estimates of typical consumption rates of the various components of food analyzed in the Total Diet Study, it is possible to estimate typical daily toexposure individual pesticides for the general population as well as for specific population subgroups defined by such factors as age, gender, and geographical location.

U.S. Department of Agriculture

A.

The USDA is another federal agency responsiblefor analyzing foods for pesticide residues. Specifically, its National Residue Program analyzes meat, poultry, and raw egg products for pesticide residues, animal drugs, and environmental contaminants. As with the FDA, samples include both domestic and imported food products. The USDA uses multiresidue analysis methods that detect each major insecticide class (chlorinated hydrocarbons, chlorinated organophosphates, organophosphates, and carbamates) and also detect 40 individual pesticides (10). The USDA’s residue program was expanded to include collection of data on pesti(AMs)was appointed cide residues in food. The USDA’s Agricultural Marketing Service to undertake the creation and implementation of such a program, currently known as the Pesticide Data Program (PDP). PDP has been in operation since May 1991 and has published findings for calendar years 1991 through 1995. ofTen the 50 states currently participate: California, Florida, Michigan, NewYork, Maryland, Ohio, Texas, Wisconsin, Colorado, and Washington (26). In 1996 the PDP collected and analyzed 5771 samples originating in 35 states and 10 foreign countries (26). Fig. 7 summarizes the 1996 PDP results in terms of origin of sample (domestic versus import) and residue findings. Eight fresh fruit and vegetable

24.8% 12%

87.8%

Domestic

5771 Samples

I

No doteetablo rerlduea

\

3.4%

Vlolathre Reslduea

71.8%

Detectable residues

Pesticide Residue

Fig. 7 SummaryofUSDA’spesticidedataprogrammonitoring, (261.)

Results 1996. (AdaptedfromUSDA

Browning and Wfnter

550

commodities were collected, including apples, carrots, grapes, oranges, peaches, spinach, sweet potatoes, and tomatoes. In addition, four processed fruit and vegetable commodities were collected, including apple juice, canned and frozen green beans, sweet corn, and 3.4% sweet peas. Remaining samples were drawn from wheat and whole milk. Of the violative residues detected, most (96.3%)of the violations occurred when a residue was found not licensed for that product(16). The PDP monitoring program shows a higher percentageof detected residues than does the FDA’s surveillance monitoring program. The major reason for the difference is that analytical methods used in the PDP are generally far more sensitive than the FDA’s methods. By incorporating the PDP data with the Total Diet Study data, the EPA can more accurately assess the dietary risks posed by pesticides. The PDP’s sampling procedures are designed to provide more realistic estimates of pesticide residues as close as possible to the point of consumption, thereby improving the reliability and extent of information to the actual percentage available for risk assessment. This gives numerical representation of crop treated with a pesticide and considerations such as washing, cooking, processing, and storage of commodities. Most recent changes in the PDP have led to a greater focus on monitoring foods consumed most frequently by infants and children in response to recommendations of the National Research Council’s 1993 report on pesticides in the diets of.infants and children.

B. State Programs In addition to the FDA’s monitoring system, individual monitoring programs exist in 38 states; such state programs vary considerably with respect to focus and sampling rates. California currently has the largest state monitoring program and typically spends more that $40 million each year to regulate pesticide use. Results of California’s Routine Marketplace Surveillance program for 1995 are shown in Fig.8; of the5502 samples analyzed, the vast majority (64.7%)showed no detectable residues, while illegal residues were de-

Residue less than 10% of tolerance 24.5%

Residue between 10% and 5 0 % of tolerance

Residue between 50% and 100% of tolerance 1 .O%

\

\No Illegal residue

Resldue Detected

64.7%

1.6Yo

Fig. 8 Summary of California’sroutinemarketplacesurveillancemonitoringprogram, (Adapted from California Department of Pesticide Regulation (15).)

1995.

Agricultural Chemicals

551

tected in 1.6% of the samples ( 15). Approximately 75%)of illegal residues occurred when pesticides were detected on commodities for which they were not registered for use; only about 25% of the illegal residues represented tolerance violations. Results also indicated that legal residues. when detected, typically existed at a small fraction of established tolerances, with the majority of detected residues present at less than 10% of tolerance (IS).

IX.

DIETARY RISKS

Calculating the dietary risks I‘aced by consumers from pesticide residues is a challenging task that requires a variety of assumptions to be made (18,27). The types of assumptions made may dramatically influence the nnagnitutles of estimated risks; the use of conservative assumptions Inay produce much higher risk estimates than those obtained using less conservative assumptions. As an example, the NRC estimated oncogenic risks for a number of pesticides by assuming exposure at the TMRC level, which resulted in the finding that most of the pesticides studied generated oncogenic risks far in excess of the negligible (28). The use of more accurate human risk standard of one excess cancer per million exposure data reduced the oncogenic risks for most of the pesticides to levels far below the same negligible risk standard (10). A common method used to address the magnitude of dietary pesticide risks is to present the resultsof regulatory monitoring programs by providing information concerning the percentages of samples found to have either no detectable residues, detectable but legal residues, and violative residues. Unfortunately such findings are of little use in the risk assessment arena since tolerances are not directly related to safety levels, as has been described prcviously, and illegal residues should not be construed as “unsafe” residues in most cases. A Inore accurate approach to estimate risks from pesticides is to use the exposure data developed from market basket surveys such as the FDA’s Total Diet Study and relate that data to reference doses and/or calculate oncogenic risks. While several assumptions relating to food consumption patterns and toxicological potencies still must be made, this type of approach precludes the need to estimate pesticide use patterns, residue levels on raw commodities, or postharvest effects on residue levels. 1991 TotalDietStudyarecompared Exposure estimates developed from FDA’s with EPA’s established reference doses and the analogous acceptable daily intakes estabi n allthree lished by WHO in Table 2 (29). Resultsindicatethatformostpesticides population subgroups, exposure estimates represent only a small fraction (often less than 1%) of the corresponding reference doses or acceptable daily intakes. I n the typical case where reference doses are derived using a 100-fold uncertainty factor when extrapolating from the highest level that does not produce a noticcable effect in animal studies to estimate an appropriate human daily exposure level, exposure at the reference dose would constitute a level 100 times lower than that of the no observed effect level i n animals. Exposure at a level of 1% of the reference dose corresponds to an exposure 10,000 times below the level that does not produce noticeable effects in the animals. Such findings provide an illustration of why the majority of health professionals consider the risksof pesticide residues i n foods tobe far lowerthan a number of other food safety risks such as those posed by nlicrobiological contamination, nutritional imbalance, environmental contaminants, and naturally occurring toxins (30). Even so. these findings

552 Table 2

Browning and Winter

Pesticide Intakes Estimatcd From FDA's Total Diet Estimated exposure (Kglkglday)

Pesticide

0.0002

alpha

Acrphate Azinphos-methyl BHC, BHC, gamma (Lindane) Captan Carbaryl Carbofuran, total Chlordanc. total Chlorpyrifos Chlorpyrifos-methyl DCPA DDT, t o t a l DEF Dcmeton Diazinon Dichlorvos Dicloran, total Dicofol, total Dieldrin Dimethoate Endosulfan, total Endrin Ethion Fenitrothion Fcnuron' Fonofos

Heptachlor, total Hexachlorobcnzene Iprodionc, total Linuron Malathion Mcthamidophos Methomyl Methoxychlor, p,p' Metobromuron" Mcvinphos. total Ncburon' Omthoate Parathion Paration-methyl Pentachlorophenol Permethrin, total Phosalonc Phoslnct Pirimiphos-methyl

6- I 1 months 0.0089 0.0028

14- l6 yearold male 0.01 l 3 0.0033

0.0004 0.0004 0.0478 0.1801 0.0002 0.0001

0.0082 0.0 104 0.0002 0.0095