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Mineral Tolerance of Animals: Second Revised 
Edition 

Committee on Minerals and Toxic Substances in Diets 
and Water for Animals, National Research Council 

ISBN:0-309-55027-0, 510pages, 8 1/2x11, (2005) 

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THE NATIONAL ACADEMIES 

Advisers to the Nation on Science, Engineering, and Medicine 



MinemI Tnlemnce nf Animals- Sennnd Revised Edition 
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MINERAL TOLERANCE 
OF ANIMALS 

SECOND REVISED EDITION, 2005 



Committee on Minerals and Toxic Substances in 
Diets and Water for Animals 

Board on Agriculture and Natural Resources 

Division on Earth and Life Studies 



NATIONAL RESEARCH COUNCIL 

OF THE NATIONAL ACADEMIES 



THE NATIONAL ACADEMIES PRESS 
Washington, D.C. 
vnwN^.nap.edu 



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THE NATIONAL ACADEMIES PRESS • 500 Fifth Street, N.W. • Washington, DC 20001 

NOTICE: The project that is the subject of this report was approved by the Governing Board of the 
National Research Council, whose members are drawn from the councils of the National Academy of 
Sciences, the National Academy of Engineering, and the Institute of Medicine. The members of the 
committee responsible for the report were chosen for their special competences and with regard for appro- 
priate balance. 

This study was supported by Contract/Grant No. 223-01-2460 between the National Academy of 
Sciences and the Center for Veterinary Medicine of the U.S. Food and Drug Administration, Department 
of Health and Human Services. Any opinions, findings, conclusions, or recommendations expressed in 
this publication are those of the author(s) and do not necessarily reflect the views of the organizations or 
agencies that provided support for the project. 

Library of Congress Cataloging-in-Publication Data 

Mineral tolerance of animals / Committee on Minerals and Toxic Substances, Board on Agriculture and 
Natural Resources, Division on Earth and Life Studies. — 2nd rev. ed. 
p. cm. 

Rev. ed. of: Mineral tolerance of domestic animals / National Research Council (U.S.). Subcommittee on 
Mineral Toxicity in Animals. 1980. 

Includes bibliographical references and index. 

ISBN 0-309-09654-5 (pbk.) — ISBN 0-309-55027-0 (pdf) 1. Veterinary toxicology. 2. Minerals in 
animal nutrition. I. National Research Council (U.S.). Committee on Minerals and Toxic Substances. II. 
National Research Council (U.S.). Subcommittee on Mineral Toxicity in Animals. Mineral tolerance of 
domestic animals. 

SF757.5M56 2005 

636.089'59— dc22 

2005024930 
Additional copies of this report are available from the National Academies Press, 500 Fifth Street, N.W., 
Lockbox 285, Washington, DC 20055; (800) 624-6242 or (202) 334-3313 (in the Washington metropoli- 
tan area); Internet, http://www.nap.edu 

Copyright 2005 by the National Academy of Sciences. All rights reserved. 

Printed in the United States of America 



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THE NATIONAL ACADEMIES 

Advisers fo fhe Nation on Science, Engineering, and Medicine 



The National Academy of Sciences is a private, nonprofit, self-perpetuating society of 
distinguistied schiolars engaged in scientific and engineering research, dedicated to the fur- 
therance of science and technology and to their use for the general welfare. Upon the 
authority of the charter granted to it by the Congress in 1863, the Academy has a mandate 
that requires it to advise the federal government on scientific and technical matters. Dr. 
Ralph J. Cicerone is president of the National Academy of Sciences. 

The National Academy of Engineering was established in 1964, under the charter of the 
National Academy of Sciences, as a parallel organization of outstanding engineers. It is 
autonomous in its administration and in the selection of its members, sharing with the Na- 
tional Academy of Sciences the responsibility for advising the federal government. The 
National Academy of Engineering also sponsors engineering programs aimed at meeting 
national needs, encourages education and research, and recognizes the superior achieve- 
ments of engineers. Dr. Wm. A. Wulf is president of the National Academy of Engineering. 

The Institute of Medicine was established in 1970 by the National Academy of Sciences to 
secure the services of eminent members of appropriate professions in the examination of 
policy matters pertaining to the health of the public. The Institute acts under the responsibil- 
ity given to the National Academy of Sciences by its congressional charter to be an adviser 
to the federal government and, upon its own initiative, to identify issues of medical care, 
research, and education. Dr. Harvey V. Fineberg is president of the Institute of Medicine. 

The National Research Council was organized by the National Academy of Sciences in 
1916 to associate the broad community of science and technology with the Academy's 
purposes of furthering knowledge and advising the federal government. Functioning in 
accordance with general policies determined by the Academy, the Council has become the 
principal operating agency of both the National Academy of Sciences and the National 
Academy of Engineering in providing services to the government, the public, and the scien- 
tific and engineering communities. The Council is administered jointly by both Academies 
and the Institute of Medicine. Dr. Ralph J. Cicerone and Dr. Wm. A. Wulf are chair and vice 
chair, respectively, of the National Research Council. 

www.national-academies.org 



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COMMITTEE ON MINERALS AND TOXIC SUBSTANCES IN 
DIETS AND WATER FOR ANIMALS 

KIRK C. KLASING, Chair, University of California, Davis 

JESSE P. GOFF, U.S. Department of Agriculture, Agricultural Research Service, 

Ames, Iowa 
JANET L. GREGER, University of Connecticut, Storrs 

JANET C. KING, Children's Hospital Oakland Research Institute, Oakland, California 
SANTOSH P. LALL, Institute for Marine Biosciences, National Research Council of 

Canada, Halifax, Nova Scotia 
XINGEN G. LEI, Cornell University, Ithaca, New York 
JAMES G. LINN, University of Minnesota, St. Paul 
FORREST H. NIELSEN, U.S. Department of Agriculture, Agricultural Research Service, 

Grand Forks, North Dakota 
JERRY W. SPEARS, North Carolina State University, Raleigh 

Staff 

AUSTIN J. LEWIS, Study Director 

JAMIE S. JONKER, Study Director* 

DONNA LEE JAMEISON, Senior Program Assistant 

PEGGY TSAI, Research Associate 



*Througli June 2004 



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BOARD ON AGRICULTURE AND NATURAL RESOURCES 

MAY R. BERENBAUM, Chair, University of Illinois, Urbana-Champaign 

SANDRA J. BARTHOLMEY, University of Illinois at Chicago 

ROGER N. BEACHY, Donald Danforth Plant Science Center, St. Louis, Missouri 

H. H. CHENG, University of Minnesota, St. Paul 

W. R. (REG) GOMES, University of California, Oakland 

ARTURO GOMEZ-POMPA, University of California, Riverside 

PERRY R. HAGENSTEIN, Institute for Forest Analysis, Planning, and Policy, 

Wayland, Massachusetts 
JEAN HALLORAN, Consumer Policy Institute/Consumers Union, Yonkers, New York 
HANS R. HERREN, Millennium Institute, Arlington, Virginia 
DANIEL P. LOUCKS, Cornell University, Ithaca, New York 
WHITNEY MACMILLAN, Cargill, Incorporated, Minneapohs, Minnesota 
BRIAN W. MCBRIDE, University of Guelph, Guelph, Canada 
TERRY L. MEDLEY, E. I. duPont de Nemours & Co., Wilmington, Delaware 
OLE NIELSEN, Ontario Veterinary College, Spruce Grove, Canada 
ROBERT PAARLBERG, Wellesley College, Watertown, Massachusetts 
ALICE N. PELL, Cornell University, Ithaca, New York 
BOBBY PHILLS, Florida A&M University, Tallahassee 

PEDRO A. SANCHEZ, The Earth Institute at Columbia University, Pahsades, New York 
SONY A B. SAL AM ON, University of Illinois, Urbana-Champaign 
B. L. TURNER, II, Clark University, Worcester, Massachusetts 
TILAHUN D. YILMA, University of California, Davis 
JAW-KAI WANG, University of Hawaii, Honolulu 

Staff 

ROBIN A. SCHOEN, Director 
CHARLOTTE KIRK BAER, Director* 
KAREN L. IMHOF, Administrative Assistant 



*Through October 2004 



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Acknowledgments 



The committee would like to thank Charlotte Kirk Baer 
and Robin Schoen, board directors, and Austin Lewis and 
Jamie Jonker, program officers, for their cheerful and expert 
guidance during this project. The committee also wishes to 
thank Gretchen Hill and Marcia Carlson Shannon for assis- 
tance with the chapters on iron and zinc and Katherine 
Mahaffey for helpful discussions on levels of minerals that 
are of concern for human health. Finally, we greatly appreci- 
ate the help of Donna Jameison, Marina Peunova-Conner, 
and Peggy Tsai for their support of our communications, 
meetings, and writings. 

The sponsorship of this study by the Center for Veteri- 
nary Medicine of the U.S. Food and Drug Administration 
(FDA) is gratefully acknowledged. The support of the FDA 
liaison, William D. Price, was especially appreciated. 

This report has been reviewed in draft form by individu- 
als chosen for their diverse perspectives and technical exper- 
tise, in accordance with procedures approved by the NRC's 
Report Review Committee. The purpose of this independent 
review is to provide candid and critical comments that will 
assist the institution in making its published reports as sound 
as possible and to ensure that the report meets institutional 
standards for objectivity, evidence, and responsiveness to 
the study charge. The review comments and draft manu- 
script remain confidential to protect the integrity of the de- 
liberative process. We wish to thank the following individu- 
als for their review of this report: 

Clarence B. Ammerman, University of Florida, 
Gainesville, FL 



Dennis J. Blodgett, Virginia Tech University, 

Blacksburg, VA 
Gerry F. Combs, USDA, Grand Forks, ND 
Gail L. Czarnecki-Maulden, Nestle Purina Research, 

St. Joseph, MO 
L. Wayne Greene, Texas A&M University, 

Amarillo, TX 
Pamela Henry Miles, University of Florida, 

Gainesville, FL 
Mark P. Richards, USDA, ARS, Beltsville, MD 
Kristi Smedley, Center for Regulatory Services, 

Woodbridge, VA 
Patricia A. Talcott, University of Idaho, Moscow, ID 
Duane E. Ullrey, Michigan State University, East 

Lansing, MI 
William P. Weiss, The Ohio State University, 

Wooster, OH 

Although the reviewers listed above provided many con- 
structive comments and suggestions, they were not asked to 
endorse the conclusions or recommendations nor did they 
see the final draft of the report before its release. The review 
of this report was overseen by Gary L. Cromwell, University 
of Kentucky. Appointed by the National Research Council, 
he was responsible for making certain that an independent 
examination of the report was carried out in accordance with 
institutional procedures and that all review comments were 
carefully considered. Responsibility for the final content of 
this report rests entirely with the authoring committee and 
the institution. 



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Preface 



In 2003, the National Research Council's Committee on 
Animal Nutrition convened an ad hoc committee to conduct 
a thorough review of the scientific literature related to min- 
erals and toxic substances in the diets and water for animals 
and to update the 1980 edition of Mineral Tolerance of Do- 
mestic Animals. In particular, the committee was asked to 
provide recommendations on animal tolerances and toxic 
dietary levels, and identify minerals that pose potential hu- 
man health concerns. A nine-person committee of scientists 
specializing in nutrition, toxicology, and veterinary medi- 
cine accepted this task. Individual members brought to this 
project diverse expertise and perspective on the impact of 
nutrition on the health of fish, poultry, livestock, compan- 
ion animals, and humans. The committee met three times 
and had monthly teleconferences over a period of more than 
a year. 

The challenge of making recommendations on the toler- 
able and toxic levels of close to 40 elements for a variety of 
species is daunting. Each of these elements exists in many 
chemical forms, each with differing properties and toxicity 
profiles, further complicating the task. The toxicity profile 
of some minerals is very well described, whereas the toxic- 
ity of other minerals, especially to livestock and companion 
animals, has received little attention. A wide variety of in- 
formational sources, ranging from government surveys to 
professional experiences, was considered for this report. 
Expert reports on several minerals were also solicited. How- 
ever, the backbone of this report is the primary literature of 
peer-reviewed journal publications. The committee recog- 
nized that much of the information in the 1980 publication 



was still relevant, but that this historic foundation needed to 
be reevaluated in the context of newer infonnation on the 
methods of mineral analysis, mechanisms of homeostasis 
and toxicity, and appropriate indices of animal health and 
well-being. Consequently, a reanalysis of the historic litera- 
ture is synthesized with the recent literature to form the rec- 
ommendations in this report. This edition considers a greater 
breadth of animal species than the past edition and expands 
the coverage on the metabolism and mechanisms of toxicity 
of minerals, methods and problems in mineral analysis, and 
the relationships between mineral exposure of animals and 
the mineral levels in animal products destined for human 
consumption. New chapters provide additional focus on 
acid-base balance, nitrates, and water quality. Finally, this 
edition has placed increased emphasis on the safety of ani- 
mal products in the human diet as criteria for setting maxi- 
mum tolerable levels of minerals in the feed and water of 
farm animals. 

The recommendations in the 1980 report have been 
widely cited and served as the basis of decisions made by 
regulatory agencies and by practicing nutritionists respon- 
sible for the formulation of animal diets. The previous report 
has also been used extensively in teaching, research, and 
veterinary practice. We hope that the utility of this new edi- 
tion equals or surpasses its predecessor. 



KIRK C. KLASING, Chair 

Committee on Minerals and Toxic Substances in Diets 

and Water for Animals 



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Contents 



SUMMARY 1 

1 INTRODUCTION 7 

2 MAXIMUM TOLERABLE LEVELS 10 

3 ALUMINUM 15 

4 ARSENIC 31 

5 BARIUM 46 

6 BISMUTH 54 

7 BORON 60 

8 BROMINE 72 

9 CADMIUM 79 

10 CALCIUM 97 

11 CHROMIUM 115 

12 COBALT 124 

13 COPPER 134 

14 FLUORINE 154 

15 IODINE 182 

16 IRON 199 

17 LEAD 210 

18 MAGNESIUM 224 

19 MANGANESE 235 

20 MERCURY 248 

21 MOLYBDENUM 262 

22 NICKEL 276 

23 PHOSPHORUS 290 

24 POTASSIUM 306 

25 SELENIUM 321 

26 SILICON 348 

27 SODIUM CHLORIDE 357 

28 SULFUR 372 

29 TIN 386 

30 VANADIUM 398 

31 ZINC 413 

32 OTHER MINERALS 428 

33 MINERALS AND ACID-BASE BALANCE 449 

34 NITRATES AND NITRITES 453 

35 WATER AS A SOURCE OF TOXIC SUBSTANCES 469 

ABOUT THE AUTHORS 477 

INDEX 479 

BOARD ON AGRICULTURE AND NATURAL RESOURCES PUBLICATIONS 495 

xi 



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Tables and Figures 



TABLES 

S-l Summary of Minerals Reviewed in This Report 6 

2-1 Maximum Tolerable Levels of Minerals in the Feed (mg/kg or % of the DM) of Animals Based on 

Indexes of Animal Health 13 

2-2 Toxicity Assessments of Nutrients for Humans Based on Food and Nutrition Board Assessments 14 

3-1 Effects of Aluminum Exposure in Animals 27 

3-2 Aluminum Concentrations in Fluids and Tissues of Animals 30 

4-1 Effects of Arsenic Exposure in Animals 42 

4-2 Arsenic Concentrations in Fluids and Tissues of Animals 45 

5-1 Effects of Barium Exposure in Animals 51 

5-2 Barium Concentrations in Fluids and Tissues of Animals 53 

6-1 Effects of Bismuth Exposure in Animals 58 

6-2 Bismuth Concentrations in Fluids and Tissues of Animals 59 

7-1 Effects of Boron Exposure in Animals 68 

7-2 Boron Concentrations in Fluids and Tissues of Animals 71 

8-1 Effects of Bromine Exposure in Animals 77 

8-2 Bromine Concentrations in Fluids and Tissues of Animals 78 

9-1 Effects of Cadmium Exposure in Animals 91 

9-2 Cadmium Concentrations in Fluids and Tissues of Animals 94 

10-1 Effects of Calcium Exposure in Animals 109 

10-2 Calcium Concentrations in Fluids and Tissues of Animals 1 14 

11-1 Effects of Chromium Exposure in Animals 121 

11-2 Chromium Concentrations in Fluids and Tissues of Animals 123 

12-1 Effects of Cobalt Exposure in Animals 130 

12-2 Cobalt Concentrations in Fluids and Tissues of Animals 133 



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TABLES AND FIGURES 



13-1 Effects of Copper Exposure in Animals 147 

13-2 Copper Concentrations in Fluids and Tissues of Animals 153 

14-1 Effects of Fluorine Exposure in Animals 169 

14-2 Fluorine Concentrations in Fluids and Tissues of Animals 181 

15-1 Effects of Iodine Exposure in Animals 194 

15-2 Iodine Concentrations in Fluids and Tissues of Animals 198 

16-1 Effects of Iron Exposure in Animals 208 

16-2 Iron Concentrations in Fluids and Tissues of Animals 209 

17-1 Effects of Lead Exposure in Animals 221 

17-2 Lead Concentrations in Fluids and Tissues of Animals 223 

18-1 Effects of Magnesium Exposure on Animals 232 

18-2 Magnesium Concentrations in Fluids and Tissues of Animals 234 

18-3 Tissue Magnesium Concentrations in Steers Fed Different Dietary Levels of Magnesium 234 

19-1 Effects of Manganese Exposure in Animals 243 

19-2 Manganese Concentrations in Fluids and Tissues of Animals 247 

20-1 Effects of Mercury Exposure in Animals 259 

20-2 Mercury Concentrations in Fluids and Tissues of Animals 261 

21-1 Effects of Molybdenum Exposure in Animals 271 

21-2 Molybdenum Concentrations in Fluids and Tissues of Animals 274 

22-1 Effects of Nickel Exposure in Animals 284 

22-2 Nickel Concentrations in Fluids and Tissues of Animals 288 

23-1 Effects of Phosphorus Exposure in Animals 300 

23-2 Phosphorus Concentrations in Fluids and Tissues of Animals 303 

24-1 Effects of Potassium Exposure in Animals 314 

25-1 Effects of Selenium Exposure in Animals 337 

25-2 Selenium Concentrations in Fluids and Tissues of Animals 344 

26-1 Effects of Silicon Exposure in Animals 355 

26-2 Silicon Concentrations in Fluids and Tissues of Animals 356 

27-1 Single Oral Dose Salt Toxicity 367 

27-2 Acute Salt Toxicity 368 

27-3 Chronic Salt Toxicity 369 

27-4 Sodium and Chloride Concentrations in Fluids and Tissues of Animals 371 

28-1 Effects of Sulfur Exposure in Animals 381 

28-2 Sulfur Concentrations in Tissues of Sheep 385 

29-1 Effects of Tin Exposure in Animals 395 

29-2 Tin Concentrations in Fluids and Tissues of Animals 397 

30-1 Effects of Vanadium Exposure in Animals 405 

30-2 Vanadium Concentrations in Selected Tissues of Animals 410 



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xiv TABLES AND FIGURES 

30-3 Vanadium Concentrations in Otlier Fluids and Tissues of Animals 412 

31-1 Effects of Zinc Exposure in Animals 423 

31-2 Zinc Concentrations in Fluids and Tissues of Animals 426 

34-1 Effects of Nitrate or Nitrite Exposure in Animals 461 

35-1 Sources and Normal Ranges of Minerals in Unpolluted Fresh Water 473 

35-2 Range or Mean of Mineral Concentrations Found in Sea Water 473 

35-3 Major Mineral Species Found in Fresh and Sea Water 474 

35-4 Drinking Water Standards for Humans and Livestock 475 

35-5 Range or Mean of Mineral Concentrations Found in Fresh and Salt Water 476 

FIGURE 

1-1 Mineral Concentrations and Animal Health 8 



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Summary 



BACKGROUND 

Inorganic elements found in the Eartli's crust are often 
referred to as minerals. Some minerals are essential for 
health and productivity of animals and have well-defined 
nutritional and biochemical roles. Many other minerals 
naturally occur at trace levels in the foods and tissues of 
animals but are not typically suspected to play a useful nu- 
tritional purpose and are considered incidental contami- 
nants. However, all minerals, whether essential or nones- 
sential, can adversely affect an animal when amounts in the 
diet and water become excessive, so the prevention of min- 
eral toxicosis is a fundamental part of animal nutrition and 
management. Establishing the levels at which each mineral 
becomes toxic to animals aids nutritionists, veterinarians, 
toxicologists, government regulators, and ranchers and other 
animal owners to safeguard the feed that animals consume 
and the water that they drink to optimize animal health and 
minimize tissue residues. 

In 1980, the National Research Council published Min- 
eral Tolerance of Domestic Animals, which reviewed and 
evaluated the literature relating to mineral tolerance of do- 
mestic animals and set maximum tolerable levels of dietary 
minerals in feeds. Since that time, there have been important 
developments in nutrition and toxicology that compel a re- 
evaluation of mineral tolerances of animals. These develop- 
ments include the following: 

• Improvements in the sensitivity and specificity of 
analysis of mineral concentrations in feeds and animal 
tissues; 

• New information on the bioavailability, homeostasis, 
and mechanism of toxicity of minerals; 

• New understandings of appropriate indices of animal 
health and well-being; 

• Increased disposal of municipal and animal wastes on 
crop lands and pastures, potentially resulting in greater ex- 
posure of animals to certain minerals; 



• Increased feeding of recycled animal by-products and 
wastes that contain high levels of certain minerals; 

• Emergence of the aquaculture industry; 

• Increased evidence for potentially toxic levels of mer- 
cury, cadmium, lead, and other minerals in the diet of hu- 
mans and companion animals; 

• Refinements in the Recommended Dietary Allowances 
and the Tolerable Upper Intake Levels of minerals for hu- 
mans that permit more precise evaluation of potentially toxic 
mineral levels in foods of animal origin; 

• New appreciation of the impact of minerals on environ- 
mental quality. 



Preventing adverse effects of minerals on the health of 
animals, consumers, and the environment requires the appli- 
cation of appropriate nutritional and toxicological principles 
to set limits on mineral exposure to animals. To address this 
need, the Food and Drug Administration of the U.S. Depart- 
ment of Health and Human Services asked the National 
Academy of Sciences to convene a committee of scientific 
experts to make recommendations on animal tolerances and 
toxic dietary levels. A subcommittee of the Committee on 
Animal Nutrition undertook this task. The subcommittee 
consisted of nine scientists specializing in nutrition, toxicol- 
ogy, and veterinary medicine, with diverse expertise and 
perspectives on the impact of nutrition on the health of fish, 
poultry, livestock, companion animals, and humans. 

COMMITTEE CHARGE AND APPROACH 

The committee was given the following task: "An ad hoc 
committee of the standing Committee on Animal Nutrition 
will be convened to conduct a thorough review of the scien- 
tific literature on trace elements and macro minerals, includ- 
ing an analysis of the effects of exposure and toxic levels in 
animal diets; provide recommendations on animal tolerances 
and toxic dietary levels, and identify elements that pose po- 



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MINERAL TOLERANCE OF ANIMALS 



tential human health concerns. The report will address re- 
cent research on tolerance and toxicity of minerals in animal 
diets including the following areas: general considerations; 
mineral sources, discrepancies and difficulties in methods of 
analyses and evaluation of biological status; metabolic min- 
eral interactions; assessments of form and species interac- 
tions; supplementation considerations; bioavailability of dif- 
ferent mineral forms and sources; maximal tolerable levels; 
and effects of diet composition, stressors, and animal physi- 
ological status on mineral utilization; and environmental 
exposure considerations. The report will include all species 
for which adequate information is available — updating the 
previous report, greatly expanding the topics covered, and 
increasing the usefulness of the report. Recommendations 
will be provided on maximum tolerable and toxic dietary 
levels of minerals in animal diets. Potential for toxic expo- 
sure, toxicosis, factors affecting toxicity, and essentiality of 
dietary minerals in various animal species will be discussed." 
This report is based mostly on the primary literature of 
peer-reviewed journal publications. The committee recog- 
nized that much of the information in the 1980 publication 
was still relevant, but that this historic foundation needed to 
be reevaluated in the context of newer infonnation on the 
methods of mineral analysis, mechanisms of homeostasis and 
toxicity, and appropriate indices of animal health and well- 
being. Consequently, a reanalysis of the historic literature is 
synthesized with the recent literature to form the recommen- 
dations in this report. In addition, this report considers a greater 
breadth of animal species than the past edition and expands 
the coverage on the metabolism and mechanisms of toxicity 
of minerals, methods and problems in mineral analysis, and 
the relationships between mineral exposure of animals and the 
mineral levels in animal products destined for human con- 
sumption. New chapters provide additional focus on acid-base 
balance, nitrates, water quality, and rare earth metals. 

ESSENTIALITY 

The essential minerals are those that have well-defined bio- 
chemical roles and must be in the diet of vertebrates for opti- 
mum health and productivity. Minerals play many vital roles: 
for example, they activate many proteins including enzymes, 
maintain the ionic and pH balance, provide for the structural 
rigidity of bones and teeth, and serve as regulatory signals in 
metabolic homeostasis. Inadequate dietary concentrations of 
essential minerals compromise animal growth, reproduction, 
and health. As shown in Table S-1, the committee identified 

• Seventeen minerals that are required by vertebrates: 
calcium, chlorine, chromium, cobalt, copper, fluorine, io- 
dine, iron, magnesium, manganese, molybdenum, phospho- 
rus, potassium, selenium, sodium, sulfur, and zinc; 

• Six additional minerals that may be required based on 
experiments that indicate beneficial effects when supple- 



mented to the diet: arsenic, boron, nickel, rubidium, silicon, 
and vanadium. However, specific biochemical functions 
have not been identified for these six, and there is not a con- 
sensus among nutritionists that these minerals are essential. 

EXPOSURE 

Animals may be exposed to toxic levels of minerals from 
a wide variety of sources. Feedstuffs, especially those de- 
rived from plants, are a common source of potentially toxic 
levels of minerals. Molybdenum and selenium occur natu- 
rally in soils of some regions at concentrations sufficient to 
cause certain plants to accumulate levels that can be toxic 
for animals. High soil, and consequently plant, concentra- 
tions of cadmium, lead, molybdenum, copper, and zinc are 
the primary minerals of concern from the application of 
municipal wastes and other biosolids to the land. Mining, 
smelting, and other industries are often associated with local 
areas of mineral contamination to the water, soil, and air, 
and, ultimately, the plants grown in that area. Feedstuffs of 
animal origin may also be sources of toxic levels of miner- 
als. For example, some types of fishmeals may be high in 
mercury because mercury bioconcentrates through the 
aquatic food chain. 

Mineral supplements are commonly added to animal di- 
ets to correct deficiencies found in pastures, forages, and 
other dietary ingredients. Some mineral supplements may 
contain potentially toxic levels of contaminating minerals, 
depending upon the source of the supplement and the method 
of its processing. Toxic levels of minerals may accidentally 
occur due to mistakes in feed formulation and manufactur- 
ing, or from contamination during storage or transportation. 
Such accidental administration can result in very high min- 
eral levels and cause acute toxicosis and death, whereas most 
other modes of introduction typically cause toxicosis only 
after chronic exposure. Surface water and occasionally even 
deep-well or domestic water supplies may contain excessive 
levels of certain minerals due to naturally high levels in the 
ground. Sulfur, sodium, manganese, selenium, and fluorine 
are among the minerals most likely to reach toxic levels in 
natural water supplies. Minerals may also be introduced into 
water supplies from industrial wastes, pesticide contamina- 
tion, and other sources of pollution. Finally, minerals such 
as arsenic, bromine, bismuth, copper, lithium, magnesium, 
silver, zinc, and some of the rare earths are sometimes added 
to feed or water as therapeutics or for growth promotion. 
Mistakes in use of these minerals have occasionally resulted 
in toxicoses. 

TOXICITY 

The committee looked at two main aspects of the effects 
of toxicity on animals: the mechanisms of toxicity of each 
mineral and the maximum tolerable level that will not impair 



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SUMMARY 



animal health or performance. The individual chapters de- 
scribe findings in each of these areas. Some general conclu- 
sions are summarized below. 



Mechanisms of Toxicity 

The adverse effects of minerals depend on the dietary 
concentration and length of exposure and range from subtle 
effects on homeostatic processes, to impairments in animal 
growth or reproductive rates, to specific pathologies and 
death. An understanding of the biochemical or physiological 
mechanism by which a mineral exerts its detrimental effect 
is useful in diagnosing toxicity problems and in designing 
research to identify the level at which that mineral becomes 
toxic. Although the means by which minerals cause their 
toxic effects are diverse, several general mechanisms are 
common. Some minerals cause oxidative damage to cellular 
macromolecules, either by their propensity to undergo redox 
reactions or by binding to and deactivating antioxidant mol- 
ecules or enzymes. This property contributes to the toxicity 
of arsenic, cadmium, chromium, copper, cobalt, lead, iron, 
mercury, nickel, selenium, and vanadium. 

Many minerals antagonize chemically related minerals 
that are nutritionally essential by impairing their absorption, 
transport, excretion, or incorporation into active sites of 
molecules. Aluminum, arsenic, bromide, cadmium, calcium, 
copper, lead, manganese, mercury, molybdenum, nickel, 
phosphorus, selenium, silver, strontium, sulfur, tin, tungsten, 
and zinc antagonize the homeostasis of at least one other 
mineral. Minerals that exert toxic effects by disturbing acid- 
base homeostasis include calcium, chloride, magnesium, 
phosphorus, potassium, sodium, and sulfur. Electrolyte bal- 
ance can be disrupted by magnesium, potassium, sodium, 
bromine, and chloride. The toxic effects of chromium and 
vanadium are at least partly due to their ability to mimic or 
potentiate the action of hormones. 

Individuals are often most sensitive to toxicity during 
embryonic development, growth, and periods of stress such 
as infections or trauma. Tolerance to minerals usually in- 
creases with age. Healthy, mature animals are often most 
resistant to mineral toxicoses because they have passed im- 
portant developmental events, their homeostatic mechanisms 
are well developed, and their relative rates of feed intake are 
low. However, cadmium and mercury are not easily excreted, 
and they can accumulate during an animal's lifetime result- 
ing in toxic effects in older animals. 

IVIaximum Tolerable Levels 

The "maximum tolerable level" (MTL) of a mineral is 
defined as the dietary level that, when fed for a defined pe- 
riod of time, will not impair animal health or performance). 
Tolerable mineral levels are typically distinguished from 
toxic levels in experiments that use incremental additions of 



the mineral of concern to the diet or the water and measure 
the impact on performance and pathological signs of toxico- 
sis. The duration of exposure to the test mineral markedly 
influences the level that causes toxicosis. The committee 
considered three exposure durations: a single dose, acute 
exposure, and chronic exposure. A single dose is defined as 
exposure due to the consumption of a single meal or by a 
single gavage of the mineral. Acute exposure is defined as 
an intake of 10 days or less. Chronic exposure is set as an 
exposure of 10 days or more but emphasis is given to the 
studies that had the longest durations of exposure. In this 
report, MTL recommendations for 38 minerals are provided, 
which is seven more than included in the 1980 Mineral Tol- 
erance of Domestic Animals. When information is available, 
MTL are given for fish, rodents, and companion animals, in 
addition to the poultry and livestock species considered in 
the previous report. Research on mineral toxicities in do- 
mestic animals conducted during the past 25 years has re- 
sulted in adjustments to many of the MTL provided in the 
1980 report. The previous report adjusted the MTL for lead 
and mercury in order to decrease tissue residues, but those 
recommendations were not based on animal health. Further- 
more, the rationale for those adjustments was not provided. 
The current MTL are based solely on considerations of ani- 
mal health. As compared to the 1980 report, the recom- 
mended MTL based on indices of animal health were 

• Not changed appreciably for 8 minerals: boron, bro- 
mine, iodine, silicon, silver, sulfur, tungsten, and vanadium; 

• Increased for 13 minerals: aluminum (nonruminants), 
barium, bismuth, cadmium, calcium (poultry), cobalt, fluo- 
rine (cattle), lead (ruminants), magnesium, manganese (ru- 
minants and swine), molybdenum (swine), nickel (ruminants 
and swine), and selenium; 

• Decreased for sodium chloride and 12 minerals: ar- 
senic, calcium (ruminants), chromium, copper, iron (cattle 
and poultry), lead (nonruminants), mercury, molybdenum 
(ruminants), phosphorus (cattle and swine), potassium, stron- 
tium (swine and poultry), and zinc (poultry). 

The recommended MTL in this report do not include a 
built-in safety factor. Consequently, the recommended val- 
ues should be adjusted according to their intended use, and 
each chapter contains relevant information on modifying fac- 
tors for each mineral. Animals that are very young, old, re- 
producing, sick, exposed to stressful environments, or con- 
suming nutritionally imbalanced diets may be especially 
sensitive to toxicoses, and each chapter provides additional 
information on the impact of these factors. In practice, the 
MTL is highly dependent upon the form of the mineral to 
which the animal is exposed. Important chemical factors that 
determine the bioavailability of the mineral sources include 
the solubility of a mineral compound in the digestive tract, 
its valence state, and whether the mineral is in an organic. 



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MINERAL TOLERANCE OF ANIMALS 



metallic, or other inorganic form. For some minerals, such 
as silica, chromium, iron, tin, lead, aluminum, and barium, 
the MTL may vary by several orders of magnitude depend- 
ing upon the chemical form of the mineral. The individual 
chapters provide further information on the bioavailabilities 
of common sources of minerals. Each chapter contains a 
table that summarizes the details on the levels, chemical 
form, duration, animal ages, and signs of toxicosis used in 
the studies reviewed to establish the recommendations. In 
some cases, there is a wide interval between the MTL and 
the levels that are toxic because research is insufficient to 
make finer discrimination. The summary tables provided in 
each chapter give insight into the level of uncertainty. 

While all minerals can become toxic when exposure lev- 
els are sufficiently high, the frequency that animals are ex- 
posed to excessively high levels differs greatly for each min- 
eral. As shown in Table S-1, the committee identified 

• Some minerals where toxicosis is not normally of con- 
cern because levels in feed and water are unlikely to be ex- 
cessive: aluminum, antimony, barium, bismuth, chromium, 
cobalt, germanium, iodone, lithium, magnesium, manganese, 
nickel, rare earth elements, rubidium, silicon, silver, stron- 
tium, tin, titanium, tungsten, and uranium; 

• Eight minerals as being of occasional concern for ani- 
mal toxicosis: arsenic, boron, bromine, calcium, iron, potas- 
sium, phosphorus, and zinc; 

• Sodium chloride and nine minerals as being of frequent 
concern for animal toxicosis: cadmium, copper, fluorine, 
lead, mercury, molybdenum (ruminants), selenium, sulfur 
(ruminants), and vanadium (poultry). 

ENVIRONMENTAL HEALTH 

Although this report focuses predominantly on levels of 
minerals that are toxic for animals, there are some cases 
where environmental factors may be the primary consider- 
ations that limit the acceptable levels of minerals in the feed 
and water of animals. The concentration of some minerals in 
excreta is greater than that in the feed consumed, and this 
relationship is magnified by high dietary levels. In some situ- 
ations, application of animal wastes to the land as fertilizer 
can reduce crop yields, result in high residue levels in crops, 
or cause environmental or human health concerns. 

The committee identified 10 minerals that could be of 
concern because of their potential effects on crop yields or 
the environment: cadmium, copper, iron, mercury, phospho- 
rus, potassium, sodium, selenium, sulfur, and zinc. Of these, 
phosphorus is often the primary concern. 

Depending upon manure management, location of dis- 
posal, and climate factors, environmental considerations may 
limit the levels of these minerals that can appropriately be 
fed to animals. In some regions, environmental issues should 
be considered along with levels that are tolerated by animals 
in regulation of the concentrations of these minerals in the 



feed and water of animals. However, the maximum tolerable 
levels recommended in this report are based solely on indi- 
ces of animal health and productivity. 

HUMAN HEALTH 

Meat, milk, and eggs are an important part of the human 
diet, in part, because they supply highly bioavailable fonns 
of minerals. Often animals can serve as an important buffer 
for the high mineral concentrations found in some plants or 
supplements, thereby reducing human exposure to poten- 
tially toxic minerals. However, levels of some minerals may 
accumulate in animal tissues intended for human consump- 
tion to concentrations that might adversely affect human 
health, even when animals are exposed to safe levels (i.e., 
levels at or below their respective MTL). Consequently, ac- 
ceptable concentrations of minerals in feeds and water of 
animals raised for food must consider the health of the hu- 
man consumer as well as the health of the animal itself. 

The committee identified minerals of concern for human 
health by a three-step process. First, the amount of a mineral 
that accumulates in meat, milk, bone, and eggs in animals 
fed their MTL was estimated. Second, acceptable safety stan- 
dards for mineral intake by humans were identified. Third, 
the maximum concentrations in animal tissues were com- 
pared to levels known to be safe for humans. Individual chap- 
ters in this report provide information on the dose-response 
relationship between feed mineral levels and tissue levels. 
The committee relied on recent recommendations by the 
Food and Nutrition Board of the National Academies and 
other national or international organizations. Recent data on 
food consumption trends in the United States were used to 
estimate daily intake of animal meat, milk, and eggs. It was 
assumed that all of the protein-rich foods consumed by an 
individual came from animals consuming minerals at their 
MTL. Using this process, the committee identified minerals 
for which levels that are tolerated by animals could result in 
unacceptably high mineral concentrations in tissues used for 
human foods. 

• Cadmium, lead, mercury, and selenium could accumu- 
late to excessive levels in skeletal muscle. If it is assumed 
that 5 percent of meat was bone fragments (due to inappro- 
priate processing), barium, and fluorine might also be exces- 
sive in some cases. 

• Cadmium, iodine, lead, and mercury might, in some 
cases, become excessive in milk. 

• Arsenic, cadmium, copper, lead, mercury, selenium, 
and possibly iron could become excessive in liver. 

• Arsenic, bisrnuth, cadmium, chromium, cobalt, fluo- 
ride, lead, mercury, and selenium could become excessive in 
kidney. 

Establishing specific recommendations for mineral lev- 
els in animal feeds that are safe for human health was be- 



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SUMMARY 



yond the charge of the committee. However, for arsenic, 
barium, bismuth, cadmium, chromium, cobalt, copper, fluo- 
ride, iron, lead, mercury, and selenium, the MTL in this 
report, while safe for animals, could result in unacceptably 
high levels of the mineral in some types of foods derived 
from these animals. 

REMAINING QUESTIONS AND RESEARCH NEEDED 

Finally, the committee was charged with identifying gaps 
in knowledge that would benefit from further research. The 
individual chapters fairly consistently highlight three main 
areas across the minerals considered: 

• The bioavailability of minerals that animals commonly 
encounter in feedstuffs is not well characterized, especially 
when fed at concentrations near the MTL. Identifying the 
bioavailability of minerals in the form that animals would 
likely be exposed to and at moderately toxic levels is needed 
for nutritionists and veterinarians to use the MTL recom- 
mendations in this report. The MTL are often based on 



reagent-grade, highly available forms. Research on the 
bioavailability of minerals at dietary levels near the MTL 
would also permit refinement of the recommended MTL. 

• The relationship between mineral concentrations in 
feed and water and the levels in meat, milk, and eggs is not 
well characterized for most minerals, particularly at levels 
that are near the MTL of the animal. For most minerals, the 
available information was determined during relatively 
short-term studies using unrealistically high levels of expo- 
sure. Information on mineral accumulation in tissues follow- 
ing lifetime exposure to minerals is needed to evaluate tissue 
residue levels and impacts on human health. 

• Relevant information for predicting the MTL of miner- 
als for aquatic and companion animals is relatively incom- 
plete. Mineral absorption and excretion in aquatic animals is 
often considerably different from that in terrestrial animals. 
Studies designed specifically to determine the MTL in 
aquatic species are needed. Companion animals have long 
life spans and there are few studies on chronic mineral toxi- 
coses in these species. 



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MINERAL TOLERANCE OF ANIMALS 



TABLE S-1 Summary of Minerals Reviewed in Ttiis Report 



Element 


Required 

Nutrient" 


Concern for 
Animal Health 


MTL Relative to 1980 
Recommendations* 




Aluminum ^^^^h 


^^^ No Jl^^^^ 


^^H Low ^^^^^^^^H 


^^H Increased (nonruminants) ^ 


^ 


Antimony 


No 


Low 


New 




Arsenic ^^^^| 


^^^^P Possibly 


Medium 


Decreased'' 




Barium 


No 


Low 


Increased'' 




Bismuth 


No 


Low 


Increased^ 


J 


Boron 


Possibly 


Medium 


Similar 




Cadmium 


No 


^^^^B Medium ^^^^^^H 
High 


Increased'' 




^^^^^^^B 


^^H Yes ^^^1 


1 Medium 


Decreased (ruminants)"^ 
^^ft Increased (poultry)^ ^^H 


^ 


Chromium 


Yes 


Low 


Decreased'' 




Cobalt '^^^ 


^^^ Yes ^^^1 


P Low 


Increased^ 




Copper 


Yes 


High 


Decreased' 




Fluorine 


Yes 


Hiah 


Increased (cattle')' 


^J 


Germanium 


No 


Low 


New 




^^^^^^^M 




^^^ Low 


Similar 


^^ 


Iron 


Yes 


Medium 


Decreased (cattle and poultry)' 




^^^^^M 


^^H ^^^H 


^B 


Increased (ruminants)'' 
Decreased (nonruminants)"^ 


t 


Lithium 


No 


Low 


New 




Magnesium 


Yes 


Low 


Increased' 




Manganese 


Yes 


Low 


Increased (ruminants, swine)"^ 




Mercury ^^^^^^^B 


^^0 ^^B 


^F High 


Decreased'^ 




Molybdenum 


Yes 


High-' 


Decreased (ruminants)"^ 
Increased (swine)'" 




Nicliel 


Possibly 


Low 


Increased (ruminants, swine)' 




Phosphorus 


Yes 


Medium 


Decreased (cattle, swine)'^ 




^^^^^^^H 


^^^^^Yes ^^^B 


^^^ Medium ^^^^^^ 


^^^ Decreased' 


\ 


Rare earths 


Possibly 


Low 


New 




Rubidium 


Possibly 


Low 


New ^^^^^H 


^^ 


Selenium 


Yes 


High 


Increased' 




Silicon 


Possibly 


Low 


^^^^^H 


I 


Silver 


No 


Low 


Similar 




Sodium Chloride ^^^^^ 


^^^ Yes ^^^ 


■■ High 


Decreased' ^^^ 


^^ 


Strontium 


No 


Low 


Decreased (swine, poultry)"^ 




Sulfur 


Yes 


High'' 


Similar 




Tin 


No 


Low 


New 




Titanium 


No 


Low 


New 




Tungsten 


No 


Low 


Similar 




^^^^^^^H 




^^^ Low ^^^^^^^^1 


^^^ New ^^^^^^^^^^^H 


^1 


Vanadium 


Probably 


High^ 


Similar 




Zinc 


Yes 


Medium 


Decreased (poultry)' 







''Possibly: Indicates that circumstantial data indicate the possibility that the mineral is essential but mechanistic information is lacliing. See specific chapters 

for supportive information. 

''Similar: Recommended Maximum Tolerable Levels for poultry and livestocl^ in this report are not appreciably different than in the 1980 report; Decreased: 

Recommendations in this report are lower than the previous recommendations; Increased: Recommendations in this report are higher than the previous 

recommendations: New: Mineral was not reviewed in 1980 or no recommendation was provided. 

'MTL changed due to new information. 

''MTL changed because the 1980 report was based on human health concerns and not on toxicity to animals. 

■"Ruminants. 

Poultry. 



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1 



Introduction 



Inorganic elements found in the Eartli's crust are often re- 
ferred to as minerals. The essential minerals are those that 
have well-defined biochemical roles and must be in the diet of 
vertebrates for optimum health and productivity. There are 
more than a dozen essential minerals and, for each, the amount 
needed for optimal health and productivity is referred to as the 
dietary requirement. Close to a dozen other minerals are sus- 
pected to be essential because they increase weight gain or 
efficiency of feed use but definitive biochemical or physiologi- 
cal roles have yet to be determined. Dozens of other minerals 
naturally occur at trace levels in foods and tissues of animals 
but are not typically suspected to play a useful nutritional pur- 
pose and are considered incidental contaminants. However, 
all minerals, whether essential or nonessential, can adversely 
affect an animal when amounts in the diet and water become 
excessive (Figure 1-1); so the prevention of mineral toxicoses 
is a fundamental part of animal nutrition and management. 
The concentration at which a specific mineral becomes toxic 
varies greatly. The most highly toxic minerals, such as mer- 
cury, can cause impairments in health and productivity of ani- 
mals when present in the diet at concentrations of less than a 
few milligrams per kilogram, whereas such low levels would 
be well below the dietary requirement of most essential min- 
erals. It is generally thought that those minerals that were en- 
riched in the aquatic environment of early evolution became 
essential and are tolerated at much higher levels than those 
that are nonessential and were historically present at very low 
concentrations. 

EXPOSURE 

Animals may be exposed to toxic levels of minerals from a 
wide variety of sources. Feedstuffs derived from plants are a 
common source of minerals. Levels of minerals in plant tis- 
sues vary greatly due to the natural variation in soil minerals. 
Molybdenum and selenium may naturally occur in soils of 
some regions at levels sufficient to cause certain plants to ac- 
cumulate levels that can be toxic for animals. High soil and 



consequently plant concentrations of cadmium, lead, copper, 
and zinc can occur from the application of municipal wastes 
and other biosolids to the land. Mining, smelting, and other 
industries are often associated with local areas of mineral con- 
tamination to the water, soil, and air, and ultimately the plants 
that grow in the area. Plants can accumulate toxic levels of 
minerals by deposition into their tissues or as the result of 
surface contamination with soil or dust. Some aquatic plants 
accumulate iodine from seawaterto toxic levels. Feedstuffs of 
animal origin may also be sources of toxic levels of minerals. 
For example, some types of fish meal may be high in mercury 
because it bioconcentrates through the aquatic food chain. 
Mineral supplements are commonly added to animal diets to 
correct deficiencies found in pastures, forages, and feed ingre- 
dients. Some sources of mineral supplements may contain 
potentially toxic levels of contaminating minerals, depending 
upon the source of the supplement and the method of its pro- 
cessing. For example, some rock phosphate deposits may be 
naturally high in fluoride or vanadium and cause toxicosis 
when supplemented in animal diets to meet their phosphorus 
requirement. Toxic levels of minerals may accidentally occur 
due to mistakes in feed formulation and manufacturing, or 
from contamination during storage or transportation. Such 
accidental administration can potentially result in very high 
mineral levels and cause acute toxicosis and death, whereas 
most other modes of introduction typically cause toxicosis 
only after chronic exposure. Surface water and occasionally 
even deep-well or domestic water supplies may contain ex- 
cessive levels of some minerals due to naturally high levels in 
the ground. Sulfur, sodium, iron, magnesium, selenium, and 
fluoride are among the minerals most likely to reach toxic 
levels in natural water supplies. Minerals may also be intro- 
duced into water supplies from industrial wastes, pesticide 
contamination, and other sources of pollution. Finally, miner- 
als such as copper, zinc, bromine, bismuth, and some of the 
rare earths are sometimes added to feeds or water as therapeu- 
tics or growth promoters and their potential for toxicity is 
accentuated. 



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MINERAL TOLERANCE OF ANIMALS 



Dietary Essential Minerals 



Optimal 
health 



Death 



Dietary IVIaximum 

Requirement Tolerable Level 





4- 


■^ 






c 


Optimal! 




^ \ 


/ — 

/ o 




Level i 


to 

=3' 


X \ 

o" \ 



Dietary concentration 



High 



Optimal 
health 



Death 



Maximum 
Tolerable Level 



Nonessential Minerals 




Dietary concentration 



High 



FIGURE 1-1 Mineral concentrations and animal health. The relationship between dietary mineral concentrations and animal health is very 
different for essential versus nonessential minerals. For essential minerals (top graph), increasing amounts of a mineral in the diet are highly 
beneficial up to a point (requirement), beyond which additional amounts have little additional value. At some point (maximum tolerable level), 
higher dietary concentrations become detrimental to the animal's health. In the case of nonessential minerals (bottom graph), low levels are 
tolerated without detrimental effects on the health of the animal. At some point, higher concentrations become detrimental to the animal. 



Mineral exposure is commonly reported in several differ- 
ent ways. Expressing exposure as an amount per day (e.g., 
mg mineral consumed/day or mg/kg BW/day) is very pre- 
cise and especially useful when intake is variable, such as 
the case when the mineral is delivered in water or in a free- 
choice supplement. Expressing exposure as a proportion of 
the feed or water (e.g., percent or mg/kg of feed) has many 
obvious practical advantages in feed formulation and gov- 
ernment regulation. These two methods of expression can be 
interconverted when the rate of consumption is known. To 
the extent possible, tables in this report express exposure as 
the concentration in feed or water. Feeds that contain for- 



ages or fresh feedstuffs have extremely variable moisture 
contents, so their mineral contents are best expressed on a 
dry matter (DM) basis whenever possible. 

TOXICITY 

The adverse effects of minerals are dependent on the in- 
tensity of exposure, and they range from subtle effects on 
homeostatic processes, to impairments in animal growth or 
reproductive rates, to specific pathologies and death. An 
understanding of the biochemical or physiological mecha- 
nism by which a mineral exerts its detrimental effect is use- 



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INTRODUCTION 



ful in diagnosing toxicosis problems and in designing re- 
searcii to identify the level at which that mineral becomes 
toxic. For example, some minerals owe their toxicities to 
displacing a nutritionally essential mineral from its normal 
biochemical and physiological role. This is illustrated by 
bromine, which has physical characteristics that are very 
similar to those of chlorine, and which displaces chlorine 
from its important functions as an electrolyte. Other ex- 
amples of this phenomenon include the displacement of cop- 
per by molybdenum, the displacement of sulfur by selenium, 
and the ability of lead to displace zinc and calcium. Toxicity 
experiments designed to exploit these relationships are most 
infonnative. 

The toxicity threshold of each mineral is dependent on its 
chemical form, enteric absorption, metabolism, duration of 
consumption by the animal, levels of other minerals in the 
diet or water, and the species and physiological state of the 
animal. Important chemical factors that determine the 
bioavailability of the mineral sources include the solubility 
of a mineral compound in the digestive tract, its valence state, 
and whether the mineral is in an organic, metallic, or other 
inorganic form. Although extensive research has described 
the bioavailability of minerals in feedstuffs and supplements 
when fed at concentrations below the dietary requirement, 
much less research has been directed toward distinguishing 
the bioavailability of different mineral sources at toxic di- 
etary concentrations. 

Characteristics of different animal species that are impor- 
tant modifiers of toxicosis include the efficiency of homeo- 
static mechanisms such as absorption or excretion pathways, 
presence of a rumen containing microflora that modify the 
chemical form of the mineral, and the rate of feed or water 
consumption. For example, fish use their gills to both absorb 
and excrete minerals and often have very different toxicosis 
profiles than terrestrial animals. Among domestic animals, 
certain ruminants are sometimes more tolerant of high levels 
of dietary minerals than nonruminants. This has been attrib- 
uted to their ruminal microflora as well as their relatively 
low rate of feed intake. 

Individuals are often most sensitive to toxicity during 
embryonic development, growth, and periods of stress such 
as infections or trauma. Some minerals, such as mercury and 
lead, affect development of complex biological systems of 
the brain and the immune system. Development of these or- 
gans occurs in the late embryonic and early neonatal period, 
which is when toxicoses of these minerals are often most 
evident. Tolerance to minerals usually increases with age. 



and healthy, mature animals are often most resistant to min- 
eral toxicoses because they have passed important develop- 
mental events, their homeostatic mechanisms are well de- 
veloped, and their relative rates of feed intakes are low. 
However, some minerals are not easily excreted and they 
accumulate during an animal's lifetime, resulting in toxic 
levels in older animals. Cadmium and mercury are two such 
minerals, and their accumulation in tissues over time has 
important implications for the safety of foods derived from 
animals that have consumed high levels of these minerals. 

ENVIRONMENTAL AND HUMAN HEALTH 

Although this report focuses predominantly on levels of 
minerals that are toxic for animals, there are some cases 
where environmental factors or the wholesomeness of foods 
for human consumption derived from animals are the pri- 
mary considerations that limit the acceptable levels of min- 
erals in the feed and water of animals. High levels of copper, 
zinc, phosphorus, and heavy metals in animal excreta can 
cause environmental damage. Depending upon manure man- 
agement, location of disposal, and climate factors, environ- 
mental considerations may limit the levels of these minerals 
that can appropriately be fed to animals. The text of this 
report provides additional information on environmental is- 
sues and key references to allow a detailed assessment of 
these concerns. However, the maximum tolerable levels rec- 
ommended in this report are based solely on indexes of ani- 
mal health and productivity. 

Even at dietary levels that are apparently safe for animals, 
some minerals accumulate in their tissues to concentrations 
that are unsafe for human consumption. Liver, kidney, and 
spleen are the tissues of maximal accumulation of cadmium, 
lead, and mercury and limit the dietary concentrations that 
can be safely fed to animals destined for human consump- 
tion. Organic forms of selenium and mercury accumulate in 
all tissues, including muscle. Health issues related to high 
levels of these minerals in human foods have been consid- 
ered in detail by many national and international commit- 
tees, and their recommendations are referenced in this re- 
port. Levels of cadmium, lead, and mercury in the feeds 
consumed by animals are normally regulated based on con- 
cerns related to human health. The recommendations for 
maximum tolerable levels of these minerals in this report are 
based solely on indexes of animal health and productivity 
and would likely result in food products that exceed recom- 
mendations for humans. 



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Maximum Tolerable Levels 



The "maximum tolerable level" (MTL) of a mineral is 
defined as the dietary level that, when fed for a defined period 
of time, will not impair animal health and performance. Toler- 
able mineral levels are typically distinguished from toxic lev- 
els by experiments that add incremental amounts of a mineral 
to the diet or the water and measure the impact on perfor- 
mance and pathological signs of toxicosis. The duration of 
exposure to the test mineral markedly influences the level that 
causes toxicosis. Long-term and multigenerational studies are 
usually most informative, but, in practice, short-term studies 
are most commonly conducted. The committee considered 
three exposure durations: a single dose, acute exposure, and 
chronic exposure. A single dose is defined as exposure due to 
the consumption of a single meal or by a single gavage of the 
mineral. Acute exposure is defined as an intake of 10 days or 
less. Chronic exposure is set as an exposure of 10 days or 
more. However, in making recommendations, emphasis was 
given to the studies that had the longest durations of exposure. 
A "toxic level" is defined as the minimal level that, when fed 
for a defined period, impairs animal health or performance 
(rate of production of milk, meat, or eggs; growth rate; repro- 
ductive capacity; disease resistance). Table 2-1 summarizes 
the committee's recommendations for MTL of dietary miner- 
als for domestic animals following chronic consumption. Prior 
to applying these recommendations, the accompanying text 
should be consulted for exceptions and for additional infor- 
mation on the ages of animals, their physiological state, and 
the length of the studies used to establish these values. Each 
chapter also provides information on the MTL following 
single and acute exposures to minerals as well as exposure 
through water. Information on the toxicity of minerals to dogs 
and cats is generally lacking. In cases where information is 
available, it is often a single study using only a single toxic 
level. Providing a summary of the MTL for dogs and cats in 
Table 2-1 without accompanying text would be misleading. 
Individual chapters should be consulted for specific informa- 
tion on mineral toxicities to dogs and cats, as well as addi- 
tional species. 



The MTL in Table 2-1 and in each individual chapter do 
not include built-in safety factors because the magnitude of 
the safety factor varies with each specific situation. Conse- 
quently, the recommended values should be adjusted accord- 
ing to their intended use, and each chapter contains relevant 
information on modifying factors for each mineral. Animals 
that are very young, old, reproducing, sick, exposed to stress- 
ful environments, or consuming nutritionally imbalanced di- 
ets may be especially sensitive to toxicoses, and individual 
chapters provide additional information on the impact of these 
factors. In practice, the MTL is highly dependent upon the 
form of the mineral to which the animal is exposed. For some 
minerals, such as silica, chromium, iron, tin, lead, and barium, 
the MTL may vary by more than an order of magnitude de- 
pending upon the chemical form of the mineral, and the indi- 
vidual chapters provide further information on the relative tox- 
icities (i.e., bioavailabilities) of common sources of minerals. 
Each chapter contains a table that summarizes the details on 
the levels, chemical form, duration, animal ages, and signs of 
toxicosis used in the studies reviewed to establish the recom- 
mendations. In some cases, there is a wide interval between 
the MTL and levels that are toxic because research is insuffi- 
cient to make finer discrimination. Inspection of the summary 
tables presented in each chapter provides insight into critical 
experiments and the level of uncertainty. 

Research on mineral toxicoses in domestic animals con- 
ducted during the past 25 years has resulted in adjustments 
to many of the MTL provided in the NRC report Mineral 
Tolerance of Domestic Animals, which was published in 
1980. Also, an appreciation for the limitations and pitfalls 
inherent in mineral analysis has changed interpretation of 
some older studies. In some cases (e.g., aluminum, boron, 
mercury, and vanadium), the dietary concentrations of min- 
erals reported in older literature are suspect because of inad- 
equacies in sample preparation or analytical procedures; thus 
greater reliance is placed on more recent publications. For 
some minerals, the previous report necessarily relied on in 
vitro experiments or studies in which animals were injected 



10 



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MAXIMUM TOLERABLE LEVELS 



11 



with minerals, bypassing homeostatic digestive processes. 
Newer information from feeding studies iias eliminated reli- 
ance on injection studies and often resulted in marked 
changes in recommendations. 

HUMAN HEALTH 

Meat, milk, and eggs are an important part of the human 
diet because they supply highly bioavailable forms of miner- 
als. Animals, via homeostatic control of gastrointestinal ab- 
sorption, can often serve to diminish the high mineral con- 
centrations found in some plants or supplements, thereby 
reducing human exposure to potentially toxic minerals. 
However, levels of some minerals may accumulate in ani- 
mal tissues intended for human consumption to concentra- 
tions that might adversely affect human health even when 
animals are exposed to safe levels (i.e., levels at or below 
their respective MTL). Consequently, in the case of animals 
raised to supply human food, acceptable concentrations of 
minerals in feeds and water must consider the health of those 
consuming food products derived from these animals as well 
as the health of the animal itself. 

The committee identified minerals of concern for human 
health by a three-step process. First, the amount of a mineral 
that accumulates in meat, milk, bone, and eggs when ani- 
mals are fed their respective MTL was estimated. Second, 
acceptable safety standards for mineral intake by humans 
were identified. Third, the maximum concentrations in ani- 
mal tissues were compared to levels known to be safe for 
humans. Individual chapters in this report provide informa- 
tion on the dose-response relationship between feed mineral 
levels and tissue levels. Recently the NRC (2000; 2001a,b) 
has extensively reviewed the scientific literature on mineral 
toxicosis in humans due to the ingestion of high levels of 
minerals in foods. The human toxicological standards based 
on these reviews and recent data on food consumption trends 
in the United States were used to estimate the potential for 
mineral toxicosis among humans consuming tissues and flu- 
ids from animals exposed to mineral levels at their respec- 
tive MTL based on animal health. 

Toxicological Standards for Minerals in Humans 

The NRC (2000; 2001a,b) set Tolerable Upper Intake 
Levels (UL) for minerals that are nutritionally required for 
humans (Table 2-2). The UL was defined as "the highest 
average daily nutrient intake that is likely to pose no risk of 
adverse effects to almost all (human) individuals in the gen- 
eral population." The UL were based on lowest-observed- 
adverse-effect levels (LOAEL) and no-observed-adverse- 
effect levels (NOAEL) that were calculated using primarily 
data from studies with humans. 

In several cases, the NRC could not identify studies in 
humans or sometimes even laboratory animals in which the 
mineral as it occurred naturally in the diet was toxic. Thus, 



the NRC did not set UL for several minerals (i.e., arsenic, 
chromium, silicon), explicitly stated that the ULs applied 
only to supplemental (not dietary) forms of two minerals 
(i.e., magnesium, nickel), and based UL on data from only 
laboratory animal studies for other minerals (i.e., boron, 
molybdenum, nickel, vanadium). Furthermore, the NRC 
noted in the text that the UL for iron and zinc were based on 
studies with humans in which the primary source of the min- 
eral was from supplements. 

These decisions by the NRC (2000; 200Ia,b) reflect a 
number of factors. One is that the form of minerals in food is 
sometimes different from that in supplements (e.g., heme- 
iron from meat versus supplemental inorganic iron). Another 
is that the total milieu of factors (especially protein, phytate, 
fiber, and other minerals) in foods can reduce the potential 
of toxicosis of high levels of dietary minerals in those foods. 
For example, there are no reports of adverse effects from 
consuming high levels of calcium and phosphorus in milk or 
manganese from cereal products. 

The NRC did not set unacceptable levels of several other 
minerals (aluminum, barium, bismuth, bromine, cobalt, and 
tin). Generally, diet is a minor source of these elements as 
compared to pharmaceuticals. The Food and Agriculture 
Organization/World Health Organization (FAO/WHO) 
Pesticide committees (1967) set the acceptable daily intake 
of 1 mg bromide/kg BW/day. Minimal effects (i.e., reduc- 
tions in the absorption of essential minerals) were the major 
signs when human diets were supplemented with aluminum 
salts (125 mg Al/day; Greger and Baier, 1983) or tin salts 
(50 mg Sn/day; Johnson et al., 1982). Cadmium, mercury, 
and lead are generally considered the elements most apt to 
produce toxic effects when ingested. The minimum risk level 
for mercury, which is the dose that can be ingested daily for 
a lifetime without a significant risk of adverse effects, was 
set at 0.0003 mg Hg/kg BW/day for a 70-kg person, based 
on neuro-developmental outcomes in children exposed in 
utero to methylmercury from maternal fish ingestion (ATSDR, 
1999). For pregnant women the suggestion is not to con- 
sume fish containing greater than 0.25 mg Hg/kg fresh 
weight. Research has demonstrated that blood lead levels 
>10 |Jg/dL whole blood were associated with adverse neuro- 
logical and hematological effects in children and that blood 
lead levels of two-year olds declined when dietary intake of 
lead was reduced from 0.053 to 0.013 mg of lead per child 
daily (NRC, 1993). The individual mineral chapters in this 
report contain additional information on toxic levels of 
specific minerals in human diets. 

Maximum Exposure to Minerals from Consumption of 
Animal Products 

Americans consumed an average of 105.7 kg of meat, 
fish, and poultry; 228.6 kg of milk and milk-derived prod- 
ucts (on a calcium equivalent weight basis, which converts 
cheese and other products into an equivalent amount of 



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12 



MINERAL TOLERANCE OF ANIMALS 



milk); 14 kg of eggs; and 9.3 kg of legumes, nuts, and soy 
(high-protein alternative to meat) per year during 1990-1999 
(Gerrior and Bente, 2002). These data are based on USDA 
figures on the disappearance of food available for human 
consumption. Accordingly, the average American poten- 
tially consumes 0.95 kg, fresh weight, of animal products 
per day or 0.98 kg of "protein-rich" foods per day. The ac- 
tual average consumption is likely to be considerably less 
than 0.98 kg because this estimate is based on disappearance 
values and not actual intakes. However, for the purposes of 
identifying minerals that might be of concern for human 
health, consumption data provide a reasonable margin of 
safety. A more extensive risk analysis should be conducted 
for those minerals that are identified below as being of con- 
cern for human health. 

The committee used this information to establish relative 
risks. The first question asked was this: How much of a min- 
eral would an individual ingest if the individual consumed 
that entire kilogram of "protein-rich" food daily as the 
muscles from an animal fed at the MTL for that mineral? 
Based on the highest concentrations of minerals found in 
muscles of livestock and fish fed at the respective MTL (see 
individual mineral chapters for data on tissue levels), we 
would not expect humans ingesting these muscles to con- 
sume excessive amounts of minerals, except for cadmium, 
lead, mercury, and selenium. If it is assumed that 5 percent 
of the kilogram of "protein-rich" food were bone fragments 
(due to incorrect processing), intake of barium and fluoride 
might be excessive in some cases. 

If the kilogram of "protein-rich" food consumed daily was 
milk, intake of cadmium, iodine, lead, and mercury might in 
some cases be excessive. The calculated exposures become 
of greater concern if the kilogram of "protein-rich" food con- 
sumed daily is from the livers or kidneys of livestock and 
fish fed toxic levels of minerals. Potentially, humans ingest- 
ing 1 kg of liver daily could ingest excessive amounts of 
arsenic, cadmium, copper, iron (see below), lead, mercury, 
and selenium. Potentially, humans ingesting 1 kg of kidney 
daily could ingest excessive amounts of arsenic, bismuth, 
cadmium, chromium, cobalt, fluoride, lead, mercury, and 
selenium. Generally, the organic forms of arsenic and iron in 
tissues are assumed to be relatively less toxic. 



These calculations underscore the concerns of the FDA 
about certain elements in animal products, namely iodine in 
milk (Hemken, 1980), mercury in fish, cadmium in kidneys, 
and lead and fluoride in bone. Establishing specific recom- 
mendations for mineral levels in animal feeds that are safe 
for human health was beyond the charge of the committee. 
However, for arsenic, barium, bismuth, cadmium, chro- 
mium, cobalt, copper, fluoride, iron, lead, mercury, and se- 
lenium, the MTL in Table 2-1, while safe for animals, could 
result in unacceptably high levels of the mineral in some 
types of foods derived from these animals 

REFERENCES 

ATSDR (Agency for Toxic Substances and Disease Registry). 1999. Toxi- 
cological profile for mercury. Atlanta, GA: U.S. Department of Health 
and Human Services. 

ATSDR (Agency for Toxic Substances and Disease Registry). 2000. Toxi- 
cological profile for arsenic. Atlanta, GA: U.S. Department of Health 
and Human Services. 

FAOAVHO Pesticide Committees. 1967. Evaluation of some pesticide resi- 
dues in food. Rome. Information about meeting available at http:// 
www.fao.org/docrepAV5897E/w5897e6a.htm 

Gerrior, S., and L. Bente. 2002. Nutrient content of the U.S. food supply, 
1909-99: a summary report. Home Economic Research Report No. 55. 
USDA. Online. Available at http://www.usda.gov/cnpp/Pubs/ 
Food%20supply/foodsupply09_99.pdf. Accessed November 7, 2004. 

Greger, J. L., and M. J. Baier. 1983. Effect of dietary aluminum on mineral 
metabolism of aduh males. Am. J. Clin. Nutr. 38:41 1^19. 

Hemken, R. W. 1980. Milk and meat iodine content: relation to human 
health. J. Am. Vet. Med. Assoc. 176:1119-1121. 

Johnson, M. A., M. J. Baier, and J. L. Greger. 1982. Effects of dietary tin on 
zinc, copper, iron, manganese, and magnesium metabolism of adult 
males. Am. J. Clin. Nutr. 35:1332-1338. 

NRC (National Research Council). 1980. Mineral Tolerance of Domestic 
Animals. Washington, D.C.: National Academy Press. 

NRC. 1993. Measuring Lead Exposure in Infants, Children, and Other Sen- 
sitive Populations. Washington, D.C.: National Academy Press. 

NRC. 2000. Dietary Reference Intakes for Vitamin C, Vitamin E, Sele- 
nium, and Carotenoids. Washington, D.C.: National Academy Press. 

NRC. 2001a. Dietary Reference Intakes for Calcium, Phosphorus, Magne- 
sium, Vitamin D, and Fluoride. Washington, D.C.: National Academy 
Press. 

NRC. 2001b. Dietary Reference Intakes for Vitamin A, Vitamin K, Ar- 
senic, Boron, Chromium, Copper, Iodine, Iron, Manganese, Molybde- 
num, Nickel, Silicon, Vanadium, and Zinc. Washington, D.C.: National 
Academy Press. 



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MAXIMUM TOLERABLE LEVELS 



13 



TABLE 2-1 Maximum Tolerable Levels of Minerals in the Feed (mg/kg or % of the DM) of Animals Based on Indexes of 
Animal Health"'* 



Element 


Rodents 


Poultry 




Swine 


Horse 


Cattle 


Sheep 


Fish 


Aluminum, mg/kg 


200 


1,000 




(1,000) 


(1,000) 


1,000 


1,000 


— 


Antimony, mg/kg 


70-150 


— 




— 


— 


— 


— 


— 


Arsenic, mg/kg"^ 


30 


(30) 




(30) 


(30) 


(30) 


(30) 


5 


Barium, mg/kg"^ 


250 


100 




(100) 


(100) 


— 


— 


— 


Bismuth, mg/kg'' 


(500) 


1,000 




500 


(500) 


— 


— 


— 


Boron, mg/kg 


150 


(150) 




(150) 


(150) 


150 


(150) 


— 


Bromine, mg/kg 


300 ^^ 


^ 2,500 




200 


(200) 


200 


(200) 


— 


Cadmium, mg/kg'' 


10 


10 




10 


10 


10 


10 


10 


Calcium, %<> ^^M 


^H 


^H 1.5, growing 
H birds; 5, 
^^ laying hens 


^^H 




1 1.5 


1.5 


0.9 


Chromium, mg/kg^ 
soluble Cr+++ 
CrO 


100 

30,000 


500 

3,000 




(100) 
(3,000) 


(100) 
(3,000) 


(100) 
(3,000) 


(100) 
(3,000) 


3,000 


Cobalt, mg/kg^ ^^^^H 


■ 25 


25 




100 


(25) 


25 


25 




Copper, mg/kg'^ 


500 


250"^ 




250 


250 


40'' 


15" 


100 


Fluorine, mg/kg'' 


150 


150 




150 


(40) 


40 


60 


— 


Germanium, mg/kg 


30 


— 




— 


— 


— 


— 


— 


Iodine, mg/kg'^ 


— 


300 




400 


5 


50 


50 


— 


Iron, mg/kg'" 


(500) 


500 




3,000 


(500) 


500 


500 


— 


Lead, mg/kg'' ^^^| 


^pio 


10 




10 


10 


100 


100 


10 


Lithium, mg/kg 


(25) 


25 




25 


(25) 


25 


25 


— 


Magnesium, % 


0.5 


0.5, growing 
birds; 0.75, 
^^^ laying hens 


0.24 


0.8 


0.6 


0.6 


0.3 


Manganese, mg/kg 


2,000 


2,000 




1,000 


(400) 


2,000 


2,000 ^ 




Mercury, mg/kg"^ 
inorganic 
organic/^ 


0.2 
1 


0.2 
1 




(0.2) 
(2) 


(0.2) 
(1) 


2 


- ■ 

2 


1 


Molybdenum, mg/kg 


7 


100 




(150) 


(5) 


5 


5 


10 


Nickel, mg/kg 


50 


250 




250 


(50) 


100 


(100) 


50 


Phosphorus, %'' 


0.6 


1, growing 
birds; 0.8, 
laying hens 


1.0 


1 


0.7 


0.6 


1 


Potassium, % 


1 


1 




1 


1 


2 


2 


- 1 


Rare earth mineral, mg/kg 


100 


— 




— 


— 


— 


— 


— 


Rubidium, mg/kg 


200 


— 




— 


— 


— 


-^^^1 


i - 


Selenium, mg/kg'^ 


(5) 


3 




4 


(5) 


5 


5 


(2) 


Silicon, % 


— 


— 




— 


— 


(0.2) 


0.2 


— 


Silver, mg/kg 


— 


100 




(100) 


— 


— 


— 


>3 


Sodium chloride, % 


4 


1.7 




3 


6 


4.5, growing 


4 


— 



animals 
3.0, lactating 
cows 



Strontium, mg/kg 


1,000 


2,000 


2,000 


(2.000) 


2,000 


(2.000) 


— 


Sulfur, % 


(0.5) 


0.4 


0.4 


(0.5) 


0.30, high- 
concentrate 
diet; 0.50, 
high-forage 
diet 


0.30, high- 
concentrate 
diet; 0.50, 
high-forage 
diet 





Tin, mg/kg 


100 


(100) 


(100) 


(100) 


(100) 


(100) 


— 


Titanium, mg/kg 


— 


— 


— 


— 


— 


— 


— 


Tungsten, mg/kg 


(20) 


20 


(20) 


(20) 


(20) 


(20) 


— 


Uranium, mg/kg 


100 


— 


— 


— 


— 


— 


< 100 



continued 



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14 



MINERAL TOLERANCE OF ANIMALS 



TABLE 2-1 Continued 



Element 


Rodents 


Poultry 


Swine 


Horse 


Cattle 


Sheep 


Fish 


Vanadium, mg/kg 


— 


25, growing 
birds; <5, 
laying hens 


(10) 


(10) 


50 


50 


— 


Zinc, mg/kg 


(500) 


500 


1,000S 


(500) 


500 


300 


250 



"The accompanying text must be consulted for additional details about factors that might increase or decrease the tolerable levels. The levels in parentheses 

were derived from interspecies extrapolation. Dashes indicate that data were insufficient to set a maximum tolerable level (MTL). Individual chapters provide 

further information on human safety and environmental problems related to feeding high levels of the mineral. 

''The MTL are based on highly soluble forms of the mineral and other forms may have lower bioavailabilities. MTL are also based on the assumption that 

exposure of the animal to minerals from water and other environmental sources is minimal and that the diet is nutritionally complete, especially in regard to 

other minerals that might be antagonized. 

''The MTL provided for this nutrient is based on animal health and not human health. Lower levels ai'e necessaiy to avoid excessive accumulation in edible tissues. 

'^'The MTL are based on diets with phosphorus levels at, but not above, the animal's requirement. Considerably higher levels can be tolerated if phosphorus 

levels are increased sufficiently to maintain an appropriate calcium:phosphorus ratio. 

Tor ducks the MTL for copper is 100 mg/kg diet. 

fA margin of safety should be added for pregnant animals to assure normal neurodevelopment of the fetus. 

^Higher levels of zinc as zinc oxide (2,000 to 3,000 mg/kg diet) are tolerated for several weeks and may provide growth promotion in weanling piglets. 

''Assuming normal concentrations of molybdenum (1-2 mg/kg diet) and sulfur (0.15-0.25%). At molybdenum and sulfur concentrations below these, copper 

may become toxic at lower levels. 

TABLE 2-2 Toxicity Assessments of Nutrients for Humans Based on Food and Nutrition Board Assessments (NRC, 2000; 
NRC, 2001a,b) 



Lowest (or No)-Observed- 
Adverse-Effect Level 
(LOAEL/NOAEL) 



Tolerable Upper 
Intake Level 
(UL) 



Arseni" 


Not set 


Not set 


I 


Boron'' 


9.6 mg/kg/d 


20 mg/d 




^^^^^^^H 




^^^f 2,500 mg/d 




Chromium'^' 


Not set 


Not set 




Copper"" 


10 mg/d 


10 mg/d 




Fluorid f 


0.10 mg/kg/d 


10 mg/d 










J 


Iron« 


70 mg/d (primarily for inorganic 
salt supplement) 


45 mg/d 




Magnesium'' ^^^^H 


^^^^^^V 360 mg from non food sources/d ^^^M 


^^^^V 350 mg/d from supplement 


1 


Manganese"" 


1 1 mg/d 


1 1 mg/d 




Molybdenum'' ^^^^^^^^ 




^^^^^B 2.0 mg/d 




Nickel'' 


5 mg/kg/d 


1.0 mg/d of soluble nickel salts 




Phosphorus' ^^^^^H 


^^^^1 g/d ^^^^^^^^B 


^^^ g/d 




Selenium'' 


800 ng/d 


400 ng/d 




Silicoiy 


Not set 


Not set 




Vanadium* 


7.7 mg/kg/d 


1.8 mg/d 




^^^^^^^H 


^^^^^^^^^V 60 mg/d (primarily for inorganic ^^^^^^H 


^^^^^V 40 mg/d ^^^^^^^H 


1 



"Chronic intake of 10 |jg/kg/d of inorganic arsenic produces arsenicism with skin changes in humans (ATSDR, 2000). 

''NOAEL based on data in rodents or humans (selenium); UL for >19 years of age. 

"LOAEL for adults: UL for 19-70 years of age. 

''Insufficient data to establish a UL for soluble chromium III salts. Concern only for Cr in supplements. 

""NOAEL for adults: UL for >19 years of age. 

tOAEL for children through 8 years: UL for >8 years of age. 

SLOAEL for adults: UL >19 years of age. 

''LOAEL for adults: UL >8 years of age. 

'NOAEL for adults: UL for 19-70 years of age. 

JNo evidence that Si naturally occurring in food or water produced adverse health effects. 

*LOAEL based on data in rats: UL for >19 years of age. 



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Aluminum 



INTRODUCTION 

Aluminum (Al) has an atomic number of 13 and is one of 
the lightest metals. It has two oxidation states: Al (0) and Al 
(+3). However, aluminum does not occur naturally in the el- 
emental state, but rather in combination with oxygen, silicon, 
and fluorine primarily. The most important raw material for 
the production of aluminum metal is bauxite, which contains 
40 to 60 percent alumina (aluminum oxide) (ATSDR, 1999). 

Aluminum is the third most abundant element in the 
Earth's crust after oxygen and silicon. Although it has been 
produced in commercial quantities for just over 100 years, it 
is used more today than any other metal, except iron. In 2000, 
the United States' estimated consumption of aluminum was 
7.5 million metric tons (Plunkert, 2002). Aluminum recov- 
ered from discarded aluminum products accounts for about 
20 percent of estimated consumption of aluminum in the 
United States (Plunkert, 2002). 

Aluminum is used industrially because it is malleable, 
ductile, durable, and easily machined and cast, and has ex- 
cellent corrosion resistance. The following areas constitute 
the major uses of aluminum in the United States: transporta- 
tion, 35 percent; packaging, 25 percent; building, 15 per- 
cent; consumer durables, 8 percent; electrical, 7 percent; and 
other, 10 percent (Plunkert, 2002). 

ESSENTIALITY 

Horecker et al. (1939) suggested that aluminum promoted 
the reaction between cytochrome C and succinic dehydroge- 
nase in vitro. The activation of purified guanine nucleotide 
binding protein, the regulatory component of adenylate cy- 
clase, by fluoride was shown to require the presence of Al"^-' 
in vitro (Sternweis and Oilman, 1982; Kahn, 1991). The sig- 
nificance of these observations in vivo is not known. No 
conclusive evidence exists to suggest that aluminum is es- 
sential for growth, reproduction, or survival of animals 
(Alfrey, 1986;Ganrot, 1986;Greger, 1993). However, it may 



be a cariostatic agent by itself and in combination with fluo- 
ride (Kleber and Putt, 1984). 



DIFFICULTIES IN METHODS OF ANALYSIS AND 
EVALUATION 

Monitoring the tissue levels of aluminum and the absorp- 
tion and excretion of aluminum is very difficult for a variety 
of methodological reasons (Kostyniak, 1983; Ganrot, 1986; 
Savory and Wills, 1988; Greger and Sutherland, 1997; 
ATSDR, 1999). Estimated amounts of aluminum in plasma 
decreased by more than 50-fold between 1950 and 1980 be- 
cause of improvements in methodology (Versieck and 
Cornells, 1980). Accordingly, this review emphasizes litera- 
ture published after 1980. 

One reason for the analytical problems is that aluminum 
is ubiquitous in the environment and contamination of bio- 
logical samples is difficult to prevent. When contamination 
from anticoagulants and glassware was prevented, serum 
aluminum concentrations were between 1 to 5 |Jg/L 
(Kostyniak, 1983; Ganrot, 1986; Savory and Wills, 1988). 

A second limitation is that flame atomic absorption spec- 
troscopy is not sensitive enough for analyses of biological 
samples. Matrix effects decrease the sensitivity of several 
methods and hence preparation of samples is often time-con- 
suming (ATSDR, 1999). The most commonly used method 
is graphite furnace atomic absorption spectrometry. Re- 
searchers must correct for interfering interaction with phos- 
phorus and silicon when using neutron activation analysis to 
determine aluminum because all three produce ^^Al when 
activated. A major problem with inductively coupled 
plasma-atomic emission spectrometry is that calcium in the 
sample raises the limit of detection for aluminum. Two other 
techniques have improved sensitivity, but are expensive and 
require highly trained personnel. Inductively coupled 
plasma-mass spectrometry has sensitivity down to 1 mg/L 
of aluminum in blood. Laser ablation microprobe mass 



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MINERAL TOLERANCE OF ANIMALS 



analysis (LAMM A) has been used for analyses of aluminum 
in brains where sensitivity and location are important. 

A third methodological limitation is the lack of useful- 
ness of aluminum isotopes for metabolic studies (Greger and 
Sutherland, 1997). Most aluminum isotopes have very short 
half-lives; ^^A[, ^^Al, ^^A[, ^^Al, and ^OAI have half-lives of 
less than 6.6 minutes (Bureau of Radiological Health and 
Training Institute, 1970). Thus they cannot be used in vivo 
to monitor absorption and excretion processes. The only alu- 
minum isotope with a biologically usable half-life is ^^Al 
(7.2 X 10^ years), but it is very scarce and hence the study 
designs and replicates are often limited (Jouhanneau et al., 
1993; Priest et al., 1995; Schonholzer et al., 1997; Yokel et 
al., 2001). Other investigators attempted to use ^^Ga (a type 
Ilia element like aluminum) as a marker for aluminum 
(Greger et al., 1994, 1995; Priest et al., 1995). These investi- 
gators concluded that "^^Ga was an unacceptable marker for 
aluminum because ^^Ga did not distribute in tissues like alu- 
minum and was more sensitive to iron intake and status than 
was aluminum. 

Finally, absorption of aluminum is difficult to quantitate 
because absorption is generally <1 percent (Alfrey 1989; 
Greger and Sutherland, 1997). In response to this method- 
ological quandary, Ganrot (1986) suggested that urinary alu- 
minum excretion could be assumed to equal aluminum ab- 
sorption. This assumption is faulty because animals 
accumulate aluminum in tissue with continued oral expo- 
sure to aluminum; this would not occur if absorption and 
urinary excretion were equal. Moreover, the relative percent- 
age of absorbed aluminum retained (not excreted in urine) 
has been found to vary with a variety of factors, including 
renal function, age, and disease states (Ecelbarger et al., 
1994a; Greger and Radzanowski, 1995). Thus one alternate 
(but time-consuming and difficult) way to estimate relative 
aluminum absorption from diet is to compare tissue accumu- 
lation of aluminum in relation to the dose in animals fed 
aluminum and in animals matched for age and weight that 
are injected with aluminum (Greger and Powers, 1992). 
Another alternate (but also time-consuming and difficult) 
way to estimate absorption from a single large dose is to sum 
all aluminum in the carcass, urine, and bile (Sutherland and 
Greger, 1998). 



equal doses of aluminum were ingested and injected, and 
summation of the increase in aluminum excretion and tissue 
retention after a dose), aluminum absorption has been esti- 
mated to be 0.01 to 0.8 percent of intake when aluminum 
was ingested in diet (Ganrot, 1986; Greger and Powers, 
1992; Jouhanneau et al. 1993) and 0.3 percent from water 
(Yokel et al., 2001). However, fasted rats absorbed 2 to 5 
percent of the small amounts of aluminum administered by 
gavage in a citrate solution (Froment et al., 1989a; 
Schonholzer et al., 1997; Sutherland and Greger, 1998). 

Aluminum absorption occurs to the greatest extent in the 
proximal intestine. Plasma aluminum and labeled plasma 
glucose levels peaked simultaneously 45 minutes and 30 
minutes after aluminum citrate and glucose were adminis- 
tered together by gavage to rats (Froment et al., 1989a,b) and 
humans (Nagy and Jobst, 1994), respectively. 

The mechanisms of aluminum absorption include both 
paracellular passage of aluminum through tight junctions by 
passive processes (Provan and Yokel, 1988b; van der Voet, 
1992) and transcellular passage through enterocytes, involv- 
ing passive, facilitated, and active transport processes (van 
der Voet, 1992). The active transcellular processes involved 
in aluminum absorption are saturable (Feinroth et al., 1982). 
At least some of the active processes (particularly the trans- 
port of aluminum into the serosal fluid) are shared with pro- 
cesses for active absorption of calcium (Feinroth et al., 1982; 
Provan and Yokel, 1988a). Some of these processes are de- 
pendent on vitamin D metabolites (Ittel et al., 1988, 1990). 
Aluminum did not appear to affect the ability of calbindin- 
D9K (the diffusional translocator of calcium across mucosal 
cells) to bind calcium in rats (Adler et al., 1991) but reduced 
the transcription and/or stability of calbindin-D28K in chicks 
(Cox and Dunn, 2001). 

Adler and Berlyne (1985) perfused in situ guts with alu- 
minum chloride at pH 2 and estimated that 23 percent of 
aluminum absorption in rats was due to non-saturable pro- 
cesses and the rest was due to active processes. However, 
the relative importance of absorptive processes varies with 
the concentration and speciation of aluminum in the gut and 
other dietary factors (van der Voet, 1992; Greger and 
Sutherland, 1997). These factors are discussed in the section 
further below on Bioavailability. 



REGULATION AND METABOLISM 
Absorption 

The gastrointestinal tract is the most important body sys- 
tem that protects mammals against accumulating aluminum 
in tissues. As noted in the previous section on Difficulty in 
Methods of Analysis and Evaluation, absorption of alumi- 
num is difficult to determine, partially because fecal losses 
approximate intakes. Using a variety of alternate method- 
ologies (measurement of aluminum in urine, usage of iso- 
topes, comparison of tissue aluminum accumulation when 



Urinary Excretion 

Urinary excretion of aluminum is proportional to alumi- 
num intake under controlled conditions (Ganrot, 1986; 
Greger and Powers, 1992). Approximately half of the ab- 
sorbed aluminum is estimated to be excreted in urine 
(Jouhanneau et al., 1993). Thus humans with normal renal 
function who consume typical diets with no medications 
excrete less than 50 |Jg/day of aluminum in urine (Ganrot, 
1986; Alfrey, 1986; Greger, 1993). This primarily reflects 
the protective effect of the gut. When patients were infused 
with parenteral solutions contaminated with aluminum, sub- 



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ALUMINUM 



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jects excreted 0.1 to 3.8 mg/day of aluminum (Klein et al., 
1982). 

The importance of renal excretion of aluminum has been 
demonstrated many times in humans with reduced renal 
function, even in the absence of parenteral exposure to alu- 
minum (King et al., 1981; Committee on Nutrition, 1986; 
Hewitt et al., 1990; Strong et al., 1996; ATSDR, 1999). 
Alfrey et al. (1980) found that patients with undialyzed ure- 
mia had 12-fold more aluminum in bone, and more than 6- 
fold more aluminum in liver and spleen than renally compe- 
tent patients. Rats given unilateral nephrectomies (that 
reduced renal excretion only moderately as might occur natu- 
rally with age) accumulated more aluminum in bone and 
liver than sham-operated rats (Ecelbarger and Greger, 1991; 
Ecelbarger et al., 1994a). Some of this increased accumula- 
tion of aluminum by uremic animals could reflect increased 
gut permeability to aluminum in uremic animals (Ittel et al., 
1988, 1992). 

Biliary Excretion 

Biliary excretion is less important than urinary excretion 
in preventing accumulation of aluminum in tissues. Dogs 
and rats given intravenous doses of aluminum excreted from 
0.1 to 7 percent of the doses in bile (Kovalchik et al., 1978; 
Klein etal., 1988; Xu et al., 1991). In contrast, biliary alumi- 
num excretion sometimes exceeded urinary excretion of alu- 
minum in humans whose main source of aluminum expo- 
sure was diet (Ishihara and Matsushiro, 1986). Although rats 
exposed to low (6.9 to 10.3 |Jg/g diet of aluminum) levels of 
aluminum in diet excreted more aluminum in bile than in 
urine, rats given moderate (0.25 to 1 mmole/kg BW of alu- 
minum) amounts of aluminum by gavage excreted more alu- 
minum in urine than bile (Sutherland and Greger, 1998). This 
reflected that the biliary excretion of aluminum was rapid 
(within 15 minutes after a gavage dose in rats) and saturated 
by doses as low as 0.2 mmole of aluminum (Sutherland et 
al., 1996; Sutherland and Greger, 1998). 

SOURCES AND BIOAVAILABILITY 
Dietary Sources 

Grain and vegetable products contain more aluminum 
naturally than animal products (Schlettwein-Gsell and 
Mommsen-Straub, 1973; Sorenson et al., 1974; Greger, 
1985; Greger et al., 1985b; Pennington and Jones, 1989). A 
few plants, such as tea and herbs (e.g., bay, oregano, and 
thyme) often "accumulate" more than 500 mg/kg dry weight 
of aluminum. The aluminum content of vegetable and grain 
products varies with plant varieties and soil conditions, in- 
cluding pH (Hopkins and Eisen, 1959; Eden, 1976). 

Water is generally not a major source of aluminum. The 
amount of aluminum in surface and ground water varies 
from <12 mg to 2.25 mg/L of aluminum in the rivers of 



North America (Jones and Bennett, 1986). Platen (1995) 
estimated that average aluminum concentrations of water 
supplies in Europe ranged from 6 to 60 |Jg/L of aluminum. 
Miller et al. (1984) estimated that there was a 40-50 per- 
cent chance that aluminum containing flocculants added to 
clarify municipal water supplies increased the aluminum 
concentration of finished water above that naturally present 
in water. They reported that the median concentration of 
aluminum in "finished" water supplied by water utilities 
was 17 |Jg/L of aluminum (range <14 |Jg/L to 2.67 mg/L of 
aluminum). However, very high concentrations (30-620 
mg/L) of aluminum have been found in "finished" water 
when processing plants made serious errors in England 
(Eastwood et al., 1990). 

The biggest source of aluminum in the diets of most 
Americans is food additives, although aluminum-contain- 
ing additives are present in only a limited number of foods 
(i.e., chemically leavened baked goods and processed 
cheese) (NRG, 1984; Greger, 1985; Pennington and Jones, 
1989). It is unlikely that aluminum-containing food addi- 
tives are intentionally added to the diets of livestock and 
pets. However, aluminum contamination of some additives 
used in livestock and pet feed is possible. For example, 
aluminum concentrations were found to be 3- to 10-fold 
higher in soy-based infant formulas than in milk-based in- 
fant formulas (Committee on Nutrition, 1986). Calcium and 
phosphate additives (with unintentional aluminum con- 
tamination) were likely sources of the aluminum in the soy 
formulas (Burgoin, 1992). 

Packaging and utensils can also be a source of dietary 
aluminum to humans. Most foods stored or cooked in alumi- 
num pans, trays, or foil accumulated less than 2 mg/kg food 
of aluminum when frozen, refrigerated, and stored at ambi- 
ent temperature (Ondreicka etal., 1971; Greger etal., 1985b; 
Liukkonen-Liija and Piepponen, 1992). A few acidic foods 
(e.g., tomato products, applesauce), when cooked for long 
periods of time, accumulated as much as 17 mg/100 g serv- 
ing of aluminum. However, packaging is probably not an 
important source of aluminum in the diets of livestock or 
even pets, as aluminum cans are lined to prevent seepage of 
aluminum into foods. 

Ptiarmaceutical and Otiier Sources 

Lione (1983, 1985) estimated that humans could ingest as 
much as 5 g/day of aluminum in pharmaceuticals such as 
antacids, buffered analgesics, antidiarrheals, and certain an- 
tiulcer drugs. Only the antidiarrheals (e.g., kaolin, 
attapulgite, and aluminum magnesium silicate) are apt to be 
used in veterinary practice. 

Aluminum hydroxide, aluminum phosphate, aluminum 
potassium sulfate (alum), and aluminum silicates (zeolite) 
are used in the preparation of a number of vaccines and other 
injectants to adsorb antigenic components and to serve as 
adjuvants that enhance immune response (Lione, 1985). The 



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MINERAL TOLERANCE OF ANIMALS 



importance of adjuvants as a source of aluminum is debat- 
able (Malakoff, 2000). Keith et al. (2002) estimated that the 
body burden of aluminum for human infants is greater from 
vaccinations than from dietary sources, but is still at a safe 
level. More aluminum was absorbed into blood by rabbits 
injected intramuscularly with aluminum phosphate than with 
aluminum hydroxide adjuvant (Hem, 2002). It is unlikely 
that injectables are a major source of aluminum exposure to 
livestock or even most pets. 

Feed or water for livestock could be contaminated with 
aluminum when aluminum sulfate and zeolite are applied to 
litter and waste lagoons to reduce phosphorus run-off from 
lands fertilized with these wastes and to reduce ammonia 
volatilization in facilities (Moore et al., 1999, 2000; Codling 
et al., 2002). Alum has also been added to dairy slurry to 
reduce ammonia emissions (Lefcourt and Mesinger, 2001). 

Bioavailability 

A variety of dietary factors affect absorption of 
aluminum. 



Speciation and Solubility 

Speciation of aluminum in water is complex and changes 
dramatically with pH (Martin, 1991). The net result is that 
aluminum is less soluble between pH 5 to 7 than at higher or 
lower pHs. Accordingly, rats absorbed more aluminum from 
in situ perfusions at pH 4 than at pH 7 (van der Voet, 1992). 

The solubility of aluminum compounds also affects their 
efficiency of absorption. Patients' serum aluminum levels 
tended to be higher after they were dosed with sucralfate in 
suspension rather than in tablet or granular form (Conway et 
al., 1994; Schiitze et al., 1995). Berthon and Dayde (1992) 
noted that aluminum phosphate was less toxic than alumi- 
num hydroxide because aluminum phosphate is virtually in- 
soluble at acidic pHs. However, Yokel and McNamara 
(1988) found no correlation between the percent 
bioavailability of aluminum from eight aluminum-contain- 
ing compounds to rabbits and the compounds' solubility. 

Citric Acid 

Citrate in diet and pharmaceuticals has been repeatedly 
found to increase aluminum absorption in humans (Slanina 
et al., 1986; Greger and Sutherland, 1997). Citrate has been 
found to increase aluminum retention in tissues of labora- 
tory animals when administered in drinking water (Fulton et 
al., 1989; Fulton and Jeffery, 1990), diet (Ecelbarger et al., 
1991, 1994b; Greger and Powers, 1992; Owen et al., 1994), 
or gavage solutions (Slanina et al., 1984, 1985; Yokel and 
McNamara, 1988; Froment et al., 1989 a,b; Quartley et al., 
1993). Generally animals fed aluminum and citrate in diets 
retained more aluminum than animals given citrate with alu- 
minum in drinking water, but less aluminum than animals 



dosed with only citrate and aluminum by gavage. The pres- 
ence of other substances (i.e., fluoride, phosphorus, calcium) 
in the gut, as occurs when aluminum and citrate were added 
to feed and water, may have moderated the effect of citrate 
on aluminum absorption (Greger, 1993; Glynn et al., 1995). 

Other Organic Acids and Anions 

Although the interaction of citrate and aluminum is prac- 
tically important, the uniqueness of the interaction has been 
overstated. A variety of organic acids found in food beside 
citric acid (including ascorbic, gluconic, lactic, malic, ox- 
alic, and tartaric acids) have been found to increase the solu- 
bility of aluminum and to increase tissue retention of alumi- 
num in rats orally dosed with aluminum (Domingo et al., 
1991, 1994). Limited data indicate that dietary factors (e.g., 
phytate and polyphenols) that reduced the absorption of other 
minerals also reduced the absorption of aluminum (Powell 
and Thompson, 1993; Powell et al., 1993). 

Several investigators have compared the biological ef- 
fects of various aluminum salts. Large (aluminum as 0.5 per- 
cent of diet) oral doses of aluminum as acetate, chloride, 
nitrate, and sulfate salts were more toxic to chicks than simi- 
lar doses of aluminum as phosphate salt (Storer and Nelson, 
1968). Similarly, human subjects had less aluminum in se- 
rum and urine after being dosed with 2.2 g aluminum daily 
as aluminum phosphate than after being dosed with alumi- 
num hydroxide, aluminum carbonate, or dihydroxyalumi- 
num aminoacetate (Kaehny et al., 1977). Rabbits dosed with 
aluminum nitrate or citrate experienced greater increases in 
serum aluminum than those dosed with aluminum as borate, 
hydroxide, chloride, glycinate, sucralfate, and acetate salts 
(Yokel and McNamara, 1988). No differences in tissue alu- 
minum concentrations were noted when rats were dosed with 
moderate levels (205 to 278 mg/kg diet of aluminum) of 
various aluminum hydroxide compounds (reagent and 
dessicated gel) and aluminum as lactate, palmitate, and phos- 
phate (Greger et al., 1985a). However, bone phosphorus lev- 
els were only affected by aluminum hydroxide. 

Silicates 

Dietary silicates may reduce the absorption of aluminum 
because silicates replace phosphates as the primary 
complexors of aluminum in solutions that are more alkaline 
than pH 6.6 (Birchall and Chappell, 1988; Birchall, 1992). 
Edwardson et al. (1993) noted that plasma ^^Al concentra- 
tions one hour after ingestion of ^®A1 and silicon in orange 
juice were only 15 percent of those observed when silicon 
was not consumed with aluminum. Quartley et al. (1993) 
demonstrated that oral consumption of silicic acid previously 
to and concurrently with aluminum reduced tissue alumi- 
num concentrations in rats four hours after dosing. 
Jugdaohsingh et al. (2000) demonstrated that oligomeric 
silica, but not monomeric silica, reduced dietary aluminum 



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ALUMINUM 



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bioavailability by 67 percent in humans. The oligomeric 
silica was not absorbed; the monomeric silica was. Addition 
of silicon (6.5 mg/L of silicon) to water containing 527 |jgl/ 
L of aluininum resulted in the formation of hydro xyl-alumi- 
nosilicates and decreased lysosomal accumulation of alumi- 
num by freshwater snails, presumably due to poorer absorp- 
tion of aluminum (Desouky et al., 2002). 

Desouky et al. (2002) hypothesized that the interaction of 
aluminum and silica may extend beyond absorption because 
snails exposed to aluminum (without supplemental silicon) 
accumulated more silica in their lysosomes as hydroxyl-alu- 
minosilicates. 

Calcium and Magnesium 

Aluminum may share some of the active processes in- 
volved in calcium absorption (Feinroth et al., 1982; Provan 
and Yokel, 1988a). Accordingly, several investigators have 
attempted to determine whether calcium- and magnesium- 
deficient animals were more sensitive to oral aluminum ex- 
posure. Yasui et al. (1991) found that rats deficient in cal- 
cium and magnesium retained more aluminum in tissues. 
Ecelbarger and Greger (1991) found that increasing the di- 
etary calcium levels 4-fold (67 to 250 mmol/g diet of cal- 
cium) resulted in reduced bone aluminum concentrations in 
rats. However, Yokel et al. (2001) suggested that the pres- 
ence of calcium and magnesium in the gut delayed but did 
not reduce the absorption of aluminum. 

Iron 

Interactions between aluminum and iron theoretically 
occur in a variety of tissues because aluminum is at least 
partially transported in plasma by transferrin. The impor- 
tance and the mechanism(s) of the interactions are contro- 
versial. The stability constants for transferrin binding to Al"''-' 
are 10 log scales lower than for iron (i.e., log K of 22 versus 
12, respectively) (Martin et al., 1987). Fatemi et al. (1991) 
estimated that 60 percent of the aluminum in human plasma 
(at pH 7.4 with 5 |jM aluminum and 25 mM HCO3") would 
be bound to transferrin, 34 percent to albumin, and the re- 
mainder to citrate. 

It is unlikely that aluminum and iron compete directly for 
absorption. The addition of equimolar quantities of iron to a 
gavage dose of aluminum did not affect tissue aluminum 
levels of rats (Greger et al., 1995). The presence of Fe III in 
in situ perfusion systems did not affect the luminal disap- 
pearance or the intestinal absorption of aluminum (van der 
Voet and de Wolff, 1987). However, the presence of Fe II 
decreased the intestinal absorption of aluminum by decreas- 
ing the release of aluminum into the blood from mucosal 
cells. 

Fernandez-Menendez et al. (1991) proposed that in iron 
deficiency the decreased saturation of transferrin resulted in 
greater aluminum absorption. In two studies, anemic rats 



orally administered aluminum for 30 to 42 days excreted 
more aluminum in urine and retained more aluminum in tis- 
sues than normal rats (Cannata et al., 1991; Brown and 
Schwartz, 1992). However, the ingestion of ferrofumarate 
with aluminum aminoacetate for 90 days by renal patients 
did not decrease plasma aluminum levels (Andersen et al., 
1987). Moreover, transferrin receptors are not present on the 
apical surfaces of mucosal cells (Anderson et al., 1990). 

An alternate explanation for the effect of dietary iron on 
tissue aluminum levels involves the effect of iron deficiency 
on the tissue turnover of aluminum. The half-lives of alumi- 
num in liver (56 versus 17 days), muscle (33 versus 16 days), 
spleen (29 versus 24 days), and serum (12 versus 8 days) 
were longer in iron-deficient anemic rats than in controls 
following a single oral aluminum dose (Greger et al., 1994). 

TOXICOSIS 

There are no practically important studies reporting the 
toxic effects of a single oral dose of aluminum (ATSDR, 
1999). The toxic effects of chronic (>2 weeks) oral expo- 
sure to aluminum in livestock appear to be related to alumi- 
num effects on general growth and longevity or on the 
utilization of essential elements (NRG, 1980). Hence the 
studies are presented accordingly. Table 3-1 summarizes 
their findings. 

Growth and Longevity 

Few gross changes have been observed in normal animals 
exposed to aluminum orally in long-term ( 6 months) stud- 
ies in which nothing was done to enhance aluminum absorp- 
tion. The longevity of rats exposed to aluminum (5 mg/L of 
aluminum) in their drinking water throughout their life was 
unaffected (Schroeder and Mitchener, 1975). Rabbits admin- 
istered aluminum in water (5 mg/L of aluminum) for 12 
months gained weight more slowly than controls but demon- 
strated no histological changes except for elevation of tissue 
aluminum concentrations (Wills et al., 1993). The food in- 
take and weights of dogs were not adversely affected when 
sodium aluminum phosphate (up to 3 percent of diet was 
sodium aluminum phosphate) was added to their diets for six 
months (Katz et al., 1984). 

Similarly, Berlyne et al. (1972) observed that adding 1 
percent or 2 percent aluminum chloride or aluminum sulfate 
to the drinking water or administering aluminum hydroxide 
(150 mg/kg BW/day of aluminum) by gavage to rats had no 
adverse effects on the growth or organs of normal rats, ex- 
cept to elevate tissue aluminum concentrations. However, 
the administration of the same doses of aluminum to rats that 
had five-sixths nephrectomies increased the death rate of 
animals (Berlyne et al., 1972). 

Neonatal animals may be more sensitive to aluminum. 
Administration of aluminum chloride or aluminum lactate 
(100 and 200 mg/kg BW/day of aluminum) by gastric 



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MINERAL TOLERANCE OF ANIMALS 



intubation to young (5- to 14-day-old) rats reduced body 
weight and decreased plasma protein and albumin but in- 
creased al globulins (Cherroret et al., 1995). 

Bone Metabolism 

More than 100 papers have been published on the impor- 
tance of aluminum in the etiology of dialysis osteodystrophy 
in human patients with uremia. There are two etiologies for 
the osteodystrophy: contamination of dialysates with alumi- 
num and the use of pharmaceutical levels of aluminum to 
treat the hyperphosphatemia associated with renal failure, 
especially in children (Alfrey, 1986; Committee on Nutri- 
tion, 1986; Hewitt et al., 1990; Jeffery et al., 1996; ATSDR, 
1999). Neither of these practices is important in the treat- 
ment of uremic livestock, and references to the studies in 
humans will not be included here. 

Animal models (e.g., rats, dogs, mice) for renal osteodys- 
trophy were generally created by injecting aluminum into 
animals. In those cases, aluminum was deposited at osteoid 
bone interfaces (Quarles et al., 1985) and inhibited osteo- 
blast and osteoclast activities (Jeffery et al., 1996). Feeding 
aluminum (37.6 pmol/g diet of aluminum) to normal rats for 
6 or 9 months did not affect urinary excretion of hydrox- 
yproline (a marker of bone resorption), but feeding alumi- 
num to rats that had one kidney removed elevated hydrox- 
yproline excretion (Ecelbarger et al., I994a,b). This suggests 
that reductions in kidney function (as occurs with age) may 
cause animals to be more sensitive to aluminum exposure in 
terms of bone metabolism. 



Phosphorus Metabolism 

Many of the symptoms associated with aluminum toxic- 
ity in bone and the occasional effects of aluminum on growth 
reflect the effects of oral aluminum exposure on phosphorus 
utilization. A series of case studies demonstrated that the 
chronic ingestion of aluminum in antacids (as aluminum 
hydroxide gels or magnesium aluminum hydroxide) 
caused phosphorus depletion (with hypophosphatemia, 
hypophosphaturia, hypercalciuria) and osteomalacia (bone 
loss of calcium and phosphorus, fractures, bone pain, el- 
evated alkaline phosphatase levels) in non-uremic human 
patients (Bloom and Flinchum, 1960; Lotz et al., 1968; 
Insogna et al., 1980). The symptoms appeared in only a small 
number of subjects ingesting pharmaceutical levels of antac- 
ids (0.5 to 9.6 g/day of aluminum) chronically (more than 6 
months). Moreover, healthy adult males fed 125 mg/day of 
aluminum as aluminum lactate experienced only a transient 
(first 12 days) decrease in phosphorus absorption with no 
other changes in phosphorus or calcium utilization (Greger 
and Baler, 1983a). 

Aluminum (>200 mg/kg of aluminum diet as aluminum 
sulfate or hydroxide salts) has been shown to depress phos- 
phorus utilization by rats (Ondreicka et al., 1966, 1971; 



Greger et al., 1985). Ingestion of 2,000 mg/kg of aluminum 
diet as aluminum chloride depressed growth, feed intake, 
plasma phosphorus levels, and phosphorus absorption in 
lambs, especially those fed low levels of phosphorus 
(Valdivia et al., 1982). Chicks fed 0. 15 or 0.3 percent alumi- 
num had depressed growth, reduced feed efficiency, and 
decreased bone ash. Only the higher level of aluminum re- 
duced plasma phosphorus levels and reduced egg production 
(Wisser et al., 1990). 

Another source of exposure to aluminum is when alum is 
applied to poultry litter or dairy slurry. Moore et al. (1999, 
2000) reported that broilers grown on alum-treated poultry 
litter were significantly heavier than control (1.73 versus 
1.66 kg) and had less mortality than controls (3.9 versus 4.2 
percent). Not only did the application of the alum not ad- 
versely affect the livestock, but also it had minimal negative 
effects on crops grown on the land. Tall fescue yields on 
fertilized fields were improved due to increased nitrogen 
availability presumably because of less ammonia volatiliza- 
tion (Moore et al., 1999). Soluble reactive phosphorus run- 
off from treated pastures were greatly reduced (Codling et 
al., 2002; Moore et al., 2000) without severely impacting 
soil fertility, although phosphorus concentrations in plants 
were progressively reduced during the three-year study (Co- 
dhng et al., 2002). 

Fluoride Utilization 

The oral administration of high (1.8 g/day of aluminum 
as antacids) and moderate (125 mg/day of aluminum as alu- 
minum lactate) amounts of aluminum has been found to de- 
press serum and urinary fluoride levels in healthy adult hu- 
mans (Spencer et al., 1981; Greger and Baler, 1983a). 
Similarly rabbits given aluminum in their drinking water 
(100 and 500 mg/g water of aluminum as aluminum chlo- 
ride) accumulated less fluoride in their tissues (Ahn et al., 
1995). However, rabbits accumulated more aluminum in 
bone when the fluoride content of the drinking water was 
increased. 

Animal scientists have used knowledge of the interaction 
between aluminum and fluoride to reduce fluoride toxicity. 
Aluminum has been intentionally added to the diets of sheep 
(3.3 g aluminum chloride/day) to reduce the symptoms of 
fluorosis (mottling of teeth) in animals provided with 30 mg/ 
kg water of fluoride (Said et al., 1977). Although the inges- 
tion of aluminum increased fecal fluoride and reduced uri- 
nary and tissue fluoride levels, the incidence and severity of 
mottling of teeth was not reduced. Similarly, aluminum (295 
or 550 mg/kg diet of aluminum) was added to the diets of 
turkeys fed fluoride-contaminated phosphate fertilizers as a 
phosphorus source (Cakir et al., 1977). The aluminum par- 
tially prevented the toxic effects of the fluoride (i.e., reduced 
bone fluoride concentrations and increased weight gain). The 
addition of aluminum ( 1 ,040 mg/kg diet of aluminum as alu- 
minum sulfate) to diets reduced the toxic effects (i.e.. 



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ALUMINUM 



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reduced feed intake, reduced egg production, and increased 
tissue fluoride levels) of dietary fluoride (1,300 mg/kg of 
fluoride) in laying hens (Hahn and Guenter, 1986). 

Calcium and Other Minerals 

The effect of dietary aluminum on the utilization of other 
minerals is less consistent in livestock. Lambs fed aluminum 
(> 1,450 mg/kg diet aluminum as aluminum chloride) had 
reduced calcium absorption, but no changes in tissue cal- 
cium concentrations; lower concentrations of magnesium in 
some tissues; and minor changes in tissue iron and copper 
concentrations in two studies (Valdivia et al., 1982; Rosa et 
al., 1982). Although cows with clinical grass tetany had el- 
evated concentrations of aluminum in their ruminal dry mat- 
ter than did control cows or asymptomatic, hypomagnesemic 
cows, the administration of aluminum orally to lactating 
cows did not reduce plasma magnesium concentrations 
(Kappel et al., 1983). Similarly, dietary aluminum had small 
inconsistent effects on tissue concentrations of calcium, mag- 
nesium, and trace elements in rats fed generally balanced diets 
(Greger et al., 1985; Ecelbarger and Greger, 1991; Ecelbarger 
et al., 1994b; Belles et al., 2001; Yasui et al., 1990). 

Neurological Symptoms 

Aluminum is known to be part of the etiology of dialysis 
dementia and hypothesized to be part of the etiology of a 
progressive dementing encephalopathy reported in occa- 
sional industrial workers and in patients with amyotrophic 
lateral sclerosis with Parkinson's dementia in Guam. Brain 
accumulation of aluminum has also been associated with the 
development of Alzheimer's disease in some patients (King 
et al., 1981; Committee on Nutrition, 1986; Ganrot, 1986: 
Strong et al., 1996; Savory et al., 1996). Injection of alumi- 
num, especially intracisternally, will induce neurological 
changes in animal models (Wisniewski et al., 1980; ATSDR, 
1999). The effects of orally administered aluminum on the 
brain are more debatable. Only those studies in which alumi- 
num was provided orally are cited here. 

Administration of aluminum (0.3 percent Al as aluminum 
sulfate) in drinking water for 30 days resulted in impairment 
of both consolidation and extinction of passive avoidance 
tasks in rats with a slight increase in hippocampal muscar- 
inic receptor numbers (Connor et al., 1988). Rats orally ad- 
ministered aluminum (100 mg/ kg BW/day of aluminum) 
for 90 days accumulated more aluminum in their brains, were 
slower to learn a labyrinth, had increased brain acetylcho- 
linesterase activity, and had decreased brain choline- 
acetyltransferase activity (Bilkei-Gorzo, 1993). 

Mice fed high aluminum levels (1,000 mg/kg diet of alu- 
minum as aluminum lactate) were less active than controls 
after 6 weeks (Golub et al., 1989) and were less active, had 
decreased grip strength, and increased startle responses after 
90 days (Golub et al., 1992). When mice were fed aluminum 



(500 or 1,000 mg/kg diet of aluminum as aluminum lactate 
to dam and then offspring) from conception to weaning or to 
adulthood, mice attained training faster but had reduced grip 
strength (Golub et al., 1995). The effects were not greater in 
mice fed aluminum to adulthood than just to weaning. 

Other research groups fed animals diets that maximized 
the bioavailability of aluminum to potentially increase the 
absorption and accordingly the neurological toxicity of di- 
etary aluminum. For example, Oteiza et al. (1993) observed 
that mice fed diets containing 1 ,000 mg/kg diet of aluminum 
as aluminum chloride with sodium citrate accumulated more 
aluminum in the brain nuclear fraction and spinal cord, had 
lower grip strength, and greater startle responsiveness after 
5 and 7 weeks. Old (18 months of age) rats exposed to alu- 
minum (100 mg/kg BW/day of aluminum) in drinking water 
with citrate (356 mg/kg BW/day of citrate) had decreased 
numbers of synapses and a greater percentage of perforated 
synapses than controls, but no changes in behavior 
(Colomina et al., 2002). 

Several groups fed low calcium levels to increase ani- 
mals' sensitivity to aluminum. Garruto etal. (1989) observed 
that cynomolgus monkeys fed a low calcium (3,200 mg/kg 
diet) diet with aluminum (125 mg/day of aluminum) and 
manganese (50 mg/day of manganese) for 41 to 46 months 
had more degenerative changes (that were consistent with 
early Alzheimer's disease or Parkinson's dementia in Guam) 
in the central nervous system than control monkeys. Golub 
andGermann (2001) observed growth depression and poorer 
performance on standardized motor tests in mice as adults 
when dams were exposed to aluminum (1,000 mg/kg diet of 
aluminum as aluminum lactate) with marginal levels of cal- 
cium and magnesium during pregnancy and lactation. Kihira 
et al. (2002) observed that mice fed a low rather than recom- 
mended levels of calcium (2,500 versus 5,000 mg/kg diet of 
calcium) with aluminum (15,600 mg/kg diet of aluminum 
hydroxide) for 11 to 25 months accumulated more 
hyperphosphorylated tau protein in the cortical neurons and 
had more atrophic neurons in the central nervous system. 

Pratico et al. (2002) reported that transgenic mice that 
over-expressed human amyloid precursor protein (but not 
wild-type littermates) had increased brain isoprostane levels 
and more amyloid b peptide formation and deposition when 
aluminum was added to their diets. The effects of aluminum 
were reversed by additional dietary vitamin E. 

In general, experimental animals did not exhibit large 
behavioral changes because of oral aluminum exposure even 
when fed diets that augmented the bioavailability of alumi- 
num. It is unlikely that aluminum is part of any neurological 
syndromes in livestock. 

Fish and Mollusks 

The effects of aluminum on fish and mollusks appear to 
be unique and worthy of a separate discussion. Acidification 
of the aquatic environment often causes aluminum to be 



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MINERAL TOLERANCE OF ANIMALS 



mobilized into tiie aqueous environment. Atlantic salmon 
(Salmo salar) exposed to acidified water (pH 5.2) with alu- 
minum (50 |Jg/L of aluminum as aluminum chloride) for 36 
days reduced their feed consumption at first but lost weight 
and increased their swimming activity throughout the test 
period (Brodeur et al., 2001). Rainbow trout fry avoided alu- 
minum at concentrations as low as 1 |imole/L of aluminum 
at pH 5 but were insensitive to aluminum at pH 6 (Exiey, 
2000). 

Temperature may also affect the sensitivity of fish to alu- 
minum. Graylings (Thymallus thymallus) were exposed to 
water containing 1 mg/L of iron and 100 (ig/L of aluminum 
as aluminum sulfate at pH 5.5 and at pH 6.9 (control). Fifty 
percent of the fish exposed to the aluminum at pH 5.5 died 
after 2 weeks when the temperature was 13°C but none died 
at 3°C. Surviving fish at both test temperatures suffered gill 
damage (adherence of lamellae), increased hematocrits, re- 
duced plasma chloride levels, and reduced oxygen consump- 
tion. The fish exposed to aluminum at the higher tempera- 
ture recovered more completely than those exposed at the 
lower temperature (Peuranen et al., 2003). 

Acclimation to aluminum may also affect the severity of 
sjTTiptoms when fish are exposed to toxic levels of alumi- 
num. Juvenile rainbow trout {Oncorhynchus mykiss) were 
exposed to a pulse of aluminum (36 |Jg/L of aluminum at pH 
5.2) for 4 days. One group was exposed to a lower level of 
aluminum (24 pg/L of aluminum) for 16 days before and 10 
days after the pulse; another was acclimated only to the pH. 
Fish became hypoactive upon exposure to the lower level of 
aluminum but recovered after 4 days. Exposure to the higher 
level of aluminum by the acclimated fish resulted in 
hypoactivity, but they quickly recovered normal swimming 
behavior during the 4-day pulse; mortality was 4 percent. In 
contrast, the fish not acclimated to the lower level of alumi- 
num became hypoactive when exposed to 36 |Jg/L of alumi- 
num and did not recover in the 6 days after the pulse; mortal- 
ity was 26 percent (Allin and Wilson, 2000). 

Mollusks appear to have several ways to avoid exposure 
to aluminum. Freshwater mussels (Anodonta cygnea L) re- 
duced their filtering activity by reducing shell opening by 50 
percent when exposed to 500 |Jg/L of aluminum as alumi- 
num nitrate at neutral pH for 15 days (Kadar et al., 2001). 
Freshwater snails (Lymnaea stagnalis) accumulated alumi- 
num, silicon, phosphorus, and sulfur in non-absorbable gran- 
ules in their digestive cells when exposed to aluminum 
(Elangovan et al., 2000). 

TISSUE LEVELS 

Humans do not accumulate large body burdens of alumi- 
num because of oral exposure to dietary aluminum from a 
variety of undefined sources. Total body aluminum loads for 
healthy adults have been estimated to range from 30 to 300 
mg aluminum (Skalsky and Carchman, 1983; Ganrot, 1986). 
Alfrey (1986) estimated that normal tissue (bone and soft 



tissue, except lungs) levels are 1 to 4 mg/kg dry weight of 
aluminum. Concentrations of aluminum in serum are lower 
than in other tissues; 1 to 5 |Jg/L of aluminum are typical for 
serum samples drawn from fasted, normal human subjects 
(Versieck and Cornells, 1980; Greger and Baler, 1983b; 
Savory and Wills, 1988). 

Rodents exposed to aluminum (as aluminum sulfate, chlo- 
ride, hydroxide, palmitate, lactate, phosphate, or citrate) 
orally accumulated more aluminum in bone than soft tissues 
(Ondreicka et al., 1966, 1971; Greger et al., 1985, 1986; 
Slanina et al., 1984, 1985). Rats fed purified control diets 
(that contained less than 15 mg/kg diet of aluminum) re- 
tained less than 0.3 pg/g wet tissue of aluminum in their soft 
tissues (kidneys, livers, brains, and muscles) and less than 
0.9 |ig/g wet tissue of aluminum in bone (Greger and Pow- 
ers, 1992; Ecelbarger et al., 1994a,b; Sutherland and Greger, 
1998). When rats were fed 1,000 mg/kg diet of aluminum in 
these studies, the concentrations of aluminum in soft tissues 
sometimes increased significantly but were generally less 
than 0.7 |jg/g wet tissue of aluminum and in bone were less 
than 2.0 |Jg/g wet tissue of aluminum. 

Similarly steers (Valdivia et al., 1978), sheep (Valdivia et 
al., 1982), and chickens (Wisser et al., 1990) fed 800-3,000 
mg/kg diet of aluminum accumulated aluminum in their tis- 
sues, but the concentrations of aluminum in their tissues were 
still <1 1 |jg/g dry tissue of aluminum. 

The amounts of aluminum in tissues were much greater if 
kidney function of the rats had been reduced or aluminum 
had been given orally by gavage to fasted animals. In those 
cases, soft tissues and bone concentrations of aluminum were 
as great as 6 and 27 |ig/g wet tissue of aluminum, respec- 
tively (Greger and Powers, 1992; Domingo et al., 1994; 
Greger etal., 1994). 

Table 3-2 summarizes the effect of aluminum on selected 
tissues. 

MAXIMUM TOLERABLE LEVELS 

The Agency for Toxic Substances and Disease Registry 
(ATSDR, 1999) noted that toxicity to aluminum had been 
reported many times in patients with reduced kidney func- 
tion, but limited data were available about toxic responses to 
aluminum in normal humans. They suggested a LOAEL 
(lowest-observed-adverse-effect level) of 130 mg/kg/day of 
aluminum on the basis of the observation that adult mice 
exposed to dietary aluminum lactate for 6 weeks decreased 
their spontaneous motor activity (Golub et al., 1989). This 
would mean a 70-kg human should be unaffected by inges- 
tion of 9.1 g of aluminum daily (18,000 mg/kg dry diet of 
aluminum if 0.5 kg dry diet is consumed daily). The occa- 
sional case histories of adverse responses to aluminum-con- 
taining antacids among adults with normal kidney function 
suggest that this LOAEL is too high. 

Ingestion of 2,000 and 1,450 mg/kg diet of aluminum 
depressed growth, feed intake, plasma phosphorus levels. 



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ALUMINUM 



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and/or phosphorus absorption in lambs (Valdivia et al., 1982; 
Rosa et al., 1982, respectively). Laying hens (White Leg- 
horn) fed 1,500 mg/kg diet of aluminum had depressed 
growth production (Wisser et al., 1990). Thus, a cautious 
maximum tolerable level of aluminum in the diets of live- 
stock would be 1,000 mg/kg diet of aluminum. 

The NRC (1982) considered the minimum toxic effect 
dose of aluminum in water for rats to be 1,000 mg/kg/day of 
aluminum. If 200-g rats consumed 20 ml water daily, the 
maximum toxic level would be 10,000 mg/L water of alumi- 
num (which exceeds the solubility of aluminum in non-acidic 
solutions). Brodeur et al. (2001) observed toxic effects in 
fish exposed to 36 |Jg/L water of aluminum at pH 5.2. These 
data suggest that fish are more sensitive to aluminum expo- 
sure than are mammals. 

FUTURE RESEARCH NEEDS 

Research on the role of aluminum in the etiology of dis- 
ease syndromes in humans has little significance to livestock 
production industry but could be of interest to veterinarians 
treating old and/or diseased pets. The relationship of tissue 
aluminum concentration and neurological problems in pets, 
especially those with reduced renal function, deserves con- 
sideration. 

The effects of acid rain and the resulting aluminum con- 
tamination of aquatic environments will continue to deserve 
further study. In both cases, the mediation of the effects of 
aluminum exposure by other dietary elements is likely to be 
important and worthy of further study. 

SUMMARY 

Aluminum is a ubiquitous element that is not essential to 
mammals. Livestock are occasionally exposed to high levels 
of aluminum, but toxicity to orally administered aluminum 
is not a problem as long as gut and kidney functions are 
normal. The first signs of aluminum toxicity in normal ani- 
mals reflect the adverse effect of dietary aluminum on phos- 
phorus utilization. On the other hand, fish are very sensitive 
to aluminum in acidic water. 

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25 



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MINERAL TOLERANCE OF ANIMALS 



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Copyright © National Academy of Sciences. All rights reserved. 



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inemi Tnlemnce nf Animals- Sennnd Revised Edition 



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28 



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Copyright © National Academy of Sciences. All rights reserved. 



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inemi Tnlemnce nf Animals- Sennnd Revised Edition 



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29 















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30 



MINERAL TOLERANCE OF ANIMALS 



TABLE 3-2 Aluminum Concentrations in Fluids and Tissues of Animals (mg/kg) 



Species 



Quantity" 



Source 



Duration 



Route Tissue Concentrations (mg/kg)'' 



Reference 



Humans 
Humans 



Rats 



Cliicisens 



Steers 



Sheep 



Uniinown 

5 mg/d 
125 mg/d 



Unknown 

Unknown 
Al lactate 



Lifetime Diet All tissues; 1^ (dry weight) 

20 d Diet Serum: 4 ng/L 

20 d Diet Serum: 7 ng/L 



11 



1,005 



160 

1,500 

3,000 

210 
510 
810 
1,410 

168 
2,168 



Unknown 

Al hydroxide 
with and without 
citrate 

Unknown 
Al sulfate 
Al sulfate 

Unknown 
Al chloride 
Al chloride 
Al chloride 

Unknown 
Al chloride 



6-7 mo 



6—7 mo 



17 wk 
17 wk 
17 wk 

84 d 
84d 
84 d 
84d 

56 d 
56 d 



Diet 



Diet 



Diet 
Diet 
Diet 

Diet 
Diet 
Diet 
Diet 



Diet 
Diet 



Liver: <0.3; muscle: <0.4; 

bone: <0.8 
Liver: <0.3; muscle: <0.7; 

bone: 1-1.4 

Bone: <2 (di-y weight) 
Bone: 6 (dry weight) 
Bone: 10 (dry weight) 

Soft tissues: 4—8 (dry weight) 
Soft tissues: 4—8 (dry weight) 
Soft tissues: 5—10 (dry weight) 
Soft tissues: 5— ll(dry weight) 

Soft tissues: 2—5 (dry weight) 
Soft tissues: 4—6 (dry weight) 



Alfrey, 1986 

Greger and Baier, 1983b 



Mice 


25 


Unknown 


=50 d 


Diet 


Liver: 0.01; bone: 3 


Donald et al., 1989 




500 


Al lactate 


«50d 


Diet 


Liver: 0.03; bone: 5 






1,000 


Al lactate 


=50 d 


Diet 


Liver: 0.05; bone: 6 





Ecelbarger and Greger, 
1994b 



Wisser et al., 1990 



Valdivia et al., 1978 



Valdivia et al, 1982 



"Quantity of exposure reported as mg/kg diet of aluminum unless noted otherwise. 
''Concentrations reported as mg/kg wet tissue unless noted otherwise. 



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Arsenic 



INTRODUCTION 

Arsenic (As) is a solid, brittle metalloid and thus has both 
metallic and nonmetallic properties. The most common 
stable form of arsenic at room temperature is metallic or 
gray arsenic. Another form of elemental arsenic, yellow ar- 
senic, occurs when arsenic vapors are cooled suddenly to 
below 0°C (Stoeppler, 2004). It is unstable and more volatile 
than gray arsenic. Arsenic is the 20th most common element 
in the Earth's crust with an average natural abundance of 
about 1.5-3 mg/kg (Mandal and Suzuki, 2002). Arsenic natu- 
rally occurs in over 200 different forms with approximately 
60 percent as arsenates, 20 percent as sulfides and sulfur- 
salts, and the remainder as arsenides, arsenites, oxides, sili- 
cates, and the elemental form (Mandal and Suzuki, 2002). 
The most common arsenic mineral is arsenopyrite. Other 
common minerals of arsenic are orpiment and realgar 
(natural sulfides) and arsenolite. However, arsenic in its most 
recoverable form is found in various types of metalliferous 
minerals (Mandal and Suzuki, 2002) such as iron pyrite, 
galena, chalcopyrite, and sphalerite. Arsenic trioxide 
(AsjOj), the common commercial form of arsenic, is pro- 
duced as a by-product of roasting various ores. 

For more than 50 years, chromated copper arsenate 
(CCA) has been the main preservative for wood products 
used outdoors. In response to consumer concerns about ar- 
senic toxicity, a voluntary phase-out of CCA-treated wood 
for certain residential uses, such as play structures, picnic 
tables, decks, and fencing, was to be completed before 2004. 
Industrial-use wood products, such as marine pilings, utility 
poles, roofing shakes, and shingles, still can be treated with 
CCA. Because of reduced use of CCA, the demand for ar- 
senic, as AsjOj, has markedly dropped. Still, about 90 per- 
cent of arsenic trioxide produced is used to make CCA. The 
balance of arsenic trioxide is mainly used in agricultural 
chemicals such as insecticides, herbicides, and coccidiostats. 
Uses of other arsenic compounds and metallic arsenic in- 
clude electronics, pigments, metal alloys, and as a bubble 



dispersant or decoloring agent in glassmaking (Brooks, 
2002). 

Arsenic exists predominantly in nature as an oxyanion 
with an oxidation state of either 3"*" or S"*", but arsenic also 
forms compounds where it has an oxidation state of 3" 
(Hindmarsh et al., 2002). Arsenic binds covalently with 
most metals and nonmetals, and forms stable organic com- 
pounds. In animals, arsenic occurs mainly as inorganic 



arsenate [0=As(0H)3; H2ASO4I-; 
arsenite [OH-As(OH)2; H2ASO3 



HASO42-; 



ASO43-] 



and 



1 • HAsO,--; AsO 3], 



'3 ' ^='-'3 
and in the methylated form, mainly dimethylarsinic acid 

[(CH3)2AsO(OH)] and monomethylarsonic acid 
[CH3AsO(OH2)]. The major arsenic species in fish, crusta- 
ceans, and mollusks apparently have the tetraalkylarsonium 
structure (R4AS"'"), and in marine algae and bivalves the ma- 
jor arsenic species have the trialkylarsine oxide structure 
(R3ASO) (McSheehy et al., 2003). Thus, arsenobetaine 
[(CH3)3As+CH2-COO-] and arsenocholine [(CH3)3As+CH2- 
CH,-OH] are found in sea animals and arsenosugars are 
found in marine algae and seaweeds. 

ESSENTIALITY 

Arsenic is generally not accepted as an essential nutrient 
for higher animals. However, the large number of responses 
to apparent arsenic deprivation (e.g., <12 |Jg/kg diet for rats 
and chicks; <35 |Jg/kg diet for goats) reported for a variety of 
animal species by more than one research group suggests 
that it may have an essential or beneficial function in ultra 
trace amounts (Anke, 1986; Uthus, 1994; Nielsen, 1998). In 
the goat, pig and rat, the most consistent signs of apparent 
arsenic deprivation have been depressed growth and abnor- 
mal reproduction characterized by impaired fertility and in- 
creased perinatal mortality. Other notable signs include de- 
pressed serum triglyceride concentrations and death with 
myocardial damage during lactation in goats (Anke, 1986), 
and depressed hepatic S-adenosylmethionine and elevated 
S-adenosylhomocysteine in rats and hamsters (Uthus, 1994). 



31 



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32 



MINERAL TOLERANCE OF ANIMALS 



Many of the responses of experimental animals to arsenic 
deprivation and other in vitro or cell culture findings suggest 
that arsenic affects the utilization of labile methyl groups 
arising from methionine in higher animals. Thus, arsenic 
may affect the methylation of metabolically or genetically 
important molecules, whose functions are dependent on or 
influenced by methyl incorporation. It has been suggested 
that rats and chicks have an arsenic requirement that is 
greater than 12 but less than 50 |Jg/kg diet (Nielsen, 1998). 

In vitro findings suggest that arsenic also has an essential 
or beneficial action in low amounts. For example. Snow et 
al. (2001) found that sub-toxic arsenite induces a multi-com- 
ponent protective response against oxidative stress in cul- 
tured human keratinocytes and fibroblasts. 

Some forms of arsenic have beneficial effects in 
supranutritional amounts. Some organic arsenicals, because 
of their antibiotic and anticoccidial properties, were used 
extensively in the past as growth promoters for swine and 
poultry (Anderson, 1983); this use has been largely sup- 
planted by newer antibiotics. However, roxarsone (3-nitro- 
4-hydroxyphenylarsonic acid; CgHgAsNOg) is still used ex- 
tensively in the feed of broiler poultry to control coccidial 
intestinal parasites, improve feed efficiency, and promote 
rapid growth. Also, melarsoprol {2-[4-[(4,6-diamino-l,3,5- 
triazin-2-yl)amino]phenyl]-l,3,2dithiarsolane-4-methanol; 
CpHijAsNgOSj} is still used to treat trypanosomiasis in hu- 
mans. High doses of arsenic trioxide recently have been 
found to be an effective treatment for acute promyelocytic 
leukemia through apototic, not necrotic, mechanisms (Chen 
et al., 2001). Arsenic trioxide also apparently is an effective 
treatment for other malignancies including megakaryocytic 
leukemia (Tallman, 2001) and lymphoma (Dai et al., 1999). 
Organic arsenicals such as melarsomine dihydrochloride 
given by intramuscular or intravenous injection are used to 
treat heartworm infection in dogs. 

DIFFICULTIES IN METHODS OF ANALYSIS AND 
EVALUATION 

The most common methods for determining total arsenic 
in biological samples after decomposition to mineral ash in 
solution are hydride atomic absorption spectrometry (AAS), 
inductively coupled plasma mass spectrometry, and graphite 
furnace AAS (Stoeppler, 2004). However, the silver 
diethyldithiocarbamate colorimetric method, which results 
in a very stable red color, is still in use to determine total 
arsenic, after conversion into arsine, in polluted ground wa- 
ter (Tareq et al., 2003). Converting biological samples to ash 
that can be solubilized has been accomplished by a number 
of different methods including dry ashing, wet or micro- 
wave-assisted ashing with acid mixtures, or ashing with ni- 
tric acid alone in open and closed (often pressurized) sys- 
tems (Stoeppler, 2004). An optimal sample decomposition 
method that avoids loss of analyte is high-pressure ashing. 
This method uses concentrated nitric acid in closed quartz 



vessels at temperatures up to 320° C (Wijrfels, 1989; Knapp, 
1990). A microwave-assisted procedure attaining >300° C 
also has been found to result in complete sample decomposi- 
tion (Goessler and Pavkov, 2003). Complete decomposition 
is of utmost importance when the hydride AAS method of 
analysis is used because traces of organic compounds inter- 
fere with the analysis. 

Because of the widely varying toxicities of different ar- 
senic species, the development of analytical methods to de- 
termine the chemical forms of arsenic and their concentra- 
tions in biological and environmental samples have become 
of interest. Collection of samples for arsenic speciation must 
be done with care and kept at a low temperature to prevent 
the modification of arsenic species by contaminating bacteria 
or inherent biological activity of the sample. The methods 
used to determine arsenic species require an extraction step. 
Tissues with high fat content may need to be defatted with a 
solvent, such as ether or acetone, before the extraction of 
arsenic. Most arsenic species in biological tissues are water 
soluble and thus can be extracted with water alone or with a 
mixture of water and methanol (McSheehy et al., 2003). Af- 
ter extraction, arsenic species are determined by a combina- 
tion of separation techniques such as high-performance liq- 
uid chromatography and capillary zone electrophoresis, and 
detection techniques that are specific and highly sensitive for 
arsenic such as inductively-coupled plasma mass spectrom- 
etry and electrospray mass spectrometry with tandem mass 
spectrometry (McSheehy et al., 2003). The difficulties en- 
countered in the determination of arsenic species in biologi- 
cal samples have been reviewed (McSheehy et al., 2003) and 
include incomplete extraction, retention time irrepro- 
ducibility, co-elution of species, presence of unidentified spe- 
cies, the lack of standards, and detection interference. An 
example of arsenic species detennination in the domestic ani- 
mal situation is that of Pavkov and Goessler (200 1), who iden- 
tified and quantified organoarsenic compounds in finishing 
chicken feed and chicken litter. 

REGULATION AND METABOLISM 
Absorption and Metabolism 

There apparently are two components to the absorption of 
arsenic (Fullmer and Wasserman, 1985). Initially, arsenate 
becomes sequestered primarily in or on the mucosal tissue. 
Eventually the sites of sequestration become filled, with con- 
comitant movement down a concentration gradient into the 
body. In rats, some forms of organic arsenic are absorbed at 
rates directly proportional to their intestinal concentration 
over a 100-fold range (Hwang and Schanker, 1973). This 
finding suggests that organic arsenicals are absorbed mainly 
by simple diffusion. The absorption and metabolism of ar- 
senic may be influenced by intestinal bacteria that can me- 
thylate arsenic or metabolize methylated arsenic (Hall et al., 
1997; Kurodaetal., 2001). 



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ARSENIC 



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Once absorbed, inorganic arsenic is transferred to various 
tissues including thie liver and testis where arsenic is methy- 
lated with S-adenosylmethionine as the methyl donor (Healy 
et al., 1999). Before arsenate is methylated, it is reduced to 
arsenite; this reduction is facilitated by glutathione (Vahter, 
1994). The conversion of arsenate into arsenite may also be 
accomplished in the animal gastrointestinal tract by bacteria 
with arsenate reductase activity (Herbel et al., 2002). Arsen- 
ite methyltransferase methylates arsenite to form mono- 
methylarsonic acid, which is then reduced to monomethyl- 
arsonous acid [CH3As(OH)2]. Monomethylarsonous acid, a 
relatively toxic form of arsenic, is rapidly methylated by a 
methyltransferase to form dimethylarsinic acid; in humans, 
monomethylarsonous acid is found in the urine only when 
excessive inorganic arsenic is consumed (Aposhian et al., 
2001). Dimethylarsinic acid can be reduced to dimethyl- 
arsinous acid [(CH3)2AsOH], which is a relatively toxic form 
of arsenic. However, the formation of dimethylarsinic acid 
usually is the final step in the metabolism of arsenic in hu- 
mans and most animals, and thus is the major form of ar- 
senic in urine. Inorganic and methyl arsenic compounds ap- 
parently are protein-bound when they are substrates for 
enzymatic biomethylation (Styblo and Thomas, 1997). Preg- 
nancy increases arsenic methylation, especially late in gesta- 
tion; dimethylarsinic acid is the main form of arsenic trans- 
ferred to the fetus (Vahter et al., 2000). In addition to the 
methylated forms, inorganic arsenic bound to transferrin is 
found in plasma (Zhang et al., 1998). Trimethylated organic 
forms of arsenic stay trimethylated after absorption. Arseno- 
betaine passes through the body into urine without biotrans- 
formation. Some orally ingested arsenocholine appears in 
the urine, and some can be incorporated into body phospho- 
lipids in a manner similar to choline; however, most is trans- 
formed into arsenobetaine before being excreted in the urine. 
Absorbed tetramethylarsonium also is not biotransformed 
before excretion (Yoshida et al., 1998). Unlike arsenobetaine 
and tetramethylarsonium, arsenosugars are transformed to 
many different arsenic species after absorption. Francesconi 
et al. (2002) found at least 12 arsenic metabolites in the urine 
of humans after they ingested an oral dose of a synthetic 
arsenosugar. The metabolism of arsenic in some animal spe- 
cies is quite unusual. For example, unlike other mammals, 
rats concentrate arsenic in their erythrocytes. Chimpanzees, 
marmosets, squirrel monkeys, tamarins, and guinea pigs are 
unable to methylate arsenic. These species apparently have 
other mechanisms for facilitating arsenic excretion (Vahter, 
1994; Healy et al., 1999; Vahter et al., 2000). 

Excretion of ingested arsenic is rapid, principally in the 
urine. In some species, significant amounts of arsenic are 
excreted in the bile in association with glutathione (Vahter, 
1994; Kala et al., 2000). The usual proportions of the forms 
of arsenic in human urine are about 20 percent inorganic 
arsenic, 15 percent monomethylarsonic acid, and 65 percent 
dimethylarsinic acid (Vahter et al., 2000). The proportions 
are quite different, however, with consumption of organic 



arsenic in forms found in seafood (mostly trimethylated ar- 
senic, e.g., arsenobetaine). For example, the urine of Japa- 
nese students who consumed high amounts of organic ar- 
senic in seafood contained 9.4 percent inorganic arsenic, 3.0 
percent monomethylarsonic acid, 28.9 percent dimethyl- 
arsinic acid, and 58.2 percent trimethylated arsenic com- 
pounds (Yamato, 1988). 

Metabolic Interactions 

Both arsenic deprivation and toxicity are influenced or 
affected by factors that change labile methyl metabolism. 
Generally, factors that reduce the availability of labile me- 
thyl groups exacerbate arsenic deprivation and toxicity. For 
example, methionine or choline deficiency, excessive dietary 
arginine, and dietary guanidoacetic acid supplementation 
enhanced the response to arsenic deprivation in chicks and 
rats (Uthus, 2003). Treatment with periodate-oxidized ad- 
enosine, a methylation inhibitor, exacerbated the toxic ef- 
fects of inorganic arsenic on development in mice (Lammon 
et al., 2003). Arsenic toxicity is also influenced by factors 
that affect reactive oxygen metabolism. For example, ascor- 
bic acid and a-tocopherol alleviated arsenic toxicosis signs 
in rats of lipid peroxidation and enzyme inhibition in mito- 
chondria of rats fed 100 mg As/L drinking water as sodium 
arsenite (Ramanathan et al., 2003). Other nutrient deficien- 
cies that can affect arsenic toxicity or deficiency through 
affecting labile methyl or oxidative metabolism include cys- 
teine (Siewicki and Leffel, 1980; Czarnecki et al., 1984), 
folic acid (McDorman et al., 2002), pyridoxine (Uthus and 
Poellot, 1991-1992), and zinc (Uthus, 1994). 

A well-established metabolic interaction is the multifac- 
eted and complex interaction between arsenic and selenium. 
Both methylation and oxidation could be involved in the in- 
teraction, possibly through competition for the methyl do- 
nor, S-adenosylmethionine, and competition for the antioxi- 
dant, glutathione (Kenyon et al., 2001). Interaction at the 
level of methylation is plausible because arsenite inhibits 
selenium methylation both in vivo and in vitro, and selenium 
is a potent inhibitor of arsenic methylation in vitro (Kenyon 
et al., 2001). However, most findings indicate that selenium 
deficiency (not excess) exacerbates arsenic toxicity and im- 
pairs arsenic metabolism. Also, supplementing with arsenic 
as arsenite or arsenate alleviates the toxicity of most forms 
of selenium (e.g., selenate, selenite, selenocysteine, seleno- 
methionine) (Levander, 1977), and selenium supplementa- 
tion as sodium selenate apparently can alleviate the toxicity 
of arsenic as arsenate (Biswas et al., 1999). These actions are 
opposite of those expected if there was a competition for 
factors involved in the metabolism and excretion of methy- 
lated arsenic and selenium. Possible mechanisms for the ap- 
parent dichotomy involve metabolism and distribution an- 
tagonism and effects on oxidative metabolism. Levander 
(1977) suggested that arsenic enhances the biliary excretion 
of selenium via the formation of a detoxification conjugate. 



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MINERAL TOLERANCE OF ANIMALS 



The identification of tlie seleno-bis(S-glutatfiionyl) arsinium 
ion in tfie bile of rabbits injected with selenite followed by 
arsenite (Gailer et al., 2000) supports this suggestion. Also, 
arsenic and selenium can combine directly to form AsjSe, 
which becomes concentrated and precipitated in renal lyso- 
somes (Berry and Galle, 1994). The oxidative metabolism 
suggestion is based on selenium, in the form of seleno- 
cysteine, being a critical component of several enzymes that 
maintain intracellular redox balance, including thioredoxin 
reductase and glutathione peroxidase. Selenium deficiency 
would decrease the antioxidant capacity to antagonize the 
oxidative stress induced by excessive arsenic, and thus exac- 
erbate arsenic toxicity (Kenyon et al., 2001). 

Another established interaction is between arsenic and 
copper. Pharmacologic or toxicological amounts of arsenic 
50 mg/kg diet as sodium arsenate and 100 mg/kg diet as 
sodium arsenite induce copper accumulation in the kidney 
of rats (Mahaffey et al., 1981; Uthus, 2001; Yu and Beynen, 
2001), chicks (100 mg roxarsone/kg diet) (Czarnecki and 
Baker, 1985), and guinea pigs, but not in mice (100 mg/kg 
diet as sodium arsenate) (Hunder et al., 1999). High dietary 
arsenic does not increase liver copper, but may slightly re- 
duce it (Yu and Beyen, 2001). High dietary arsenic decreases 
plasma copper concentrations (Yu and Beynen, 2001; 
Schmolke et al., 1992). High dietary arsenic also exacer- 
bates copper deficiency in rats (Uthus, 2001), and high di- 
etary roxarsone (3-nitro-4-hydroxyphenylarsonic acid) en- 
hances copper toxicity in pigs (Edmonds and Baker, 1986). 
On the other hand, low-dose roxarsone administration ap- 
parently ameliorates copper toxicity (Edmonds and Baker, 
1986). The mechanism through which arsenic affects copper 
distribution in the body has not been established. However, 
Yu and Beynen (2001) suggested that arsenic toxicosis de- 
creases the excretion of copper through the predominant site 
of excretion, the bile, by reducing hepatic copper. Biliary 
copper excretion is detennined by the copper concentration 
in liver. As a result of the change in biliary excretion, copper 
is directed to the renal cortex where it accumulates. 

Mechanisms of Toxicity 

As indicated above, three mechanisms of action through 
which arsenic may be toxic are oxidative stress, altered me- 
thylation, and altered metabolism of other essential miner- 
als. Oxidative stress and altered methylation are the bases 
for arsenic being categorized as a human carcinogen 
(Kitchin, 2001; Thomas et al., 2001; Hughes, 2002). Other 
mechanisms of toxicity are more specific to pentavalent and 
trivalent forms of arsenic. Arsenate apparently can replace 
phosphate in some biochemical reactions because they have 
similar structure and properties (Hughes, 2002). This re- 
placement uncouples oxidative phosphorylation and thus can 
result in the depletion of adenosine triphosphate (ATP). 
However, the toxicity of arsenate may be mainly the result 
of its conversion into arsenite. Trivalent arsenic (e.g., arsen- 



ite) readily binds with thiols and vicinal sulfhydryls that are 
specific functional groups within enzymes, receptors, and 
co-enzymes (Thomas et al., 2001; Hughes, 2002). This bind- 
ing can result in the inhibition of critical biochemical func- 
tions or reactions. It should be noted, however, that the bind- 
ing of arsenite at nonessential sites in proteins may be a 
detoxification mechanism (Aposhian, 1989). 

SOURCES AND BIOAVAILABILITY 

Sea plant and fish products and supplemental minerals 
supply most of the arsenic found in animal feeds. The con- 
centration of arsenic has been found to range between 1 and 
180 mg/kg dry weight for various marine macro algae, <2 
and 170 mg/kg fresh weight in marine fish and bivalves, and 
<0. 1 and 3 mg/kg fresh weight for freshwater fish (Stoeppler, 
2004). The arsenic content of commercial fishmeals used for 
livestock was found to range between 2.9 and 9.1 mg/kg dry 
weight (Lunde, 1968). Most arsenic in fish and algae is in 
the relatively nontoxic organic form (e.g., arsenobetaine, 
arsenosugars). Grains have measurable amounts of arsenic; 
reported mean concentrations (in |Jg/kg fresh weight) include 
oats, 189; barley, 67; and wheat, 45 (Wiersma et al., 1986). 
Grass species contain about 100 |Jg/kg dry weight (Stoeppler, 
2004). Grass from areas close to industrial sites, or grown on 
high-arsenic soils, can be markedly higher (up to 62 mg/kg 
dry weight near a lead smelter) (Woolson, 1983). Some of 
the arsenic in plants growing near mining and smelting op- 
erations comes from aerial deposition, but much comes from 
root uptake (Woolson, 1983). Straw from rice grown in a 
greenhouse in pots flooded with water containing 8 mg As/L 
accumulated about 100 mg As/kg, mostly in the inorganic 
form (Abedin et al., 2002). Interestingly, arsenic increased 
only slightly in the rice grain (from 0.15 mg/kg to 0.42 mg/ 
kg) with the arsenic treatment, and thus did not exceed the 
food hygiene limit of 1 mg/kg. Sheep and cattle do not find 
arsenic distasteful and actually may develop a taste for it 
(Clarke and Clarke, 1975). Ruminants apparently will graze 
selectively on contaminated forage. The arsenic content in 
muscle of terrestrial animals (e.g., cattle, swine, and poultry) 
is generally below 20 |Jg/kg fresh weight (Michels, 1986). 
Drinking water can be a major source of arsenic, especially 
in the inorganic form. Arsenic concentrations in unpolluted 
fresh waters, mainly as arsenate, generally range between 1- 
10 |Jg/L. However, the arsenic content can be much higher in 
waters in some geochemical environments. These include 
aquifers under strongly reducing conditions; aquifers under 
oxidizing, high-pH (>8) conditions; areas of sulfide mineral- 
ization and mining; and geothermal areas (Smedley et al., 
2001). Waters that may be used for drinking purposes have 
been found to be as high as 0.1-5 mg As/L (Smedley et al., 
2001; Mandal and Suzuki, 2002). 

The bioavailability of inorganic arsenic from the gas- 
trointestinal tract correlates well with the solubility of the 
compound ingested (Vahter, 1983; Marafante and Vahter, 



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ARSENIC 



35 



1987). In humans and most laboratory animals, >90 percent 
of inorganic arsenate and arsenite in a water solution is ab- 
sorbed. However, only 20-30 percent of arsenic in arsenic 
trioxide and lead arsenate, which are only slightly soluble in 
water, is absorbed by hamsters, rats, and rabbits. In a dog 
study, the bioavailability, or the amount absorbed, of inor- 
ganic arsenic from bog ore-containing soil was found to be 
only 8.3 percent (Groen et al., 1994). Other studies found 
similar low bioavailability of soil arsenic. Rabbits absorbed 
24 percent and cynomolgus monkeys absorbed 19 percent of 
the arsenic in soils near smelters (Freeman et al., 1993, 
1995). About 60-75 percent of inorganic arsenic ingested 
with food is absorbed by humans (Hopenhayn-Rich et al., 
1993). 

The form of organic arsenic also determines it 
bioavailability. When orally dosed, >90 percent of arseno- 
betaine was recovered in the urine of hamsters; 70-80 per- 
cent of arsenocholine was recovered in the urine of mice, 
rats, and rabbits; and 45 percent of dimethylarsinic acid was 
recovered in the urine of hamsters (Marafante et al., 1984; 
Yamauchi and Yamamura, 1984; Yamauchi et al., 1986). In 
contrast, >90 percent of an oral dose of sodium-/?-N- 
glycolylarsenilate was recovered in the feces of rats or hu- 
mans within 3 days of administration; urinary excretion ac- 
counted for only 4-5 percent of the dose (McChesney et al., 
1962). The bioavailability of organic arsenic as found in fish 
is highly available. Rats absorbed 98-99 percent of the ar- 
senic in flounder (Siewicki and Sydlowski, 1981), and hu- 
mans absorbed 66-86 percent of arsenic in fish cakes and 
flounder (Freeman et al., 1979; Tam et al., 1982). The 
bioavailability of arsenosugars as found in seaweeds has not 
been well established. Shiomi (1994) found that in mice, 
orally administered partially purified arsenosugars from the 
red alga Porphyra yezoensis were excreted mainly through 
the feces; this suggests that the arsenosugars were not very 
bioavailable. In contrast, Francesconi et al. (2002) found that 
80 percent of an orally administered synthetic arsenosugar 
to humans was excreted in the urine within 4 days. Further 
evidence that arsenosugars in seaweed are absorbed and 
metabolized is that sheep living largely on seaweed have 
elevated wool, blood, and urinary arsenic concentrations and 
no arsenosugars in the urine (Feldmann, 2000). 

TOXICOSIS 

Considering its reputation as a poison, it may be surpris- 
ing to some individuals that arsenic has a low order of toxic- 
ity, especially when it is in the pentavalent oxidation state. 
The ratio of the toxic to the apparent nutritional dose of inor- 
ganic arsenic for rats is near 1,250 (Nielsen, 1996). The le- 
thal dose in domestic animals ranges from 1 to 25 mg/kg 
body weight as sodium arsenite, which is 3-fold to 10-fold 
more toxic than arsenic trioxide (Stoeppler, 2004). Some 
forms of organic arsenic, particularly those found in sea- 
food, are virtually nontoxic; for example, a 10 g/kg BW dose 



of arsenobetaine depressed spontaneous motility and respi- 
ration in male mice, but these signs disappeared within one 
hour (Kaise et al., 1985). Arsenocholine is slightly more 
toxic than arsenobetaine; a dose of 5.8 g/kg BW caused death 
in some rats, but a dose of 4.8 g/kg BW did not (Kaise et al., 
1992). 

Acute 

The acute toxicity of arsenic is determined by its chemi- 
cal form and oxidation state. Generally, the acute toxicity of 
trivalent arsenic is greater than pentavalent arsenic (Thomas 
et al., 2001). Most pentavalent organic arsenic species are 
relatively nontoxic. In contrast, some organic forms of triva- 
lent arsenic (e.g., monomethylarsonous acid) are more toxic 
than inorganic arsenite, and the toxicity of some others (e.g., 
dimethylarsinous acid) is similar to inorganic arsenite (Tho- 
mas et al., 2001; Hughes, 2002). As a result, providing one 
LDjg value for arsenic cannot be done. This is demonstrated 
by the reported LDjq for various forms of arsenic in mice (in 
mg As/kg body weight): arsenic trioxide, 26; mono- 
methyarsonic acid, 916; dimethylarsinc acid, 648; tri- 
methylarsine oxide, 5,500; arsenocholine, 6,500; and 
arsenobetaine, >4,260 (Kaise et al., 1985, 1989, 1992). The 
reported LD^q of arsenic trioxide for the rat is 15 mg/kg body 
weight (Harrison et al., 1958). The lethal range of inorganic 
arsenic (i.e., arsenic trioxide) for humans has been estimated 
to be 1-3 mg/kg body weight (Ellenhorn, 1997). The signs 
of acute arsenic toxicosis include intense abdominal pain, 
vomiting, diarrhea, weakness, staggering gait, hypothermia, 
and death (Stoeppler, 2004). 

Fish also have a fairly high tolerance to arsenic in their 
environment. In a study testing seven fish species, the 96-hr 
LCjQ values ranged from 13.3 mg/L for rainbow trout to 41.5 
mg/L for bluegill (National Research Council of Canada, 
1978). The LCjq values for tilapia were determined to be (in 
mg/L pond water) at 24 hr, 69; 48 hr, 51; 72 hr, 38; and 96 
hr, 28 (Liao et al., 2003). 

Chronic 

Chronic oral arsenic toxicosis in domestic animals is sel- 
dom reported. The reason for this may be the fact that ar- 
senic is relatively nontoxic to domestic animals. For ex- 
ample, sheep chronically consuming about 35 mg daily in 
the form of seaweed (mostly dimethylated arsenoribosides) 
exhibited no abnormalities or signs of arsenic toxicosis 
(Hansen et al., 2002). The fatal dose of arsanilic acid for 
horses and cows was reported to be 40 mg/kg BW and 6-12 
mg/kg BW for sheep. Horses and cattle ingested 2.66 to 4 
mg arsanilic acid per kg BW daily for 18 months without 
any discernible injury (Reeves, 1925). The National Toxi- 
cology Program (1989) reported no significant toxic effects 
in mice fed 200 mg roxarsone (57 mg As)/kg diet for two 
years. Table 4-1 summarizes some reported doses that cause 



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MINERAL TOLERANCE OF ANIMALS 



arsenic toxicosis and thie effects of chronic consumption of 
these high amounts of arsenic by various animals. Signs of 
chronic arsenic intoxication usually reported were depressed 
growth, feed efficiency, feed intake, and, for some species, 
convulsions, uncoordinated gait, and decreased hemoglobin. 
Chronic high exposure to arsenic can induce numerous bio- 
chemical changes. For example, in rats and guinea pigs, 
dosing the drinking water with 10 or 25 mg of As^+/L for 16 
weeks resulted in reduced blood 5-amino-levulinic acid 
dehydratase activity, increased urinary 5-aminolevulinic 
acid, and significant changes in whole-brain concentrations 
of neurotransmitters, 5-hydroxytryptamine, dopamine, and 
norepinephrine (Kannan et al., 2001). In humans, chronic 
exposure to arsenic has been associated with various fonns 
of cancer (Wang et al., 2002). Animal models showing that 
arsenic promotes or induces cancer have been difficult to 
establish; special strains of experimental rodents, non-oral 
routes of administration, and stressors such as a carcinogen 
generally have to be employed (Wang et al., 2002). Because 
arsenic carcinogenicity apparently is not an issue for domes- 
tic animals, it will not be reviewed here. 

Prenatal death, malformation, and inhibition of growth 
can result from exposure to arsenite, arsenate, dimethyl- 
arsinate, and monomethylarsonate in hamsters, mice, and 
rats (Hood, 1983). Such effects generally are seen only at 
doses causing maternal toxicity including death. To induce 
malformation in animals, the arsenic typically has to be ad- 
ministered intraperitoneally or intravenously rather than 
orally (Hood, 1983; Thomas et al., 2001; DeSesso, 2001). A 
few studies have shown that extremely high single oral doses 
of inorganic arsenic (40-125 mg/kg BW as sodium arsenate) 
will cause some fetal stunting and malformations (Hood, 
1983). Male mice fed 40 mg As/L of drinking water as so- 
dium arsenite for 35 days exhibited decreased sperm count 
and motility and increased abnormal sperm; no effect was 
seen at 20 mg/L (Pant et al., 2001). Published reproductive 
findings do not appear practically relevant; thus, reproduc- 
tive toxicity of arsenic under natural conditions is unlikely. 

Factors Influencing Toxicity 

In addition to those indicated in the metabolic interac- 
tions section, other factors that may affect arsenic toxicity 
include vitamin Bjj status (Zakharyan and Aposhian, 1999) 
and nutritional factors affecting the 5'-adenosylmethionine- 
mediated transmethylation/transulfuration pathway, includ- 
ing niacin, riboflavin, folate, and iron (Donohue and 
Abernathy, 2001). Age, sex, and pregnancy apparently af- 
fect arsenic toxicity. Vahter et al. (2000) reported findings 
indicating that children are more sensitive to arsenic toxicity 
than adults, and that pregnancy may increase the ability to 
metabolize or detoxify arsenic. Even when not pregnant, 
women may be less susceptible to arsenic toxicity. Dose 
level, ethnicity, and genetic polymorphism also are factors 
influencing arsenic toxicity (Vahter et al., 2000). 



TISSUE LEVELS 

If the ingestion of arsenic is low, no tissue significantly 
accumulates arsenic; most tissues contain less than 50 |-ig/kg 
wet weight. However, high intakes of arsenic can markedly 
increase the arsenic content of tissues; this is indicated by 
the data in Table 4-2. Arsenic is widely distributed in the 
body with the highest amounts in skin, hair, and nails, prob- 
ably the result of arsenite binding to SH groups of proteins 
such as keratin that are relatively plentiful in these tissue. 
Organs highest in arsenic are kidney and liver; these organs 
also accumulate the highest amount of arsenic when exces- 
sive amounts are ingested (see Table 4-2). Although tissue 
arsenic can be increased to relatively high amounts in sea- 
food (e.g., shrimp) and fish, the toxicity of the organic forms 
of arsenic found in the edible parts of these animals is essen- 
tially nontoxic. Even with high intakes of arsenic, the data in 
Table 4-2 suggest that no domestic animal tissue, except 
perhaps liver and kidney, used as a food will contain enough 
arsenic to be of toxicological concern for humans. 

MAXIMUM TOLERABLE LEVELS 

The highest dietary level at which arsenic has no adverse 
effect, and the lowest level that induces toxicosis, varies with 
species. The data in Table 4-1 indicate that, in the inorganic 
form, 50 mg As/kg diet is toxic to rats and 100 mg As/kg diet 
is toxic to chicks; other animals are apparently more tolerant 
to inorganic arsenic. Pigs fed roxarsone may be the best indi- 
cator of a species sensitive to organic arsenic; 187.5 mg/kg 
diet of roxarsone (53.4 mg As/kg) was toxic to pigs (Rice et 
al., 1985; Kennedy et al., 1986). The preceding indicates 
that the maximum tolerable levels set for domestic animals 
in the previous edition of this book (NRC, 1980) of 50 mg/ 
kg diet for inorganic arsenic and 100 mg/kg diet for organic 
arsenic may be too high. Because a 12.5 mg As/kg diet ap- 
parently is not toxic to rats (Hisanaga, 1982), a more appro- 
priate maximum tolerable limit for arsenic would be between 
12.5 and 50 mg/kg diet; the midpoint, or 30 mg/kg diet, may 
be a reasonable maximum tolerable level for domestic ani- 
mals. Based on the data in Table 4-1, fish are less tolerant to 
dietary inorganic arsenic than mammals; diets as low as 10 
mg As/kg induced toxicosis in rainbow trout. Thus, a maxi- 
mum tolerable level for inorganic arsenic of 30 mg/kg diet 
for fish is inappropriate. A maximum tolerable limit for fish 
may be in the range of 5 mg As/kg diet. 

The European Communities (2002) issued a directive that 
set the maximum arsenic contents allowable in products in- 
tended for animal feed with a moisture content of 12 percent 
at much lower levels than 30 mg/kg diet. The scientific basis 
for setting these low values was not given in the directive. 
The level set for animal feeds was 2 mg/kg, with the excep- 
tion of meals made from grass, dried lucerne, dried clover, 
dried sugar beet pulp, and dried molasses sugar beet pulp 
(set at 4 mg/kg), as well as phosphates and feedstuffs ob- 



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37 



tained from the processing of fish or other marine animals 
(set at 10 mg/kg). The maximum for complete feedstuffs for 
fish was set at 4 mg/kg, which is near the maximum toler- 
able limit of 5 mg/kg suggested above. The maximum for 
complementary feedstuffs for fish was set at 4 mg/kg with 
the exception of complementary mineral feedstuffs (set at 12 
mg/kg). 

HUMAN HEALTH 

Generally, in human health, arsenic is considered only as 
a toxicant and it is classified as a carcinogen. Thus, recent 
efforts have been directed towards decreasing the ingestion 
of arsenic, particularly through drinking water. Under the 
authority of the Safe Drinking Water Act, the U.S. Environ- 
mental Protection Agency (EPA) in 200 1 reduced the drink- 
ing water standard from 50 |-ig/L to 10 |ig/L; public water 
systems have until January 2006 to comply (Brooks, 2002). 
There are epidemiological findings showing that some can- 
cer is correlated with markedly decreased serum arsenic con- 
centrations (Mayer et al., 1993). Also, it has been noted that 
the incidence of some forms of cancer were higher when 
drinking water contained very low amounts of arsenic than 
when it contained reasonable amounts of arsenic (Guo et al., 
1995). These findings are consistent with the report that an 
exposure to drinking water containing 20 to 50 |-ig/L appar- 
ently does not affect mortality (Buchet and Lison, 1998). 
Biochemical evidence suggesting that low dietary arsenic 
can increase cancer susceptibility includes the finding that 
low and excessive amounts of arsenic, compared to control 
amounts, significantly decreased global methylation of DNA 
in cultured Caco-2 cells (Davis et al., 2000). DNA hypo- 
methylation is associated with some types of cancer (Dizik 
et al., 199 1 ; Zapisek et al., 1992). This finding, in addition to 
those described in the essentiality and metabolic interactions 
sections above, suggests that an inadequate arsenic status 
may cause hypomethylation because of a depressed methy- 
lation function and excessive arsenic may cause hypo- 
methylation through an increased need for methyl groups for 
its elimination, and thus making less available for DNA me- 
thylation. In both cases, the hypomethylation could result in 
an increased susceptibility to cancer. 

FUTURE RESEARCH NEEDS 

Although the toxicity of arsenic, especially its carcino- 
genic properties, for humans has been extensively studied, 
there are some research needs. Establishing the basis for 
the apparent essential or beneficial action of arsenic, and 
the intake below which this action is compromised, would 
be helpful in setting the lower limits of toxicity standards 
for arsenic. Mechanisms through which the various forms 
of arsenic are absorbed need to established. Speciation of 



arsenic found in animal feedstuffs would help in determin- 
ing the toxicity potential of those containing relatively high 
amounts of arsenic. The toxicity of arsenic to some domes- 
tic animals (e.g., cattle, goats) need to be better defined. 
The basis for the apparent difference in arsenic toxicity for 
humans (cancer, heart disease, diabetes, etc., thought to oc- 
cur at relatively low intakes) and domestic animals (lim- 
ited signs of toxicosis at relatively high intakes) should be 
determined. 

SUMMARY 

Arsenic is widely distributed in the biosphere and exists 
predominantly as an oxyanion in an oxidation state of 3+ or 
5+. In animals, arsenic occurs mainly as inorganic arsenate 
or arsenite, monmethylarsonic acid, and dimethylarsinic 
acid. The major form of arsenic in sea animals is arseno- 
betaine. In seaweeds, arsenic is found in arsenosugars. Ar- 
senic generally is not accepted as an essential element, but 
studies with goats, chicks, hamsters, and rats suggest that it 
may have an essential or a beneficial function in ultra trace 
amounts (micrograms per kg diet). Some organic arsenicals 
(e.g., roxarsone) are still used for antibiotic or anti-coccidial 
purposes in poultry. Arsenic trioxide has been found to be an 
effective treatment for some forms of cancer in humans. 
Soluble inorganic arsenic and organic arsenic as found in sea 
foods are highly absorbed and excreted mainly in the urine. 
Most inorganic arsenic is enzymatically methylated before 
being excreted. Arsensobetaine is not, but arsenosugars are 
transformed before being excreted. Sea plant and fish prod- 
ucts are sources of arsenic for animal feeds; this arsenic is 
relatively nontoxic. Contaminated drinking water and foli- 
age are major sources of inorganic arsenic. Mechanisms 
through which arsenic may be toxic are altered methyl me- 
tabolism, oxidative stress, altered metabolism of other es- 
sential minerals, replacement of phosphate in biochemical 
reactions, and inhibition of critical biochemical functions by 
binding thiols and vicinal sulfhydryls. Arsenic is relatively 
nontoxic to domestic animals. The toxic dietary concentra- 
tions of arsenic are generally between 500 and 1,000 times 
greater than the concentrations normally found in animal 
feeds. Signs of chronic arsenic intoxication include de- 
pressed growth, feed intake, feed efficiency, and, for some 
species, convulsions, uncoordinated gait, and decreased he- 
moglobin. The suggested maximum tolerable level for do- 
mestic animals is 30 mg/kg diet. The suggested maximum 
tolerable level for fish is 5 mg/kg diet. Thus, except for lo- 
calized areas where arsenic is extremely high in drinking 
water, major arsenic contamination by mining and smelting 
industries has occurred, or old arsenic pesticides or ashes of 
CCA lumber are accessible, arsenic toxicosis is not a con- 
cern for domestic animals. 



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Barium 



INTRODUCTION 

Barium (Ba) is a dense alkaline metal in Group IIA of the 
periodic table and is the 16th most abundant element of the 
Earth's crust, constituting about 0.04 percent of it. Barium 
occurs as a divalent cation in combination with other ele- 
ments and is chiefly found in underground ore deposits of 
barite and witherite. Barite is predominantly BaSO^ and 
witherite is predominantly BaCOj. Barite occurs in abun- 
dance in Alaska, Arkansas, California, Georgia, Missouri, 
Nevada, and Tennessee, as well as in Canada and Mexico 
(USGS, 2000). Worldwide, China leads in barite ore mining 
followed by the United States, with production predominat- 
ing in Nevada and Georgia. BaS04 is used as raw material 
for producing BaClj, BaOH, and other compounds. 

The primary use of BaSO^ is by the oil and gas industries 
as drilling muds. Additionally, barium compounds are used 
to make ceramics, brake linings, paints, bricks, tiles, glass, 
rubber, inks, adhesives, additives for fuels and oils, and ro- 
denticides. Barium nitrite and sulfide are used in fireworks 
and depilatories, respectively. Medically, BaS04is used as a 
radiocontrast medium for x-rays of the gastrointestinal tract. 
The natural forms of barium have poor water solubility: 
0.001 g/L for BaS04 and 0.025 g/L for BaCOj. Commer- 
cially produced salts are usually much more soluble in wa- 
ter. For example, BaCU has a solubility of 375 g/L and con- 
sequently exhibits a very different toxicological profile than 
BaS04. 

ESSENTIALITY 

Barium is not considered as an essential nutrient for 
plants or animals. The primary beneficial action attrib- 
uted to barium is reduction of dental carries (Zdanowicz 
et al., 1989). Several thorough reviews are available on 
barium and its toxicological profile in human and envi- 
ronmental health (ATSDR, 1992; NTP, 1994; EPA, 1998; 
IPCS, 2001). 



DIFFICULTIES IN METHODS OF ANALYSIS AND 
EVALUATION 

Total barium concentrations in water, feed, and tissues 
can be determined by atomic absorption spectrophotometry 
(AAS) after preparing samples by digestion with acid (Sharp 
and Knevel, 1971; EPA, 1974). Graphite furnace AAS has 
detection limits below one part per billion, but flame AAS is 
sufficiently sensitive for biological samples that have toxic- 
ity potential. Inductively coupled plasma-atomic emission 
spectrometry is also a sensitive method for measuring barium 
in biological samples; however there is potential for interfer- 
ence from spectral bands from other compounds, such as 
boric acid or sodium borate (IPCS, 2001). Standard tech- 
niques for barium determination give total barium concen- 
tration in the sample. However, measuring soluble barium 
concentrations is more meaningful for toxicological evalua- 
tion because the common but insoluble BaS04 form is rela- 
tively nontoxic compared to soluble forms such as BaClj or 
barium acetate. Unfortunately, standardized techniques for 
extracting soluble compounds have yet to be developed. 

REGULATION AND METABOLISM 

Due to its insolubility, BaS04 is poorly absorbed from 
the gastrointestinal tract. In chickens with free access to feed 
and given a single oral dose of BaS04, 97.8 percent of the 
dose was recovered from the feces within 48 hours (Vohra 
and Kratzer, 1967). Healthy humans absorb a similarly small 
proportion of BaS04 even when a high dose is given (Mauras 
et al., 1983). A very wide range (0.7 to 85 percent) of ab- 
sorption efficiencies of soluble barium compounds have been 
reported in animals dosed orally, depending upon the chemi- 
cal form, species, age, and fasting status of the animal. In 
rats, young animals (22 days old) absorb about 10 times more 
barium chloride than older animals. Consumption of barium 
following fasting increases absorption by about 20 percent 
(ATSDR, 1992; IPCS, 2001). 



46 



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BARIUM 



47 



Barium in blood is quickly transported into cells where it 
blocks potassium channels and in some systems has Ca++ 
agonist properties. When barium is transported into muscle 
cells, it blocks the exit of potassium, causes depolarization, 
and stimulates muscle contraction. Barium can substitute for 
calcium ions in stimulating nitric oxide production in the 
endothelium of blood vessels and changes vascular tone 
(Yamazaki et al., 1995). These effects on calcium and potas- 
sium metabolism appear to mediate the toxic effects of 
barium. 

About 90 percent of total body barium is found in bone 
and teeth. The efficiency of bone uptake of barium is 1.5 to 
5 times higher than uptake of calcium or strontium (IPCS, 
2001). Barium is primarily excreted in the feces after oral, 
inhalation, or parenteral exposure. Bile is not an important 
mechanism of excretion and other routes of loss into the in- 
testines must be important in elimination of systemic barium 
(Edel et al., 1991). In humans, sweat accounts for a greater 
proportion of the daily loss than urine (Schroeder et al., 
1972). The biological half-time of BaClj in beagle dogs was 
estimated to be 12.8 days (Cuddihy and Griffith, 1972). 

SOURCES AND BIOAVAILABILITY 

Barium is not typically supplemented to the diets of ani- 
mals, and intake is due to background levels in feed ingredi- 
ents and water. Some commercially available sources of cal- 
cium phosphate used in animal production may have high 
levels of barium (Fernandes et al., 1999), but levels of this 
mineral are not typically monitored because of its low toxic- 
ity profile. The form of barium in calcium phosphate sources 
has not been determined, but would likely be present as 
BaS04 and BaCOj. 

Barium concentrations in surface waters are extremely 
variable and depend on local geography, depth that the 
sample was taken, and water hardness (NRC, 1977). Con- 
centrations between 43 and 58 mg/L are typical in U.S. sur- 
face waters although levels as high as 1,000 mg/L have been 
reported. Water from deep rock and drift wells may have 
barium levels that exceed 20 mg/L. Seawater contains be- 
tween 0.02 and 25 mg/L depending upon the ocean, latitude, 
and depth. High levels of barium in water are often due to 
the presence of insoluble barium attached to suspended par- 
ticles, which are typically removed during water purifica- 
tion. Domestic drinking water supplies normally contain less 
than 1 mg/L barium. In seawater, the limit of soluble barium 
is about 1 mg/L because above this level BaS04 forms and 
precipitates out of solution (Spangenberg and Cherr, 1996). 

Barium levels in soils range from 50 to 3,000 mg/kg 
(IPCS, 2001). Soils formed from limestone, feldspar, and 
biotite are highest in barium, which is mostly in the barium 
sulfate form. In general, the relationship between soil barium 
level and plant barium level is not very strong, but the corre- 
lation is greatly increased if soil levels are expressed as ex- 
changeable barium (Robinson et al., 1950). An average 



bioconcentration factor (BCF = barium in organism/barium 
in soil) of 0.4 was found for a large variety of plant species 
living at a site with a soil barium concentration of 104 mg/kg 
(Hope et al., 1996). In the same environment, insects and 
rodents had a BCF factor of 0.02. Wheat stalks, wheat grain, 
corn grain, and alfalfa have BCFs of 0.31, 0.01, 0.03, and 
0.93 of the extractable barium, respectively (Robinson et al., 
1950). Leaves from red ash {Fraxinus pennsylvanica), black 
walnut (Juglans nigra), and hickory {Carya sp.) have BCFs 
above 1.0. Brazil nuts from regions with high soil levels of 
barium have the highest plant barium concentrations mea- 
sured at 1,500 to 3,000 mg/kg (Smith, 1971). 

Few studies have examined the bioavailability of barium 
in foodstuffs or water. In one study, rats fed a diet containing 
Brazil nuts supplying 249 mg/kg barium for 29 days had 
bone barium levels that were 78 percent of control rats fed 
an equal level of BaClj (Stoewsand et al., 1988). This sug- 
gests that most of the barium in Brazil nuts is considerably 
more bioavailable than BaS04. Nutritional bioavailability 
tests have not been done for any other food or water source. 

TOXICOSIS 

The toxicity of barium is dependent upon its solubility. 
Barium sulfate is practically insoluble and virtually nontoxic 
so it has been used for decades in human and animal medi- 
cine as a radiocontrast medium. Toxicity of more soluble 
barium salts is due to their calcium agonist properties and 
their ability to block potassium transport. Consequently, 
therapy includes administration of calcium antagonists and 
potassium. Other toxic mechanisms have not been found and 
barium salts are not currently considered genotoxic, carcino- 
genic, or teratogenic, though extensive research has yet to be 
conducted (NTP, 1994; IPCS, 2001). 

Single Dose 

Barium sulfate is routinely dosed at up to 8 g/kg BW to 
adult humans prior to gastrointestinal x-rays and is generally 
considered safe. In humans, ingestion of soluble barium 
compounds (e.g., BaClj) causes vomiting, diarrhea, abdomi- 
nal pain, hypokalemia, cardiac arrhythmias, respiratory 
weakness, renal failure, and skeletal muscle paralysis 
(ATSDR, 1992; IPCS, 2001). In dogs, intravenous infusion 
of BaClj causes salivation, diarrhea, hypertension, ventricu- 
lar tachycardia, skeletal muscle paralysis, and, finally, respi- 
ratory arrest and ventricular fibrillation (Roza and Berman, 
1971). Potassium administration prevents all of these effects 
except hypertension. In rats, a single gavage of 300 mg Ba/ 
kg BW as BaClj causes ocular discharge, fluid in the tra- 
chea, darkened liver, and intestinal inflammation (Borzelleca 
et al., 1988). The LDjq of BaClj in adult rats after a single 
dose was 132 mg Ba/kg BW in one experiment, but about 
twice this level in two other experiments (Table 5-1). 



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MINERAL TOLERANCE OF ANIMALS 



The relative toxicity of different soluble barium com- 
pounds has received little attention. BaCOj is considerably 
less soluble than BaClj, but is only slightly less toxic when 
administered in water to rats (Schwartze, 1920). However, 
toxicity of BaCOj is decreased considerably if it is provided 
in alkaline bicarbonate buffer (McCauley and Washington, 
1983) or in feed (Schwartze, 1920). 



Acute 

In a 10-day acute toxicity experiment using rats, daily 
oral gavage of 209 mg Ba/kg BW as BaCl, in water did not 
cause changes in body weight, tissue weights, histopathol- 
ogy, or clinical chemistry except a decrease in blood urea 
nitrogen (Borzelleca et al., 1988). Barium at 300 mg/kg BW 
resulted in increased mortality in females, but not in males. 
The acute LD^q of BaCOj in the diet of rats is about 10,000 
mg/kg (Schwartze, 1920). 

Chronic 

Rats and mice given water with up to 1,318 mg/L of 
barium as BaCU for 13 weeks were clinically, behaviorally, 
and histopathologically normal, but those fed 2,636 mg/L 
exhibited increased mortality (Dietz et al., 1992; NTP, 1994). 
This indicates that the NOAEL of highly soluble barium 
from BaClj in rodents is 1,318 mg/L, although a reanalysis 
of the organ weight data suggest a NOAEL of 659 mg/L 
(EPA, 1998). Similar results were found in a 2-year study in 
rats and mice (NTP, 1994). 

The primary clinical signs of chronic barium intake at high 
levels are cardiomyopathies and hypertension that mimic the 
effect of digitalis. Rats chronically exposed via the water to 
100 mg/L of barium from BaClj for 1 month or 10 mg/L for 8 
months develop a modest increase in systolic pressure (Perry 
et al., 1989). The lower level did not have functional conse- 
quences on the heart but 100 mg/L resulted in depressed rates 
of cardiac contraction and electrical excitability. The kidney 
is also sensitive to barium and very high levels of BaCL (2,636 
mg Ba/L in water) cause nephrosis in rodents characterized by 
tubular dilation and deposition of refractile crystals in the lu- 
men of the tubules (Dietz et al., 1992). 

Chronic toxicity studies using barium delivered in the diet 
at toxic levels are not available. Rats fed diets containing 
249 mg/kg of barium from BaClj or the same amount from 
Brazil nuts did not cause observable effects (Stoewsand et 
al., 1988), but a toxic level has not been established. 

The toxicity of barium in livestock and poultry has re- 
ceived little attention and the few studies that have been con- 
ducted used insufficient dosage levels to establish a toxicity 
threshold (Table 5-1). Similarly, aquatic systems have had 
little study. The 30-day LCjg values for two species of cray- 
fish {Austropotamobius pallipes pallipes and Orconectes 
limosus) were 46 and 78 mg/L of barium as BaClj, respec- 
tively (Boutet and Chaisemartin, 1973). Highly soluble 



barium acetate interferes with calcification of the shell and 
delayed development of marine mussel larvae (Mytilus 
calif ornianus). Interestingly, this developmental defect was 
observed only at levels between 200 and 800 |Jg/L because 
additions of 1 mg/L and above caused precipitation of 
BaS04, which apparently resulted in the formation of crystal 
seeds that rapidly precipitated virtually all of the barium from 
the seawater (Spangenberg and Cherr, 1996). Exposure of 
unspecified species of marine fish, crustaceans, and mollusks 
to drilling mud containing 7,500 mg/kg of barium as BaSO^ 
did not cause mortality (Daugherty, 1951). 

Factors Influencing Toxicity 

The toxicity of barium is dependent upon its solubility. It 
has been hypothesized that insoluble BaSO^ can be solubi- 
lized by the acid conditions of the stomach, especially if 
given in unbuffered water to fasted animals (McCauley and 
Washington, 1983). The water solubility of BaCOj and its 
toxicity when dosed in buffered water depend on the pH. 

Barium competes with calcium for uptake into bone, but 
it is not clear if high dietary levels of calcium decrease the 
toxicity of barium. Barium blocks potassium transport across 
cell membranes, and potassium administration decreases 
many of the signs of barium toxicosis (Roza and Berman, 
1971). 

TISSUE LEVELS 

Barium is found predominantly in the bone and teeth, 
where it substitutes for calcium. Muscle levels of barium are 
below dietary levels regardless of the level or type of barium 
salt consumed (Table 5-2), and bioaccumulation of barium 
through the food chain is not expected to be a problem. 
Barium levels in meat, milk, and eggs of poultry and live- 
stock exposed to high levels of barium by diet or water have 
not been determined. However levels in soft tissues (e.g., 
muscle, kidney, heart, liver) of rats fed high levels of BaClj 
are low (<1 mg/kg fresh weight) (Perry et al., 1989; Tardiff 
etal., 1980). 

There has been concern that the buildup of barium in the 
aquatic environment from use of oil drilling muds high in 
BaS04 could result in high levels of barium in aquatic or- 
ganisms harvested for human consumption. However, 
barium in clams (Maritrix maritrix) collected from oil fields 
in the Arabian Gulf averaged only 0.99 mg/kg wet weight 
and the concentration was not significantly correlated with 
the level in the sediment (Sadiq et al., 1990). Presumably, 
this is because of the very low solubility of barium in seawa- 
ter due to its high sulfate content. 

MAXIMUM TOLERABLE LEVELS 

The Maximum Tolerable Level (MTL) of barium is de- 
fined as the dietary level that, when fed for a defined period 



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BARIUM 



49 



of time, will not impair accepted indices of animal health or 
performance. The MTL of barium is highly dependent upon 
its chemical form, which is not known for most foodstuffs or 
water supplies. The most common form in the environment, 
BaS04, is very poorly absorbed and very high levels ( 1 g/kg 
BW/d) in the diet or drinking water are tolerated by rodents 
and humans. Soluble forms of barium, such as barium chlo- 
ride, acetate, or nitrate have a much different toxicity pro- 
file. In rats, the level at which BbCLt becomes toxic is simi- 
lar for acute and chronic exposure. The cardiovascular 
system is the most sensitive indicator of barium toxicosis in 
rats, and modest but measurable functional changes occur at 
barium levels of 100 mg/L water as BaCl, (Perry et al., 
1989). However giving 1,250 mg/L for 2 years does not re- 
sult in observable pathology to any organ system (NTP, 
1994). In rats and mice, organ pathology occurs at barium 
levels of 2,500 mg/L. A dietary barium level of 249 mg/kg 
diet from BaClj does not cause an observable effect on rats 
(Stoewsand et al., 1988), but 218 mg/kg decreases the rate of 
weight gain of chicks (Taucins et al., 1969). As BaClj does 
not occur naturally in foods or water, these levels are most 
applicable to barium arising from accidental contamination 
of water or feed with soluble forms of barium. Estimates of 
the bioavailability of intrinsic barium in feedstuffs and water 
are needed in order to determine the MTL arising from nor- 
mal dietary sources. Based on only one study in rats, a di- 
etary barium level of 249 mg/kg diet from Brazil nuts does 
not cause observable toxic effects (Stoewsand et al., 1988). 
The NOAEL for barium salts or natural forms of barium is 
not known for poultry or livestock but, based on rodent stud- 
ies, it is unlikely that barium naturally found in feedstuffs 
reaches sufficiently high levels to be toxic. Based primarily 
on rodent studies, the MTL for poultry, swine, and horses is 
set at 100 mg/kg diet. 

HUMAN HEALTH 

The relationship between dietary barium levels and the 
levels in edible tissues of animals is not known. However, 
the levels in soft tissues (e.g., muscle, kidney, heart, liver) of 
rats fed high levels of BaClj, which is a highly bioavailable 
form of barium, are low (<1 mg/kg fresh weight) (Tardiff et 
al., 1980; Perry et al., 1989). This level would not be ex- 
pected to be of concern for human health (ATSDR, 1992; 
NTP, 1994; EPA, 1998; IPCS, 2001). However, barium ac- 
cumulates in bone, and bones from animals fed at their MTL 
could have excessive levels of barium. 

FUTURE RESEARCH NEEDS 

Future research on the toxicity of barium should focus on 
two areas. First, the bioavailability of barium that naturally 
contaminates water and feedstuffs should be determined be- 
cause it is not known if the bioavailability in these sources is 
similar to that of insoluble salts (e.g., BaS04), soluble salts 



(e.g., BaClj), or is intermediate to these salts. Second, the 
NOAEL of barium to poultry, livestock, companion animals, 
and aquatic species needs to be determined. At this time rec- 
ommendations must be based on toxicity experiments done 
in rats and mice that use salts not normally found in nature. 

SUMMARY 

There are no known essential biochemical functions of 
barium, and it is not usually considered as an essential nutri- 
ent. The natural forms of barium have poor water solubility 
and have very low toxicity. Commercially produced barium 
salts, such as BaClj, are usually much more soluble in water 
and are considerably more bioavailable and toxic. Once ab- 
sorbed, barium is deposited predominantly in bone and teeth. 
Toxic properties of barium are attributed to blocking potas- 
sium channels and Ca++ agonist properties. Barium is not 
typically supplemented to the diets of animals. Dietary lev- 
els result from background levels in feed ingredients and by 
accidental contamination. 



REFERENCES 

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50 



MINERAL TOLERANCE OF ANIMALS 



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259-262. 
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Taucins, E., A. Svilane, A. Valdmains, A. Buike, R. Zarina, and E. 

Fedorova. 1969. Barium, strontium, and copper salts in chick nutrition. 

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commodity. Accessed on November 20, 2003. 
Vohra, P., and F. H. Kratzer. 1967. Absorption of barium sulfate and chro- 
mic oxide from the chicken gastrointestinal tract. Poult. Sci. 46:1603— 

1604. 
Yamazaki, J., F. Ohara, Y. Harada, and T. Nagao. 1995. Barium and stron- 
tium can substitute for calcium in stimulating nitric oxide production 

in the endothelium of canine coronary arteries. Jpn. J. Pharmacol. 68: 

25-32. 
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23:65-69. 



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57 



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53 



TABLE 5-2 Barium Concentrations in Fluids and Tissues of Animals (mg/kg or mg/L) 



Animal 



Quantity Source 



Duration 



Route 



Muscle Kidney 



Bone 



Reference 



Rat 


Omg/L 
10 mg/L 
50 mg/L 
250 mg/L 


BaClj 


13 wk 


Drinking 
water 


0.19° — 

0.48 

0.58 

0.44 


9. 1° 
14.5 
50.5 
220 


Tardiff et al., 


1980 



Sheep (wethers) 7 mg/kg 



Pasture 



Weaning to 
30 months 



Feed 



0.15" 



60* 



Healy andLudwig, 1968 



35 mg/kg Pasture 



0.61 



250 



"Data are on a fresh tissue basis. 
*Data are on a dry tissue basis. 



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Bismuth 



INTRODUCTION 

Bismuth (Bi) is a pinkishi-white, lustrous, soft mineral and 
is widely distributed in small quantities throughout the 
world. Although it has the crystal structure of a semi-metal, 
bismuth is often considered a metal. Of all the metals, bis- 
muth has the widest range between melting and boiling 
points, the lowest thermal and heat conductivity, the highest 
diamagnetic index, and the lowest absorption for neutrons. 
Its crystal structure along with several of its other physical 
properties makes it a substitute for lead in some applica- 
tions. Bismuth forms compounds in oxidation states +3 and 
+5, and its chemistry is diverse and poorly understood 
(Briand and Burford, 1999). Bismuth nitrate is the initial 
material used for the production of many bismuth com- 
pounds used in industry and medicine. 

World reserves of bismuth are usually associated with 
lead deposits, except in Asia, where economically recover- 
able bismuth is found with tungsten ores and some copper 
ores, and in Australia, where bismuth is found with copper- 
gold ores. Currently, bismuth is not refined in the United 
States. Worldwide production is dominated by China, 
Mexico, Peru, and Canada (USGS, 2003). 

In 2002 about 42 percent of the bismuth used in the United 
States was in fusible alloys, solders, and cartridges; 37 per- 
cent in pharmaceuticals and chemicals; 19 percent in metal- 
lurgical additives; and 2 percent in other uses (USGS, 2003). 
Bismuth is used in several applications designed to provide 
nontoxic substitutes for lead, including shot for waterfowl 
hunting, fishing weights, plumbing fixtures, solders, and lu- 
bricating greases. Other applications of bismuth chemicals 
and compounds include uses in superconductors and pearl- 
colored pigments for cosmetics and paints. Pharmaceuticals 
based on trivalent bismuth have been used for more than a 
century and include a variety of compounds to treat gas- 
trointestinal ailments including diarrhea, upset stomach (bis- 
muth subsalicylate, bismuth subnitrate, and bismuth subcar- 
bonate), and ulcers and gastritis caused by Helicobacter 
pylori (bismuth subcitrate). 



ESSENTIALITY 

Bismuth is not considered an essential nutrient for plants 
or animals and has no known essential biochemical func- 
tions in normal metabolism. 

DIFFICULTIES IN METHODS OF ANALYSIS AND 
EVALUATION 

Bismuth can be measured by atomic absorption spectro- 
photometry (AAS). Graphite furnace AAS has greater sensi- 
tivity than flame AAS. The primary difficulty in analysis is 
volatilization, but this can be prevented by use of a platinum 
matrix modifier (Slikkerveer et al., 1993). Sample prepara- 
tion by acid digestion is necessary due to evaporation of bis- 
muth during ashing. Other methods of analysis include 
particle-induced x-ray emission (Canena et al., 1998), in- 
ductively coupled plasma-mass spectrometry, and atomic 
fluorescence spectrophotometry (Cava-Montesinos et al., 
2003). These other methods are rarely used because expen- 
sive equipment is needed. 

REGULATION AND METABOLISM 

Bismuth compounds are very poorly absorbed in the in- 
testinal tract. In rats and humans, greater than 99 percent of 
an oral dose of a variety of bismuth compounds is lost in the 
feces without absorption (Dresow et al., 1992; Slikkerveer et 
al., 1995). This poor absorption is probably due to poor solu- 
bility in both gastric juice (pH 1.2) and duodenal juice (pH 
6.8). Bismuth citrate is more soluble at neutral pH (35 pmol/ 
L) than bismuth salicylate, gallate, aluminate, chloride, or 
nitrate (<2 |jmol/L) and is slightly better absorbed. In dogs, 
absorption in the small intestine appears to be greater than 
absorption from the stomach (Hespe et al., 1993). 

Once absorbed, bismuth binds to transferrin at specific 
iron binding sites (70 percent) and to albumin (30 percent). 
Presumably, these transport proteins deliver bismuth to tis- 
sues (Sun and Szeto Ka, 2003). The tissue distribution of 



54 



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BISMUTH 



55 



absorbed bismuth is kidney»bone>red blood cells»liver, 
heart, spleen, muscle » serum, and fat. Because of this dis- 
tribution, bismuth concentrations in whole blood are a much 
better indicator of bismuth exposure than levels in serum. 
Bismuth is excreted relatively quickly by the kidney and to a 
lesser extent in bile (Dresow et al., 1991). 

SOURCES AND BIOAVAILABILITY 

Bismuth is a rare element, occurring at about 200 to 350 
|jg/kg in soils and 20 |Jg/L in fresh water and below 1 |Jg/L in 
seawater (Lee, 1982). Bismuth levels in plants grown on 
uncontaminated land are usually less than 60 |Jg/kg dry 
weight, and levels in plants grown on contaminated soils do 
not usually exceed 200 |Jg/kg dry weight (Bowen, 1979). 
Levels in leaves are higher than in fruits or seeds (Jung et al., 
2002). 

Bismuth is not normally added to the diet of animals and 
is not typically a quantitatively important contaminant of 
feedstuffs or additives. Consequently, the primary exposure 
of animals is use of bismuth-based medications or accidental 
contamination. A variety of over-the-counter and prescribed 
preparations of bismuth compounds (e.g., Pepto Bismol®) 
are used to treat animals for intestinal disorders ranging from 
ulcers, gastritis, irritable colon, and constipation, to diarrhea 
(Lambert, 1991; Roussel and Brumbaugh, 1991). They are 
also used to improve the consistency and odor of stools. 

TOXICOSIS 

A characteristic sign that high levels of trivalent bismuth 
compounds have been consumed is dark- or black-colored 
feces, which is due to bismuth sulfide produced in the large 
intestine. Toxic levels of bismuth may affect several target 
organs depending upon dose, duration, and type of com- 
pound (Table 6-1). In humans, encephalopathy is the most 
life-threatening consequence of long-term exposure to bis- 
muth subnitrate or subgallate, whereas bismuth subcitrate 
results in nephrotoxicity. The nephrotoxicity is character- 
ized by necrotic death of proximal tubular epithelial cells, 
apparently because bismuth destabilizes the cell membrane 
(Leussink et al., 2002). Bismuth compounds are not thought 
to be carcinogens (Preussmann and Ivankovic, 1975). 

Bismuth toxicities due to high levels in drinking water 
have not been reported. Most bismuth compounds are not 
sufficiently soluble in water to result in toxic levels. Suspen- 
sions or colloids of bismuth compounds administered as 
pharmaceuticals can be toxic if consumed at relatively high 
levels. 



Single Dose 

Clinical signs of acute bismuth toxicosis in rats consist of 
erect hairs and hunched posture within 6 hours of adminis- 
tration of 627 mg/kg BW of bismuth as bismuth subcitrate; 



at 48 hours rats have swollen ceca filled with gas and black 
fluid (Leussink et al., 2001). Rats gavaged with 314 mg/kg 
as bismuth subcitrate appeared visually normal but had im- 
paired kidney function, which returned to normal within 48 
hours. Absorption and toxicity of bismuth is enhanced when 
it is complexed with cysteine. Such complexes have an LD^q 
of 156 mg Bi/kg BW in rats (Chaleil et al., 1981). 

Shot designed for waterfowl hunting given as a single 
oral dose of 460 mg of bismuth metal to adult mallards did 
not adversely affect food intake, body weight gain, hematol- 
ogy, clinical chemistry, and histopathology (Ringelman et 
al., 1993). Furthermore, bismuth levels in tissues were low 
and not different from control birds. 

Chronic 

In humans the primary behavioral symptoms of chronic 
consumption of bismuth subgallate or subnitrate is apathy, 
mild ataxia, and headaches. More severe cases present with 
dysarthria, confusion, hallucinations, epileptic seizures, and 
occasionally death (Tillman et al., 1996). In the majority of 
cases of bismuth toxicosis in humans, symptoms occurred 
after consumption of doses that often exceeded 10 g of bis- 
muth per day for 2 to 20 years. The pathogenesis of bismuth- 
induced neurotoxicity is unknown, although it has been sug- 
gested that the conversion of the original compounds into 
more soluble or more toxic forms by intestinal bacteria is 
involved (Bader, 1987). This is supported by the observation 
that patients taking bismuth because of intestinal bacterial 
overgrowth were most likely to suffer toxicosis. Bismuth 
subcitrate and subsalicylate do not usually result in severe 
neurological symptoms, but cause nephrotoxicity (Gorbach, 
1990). 

Pigs fed 333 mg/kg BW daily of bismuth from bismuth 
subnitrate, and chickens fed diets containing 1 g/kg bismuth 
from bismuth trioxide, did not display signs of toxicosis af- 
ter exposure for 8 or 9 weeks, respectively (Hermayer et al., 
1977; PoUet et al., 1979). Bismuth subcitrate is the most 
bioavailable of the common forms of bismuth and appears to 
be the most toxic. Rats fed 314 mg/kg BW daily of bismuth 
as bismuth subcitrate displayed functional and histopatho- 
logical signs of toxicosis (Leussink et al., 2001). 

Factors Influencing Toxicity 

Absorption of bismuth in rats is increased by compounds 
containing sulfhydryl groups, such as cysteine, and by 
complexing agents, such as EDTA or citrate (Allain et al., 
1991; Chaleil et al., 1981). Cysteine increases the acute tox- 
icity of bismuth (Chaleil et al., 1981; Krari et al., 1995). 

Selenium affects the metabolic fate of bismuth 
(Szymanska et al., 1978), and bismuth affects selenium me- 
tabolism (Gregus et al., 1998), but the impact on toxicity is 
not clear. In rats, bismuth diminishes both the biliary excre- 
tion of selenium and its accumulation in erythrocytes. Bis- 



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56 



MINERAL TOLERANCE OF ANIMALS 



muth also induces the expression of metallotliionein in tlie 
liver and especially the kidney (Szymanska et al., 1993). 
Bismuth also can displace zinc and cadmium from existing 
metallothionein (Sun et al., 1999) and augments the excre- 
tion of zinc and copper (Szymanska et al., 1993). However, 
the effect of bismuth on the toxicity or the requirement of 
these metals is not known. 

TISSUE LEVELS 

Normal bismuth levels in tissues are low, typically below 
0.2 mg/kg (AUain et al., 1991). Although the relationship 
between bismuth intake and bismuth accumulation in tissues 
of production animals is not known, this metal is not 
bioaccumulated in rats or dogs (Table 6-2). Even at high 
daily doses of bismuth, well above therapeutic levels, muscle 
levels are relatively low (<1 mg/kg). The kidney is the pri- 
mary organ that accumulates bismuth. Dogs dosed daily with 
108 mg/kg BW daily of bismuth ammonium citrate, which is 
a relatively soluble form, accumulated 58 mg/kg bismuth 
(wet weight) in their kidneys. Such levels in animal products 
would not be expected to be of concern for human health. 

MAXIMUM TOLERABLE LEVELS 

The MTL of bismuth is defined as the dietary level that, 
when fed for a defined period of time, will not impair ac- 
cepted indices of animal health or performance. 

Acute 

Dogs can tolerate a daily dose of 108 mg/kg BW daily of 
bismuth from bismuth subcarbonate or bismuth ammonium 
citrate for 2 weeks without clinical signs of toxicosis (Hall 
and Farber, 1972). Similarly, rats can tolerate 157 mg/kg 
BW daily of bismuth from bismuth subcitrate for 2 weeks 
(Leussink et al., 2001). 

Chronic 

Pigs tolerate 333 mg/kg BW daily of bismuth from bis- 
muth subnitrate (Pollet et al., 1979), and chickens tolerate 
1,000 mg/kg diet of bismuth from bismuth trioxide 
(Hermayer et al., 1977). 

Via Water 

Most bismuth compounds are not sufficiently soluble in 
water to cause toxicity. 

FUTURE RESEARCH NEEDS 

Little is known about the relationship between bismuth 
intake and the levels in edible products, such as meat, milk, 
and eggs. Very high levels of bismuth are sometimes admin- 



istered to farm animals, and research is needed to determine 
if food residue problems could occur. 

SUMMARY 

Bismuth is a rare element that normally occurs at low lev- 
els in soil and water and is not bioaccumulated by plants or 
animals. It is not normally added to the feed of animals and 
occurs at very low levels in feedstuffs and mineral supple- 
ments. Bismuth metal and its compounds are poorly absorbed 
by the gastrointestinal tract and are relatively nontoxic. Toxi- 
cosis is likely only if pharmaceutical preparations containing 
bismuth are chronically administered at high doses. 



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Bader, J. P. 1987. The safety profile of De-Nol. Digestion 37 (Suppl. 2): 
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Bowen, H. J. M. 1979. Environmental Geochemistry of the Elements. New 
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BISMUTH 



57 



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bismuth in tissue by electrothermal atomic absorption spectrometry. 
Clin. Chem. 39:800-803. 

Slikkerveer, A., R. B. Helmich, G. B. van Der Voet, and F. A. de Wolff. 
1995. Absorption of bismuth from several bismuth compounds during 
in vivo perfusion of rat small intestine. J. Pharm. Sci. 84:512-515. 

Sun, H., and Y. Szeto Ka. 2003. Binding of bismuth to serum proteins: 
implication for targets of Bi(lll) in blood plasma. J. Inorg. Biochem. 
94:114-120. 

Sun, H., H. Li, 1. Harvey, and P. J. Sadler. 1999. Interactions of bismuth 
complexes with metallothionein(ll). J. Biol. Chem. 274:29094-29101. 

Szymanska, J. A., J. Chmielnicka, A. Kaluzynski, and W. Papierz. 1993. 
Influence of bismuth on the metabolism of endogenous metals in rats. 
Biomed. Environ. Sci. 6:134-144. 

Szymanska, J. A., M. Zychowicz, A. J. Zelazowski, and J. K. Piotrowski. 
1978. Effect of selenium on the organ distribution and binding of bis- 
muth in rat tissues. Arch. Toxicol. 40:131-141. 

Tillman, L. A., F. M. Drake, J. S. Dixon, and J. R. Wood. 1996. Review 
article: safety of bismuth in the treatment of gastrointestinal diseases. 
Aliment Pharmacol. Ther. 10:459-^67. 

USGS (U.S. Geological Survey). 2003. Mineral Commodity Summaries: 
Bismuth. Available at http:/minerals. usgs.gov/minerals/pubs/ 
commodity /bismuth/. Accessed November 25, 2003. 

Zommer-Urbanska, S., E. Bulska, R. Pawlaczyk, and M. Kuklinski. 1994. De- 
termination of bismuth in rabbit blood serum and tissues after administra- 
tion of pharmaceuticals containing BijO,. Acta. Pol. Pharm. 51:7-10. 



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59 



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Boron 



INTRODUCTION 

Elemental boron (B) is a relatively inert metalloid ttiat 
exists as either black monoclinic crystals or yellow-brown 
amorphous powder when impure at room temperature. 
However, boron as an element does not occur in nature; it is 
always found bound to oxygen or in the borate form. The 
most common commercial compounds of boron are anhy- 
drous, pentahydrate and decahydrate (tincal) forms of 
disodium tetraborate (borax, Na2B407), colemanite 
(2CaO-3B203-5H20), ulexite (Na20-2CaO-5B203l6H20), 
boric acid (H3BO3), and monohydrate and tetrahydrate 
forms of sodium perborate (NaB03) (Woods, 1994). The 
borate industry began in 1865 with the mining of borate 
pandermite (priceite, 4CaO-5B,03-7H20) in Turkey. 
Shortly thereafter, several borate deposits were found in 
California and Nevada, including ulexite and colemanite in 
Death Valley. Subsequently, tincal, colemanite and kernite 
(Na,0-2B203-4H20) were found and mined in the Mojave 
Desert (Woods, 1994). In addition to Turkey and the United 
States, other countries producing borates are Peru, Chile, 
Russia, and China. Sodium perborates are hydrolytically 
unstable compounds containing boron-oxygen-oxygen 
bonds; they are used as bleaches in detergents. The end uses 
of boric acid and borates are diverse and include glass, 
enamel, and synthetic gems manufacturing; wood and 
leather preservatives; flame retardants; cosmetics; medical 
products; detergents; insecticides; fertilizers; and neutron 
absorbers for the nuclear industry. Boron halides and hy- 
drides are used as catalysts and in jet and rocket fuels. El- 
emental boron and its carbides and nitrides are used in high- 
temperature abrasives and in steelmaking (Larsen, 1988). 

Boric acid is thought to be the most prevalent form of 
boron in animals and humans. Boric acid is colorless and 
odorless with either a white granular powder or transparent 
crystalline form, which are readily soluble in water. It is a 



weak Lewis acid, accepting OH to form the tetrahedral, 
tetrahydroxy borate anion at a pK^^ of 9.2 (Coughlin, 1996). 

ESSENTIALITY 

In 1923, signs of boron deficiency for several leguminous 
plants were reported (Warington, 1923). Shortly thereafter, 
Sommer and Lipman (1926) reported that boron was essential 
for the completion of the life cycle of a number of plants. This 
evidence was enough for wide acceptance of boron essentiality 
for plants. More than 70 years elapsed before a biochemical 
function for boron in plants was described. Boron cross-links 
two dimers of rhamnogalacturonan-II, a small structurally com- 
plex pectic polysaccharide found in primary cell walls of plants 
(Matoh and Kobayashi, 2002). This function is not adequate to 
explain all boron deficiency signs in plants, which most often 
occur in dicotyledons raised on light-colored sands and silt 
loams in humid regions of the world including the eastern 
United States (Sprague, 1972). Boron stimulates the growth of 
yeast (Saccharomyces cerevisiae) (Bennett et al., 1999) and is 
an essential component of the sensor protein of autoinducer-2, 
an extracellular signaling molecule used for cell-to-cell com- 
munication by some bacteria (Chen et al., 2002). Boron has 
been found to be essential for frogs (Fort et al., 1999a,b) and 
zebrafish (Eckhert and Rowe, 1999) to complete their life cycle. 
Substantial evidence from experiments comparing very low in- 
takes (<100 Mg/kg diet for animals, 0.25 mg/d for humans) with 
physiological intakes (1-3 mg/kg diet) of boron suggests that 
boron is needed for optimal bone health, brain function, and 
immune function in higher animals and humans (Nielsen, 1996, 
2002a). However, although the evidence is similar, boron is not 
consistently accepted as an essential nutrient for higher animals 
as it is for plants. Apparently, the lack of a clearly defined spe- 
cific biochemical function is a major obstacle to wide accep- 
tance of boron essentiality by animal and human nutritionists. 



60 



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BORON 



61 



DIFFICULTIES IN METHODS OF ANALYSIS AND 
EVALUATION 

Less modern methods involving colorimetric, gravimet- 
ric, and titrametric tecliniques can adequately determine bo- 
ron in materials that contain high amounts such as fertilizer, 
soils, and some botanical materials. The spectrophotometric 
method whereby the colored complex of Azomethine-H and 
boron is measured is an international standard method and 
still used (Lopez et al., 1993). Accurate analysis of boron in 
amounts found in most biological materials requires more 
modern techniques and instrumentation, which indicate that 
boron concentrations reported in the older biological and 
food composition literature often may be too high. Analyti- 
cal techniques that utilize inductively-coupled plasma, such 
as inductively-coupled argon plasma spectrometry (ICAP), 
inductively-coupled plasma mass spectrometry (ICP-MS, 
probably the most sensitive technique), and inductively- 
coupled plasma emission spectroscopy (ICP-ES) have be- 
come the instruments of choice for boron analysis. How- 
ever, other methods including neutron-activation-mass 
spectrometry and prompt gamma-activation analysis have 
been used to accurately measure boron in biological materi- 
als. Most of the difficulties encountered in boron analysis 
occur during sample preparation (Downing et al., 1998). 
Because boron is so prevalent in the environment, contami- 
nation is a major problem in analysis of boron in low 
amounts. Sources of this contamination have been listed 
(Downing et al., 1998). Among those listed are sample con- 
tact with borosilicate glass and ashing procedures using the 
same digestion vessels for both high and low boron-contain- 
ing samples, which can result in cross contamination because 
of the difficulty in removing all boron that has bound to or 
migrated into the vessel surface. Another significant con- 
cern in boron analysis is its volatility and mobility. Because 
some boron in biological materials is water soluble, it is 
mobile and could be lost if a sample is slowly frozen for 
storage or freeze-drying; quick freezing in liquid nitrogen 
will reduce the redistribution or loss of boron. Many boron 
compounds volatilize at temperatures far below those re- 
quired for dry ashing (Hunt and Shuler, 1989). Ashing a neu- 
tral or acidic sample requires boron to be quickly converted 
into a nonvolatile compound. This can be accomplished by 
ashing at low temperatures or by making the sample basic 
(Downing et al., 1998). Because of the contamination and 
loss concerns, the validity of any boron analysis can only be 
assured by the use of quality control procedures such as the 
concomitant analysis of standard reference materials. 

REGULATION AND METABOLISM 
Absorption and Metabolism 

About 85-90 percent of ingested borax, boric acid, and, 
apparently, dietary boron is rapidly absorbed and excreted 



mostly in the urine shortly after ingestion (Jansen et al., 1984; 
Hunt et al., 1997; Nielsen and Penland, 1999; Sutherland et 
al., 1999). Because there is no usable radioisotope of boron, 
the study of its metabolism is difficult. It is likely, however, 
that most ingested boron is converted into boric acid, the 
dominant species of boron compounds hydrolyzed at the pH 
of the gastrointestinal tract, and then absorbed and excreted 
mainly in that form. Absorption of boron through the intact 
skin is negligible, and, thus, is not a toxicological concern 
(Wester et al., 1998). However, toxic amounts can be ab- 
sorbed through damaged skin (Nielsen, 1970; Draize and 
Kelley, 1959). Several cases of boron toxicosis, some result- 
ing in death, have been reported in humans who had open 
skin lesions treated with medications containing high 
amounts of boric acid (Pfeiffer et al., 1945); such medica- 
tions are no longer in use. The boron hydrides (boranes), 
which are highly toxic, can be absorbed by the lungs (NRC, 
1980). Inhaled borates also can be absorbed by the lung but 
are relatively nontoxic. 

IVIetabolic Interactions 

The most relevant property of boric acid, the prevalent 
form of boron in vivo, is its ability to form complexes with 
substances containing c«-hydroxyl groups (Hunt, 2002). 
During transport in the body, boric acid most likely is weakly 
attached to organic molecules containing cw-hydroxyl 
groups. The interaction with organic compounds containing 
hydroxyl groups most likely also affects the response to de- 
ficient and toxic intakes of boron. For example, boron toxic- 
ity is alleviated by riboflavin (Roe et al., 1972). It is also 
hypothesized that boron can enhance the function of other 
nutrients and hormones acting at the cell membrane level 
(Nielsen, 2002a). Thus, changes in the status of some nutri- 
ents and hormones that act at the cell membrane level appar- 
ently can affect the response to various intakes of boron. 
Among the nutrients that affect the response to dietary boron 
are vitamin D, magnesium, molybdenum, calcium, selenium, 
protein, and omega-3 fatty acids (Hoffman et al., 199 1 ; Hunt, 
1994; Nielsen, 2002b). Estrogen status apparently affects the 
response to boron deficiency: postmenopausal women on 
hormone replacement therapy showed changes in plasma 
copper and serum I7p-estradiol concentrations when de- 
prived of boron, but those not on hormone replacement 
therapy did not (Nielsen et al., 1992). High intakes of boron 
have been investigated as antidotes to fluoride toxicity in 
rabbits (Baer et al., 1977; Elsair et al., 1980), pigs (Seffner 
and Teubener, 1983), and sheep (Wheeler et al., 1988). 



IVIechanisms of Toxicity 

The mechanism of toxicity for boron has not been firmly 
established. Ku and Chapin (1994) studied the possible 
mechanisms through which boric acid causes toxicologic 
manifestations in testicular function of the rat. Their find- 



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62 



MINERAL TOLERANCE OF ANIMALS 



ings indicated ttiat boron toxicosis did not occur througli in- 
ducing a riboflavin deficiency or impairing pliospfiorus, cal- 
cium, and zinc metabolism or utilization. They did obtain 
findings suggesting that boron toxicosis may be the result of 
an impairment of energy metabolism through the inhibition 
of some enzymes that produce utilizable energy substrates. 
Also, they suggested that toxic amounts of boric acid af- 
fected the DNA synthetic activity of both mitotic (sper- 
matogonial) and meiotic (post-spermatogonial) germ cells 
of the rat. 

SOURCES AND BIOAVAILABILITY 

Sources of exposure to boron have been reviewed (Howe, 
1998; IPCS, 1998). Boron enters the environment mainly 
through the weathering of rocks; volatilization from seawa- 
ter; burning of oil, coal, and wood; industrial and household 
use of boron-containing products (especially soaps and de- 
tergents); fertilizers; and sewage and sludge disposal. Bo- 
ron-containing vitreous materials such as fiberglass, boro- 
silicate glass, enamels, frits, and glazes are not significant 
sources of exposure because the boron is tightly bound in the 
glassy structure. Significant amounts of boron are not present 
in the atmosphere (average of 20 ng/m-' over continents). 
The Earth's crust contains about 10 mg B/kg, ranging from 5 
mg/kg in basalts to 100 mg/kg in shales. Boron occurs in 
soils at concentrations ranging from 10 to 300 mg/kg (aver- 
age 30 mg/kg). Concentrations in surface waters range 
widely from 0.001 to 150 mg/L (Coughhn, 1998). The aver- 
age boron concentration in the oceans is 4.6 mg/L. Most 
freshwater concentrations of boron (mostly in the boric acid 
form) are below 0.4 mg/L and not lowered by treatment for 
drinking water. Boron accumulates in aquatic and terrestrial 
plants but does not magnify through the food chain. On a dry 
weight basis, boron concentrations range between 26 and 
382 mg/kg in submerged aquatic freshwater plants, 11 and 
57 mg/kg in freshwater emergent vegetation, and 2 and 95 
mg/kg in terrestrial plants (IPCS, 1998). Among plants, 
monocotyledons generally contain less boron than dicotyle- 
dons. Boron deficiency occurs in a wide variety of plants 
when their dry matter concentrations are less than 10 mg/kg 
(Bell, 1997). A rough method of diagnosing boron toxicity 
in plants is determining boron in plant components; dry tis- 
sue boron concentrations exceeding 250 mg/kg often indi- 
cate boron toxicity (Nable et al., 1997). On a wet weight 
basis, boron concentrations in marine invertebrates and fish 
are similar to their environment, or between 0.5 and 4 mg/kg 
(IPCS, 1998). The preceding values indicate that diet and 
sometimes drinking water are the major sources of exposure 
to boron. For example, the daily boron intakes for humans 
were estimated to be 0.44 |jg from ambient air, 0.5 \ig from 
soil, 0.1 mg from consumer products, 0.2-0.6 mg from drink- 
ing water, and 1.2 mg from the diet (IPCS, 1998). 

Boron supplements for the alleviation of arthritis in hu- 
mans appeared in 1976 (Newnham, 1994). Shortly after bo- 



ron was indicated as being beneficial to postmenopausal 
women (Nielsen et al., 1987), the presence of boron in over- 
the-counter supplements increased markedly. Most supple- 
ments provide boron in amounts that would not be toxic for 
humans, usually 3 mg or less daily. Although boron supple- 
ments for animals are not approved in the United States, one 
report (Newnham, 2002) indicates that they have been used 
in other parts of the world. Boron supplements reportedly 
are effective in the treatment of arthritic dogs, horses, and 
cattle; the dose administered was 3 mg B/day per 25 kg BW 
for 2 to 4 weeks (Newnham, 2002). It would not be surpris- 
ing to find that boron supplementation to poultry may be 
considered because of reports showing that this improved 
bone strength and growth (Rossi et al., 1993; Wilson and 
Ruszler, 1997; Kurtoglu et al., 2001). Boron supplementa- 
tion of pigs may be considered because it may increase body 
weight, bone strength, and immune function (Armstrong et 
al., 2002; Armstrong and Spears, 2003). 

As indicated in the absorption discussion above, boron in 
foods, feeds, water, and supplements are generally highly 
available. 

TOXICOSIS 
Single Dose 

The oral LD^q for boron (provided as boric acid and bo- 
rax) varies among animal species. Values in mg B/kg BW 
are in the range of 400-700 for mice and rats (Pfeiffer et al., 
1945; Weir and Fisher, 1972); 210 for guinea pigs 
(Verbitskaya, 1975); and 250-350 for dogs, rabbits, and cats 
(Pfeiffer etal., 1945; Verbitskaya, 1975). The signs of toxic- 
ity in rats, mice, and guinea pigs upon administration of large 
single doses of borax and boric acid used to determine the 
LDjQ were depression, ataxia, occasional convulsions, de- 
creased body temperature, and violet-red color of skin and 
all mucous membranes (Pfeiffer et al., 1945; Weir and 
Fisher, 1972). Goats dosed with 2 g soluble borate fertilizer 
had increased cerebrospinal fluid monoamine metabolites 
and exhibited seizure-like activity suggesting a central ner- 
vous system effect (Sisk et al., 1990). Necropsy signs of 
boron toxicity in mice, rats, and dogs included glomerular 
damage (altered permeability of capillaries) and tubular dam- 
age (cellular vacuolization and shedding of cells into the tu- 
bular lumen) in the kidney, and an increase in small dark 
cells (probably microglia) in the spinal cord and in the gray 
matter of the brain cortex (Pfeiffer et al., 1945). The reported 
single toxic dose of boric acid for humans varies from 20 to 
45 g (3.5 to 7.9 g boron) for adults (Potter, 1921) and 1 to 
6 g (0.175 to 1.05 g boron) for infants (McNally and Rust, 
1928; Young et al., 1949). However, these values are based 
on old reports and may be questionable. Of the 784 poison- 
ing cases examined by one group (Litovitz et al., 1988), no 
deaths or severe manifestations of toxicity were found in 
patients ingesting boric acid in single doses ranging from 10 



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BORON 



63 



mg to 88.8 g (1.75 mg to 15.54 g boron); 88 percent did not 
exhibit any toxicity symptoms in this group whose median 
age was 2 years. In the patients that showed symptoms, the 
most common were vomiting, abdominal pain, and diarrhea. 
Lethargy, headache, lightheadedness, and atypical rash were 
seen less frequently. Similar findings were obtained from 
the records of another 300 cases of single, acute ingestions 
of 10 to 297 g of boric acid (1.75 to 52 g of boron) (Linden, 
et al., 1986). The initial responses to the lethal doses re- 
ported in the older literature included nausea, vomiting 
(sometimes with blood), abdominal pain, and diarrhea. 
Shock with low blood pressure, tachycardia, and cyanosis 
sometimes occurred. Death apparently was the result of cen- 
tral nervous system depression. Autopsy signs of toxicity 
included a cloudy swelling of the kidneys, centrolobular he- 
patic necrosis, and hemorrhagic enteritis. It should be noted 
that no human deaths from large single dose boric acid in- 
gestion were reported between 1928 and 1988 (Litovitz et 
al., 1988). 

Acute 

The topic of the first publication in the American Journal 
of Physiology was boron toxicity in dogs (Chittenden and 
Gies, 1898). This study found that daily doses of 3 g of boric 
acid or 5 g of borax for 8-10 days had no physiological or 
pharmacological effect in adult dogs weighing 8 to 12 kg. 
However, doses of 5 to 10 g B/d resulted in an increase in 
urinary nitrogen, sulfur and phosphorus excretion, and doses 
of borax and boric acid equal to 1.5-2.0 percent of daily diet 
intake induced nausea and vomiting. Rabbits orally adminis- 
tered 800 mg boric acid/kg BW/day for 4 days exhibited 
anorexia, weight loss, and diarrhea; 850 and 1,000 mg boric 
acid/kg BW resulted in 100 percent mortality (Draize and 
Kelley, 1959). Cattle consuming water containing 150 or 
300 mg B/L as boric acid for 30 days exhibited lethargy, 
inflammation, and edema in the legs and around the dew- 
claws; occasional diarrhea; and decreased food consump- 
tion, weight gain, hematocrit, and hemoglobin concentration 
(Green and Weeth, 1977). Rats fed 262.5 mg B/kg BW/day 
as boric acid or borax in the diet exhibited reduced weight 
gain and died within 3 to 6 weeks (Weir and Fisher, 1972). 
Administration of 250 mg boric acid/kg BW on gestational 
days 6-19 to rabbits resulted in increased prenatal mortality, 
decreased litter size, and malformed fetuses (primarily 
cardiovascular defects) (Price et al., 1996). Fish are not 
especially sensitive to boron as borate or boric acid. The 
concentration of borate acutely toxic to freshwater fish (le- 
thal to 50 percent in 1 to 6 days) ranges from 14 to 3,400 mg/ 
L of water (ECETOC, 1996). Rainbow trout and zebra fish 
are the most sensitive species tested to date. The LCjq for 
most fish ranged between 200 and 1,000 mg/L of water. 
Eleven infants fed formulae that were accidentally prepared 
with a 2.5 percent boric acid solution for 1 to 5 days (2 to 
14.06 g boric acid, or (0.350 to 2.458 g boron total) exhib- 



ited diarrhea, vomiting, erythema, exfoliation, desquamation 
of the skin, and central nervous system irritation; five of the 
infants died (Wong et al., 1964). 

Chronic 

Table 7-1 summarizes the doses and effects of a chronic 
consumption of high or nonphysiologic amounts of boron by 
various animals and humans. Table 7-1 indicates that intakes 
of boron in the form of boric acid and borates that are approxi- 
mately 100- to 1,000-fold greater than normal are needed to 
induce reproductive and developmental toxicity in animals. 
Thus, other than waterfowl in specific habitats, it is unlikely 
that boron toxicity under normal environmental conditions is 
a concern for animals. High concentrations of boron have been 
found in irrigation drainwaters and food consumed by water- 
fowl in certain areas of the western United States, such as the 
San Joaquin Valley and Kesterson National Wildlife Refuge 
in California. Reported waterfowl food boron concentrations 
(mg/kg dry weight) in these regions include aquatic insects, 
150; algae, 400; wetland plants, up to 1,860; and seeds, up to 
3,500 (ECETOC, 1996). An excellent summary of the fairly 
extensive human toxicity reports has been given by Culver 
and Hubbard (1996). Prolonged consumption of boric acid by 
humans has been reported to result in mild gastrointestinal 
irritation, anorexia, disturbed digestion, nausea, vomiting, and 
an erythematous rash. Seven infants provided soothers dipped 
in a proprietary borax and honey mixture containing about 3 g 
B/28 mL (23 mg boron/mL) for 4 to 10 weeks (calculated to 
be about 30 to 90 mg B/day) suffered from seizures that 
stopped upon cessation of use of the mixture (O'Sullivan and 
Taylor, 1983). 

Factors Influencing Toxicity 

Interaction with polyhydroxyl compounds can affect the 
response to high intakes of boron. Substances possessing cis- 
hydroxyl groups on adjacent carbons fonn very stable diesters 
with boron that are almost undissociable in water. This indi- 
cates why supplementing the diet with riboflavin, a dihydroxy 
compound, protects chickens against boric acid toxicity (Roe 
et al., 1972). The toxicity of boron to fish is increased with 
increasing water hardness (Birge and Black, 1977). High di- 
etary selenium (15 and 60 mg/kg diet) and low dietary protein 
(7 percent compared to 22 percent of the diet) increased the 
toxicity of boron in mallard ducklings. Feeding 1 g B/kg diet 
to ducks enhanced the accumulation of toxic amounts of sele- 
nium in liver caused by low dietary protein when dietary sele- 
nium was high (Hoffman et al., 1991). 

TISSUE LEVELS 

Boron is distributed throughout the soft tissues and fluids 
at concentrations mostly between 0.015 and 2.0 mg/kg wet 
weight. Table 7-2 shows representative values in various 



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MINERAL TOLERANCE OF ANIMALS 



organs and fluids from various animals and humans. The 
values show that tissues from animals fed nontoxic amounts 
of boron are not of potential concern for human boron toxic- 
ity. As with other mineral elements, overcoming homeostatic 
mechanisms by high boron intakes will elevate tissue and 
blood boron concentrations. However, the values in Table 7- 
2 indicate that no animal tissue or fluid used as a food will 
accumulate boron to the extent that it would be of potential 
toxicological concern for humans. The uptake and retention 
of boron are highest in bone. 

MAXIMUM TOLERABLE LEVELS 

The most sensitive indicators of boron toxicity in animals 
have been decreased weight and rib anomalies in the devel- 
oping rat fetus which occurs with intakes of about 13 mg 
B/kg BW/day (IPCS, 1998). Thus, 10 mg B/kg BW/day may 
be a reasonable MTL for animals; this translates into about 
135 mg/kg diet. Other effects of boron toxicity and the dose 
at which they occur in mg/kg BW/day include further rib 
anomalies in fetuses and testicular pathology in growing rats 
at about 25; decreased fetal body weight and increased fetal 
cardiovascular malformations in the rabbit, and severe tes- 
ticular pathology in the rat at about 40; testicular atrophy 
and sterility in the rat at about 55; and decreased fetal body 
weight in the mouse at 80 (IPCS, 1998). Mallard duckling 
growth was adversely affected with dietary boron concentra- 
tions of 30 and 300 mg/kg, and survival was reduced at 1,000 
mg/kg (IPCS, 1998). Based on studies with reconstituted 
water, the NOAEL concentration for early life stages of rain- 
bow trout, considered the most sensitive fish species, has 
been found to range from 0.009 to 0.103 mg B/L. However, 
studies using natural water collected from three sources con- 
taining a variety of boron concentrations, including 0.75 mg/ 
L in the Firehole River in Yellowstone National Park, 
showed no adverse effects on embryo-larval stages of rain- 
bow trout (Black et al., 1993). Lowest observed effect (de- 
tectable change in embryo-larval morphology or survival) 
concentrations ranged from 1.10 to 1.73 mg B/L for the three 
sources of natural water. Therefore, under natural conditions, 
the no-effect-level for fish is apparently around 1 mg B/L 
(ECETOC, 1996). 

HUMAN HEALTH 

Based on developmental effects in rats, mice, and rabbits, 
the acceptable upper limit of daily intake of boron has been 
estimated to be in the range of 1 1 to 20 mg/day for adults 
(Murray, 1996; WHO, 1996; NRC, 2001). However, the 
NOAEL for humans can be established at 1 g boric acid (175 
mg B)/day for adults, or about 2.5 mg B/kg BW/day, and the 
chronic-adverse-effect level is 5 mg B/kg BW/day (Culver 
and Hubbard, 1996). These relatively high values indicate 
why boric acid and borates were used for many years as food 
preservatives without apparent widespread toxicity. This 



practice stopped when boric acid and borates became con- 
sidered as poisons because of numerous reports of toxic ef- 
fects after accidental and inappropriate domestic and medi- 
cal misuse of these compounds. Thus, intakes of boron at or 
below the MTL suggested above for animals would not be 
expected to result in tissue boron concentrations that would 
be of concern for human health. The guide level for drinking 
water in Europe of 1 mg B/L is considered conservative with 
an ample safety margin (ECETOC, 1994). Respiratory ex- 
posure to boron in industry has not caused chronic pulmo- 
nary effects, and skin exposure does not cause dermatitis 
(Culver and Hubbard, 1996). 

FUTURE RESEARCH NEEDS 

The toxicity of boron for captive animals has been rela- 
tively well studied. However, there is a need to more clearly 
define the mechanisms of boron toxicity. Also, more needs 
to be known about the effects of boron at low levels of in- 
take, including establishing the basis for its apparent essen- 
tial or beneficial actions, and the intake below which these 
actions are compromised. The mechanisms through which 
boron is absorbed from the gastrointestinal tract need further 
definition. Further knowledge in these areas should help 
identify interactions with other nutrients and the environ- 
ment that could modify the toxic and beneficial actions of 
boron. 

SUMMARY 

Boron is always found in nature bound to oxygen or as 
borates. Boric acid is the prevalent form of boron in animals 
and humans. It is a weak Lewis acid that forms complexes 
with biological substances containing c«-hydroxyl groups. 
Boron is accepted as essential for plants because it is re- 
quired to complete their life cycle. To date, only one essen- 
tial function has been found for boron in plants; it cross- 
links two dimers of rhamnogalacturan-II, a small structurally 
complex pectic polysaccharide found in primary cell walls. 
Although boron has been found essential to complete the life 
cycle of some animals (frog, zebrafish), it is not generally 
accepted as essential, apparently because it lacks a defined 
biochemical function, for higher animals and humans. Ana- 
lytical techniques that utilize inductively-coupled plasma 
have become preferred for the analysis of boron in biologi- 
cal materials. Ashing of samples is a critical step in the accu- 
rate analysis of boron in low amounts because of contamina- 
tion and volatilization concerns. About 85-90 percent of 
dietary boron is rapidly absorbed and excreted mostly in the 
urine. Toxic amounts of boron can be absorbed through dam- 
aged, but not intact, skin. The diet and sometimes drinking 
water are the major sources of exposure to boron. Boron is a 
relatively nontoxic element. Reported oral LD^q values for 
boron in mg/kg BW are in the range of 400-700 for mice and 
rats, 210 for guinea pigs, and 250-350 for dogs. Cattle con- 



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suming water containing 150-300 mg B/L exhiibited toxicity 
signs including decreased food consumption and weigtit. 
Boron concentrations found lethal to 50 percent of tested 
freshwater fish ranged from 14 to 3,400 mg/L of water. This 
large range occurred because rainbow trout and zebrafish 
were much more sensitive than most other fish tested, and 
one species was very nonsensitive. The LC^q for most fish 
ranged between 200 and 1,000 mg/L of water. Chronic tox- 
icity studies indicate that intakes of boron in the form of 
borates and boric acid approximately 100- to 1,000-fold 
greater than normal are needed to induce reproductive and 
developmental toxicity in animals. The most sensitive indi- 
cators of boron toxicity in animals have been decreased 
weight and rib anomalies in the developing rat fetus which 
occurs with intakes of about 13 mg/kg BW/day. Thus, other 
than for waterfowl and fish in some specific habitats, it is 
unlikely that boron toxicity under nonnal environmental con- 
ditions is a concern for animals. 

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poison. Can. Med. Assoc. J. 61:44. 



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8 



Bromine 



INTRODUCTION 

Bromine (Br) is a lialogen and thie only nonmetallic ele- 
ment that is a liquid at ambient temperature and pressure. 
Naturally occurring bromine is found bound to metals in the 
form of inorganic salts — the bromides. Seawater is the great- 
est reservoir for bromine on Earth and contains 65 mg/L. 
The concentration in brine or salt lakes may be enriched by 
up to 100-fold. Bromine is abundant in saline deposits origi- 
nating from evaporated lakes. Bromine levels in soils typi- 
cally range from 1 to 20 mg/kg, although some volcanic soils 
have considerably higher levels. 

Bromine-containing compounds have a variety of agri- 
cultural, medicinal, and industrial uses (Lyday, 2002). Me- 
thyl bromide is used to fumigate the soil for controlling in- 
sects, nematodes, weeds, and pathogens in more than 100 
food crops and in forest and ornamental nurseries. This gas- 
eous form of organic bromide is also used for postharvest 
protection and quarantine treatments of foods, feedstuff s, and 
wood products in warehouses, ship holds, freight cars, and 
other areas of storage. Methyl bromide was defined by the 
Montreal Protocol of 1991 as a chemical that contributes to 
depletion of the Earth's ozone layer, so most countries have 
agreed to phase out its use. Potassium bromate is used in the 
baking industry to help bread rise in the oven and to create a 
good texture in the finished product. Bromine is also used in 
the production of brominated vegetable oils that are used as 
emulsifiers in some soft drinks and fruit drinks. 

Many bromide-containing drugs were developed in the 
early 20th century and used as sedatives and anticonvulsants, 
but they have mostly been replaced by other drugs. How- 
ever, bromide salts are still commonly used to treat epilepsy 
in dogs (March et al., 2002). Bromine is used to estimate the 
extracellular space in physiology research. Industrial uses of 
bromine-containing compounds include drilling fluids, fuel 
additives, photographic chemicals, rubber additives, and 
polybrominated biphenyls used as flame retardants. These 
industrial compounds are not expected to be found in feed 



ingredients or water consumed by animals and are not con- 
sidered in this analysis. 

ESSENTIALITY 

Several studies have demonstrated improved growth in 
chicks and mice fed diets supplemented with bromide, but 
evidence is conflicting and not robust. There are no known 
essential biochemical functions of bromine and it is not usu- 
ally considered as an essential nutrient (NRC, 1980). 

DIFFICULTIES IN METHODS OF ANALYSIS AND 
EVALUATION 

Total bromide in foods is usually measured following 
ashing in alkali (Greve, 1983) and determining the bromide 
ion content using gas chromatography or high performance 
liquid chromatography (HPLC). Ashing converts organic 
forms of bromine into inorganic salts. 

Bromine introduced into foodstuffs by fumigation with 
methyl bromide is unstable and converts to inorganic forms 
rapidly over time. However, some conditions may result in 
trace levels of unconverted fumigant. Given the differing 
toxicological profiles of organic and inorganic forms, their 
analytical differentiation may be desirable. Procedures for 
the separation of organic bromides by extraction permit the 
measurement of both organic and inorganic bromide con- 
centrations (Getzendaner, 1975). 

REGULATION AND METABOLISM 

Bromide has very similar physiochemical properties as 
chloride and has a comparable metabolic profile (reviewed 
by van Leeuwen and Sangster, 1987). Like chloride, bro- 
mide is very efficiently absorbed from the gastrointestinal 
tract and found predominantly in the extracellular fluids, 
gastric secretions, and saliva. Urine is the primary excretory 
pathway for bromide (Pavelka et al., 2000). The glomerulus 



72 



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BROMINE 



73 



filters bromide, which competes with chloride for reabsorp- 
tion in the tubules. Most of the bromide in saliva and gastric 
juices is reabsorbed in the small intestine, and little bromide 
is found in the feces. 

Steady state pharmokinetics of bromide can be described 
by a simple one-compartment model with a linear relation- 
ship between plasma bromide concentrations and the in- 
gested dose (van Logten et al., 1973; van Logten et al., 1974; 
Rauws, 1975). The half-life of bromide at normal dietary 
chloride levels is 10-12 days in humans (van Leeuwen and 
Sangster, 1987), 15 days in dogs (March et al., 2002), and 3 
days in rats (Rauws and Van Logten, 1975). The half-life is 
strongly dependent upon the dietary chloride level and is 
increased IG-fold by a chloride deficiency and decreased 
similarly by dosing chloride. Thus, sodium chloride is used 
to treat bromide toxicosis. 

Bromide replaces chloride in intra- and extracellular flu- 
ids, and hypochloremia is diagnostic of bromide toxicity. 
The hydrated bromide ion is slightly larger than the hydrated 
chloride ion and has slightly different transport kinetics 
across the cell membrane. Bromide causes hyperpolariza- 
tion of nerve cell membranes, which is thought to be the 
basis of its pharmacological properties on the central ner- 
vous system. 

The thyroid gland partially discriminates between iodine 
and bromide, but bromide levels are often higher in this tis- 
sue than others, especially during an iodine deficiency. Thy- 
roid peroxidase does not oxidize bromide, and bromine does 
not substitute for iodine in thyroxine (Hosoya, 1963). How- 
ever, high levels of bromide decrease iodine uptake by the 
thyroid and may exacerbate an iodine deficiency (Pavelka et 
al., 2002). Conversely, dietary bromide reduces the toxicity 
of iodine in chicks (Baker et al., 2003). 

SOURCES AND BIOAVAILABILITY 

Bromine is not typically supplemented to the diets of ani- 
mals. Dietary levels result from background levels in feed 
ingredients and contaminating levels introduced by the use 
of bromine-based fumigants and disinfectants. 

Natural bromine levels in crops range from approximately 
8 to 50 mg/kg dry matter (summarized in van Leeuwen and 
Sangster, 1987). Marine plants are the exception and some 
types of algae can concentrate very high levels of bromine. 
Fishmeal has the highest natural bromide level (12.6 mg/kg) 
of feedstuffs normally used in animal diets (Greve, 1983). 

Fruits, grains, and vegetables grown in soils fumigated 
with methyl bromide may have higher levels of bromide. 
Bromide residues are especially high when planting occurs 
closely after soil fumigation (Roughan and Roughan, 1984) 
and can reach levels up to 4,037 mg/kg dry weight in foliage 
vegetables (Maw and Kempton, 1982). However, when good 
agricultural practices are followed, most foods have levels 
below 40 mg/kg (Greve, 1983). 



Postharvest fumigation of foods and feed ingredients can 
result in high levels of bromide residues. Fumigation of feed 
grains and hay, especially during shipment or storage, is cur- 
rently a common practice. Residue levels in fumigated wheat 
of 70 mg/kg (Corvi et al., 1977) and in corn fumigated in a 
ship's hold of 200 mg/kg have been reported (Thompson 
and Hill, 1969). 

Other dietary sources of bromine include salt that has 
been prepared from brines containing high levels of bromide. 
Bakery wastes may contain trace levels of potassium bro- 
mate added in the baking process. Polybrominated biphe- 
nyls and other industrial chemicals have contaminated ani- 
mal feeds due to mislabeling or mishandling; these isolated 
events are increasingly rare due to improvements in material 
handling practices (Reich, 1983). 

Bromide salts are presumed to be rapidly and completely 
absorbed in the gastrointestinal tract using transport systems 
for the chloride ion. Bioavailability studies have not been 
formally conducted, but 96 percent of an oral bromide dose 
is absorbed in humans (Vaiseman et al., 1986). 

TOXICOSIS 

The toxicological profile of bromine depends on its form. 
Currently, most toxic studies on bromine concern inhalation 
toxicity of methyl bromide gas to human applicators. Though 
this gas is highly toxic, it is not a known contaminant of 
animal feeds or water and is not currently of concern for 
animal safety. Inorganic bromide salts that results from the 
decomposition of methyl bromide following fumigation of 
hay, feed ingredients, or occasionally complete feeds is the 
primary source of bromine in animal diets (Knight and 
Costner, 1977; Knight and Reina-Guerra, 1977). Data on 
which to base the toxicity of bromide in animal feeds come 
mostly from studies in humans and laboratory animals, with 
only a few studies available with production or companion 
animals (Table 8-1). The historical medical use of bromide 
salts at relatively high levels compelled extensive research 
in the first half of the 20th century on bromide intoxication 
(bromism). Toxicity of bromide depends on its replacement 
of chloride in body fluids. Because of the large pool size of 
body chloride, expression of bromide toxicosis typically oc- 
curs only after chronic exposure to relatively high levels of 
bromide (Trump and Hochberg, 1976). 

Potassium bromate is more toxic than bromide salts due 
to its oxidizing properties. It also is a carcinogen (Kurokawa 
etal., 1986; lARC, 1986; DeAngelo etal., 1998), but it is not 
a known contaminant of animal feeds. Polybrominated bi- 
phenyls are known liver carcinogens in rats at levels of 
around 10 mg/kg (NTP, 1993). 

Single Dose 

Bromide salts irritate the gastric mucosa and cause nau- 
sea and vomiting (Trump and Hochberg, 1976). The result- 



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74 



MINERAL TOLERANCE OF ANIMALS 



ing feed refusal coupled with the pharmokinetics of bromide 
in the body make it unlikely that systemic toxicosis would 
occur when animals are presented with a single meal of a 
feed containing very high levels of bromine. 

Acute 

The acute (48-hour) NOAEL for bromide as sodium bro- 
mide in water for guppies and medaka is 78 and 250 mg/L 
(Canton et al., 1983). Lethality (LC,,,) occurs at 16,000 and 
25,000 mg/L, respectively. Orally dosed bromide salts have 
very low acute toxicity in rodents and an LDjq of 3.5 to 7 g 
Br/kg BW has been reported (Smith and Hambourger, 1925; 
Vossetal., 1961). 

Chronic 

In humans, symptoms of chronic bromide intoxication are 
apathy, headaches, ataxia, memory loss, drowsiness, and loss 
of emotional control. Dermal symptoms, including acne or a 
nodular rash, may also occur. Tremors, hallucination, stupor, 
and coma occur in severe cases (reviewed in van Leeuwen and 
Sangster, 1987). Shifts in EEG activities, increased circulating 
thyroxine, and other endocrine changes are the primary physi- 
ological disruptions of a chronic toxicosis (Sangster et al., 
1983). A summary of studies in humans indicates that the daily 
NOAEL for bromide in humans is 4 mg/kg BW (van 
Leeuwen and Sangster, 1987). The Food and Agriculture 
Organization/World Health Organization (FAO/WHO 
Pesticide Committees, 1967) applied a margin of safety and 
set the acceptable daily intake of bromide at 1 mg/kg. 

The most sensitive indicators of bromide toxicosis in ro- 
dents are changes in behavior and weight gain (Hansen and 
Hubner, 1983). In mice, the NOAEL is between 400 and 
1,200 mg/kg diet (Hansen and Hubner, 1983). In rats, distur- 
bances in thyroid and renal function occur at dietary levels 
between 1,200 and 19,200 mg/kg diet (Loeber et al., 1983) 
and a decrease in fertility occurs at 1,200 mg/kg (van 
Leeuwen et al., 1983). A chronic 2-year study using feed 
fumigated with methyl bromide found that residual bromide 
levels of 500 mg/kg diet were not toxic to rats as indicated 
by normal livability and normal histopathology, but this level 
did cause a small reduction in body weight; 200 mg/kg had 
no effect on body weight (Mitsumori et al., 1990). 

Potassium bromide is frequently used as therapy for epi- 
leptic dogs, and toxicosis occurs occasionally (Yohn et al., 
1992; Podell, 1998). Characteristic clinical signs are ataxia 
and lameness, especially of the pelvic limbs, but the maxi- 
mum tolerable level has not been determined. 

Bromide toxicity is treated by removal of the bromide 
source and administering sodium chloride. In humans, com- 
plete recovery follows normalization of blood bromide levels. 



Factors Influencing Toxicity 

The primary factor affecting toxicity of bromide is the 
dietary chloride level because high dietary chloride increases 
the renal excretion of both chloride and bromide. For ex- 
ample, the daily bromine intake needed to maintain near 
toxic levels of serum bromide is about twice as high for dogs 
fed 1.3 percent dietary chloride as those fed 0.4 percent and 
three times as high as those fed 0.2 percent (Trepanier and 
Babish, 1995). In rats the biological half-life of bromide in- 
creases from 3 days at normal dietary chloride levels to 25 
days when sodium chloride is excluded from the diet (Rauws 
and Van Logten, 1975). 

TISSUE LEVELS 

In rats, plasma and tissue bromine levels increase linearly 
with dietary levels. Bromide replaces about half of the chlo- 
ride in plasma and tissues at a dietary level of 19,200 mg/kg 
(van Logten et al., 1974). Lynn et al. (1963) found that dairy 
cows fed 9.5 to 38 mg/kg dietary bromide as NaBr have milk 
levels ranging from 1 to 12 mg/kg on a wet weight basis 
(Table 8-2). Bromide in cow's milk was proportional to the 
level in the blood, which was proportional to the level in the 
feed. More recently, Vreman et al. (1985) reexamined the 
relationship between dietary and milk bromide levels. Diets 
contained 22, 69, or 115 mg/kg inorganic bromide residue 
that resulted from the decomposition of methyl bromide fu- 
migate and were fed for 5 weeks. Milk bromide increased 
linearly with dietary bromide, and the average ratio between 
dietary bromine concentration and milk bromine concentra- 
tion was 0.27. At the end of the study, tissues were sampled 
and muscle bromide concentrations increased linearly with 
dietary bromide, with the 115 mg/kg dietary level giving 
20.8 mg/kg fresh muscle. 

MAXIMUM TOLERABLE LEVELS 

The MTL of bromine is defined as the dietary level that, 
when fed for a defined period of time, will not impair ac- 
cepted indices of animal health or performance. Bromide is 
the mostly likely form of bromine that animals are exposed 
to through their feed and water. No adverse effects of bro- 
mide at levels of 300 mg/kg diet or below have been re- 
ported in chronic feeding studies in rodents, and levels of 
500 mg/kg slightly reduce growth but do not cause pathol- 
ogy. Hatchling chicks tolerate 5,000 mg/kg for 4 weeks with- 
out impaired growth or efficiency of feed conversion. Dogs 
tolerate 20 mg/kg BW/day with no adverse effects. Maxi- 
mum tolerable levels have not been determined for other 
animal species. 



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BROMINE 



75 



HUMAN HEALTH 

Tissue levels of bromine in farm animals have not been 
determined at dietary levels that are toxic. However, extrapo- 
lation of muscle bromine concentrations known to occur in 
animals fed subtoxic levels to animals fed maximum toler- 
able levels indicates that residue levels would not be of con- 
cern for human health. The daily NOAEL for bromide in 
humans is 4 mg/kg BW (van Leeuwen and Sangster, 1987). 

FUTURE RESEARCH NEEDS 

Bromine toxicity has not been a problem in animal nutri- 
tion so very little research has been done. Accurate estimates 
of maximum tolerable dietary levels cannot be made for any 
species except rodents, and more research is needed. Resi- 
due levels in meat, milk, and eggs of animals fed maximum 
tolerable dietary levels of bromide are also needed. Finally, 
there are insufficient data available to estimate maximum 
tolerable bromide levels in water of any species. 

SUMMARY 

Bromine is widely distributed in nature and is found al- 
most exclusively as bromide salts. There are no known es- 
sential biochemical functions of bromine, and it is not usu- 
ally considered as an essential nutrient. Bromine is not 
typically supplemented to the diets of animals and dietary 
levels result from background levels in feed ingredients and 
contaminating levels introduced by the use of bromine-based 
fumigants and disinfectants. Bromide has very similar 
physiochemical properties as chloride and has a comparable 
metabolic profile and tissue distribution. Bromide competes 
with chloride in metabolism and causes hyperpolarization of 
nerve cell membranes, which is thought to be the basis of its 
pharmacological properties on the central nervous system. 
Because of the large pool size of body chloride, expression 
of bromide toxicosis typically occurs only after chronic ex- 
posure to relatively high levels of bromide. It is rapidly ex- 
creted, primarily in the urine, so toxic levels decline quickly 
after the source has been removed. 

REFERENCES 

Baker, D. H., T. M. Pan', and N. R. Augspurger. 2003. Oral iodine toxicity 
in ctiiciis can be reversed by supplemental bromine. J. Nutr. 133:2309— 
2312. 

Barber, R. S., R. Braude, and K. G. Mitchell. 1971. Arsanilic acid, sodium 
salicylate and bromide salts as potential growth stimulants for pigs re- 
ceiving diets with and without copper sulfate. Br. J. Nutr. 25:381-389. 

Bosshardt, D. K., J. W. Huff, and R. H. Barnes. 1956. Effect of bromide on 
chicii growth. Proc. Soc. Exp. Biol. Med. 92:219-224. 

Canton, J. H., P. W. Wester, and E. A. Mathijssen-Spiekman. 1983. Study 
on the toxicity of sodium bromide to different freshwater organisms. 
Food Chem. Toxicol. 21:369-378. 



Corvi, C, I. Dallmayr, M. Meyer, and I. Vogel. 1977. Bromine residues in 
foods after use of methyl bromide. Mitt. Geb. Lebensmittelunters. Hyg. 
68:431^t41. 

DeAngelo, A. B., M. H. George, S. R. Kilburn, T. M. Moore, and D. C. 
Wolf. 1998. Carcinogenicity of potassium bromate administered in the 
drinking water to male B6C3F1 mice and F344/N rats. Toxicol. Pathol. 
26:587-594. 

Doberenz. A. R., A. A. Kurnick, B. J. Hulett, and B. L. Reid. 1965. Bromide 
and fluoride toxicities in the chick. Poult. Sci. 67:32-37. 

FAOAVHO Pesticide Committees. 1967. Evaluation of some pesticide resi- 
dues in food. Rome: Food and Agriculture Organization of the United 
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Getzendaner, M. E. 1975. A review of bromine determination in foods. J. 
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Greve, P. A. 1983. Bromide-ion residues in food and feedstuffs. Food Chem. 
Toxicol. 21:357-359. 

Hansen, K., andH.Hubner. 1983. Effectsof bromide on behaviour of mice. 
Food Chem. Toxicol. 21:405^08. 

Hosoya. T. 1963. Effect of various reagents including antithyroid com- 
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lARC. 1986. Potassium bromate. lARC Monogr. Eval. Carcinog. Risk 
Chem. Hum. 40:207-220. 

Knight, H. D., and G. C. Costner. 1977. Bromide intoxication of horses, 
goats, and cattle. J. Am. Vet. Med. Assoc. 171:446^148. 

Knight, H. D., and M. Reina-Guerra. 1977. Intoxication of cattle with so- 
dium bromide-contaminated feed. Am. J. Vet. Res. 38:407^09. 

Kurokawa, Y., S. Aoifi, Y. Matsushima, N. Taiiamura, T. Imazawa, and Y. 
Hayashi. 1986. Dose-response studies on the carcinogenicity of potas- 
sium bromate in F344 rats after long-term oral administration. J. Nat. 
Cancer Inst. 77:977-982. 

Loeber, J. G., M. A. Franken, and F. X. van Leeuwen. 1983. Effect of 
sodium bromide on endocrine parameters in the rat as studied by immu- 
nocytochemistry and radioimmunoassay. Food Chem. Toxicol. 21: 
391^04. 

Lyday, P. A. 2002. Bromine. USGS, Minerals Yearbook. Available at http: 
//minerals. usgs.gov/minerals/pubs/commodity /bromine/index. html. 

Lynn, G. E., S. A. Shrader, O. H. Hammer, and C. A. Lassiter. 1963. Occur- 
rence of bromides in the milk of cows fed sodium bromide and grain 
fumigated with methyl bromide. J. Agric. Food Chem. 1 1:87-92. 

March, P. A., M. Podell, and R. A. Sams. 2002. Pharmacokinetics and tox- 
icity of bromide following high-dose oral potassium bromide adminis- 
tration in healthy beagles. J. Vet. Pharmacol. Ther. 25:425^32. 

Maw, G. A., and R. J. Kempton. 1982. Bromine in soils and peats. Plant and 
Soil 65:103-110. 

Mitsumori, K., K. Maita, T. Kosaka, T. Miyaoka, and Y. Shirasu. 1990. 
Two-year oral chronic toxicity and carcinogenicity study in rats of diets 
fumigated with methyl bromide. Food Chem. Toxicol. 28:109— 1 19. 

NRC (National Research Council). 1980. Mineral Tolerance of Domestic 
Animals. Washington, D.C.: National Academy Press. 

NTP (National Toxicology Program). 1993. Toxicology and Carcinogen- 
esis Studies of Polybrominated Biphenyls. NTP TR 398. Research Tri- 
angle Park, NC: U.S. Department of Health and Human Services. 

Pavelka, S., A. Babicky, M. Vobecky, and J. Lener. 2000. Bromide iiinetics 
and distribution in the rat. II. Distribution of bromide in the body. Biol. 
Trace Elem. Res. 76:67-74. 

Pavelka. S., A. Babicky, J. Lener, and M. Vobecky. 2002. Impact of high 
bromide intake in the rat dam on iodine transfer to the sucklings. Food 
Chem. Toxicol. 40:1041-1045. 

Podell, M. 1998. Antiepileptic drug therapy. Clin. Tech. Small Anim. Pract. 
13:185-192. 

Rauws, A. G. 1975. Bromide pharmacokinetics: a model for residue accu- 
mulation in animals. Toxicology 4:195-202. 

Rauws, A. G., and M. J. Van Logten. 1975. The influence of dietary chlo- 
ride on bromide excretion in the rat. Toxicology 3:29-32. 



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76 



MINERAL TOLERANCE OF ANIMALS 



Reich, M. R. 1983. Environmental politics and science: the case of PBB 
contamination in Michigan. Am. J. Public Health 73:302-313. 

Sangster, B., J. L. Blom, V. M. Sekhuis, J. G. Loeber, A. G. Rauws, J. C. 
Koedam, E. 1. Kiajnc, and M. J. van Logten. 1983. The influence of 
sodium bromide in man: a study in human volunteers with special em- 
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Toxicol. 21:409^19. 

Smith, P. K., and W. E. Hambourger. 1925. Antipyretic toxic effects of 
combinations of acetanilide with sodium bromide and with caffeine. J. 
Pharmacol. Exp. Ther. 55:200-206. 

Thompson, R. H., and E. G. Hill. 1969. Pesticide residues in foodstuffs in 
Great Britain. X. Bromide residues in maize, pulses and nuts. J. Sci. 
Food Agric. 20:287-292. 

Trepanier, L. A., and J. G. Babish. 1995. Effect of dietary chloride content 
on the elimination of bromide by dogs. Res. Vet. Sci. 58:252-255. 

Trump, D. L., and M, C. Hochberg. 1976. Bromide intoxication. Johns 
Hopkins Med. J. 138:119-123. 

Vaiseman, N., G. Koren, and P. Pencharz. 1986. Pharmacokinetics of oral 
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24:403-413. 



vanLeeuwen, F. X., andB. Sangster. 1987. The toxicology of bromide ion. 
Crit. Rev. Toxicol. 18:189-213. 

van Leeuwen, F. X., E. M. den Tonkelaar, and M. J. van Logten. 1983. 
Toxicity of sodium bromide in rats: effects on endocrine system and 
reproduction. Food Chem. Toxicol. 21:383-389. 

van Logten, M. J., M. Wolthuis, A. G. Rauws, and R. Kroes. 1973. Short- 
term toxicity study on sodium bromide in rats. Toxicology 1:321-327. 

van Logten, M. J., M. Wolthuis, A. G. Rauws, R. Kroes, E. M. den 
Tonkelaar, H. Berkvens, and G, J, van Esch. 1974. Semichronic toxicity 
study of sodium bromide in rats. Toxicology 2:257—267. 

Voss, E., A. R. Haskell, and L. Gartenberg. 1961. Reduction of tetramicine 
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Vreman, K., A. H. Roos, and G. M. Tuinstra. 1985. The excretion of inor- 
ganic bromide into milk of dairy cows after oral administration. Neth. 
MilkDairy J. 39:173-181. 

Yohn, S. E., W. B. Morrison, and P. E. Sharp. 1992. Bromide toxicosis 
(bromism) in a dog treated with potassium bromide for refractory sei- 
zures. J. Am. Vet. Med. Assoc. 201:468^70. 



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77 



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78 



MINERAL TOLERANCE OF ANIMALS 



TABLE 8-2 Bromine Concentrations in Fluids and Tissues of Animals (mg/kg or mg/L)" 



Animal 



Quantity'' Source 



Duration 



Route 



Plasma/ 
Serum 



Muscle 



Liver Kidney Milk Reference 



Dogs 
Rats 



20 mg/kg/d KBr 





300 

1,200 

4,800 

19,200 



NaBr 



115d 



28 d 



Diet 



Diet 



200 

16 

160 

647 

1,998 

4,474 



Cattle, 


10 


NaBr 


22 d 


lactating. 


19 




19 d 


dairy 


43 




28 d 




10 


Inorganic 


39 d 




19 


bromide"^ 


39 d 




43 




39 d 



Diet 



15 
33 
68 



8 


56 


van Logten et al. 


96 


112 


1973 


248 


415 




719 


1,278 




1,678 


2,716 





Rats 





NaBr 


28 d 


Diet 


16 


' ■ 


^^^^ van Logten et al.. 




75 








48 


24 ■ 


H 1974 




300 








168 


■ 


^^^^^^^^^^^^^ 




1,200 








615 


336 H 


^^^^^^^^^^^^^ 




4,800 








1,998 


H 


^^^^^^^^^^^1 




19,200 


^ 




^^^^ 


4,075 ^^^_ 


^^^_ 2,397 


^^^^^^^^^ 



3 

5 

12 

7 

8 

15 



Lynn et al., 1963 



Cattle, 


22 ^M 


H NaBr 


30 d 


Diet 


21 


3 


4 


14 


6 


Vreman et al., 


lactating. 


69 ^^ 


^v 






58 


9 


12 


31 


17 


1985 , 


dairy 


115 








95 


2 


27 


88 


31 


^™ 



"Values are on a wet weight basis. 

''Quantity in mg Br/kg of feed (dry matter basis) unless otherwise noted. 

"^Inorganic salts from the decomposition of methyl bromide used to fumigate the feed. 



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Cadmium 



INTRODUCTION 

Cadmium (Cd) is a soft metal with a silver-white color. It 
has an atomic number of 48, a molecular weight of 1 12.41, 
and eight naturally occurring isotopes. It is a divalent 4d 
transition metal with chemical properties that are similar to 
zinc. The pure metal crystallizes in distorted but closely 
packed structures with relatively weak bonding. Conse- 
quently, it is soft enough to cut with a knife and for a metal 
has a low melting point of 321°C. Cadmium has a relatively 
high vapor pressure, and the vapor released from cadmium 
metal quickly forms a surface oxide layer when exposed to 
air. Cadmium is reactive with halides, sulfur, oxygen, and 
phosphorus, but is unreactive toward carbon, hydrogen, or 
nitrogen (Butterman and Plachy, 2004). 

Cadmium is the 63rd most abundant element in the 
Earth's crust (0.000016 percent) and is naturally found in 
association with zinc and, to a lesser extent, with lead and 
copper. It is extracted mainly as a by-product of zinc min- 
ing and processing. In 2000, it was refined in 27 countries, 
with production dominated by Asia, followed by Europe 
and then North America. World production in 2000 was 
19,700 metric tons, but production is declining due to an 
increase in the recovery of cadmium by recycling and lower 
demand because of regulatory pressure to find less toxic 
substitutes (Plachy, 2002). 

Cadmium forms numerous alloys and compounds with 
other elements and is used in batteries, solders, semiconduc- 
tors, solar cells, plastic stabilizers, and to plate iron and steel. 
Cadmium salts have a wide spectrum of vivid colors and are 
used as pigments in a variety of applications such as color- 
ing plastics and ceramics. However, this use is declining 
because of replacement by less toxic pigments. End uses for 
cadmium in 2002 were batteries, 78 percent; pigments, 12 
percent; coatings and platings, 8 percent; all other, 2 percent 
(Plachy, 2002). Cadmium can enter the environment from a 
variety of anthropogenic sources including by-products from 
zinc refining, coal combustion, mine wastes, electroplating 



processes, iron and steel production, pigments, fertilizers, 
and sewage sludges. 

Sulfate, nitrate, chloride, and acetate salts of cadmium 
are very soluble in water. Cadmium oxide, hydroxide, and 
sulfide have very low solubilities. Organo-cadmium com- 
pounds are not known to occur in nature; however, cadmium 
readily forms complexes with proteins, organic acids, and 
other organic compounds. 

ESSENTIALITY 

Cadmium is not considered an essential nutrient for ani- 
mals. However, a number of studies with rodents, chickens, 
and livestock have reported increased weight gain when low 
levels of cadmium were added to the diets (Bokori and 
Fekete, 1995). The bases for these effects are unknown and 
may be the result of antibiotic or pharmacologic actions. 
Cadmium is an essential nutrient for Thalassiosira 
weissflogii, a common marine diatom. In this organism cad- 
mium is a cofactor for an isoform of carbonic anhydrase that 
is needed under conditions of low zinc, which are typical of 
many marine environments (Lane and Morel, 2000). Higher 
plants have not been shown to need cadmium. 

DIFFICULTIES IN METHODS OF ANALYSIS AND 
EVALUATION 

Cadmium levels in feeds and tissues are most commonly 
measured by atomic absorption spectrophotometry or 
inductively-coupled atomic emission spectroscopy. Both of 
these techniques have sufficient sensitivities to monitor cad- 
mium levels of concern in toxicosis (ATSDR, 1999). Atomic 
absorption spectrometry can detect cadmium in foods and 
tissues at approximately 10 |Jg/kg. Samples are usually pre- 
pared by digestion with nitric acid and microwave digestion 
techniques give excellent recoveries. Dry ashing is also com- 
monly used for preparing foods for cadmium analysis and little 
loss of cadmium occurs at temperatures below 450°C (IPCS, 



79 



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1992a). Cadmium is a relatively ubiquitous element and care- 
ful attention to possible contaminating sources is needed. In 
particular, colored plastics such as pipet tips can be a source of 
cadmium contamination. Determining low levels of cadmium 
in biological specimens requires special attention to back- 
ground detection (Cerny and Bhattacharyya, 2003). 

Cadmium concentrations in tissue of living animals can 
be measured by neutron activation analysis or x-ray fluores- 
cence. These techniques take advantage of the capture char- 
acteristics of the thermal neutrons of the naturally occurring 
stable isotope I'^Cd (IPCS, 1992a). 

REGULATION AND METABOLISM 
Absorption 

Cadmium can be absorbed in toxicologically relevant 
amounts from oral or inhalation exposure. Absorption by 
dermal exposure is relatively insignificant unless exposure 
time is very long (Wester et al., 1992). Intestinal absorption 
of cadmium is relatively low compared to similar divalent 
cations such as zinc and iron. Absorption of an experimental 
dose of cadmium after dosage of soluble salts is about 1-2 
percent in mice and rats, 0.5-3.0 percent in monkeys, 2 per- 
cent in goats, 5 percent in pigs, 5 percent in lambs, and 16 
percent in cattle (ATSDR, 1999). Cadmium is absorbed pre- 
dominantly in the duodenum and proximal jejunum. Spe- 
cific cellular importers for cadmium have not been identi- 
fied, and inadvertent uptake through pathways intended for 
the essential nutrients iron, zinc, manganese, and cysteine 
seem to mediate cadmium transport across the brush border 
(Zalups and Ahmad, 2003). For example, cadmium can be 
absorbed by the divalent metal transporter- 1, which nonnally 
mediates dietary iron absorption (Bressler et al., 2004). Cad- 
mium bound to peptides and proteins may also enter the 
enterocyte via endocytosis. 

The proportion of cadmium absorbed by the intestines is 
dependent on the dose, with higher doses being absorbed 
more efficiently (Matsuno et al., 1991a). High levels of cad- 
mium disrupt tight junctions between intestinal enterocytes 
and impair epithelial barrier function (Duizer et al., 1999). 
Thus, cadmium may increase its own bioavailability by caus- 
ing disruption of the intestinal paracellular barrier. Absorp- 
tion decreases markedly with chronic exposure (Muller et 
al., 1986), apparently due to the induction of protective 
mechanisms such as intraepithelial metallothionein. 

The bioavailability of cadmium appears to be related to 
its solubility in the digestive tract. Highly soluble salts of 
cadmium, such as chloride, nitrate, acetate, and sulfate, are 
absorbed much better than poorly soluble forms, such as cad- 
mium sulfide and cadmium sulfoselenide pigments 
(ATSDR, 1999). Cadmium bound to metallothionein or the 
cadmium incorporated into pig liver is absorbed by rats less 
efficiently than cadmium chloride, indicating that cadmium 
in foods of animal origin may be less available than soluble 



salts (Groten et al., 1990, 1994), although at low levels, 
inorganic and organic forms appear to be absorbed at similar 
efficiencies. 

The efficiency of cadmium absorption increases with iron 
or calcium deficiency (Brzoska and Moniuszko-Jakoniuk, 
1998). High dietary levels of calcium, chromium, magne- 
sium, or zinc decrease cadmium uptake (Foulkes, 1985). 
Cadmium can also decrease the absorption of calcium and 
exacerbate a deficiency (Fullmer et al., 1980). 

Transport and Distribution 

After absorption, cadmium is transported in the blood 
bound primarily to albumin, with lesser amounts bound to 
globulins, metallothionein, cysteine, glutathione, or directly 
to cells (Zalups and Ahmad, 2003). It distributes throughout 
the body with highest concentrations in the kidney and liver, 
which account for more than half of the total body burden. 
Initially, the liver takes up the highest amount of cadmium, 
but over a few days cadmium in the liver is released and 
taken up by the kidney. Thus, the kidney usually has the 
highest concentration of cadmium of any tissue in the body. 
Blood cadmium levels are indicative of recent exposure to 
cadmium but are not especially indicative of total body bur- 
den. Urine levels are a better indicator of total body burden 
(ATSDR, 1999). 

Cadmium is not transported efficiently into milk or eggs. 
Little cadmium is incorporated into hair, and levels in hair 
are not sensitive indicators of exposure (Combs et al., 1983). 
The transport of cadmium across the placenta is also ineffi- 
cient and fetal levels are considerably lower than maternal 
levels (Smith et al., 1991a). Pregnant goats fed diets with 
high levels of cadmium do not pass on sufficient levels of 
cadmium to their kids during gestation and lactation to raise 
their offsprings' tissue cadmium levels above normal 
(Telford et al., 1984b). 

Cadmium (+2) readily complexes to anionic groups, es- 
pecially sulfhydryl groups, in proteins and other molecules. 
Metallothionein, a low molecular weight protein that is very 
high in cysteine, is capable of binding up to seven cadmium 
atoms per molecule. Cadmium is a strong inducer of 
metallothionein synthesis and cadmium also decreases the 
degradation rate of this intracellular chelator (Laurin and 
Klasing, 1990). Cadmium displaces zinc and copper from 
previously synthesized metallothionein. Metallothionein is 
important in modulating the fate of cadmium in tissues by 
affecting the duration of its retention in tissues and reducing 
its cellular toxicity (Klaassen and Liu, 1998). The kidney is 
a major site of metallothionein synthesis and consequently 
accumulates cadmium. 

Excretion 

Absorbed cadmium is excreted slowly with daily losses 
of approximately 0.009 percent of the total burden via the 



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CADMIUM 



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urine and 0.007 percent via feces. Most of the cadmium loss 
into ttie intestines is derived from iiepatocellular secretion of 
cadmium into bile. Circulating cadmium-protein complexes 
are filtered at the glomerulus and then resorbed from the 
glomerular filtrate by the cells in the SI and S2 segments of 
the proximal convoluted tubules (Dorian et al., 1995). This 
region of the renal cortex concentrates cadmium and is sus- 
ceptible to necrosis. The half-life of cadmium in rodents is 
very long and estimates vary from several months to a year or 
more (ATSDR, 1999). In beagles, the biological half-life of 
cadmium fed at either 1, 3, 10, or 50 mg/kg diet is between 1 
and 2 years (Matsuno et al., 1991b). Because of this low excre- 
tory rate, cadmium accumulates with age, and older animals 
have significant levels of cadmium in their kidneys even if the 
levels in their diets and water were consistently low. 

Mechanism of Toxicity 

The toxic effects of cadmium are thought to be caused by 
free cadmium ions, and cadmium bound to metallothionein 
is usually less active (Goyer et al., 1989). Specific mecha- 
nisms of toxicity vary according to cadmium concentration 
and cell type but generally are linked to cadmium's disrup- 
tion of the cellular redox state and to its structural similari- 
ties with zinc, calcium, and other divalent cations (Pinot et 
al., 2000). The redox activity of cadmium depletes antioxi- 
dants and glutathione, causes oxidative stress, enhances lipid 
peroxidation, and perturbs the lipid composition of mem- 
branes (Gill et al., 1989; Xu et al., 2003). Cadmium affects 
the function of glomerular membranes by depleting 
polyanions and interfering with membrane charge (Cardenas 
et al., 1992). Cadmium-induced reactive oxygen intermedi- 
ates can lead to decreased DNA synthesis and strand breaks. 

Cadmium ions displace zinc and other metals from the 
binding sites of many metalloproteins, perturbing their ar- 
chitecture and functions. In the testes, cadmium interferes 
with zinc -protein transcription factors leading to apoptosis 
(Xu et al., 1999). Cadmium binding to metal transcription 
elements induces expression of a variety of genes including 
metallothionein, heme oxygenase, and heat shock proteins. 
Cadmium also has estrogenic effects by binding to the estro- 
gen receptor (Stoica et al., 2000), causing reproductive dis- 
ruption and accelerated puberty in mammals (Johnson et al., 
2003). Cadmium may permeate or block calcium channels 
and acts to mobilize free-calcium in the cell, causing cell- 
specific disregulation in function. 

Mitochondria are especially susceptible to cadmium tox- 
icity. Cadmium binds to thiol groups of proteins in the mem- 
brane of this organelle. Thiol group inactivation leads to 
oxidative stress and membrane depolarization. Impairment 
in mitochondrial function results in ATP depletion and ne- 
crotic death of the cell. In some cell types, cadmium-induced 
leakage of mitochondrial enzymes leads to apoptotic death 
(Pinot et al., 2000). Thus, depending on the cell type, cad- 
mium may cause either necrotic or apoptotic cell death. 



SOURCES AND BIOAVAILABILITY 
Sources 

Cadmium is sparsely distributed in the environment and 
normally ranges in concentrations between 0.1 and 1 mg/kg 
in the Earth's crust (Blinder, 1992). Levels 10- to 20-fold 
higher may accumulate in some types of sedimentary rocks. 
Levels in the oceans range from 0.001 to 0.1 |Jg/L, with 
higher levels found in phytoplankton-rich areas. Levels in 
natural surface and ground water are usually less than 1 |Jg/L 
(ATSDR, 1999; Pinot et al., 2000). However, spillage or 
leaching of water from mines can contaminate surface or 
ground waters, resulting in much higher levels. Levels in 
topsoil are usually higher than in subsoils due to atmospheric 
fallout and contamination. Unpolluted soils in the United 
States contain an average of 0.25 mg/kg (EPA, 1985). Higher 
concentrations of cadmium usually result from human ac- 
tivities such as mining, smelting, fuel combustion, and dis- 
posal of batteries and other cadmium-containing products. 
For example, soil levels within 1 kilometer of a smelter were 
72 mg/kg, compared with 1.4 mg/kg between 18 and 60 ki- 
lometers away. In 1998, there were 776 hazardous waste sites 
in the United States with focally high levels of cadmium 
contamination; animals grazing near these sites may con- 
sume high levels of cadmium via food or water. 

The most likely source of high cadmium exposure for 
most animals kept in captivity is the mineral supplement used 
in the feed. In particular, phosphate and zinc sources can be 
important contributors (King et al., 1992; Linden et al., 1999; 
Nicholson et al., 1999; Sapunar-Postruznik et al., 2001; Lin- 
den et al., 2003). Natural cadmium levels found in phos- 
phates from sedimentary rocks range from 3 to 100 mg/kg, 
depending upon their location (Singh, 1994). Phosphates 
from the southeastern United States have approximately 35 
mg/kg (IPCS, 1992a). Analysis of 16 calcium phosphates 
used in animal feeds in the United States found cadmium 
levels ranging from <1 to 67.3 mg/kg (Sullivan et al., 1994). 
Calcium phosphate is often added to animal feeds at levels 
greater than 1 percent of the diet, which can result in dietary 
cadmium levels that are high enough to cause unacceptably 
high levels in livers and kidneys intended for human 
consumption. 

Cadmium accumulates in plants grown in high-cadmium 
soils. Its mobility and bioavailability to plants is highest in 
acid soils. Cadmium taken up by plants is concentrated in 
the leaves with lower levels in seeds and roots (He and Singh, 
1994), so grazing animals receive the highest exposure 
(Hansen and Hinesly, 1979). Application of manure, sewage 
sludge, or phosphate fertilizers can enrich soils in cadmium. 
Consequently, limits on the maximum cadmium concentra- 
tion in fertilizers and biosolids applied to land, the maximal 
loading rate (kg/ha/y) of cadmium applied to land, and moni- 
toring procedures are set by the EPA and many state agen- 
cies (ATSDR, 1999; NRC, 2002). 



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MINERAL TOLERANCE OF ANIMALS 



In aquatic and benthic environments, cadmium concen- 
trations are relatively uniform tliroughiout tlie food web and 
cadmium does not appear to biomagnify as it moves up the 
food chain. In freshwater, the cadmium level in animals is 
dependent on their ability to absorb it from the water rather 
than their trophic level (IPCS, 1992b). 

Cadmium levels in human foods are highest in shellfish, 
oysters, salmon, and fungi, and are lowest in fruits, dairy 
products, legumes, meat, and poultry, with the exception of 
liver and kidney products . Potatoes and leafy vegetables have 
higher cadmium levels than most other foods of plant origin 
(ATSDR, 1999). 

The toxicity of cadmium is well recognized, and its con- 
centrations are monitored and regulated by a wide variety of 
government agencies (ATSDR, 1999). Cadmium is consid- 
ered a hazardous waste, and the EPA regulates levels in in- 
dustrial and municipal wastewaters, sewage sludge, and air 
emissions. State public health departments or departments 
of natural resources monitor cadmium levels in fish and wild- 
life that are potentially consumed and issue advisories to 
limit or prevent consumption of species of concern found in 
contaminated environments. The World Health Organization 
has set a 1 mg/kg upper limit for cadmium in complete feeds 
for animals in order to prevent high levels in food products 
(IPCS, 1992a). 

Bioavailability 

The bioavailability of various chemical forms of cadmium 
is dependent upon its chemical form. Cadmium is likely to 
be in the free ionic form in fresh water, whereas in foods it 
generally exists in a complex with a variety of ligands, in- 
cluding proteins such as metallothionein. The toxicological 
properties of cadmium ions do not seem to be dependent on 
the anion present. Cadmium chloride is the most common 
form used in animal experimentation because it is highly 
soluble and has a high bioavailability. In general, the 
bioavailability of cadmium chloride supplemented to water 
is similar to that supplemented to food (Ruoff et al., 1994). 
The cadmium found in a commercial calcium phosphate was 
found to be somewhat less bioavailable than cadmium chlo- 
ride to pigs (King et al., 1992). The bioavailability of cad- 
mium in animal tissues is usually less than that of cadmium 
chloride. For example, cadmium-metallothionein and cad- 
mium in pig liver have lower bioavailabilities and toxicities 
than cadmium chloride (Groten et al., 1990, 1994). The 
bioavailability of cadmium in horse kidney is lower than that 
of cadmium chloride and is not affected by cooking (Lind et 
al., 2001). The cadmium in the hepatopancreas of crab has 
lower bioavailability than cadmium chloride (Lind et al., 
1995). The decreased bioavailability of cadmium associated 
with high protein foods may be due to the ability of undi- 
gested oligopeptides to decrease the amount of free cadmium 
available to be absorbed from the intestine by complexing 
cadmium (Kojima et al., 1985). The bioavailability of 



cadmium in foods of plant origin such as mushrooms and 
lettuce is similar to that in cadmium salts (McKenna et al., 
1992; Lind et al., 1995). Phytate, which decreases the 
bioavailability of many divalent cations, appears to increase 
cadmium bioavailability to rats (Rimbach et al., 1995). 

The bioavailability of cadmium in water to aquatic organ- 
isms decreases with increasing organic carbon and oxygen- 
ation. Dissolved organic matter and particulates may absorb 
and render unavailable substantial amounts of cadmium. For 
these reasons, the effective cadmium content of water is of- 
ten expressed as the dissolved metal concentration, which is 
defined as the amount of metal in solution that passes through 
a 0.45 pm filter (EPA, 2001). Bioavailability of cadmium to 
aquatic organisms in freshwaters decreases with increasing 
water hardness and especially calcium content. In saltwaters 
with salinities above 10 g/kg, insoluble complexes of cad- 
mium chloride predominate and these are of considerably 
lower availability than ionic cadmium (Kramer et al., 1997). 
Thus, toxicity thresholds for aquatic animals must be inter- 
preted in the context of the characteristics of the water. 

TOXICOSIS 

Repeated exposure of humans to excessive levels of 
cadmium-containing dust or fumes, found usually at 
cadmium-producing and/or -consuming factories, can have 
irreversible effects on kidneys and lead to reduced lung ca- 
pacity and emphysema. High levels of cadmium in dust and 
soils found in or near toxic waste sites may also pose an 
inhalation threat, but this route of exposure is not likely to be 
of concern in animal husbandry and is not considered in this 
report. Toxicological profiles following exposure by this 
route have been extensively reviewed (IPCS, 1992a; Jarup et 
al., 1998; ATSDR, 1999). 

Virtually all major organ systems are affected by chronic 
consumption of foods or water containing high levels of cad- 
mium, with the kidney and the liver as the primary target or- 
gans in most species (Swiergosz-Kowalewska, 2001). Usu- 
ally, nephrotoxicity is the primary pathology leading to initial 
signs of toxicosis. Cadmium causes damage to proximal tu- 
bule cells and interstitial fibrosis in the kidney cortex, result- 
ing in proteinuria, glycosuria, amino aciduria, and polyuria. In 
dogs, mesangium cells of the glomerulus also become necrotic 
and the glomerular basement membrane thickens in rats. Cad- 
mium causes renal damage in rats when its concentration in 
the kidney is 10 mg/kg regardless of the type of cadmium 
complex that is administered (Min et al., 1986). 

Histopathological changes in the liver induced by chronic 
cadmium exposure include intralobular fibrosis, cirrhosis, 
focal mononuclear infiltration, and proliferation of the 
smooth endoplasmic reticulum. A mild osteomalacia has fre- 
quently been reported after chronic cadmium exposures. This 
may be due to direct effects on the turnover of bone matrix 
by stimulation of bone remodeling pathways (Regunathan et 
al., 2002), though it can also be secondary to decreased cal- 



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CADMIUM 



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cium and phosphorus absorption (Sugawara and Sugawara, 
1974). The effect on bone is exacerbated by multiparity 
(Whelton et al., 1994). In multiparous mice, decreased bone 
calcium is a more sensitive indicator of cadmium toxicosis 
than changes in renal function, but this is not seen in 
nonreproducing females (Bhattacharyya et al., 1988a). Ane- 
mia is also a commonly reported sign of cadmium toxicosis, 
though it may be secondary to decreased iron absorption. 
Cadmium causes decreased hematopoiesis and the resulting 
anemia is often one of the first detectible changes in toxico- 
sis. Cadmium-induced infertility, pancreatic necrosis, and 
decreased immunocompetence also contribute to the toxic- 
ity profile of cadmium. After withdrawal of cadmium, tissue 
cadmium levels decay only very slowly, and the signs of 
toxicosis require a half year or more to resolve (Nomiyama 
and Nomiyama, 1984). 

Because cadmium is poorly excreted and builds up over 
time, animals with higher rates of food intake appear to be 
affected at lower levels of dietary cadmium than animals 
with lower food intakes. Thus, the dietary concentration at 
which cadmium becomes toxic appears to be higher in small 
animals, such as rodents and quail, than in larger animals, 
such as monkeys and pigs, because mass-specific metabolic 
rate and food intake decrease with body size. 

In experimental animals, cadmium is carcinogenic after 
oral exposure. This is due to cadmium's prooxidant proper- 
ties, effects on cell proliferation, differentiation, and 
apoptosis, aberrant activation of protooncogenes, and inhi- 
bition of DNA repair and DNA methylation (Waalkes, 2003; 
Waisberg et al., 2003). In humans, occupational exposure by 
inhalation of cadmium dusts and fumes has been related to 
lung cancer; however, carcinogenic activity in human popu- 
lations exposed to high dietary levels of cadmium is not well 
documented by epidemiological studies (Pinot et al., 2000; 
Satoh et al., 2002). In 1993, cadmium was classified as a 
human carcinogen by the International Agency for Research 
on Cancer. Cadmium is teratogenic in birds and mammals 
(ATSDR, 1999; Kertesz and Fancsi, 2003). 

The rate of cadmium accumulation in the kidney and liver 
of rats is very similar for soluble cadmium delivered via the 
feed versus the water. When cadmium dosage is expressed 
on a daily intake basis (mg/kg BW/day), toxicity does not 
differ between food and water exposure (Ruoff et al., 1994). 
Because the data on the toxicity of cadmium in water are 
very limited in terrestrial animals other than rodents, it is 
useful to assume that intakes determined to be toxic when 
consumed in feeds are appropriate for estimating toxicity of 
cadmium in water. 

Toxicity of cadmium to fish and aquatic invertebrates is 
variable across species. Among fish, salmonids are particu- 
larly sensitive (EPA, 2001). Cadmium interferes with cal- 
cium uptake from water and causes hypocalcemia, malfor- 
mations of the spine, and infertility. The most sensitive life 
stages are the embryo and early larva. The EPA has con- 
ducted an extensive review of the published literature on 



cadmium toxicity to 125 species of aquatic animals (EPA, 
2001). Table 9-1 includes data from some of the studies on 
economically important species; however, the EPA docu- 
ment should be consulted for additional information. The 
acute toxicity of cadmium to freshwater fish is highly depen- 
dent on the hardness of the water. For example, the LC 50 of 
cadmium chloride to rainbow trout is 0.83 |Jg/L of cadmium 
at 30 mg/L hardness, but 5.23 pg/L at 90 mg/L hardness 
(Hansen et al., 2002a). Water used in aquaculture normally 
has a hardness considerably >50 mg/L. Because water hard- 
ness has such a marked impact on cadmium toxicity in 
aquatic environments, EPA toxicity criteria are standardized 
to a hardness of 50 mg/L and include a regression equation 
to adjust for other water hardness values. 

Studies on cadmium toxicity to animals since 1979 are 
listed in Table 9-1. Studies before 1980 are summarized in 
the previous version of this publication (NRC, 1980). Only 
selected studies in mice and fish are provided in Table 9-1, 
but thorough reviews of this extensive literature are avail- 
able elsewhere ( IPCS, 1992a,b; ATSDR, 1999; EPA, 2001). 

Single Dose 

Numerous human and animal studies indicate that a single 
oral dose of cadmium causes severe irritation to the gas- 
trointestinal epithelium (Andersen et al., 1988). Symptoms 
include nausea, vomiting, salivation, abdominal pain, and 
diarrhea (ATSDR, 1999). Desquamation and necrosis of the 
gastric and intestinal mucosa and dystrophy of the liver, 
heart, and kidneys are the primary pathological indications 
(Tarasenko et al., 1974). The single oral dose LD^q for cad- 
mium in rodents is dependent on the solubility of the source. 
Cadmium oxide and the highly soluble sulfate, chloride, 
nitrate, and iodide salts of cadmium have an LD^q of about 
50 mg Cd/kg BW, whereas insoluble cadmium metal and 
cadmium sulfide have an LD^q of about 900 mg/kg BW in 
mice (ATSDR, 1999). A single oral dose of 15.7 mg Cd/kg 
BW as cadmium chloride in water to mice does not cause 
histopathology of the stomach or liver (Andersen et al., 
1988). 

Acute 

In rats, an acute dose of cadmium (30 mg/kg BW/day) 
administered by gavage causes severe necrosis, hemorrhage, 
and ulcers in the gastrointestinal epithelium (Andersen et al., 
1988; Basinger et al., 1988). The pain of the gastroenteritis 
induced by high cadmium intake usually limits consumption 
and limits toxicity. In Japanese quail, feeding a diet contain- 
ing 75 mg/kg cadmium as cadmium sulfate results in de- 
creased egg production within 10 days of exposure (Bokori 
et al., 1995). In chickens, 50 mg/kg cadmium in the diet as 
cadmium chloride impairs egg production and egg weight 
(Hennig et al., 1968). 

The EPA (2001) has summarized all studies that examined 



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MINERAL TOLERANCE OF ANIMALS 



the acute toxicity of cadmium for more tfian 100 species of 
aquatic animals and then calculated the geometric mean toxic- 
ity for individual species (Species Mean Acute Value, or 
SMAV). These SMAV for cadmium are based on mortality 
(LC 50) and range from 1 1,859 |Jg/L for crayfish to 1.6 |ig/L 
for brown trout. The SMAV for other economically important 
freshwater animals are tilapia, 10,633; channel catfish, 5,055; 
common carp, 4,238; coho salmon, 6.2; rainbow trout, 2.1; 
striped bass, 2.9; and brook trout, 1 .8 |Jg/L. All of these values 
are adjusted to a water hardness of 50 mg/L. Tolerance to 
cadmium increases with increasing hardness with a slope of 
1.01; consequently fish in water with a hardness of 200 mg/L 
will be about four times less sensitive (EPA, 2001). 

Cadmium is much less toxic in saltwater than freshwater, 
and coho salmon have a SMAV of 1 ,500 |Jg/L when raised in 
saltwater compared to 6.2 |ig/L in freshwater. The SMAV 
for other saltwater species are sheepshead minnows, 50,000; 
striped mullet, 7,079; blue crab, 2,594; Pacific oyster, 228; 
American lobster, 78; and striped bass, 75 |Jg/L. 

Chronic 

Ruminants including cattle, sheep, and goats have been 
fed crops grown in high-cadmium soils or diets supple- 
mented with sewage sludge resulting in dietary cadmium 
levels of up to 13 mg/kg dry matter (Tables 9-1 and 9-2). In 
these studies, decreased performance was not reported and 
in some cases performance increased. Goats fed 28.5 mg/kg 
cadmium as cadmium chloride did not have significant 
changes in growth, feed intake, feed efficiency, or organ 
weights (Combs et al., 1983). A diet containing 10 mg/kg 
cadmium from cadmium chloride did not influence milk pro- 
duction in Holstein cows, but only four animals were used 
per treatment level (Sharma et al., 1979). A diet containing 
40 mg/kg cadmium from cadmium chloride did not influ- 
ence feed intake of calves, but 160 mg/kg resulted in de- 
creased feed intake (Powell et al., 1964). Unfortunately, sen- 
sitive measures of toxicosis, such as renal pathology or 
proteinuria, have not been measured in ruminants so a 
LOAEL cannot be accurately determined, but 10 mg/kg dry 
matter seems to be well tolerated using growth and general 
health as criteria. Several experiments have examined the 
effects of cadmium chloride in the water on sheep. In one 
experiment, cadmium at 3 mg/kg BW resulted in decreased 
testes weight, decreased semen volume, and lesions in the 
Sertoli cells, seminiferous tubules, and primary and second- 
ary spennatocytes (Lymberopoulos et al., 2000). Cadmium 
at 2.6 mg/kg BW given in water to lactating ewes resulted in 
pathological changes in the liver, kidney, and mammary 
gland. 

Among nonruminants, pigs fed 10 mg/kg cadmium from 
cadmium chloride have normal growth rates, but those fed 
50 mg/kg develop anemia (Cousins et al., 1973, Sharma et 
al., 1979). Dogs given 5 mg/L cadmium in water for 4 years 



develop signs of glomerular and tubular pathology, but those 
given 2.5 mg/L are normal (Anwar et al., 1961). Functional 
indications of cadmium toxicosis in rats and mice, such as 
increased blood pressure, have been observed at levels as 
low as 1 mg/kg diet, although most studies do not report 
changes at this level. Indications of pathology in the kidney 
or other tissues occur at 5 mg/kg diet, and decreased growth 
rates occur at about 30 mg/kg diet or 30 mg/L water (IPCS, 
1992a,b; ATSDR, 1999). In monkeys, a diet containing 3 
mg/kg cadmium did not cause any pathological or histo- 
pathological effects after 9 years, but 10 mg/kg resulted in 
decreased body weights (Masaoka et al., 1994). In another 
study, rhesus monkeys given 5 mg/kg cadmium BW/day 
orally developed renal failure and increased urinary excre- 
tion of total protein if they concurrently suffered from pro- 
tein calorie malnutrition (Prasad and Nath, 1995). Dogs fed 
cadmium at 1,3, 10, and 30 mg/kg from cadmium chloride 
for 3 months were clinically normal and did not have histo- 
pathological evidence of hepatic or renal damage (Loeser 
and Lorke, 1977). However, dogs fed 50 mg/kg of dietary 
cadmium from cadmium chloride for 8 years had renal atro- 
phy and renal tubule degeneration (Hamada et al., 1994) and 
diminished renal function (Kodama et al., 1992). Cadmium 
at 15 mg/L in the drinking water of ovariectomized beagles 
induced bone loss over a 7-month period (Bhattacharyya et 
al., 1992; Sacco-Gibson et al., 1992). 

Diets containing cadmium at 10 mg/kg do not decrease 
gain or cause anemia when fed to chickens, ducks, or quail. 
A diet containing cadmium at 20 mg/kg resulted in anemia 
and increased serum glutamic-pyruvate transaminase in mal- 
lards (Cain et al., 1983). Laying hens fed 3 mg/kg cadmium 
as cadmium sulfate had increased egg production, whereas 
those fed 12 mg/kg had decreased egg production in one 
experiment and decreased egg shell thickness in a second 
experiment (Leach et al., 1979). Broiler chickens fed 25 mg/ 
kg cadmium from cadmium sulfate develop focal fatty infil- 
tration of the liver, histiocytic infiltration of the jejunal mu- 
cosa, focal lymphohistiocytic interstitial infiltration, and fi- 
brosis of the kidney (Bokori et al., 1996). 

In fish, growth rate is not a good indicator of chronic 
toxicity. For example, female rainbow trout grow at a nor- 
mal rate when exposed to high levels of cadmium in the 
water, but their sexual development is markedly impaired 
and any eggs that are produced are sterile (Brown et al., 
1994). Unfortunately, the majority of toxicity studies in fish 
use growth rate and mortality as the primary endpoints. 
Based on the available literature, EPA has calculated mean 
chronic toxicity levels for a variety of aquatic animals (Mean 
Chronic Value, or MCV). The MCV for freshwater fish are 
tilapia, >23.6; smallmouth bass, 8.1; Atlantic salmon, 7.9; 
brook trout, 2.6; coho salmon, 4.2; and rainbow trout, 1.3 
|jg/L. All of these values are adjusted to a water hardness of 
50 mg/L. In these chronic studies, tolerance to cadmium in- 
creases with increasing hardness with a slope of 0.74. Con- 



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CADMIUM 



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sequently, fish in water with a hardness of 200 mg/L will be 
about three times less sensitive (EPA, 2001). 

Factors Influencing Toxicity 

The toxicity of cadmium is affected by the nutritional and 
physiological state of an animal. In Japanese quail, deficien- 
cies of zinc, iron, copper, calcium, or protein increase the 
tissue accumulation and toxicity of cadmium (Fox et al., 
1979, 1984). In rodents, physiological states that induce an 
increase in iron absorption, such as a fast growth rate or preg- 
nancy, increase cadmium absorption and toxicity (Schafer et 
al., 1990, Waalkes 1986). In humans, adult females absorb 
more cadmium than males, and this is at least partially due to 
higher incidence of anemia in females (Choudhury et al., 
2001). Low dietary calcium increases cadmium absorption 
and accumulation in tissues leading to cadmium toxicosis at 
lower levels than in well-nourished animals (Brzoska and 
Moniuszko-Jakoniuk, 1998). Furthermore, cadmium inter- 
feres with the metabolic functions of calcium, magnifying 
the deficiency. A similar situation occurs with zinc (Brzoska 
and Moniuszko-Jakoniuk, 2001). Age also is an important 
factor: in rats, cadmium is absorbed at higher rates in im- 
mature animals than in adults (Foulkes et al., 1991). Kidney 
damage from causes unrelated to cadmium toxicity, such as 
diabetes and old age, are expected to increase the sensitivity 
to nephrotoxicity caused by cadmium (Buchet et al., 1990). 

Cadmium toxicity can be markedly (>80 percent) reduced 
by high levels of several minerals, especially calcium, phos- 
phate, zinc, or iron (Groten et al., 1991). For example, a high 
level of dietary calcium decreases absorption and decreases 
the toxicity of absorbed cadmium (Brzoska and Moniuszko- 
Jakoniuk, 1998). Zinc protects against cellular toxicity of 
cadmium by reducing the accumulation of cadmium in cells 
and also by inducing metallothionein (Kaji et al., 1988). High 
levels of dietary zinc ameliorate the inhibition in egg pro- 
duction caused by cadmium in the diet of laying hens (Nolan 
and Brown, 2000). High intakes of vitamin E also reduce the 
development of toxicosis in growing chickens (Gupta and 
Kar, 1999), as does cysteine (Czarnecki and Baker, 1982). 

For aquatic organisms, the toxicity of cadmium decreases 
with increasing salinity of the water. The complexing of ionic 
cadmium with chloride and the high level of calcium in sea- 
water decrease cadmium absorption (Engel and Fowler, 
1979). Thus, the accumulation and toxicity of cadmium for a 
given species can be several orders of magnitude lower in 
ocean environments than freshwater environments. The pro- 
tective effect of calcium has been attributed to the lower 
toxicity of cadmium in hard water than in soft water (Gill 
and Epple, 1992; Brzoska and Moniuszko-Jakoniuk, 1998). 
Consequently, animals in saltwater environments receive 
most of their cadmium from their food, whereas those in 
freshwater obtain important amounts of cadmium from both 
food and water (IPCS, 1992b). 



TISSUE LEVELS 

Cadmium accumulation is greatest in the kidney, fol- 
lowed by liver, testes, pancreas, and spleen. Muscle and bone 
do not accumulate cadmium at high levels (Table 9-2). There 
are no major differences in the amount of cadmium that ac- 
cumulates across different types of muscles. Tissue levels 
increase with the time of exposure; when dietary levels are 
high, tissue levels eventually reach a plateau. For example, 
in ducks fed 20 mg/kg cadmium, muscle cadmium increases 
from 0.006 to 0.025 mg/kg fresh tissue during the first 30 
days and then further increases to 0.077 mg/kg during the 
next 30 days, but does not increase further thereafter (White 
et al., 1978). The plateau in muscle usually occurs relatively 
rapidly (months), but cadmium in kidney and liver accumu- 
lates over a much longer period of time. Once elevated lev- 
els of cadmium have accumulated, the withdrawal of cad- 
mium from the feed for a month or more does not result in 
appreciable loss of cadmium from tissues (White and Finley, 
1978; Sharma et al., 1979; Baxter et al., 1982). 

Phosphate sources used in animal diets are likely sources 
of cadmium contamination. In one study with pigs, a rock 
phosphate that contained an exceptionally high level of natu- 
ral cadmium contamination (100.3 mg/kg) was incorporated 
into growing and finishing feeds to provide 1.2 mg/kg cad- 
mium final feed. Pigs fed this diet to a 90-kg slaughter weight 
did not have detectable levels of cadmium in their muscle or 
fat (<0.3 mg/kg dry weight), but their livers and kidneys 
contained 0.35 and 1 .68 mg/kg, respectively (King et al., 1992). 

The relationship between cadmium intake and the con- 
centration in the liver and kidneys of rats is linear over a 
very wide range (Ruoff et al., 1994). At realistic levels of 
intake (<10 |Jg/kg BW/day), the slope of the relationship be- 
tween intake and tissue cadmium (wet weight) is 0.11 for 
kidney cortex and 0.04 for liver. Several dietary factors can 
modify the amount of cadmium that accumulates in tissues. 
High levels of dietary zinc, calcium, or cysteine decrease 
accumulation (Czarnecki and Baker, 1982; Lamphere et al., 
1984). 

In fish, the deposition of cadmium is usually highest in 
the gills and kidney, with lower levels in the liver. More than 
90 percent of the total whole body cadmium in rainbow trout 
exposed to high levels of cadmium is found in these three 
tissues (Kay et al., 1986). Experiments examining cadmium 
accumulation from the water typically use water with suffi- 
cient hardness to prevent acute toxicity and mortality in 
the fish. 

Cadmium transfer from the laying hen to the egg is very 
inefficient. Hens with very high cadmium burdens (100 mg/ 
kg in liver) lay eggs with undetectable levels of cadmium in 
the albumen and approximately 0.1 mg/kg dry weight in the 
yolk (Sato et al., 1997; Leach et al., 1979). Cadmium trans- 
fer to milk is similarly inefficient (Sharma et al., 1979; Smith 
et al., 1991b). Feeding heifers a diet containing 5 mg/kg cad- 



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MINERAL TOLERANCE OF ANIMALS 



mium for more than a year before parturition and tlirougfiout 
lactation did not result in significantly elevated milk cad- 
mium levels during any part of lactation. Using radioactive 
cadmium, it was found that most cadmium in milk is bound 
to casein, with lesser amounts bound to albumin and lactose, 
and none detectable in fat (Van Bruwaene et al., 1982). 

Regardless of the level of cadmium fed, the concentration 
of cadmium in meat, milk, and eggs is always lower than the 
level in the diet that the animal consumed (on a dry matter 
versus dry matter basis). Thus, foods derived from these 
products decrease exposure of humans to environmental cad- 
mium. However, levels in kidney and liver are always con- 
siderably higher than the levels in the diet and these tissues 
magnify environmental cadmium (Morcombe et al., 1994). 

Kidneys can make their way into a variety of human 
foods. The kidneys of poultry are located in recesses of the 
sacrum and ilium and remain with the carcass after the rest 
of the viscera are removed during processing. After egg pro- 
duction ceases, hens are usually more than 1 year old and 
have kidney cadmium levels of 0.80 mg/kg (Murphy et al., 
1979). In the United States, mechanically deboned meat 
made from the frames of spent hens was found to contain 
0.02 mg/kg (Murphy etal., 1979). However, if only the back, 
which contains the kidneys, was used to make mechanically 
deboned poultry meat, the cadmium concentration would be 
0.096. Consequently, a regulation was issued by the Food 
Safety and Inspection Service requiring the removal of kid- 
neys from mature chickens and turkeys at slaughter. How- 
ever, the value of this requirement has been questioned based 
on surveys of cadmium levels in products (Vos et al., 1990). 
Mechanically deboned meat from broilers should not be a 
problem. Broilers, which are usually marketed at approxi- 
mately 6-7 weeks of age, have 0.05 mg/kg cadmium in their 
kidneys and <0.005 mg/kg fresh weight in their muscle 
(Murphy et al., 1979). 

MAXIMUM TOLERABLE LEVELS 

The MTL of cadmium is defined as the dietary level that, 
when fed for a defined period of time, will not impair ac- 
cepted indices of animal health or performance. Animals are 
able to tolerate acute exposure to 25 mg/kg cadmium in the 
diet for a few days. Subtle histological signs of toxicosis in 
rodents occur after chronic consumption of 1 mg cadmium/ 
kg diet for several months. Dietary levels of 10 mg/kg are 
tolerated chronically by poultry and livestock species, but 
these levels result in unacceptable levels of cadmium in kid- 
neys, livers, and, in some cases, muscles of animals. Rhesus 
monkeys can tolerate 3 mg/kg diet for 9 years without signs 
of pathology. Dogs can tolerate 10 mg/kg diet for 8 years. 
The toxicity of cadmium in water appears to be very similar 
to that in feeds. For this reason, the total daily intake of both 
feed and water sources should be considered. The toxicity of 
cadmium to fish is extremely variable with trout being the 
most susceptible. Cadmium at a level of 1 |Jg/L is tolerated 



by all species in all water conditions. With hard water, trout 
and all other species tested can tolerate 4 |Jg/L. 

HUMAN HEALTH 

Animals are able to tolerate levels of cadmium in their 
diet that result in accumulation of cadmium in their kidneys 
and livers at concentrations that are of concern for humans 
consuming foods made from these organs. Consequently, 
maximum dietary levels in the feed of animals used for 
human food should be set on the basis of human health and 
not animal health. 

Based on residue levels, the World Health Organization 
has set a 1 mg/kg upper limit for cadmium in complete feeds 
for animals (IPCS, 1992a). 

FUTURE RESEARCH NEEDS 

The level of cadmium in the diet of agriculturally impor- 
tant animals should be based on cadmium content of tissues 
used as human foods and not on the maximum tolerable level 
for the animal itself. Few studies have examined the dose- 
response relationship between dietary cadmium and tissue 
cadmium in production animals at levels <1 mg/kg diet. 
Better definition of the dose-response relationship is required 
to establish the maximal tolerable levels of cadmium in ani- 
mal feeds based on residue levels. Because cadmium accu- 
mulates over many years, examining this relationship in rela- 
tively long-lived animals such as dairy cattle and laying hens 
is especially needed. 

Toxicity of cadmium to aquatic species has mainly uti- 
lized growth rate and mortality as endpoints. Use of more 
sensitive criteria such as pathological changes in kidneys, 
gills, and liver is needed to define more precisely chronic 
toxicities. Cadmium tolerance and signs of toxicosis in com- 
panion animal species have not been investigated. Given the 
high proportion of kidney and liver tissues in meat meals 
that are commonly fed to these species, investigation is 
warranted. 

SUMMARY 

Cadmium is industrially valuable for batteries, pigments, 
and coatings. It enters the environment by a wide variety of 
anthropogenic means and may also be high in phosphate and 
zinc supplements fed to animals. Cadmium is known to be 
essential in diatoms, but essentiality has not been established 
in animals. Cadmium is not absorbed very efficiently, but 
once it enters the body, it is excreted very slowly and accu- 
mulates according to lifetime exposure. The bioavailability 
of cadmium is mostly similar across feed types and is also 
similar between feed and water. High intakes of cadmium 
from the feed or from water can cause decreased growth 
rates, anemia, kidney damage, osteomalacia, infertility, and 
hypertension. High dietary cadmium can also interfere with 



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CADMIUM 



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the absorption and metabolism of other nutrients, especially 
zinc and calcium. Cadmium accumulates to very high lev- 
els in the kidney and liver. This accumulation limits the 
safe levels of cadmium that can be fed to animals destined 
for human consumption. Cadmium is not transferred effi- 
ciently to milk or eggs, and these foods have much lower 
levels of cadmium than those found in the diet of the cow 
or hen. Levels in muscle are also lower than dietary levels. 
Further research is needed to determine the dose-response 
relationship between dietary cadmium at low levels of 
supplementation and the levels in tissues destined for hu- 
man consumption. 

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Calcium 



INTRODUCTION 

Calcium (Ca) atoms do not exist in their free state — but 
calcium-containing minerals are common in nature. Fairly 
pure deposits of calcium carbonate in the form of limestone 
are common and are a major source of calcium used in ani- 
mal diet formulation. Nearly pure calcium sulfate is also 
found in the form of gypsum that is used in Portland cement, 
drywall, and plaster production as well as animal feeds. Cal- 
cium hydroxide (lime) is not commonly used as a dietary 
supplement but is often used as an astringent to reduce bac- 
terial loads during disinfection of animal pens. Calcium 
phosphate salts are used for the manufacture of certain types 
of glass, but they are more commonly used as a source of 
calcium and phosphate in animal feeds. Calcium chloride is 
used in animal feeds when acidification of blood and urine is 
desired. Calcium polysulfide (lime sulfur, "wettable sulfur") 
is used as a fungicide, especially in orchards and around or- 
namental shrubbery. 

Calcium is a positively charged alkaline Earth metal with 
an atomic weight of 40.08 and an equivalent weight of 20.04. 
Calcium is a divalent cation. Chloride and sulfate salts of 
calcium are water soluble; most other inorganic calcium salts 
are only slightly soluble in water. Organic salts of calcium, 
such as calcium propionate, are very soluble and are increas- 
ingly being used in diets for man and animals. 



ESSENTIALITY 

Extracellular calcium is essential for formation of skel- 
etal tissues, transmission of nervous tissue impulses, excita- 
tion of skeletal and cardiac muscle contraction, blood clot- 
ting, eggshell formation, and as a component of milk. 
Intracellular calcium, while 1/10,000 the concentration of 
extracellular calcium, is involved in the activity of a wide 
array of enzymes and serves as an important "second mes- 
senger" conveying information from the surface of the cell 
to the interior of the cell. 



About 98 percent of the calcium in the body is located 
within the skeleton where calcium, along with phosphate 
anion, serves to provide structural strength and hardness to 
bone. The other 2 percent of the calcium in the body is found 
primarily in the extracellular fluids of the body. Plasma cal- 
cium concentration is normally 90-110 mg/L or 2.25-2.75 
mM. The ionized calcium concentration of the plasma of 
mammals and birds must be maintained at a relatively con- 
stant value of 1-1.25 mM to ensure normal nerve membrane 
and muscle end plate electrical potential and conductivity, 
forcing vertebrates to evolve an elaborate system to main- 
tain calcium homeostasis. This system attempts to maintain 
extracellular calcium concentration constant by increasing 
calcium entry into the extracellular fluids whenever there is 
a loss of calcium from the extracellular compartment. When 
dietary calcium is inadequate this system may sacrifice bone 
calcium stores in an attempt to maintain nonnal blood cal- 
cium concentration. 

Most farm species will require diets that are between 0.5 
and 1 percent calcium. Optimal growth and reproduction, 
including lactation, will require more calcium than mainte- 
nance of the adult animal. Hens laying eggs require about 
3.5 percent calcium in the diet to maintain quality of the 
eggshell. 

Cold-blooded species can vary greatly in their require- 
ment for dietary calcium. Softshell turtles raised for meat 
grew best when their diet was 5.7 percent calcium (and 3 
percent phosphorus) (Huang et al., 2003). Some fish species 
reared in fresh water or seawater can obtain all the calcium 
they need from the water in which they reside. In freshwater 
species, the gills can absorb calcium from the water. Brook 
trout, for example, can obtain as much as 80 percent of their 
skeletal calcium requirement from water (McCay, 1936). Sea 
fish drink enough seawater to obtain much if not all of their 
calcium needs. However some freshwater and saltwater fish 
also require calcium supplementation for optimal growth 
(about 0.2 percent calcium added to the diet) as they cannot 
obtain enough calcium from the water alone (Robinson et 



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MINERAL TOLERANCE OF ANIMALS 



al., 1986; Chavez-Sanchez et al., 2000; Hossain and 
Furuichi, 2000). 

DIFFICULTIES IN METHODS OF ANALYSIS AND 
EVALUATION 

Determination of calcium in feeds and tissues is best ac- 
complished by wet or dry ashing of the sample followed by 
resuspension of the ash in an acidic solution for analysis by 
atomic absorption spectrophotometry. Atomic absorption is 
conducted at a wavelength of 422.7 nm and can detect as 
little as 0.01 mg Ca/L. Phosphate, sulfate, and aluminum 
that might be in the sample can interfere with calcium absorp- 
tion spectra, but their effect is masked by the addition of 
lanthanum to the standards and samples being analyzed. 
Since low calcium values result if the pH of the sample is 
above 7, both standards and samples are prepared in dilute 
hydrochloric acid solution. Concentrations of magnesium 
greater than 1,000 mg/L can also cause low calcium values. 
Concentrations of up to 500 mg/L each of sodium, potassium, 
and nitrate cause no interference. Anionic chemical interfer- 
ences can be expected if lanthanum is not used in samples 
and standards (EPA, 1983). The nitrous oxide-acetylene 
flame will provide two to five times greater sensitivity than 
an air-acetylene flame, but is not necessary for routine 
analysis of feeds or biological samples. Near-infrared spec- 
trophotometry is not a satisfactory method of determining 
calcium content of feedstuffs and forages, though it is often 
used for that purpose. 

Calcium concentrations can also be measured to ppb (ug/L) 
levels using inductively coupled plasma optical emission 
spectrophotometry (ICP-OES). ICP-OES uses radio fre- 
quency-generated plasma to excite the electrons of the 
calcium atoms, which then produce photons unique to 
calcium. Photomultiplier tubes detect and quantitate the 
photons emitted by the excited calcium atoms, allowing 
quantitation of the calcium concentration. An inductively- 
coupled plasma source atomizes and excites even the most 
refractory elements with high efficiency, so there is less 
interference and about 10-fold greater sensitivity using 
ICP-OES than using atomic absorption spectrometry. 

REGULATION AND METABOLISM 

When calcium loss exceeds entry, hypocalcemia can oc- 
cur. This results in loss of nerve and muscle function which 
can, in some instances, lead to recumbency (milk fever in 
cows) or tetany (lactating dogs, cows, pigs). During vitamin 
D intoxication, calcium can enter the extracellular compart- 
ment faster than it leaves, resulting in hypercalcemia, which 
can lead to soft tissue deposition of calcium and then eventu- 
ally necrosis of the tissues. 

Calcium leaves the extracellular fluids during bone for- 
mation, in digestive secretions, sweat, and urine. An espe- 
cially large loss of calcium occurs during lactation or egg- 



shell formation. Calcium lost via these routes can be replaced 
from dietary calcium, from resorption of calcium stored in 
bone, or by resorption of a larger portion of the calcium fil- 
tered across the renal glomerulus (i.e., reducing urinary cal- 
cium loss). Whenever calcium loss from the extracellular 
fluids exceeds the amount of calcium entering the extracel- 
lular fluids, plasma calcium concentration decreases. The 
parathyroid glands monitor carotid artery blood calcium con- 
centration and secrete parathyroid hormone when they sense 
a decrease in blood calcium concentration. Parathyroid hor- 
mone immediately increases renal calcium reabsorption 
mechanisms to reduce urinary calcium loss. This will suc- 
ceed in returning blood calcium concentration to normal if 
the loss from the extracellular compartment is small, because 
normally only small amounts of calcium are excreted in the 
urine each day. For example, a 600-kg cow typically ex- 
cretes 0.5-2 g calcium in her urine each day. When calcium 
losses are larger, parathyroid honnone will stimulate pro- 
cesses to enhance intestinal calcium absorption and resorp- 
tion of bone calcium stores. 

Bone is a living tissue that is constantly undergoing for- 
mation and resorption. In young animals the rate of forma- 
tion exceeds the rate of resorption, resulting in net bone ac- 
cretion. In mature animals, portions of the skeleton, 
presumably those traumatized with microfractures during 
normal wear and tear, are resorbed and reformed constantly. 
In humans it is estimated that the entire adult skeleton is 
rebuilt every 7 years (Frost, 1964). Parathyroid hormone can 
uncouple bone resorption from bone formation, stimulating 
resorptive mechanisms of bone osteoclasts while inhibiting 
formation mediated by bone osteoblasts. The net result is an 
efflux of calcium from bone to extracellular fluids. When 
excessive calcium enters the blood, another hormone, calci- 
tonin, produced within the thyroid gland, is secreted. Calci- 
tonin inhibits osteoclastic bone resorption and stimulates re- 
nal excretion of calcium. 

Ultimately dietary calcium must enter the extracellular 
fluids to permit optimal performance of the animal. Calcium 
absorption can occur by passive transport between epithelial 
cells across any portion of the digestive tract whenever ion- 
ized calcium concentration in the digestive fluids directly 
over the mucosa exceeds 6 mM (Bronner, 1987). These con- 
centrations are reached when calves are fed all-milk diets 
and when cows are given oral calcium drenches for preven- 
tion of hypocalcemia (Goff and Horst, 1993). In nonruminant 
species, studies suggest that as much as 50 percent of dietary 
calcium absorption can be passive (Nellans, 1988). It is 
unknown how much passive absorption of calcium occurs 
from the diets typically fed to ruminants, but the diluting 
effect of the rumen would likely reduce the degree to which 
passive calcium absorption would occur. Active transport of 
calcium appears to be the major route for calcium absorption 
in mature animals and this process is controlled by 1,25- 
dihydroxy vitamin D, the hormone derived from vitamin D. 
Vitamin D, produced within the skin or provided in the diet. 



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CALCIUM 



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is converted to 25-hydroxyvitamin D in tlie liver and can be 
further metabolized to 1,25-dihydroxyvitamin D in the kid- 
neys. Parathyroid hormone indirectly stimulates intestinal 
calcium absorption because it is the primary regulator of re- 
nal production of 1,25-dihydroxyvitamin D. The 1,25- 
dihydroxyvitamin D is released to the circulation and inter- 
acts with nuclear receptors within the intestinal epithelium, 
primarily in the small intestine, causing transcription and 
translation of calcium transport proteins. Vitamin D-depen- 
dent calcium-binding protein captures calcium at the apical 
surface of epithelial cells and ferries the calcium to the 
basolateral side of the cell. There it is pumped into the extra- 
cellular space against a concentration gradient by a 1,25- 
dihydroxyvitamin D-dependent calcium pump (Wasserman, 
1981; Bronner, 1987). By carefully regulating the amount of 
1,25-dihydroxyvitamin D produced, the amount of dietary 
calcium absorbed can be adjusted up or down to maintain 
constant extracellular calcium concentration. 

Freshwater fish can obtain calcium from the water across 
their gills. If the water in which they are reared is high in 
calcium, they have little need for dietary calcium and little 
need for vitamin D as intestinal calcium absorption is not 
critical to meeting their calcium requirement. However, 
when water calcium content is low the diet must supply cal- 
cium, and the fish require vitamin D to use intestinal calcium 
absorption efficiently (Lovell and Li, 1978). When freshwa- 
ter fish must rely on their diet to meet calcium needs, they 
also face another problem that terrestrial animals do not en- 
counter: many do not possess gastric glands and cannot se- 
crete hydrochloric acid into the ingesta. Therefore the in- 
ability to solubilize dietary calcium can reduce absorption of 
calcium incorporated into feed. Saltwater fish drink seawa- 
ter where the calcium is already in a soluble form, so the 
calcium is readily available for intestinal absorption. 

SOURCES AND BIOAVAILABILITY 

Calcium content of grains and high-starch feedstuffs is 
very low and generally of low importance as a source of 
calcium that might cause toxicity in an animal. Legumes can 
be an important source of calcium for herbivores but the 
bioavailability of calcium in plants is generally lower than 
that of mineral sources (Martz et al., 1990). Common min- 
eral sources of calcium used to supplement diets of animals 
include calcium carbonate, calcium sulfate, mono- and di- 
basic calcium phosphate, and calcium chloride. Bone meal 
and oyster shells are also good sources of calcium. In rumi- 
nants, calcium carbonate may have low bioavailability due 
to its poor solubility. However, if finely ground, the calcium 
in calcium carbonate becomes readily solubilized upon con- 
tact with the acid of the abomasum and availability can be 
quite high. In pigs, particle size of calcium carbonate had no 
effect on calcium bioavailability (Ross et al., 1984). Cal- 
cium from these mineral sources can be from 70-90 percent 
available. During positive calcium balance, intestinal mecha- 



nisms for absorption are shut down in most species. Notable 
exceptions are the horse and rabbit (and possibly other hind 
gut fermenters). In these species calcium absorption appears 
to be independent of vitamin D and a large proportion of 
dietary calcium is always absorbed (Bourdeau et al., 1986; 
Brommage et al., 1988; Maenpaa et al., 1988). 

Water can supply a small portion of the dietary calcium 
requirement of birds and mammals but does not contain 
enough calcium to cause toxicosis. Many freshwater fish 
thrive in the hardest waters (hardness up to 300 mg calcium 
carbonate/L) as it reduces the difference between the osmo- 
larity of the fish's body and the water and reduces the energy 
required by the fish to osmoregulate (Boyd, 1979). 

TOXICOSIS 
Single Dose 

If a large bolus dose of calcium is given to an animal 
orally it is possible for the concentration of calcium directly 
over the mucosa of the intestinal tract to rise to the level at 
which passive absorption of calcium occurs by paracellular 
transport (Bronner, 1987). This pathway is not regulated by 
the calcium homeostatic mechanisms. As a result, very large 
amounts of readily soluble calcium administered as a single 
dose can cause hypercalcemia for several hours (Goff and 
Horst, 1994) (Table 10-1). Moderate hypercalcemia (plasma 
calcium between 120 and 150 mg/L or 3-3.75 mM) causes 
increased urine excretion (diuresis) and depresses feed in- 
take. Severe hypercalcemia (plasma calcium between 150 
and 250 mg/L or 3.75-6.25 mM) will begin the process of 
metastatic calcification of soft tissues, which may or may 
not be reversible. In some cases blood calcium can increase 
to the point that the heart stops during systole (plasma cal- 
cium above 280 mg/L or 7 mM) (Littledike et al., 1976). 
These definitions of hypercalcemia do not apply to laying 
hens — their total plasma calcium content is typically 200- 
400 mg/L or 5-10 mM. However, their ionized calcium con- 
tent is 40-50 mg/L or 1-1.25 mM, similar to other animals 
(Puis, 1994). 

In cattle, oral calcium drenches are used to prevent and/or 
treat the hypocalcemia that is common in dairy animals 
around the time of calving and the onset of lactation. Cal- 
cium chloride and calcium propionate are very soluble cal- 
cium salts that have been used widely for this purpose 
(Jonsson and Pehrson, 1970; Goff et al., 1996). Administer- 
ing a concentrated solution that supplied 50 g of calcium 
from calcium chloride into the oral cavity increased plasma 
calcium by 30-40 mg/L or 0.75-1.0 mM, which was main- 
tained for 4-6 hours. Administering 100 g of calcium from 
calcium chloride increased plasma calcium by 60-80 mg/L 
(1.5-2.0 mM), which stayed elevated for 6-8 hours. These 
doses did not cause the toxic effects associated with severe 
hypercalcemia (Jonsson and Pehrson, 1970; Goff and Horst, 
1994). However, it was noted that repeated doses of the 



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MINERAL TOLERANCE OF ANIMALS 



higher quantities of calcium chloride induced a life-threatening 
metabolic acidosis, a result of the chloride anion being 
absorbed along with the calcium (Goff and Horst, 1993). 
The use of chloride salts of calcium has also been associated 
with erythema and necrosis of the abomasum and rumen 
(Wentink and van den Ingh, 1992). Using calcium propi- 
onate as the source of calcium has similar effects on plasma 
calcium concentration but does not cause metabolic acidosis 
(Goff and Horst, 1994; Pehrson et al., 1998). Placement of 
the calcium into the back of the pharynx can elicit closure of 
the esophageal groove in cattle, allowing a proportion of the 
solution being administered to bypass the rumen. This causes 
a greater increase in blood calcium than occurs if the cal- 
cium is placed into the rumen via use of a tube or hose end- 
ing in the rumen (Goff and Horst, 1993). Cows receiving 
146 g calcium as a drench delivered into the esophagus (be- 
yond the pharynx) on the day of calving as calcium propi- 
onate had rapid increases in plasma calcium, with no delete- 
rious effects noted. Cows receiving 219 g calcium as calcium 
propionate also had rapid increases in plasma calcium. The 
hypercalcemia was not considered severe and lasted less than 
24 hours. However, it was accompanied by profound hypo- 
magnesemia, which was considered an undesirable effect. 
Calcium propionate delivered into the esophagus at a quan- 
tity of 1.36 kg (supplying 285 g calcium) caused severe hy- 
percalcemia in one of four cows given this dose at calving. 
The same dose given to two steers was found to be lethal in 
both (Goff et al., 2002), suggesting the calcium demands of 
lactation offered some protection from the development 
of acute severe hypercalcemia following oral administration 
of calcium. 

Administering 50 g calcium from calcium carbonate to 
dairy cows did not alter blood calcium concentration, sug- 
gesting that the more soluble the form of calcium adminis- 
tered in a single oral dose the greater the risk of hypercalce- 
mia developing (Goff and Horst, 1993). 

Calcium polysulfide (lime sulfur), used to control fungal 
diseases in trees and shrubs, has the potential for accidental 
incorporation into diets as a calcium source. The animals 
could become sick from the metabolic acidosis induced by 
the anionic portion of the calcium polysulfide molecule. The 
calcium itself is not the toxic moiety (Horowitz et al., 1997). 

Acute 

In animals with intact calcium homeostatic mechanisms, 
short-term increases in dietary calcium are very well toler- 
ated. The production of 1, 25 -dihydroxy vitamin D will be 
downregulated to decrease intestinal calcium absorption of 
dietary calcium. If kidneys are functioning properly, any 
excess calcium that is absorbed will be rapidly excreted with 
the urine. In the horse and rabbit the intestine is not a regula- 
tory point of calcium homeostasis. Intestinal calcium absorp- 
tion mechanisms are always turned on in these species, and 
feeding high dietary calcium increases the amount of cal- 



cium that enters the blood. Instead, these species use renal 
calcium excretion to control blood calcium concentration. 
During renal failure these species are in danger of develop- 
ing hypercalcemia even when dietary calcium is within nor- 
mal limits. 

Young growing pigs may be more likely than other spe- 
cies to develop hypercalcemia with increasing dietary cal- 
cium (Reinhart and Mahan, 1986; Hall et al., 1991). In one 
case report, a 5.62 percent calcium diet was accidentally fed 
to piglets after weaning (the requirement was about 0.8 per- 
cent diet calcium). Several of the piglets were severely hy- 
percalcemic with blood calcium greater than 200 mg/L 
(5 mM). All grew poorly and had severely reduced feed in- 
take. They were dehydrated, had markedly dry and hard fe- 
ces, and were drowsy (Kamphues et al., 1989). In this case, 
dietary calcium concentration was high enough to permit cal- 
cium to be absorbed by the paracellular, vitamin-D indepen- 
dent pathway at a greater rate than the kidneys could excrete 
the calcium. 

Chronic 

Feeding excessive dietary calcium long term is generally 
not associated with any specific calcium-based toxicity. Hy- 
percalcemia generally does not occur if calcium homeostatic 
mechanisms are intact. The main effect directly attributable 
to calcium is a reduction in feed intake as more calcium min- 
eral is added to the diet. This may simply be a palatability 
issue or it could be mediated by calcitonin, a hormone pro- 
duced in the gut following a meal, and by thyroid C-cells, in 
response to even slight elevations of calcium concentration. 
Excessive calcitonin can inhibit feed intake (Freed et al., 
1979). A second common effect of calcium in the diet is to 
reduce the availability of other minerals in the diet, such as 
phosphorus or zinc, especially if the animal is receiving a 
diet that is marginally adequate in these other minerals. 

Rabbits can be fed as much as 4.5 percent calcium diets 
with no ill effects (Chapin and Smith, 1967). Horses fed di- 
ets that were 2.5 percent calcium for four years exhibited no 
ill effects provided the diets also had adequate phosphorus 
(Jordan et al., 1975). Renal excretion of calcium increases 
with increased dietary calcium concentration in rabbits and 
horses to prevent development of hypercalcemia and its at- 
tendant problems (Schryver et al., 1974). 

Feed intake in dogs and cats is largely dictated by energy 
content of the diet and therefore feeding standards for dogs 
and cats often express calcium and phosphorus contents of 
diets in terms of g/1,000 kcal metabolizable energy. For this 
report, the diet calcium levels will be expressed as g/kg diet 
DM. If the original literature cited expressed diet calcium 
only in terms of diet energy, the assumption was made that 
typical dog and cat diets are 4,000 kcal ME/kg DM. For 
puppies, the dietary calcium and phosphorus allowances are 
12 g Ca and 10 g P/kg DM (NRC, 2006). For kittens, the 
dietary calcium and phosphorus allowances are 8 g Ca and 



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CALCIUM 



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7.2 g P/kg DM (NRC, 2006). These allowances decrease 
with age as growth slows in the animals. 

In large-breed dogs, bone growth is especially rapid the 
first three months of life. Disturbances in endochondral ossi- 
fication are a frequent cause of clinical problems in the giant 
breed dogs, particularly in Great Danes. Giant breed dogs 
fed high calcium diets may be at greater risk of developing 
osteochondrosis, rickets, and other bone problems than dogs 
fed lower calcium diets, especially if the diet provides 
enough energy and protein to support rapid growth, or is 
marginal in phosphorus. Ad libitum feeding may increase 
the risk of osteochondrosis over feed restriction, where 
growth is slowed (Hedhammer et al., 1974). Feeding Great 
Dane puppies diets that were 2.72 percent calcium and 0.9 
percent phosphorus greatly increased the incidence of osteo- 
chondrosis and skeletal problems and caused significant hy- 
pophosphatemia in some animals (Hazewinkel et al., 1985; 
Goedegebuure and Hazewinkel, 1986). If dietary phospho- 
rus is increased along with the dietary calcium, the negative 
effects of dietary calcium are largely, but not entirely, nulli- 
fied (Weber et al., 2000; Lauten et al., 2002). Schoenmakers 
et al. (2000) concluded that feeding increased diet calcium 
alone decreased the amount of phosphorus available for bone 
formation, which resulted in rachitic lesions, while increas- 
ing both diet calcium and phosphorus increased the risk of 
osteochondrosis lesions in fast-growing Great Danes. When 
the diet calcium to phosphorus ratio exceeds 2: 1 there is risk 
of increased osteochondrosis in the Great Dane breed 
(Hazewinkel et al., 1991; Schoenmakers et al., 2000). The 
Great Dane breed may be rather unique in its sensitivity to 
dietary calcium concentration, as Giant Schnauzers fed simi- 
lar diets show no osteochondrosis bone lesions (Weber et al., 
2000). Miniature poodles fed diets that were as high as 3.3 
percent calcium and just 0.9 percent phosphorus still man- 
aged to grow well and had no bone problems, though they 
did have a temporary decrease in feed intake at 11 weeks of 
age when compared to poodle pups fed a 1.1 percent cal- 
cium, 0.9 percent phosphorus diet (Nap et al., 1993). A sum- 
mary of trials performed by a commercial pet food manufac- 
turer demonstrated that puppies across a variety of breeds 
exhibited no skeletal problems when fed up to 2.0 percent 
calcium on a DM basis — even if diet phosphorus was not 
increased. When diet calcium exceeded 2.3 percent, feed in- 
take and growth were depressed in many breeds (Laflamme, 
2000). 

Few studies have examined the effects of elevated diet 
calcium in cats. Howard et al. (1998) observed reduced feed 
intake and growth in kittens fed 2.3 percent calcium diets. 
The kittens also exhibited pronounced hypomagnesemia. 

Calves are typically fed milk (whole milk is approxi- 
mately 0.95 percent calcium on a DM basis) or milk replacer 
that is 0.9-1 . 1 percent calcium. At weaning, the diet calcium 
is generally reduced to 0.6-0.7 percent. Maintenance diets 
for adult cattle are typically 0.45-0.6 percent calcium. In 
lactating cows the diet calcium is increased to 0.7-0.9 per- 



cent calcium to accommodate lactational demands for cal- 
cium. In cattle, dietary calcium concentrations greater than 1 
percent have been associated with reduced DM intake and 
lower performance (Miller, 1983). The amount of mineral 
added to some of these rations to achieve the higher calcium 
levels is often unpalatable and also replaces energy or pro- 
tein that could be used by the animal for growth or other 
functions. Beede et al. (2001) fed 0.47, 0.98, 1.52, and 1.95 
percent calcium diets to cows in late gestation that were re- 
ceiving a high-chloride diet to prevent milk fever. Cows fed 
1.5 percent calcium diets tended to eat less than cows of- 
fered 0.98 percent or 0.47 percent calcium diets, while those 
fed the 1 .95 percent calcium diet had significantly lower feed 
intake. Dietary calcium did not influence the degree of hy- 
pocalcemia experienced at calving or milk production in the 
subsequent lactation. 

When diets suitable for lactating cows (0.85 percent cal- 
cium) were fed to mature bulls for prolonged periods, the 
bulls were reported to have developed osteopetrosis of the 
bone and ultimo branchial tumors of the thyroid gland, pre- 
sumably from excessive calcitonin production (Krook et al., 
1969). Similarly, high calcium diets fed to cows in late ges- 
tation were thought to lead to benign hyperplasia of the thy- 
roid gland and excessive secretion of calcitonin. The high- 
calcium diets were also found to depress secretion of 
parathyroid hormone prior to calving (Black et al., 1973; 
Yarrington et al., 1977). Other experiments demonstrated 
that very low calcium diets fed in late gestation could pre- 
vent milk fever, a condition of severe hypocalcemia experi- 
enced by some cows at the onset of lactation (Boda and Cole, 
1954; Goings et al., 1974; Green et al., 1981). These obser- 
vations gave rise to the theory that high calcium diets fed to 
the late gestation dairy cow caused milk fever to develop. 
Studies now suggest milk fever is instead caused by exces- 
sive dietary potassium or sodium, not calcium (Ender et al., 
1971; Goff and Horst, 1997). The high cation diets induce a 
metabolic alkalosis in the cow, which interferes with par- 
athyroid hormone function, predisposing the cow to hypo- 
calcemia (Gaynor et al., 1989; Phillippo et al., 1994). 

Calcium, fed in the form of limestone, has some rumen 
buffering activity in cattle. Wheeler and Noller (1976) fed 
lactating cows up to 1 .48 percent calcium diets. Though there 
was a small decrease in feed intake with the higher calcium 
diets, there was no effect on fat-corrected milk production 
and there was more efficient use of dietary starch. Similar 
observations were made by Clark et al. (1989). However, in 
their study, feeding a diet that was 3.5 percent calcium car- 
bonate (estimated to be 1 .7 percent calcium) did significantly 
decrease feed intake, though fat-corrected milk production 
was not affected. 

Very high diet calcium concentration can reduce digest- 
ibility of the diet. Ammerman et al. (1963) found that diets 
that were 4.4 percent calcium reduced protein and energy 
digestibility of the ration of beef steers. In another study, 
veal calves aged 8 weeks were fed iso-energetic amounts of 



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MINERAL TOLERANCE OF ANIMALS 



0.71 percent or 1.16 percent calcium milk replacers for a 
period of 10 weeks. The extra calcium was added in the form 
of calcium formate. Feces were collected during the final 
week of the trial. The high calcium diet raised the amount of 
energy contained in excreted feces by 70 percent. The extra 
fecal energy output was mostly in the form of crude fat, pre- 
sumably from formation of insoluble calcium soaps of the 
fatty acids in the diet. The high versus low calcium intake 
not only depressed apparent digestibility of total lipids but 
also that of crude protein, carbohydrates, and ash (lower ash 
digestibility was largely a result of dietary calcium that was 
not absorbed). However, the final body weight of the two 
dietary groups was similar, so it is difficult to use these data 
to suggest that 1.16 percent calcium (DM) in milk replacer is 
necessarily excessive (Xu et al., 1999). When weaned calves 
were fed diets that were 0.34 percent phosphorus and either 
0.17, 0.67, 1.31, or 2.35 percent calcium, all grew normally 
during the four-week trial, suggesting little effect on diet 
digestibility. However, in those calves fed the 1.31 or 2.35 
percent calcium diets, there was a reduction in soft tissue 
levels of trace minerals such as zinc, manganese, and cop- 
per. The 2.35 percent calcium diet also depressed blood 
phosphorus concentration to levels that might ultimately 
have led to rickets had the study gone on longer (Alfaro et 
al., 1988). Fontenot et al. (1964) demonstrated that diets con- 
taining 1.2 percent calcium could induce zinc deficiency in 
lambs receiving no supplemental zinc. However, if zinc were 
added to the diets, the lambs could be fed 2.4 percent cal- 
cium diets with no effects on growth. 

In pigs, feed intake and growth are markedly reduced 
whenever the dietary calcium:phosphorus ratio exceeds 3: 1 
(Reinhart and Mahan, 1986). Thus, starting pigs requiring 
0.60 percent dietary phosphorus would be expected to suffer 
reduced feed intake and growth when dietary calcium ex- 
ceeds 1.8 percent, and finishing pigs requiring 0.45 percent 
dietary phosphorus would suffer reduced growth rate when 
dietary calcium exceeds 1.35 percent, unless diet phospho- 
rus was also increased. High calcium content is reported to 
reduce diet digestibility in pigs. Zimmerman et al. (1963) 
and Combs et al. (1966) found that addition of calcium to the 
diet of baby pigs reduced growth, feed efficiency, DM di- 
gestibility, and bone mineralization, especially when the cal- 
cium level exceeded 1 .0 percent of the diet. These effects 
were observed even though the dietary calcium to phosphorus 
ratios were less than 2:1. The experiments of Zimmerman et 
al. (1963) and Combs et al. (1966) suggest that the maximal 
tolerable dietary calcium level is 1.0 percent, although the 
study by Reinhart and Mahan (1986) demonstrated that 
increasing dietary phosphorus could increase the tolerable 
dietary calcium level. 

Fangauf et al. (1961) and Urbanyi (1960) in separate ex- 
periments fed chicks diets that ranged from 1.2 to 6.5 per- 
cent calcium. Levels above 2 percent depressed feed intake 
and weight gain and increased mortality rate. Shane et al. 
(1969) report that visceral gout and nephrosis occurred in 



growing pullets fed a 2.5 percent calcium diet from 8-20 
weeks of age. Hurwitz et al. (1995) fed chicks of fast-grow- 
ing (Cobb) and slow-growing (Leghorn) breeds diets that 
were 0.4, 0.6, 0.8, 1.0 (considered the required level of di- 
etary calcium), 1.5, and 2.0 percent calcium with 0.7 percent 
phosphorus (considered the required phosphorus level). 
Chicks of the slow-growing breed grew equally well with 
1.0, 1.5, and 2.0 percent calcium diets. However, chicks of 
the fast-growing breed fed 2 percent dietary calcium weighed 
significantly less than fast-growing chicks fed the 1.0 per- 
cent calcium diet. When dietary phosphorus was increased 
to 0.9 percent the negative effect of the 2 percent calcium 
diet on growth was greatly ameliorated, but not eliminated. 

For hens consuming 100 g feed/day the dietary calcium 
requirement is 3.25 percent (NRC, 1994). The 1980 NRC 
Mineral Tolerance of Domestic Animals set 4 percent diet 
calcium as a maximum tolerable level in laying hens based 
largely on the work of Gutowska and Parkhurst (1942), who 
reported that laying hens fed a 3.95 percent calcium diet suf- 
fered reduced egg production and feed efficiency, though 
eggshell strength was not affected. However, in another 
study, laying hens fed diets as high as 5 percent calcium 
showed no ill effects (Harms and Waldroup, 1971). In fact, 
recent studies with modern high-producing hens suggest the 
calcium requirement of laying hens may be closer to 5 per- 
cent calcium (Bar et al., 2002). 

The greater potential risk from high-calcium diets is in- 
terference with absorption of other minerals, causing defi- 
ciency of these minerals, despite their inclusion in the diet at 
levels that would ordinarily be considered sufficient. High- 
calcium diets can interfere with the absorption of phospho- 
rus, which is of particular concern in nonruminant animals. 
Much of the phosphorus of plant seed origin fed to these 
animals is bound to an inositol backbone. Up to 6 phosphate 
molecules can be bound to each inositol molecule, forming 
inositol 1,2,3,4,5,6-hexakis dihydrogen phosphate, also 
known as phytate. Nonruminant animals vary in their ability 
to digest phytate to free up the phosphorus. Poultry can, un- 
der some extreme circumstances, use as much as 50 percent 
of dietary phytate phosphorus. However, in general, swine 
and poultry hydrolyze only a small portion of the phytate 
phosphorus they are fed. The endogenous phytase of most 
nonruminant animals exists primarily in the mucosa of the 
small intestine and the phytate must be soluble at the pH 
found in the small intestine for the phytase to effectively 
hydrolyze the phytate. Phytases originating from feed ingre- 
dients or microbial synthesis are also most active when the 
phytate is soluble. Unfortunately the phytate molecule, with 
its protruding phosphate, is a highly negatively charged par- 
ticle. Cations (and in some cases proteins or starches) can 
then form bonds with the phosphate moieties. The propen- 
sity to form bonds with a cation is so great that much of the 
feedstuff phytate ingested is already chelated to cations, such 
as potassium, iron, calcium, magnesium, zinc, and copper. 
Phytate bound to divalent cations, especially calcium, is rela- 



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lively insoluble at the pH of the small intestine and is there- 
fore resistant to intestinal mucosa phytase digestion. Phytate 
bound to calcium is soluble at the acidic pH of the stomach 
and will be digested by phytases of microbial origin that are 
active at low pH. Calcium, being the most prevalent divalent 
cation in many diets, is commonly chelated to phytate, but 
other divalent and trivalent minerals can also become bound 
to phytate. A single phytate molecule can bind more than 
one divalent cation. The more cations attached to the phytate 
the lower the solubility of the complex and the less prone it 
is to hydrolysis by phytases (Wise, 1983). For this reason, 
therefore, the greater the calcium content of the diet the less 
available phytate phosphorus is to the nonruminant animal. 
In ruminants the activity of microbial phytase, either within 
the rumen or as the ingesta passes through the abomasum or 
true stomach, does a thorough job of digesting phytate and 
freeing the bound minerals. Therefore high calcium diets fed 
to ruminants do not interfere with phosphorus and trace min- 
eral absorption to the same extent as in nonruminants 
(Kincaid, 1979). 

In nonruminants only small amounts of phytate are di- 
gested by the endogenous phytases of the gastrointestinal 
tract. Dietary calcium reduces the efficacy of intestinal 
phytase and also the efficacy of phytase of fungal origin 
added to the diet. In poultry and swine, the ability of calcium 
to interfere with phytase activity occurs even at dietary cal- 
cium concentrations considered essential to meet the calcium 
needs of the bird (0.9 percent calcium diets) (Atia et al., 
2000; Applegate et al., 2003) or pigs (0.8 percent calcium 
diets) (Sandberg et al., 1993; Lei et al., 1994). However, 
inhibition of phytate digestion becomes more critical at di- 
etary calcium levels that exceed the requirement of the ani- 
mal. For example, in fast-growing broilers, increasing di- 
etary calcium from 0.8 to 2.0 percent reduced growth rate 
and caused pronounced hypophosphatemia. This effect 
seemed to be due to decreased absorption of phosphate from 
the diet of the birds (Hurwitz et al., 1995). It was also noted 
that increasing diet calcium in broilers using calcium car- 
bonate caused hypophosphatemic rickets, while the same 
level of diet calcium achieved by supplementation with 
dicalcium phosphate did not. This suggests that the bone 
problems arose because calcium carbonate interfered with 
phosphorus metabolism rather than as a direct effect of cal- 
cium toxicity (Ogura, 1981). 

Feeding excessive calcium could interfere with trace min- 
eral absorption (especially zinc and other trace minerals bound 
to phytate) and replaces energy or protein the animal might 
better use for increased production. In swine, excess calcium 
greatly reduces zinc availability, especially if phytate is high 
(Morgan et al., 1969). A diet containing 1.71 percent calcium 
and 68 mg Zn/kg fed to growing boars induced parakeratosis 
(abnormal keratinization of the skin) due to zinc deficiency. 
The dietary zinc requirement of boars at this age is 50 mg/kg 
diet, so this diet would otherwise have been considered more 
than adequate in zinc (Beilage et al., 1992). 



High dietary calcium may also increase the requirement 
for vitamin K. Pigs fed a 2.7 percent calcium diet based on 
corn and soybean meal with no supplemental vitamin K de- 
veloped internal hemorrhaging and died within 28 days. 
Supplementing the diet with 5 mg menadione prevented fur- 
ther problems (Hall et al., 1991). 

In fish, dietary calcium can also interfere with trace min- 
eral absorption and retention (Hardy and Shearer, 1985; 
Kaushik, 1995). For example, Redlip mullet (Liza 
haematocheila) fed a 2.5 percent calcium diet were reported 
to have reduced bone zinc, manganese, and iron content 
when compared to fish fed a 0.2 percent calcium diet 
(Hossain and Furuichi, 2000). High water calcium concen- 
tration also reduces gill uptake of zinc from the water. Al- 
though this effect might induce a secondary zinc deficiency, 
it may be used as a means of protecting fish when water is 
contaminated with excessive zinc or other heavy metals 
(Barron and Albeke, 2000). 

Female rats given 0.50, 0.75, 1.00, or 1.25 percent dietary 
calcium as calcium carbonate in AIN-76A diets for 6 weeks 
before mating, during mating, and for 20 days of gestation 
had dose-related linear decreases in the iron content of the 
liver, and in the zinc, iron, and magnesium contents of the 
kidney. Even though the highest calcium diet represents only 
a moderate increase in calcium above requirement, the fe- 
tuses from these rats had dose-related decreases in their 
whole-body contents of phosphorus, iron, copper, and mag- 
nesium (Shackelford et al., 1994). 

Factors Influencing Toxicity 

One factor that can upset calcium homeostasis is exces- 
sive (toxic) intake of vitamin D, or ingestion of plants con- 
taining glycosides of 1, 25 -dihydroxy vitamin D. In this case 
the intestinal calcium absorption mechanisms are fully en- 
gaged and diets high in calcium are more rapidly able to 
induce hypercalcemia and metastatic calcification than low 
calcium diets. 

In the horse and the rabbit, compromised renal function 
will not allow excretion of excess calcium absorbed from the 
diet and hypercalcemia will develop. 

TISSUE LEVELS 

Normal total plasma or serum calcium concentrations are 
between 90 and 1 10 mg/L or 2.25-2.75 mM for many mam- 
mals and growing birds. Horses and rabbits tend to be 
slightly higher, and up to 1 30 mg Ca/L plasma or serum may 
be considered normal (Table 10-2). The rabbit, in particular, 
will exhibit higher blood calcium concentration as dietary 
calcium content increases. Laying hens have a special cal- 
cium binding protein circulating in their blood, allowing 
them to rapidly deposit calcium into the oviduct during egg- 
shell formation, and they will normally have total plasma or 
serum calcium concentrations ranging from 180-360 mg/L 



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MINERAL TOLERANCE OF ANIMALS 



or 4.5-9 mM. Ionized calcium concentration — which is cal- 
cium that is not bound to proteins or other organic moieties 
in the blood — is relatively constant in all species, including 
poultry that are laying eggs, and is usually 40-60 mg Ca/L 
(1-1.5 mM) plasma or serum. 

Elevations in blood calcium concentration are common 
in cases of acute calcium toxicity. Plasma total calcium con- 
centrations exceeding 280 mg/L will cause cardiac arrest in 
many species, since this often means ionized calcium con- 
centration is also greatly increased. Typically these high 
blood calcium values are only encountered when animals 
are receiving intravenous calcium treatments at too high a 
dose or too rapid a rate of administration. Blood calcium 
concentrations exceeding 150 mg/L (3.75 mM) for more than 
12-24 hours will begin to cause metastatic calcification of 
soft tissues, particularly the kidneys, stomach, and vascula- 
ture, which would severely compromise renal function 
within 1-2 weeks. Chronically elevated blood calcium con- 
centrations above 120 mg/L (3 mM) can also cause meta- 
static calcification of soft tissues but the process is slower 
and would occur over weeks to months. Normal liver and 
kidney calcium content is generally below 200 mg/kg wet 
weight or about 800 mg/kg on a dry weight basis. Kidney or 
liver tissue calcium contents greater than 300 mg/kg wet 
weight would indicate metastatic calcification. Chronically 
elevated blood calcium concentrations can occur with ex- 
tremely high calcium diets, but this is rare. More commonly 
it is a sequelae of vitamin D intoxication, primary hyperpar- 
athyroidism, or, in some species such as the horse and rabbit, 
a consequence of renal failure. Urine calcium concentrations 
are a poor indicator of dietary calcium excess. Bone ash and 
bone calcium are sensitive to calcium deficiency states but 
do not reflect a calcium toxicity situation. 

Cow's milk contains approximately 1.2 g Ca/L. Sheep 
milk contains approximately 2.0 g Ca/L and goat milk about 
1 .5 g Ca/L. These values change with breed slightly but they 
are not impacted by dietary calcium. Sow's milk is about 
1.3 g Ca/L. Because sow's milk is about 20 percent dry 
matter, a pig that is consuming only sow's milk is receiving 
a diet that is approximately 0.65 percent calcium on a dry 
matter basis. 

MAXIMUM TOLERABLE LEVELS 
Single Dose 

The criteria used to determine the maximum tolerable 
single oral dose of calcium is that quantity of calcium that is 
not expected to cause severe hypercalcemia, defined as 
plasma calcium greater than 150 mg/L (3.75 mM) for more 
than 12 hours, as this degree of hypercalcemia appears to 
have severe consequences on feed intake, renal function, and 
hydration state. The maximum tolerable dose of calcium that 
can be given once to adult cattle as a drench without causing 
life-threatening hypercalcemia is 150 g calcium. This would 



be approximately equivalent to 0.25 g Ca/kg BW per os. 
Data for making a recommendation for any other species do 
not exist, and the safety of 0.25 g Ca/kg is likely reduced in 
nonruminant animals. In adult ruminants, the rumen fluids 
tend to dilute the calcium administered so that the concentra- 
tion of calcium overlying intestinal mucosa is lower, render- 
ing passive transport of calcium as less efficient (Goff and 
Horst, 1994). In nonruminants, the lack of dilution of the 
administered calcium before it reaches the small intestine 
would allow a greater portion of the administered calcium 
available for passive absorption. 

In some cases, the anion accompanying the calcium will 
prove more toxic (due to induction of metabolic acidosis or 
caustic damage to tissues) than the calcium in the salt, reduc- 
ing the tolerable amount of that particular calcium salt. For 
instance, the chloride of calcium chloride can induce a meta- 
bolic acidosis at a dose below the level where the calcium 
causes life-threatening hypercalcemia. 

Acute 

The criteria used to determine the maximum tolerable 
acute dietary calcium level is that quantity of calcium that is 
not expected to cause severe hypercalcemia, defined as 
plasma calcium greater than 150 mg/L (3.75 mM), for more 
than 1 2 hours when animals are introduced to the diet. 

Very high concentrations of dietary calcium are tolerated 
by all species for short periods, provided the animal's kid- 
neys are functioning or there is not excessive stimulation of 
1, 25 -dihydroxy vitamin D receptors in the intestine of the 
animal (vitamin D intoxication, ingestion of plants contain- 
ing glycosides of 1,25 -dihydroxy vitamin D) causing uncon- 
trolled absorption of calcium across the gut. It is likely that 
there is a dietary calcium level that will cause acute toxicosis 
from excessive hypercalcemia in every species if animals 
could be induced to consume these diets. However, for most 
species, acute toxicosis due to hypercalcemia has not been 
reported and a maximal tolerable level based on this crite- 
rion cannot be supported by data. This likely reflects the 
observation that feed intake decreases as the concentration 
of calcium increases in the diet (as in the studies of Reinhart 
and Mahan, 1986, and of Beede et al., 2001). Feed intake 
often declines quickly once diet calcium levels exceed 2-3 
percent (though laying hens routinely consume 4.5 percent 
calcium diets with no ill effects). This is well below the 5 
percent calcium diet level associated with acute toxicity 
when fed to piglets, which is the only documented instance 
of a high calcium diet causing acute toxic hypercalcemia in 
any species (Kamphues et al., 1989). 

Chronic 

If feed intake is significantly reduced by addition of cal- 
cium to the diet, animal growth will suffer. In assessing the 
maximum tolerable level of chronic dietary calcium expo- 



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CALCIUM 



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sure, a reduction in feed intake accompanied by a reduction 
in performance (growtti, milk or egg production, reproduc- 
tion) will be considered the criteria comprising evidence of 
calcium toxicosis. More stringent criteria could have been 
used based on the evidence that addition of calcium to diets 
reduces the availability of phosphorus and trace minerals to 
nonruminants. Unfortunately, this effect occurs even at cal- 
cium concentrations in the diet that are suggested to be the 
required concentrations of calcium in the diet of 
nonruminants. Under these criteria, any time the calcium 
content of the diet exceeds the amount required to meet the 
calcium needs of the body there is a detrimental effect, be- 
cause the amount of phosphorus and trace mineral required 
by the animal may be increased. Though these effects are 
generally of negligible consequence, there is a point at which 
these effects of calcium excess become intolerable and 
should be reduced. In nonruminant animals with fast growth 
rates (broilers, turkey poults, pigs, rats) the maximum toler- 
able calcium would be considered, by this criteria, to be very 
close to the calcium requirement of the animal because of 
the effect diet calcium has on use of phytate-bound minerals. 
These effects were generally not considered in determining 
the maximum tolerable levels, since most practical diets pro- 
vide enough excess phosphorus or trace mineral to allow the 
animal to tolerate minor calcium effects on their availability. 
However, when studies demonstrated that the reduction in 
availability of secondary minerals caused by excess diet cal- 
cium resulted in increased clinical disease, such as rickets or 
parakeratosis, then the induction of secondary mineral defi- 
ciency was considered a significant criterion for determin- 
ing the maximum tolerable dietary calcium level. 

In preruminant calves, diet calcium concentration ex- 
ceeding 1.2 percent DM can interfere with fat use, but does 
not impair growth or feed intake. The maximum tolerable 
dietary calcium for suckling calves is at least 1.2 percent 
and likely higher. In cattle, sheep, goats (and presumably 
other ruminants) the effects on phytate digestion are incon- 
sequential. Adult cows were fed diets containing up to 1.5 
percent calcium (in several studies), with little effect on 
feed intake and no significant effect on performance. Diets 
containing 1.9 percent calcium or greater can cause a sig- 
nificant decrease in feed intake in cattle. Although high 
calcium in the diet causes zinc deficiency in sheep fed diets 
without added zinc, few data demonstrate a detrimental ef- 
fect of high calcium diets in sheep or goats. Therefore, for 
ruminants, the maximum tolerable dietary calcium level is 
set at 1.5 percent. In the horse and rabbit (and presumably 
other hind gut fermenters), 2 percent calcium is the maxi- 
mum tolerable level, provided sufficient available phospho- 
rus is included in the diet. 

For growing poultry chicks, the maximum tolerable di- 
etary calcium level is 1.5 percent. Diets containing 2 per- 
cent calcium will affect growth and bone ash of fast-grow- 
ing chicks when diets contain required levels of 
phosphorus. High-producing laying hens can tolerate 5 per- 



cent calcium diets. Growing pigs have the lowest tolerance 
for dietary calcium, and detrimental effects are possible 
whenever the dietary calcium to phosphorus ratio exceeds 
2:1. The maximum tolerable level of dietary calcium for 
pigs is 1 percent, though increasing dietary phosphorus can 
increase the maximum tolerable level to some extent. In 
fish the data are too limited to allow an estimate of the 
maximum tolerable dietary calcium concentration. The only 
data available suggest that diets that contain 2 percent or 
greater calcium interfere with use of trace minerals and 
affect growth when compared to diets that contain 0.2 per- 
cent calcium. Since fish are often fast growing, especially 
when young, and most are unable to digest phytate, it would 
seem reasonable to believe that dietary calcium levels re- 
sulting in calcium to phosphorus ratios less than 2: 1 would 
not cause any detrimental problems, similar to other single- 
stomached species. Because most fish require 0.45 percent 
phosphorus diets (Lovell, 1991), the maximum tolerable 
dietary level is presumed to be 0.9 percent. 

For most breeds of dog, the maximum tolerable dietary 
calcium concentration is at least 2 percent, including grow- 
ing puppies of larger breeds. The tolerable level can be in- 
creased somewhat for most dogs provided the diet calcium 
to phosphorus ratio does not exceed 3:1. Most breeds will 
suffer a reduction in feed intake and growth if diet calcium 
exceeds 2.3 percent. For Great Dane and perhaps some other 
large breed puppies, rickets may occur if the diet calcium to 
phosphorus ratio exceeds 4:1. The maximum tolerable 
dietary calcium concentration for these giant breeds is 1 . 1 
percent unless phosphorus is also added to the diet to bring 
diet calcium to phosphorus ratios between 1.2:1 and 1 .4: 1 . If 
phosphorus is added to maintain this ratio, even Great Danes 
can be expected to tolerate up to 2.0 percent calcium diets. 
Diets containing more than 2.3 percent calcium risk in- 
creased occurrence of osteochondrosis in these breeds, even 
if dietary phosphorus is raised. In cats 1 percent calcium 
diets are well tolerated and result in optimal bone density 
(Pastoor et al., 1994). Feeding cats 2.3 percent calcium diets 
reduced growth rate and depressed feed intake and also 
caused a negative magnesium balance (Howard et al., 1998). 
There are no data to allow a more specific recommendation. 

FUTURE RESEARCH NEEDS 

Only with extremely high dietary calcium is it likely that 
hypercalcemia and its attendant problems will become mani- 
fest. However, it is increasingly clear that feeding animals 
more calcium than they require to meet their physiological 
requirements can reduce the efficiency of absorption of sev- 
eral other minerals, which might result in deficiencies of 
these minerals secondary to excess dietary calcium. Research 
to define the calcium level at which significant interaction 
with use of these other nutrients, in particular phytate phos- 
phorus and zinc, could increase awareness of these subtle 
detrimental dietary calcium effects. Unfortunately, because 



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MINERAL TOLERANCE OF ANIMALS 



the consequences of dietary calcium insufficiency can be 
severe and because calcium is an inexpensive feed additive, 
the tendency of nutritionists formulating animal diets is to 
provide calcium in excess of levels needed to meet the 
animal's requirement. To keep calcium to phosphorus ratios 
optimal, excess diet calcium encourages oversupplementa- 
tion of dietary phosphorus, the most expensive and environ- 
mentally damaging of minerals added to animal diets. 

SUMMARY 

Animals can tolerate relatively high amounts of dietary 
calcium for relatively long periods. Using hypercalcemia as 
the primary indicator of acute toxicity resulting from exces- 
sive absorption of dietary calcium, the level of dietary cal- 
cium that would cause this effect is generally so high that 
feed intake of the animal would be greatly reduced, limiting 
the development of hypercalcemia. When animals are fed 
calcium above the maximum tolerable levels over a longer 
period of time, the main effects observed would include in- 
terference with use of other minerals, especially phosphorus 
and zinc, and/or a significant reduction in feed intake affect- 
ing performance. 



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114 



MINERAL TOLERANCE OF ANIMALS 



TABLE 10-2 Calcium Concentrations in Fluids and Tissues of Animals (mg/kg or mg/L)" 



Fisti 



Animal 


Serum 


Muscle 

(wet wt basis) 


Liver* 

(wet wt basis) 


Kidney* 
(wet wt basis) 


BonegCa/lOOg 
(fat free, dry wt basis) 


Milk (g/L) or Egg 
(g Ca/eggshell) 


Ctiicken, 
broiler 


90-130 




75-80 


170-600 


14-20 




Laying hen 


200-400 








18-22 


2.0-2.1 


Pig ^^B 


90-120 


^^^^^B 


34-63 ^^H 


60-125 


^^^1 


to^^^^l 


Cow 


90-100 


30-300 


30-200 


45-200 


24.5-26.0 


1.2 


Sheep 


90-110 


55-96 


40-80 


60-140 




1.8-2.0 



80-100 With bone 

1,900^,100 

Without bone 
<1,000 

Whole fish meaF 
40-50 g/kg dry wt 



NOTE: Dietary calcium excess does not increase the calcium content of animal tissues significantly, unless accompanied by vitamin D intoxication. 
"Data largely adapted from Puis, 1994. 

''Vitamin D intoxication can cause metastatic calcification of soft tissues increasing calcium content of these tissues several-fold above normal upper limits. 
"^Menhaden or anchovy fish meal fed to livestock. 



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11 



Chromium 



INTRODUCTION 

Chromium (Cr) is a metallic element that occurs in each 
of the oxidation states from -2 to +6. Only the 0, +2, +3, 
and +6 valence states are commonly found. Elemental chro- 
mium (0) does not occur naturally. Chromium (+2) is un- 
stable and is readily oxidized to trivalent chromium (+3). 
Chromium occurs naturally primarily in the trivalent form, 
largely as ferrochromite (FeCrjO^) ores. Trivalent chro- 
mium (h-3) forms stable complexes with both organic and 
inorganic ligands. Hexavalent chromium (-1-6) has strong 
oxidizing properties and is produced almost entirely by in- 
dustrial processes (WHO, 1988). Chromium (+6) is spon- 
taneously reduced to chromium (H-3), when present in a 
soluble form. 

The principal component of chromium ores is chromite, a 
black mineral consisting largely of iron and chromium (H-3) 
oxides, with lesser amounts of magnesium and aluminum 
oxides (Katz and Salem, 1994). Chromium has not been 
mined in the United States since 1961. Most of the chro- 
mium ores processed in the United States are imported from 
South America, Turkey, and Russia (ATSDR, 2000). 

Approximately 60 percent of the chromium processed is 
used in the production of stainless steel and other alloys. 
Ferrochromite used in the production of stainless steel and 
other alloys is prepared by the reduction of chromite with car- 
bon (Katz and Salem, 1994). Chromium (+3) is used in the 
refractory industry as a component in chrome and chrome- 
magnesite, magnesite-chrome bricks, and granular chrome- 
bearing and granular chromite, which are used as linings for 
high-temperature industrial furnaces (ATSDR, 2000). Both 
trivalent and hexavalent chromium are produced in the chemi- 
cal industry and used for chrome plating, leather tanning, and 
manufacturing of pigments and wood preservatives. 

ESSENTIALITY 

Early studies by Schwartz and Mertz (1959) showed that 
chromium was a component of a glucose tolerance factor 



that corrected impaired glucose metabolism in rats fed cer- 
tain diets. Later studies demonstrated that chromium poten- 
tiates insulin function (Offenbacher et al., 1997). Clinical 
signs of chromium deficiency, including glucose intolerance, 
weight loss, and nerve and brain disorders, have been ob- 
served in humans receiving long-term parenteral nutrition 
(Anderson, 1995). Addition of small amounts of chromium 
(+3) chloride (CrClj) to the total parenteral nutrition solu- 
tion corrected deficiency signs in all patients. Recent studies 
suggest that chromium affects insulin by binding to a low- 
molecular weight oligopeptide named chromodulin 
(Vincent, 2001). Chromodulin tightly binds four chromic 
ions and amplifies insulin receptor tyrosine kinase activity; 
thus, it enhances insulin action. Without chromium, 
apochromodulin is largely ineffective in stimulating insulin- 
dependent tyrosine kinase activity. Activation of 
apochromodulin is specific for chromium. 

Until recently, practical diets fed to domestic animals 
were assumed to provide sufficient chromium to meet ani- 
mal requirements. Although responses to chromium supple- 
mentation have been variable, a number of studies in the past 
10 years have indicated that supplementation of diets with 
organic forms of chromium may affect animal metabolism 
and production criteria as well as the composition of animal 
products produced. Addition of chromium picolinate to diets 
of calves (Bunting et al., 1994) and growing pigs (Amoikon 
et al., 1995) increased glucose clearance rate following in- 
travenous glucose administration. In swine, chromium 
picolinate has increased carcass lean and decreased carcass 
fat in growing-finishing pigs (Page et al., 1993) and in- 
creased litter size in sows (Lindemann et al., 1995; 
Lindemann, 1999). Chromium supplementation to poultry 
diets has improved growth rate in broilers (Lien et al., 1999) 
and reduced serum and egg yolk cholesterol in hens (Lien et 
al., 1996). A number of studies in cattle indicate that chro- 
mium supplementation from various sources may increase 
immune response and reduce incidence of disease, especially 
in stressed animals (Spears, 2000). Addition of chromium 



115 



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MINERAL TOLERANCE OF ANIMALS 



methionine to lactating dairy cow diets increased milk pro- 
duction and feed intalce (Hayirli et al., 2001). 

DIFFICULTIES IN METHODS OF ANALYSIS AND 
EVALUATION 

Contamination of biological samples with chromium dur- 
ing collection, storage, and preparation of samples for analy- 
sis can present a major source of error. Prior to 1978, re- 
searchers were unaware that chromium concentrations they 
measured in tissues, blood, urine, and foods far exceeded 
true concentrations, because of background and environmen- 
tal contamination (Guthrie et al., 1978). Chromium is gener- 
ally present in tissues, plasma, and urine in the ng/g or ng/L 
range. Therefore, it is essential to minimize contamination 
from dust and stainless steel material (scalpels, knives, trays, 
needles, and grinding and homogenizing equipment) used in 
collection and preparation of samples. Stainless steel instru- 
ments should be replaced with titanium, glass, quartz, or 
polyethylene instruments (Katz and Salem, 1994). Needles 
made from nickel or platinum should be used for collection 
of blood samples for chromium analysis. Reagents of the 
highest purity also must be used to avoid contamination. 
Reagent blanks should be carried through sample prepara- 
tion and analysis to correct for background contamination. 
Losses of chromium through volatilization may occur dur- 
ing heating or acid digestion of samples in open systems 
(WHO, 1988). A reference material (obtained from the Na- 
tional Institute of Standards and Technology) with a certi- 
fied value for chromium is necessary to validate analytical 
procedures. 

Low concentrations of chromium in biological samples 
can be measured using graphite furnace atomic absorption 
spectrometry, neutron activation analysis, or mass spectrom- 
etry. Graphite furnace atomic absorption spectrometry is the 
most commonly used method, and has a detection limit of 
0.005 |Jg/L when an appropriate background correction 
method is used. Flame atomic absorption spectrometry and 
inductively-coupled plasma atomic emission spectrometry 
are not sensitive enough to detect chromium concentrations 
typically found in biological samples, but can be used to 
determine potentially toxic concentrations of chromium in 
feedstuffs. 

REGULATION AND METABOLISM 
Absorption and Metabolism 

Metabolism of chromium is greatly affected by valance 
state. Hexavalent chromium is absorbed to a greater extent 
than the trivalent form (Donaldson and Barreras, 1966; 
Kerger et al., 1996) and readily enters cells through nonspe- 
cific anion carriers. In contrast, chromium (+3) is poorly 
absorbed and enters cells with very low efficiency. Insignifi- 
cant amounts of hexavalent chromium are likely to be con- 



sumed orally because chromium (-1-6) is reduced in the envi- 
ronment to chromium (-1-3), the more stable oxidation state. 
Furthermore, chromium (+6) consumed is totally or at least 
partly reduced to chromium (+3) in the acid environment of 
the stomach (Donaldson and Barreras, 1966). Hexavalent 
chromium also does not generally occur naturally and is pro- 
duced almost totally from human activities. 

Absorption of chromium by humans consuming self-se- 
lected diets ranged from 0.5 to 2.0 percent, with higher ab- 
sorption values observed in individuals consuming a low 
chromium intake (Anderson and Kozlovsky, 1985). Esti- 
mated absorption of chromium from soluble sources, such as 
CrClj and chromium acetate, in humans and rats has ranged 
from 0.4 to approximately 2.0 percent (Donaldson and 
Barreras, 1966; Juturu et al., 2003). Absorption of chromium 
from chromium (H-3) oxide was negligible in rats (Juturu et 
al., 2003). It is this property that allows use of chromic oxide 
as a nondigestible marker for digestibility studies. Gargus et 
al. (1994) reported a mean absorption of 2.8 percent in hu- 
mans administered 400 [ig Cr/day as chromium picolinate. 

Chromium is transported in the blood primarily bound to 
transferrin (Offenbacher et al., 1997). Plasma or serum chro- 
mium concentrations in humans are generally less than 
0.30 |Jg/L. Blood chromium concentrations are not in equi- 
librium with tissue chromium concentrations and do not 
reflect body stores. Tissue chromium concentrations are low 
(generally less than 100 ng/g DM) with kidney, liver, spleen, 
and bone containing the highest concentration. 

The active form of chromium that potentiates insulin 
function remains unclear. Early studies suggested that chro- 
mium was a component of a glucose tolerance factor (Mertz, 
1993). Glucose tolerance factor isolated from brewer's yeast 
contained chromium (H-3), nicotinate, glycine, cysteine, and 
glutamate (Toepfer et al., 1976). More recent research sug- 
gests that chromodulin is the active form of chromium 
involved in facilitating insulin action (Vincent, 2001). 
Chromodulin is an oligopeptide comprised primarily of gly- 
cine, cysteine, aspartate, and glutamate, and has been iso- 
lated from the liver or kidney of a number of animal species 
(Vincent, 2001). In response to insulin release, chromium 
from the blood is transported to insulin-sensitive cells where 
chromium binds to apochromodulin. When insulin concen- 
trations decrease, chromodulin is believed to be released 
from cells and excreted in the urine. Urine is the major route 
of excretion for absorbed chromium. Consumption of high 
carbohydrate diets that increase blood insulin concentrations 
increases urinary excretion of chromium (Offenbacher et al., 
1997). 

IVIetabolic Interactions and IVIechanisms of Toxicity 

Studies have indicated that chromium (+3) absorption is 
increased by ascorbic acid, amino acids, and oxalate 
(Offenbacher et al., 1997). Zinc may reduce chromium ab- 
sorption; however, increasing dietary zinc did not reduce 



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CHROMIUM 



117 



toxicosis of chiromium (+3) in chicks (Ciiung et al., 1983). 
Iron overload in humans increases saturation of transferrin 
with iron and may impair chromium transport (Offenbacher 
et al., 1997). High dietary concentrations of chromium (+3) 
alleviate signs of vanadium toxicosis in chicks (Hill and 
Matrone, 1970). 

Trivalent chromium has a low order of toxicity, because 
of low intestinal absorption and limited entry into cells. 
Hexavalent chromium is several-fold more toxic than triva- 
lent chromium (WHO, 1988; Dayan and Paine, 2001). 
Hexavalent chromium not reduced to chromium (H-3) in the 
gastrointestinal tract or blood can cross cell membranes 
where it is ultimately reduced to chromium (H-3) by reducing 
agents such as glutathione, cysteine, and ascorbic acid. The 
mechanism of chromium toxicosis is believed to be due to 
chromium (+3) and/or chromium (+5) and chromium (+4), 
intermediates in the reduction of hexavalent to trivalent chro- 
mium, binding to intracellular macromolecules, including 
DNA (ATSDR, 2000). Toxicity of chromium also may be 
related to chromium (+3, +4, or +5) induced free radical for- 
mation (Bagchi et al., 2002). 

SOURCES AND BIOAVAILABILITY 

Chromium occurs naturally in the Earth's crust and also 
is released into the environment from combustion processes 
and ore processing industries. Naturally occurring chromium 
is almost always in the trivalent state, while chromium (+6) 
is almost entirely derived from human activities (WHO, 
1988). Concentrations of chromium in the ambient air in the 
United States range from 0.005 to 0.525 |ig/m-\ In U.S. river 
and ocean waters, chromium concentrations average 10.0 
and 0.3 |ig/L, respectively (ATSDR, 2000). Soil chromium 
varies greatly with a mean concentration of 37.0 mg/kg 
(USGS, 1984). Chromium is present in soils primarily in 
carbonate and oxide forms that are not mobile in soil. Ex- 
tractable chromium, measured following extraction of soil 
with acids or chelating agents, provides a much better esti- 
mate of chromium available for plant uptake than total soil 
chromium. 

Chromium analysis of animal feedstuffs has received little 
attention. However, most human food sources are low (<0. 10 
mg/g DM) in chromium. Feed phosphate sources appear to 
be a major source of chromium in certain animal diets. Feed 
grade monocalcium phosphate and defluorinated phosphate 
sources vary in chromium content but average 83 and 110 
mg Cr/kg, respectively (Sullivan et al., 1994). At present, 
chromium picolinate, chromium propionate, and chromium 
methionine can be supplemented to swine diets, at levels 
from 0.2 to 0.4 mg Cr/kg diet, in the United States. Chro- 
mium supplementation is currently not permitted in other 
animal species. 

When given orally, hexavalent chromium has a much 
greater bioavailability than even water-soluble forms of in- 
organic chromium (H-3). However, as discussed earlier, chro- 



mium (+6) is not likely to be consumed orally. Chromium 
(+3) oxide is essentially unavailable when given to rats, com- 
pared to the more soluble CrClj and chromium (+3) acetate 
(Juturu et al., 2003). Certain organic forms of chromium (+3) 
appear to be more bioavailable than inorganic trivalent 
forms. Rats supplemented with 5 mg Cr/kg from chromium 
picolinate or a chromium dinicotinic acid-diglycine-cysteine- 
glutamic acid complex had higher chromium concentrations 
in kidney and liver than those receiving CrClj after three 
weeks (Anderson et al., 1996). When supplemented at 20 to 
100 mg Cr/kg for 20 weeks, rats fed chromium picolinate 
also had much higher liver and kidney chromium concentra- 
tions than rats fed CrClj (Anderson et al., 1997a). In contrast 
to these findings, absorption of ^'Cr from CrClj and chro- 
mium picolinate has been similar in short-term studies with 
rats (Ohn et al., 1994; Anderson et al., 1996). 

TOXICOSIS 

Chromium toxicity has been extensively reviewed (WHO, 
1988; Katz and Salem, 1994; EPA, 1998; Dayan and Paine, 
2001). Toxicosis of chromium is a major concern in humans 
exposed in occupational and industrial settings to chromium 
via inhalation and dermal contact. The major form of chro- 
mium believed to be responsible for toxicosis in humans is 
chromium (+6). Hexavalent chromium can act as a carcino- 
gen, an allergen, and an acute irritant in humans and experi- 
mental animals (Dayan and Paine, 2001). Chromates (+6) 
are used in oil fields as a corrosion inhibitor and in drilling 
muds. Toxicosis from chromates (+6) has been suspected in 
cattle exposed to oil field wastes (Kerr and Edwards, 1981). 
However, chromium (+6) is generally not consumed orally 
for reasons described earlier. This discussion will focus on 
chromium (+3). Trivalent chromium is relatively nontoxic 
via oral intake. 



Single Dose and Acute 

Large doses of chromium (+3) are required to produce 
signs of acute toxicosis. A single oral dose of 650 mg Cr/kg 
BW produced no overt toxicosis in young rats (NRC, 1980). 
The LDjQ in rats for chromium (+3) as CrCl3-6H20, 
Cr(N03)3-9H20, and Cr(CH3COO)3-H20 is approximately 
365, 422, and 2,376 mg Cr/kg BW, respectively (Katz and 
Salem, 1994). Female rats are slightly more sensitive to acute 
doses of chromium than males (ATSDR, 2000). Signs of 
acute toxicosis in rats include hypoactivity, mydriasis, lacri- 
mation, and diarrhea. Exposing carp to water containing 
20 mg Cr/L as CrCl3 for 48 hours resulted in 100 percent 
mortality (Table 1 1-1); however, 10 mg/L had no effect on 
mortality (Muramoto, 1981). Chromium sulfate caused 
lower mortality in carp than CrCl3 when supplied at 20 mg 
Cr/L. The LCjq for chromium in prawn larvae was estimated 
at 12.5 mg/L for CrCl3 (Marino-Balsa et al., 2000). 



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118 



MINERAL TOLERANCE OF ANIMALS 



Chronic 

A number of studies evaluating oral ingestion of high 
concentrations of chromium (+3) are summarized in Table 
11-1. Body weight gain in young chicks was reduced by ad- 
dition of 1,500 mg Cr/kg diet as Cr2(S04)3 (Chung et al., 
1983) or 2,000 mg/kg diet as CrClj (Hill and Matrone, 1970). 
In laying hens, 50 mg Cr/kg diet from either CrClj, chro- 
mium yeast, or a chromium aminoniacinate complex did not 
affect egg production, but reduced activity of liver cyto- 
chrome P-450 dependent monooxygenases (Guerra et al., 
2002). They suggested that 50 mg Cr/kg may impair cyto- 
chrome P-450 catalyzed drug metabolism. Considering the 
life expectancy of laying hens, the biological significance of 
this finding is unclear. 

Providing chromium (CrClj) in drinking water at 328 mg/L 
in rats (Bataineh et al., 1997) or 656 mg/L in mice (Elbetieha 
and Al-Hamood, 1997) for 12 weeks reduced body weight 
gain. In male mice, CrClj at 1,640 mg Cr/L of water reduced 
fertility, and 656 mg/L reduced implantation and viable 
fetuses in pregnant females (Elbetieha and Al-Hamood, 
1997). Mice born and raised to mice exposed to 328 mg Cr 
(CrCl3)/L of water during gestation and lactation (20 d) had 
reduced body weight at 60 days of age (Al-Hamood et al., 
1998). Fertility was impaired in female but not male mice 
exposed prenatally and postnatally to chromium (Al- 
Hamood et al., 1998). Reduced sexual behavior (measured 
by number of mounts and number of males ejaculating) was 
observed in male rats given 328 mg Cr/L of water for 12 
weeks, but fertility was not affected (Bataineh et al., 1997). 

Limited research has evaluated chronic toxicity of organic 
forms of chromium. Rats supplemented for 20 weeks with 
100 mg Cr/kg diet from chromium picolinate had chromium 
concentrations in liver and kidney that were approximately 
7-fold higher than observed in rats fed a similar concentra- 
tion of CrClj (Anderson et al., 1997a). However, no signs of 
toxicosis, including pathological changes in liver or kidney, 
were seen in rats fed either chromium source. Chromium 
(-1-3) oxide is very insoluble and did not produce toxicosis 
even when provided in the diet of rats at up to 34,215 mg Cr/kg 
for 2 years (Ivankovic and Preussmann, 1975). 

Factors Influencing Toxicity 

Soluble forms of chromium (+6) are several-fold more 
toxic than soluble forms of chromium (H-3). Animals are not 
usually exposed to chromium (H-6) orally for reasons dis- 
cussed previously. Toxicity of chromium (H-3) is affected by 
solubility of the compound. Insoluble CrjOj has not pro- 
duced toxicosis in animals (Ivankovic and Preussmann, 
1975; NRC, 1980) while soluble forms such as CrCl, and 
Cr2(S04)3 can reduce body weight gain and impair repro- 
duction when provided in feed or water at high concentra- 
tions (Table 11-1). Organic forms of chromium may be more 
toxic than CrClj. Chromium picolinate feeding resulted in 



higher tissue chromium concentrations than CrClj (Ander- 
son et al., 1997a). However, elevated tissue chromium con- 
centrations in rats supplemented with chromium picolinate 
has not caused toxicosis. 

TISSUE LEVELS 

Chromium is widely distributed in the body at very low 
(ng/g DM) concentrations. Representative values in tissues 
from various animals are shown in Table 11-2. Addition of 
100 mg Cr/kg as CrClj to turkey diets for 5 weeks greatly 
increased tissue chromium concentrations; however, even at 
the high level of supplementation, tissue chromium only ex- 
ceeded 1 mg/kg DM in kidney (Anderson et al., 1989). In 
growing pigs, supplementation with 0.2 to 0.3 mg Cr/kg as 
chromium picolinate increased chromium concentrations in 
liver, kidney, and heart, but not in muscle (Ward, 1995; 
Anderson et al., 1997b). Liver and kidney chromium con- 
centrations were much higher in rats fed chromium 
picolinate than in those fed CrCl, at high concentrations 
(Anderson etal., 1997a). Supplementation of cattle diets with 
low concentrations of chromium did not increase tissue chro- 
mium (Spears et al., 2004). Tissue chromium data are not 
available for animals fed toxic concentrations of chromium. 
It is unlikely that chromium accumulation in animal prod- 
ucts, other than possibly kidney, would cause a toxicological 
concern for humans. 

MAXIMUM TOLERABLE LEVELS 

The maximum tolerable level for chromium is defined as 
the dietary level that, when fed for a defined period of time, 
will not impair animal health and/or performance. Valance 
state and chemical form of chromium affect the maximum 
tolerable level for chromium. Hexavalent chromium (-1-6) is 
much more toxic than trivalent chromium (H-3). Maximum 
tolerable levels for chromium (+6) have not been defined for 
most domestic animals because chromium (+6) is generally 
not ingested orally. 

Chromic oxide, an insoluble form of chromium (+3) is 
very poorly absorbed, and has been widely used as an indi- 
gestible marker in animals for periods of several weeks at 
concentrations as high as 3,000 mg Cr/kg diet with no 
evidence of adverse effects (NRC, 1980 ). Feeding diets con- 
taining over 30,000 mg Cr/kg from CrjOj for two years also 
did not produce adverse effects in rats (Ivankovic and 
Preussmann, 1975). 

Soluble forms of chromium (+3), such as CrClj and 
Cr,(S04)3, reduce body weight gain in young chicks when 
fed at 1,500 to 2,000 mg Cr/kg diet. Young chicks can toler- 
ate 500 mg Cr (from soluble forms)/kg diet for 21 to 35 days 
without adverse effects (Hill and Matrone, 1970; Chung et 
al., 1983). Exposure of rats and mice to 328 to 656 mg Cr/L 
in drinking water for 12 weeks reduced body weight and 
impaired reproduction (Elbetieha and Al-Hamood, 1997; Al- 



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CHROMIUM 



119 



Hamood et al., 1998). Developmental effects were also ob- 
served in mice exposed prenatally and postnatally to 328 mg 
Cr/L of water (Al-Hamood et al., 1998). Dietary supplemen- 
tation of rats for 20 weeks with 100 mg Cr/kg from either 
CrClj or chromium picolinate did not produce toxicosis, but 
increased liver and kidney chromium concentrations 
(Anderson et al., 1997a). Based on available literature, the 
maximum tolerable level for chromium (H-3) was set at 3,000 
mg/kg for Cr^Oj. For more soluble forms of chromium (H-3) 
the maximum tolerable level was set at 500 mg/kg for poultry 
and 100 mg/kg for mammalian species. Limited data in fish 
suggest that soluble forms of chromium (-H3) can be toler- 
ated for 48 to 72 hours at 8.0 to 10.0 mg/L of water. In animal 
species other than poultry and fish, rat and mice data were 
used to establish maximum tolerable levels. At dietary con- 
centrations of chromium up to the maximum tolerable levels 
indicated, any increase in chromium concentration in edible 
tissue, with the possible exception of kidney, should not 
present a human health concern. 

FUTURE RESEARCH NEEDS 

Studies are needed to define the maximum tolerable level 
of chromium for most domestic animals. Research is par- 
ticularly needed to determine the concentration of chromium 
from various organic chromium supplements that can be tol- 
erated without adverse effects. 

SUMMARY 

Chromium occurs in a number of oxidation states, with 
chromium (H-3) being the most stable form. Trivalent chro- 
mium potentiates insulin activity and is generally recognized 
as an essential trace mineral. Chromium is found naturally 
almost entirely as chromium (+3), but both trivalent and 
hexavalent chromium are produced and used in the chemical 
industry. Hexavalent chromium is much more toxic than 
chromium (+3). However, chromium (+6) released into the 
environment is largely reduced to chromium (+3). Almost 
all of the chromium consumed orally is in the trivalent form. 
Chromium (+3) is relatively nontoxic due to its poor intesti- 
nal absorption and limited entry of absorbed chromium (+3) 
into cells. Chromium oxide even at dietary concentrations 
over 30,000 mg Cr/kg diet did not produce toxicosis in rats. 
The level of chromium (+3) in soluble forms, such as CrClj, 
needed to cause adverse effects in animals is above 100 mg/kg. 
Fish can tolerate at least 8 mg Cr/L of water for 48 to 72 hours 
without adverse effects. The amount of soluble chromium 
(+3) needed to produce adverse effects is at least 100 times 
the amount needed to meet animal requirements. 



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CHROMIUM 



123 



TABLE 11-2 Chromium Concentrations in Fluids and Tissues of Animals (mg/kg)" 





















Egg 


Egg 




Animal 


Quantity 


Source 


Duration 


Route 


Muscle 


Liver 


Kidney 


Heart 


white 


yolk 


Reference 


Cattle 


Control 
+0.4 mg/kg 
+0.8 mg/kg 


CrCl, 

Cr nieotinate 


146 d 


Diet 




25 
21 
20 


13 
12 
11 








Spears et al., 2004 


Cattle 


Control 
+0.8 mg/kg 


Cr methionine 


125 d 


Diet 


116 
71 


41 
38 


108 

127 








Spears et al., 2004 


Pigs 


Control 




80 d 


Diet 


2 


9 


10 


8 






Ward, 1995 




+0.2 mg/kg 


CrCl, 






6 


17 


15 


19 










+0.2 mg/kg 


Cr picolinate 






3 


19 


42 


12 






J 


Pigs 


Control 




50 d 


Diet 


<1.5 


21 


27 


4 






Anderson et al.. 




+0.3 mg/kg 


Cr picolinate 






<1.5 


29 


57 


11 






1997b 


Rats 


Control 
+100 mg/kg 
+100 mg/kg 


CrCl, 

Cr picolinate 


20 wk 


Diet 




6 

90 

550 


8 

700 
2,200 








Anderson et al., 1 
1997a 


Turkey hens 


Control 




35 d 


Diet 


3 


6 


14 


4 


14 


29 


Anderson et al., 1989 




+25 mg/kg 


CrClj 






4 


106 


367 


8 


9 


27 






+100 mg/kg 


CrClj 






10 


494 


933 


35 


15 


56 






+200 mg/kg 


CrCl:, 






13 


959 


2,254 


52 









"Values are on a dry matter basis. 



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12 



Cobalt 



INTRODUCTION 

Cobalt (Co) is a silvery-white, hard metal with an atomic 
number of 27 and an atomic weight of 58.93. Most common 
compounds of cobalt have an oxidation state of +2 or +3 
(Greenwood and Earnshaw, 1997). The +2 valence is stable 
in aqueous solution and is the major form of cobalt found in 
simple salts. In contrast, cobalt (+3) is a strong oxidizing 
agent and is unstable in aqueous solution. However, cobalt 
(+3) can form numerous coordination complexes with nitro- 
gen-donor ligands and is the form of cobalt present in vita- 
min Bjj. Cobalt is known for its magnetic properties. The 
element is easily magnetized and retains its magnetism un- 
der a wide range of environmental conditions. 

Linnaeite (CO3S4), safforite (CoAsj), glaucodot (CoAsS), 
and erythrite (Co3(As204)2) are the most important ores of 
cobalt (ATSDR, 2001). These ores are associated with 
nickel, copper, and lead, and most of the cobalt produced is 
recovered as a by-product of ores treated for recovery of 
nickel or copper. The largest cobalt reserves are in Zaire, 
Zambia, Morocco, Canada, and Australia. Cobalt deposits in 
the United States are not concentrated enough to allow eco- 
nomical recovery of the metal. With the exception of a neg- 
ligible amount of by-product cobalt produced from some 
mining operations, no cobalt is mined or refined in the United 
States (ATSDR, 2001). Reported U.S. use of cobalt metal in 
2003 was 8,000 metric tons (USGS, 2004). 

A major use of metallic cobalt is in the production of 
superalloys that are used in gas turbine aircraft engines 
(ATSDR, 2001). It is also used in magnetic alloys and alloys 
that require hardness, wear resistance, and corrosion resis- 
tance. In addition, cobalt compounds are used (1) as pig- 
ments (blue) in glass, ceramics, and paint; (2) as catalysts in 
the petroleum industry; and (3) to hasten the oxidation and, 
thus, drying of oil-based paints. Gamma rays from radioac- 
tive ^°Co are widely used to sterilize medical devices, as an 
external source in radiography and radiotherapy, and for food 
irradiation. 



ESSENTIALITY 

The only known function of cobalt is as an essential com- 
ponent of vitamin 6,2 (cobalamin). Vitamin Bj2 contains 
cobalt (H-3) surrounded by six coordinately linked groups to 
form an octahedron. Vitamin Bjj is a cof actor for the en- 
zymes methylmalonyl CoA mutase and methionine synthase. 
Methylmalonyl CoA mutase is responsible for conversions 
of methylmalonyl CoA to succinyl CoA and is important in 
propionate metabolism. Methionine synthase is involved in 
the regeneration of methionine following loss of its methyl 
group and in the maintenance of biologically active folate 
concentrations in tissues. 

Mammals lack the ability to synthesize vitamin B|2, and 
nonruminant animals require a dietary source of vitamin Bp. 
Vitamin B|2 can be synthesized from inorganic cobalt by 
certain bacteria and algae (Smith, 1997). Ruminal bacteria 
synthesize enough vitamin B|2 to meet the requirements of 
ruminants provided that adequate dietary cobalt is supplied. 
Ruminants require 0. 10 to 0. 15 mg Co/kg diet to allow suffi- 
cient synthesis of vitamin Bjj by ruminal bacteria to meet 
the animal's requirement. In certain areas of the world, for- 
age cobalt concentrations are well below requirements of 
ruminants due to low soil cobalt concentrations. Cobalt defi- 
ciency in ruminants, caused by low dietary intake of cobalt, 
results in loss of appetite, reduced growth rate or even loss in 
body weight in severe cases, and anemia (Underwood and 
Suttle, 1999). 

DIFFICULTIES IN METHODS OF ANALYSIS AND 
EVALUATION 

Biological samples of plant and animal origin are gener- 
ally low (<0.5 mg/kg DM) in cobalt. Graphite furnace 
atomic absorption spectrometry with Zeeman background 
correction is the method of choice for measuring low con- 
centrations of cobalt (Smith, 1997). Neutron activation 
analysis and differential pulse anodic stripping voltammetry 



124 



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COBALT 



125 



also have been used for determining low concentrations of 
cobalt. Flame atomic absorption spectrometry is not sensi- 
tive enough to detect cobalt concentrations normally found 
in biological samples. 

Contamination of samples with cobalt during collection, 
storage, and preparation of samples for analysis should be 
avoided. Disposable syringes and technical-grade anticoagu- 
lants may contaminate blood samples with cobalt. Wet 
ashing with acid is the preferred method for preparing solid 
samples for cobalt analysis. Dry ashing may result in losses 
of cobalt. 

REGULATION AND METABOLISM 
Absorption and Metabolism 

In weanling rats fed physiological concentrations of co- 
balt, from CoClj, apparent and true absorption of cobalt av- 
eraged 28 and 29.8 percent, respectively (Kirchgessner et 
al., 1994). Mice absorbed 26 percent of an oral dose of la- 
beled cobalt (Toskes et al., 1973). Absorption of cobalt was 
very high in young rats (1 to 20 days of age) and decreased 
greatly by 30 days of age (Naylor and Harrison, 1995). 
Cobalt absorption also declined with age in guinea pigs. 
However, independent of age, cobalt absorption was much 
lower in guinea pigs than in rats (Naylor and Harrison, 1995). 
In nonruminants, cobalt and iron appear to share a common 
intestinal transport system and cobalt absorption is greatly 
increased in iron deficiency (Thomson et al., 1971). Absorp- 
tion of cobalt is very low (1 to 2 percent) in ruminants 
(Looney et al., 1976; Van Bruwaene et al., 1984). Low 
absorption of cobalt in ruminants may relate to binding of 
cobalt by ruminal microorganisms. A portion of cobalt in- 
gested by ruminants is used by ruminal bacteria for synthesis 
of vitamin Bp. It has been estimated that the efficiency of 
cobalt conversion to vitamin Bj, ranges from 1.7 to 18 per- 
cent (Smith and Marston, 1970). 

Absorbed cobalt is primarily excreted in urine with small 
amounts excreted via fecal endogenous routes (Kirchgessner 
et al., 1994). Cobalt concentrations in tissues are generally 
low (1 mg/kg DM or less), but liver and kidney cobalt con- 
centrations increase dramatically when animals are fed high 
dietary cobalt (Henry et al., 1997). Liver has the highest con- 
centration of cobalt followed by the kidney and heart. Be- 
cause cobalt is required in the mammalian system in the form 
of vitamin B|2, transport and intermediatory metabolism of 
cobalt per se has received little attention. 

IVIetabolic Interactions and IVIechanisms of Toxicity 

Sulfur amino acids, especially cysteine, may form stable 
complexes with cobalt and reduce its absorption. Supple- 
menting cysteine or methionine in excess of dietary require- 
ments reduced liver and kidney cobalt concentrations in 
chicks fed high dietary cobalt (Southern and Baker, 1981). 



Iron and cobalt interact in the intestinal mucosal and are 
mutually antagonistic (Thomson et al., 1971). Supplementa- 
tion of cobalt at 100 mg/kg diet or higher reduced serum and 
liver iron in pigs (Huck and Clawson, 1976). In chicks 
supplemented with high concentrations (100 to 400 mg/kg 
diet) of cobalt, liver and kidney cobalt concentrations were 
greatly elevated when chicks were fed an iron-deficient diet 
(Blalock, 1985). The addition of 100 or 1,000 mg Fe/kg to 
the high cobalt diets greatly reduced liver and kidney cobalt 
concentrations. 

Feeding elevated but nontoxic concentrations of cobalt 
increased zinc absorption in chicks fed diets low in zinc 
(Chung et al., 1977). Similarly, growth and zinc status of 
pigs fed a low zinc diet were improved by the addition of 27 
or 54 mg Co/kg diet (Chung et al., 1976). 

A number of possible mechanisms for cobalt toxicity may 
exist. Exposure to cobalt may cause oxidative damage to 
tissues by causing generation of oxygen radicals and free 
radical-induced DNA damage (Kasprzak et al., 1994). In 
support of this hypothesis, selenium and vitamin E adminis- 
tration offered protection against cobalt-induced cardiomy- 
opathy in pigs (Van Vleet et al., 1977). Increasing dietary 
cysteine, a precursor of the antioxidant glutathione, also al- 
leviated growth depression resulting from cobalt toxicosis in 
chicks (Southern and Baker, 198 1). Another possible mecha- 
nism of CO bait toxicity relates to the ability of co bait to block 
calcium channels and thus, alter cellular calcium influx 
(Yamatani et al., 1998). Some cobalt toxicosis signs may 
relate to the effect of cobalt on heme and heme-containing 
enzymes (ATSDR, 2001). Cobalt is believed to inhibit two 
different sites in the heme biosynthetic pathway. 

SOURCES AND BIOAVAILABILITY 

Cobalt occurs naturally in the Earth's crust at an average 
concentration of 20 to 25 mg/kg (ATSDR, 2001). Soils gen- 
erally contain from 1 to 40 mg Co/kg and the average cobalt 
concentration in U.S. soils is 7.2 mg/kg. Cobalt concentra- 
tions in surface and groundwater are usually between 1 and 
10 |Jg/L. Elevated concentrations of cobalt in soil and water 
can result from activities such as mining and processing of 
cobalt-bearing ores, disposing of cobalt-containing wastes, 
burning of fossil fuels, copper and nickel smelting and refin- 
ing, and applying cobalt containing sludge or fertilizers to 
soil (ATSDR, 2001). 

Most animal feedstuffs are low in cobalt (<0.5 |ig/kg). 
Concentration of cobalt in plant material is dependent on 
soil cobalt, soil pH, and plant species. Soil uptake of cobalt 
by forages decreases as soil pH increases. Legumes are 
higher in cobalt than grasses when grown on cobalt adequate 
soil (Underwood and Suttle, 1999). Soils deficient in cobalt 
(< than 3.0 mg Co/kg soil) are found in certain areas of the 
southeastern and northeastern United States. Ruminants 
grazing or consuming forages grown in these areas require 
cobalt supplementation to prevent deficiency. Cereal grains 



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126 



MINERAL TOLERANCE OF ANIMALS 



usually contain deficient concentrations (<0.06 mg/kg) of 
cobalt. Feeds of animal origin, with the exception of liver 
products, are also low in cobalt. 

Cobalt is generally supplemented to ruminant diets as 
C0CO3, C0SO4, or various organic forms. Kawashima et al. 
(1997a) compared bioavailability of different cobalt sources 
based on tissue cobalt accumulation in lambs fed high (40 
mg Co/kg) dietary cobalt. Feed grade sources of C0CO3 and 
cobalt glucoheptonate were similar in bioavailability to re- 
agent grade CoSO^-VHjO. Cobalt oxide was approximately 
20 percent as bioavailable as CoSO^-VHjO based on accu- 
mulation of cobalt in various tissues. In vitro studies indi- 
cated that CO3O4 was totally unavailable for synthesis of vi- 
tamin Bj2 by ruminal microorganisms (Kawashima et al., 
1997b). Bioavailability of cobalt from cobalt propionate was 
similar to C0CO3 in steers based on plasma and liver vitamin 
Bj2 concentrations (Tiffany et al., 2003). Absorption of co- 
balt from cobalt naphthenate, an organometallic cobalt-con- 
taining compound used as a drier in paint, appears to be simi- 
lar to C0CI2 in rats (Firriolo et al., 1999). 

TOXICOSIS 

Cobalt toxicosis in animals is very rare because concen- 
trations of cobalt normally present in animal diets are much 
lower than those needed to cause toxicosis. Suspected cases 
of cobalt toxicosis, due to excessive cobalt addition to water, 
feed, or pastures, have been reported in cattle (Dickson and 
Bond, 1974). Cobalt toxicosis is not likely to occur in 
nonruminants unless environmental contamination of feed 
or water occurs. 

Single Dose and Acute 

High oral doses of cobalt caused sedation, diarrhea, trem- 
ors, convulsions, and death in rats (Speijers et al., 1982). 
Histological changes were observed in heart, liver and kid- 
ney of rats that died. The LD^q in rats for CoCl2-6H20 has 
ranged from 42.4 to 190 mg Co/kg BW in various studies 
(ATSDR, 2001; Speijers et al., 1982). Oral administration of 
100 mg Co/kg BW, as C0SO4, for 3 days in young pigs re- 
sulted in cardiomyopathy, listlessness, diarrhea, and high 
mortality (Van Vleet et al., 1977). Severity of cardiomyopa- 
thy was greater in pigs that were inherently susceptible to 
stress compared to nonstress-susceptible pigs (Van Vleet et 
al., 1977). 

Chronic 

Studies evaluating oral ingestion of high concentrations 
of cobalt are summarized in Table 12-1. Cobalt concentra- 
tions as low as 100 mg Co/kg diet have reduced weight gain 
in young chicks (Hill, 1974, 1979a; Ling and Leach, 1979). 
Lower concentrations of cobalt may affect disease suscepti- 
bility as chicks fed 50 mg Co/kg diet had increased mortality 



following experimental infection with Salmonella 
gallinarum (Hill, 1974). Addition of 200 mg Co/kg or higher 
to chick diets has increased mortality in noninoculated birds 
(Hill, 1974; Diaz et al., 1994) and resulted in histological 
lesions in the digestive tract, liver, heart, and skeletal muscle 
(Diaz et al., 1994). 

The amount of cobalt that can be tolerated without ad- 
verse effects in rats appears to be similar to chicks. In rats 
fed an iron-adequate diet, 50 mg Co/kg did not affect weight 
gain, but addition of 100 mg Co/kg diet or higher reduced 
weight gain and feed intake and caused some mortality 
(Huck, 1975). However, as little as 25 mg Co/kg diet re- 
duced weight gain and feed intake when rats were fed an 
iron-deficient diet (Huck, 1975). Myocardial damage has 
been seen in adult rats given 12.4 mg Co/kg BW for 21 days 
(Morvai et al., 1993). Testicular degeneration has been ob- 
served in male rats (Corrier et al., 1985; Anderson et al., 
1992) and mice (Anderson et al., 1993) given high oral doses 
of cobalt. Oral administration of cobalt at 5.2 mg Co/kg BW 
or higher during gestation has impaired fetal development in 
mice and rats, and increased mortality in pregnant rabbits 
(Szakmary et al., 2001). 

Based on limited research, higher concentrations of co- 
balt are required in pigs than in chicks and rats to cause toxi- 
cosis. Addition of up to 200 mg Co/kg diet did not cause 
observable adverse effects in young pigs (Huck and 
Clawson, 1976). Cobalt concentrations of 400 or 600 mg/kg 
diet caused anorexia, growth depression, reduced blood he- 
moglobin and hematocrit, incoordination, muscular tremors, 
stiff legs, and humped back in pigs (Huck and Clawson, 
1976). 

Older studies indicated that cattle can tolerate up to 0.86 
mg Co/kg BW without adverse effects (Ely et al., 1948; 
Keener etal., 1949). Higher concentrations of cobalt reduced 
feed intake and caused hyperchromemia (Keener et al., 
1949). No recent studies examining cobalt toxicosis in cattle 
were found in the literature. Becker and Smith (1951) re- 
ported that 4.4 mg Co/kg BW or higher, administered via 
water, decreased feed intake in sheep. A daily dose of 1 1.0 
mg Co/kg BW caused anemia and high mortality in sheep 
(Becker and Smith, 1951). In a more recent study, adult rams 
were given 3.0 or 4.5 mg Co/kg BW for 70 days (Corrier et 
al., 1986). The dose of cobalt was then increased to 10 or 15 
mg/kg BW for an additional 39 days. Tissue cobalt concen- 
trations were greatly elevated in cobalt-dosed rams, but no 
pathological or clinical manifestations of toxicosis were 
found in this study. The addition of 40 mg Co/kg diet for 60 
days also did not affect performance or general health of 
lambs (Henry et al., 1997). 

Factors Influencing Toxicity 

Iron status greatly affects the concentration of cobalt re- 
quired to produce a toxicosis. Cobalt absorption is increased 
in iron deficiency (Valberg et al., 1969; Blalock, 1985), and 



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COBALT 



127 



iron deficiency in cliicks (Chetty, 1972) and rats (Huck, 
1975) lowered the concentration of cobalt required to de- 
press growth by over 50 percent. The addition of 100 to 200 
mg Fe/kg to diets adequate in iron partially alleviated cobalt- 
induced growth depression but did not affect increased sus- 
ceptibility of chicks to Salmonella gallinarum (Hill, 1974). 

Increasing dietary concentrations of methionine or cys- 
teine alleviates cobalt toxicosis. Addition of 0.5 or 1.0 per- 
cent DL-methionine to a corn-soybean meal-based diet com- 
pletely alleviated growth depression and reduced serum iron 
concentrations in pigs fed 600 mg Co/kg diet (Huck and 
Clawson, 1976). However, serum cobalt concentrations in 
pigs were not reduced by methionine supplementation. In- 
creasing dietary methionine or cysteine partially alleviated 
reduced growth and feed efficiency noted in chicks fed 250 
or 500 mg Co/kg diet (Southern and Baker, 1981). Cysteine 
was more efficacious than methionine, when provided on an 
isosulfurous basis, in alleviating cobalt toxicosis in chicks. 
Sulfur amino acids may reduce cobalt toxicity by forming 
stable complexes with cobalt in the digestive tract that are 
poorly absorbed and/or by enhancing the detoxication of 
absorbed cobalt (Baker and Czarnecki-Maulden, 1987). In- 
creasing dietary protein from 10 to 20 or 30 percent partially 
prevented growth depression but not increased susceptibility 
to Salmonella gallinarum observed in chicks fed toxic levels 
of cobalt (Hill, 1979a). 

Selenium and vitamin E status may affect toxicity of co- 
balt. Intramuscular injection of selenium and vitamin E, one 
day prior to oral administration of 100 mg Co/kg BW, pro- 
tected against cobalt-induced myocardial damage (Van Vleet 
et al., 1977). Ascorbic acid addition to diets partially pre- 
vented growth depression observed in chicks fed 200 mg 
Co/kg (Hill, 1979b). Cobalt toxicosis in chicks was exacer- 
bated by duodenal coccidiosis (Eimeria acervulina) in chicks 
(Southern and Baker, 1982). 

Based on studies showing a much higher cobalt absorp- 
tion in young ( 1 to 20 days) rats and guinea pigs than in older 
(30 to 200 days) animals, one would expect that lower con- 
centrations of cobalt may be required to produce toxicosis in 
young animals. Young calves appeared to be affected more 
by lower concentrations of cobalt than older calves (Keener 
et al., 1949). 

TISSUE LEVELS 

Cobalt is found in tissues at low concentrations (<1.0 
mg/kg DM) when animals are fed normal dietary cobalt con- 
centrations. Exposure of animals to high dietary cobalt 
greatly increases concentrations of cobalt in a number of tis- 
sues. Representative values in tissues from various animals 
fed normal or high concentrations of cobalt are shown in 
Table 12-2. Cobalt concentrations in liver and kidney in- 
crease to the greatest extent with smaller increases in hearts 
of animals fed high concentrations of cobalt. Increases in 
muscle cobalt in animals given high concentrations of cobalt 



are relatively small, and muscle cobalt was only 0.26 mg/kg 
DM in lambs supplemented with 40 mg Co/kg diet for 60 
days (Henry et al., 1997). 

MAXIMUM TOLERABLE LEVELS 

The maximum tolerable level for cobalt is defined as the 
dietary level that, when fed for a defined period of time, will 
not impair animal health and/or performance. A number of 
factors can affect the maximum tolerable level for cobalt, 
especially iron status of the animal and dietary concentration 
of sulfur amino acids. Age and antioxidant status of the ani- 
mal also appear to affect the level of cobalt that can be toler- 
ated without adverse effects on animal performance or 
health. 

Chicks and rats are able to tolerate 50 mg Co/kg diet with- 
out adverse effects on growth (Hill, 1974; Huck, 1975). 
However, a 50 mg Co/kg diet increased disease susceptibil- 
ity in chicks experimentally inoculated with Salmonella 
gallinarum (Hill, 1974). In pigs, cobalt concentrations 
greater than 200 mg/kg diet were required to depress growth 
and feed efficiency (Huck and Clawson, 1976). Older stud- 
ies suggested that calves can tolerate up to 0.86 mg Co/kg 
BW (Ely et al., 1948). This would be 34 mg/kg diet if dry 
matter intake is assumed to be 2.5 percent of BW. Controlled 
studies to assess cobalt tolerance have not been reported in 
older cattle. Yearling sheep tolerated 3.5 mg Co/kg BW (ap- 
proximately 144 mg Co/kg diet) for 56 days (Becker and 
Smith, 1951), while adult male sheep tolerated approxi- 
mately 180 mg Co/kg diet for 109 days without adverse ef- 
fects (Corrier et al., 1986). Studies have not been conducted 
to determine if such high concentrations of cobalt can be 
safely tolerated by younger lambs or during fetal development. 

Based on available literature, the maximum tolerable lev- 
els for cobalt were set at 25 mg/kg diet for chicks, rats, sheep, 
and cattle, and 100 mg/kg diet for swine. Insufficient data 
are available to set a maximum tolerable level for cobalt for 
dogs and cats. At dietary cobalt concentrations up to the 
maximum tolerable levels indicated, increases in cobalt in 
edible tissue are not likely to present a human health con- 
cern. However, animals fed diets containing the maximum 
tolerable level of cobalt may have kidney cobalt concentra- 
tions that exceed standards for human health (see Maximum 
Tolerable Levels chapter). 

FUTURE RESEARCH NEEDS 

Additional work is needed with swine to confirm earlier 
research indicating that growing pigs could tolerate consid- 
erably higher dietary concentrations of cobalt than chicks or 
rats. The most pressing need for cobalt toxicosis research is 
in ruminants, especially cattle. Studies examining cobalt tol- 
erance in growing calves were conducted in the 1940s, and 
no research has been conducted to define levels of cobalt 
that can be safely tolerated by older cattle. In contrast, cobalt 



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128 



MINERAL TOLERANCE OF ANIMALS 



Studies in sheep have all been conducted with yearling or 
adult animals, and these studies indicate that sheep may be 
able to tolerate much higher cobalt concentrations than cattle. 
It is unclear if the discrepancy in cobalt tolerance reported 
between sheep and cattle is truly a species difference or an 
age effect. 

SUMMARY 

Cobalt functions as an essential component of vitamin 
Bj2- Nonruminant animals do not require a dietary source of 
cobalt because mammalian tissues are unable to synthesize 
vitamin Bj2 from inorganic cobalt. Ruminal microorganisms 
synthesize vitamin Bjj from cobalt, and the vitamin Bjj pro- 
duced serves to meet the animal's requirement. Therefore, 
ruminants have a dietary requirement for cobalt. Cobalt toxi- 
cosis is generally not a practical problem because concentra- 
tions of cobalt needed to cause toxicosis are much higher 
than those normally found in animal diets. Errors in formu- 
lation of mineral supplements for ruminants could result in 
cobalt toxicosis. However, toxic levels of cobalt are over 
100 times greater than dietary requirements for ruminants. 



REFERENCES 

ATSDR (Agency for Toxic Substances and Disease Registry). 2001. Toxi- 
cological profile for cobalt. Atlanta, GA: tJ.S. Department of Health 
and Human Services, Public Healtti Service. Available at http:// 
www.atsdr.cdc.gov/toxprofiles/tp33.html. 

Anderson, M. B., N. G. Pedigo, R. P. Katz, and W. J. George. 1992. Histo- 
pathology of testes from mice chronically treated with cobalt. Reprod. 
Toxicol. 6:41-50. 

Anderson, M. B., K. Lepak, V. Farinas, and W. J. George. 1993. Protective 
action of zinc against cobalt-induced testicular damage in the mouse. 
Reprod. Toxicol. 7:49-54. 

Baker, D. H., and G. L. Czarnecki-Maulden. 1987. Pharmacologic role of 
cysteine in ameliorating or exacerbating mineral toxicities. J. Nutr. 
117:1003-1010. 

Becker, D. E., and S. E. Smith. 1951. The level of cobalt tolerance in year- 
ling sheep. J. Anim. Sci. 10:266—271. 

Blalock, T. L. 1985. Studies on the role of iron in the reversal of zinc, 
cadmium, vanadium, nickel, and cobalt toxicities in broiler pullets. 
Ph.D. dissertation. North Carolina State University, Raleigh. 

Chetty, K. N. 1972. Interactions of cobalt and iron in chicks. Ph.D. disser- 
tation. North Carolina State University, Raleigh. 

Chung, A. S., W. G. Hoekstra, and R. H. Grummer. 1976. Supplemental 
cobalt or nickel for zinc deficient G. F. pigs. J. Anim. Sci. 42:1352- 
1353 (Abstr.). 

Chung, A. S., M. L. Sunde, R. H. Grummer, and W. G. Hoekstra. 1977. The 
sparing effect of cobalt in chicks. Fed. Proc. 37:668 (Abstr). 

Clyne, N., L. E. Lins. S. K. Pehrsson, A. Lundbereg, and J. Werner. 1988. 
Distribution of cobalt in myocardium, skeletal muscle and serum in 
exposed and unexposed rats. Trace Elem. Med. 5:52-54. 

Corrier, D. E., H. H. Mollenhauer, D. E. Clark, M. F. Hare, and M. H. 
Elissalde. 1985. Testicular degeneration and necrosis induced by di- 
etary cobalt. Vet. Pathol. 22:610-616. 

Corrier, D. E.. L. D. Rowe, D. E. Clark, and M. F. Hare. 1986. Tolerance 
and effect of chronic dietary cobalt on sheep. Vet. Hum. Toxicol. 
28:216-219. 



Diaz, G. J., R. J. Julian, and E. J. Squires. 1994. Lesions in broiler chickens 
following experimental intoxication with cobalt. Avian Dis. 38:308-316. 

Dickson, J., and M. P. Bond. 1974. Cobah toxicity in cattle. Aust. Vet. J. 
50:236. 

Ely, R. E., K. M. Dunn, and C. F. Huffman. 1948. Cobalt toxicity in calves 
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Firriolo, J. M., F. Ayala-Fierro, I. G. Sipes, and D. E. Carter. 1999. Absorp- 
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J. Toxicol. Environ. Health 58:383-395. 

Greenwood, N. N., and A. Earnshaw. 1997. Chemistry of the Elements. 
(2nd ed.) Oxford: Butterworth-Heinemann. 

Henry, P. R., R. C. Littell, and C. B. Ammerman. 1997. Bioavailability of 
cobalt sources for ruminants. 1 . Effects of time and dietary cobalt con- 
centration on tissue cobalt concentration. Nutr. Res. 17:947-955. 

Hill, C. H. 1974. Influence of high levels of minerals on the susceptibility of 
chicks to Salmonella gallinarum. J. Nutr. 104:1221-1226. 

Hill, C. H. 1979a. The effect of dietary protein levels on mineral toxicity in 
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Hill, C. H. 1979b. Studies on the ameliorating effect of ascorbic acid on 
mineral toxicities in the chick. J. Nutr. 109:84—90. 

Huck, D. W. 1975. The study of cobalt toxicity in pigs and rats. Ph.D. 
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Huck, D. W., and A. J. Clawson. 1976. Excess dietary cobalt in pigs. J. 
Anim. Sci. 43:1231-1246. 

Kasprzak, K. S., T. H. Zastawny, and S. L. North. 1994. Oxidative DNA 
base damage in renal, hepatic, and pulmonary chromatin of rats after 
intraperitoneal injection of cobalt (II) acetate. Chem. Res. Toxicol. 
7:329-335. 

Kawashima, T., P. R. Henry, C. B. Ammerman, R. C. Littell, and J. Price. 
1997a. Bioavailability of cobalt sources for ruminants. 2. Estimation of 
the relative value of reagent grade and feed grade cobalt sources from 
tissue cobalt accumulation and vitamin Bp concentrations. Nutr. Res. 
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Kawashima, T., P. R. Henry, D. G. Bates, C. B. Ammerman, R. C. Littell, 
and J. Price. 1997b. Bioavailability of cobalt sources for ruminants. 3. 
In vitro ruminal production of vitamin Bp and total corrinoids in re- 
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17:975-987. 

Keener, H. A., G. P. Percival, K. S. Mon-ow, and G. H. Ellis. 1949. Cobalt 
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Kirchgessner, M., S. Reuber, and M. Kreuzer. 1994. Endogenous excretion 
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Marcel Dekker. 

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Southern, L. L., andD. H. Baker. 1981. The effect of methionine or cysteine 
on cobalt toxicity in the chick. Pouh. Sci. 60:1303-1308. 



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COBALT 



129 



Southern, L. L. and D. H. Balcer. 1982. Eimeria acervuUna injection in 
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Speijers, G. J. A., E. 1. Krajnc, J. M. Berkvens, and M. J. van Logten. 1982. 
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Szakmary, E., G. Ungvary, A. Hudak, E. Tatrai, M. Naray, and V. Morvai. 
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sex-linked anemia mice. Am. J. Clin. Nutr. 26:435^37. 



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stock (3rd ed). New York: CABl Publishing. 
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Available at http://minerals.usgs.gov/minerals/pubs/commodity/cobalt. 

Accessed on March 27, 2005. 
Valberg, L. S., L. Ludwig, andD. Olatunbosun. 1969. Alteration in cobalt 

absorption in patients with disorders of iron metabolism. 

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Van Bruwaene, R., G. B. Gerber, R. Kirchmann, J. Colard, and J. Van 

Kerkom. 1984. Metabolism of ^'Cr, ^''Mn, '"'Fe and ^"Co in lactating 

dairy cows. Health Phys. 46:1069-1082. 
Van Vleet, J. F., A. H. Rebar, and V. J. Ferrans. 1977. Acute cobalt and 

isoproterenol cardiotoxicity in swine: protection by selenium- vitamin E 

supplementation and potentiation by stress-susceptible phenotype. Am. 

J. Vet. Res. 38:991-1002. 
Yamatani, K., K. Saito, and Y. Ikezawa. 1998. Relative contribution of 

Ca^+-dependent mechanism in glucagon-induced glucose output from 

the liver. Aich. Biochem. Biophys. 355:175-180. 



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132 



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COBALT 



133 



TABLE 12-2 Cobalt Concentrations in Fluids and Tissues of Animals (mg/kg) 



Animals Quantity 



Source Duration Route Serum Liver Kidney 



Muscle 



Heart 



Spleen 



Reference 



Rats 



Chicks 



Control 

4.2 mg/kg BW 

Control 
+100 mg/kg 
+200 mg/kg 
+400 mg/kg 



CoSO^- 
VH^O 

CoCL- 



56 d 



Diet 0.001 

0.12 

Diet 



0.02" 
0.48 



0.14° 
1.50 
1.11 
4.53 



0.15" 
2.83 
8.25 
10.24 



Sheep Control CoClj- 109 d Gavage 0.09 0.09" 0.09" 

3.0-10.0 mg/kg BW eH^O 0.98 8.50 2.10 

4.5-15.0 mg/kg BW ' 1.33 8.70 1.90 

Sheep Control CoS04- 60 d Diet 0.20* 0.77* 

+20 mg/kg 7H,0 3.74 3.27 

+40 mg/kg ' 7.33 4.83 



0.05" 
1.62 



Clyne et al., 
1988 

Blalock, 1985 



Pigs 


Control 


CoClj- 


84 d 


Diet 


1.16* 


0.39* 


ND^ 


0.62* 


Huck and 




+200 mg/kg 


6H,0 






8.40 


16.72 


2.90 


4.46 


Clawson, 




+400 mg/kg 








10.85 


39.73 


5.40 


5.99 


1976 




+600 mg/kg ^H 


^^^^ 


^^1 


^^^h 


12.72 


35.89 ^H 


B 9.28 


6.89 





0.10* 

0.14 

0.26 



0.13* 

0.59 

1.26 



0.10* 

0.39 

0.67 



Corrier et al., 
1986 



Henry et al., 
1997 



"Wet tissue basis. 
*Dry tissue basis. 
^Not detectable. 



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13 



Copper 



INTRODUCTION 

Copper (Cu) is one of the oldest metals known to hu- 
mans. It occurs naturally, to a small extent as the free metal, 
but most copper occurs as compounds in the +1 (cuprous) or 
+2 (cupric) oxidation state. Cuprous compounds are gener- 
ally colorless and are rapidly oxidized to the cupric form in 
aqueous solution. Cupric compounds are blue or green in 
color and +2 is the most important oxidation state of copper. 
Copper has a high electrical and thermal conductivity, and is 
highly resistant to corrosion (ATSDR, 2002). These unique 
properties have made copper one of the most important in- 
dustrial metals. 

Copper is found primarily as oxide, sulfide, or carbonate 
ores with chalcocite (CUjS), chalcopyrite (CuFeSj), mala- 
chite (CuC03(CuOH2)), and cuprite (Cu,0) ores as the most 
important sources. Chile is the world's leading copper pro- 
ducer followed by the United States. In the United States, 
1.12 million tons of copper were mined and 860,000 tons 
were imported for processing in 2003 (USGS, 2004). An 
additional 210,000 tons of copper were reclaimed in the 
United States by recycling from old scrap in 2003. 

Brass, bronze, gun metal, and monel metal are important 
alloys that contain copper. Copper alloys are used in construc- 
tion, electrical products, transportation equipment, industrial 
machinery and equipment, coins, and consumer products 
(ATSDR, 2002). Copper compounds are also used as fungi- 
cides, in fertilizers, in nutritional supplements for humans 
and animals, and as algicides in reservoirs and streams. 



(Linder, 2002). Copper-dependent enzymes function in en- 
ergy metabolism, maturation and stability of collagen and 
elastin, pigmentation, the antioxidant defense system, and 
iron metabolism, as well as other biological processes. Defi- 
ciency of copper may result in cardiovascular disorders (car- 
diac failure or rupture of the aorta), depigmentation and im- 
paired keratinization of hair and wool, anemia, reduced 
growth, neonatal ataxia, bone abnormalities, and impaired 
immune responses. Deficiency signs vary depending on ani- 
mal species and the severity of the deficiency. Copper defi- 
ciency is a practical problem in ruminants in many areas of 
the world. Most copper deficiencies in ruminants are due to 
the presence of antagonists (molybdenum, sulfur, and iron) 
in feeds that greatly impair copper metabolism. 

Nonruminant animals generally require between 4 and 8 
mg Cu/kg diet (Underwood and Suttle, 1999). Copper re- 
quirements appear to be higher in horses (Underwood and 
Suttle, 1999). It is well documented that pharmacological 
concentrations of copper (125 to 250 mg Cu/kg diet) can 
stimulate growth and feed efficiency in swine (Cromwell et 
al., 1989) and poultry (Harms and Buresh, 1987; Pesti and 
Bakalli, 1996). The mechanism responsible for the growth- 
promoting action of high dietary copper in nonruminants is 
unclear. Copper requirements of ruminants vary from ap- 
proximately 4 to over 20 mg Cu/kg diet, depending on di- 
etary concentrations of copper antagonists (Underwood and 
Suttle, 1999). Relatively low dietary concentrations of mo- 
lybdenum and sulfur can increase copper requirements by 2- 
to 3-fold. 



ESSENTIALITY 

Copper was reported to be essential for growth and he- 
moglobin formation in rats in 1928 (Underwood and Suttle, 
1999). Subsequent research indicated that copper was an es- 
sential component of a number of enzymes including cyto- 
chrome oxidase, lysyl oxidase, superoxide dismutase, tyro- 
sinase, ceruloplasmin, and dopamine [3-monooxygenase 



DIFFICULTIES IN METHODS OF ANALYSIS AND 
EVALUATION 

Because of the abundance of copper in the environment, 
precautions should be taken to avoid contamination when 
collecting samples for copper analysis (WHO, 1998). 
Vessels to be used in the collection of samples for copper 



134 



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COPPER 



135 



determination shiould be cleaned of dust and washied withi 
dilute acid. Glassware and most plastics are relatively free of 
copper contamination, but heavily pigmented plastics may 
contain copper and should be avoided. Deionized water 
should be used to dilute samples for copper analysis because 
even distilled water can be contaminated with copper 
through contact with copper plumbing and brass fixtures. 
Wet ashing with acid in an open vessel digestion system or 
closed microwave digestion system is the preferred method 
for preparing solid samples for copper analysis. Acid(s) used 
in the digestion process should be extremely low in copper, 
and a blank should be carried through all procedures to cor- 
rect for contamination. 

Atomic absorption spectrophotometric (AAS) methods 
(either flame or graphite furnace) are common methods used 
for detennination of copper. Flame AAS has a detection limit 
of 0.01-0.1 |Jg/mL, while graphite furnace AAS is much 
more sensitive with a detection limit of 0.01-1.0 ng/mL 
(Harris, 1997). Another common method for copper deter- 
mination is inductively-coupled plasma atomic emission 
spectroscopy (ICP-AES). Sensitivity of ICP-AES for copper 
is similar to graphite furnace AAS and this method extends 
linearity over a wider concentration range than AAS and al- 
lows for multielement analysis. 

REGULATION AND METABOLISM 
Absorption and Metabolism 

Major differences in copper metabolism exist between 
nonruminant and ruminant animals. Studies in humans and 
rats indicate that copper is fairly well absorbed (~30 to 75 
percent) in nonruminants (Linder, 2002). Absorption of cop- 
per occurs primarily from the small intestine. Percentage of 
dietary copper absorbed is affected by dietary copper, being 
higher when intake of copper is marginal or low relative to 
requirements. In the intestinal mucosa, a portion of the cop- 
per is bound to metallothionein or similar proteins (Linder, 
2002). Binding to metallothionein prevents transfer of cop- 
per to the intestinal serosa, and metallothionein-bound cop- 
per can be lost in the feces during normal turnover of intes- 
tinal cells. Metallothionein has a higher affinity for copper 
than zinc, and high dietary zinc can reduce copper absorp- 
tion by inducing synthesis of metallothionein (Harris, 1997). 

In ruminants, with a functional rumen, copper absorption 
is low (<1.0 to 10 percent) relative to nonruminants 
(Underwood and Suttle, 1999). Low absorption of copper in 
ruminants is largely due to complex interactions that occur 
among copper, sulfur, and molybdenum in the ruminal envi- 
ronment. Copper absorption is much higher (~75 percent) in 
young ruminants prior to establishment of a ruminal micro- 
flora (Underwood and Suttle, 1999). 

Absorbed copper in portal blood binds to albumin and 
transcuprein for transport primarily to the liver (Linder, 
2002). In the liver, copper can be excreted via the bile, stored. 



or used for synthesis of ceruloplasmin or other copper 
metalloenzymes. Biliary excretion is the major mechanism 
responsible for copper homeostasis. Copper excreted in bile 
is present in forms that are poorly reabsorbed from the small 
intestine. Liver copper concentrations (15 to 30 mg Cu/kg 
DM) are well regulated in most nonruminants; however, bil- 
iary copper excretion becomes saturated at high dietary cop- 
per concentrations, and then liver copper accumulation oc- 
curs. Genetic disorders, associated with impaired biliary 
excretion of copper, occur in humans (Wilson's disease) and 
certain breeds of dogs (Hyun and Filippich, 2004) that result 
in elevated liver copper concentrations. Normal liver copper 
concentrations in ruminants (100 to 400 mg Cu/kg DM) are 
considerably higher than in pigs and chickens (Underwood, 
1977). In fact, concentrations of copper normally found in 
the liver of most nonruminants would be indicative of cop- 
per deficiency in ruminants. Biliary copper excretion is much 
less effective in regulating liver copper concentrations in 
ruminants. This is especially true in sheep where increasing 
dietary copper does not appear to increase biliary copper 
excretion (Saylor and Leach, 1980). 

Liver is the major storage organ for copper, and stored 
copper is largely bound to metallothionein in most species 
(Bremner, 1987). When high dietary copper is ingested, 
binding to metallothionein appears to be an important cellu- 
lar detoxification mechanism in some animal species 
(Bremner, 1987). In sheep, a much smaller proportion of 
liver copper is bound to metallothionein, and sheep have a 
limited ability to increase metallothionein synthesis in re- 
sponse to increased liver copper (Saylor et al., 1980). 

Copper not excreted in bile, stored, or used for copper- 
dependent enzymes in the liver leaves the liver largely bound 
to ceruloplasmin, an oxidase and acute phase protein. In most 
species (except birds), ceruloplasmin is the major form of 
copper in plasma and is believed to be the major protein that 
transports copper to extrahepatic tissues. Ceruloplasmin is 
thought to recognize receptors on the plasma membrane of 
tissues and to release copper into cells (Harris, 1997). Free 
copper is highly toxic to cells; cytosolic proteins, referred to 
as copper chaperones, bind copper ions and deliver them to 
copper-requiring proteins without releasing free copper ions 
(Pena et al., 1999). Tissue copper concentrations are highest 
in the liver followed by the kidney and brain. 

IVIetabolic Interactions and IVIechanisms of Toxicity 

Interactions that occur among copper, sulfur, and molyb- 
denum are extremely important in ruminant nutrition. Mo- 
lybdenum in ruminant diets is frequently within the range of 
1 to 5 mg Mo/kg DM, while total sulfur usually varies from 
0.1 to 0.3 percent. Concentrations of molybdenum and sul- 
fur on the upper end of this range greatly reduce copper 
bioavailability and increase the risk of copper deficiency. In 
contrast, low dietary concentrations of molybdenum and sul- 
fur increase the risk of copper toxicosis, especially in sheep. 



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MINERAL TOLERANCE OF ANIMALS 



Sulfide, derived from sulfate reduction and degradation 
of sulfur amino acids by ruminal microorganisms, is believed 
to reduce copper absorption via formation of insoluble cop- 
per sulfide (Suttle, 1991). Increasing dietary sulfur from 0.1 
to 0.4 percent reduced copper bioavailability by 30 to 56 
percent in hypocupremic sheep fed low molybdenum diets 
(Suttle, 1974). Sulfide can also interact with molybdate in 
the rumen to form various thiomolybdates (Suttle, 1991). 
Tri-and tetrathiomolybdates reduce copper absorption by 
forming insoluble complexes with copper that do not release 
copper even under acidic conditions. When dietary sulfur 
and molybdenum are high, di- and trithiomolybdates can be 
absorbed and affect systemic metabolism of copper 
(Gooneratne et al., 1989). Systemic effects on copper me- 
tabolism attributed to thiomolybdates include (1) increased 
biliary excretion of copper from liver stores; (2) removal of 
copper from metalloenzymes; and (3) strong binding of cop- 
per to plasma albumin, which results in reduced transport of 
available copper for biochemical processes. Molybdenum 
and sulfur supplementation have been used to prevent and 
treat copper toxicosis in sheep (Howell and Gooneratne, 
1987). Intravenous administration of ammonium 
tetrathiomolybdate has effectively been used to treat copper 
toxicosis in sheep (Howell and Gooneratne, 1987). 

In nonruminants, interactions between copper and mo- 
lybdenum are minimal and only occur at very high concen- 
trations of molybdenum (Mills and Bremner, 1980). Copper 
does interact with amino acids in nonruminants. Sulfur 
amino acid requirements for maximal growth are higher in 
chicks fed pharmacological levels of copper (Robbins and 
Baker, 1980; Wang et al., 1987). Increasing the concentra- 
tion of methionine or cysteine above dietary requirements 
for growth alleviates the growth depression seen in chicks 
fed excess copper (500 mg Cu/kg) (Jensen and Maurice, 
1979; Robbins and Baker, 1980). Cysteine supplementation 
reduced liver copper concentrations in chicks fed high di- 
etary copper (Persia et al., 2003) and may chelate copper and 
reduce its absorption (Baker and Czarnecki-Maulden, 1987). 

A number of studies indicate that high dietary zinc can 
reduce copper absorption (WHO, 1998). High doses of zinc 
are effective in reducing copper absorption in patients with 
Wilson's disease. High dietary zinc (220 or 420 mg Zn/kg 
DM) was effective in preventing copper toxicosis by reduc- 
ing liver copper accumulation in lambs fed high copper diets 
(Bremner et al., 1976). High dietary iron can reduce copper 
absorption in nonruminants (Yu et al., 1994) and reduces 
copper status in cattle and sheep (Spears, 2003). 

The mechanism of chronic copper toxicity appears to re- 
late to ionic copper producing oxidative damage to tissues. 
Copper can shift between an oxidized (Cu+-) and reduced 
state (Cu"*"') in the cell, which allows copper to bind strongly 
to many types of electron-rich structures (WHO, 1998). Nor- 
mally, free copper in the cell is maintained at low concentra- 
tions by copper-binding compounds such as metallothionein, 
glutathione, and copper chaperone proteins (Viarengo et al.. 



2002). However, when the copper concentration in the cell 
overwhelms metal homeostasis, free copper can alter cellu- 
lar functions by directly binding to proteins and nucleic ac- 
ids and by formation of reactive oxygen species. Ionic cop- 
per readily participates in the Fenton reaction that results in 
the production of reactive oxygen species, including the hy- 
droxyl radical (Viarengo et al., 2002). Hydroxyl radicals can 
cause lipid peroxidation in cell membranes, cleavage of 
nucleic acids, and oxidation of cellular proteins. 

Acute copper toxicosis in several species, including hu- 
mans, results in nausea and vomiting. It is believed that the 
mechanism responsible for these signs is due to copper ions 
stimulating receptors that, in turn, stimulate the vagus nerve, 
eliciting a reflex response of nausea and vomiting (Araya et 
al., 2002). 

SOURCES AND BIOAVAILABILITY 

Copper is found naturally in the Earth's crust at an aver- 
age concentration of 50 mg/kg (ATSDR, 2002). Seawater 
contains approximately 0.15 [Jg Cu/L, and fresh water con- 
tains 1.0 to 20 |Jg Cu/L in uncontaminated areas (WHO, 
1998). Natural weathering of soil and discharges from in- 
dustries and sewage treatment plants can result in copper 
release into water. Copper compounds are also sometimes 
applied to water to kill algae. Copper released into water is 
primarily in particulate form and tends to settle out, precipi- 
tate out, or be adsorbed by organic matter, hydrous iron, 
manganese oxides, or clay in the sediment. This usually re- 
sults in low concentrations of copper downstream from the 
source of copper entry. The cuprous (H-1) ion is unstable in 
aqueous solution and tends to disproportionate to the cupric 
(h-2) form and copper metal unless a stabilizing ligand is 
present (WHO, 1998). The only cuprous compounds stable 
in water are insoluble ones such as cuprous sulfide. Cupric 
copper forms coordination compounds or complexes with 
inorganic and organic ligands. Bioavailability of copper in 
water is generally low due to adsorption to suspended par- 
ticles and complexation by dissolved organic matter or inor- 
ganic ligands such as carbonate (WHO, 1998). Copper in 
sediments also appears to be of poor bioavailability because 
of its ability to react with acid volatile sulfides and form 
insoluble precipitates (Besser et al., 1996). 

Copper concentrations in various soil types in the United 
States can vary from 1 to 700 mg/kg with an average con- 
centration of 25 mg/kg (WHO, 1998). Soil copper concen- 
trations are highest in areas in close proximity to copper 
mining and smelting activities. Application of sludge, poul- 
try or swine waste, and copper-containing fungicides can 
increase soil copper concentrations. The majority of copper 
deposited in soil will be strongly adsorbed and remain in the 
upper few centimeters of soil (Georgopoulos et al., 2002). 
Soil copper can adsorb to organic matter, clay minerals, car- 
bonate minerals, or hydrous iron and manganese oxides. 
Copper binds to soil more strongly than other divalent 



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cations (Georgopoulos et al., 2002). Because of the strong 
adsorption and complexation of soil copper, elevated soil 
copper often does not greatly increase copper uptake by 
plants (Suttle and Price, 1976; Payne et al., 1988). 

Forages generally contain 3 to 8 mg Cu/kg DM (Minson, 
1990). Legumes are usually higher in copper than grasses 
when grown under temperate conditions. Bioavailability of 
copper from forages is low in ruminants and is greatly af- 
fected by forage concentrations of sulfur, molybdenum, and 
iron (Underwood and Suttle, 1999). Normal concentrations 
of copper in cereal grains range from 3 to 8 mg Cu/kg DM, 
while leguminous and oilseed meals range from 15 to 35 
mg/kg DM. Relative to copper sulfate, copper bioavailability 
from corn gluten meal, soybean meal, and cottonseed meal 
was 48, 38, and 41 percent, respectively, in chicks (Aoyagi 
et al., 1995). Feeds of animal origin with the exception of 
liver products are low in copper. Liver is often used as an 
ingredient in pet foods. Based on chick studies, using bile 
copper excretion as a measure of bioavailability, copper in 
pork liver was essentially unavailable (Aoyagi et al., 1993). 
However, copper from chicken, turkey, beef, and sheep liver 
was similar in bioavailability to copper sulfate (Aoyagi et 
al., 1993). 

Use of poultry or swine waste in diets or as pasture fertil- 
izer can result in high intakes of copper by ruminants. Poul- 
try and swine waste are high in copper, especially if waste is 
obtained from animals fed growth stimulatory concentrations 
of copper (100 to 250 mg/kg). Copper from swine and poul- 
try waste is equal in bioavailability to copper sulfate when 
fed to hypocupremic ewes (Suttle and Price, 1976). Applica- 
tion of high copper waste to pasture (Suttle and Price, 1976) 
or crop land (Payne et al., 1988) did not increase forage or 
grain copper concentrations greatly. However, application 
of waste to pastures can result in copper toxicosis due to 
ingestion of forage contaminated with high copper waste. 
Copper toxicosis has been reported in ruminants fed poultry 
waste (Suttle et al., 1978; Tokarnia et al., 2000) or grazing 
pastures where large amounts of waste have been applied 
(Sargison and Scott, 1996). However, other studies have 
shown no ill effects from grazing sheep on pasture fertilized 
with high-copper pig manure (Prince et al., 1975). In addi- 
tion, in some studies chicks and sheep have been fed high- 
copper pig manure directly with no ill effects, apparently 
because the copper in the manure was poorly bioavailable to 
the chickens and sheep (Prince et al., 1975; Izquierdo and 
Baker, 1986). 

Another potential source of copper in ruminants is copper 
used in foot baths. Copper sulfate foot baths are used in ru- 
minants to control foot rot and other lameness-related prob- 
lems. Ingestion of copper from baths or consumption of pas- 
ture where the copper sulfate solution has been disposed of 
can lead to copper toxicosis. Copper has been used as an 
anthelmintic to control gastrointestinal parasites in ruminants 
(Bang et al., 1990). For example, high doses of copper (3 to 
4 g) from copper oxide needles have been administrated to 



sheep (Bang et al., 1990) and goats (Chartier et al., 2000) to 
control parasite loads. 

A major source of copper in animal diets is supplemental 
copper added to diets or free choice mineral supplements. 
Errors in copper formulation or excess supplementation with 
copper can result in toxicosis. Sources of copper supple- 
mented to animal diets include cupric sulfate, tribasic cupric 
chloride, copper oxide (primarily cupric oxide), cupric car- 
bonate, and various organic copper sources. Copper from 
copper oxide powder is essentially unavailable when fed to 
cattle (Kegley and Spears, 1994), hens (Jackson and 
Stevenson, 1981), chicks (Baker et al., 1991), and swine 
(Cromwell et al., 1989). Copper oxide needles supply avail- 
able copper in ruminants because the needles are retained in 
the digestive tract and release copper over several weeks. 
Apparently, copper oxide powder passes through the acid 
environment of the digestive tract before the copper can be 
solubilized (Spears, 2003). Tribasic cupric chloride is simi- 
lar in bioavailability to cupric sulfate in chicks (Miles et al., 
1998) and swine (Cromwell et al., 1998) and in cattle fed 
diets low in molybdenum and sulfur (Spears et al., 2004). 
Relative to cupric sulfate (set at 100 percent), tribasic cupric 
chloride is more bioavailable (132 to 196 percent) when 
supplemented to diets high in molybdenum and sulfur 
(Spears et al., 2004). Feed grade cupric carbonate appears to 
be somewhat less bioavailable, based on liver copper con- 
centrations, than cupric sulfate (Ledoux et al . , 1991; Ward et 
al., 1996). Results of studies evaluating the bioavailability 
of copper from different organic sources have been variable, 
with some studies indicating similar bioavailability and oth- 
ers higher bioavailability relative to cupric sulfate (Spears, 
2003). 

In field studies with fish, the bioconcentration factor for 
copper was 10 to 100, indicating a low potential for 
bioconcentration (ATSDR, 2002). Biomagnification of cop- 
per also does not occur in the food chain. 

TOXICOSIS 

A number of excellent reviews are available on copper 
toxicosis in mammals and fish (Howell and Gooneratne, 
1987; Bremner, 1998; WHO, 1998; ATSDR, 2002; 
Clearwater et al., 2002). Copper toxicosis can result from a 
single large dose of copper or from repeated exposure to 
copper concentrations that exceed animal requirements. A 
number of studies evaluating oral ingestion of high concen- 
trations of copper are summarized in Table 13-1. 

Animal species differ greatly in their ability to tolerate 
excess copper. Sheep are very sensitive to copper toxicity. 
Death due to copper toxicosis is a common problem in sheep. 
The range of dietary copper concentrations required by sheep 
under some conditions can overlap with dietary concentrations 
that cause toxicosis under other conditions. For example, 
10 mg Cu/kg diet may be required by sheep if dietary sulfur 
and molybdenum are fairly high. However, if dietary 



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MINERAL TOLERANCE OF ANIMALS 



molybdenum is low, 10 mg Cu/kg diet can cause toxicosis in 
some breeds of sheep (Hogan et al., 1968). The sensitivity of 
sheep to copper toxicity appears to relate to their inability to 
increase biliary copper excretion in response to elevated copper 
intakes (Bremner, 1998). Cattle and goats are less suscep- 
tible to copper toxicity than sheep. Young ruminants are 
more susceptible than adults because of higher absorption. 

In nonruminants, copper homeostatic control mechanisms 
generally are very efficient in preventing toxicosis. Concen- 
trations of copper needed to cause toxicosis in nonruminants 
generally exceed requirements by at least 25 -fold, and are as 
high as 50-fold in pigs. Rats can tolerate higher dietary con- 
centrations of copper than swine and poultry, and mice are 
less sensitive than rats to copper toxicity (Hebert et al., 
1993). Most fish can tolerate fairly high concentrations of 
dietborne copper (Clearwater et al., 2002). However, some 
fish species are sensitive to relatively low concentrations of 
copper in water (Taylor et al., 1996). 

Liver and kidney are target organs for copper toxicosis in 
all species. Lesions in the forestomach also occur in rats and 
mice (Hebert et al., 1993), and proventriculitis (Wideman et 
al., 1996) and gizzard erosion (Jensen and Maurice, 1978) 
are seen in poultry exposed to high dietary copper. 

Single Dose and Acute 

Acute copper toxicosis signs include nausea, vomiting, 
diarrhea, excessive salivation, abdominal pain, convulsions, 
paralysis, and sometimes death (NRC, 1980; WHO, 1998). 
Necropsy of animals with acute copper toxicosis reveals 
acute gastroenteritis, necrotic hepatitis, and splenic and re- 
nal congestion. The acute toxic level of oral copper (as 
CUSO4) is 9 to 20 mg/kg BW in sheep and approximately 
200 mg/kg BW in cattle (NRC, 1980). Administration of 
copper oxide wire or needles at a rate of 4.1 g Cu/head to 
sheep (Bang et al., 1990) or 3.4 g Cu/head to goats (Chartier 
et al., 2000) did not produce toxicosis. Copper oxide needles 
release copper slowly in the gastrointestinal tract and appear 
to be relatively safe when administered at the labeled dosage 
to ruminants with a functional rumen. However, administra- 
tion of copper oxide needles to young calves has resulted in 
mortality from copper toxicosis (Hamar et al., 1997; Steffen 
et al., 1997). In horses, a single oral dose of 125 mg CUSO4 
(in solution)/kg BW resulted in gastroenteritis, hemolysis, 
kidney and liver damage, and death within 2 weeks (Hintz, 
1987). Acute toxicosis did not occur when the same amount 
of copper was added to the feed of horses. The LD^q in rats 
for copper as CUSO4 has ranged from 120 to 244 mg Cu/kg 
BW in different studies (WHO, 1998). 

The 96-hr LCjq for Chinook salmon was 54-64 |ig Cu/L 
of water (Hamilton and Buhl, 1990). In channel catfish, the 
24-hr LC5Q was 2,500 to 3,500 pg/L depending on water tem- 
perature (Smith and Heath, 1979). In rainbow trout, the 96-hr 
LCjQ ranged from 20 |Jg Cu/L in soft acid water to 520 |-ig/L 
in hard alkaline water (Howarth and Sprague, 1978). 



Chronic 

Chronic copper toxicosis in sheep is characterized by the 
gradual accumulation of copper in the liver over a period of 
several weeks or months. The condition is most common in 
lambs fed large amounts of concentrate feeds, but can occur 
under grazing conditions if forage molybdenum concentration 
is low or if sheep are consuming plants that contain hepato- 
toxic compounds (Underwood and Suttle, 1999). Copper 
toxicosis in sheep consists of a prehemolytic, and hemolytic 
phase (Howell and Gooneratne, 1987). Although copper is 
accumulating in the liver and to a lesser extent in the kidney 
during the prehemolytic phase, the animal is clinically normal, 
and depressed animal performance is generally not evident 
until at least shortly before the hemolytic phase occurs. Dur- 
ing the prehemolytic phase, liver necrosis occurs and enzymes 
(aspartate aminotransferase, glutamate dehydrogenase, and 
sorbitol dehydrogenase) indicative of liver damage may be- 
come elevated in serum, but blood copper is generally nor- 
mal (Howell and Gooneratne, 1987). Liver concentrations of 
copper in all cellular fractions are increased, but the propor- 
tion of cellular copper in the nuclear fraction is increased 
during copper loading (Gooneratne et al., 1979). 

Sheep show histological and biochemical evidence of 
liver damage at liver copper concentrations as low as 350 
mg Cu/kg DM; however, clinical signs of toxicosis do not 
usually occur until liver concentrations of 1,000 mg Cu/kg 
DM or higher are reached (Underwood and Suttle, 1999). 
Animals that die from copper toxicosis often have liver con- 
centrations that exceed 2,000 mg Cu/kg DM. The hemolytic 
phase (referred to as hemolytic crisis) is associated with rapid 
release of copper from the liver into the blood and is charac- 
terized by hemolysis, hemoglobinemia, and hemoglobinuria. 
Clinical signs may include dullness, anorexia, excessive 
thirst, jaundice of mucous membranes, dark-colored urine, 
sunken eyes with blood vessels on the surface of the sclera 
showing a chocolate-brown color, and death (Howell and 
Gooneratne, 1987). Observed hemolysis is due to entry of 
excessive copper into erythrocytes. The mechanism whereby 
copper induces hemolysis is unclear, but may be due to cop- 
per inducing production of superoxide radicals that cause 
erythrocyte membrane damage (Howell and Gooneratne, 
1987). Sheep with a mild hemolysis may survive and re- 
cover, especially if treated with copper chelating agents such 
as ammonium tetrathiomolybdate or penicillamine that in- 
crease copper excretion. 

Sheep that are killed during the hemolytic phase or those 
that die from toxicosis show major liver and kidney damage 
(Howell and Gooneratne, 1987). Livers contain areas of ne- 
crotic parenchymal cells and swollen copper-containing 
Kupffer cells high in acid phosphatase. Kidney copper is 
greatly elevated following the hemolytic phase. The elevated 
copper causes necrosis in the proximal convoluted tubules and 
impaired glomerular and tubular function. In addition, kid- 
neys have a characteristic black or dark brown color. 



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The concentration of dietary copper needed to cause toxi- 
cosis in siieep lias varied greatly among studies (Table 13-1) 
and is greatly affected by genetics and dietary factors that will 
be discussed in the next section. In several studies (Hill and 
Williams, 1965; Pond, 1989; Zervas et al., 1990) feeding total 
(diet plus supplemental) dietary copper concentrations of 30 
to 40 mg/kg DM resulted in some mortality from toxicosis. 
Death losses occurred after 3 to 12 weeks of feeding the high 
copper diets. Elevated plasma aspartate aminotransferase ac- 
tivity was observed in some lamb breeds fed a total dietary 
copper concentration of 20 mg/kg DM for 9 weeks 
(Woolliams et al., 1982). Long-term studies have indicated 
that copper toxicosis can occur in housed sheep fed diets con- 
taining approximately 10 mg Cu/kg (Hogan et al., 1968) and 
in ewes grazing forage containing 7.5 to 15.0 mg Cu/kg DM 
(MacPherson et al., 1997) if dietary molybdenum is low. 

Cattle can tolerate higher dietary concentrations of cop- 
per than sheep. However, some breeds of cattle may be more 
susceptible to copper toxicosis than others. Clinical signs of 
copper toxicosis in cattle are similar to those described for 
sheep. The preruminant calf is more susceptible to copper 
toxicosis than older cattle during relatively short-term cop- 
per exposure. Clinical cases of copper toxicosis occurred in 
10- to 12-week-old calves fed milk replacer containing 115 
mg Cu/kg DM (Shand and Lewis, 1957). In calves (Holstein 
and Ayrshire-Holstein crossbreds), supplementation of milk 
replacer with 50 mg Cu/kg DM for 6 weeks reduced intake 
(Jenkins and Hidiroglou, 1989). Copper addition to milk re- 
placer at 200 mg/kg DM reduced gain and intake while 1,000 
mg Cu/kg DM resulted in a 43 percent mortality in calves 
(Jenkins and Hidiroglou, 1989). 

Older cattle (Bonsmara) receiving 10 or 20 mg Cu/kg 
BW, in an oral drench, for 5 days per week showed clinical 
signs of copper toxicosis by day 679 after initial dosing 
(Gummow, 1996). However, liver damage occurred much 
sooner based on elevated serum asparate aminotransferase 
activity. A number of factors may influence the ability of 
older cattle to tolerate copper. In some studies (Chapman et 
al., 1962; Felsman et al., 1973), cattle have tolerated high 
doses of copper without showing signs of toxicosis, and un- 
der certain conditions (that are not well defined) cattle ap- 
pear to regulate liver copper concentrations once concentra- 
tions reach a certain level. Steers (Hereford x Brahman) 
dosed daily with 0.13 to 2.0 g Cu/d, via gelatin capsule, for 
16 months showed no clinical signs of toxicosis (Chapman 
et al., 1962). Liver copper concentrations increased during 
the study, but final liver copper concentrations were approxi- 
mately 2,000 mg/kg DM regardless of the quantity of copper 
dosed. Steers (Hereford) administered 3 g Cu/d, via a gelatin 
capsule, also did not show adverse effects by 64 days; how- 
ever, this quantity of copper given in water resulted in clini- 
cal signs of copper toxicosis, including death, and much 
higher liver copper concentrations (Chapman et al., 1962). 
No signs of copper toxicosis were observed in weaned Hol- 
stein calves fed a high concentrate diet supplemented with 



125 to 900 mg Cu/kg DM for 98 days (Felsman et al., 1973). 
Liver copper concentrations at the end of the study in cop- 
per-supplemented calves were approximately 900 mg/kg 
DM, regardless of dietary copper (Felsman et al., 1973). 

Long-term supplementation of lower dietary copper con- 
centrations than those described above can cause toxicosis 
in cattle. Bradley (1993) reported the death of 9 of 63 Hol- 
stein cows in a herd from copper toxicosis. For over two 
years, the cows received a diet that analyzed 37.5 mg Cu/kg 
DM during lactation and 22.6 mg Cu/kg DM during the dry 
period. Cows exhibited clinical signs of copper toxicosis 
prior to death and had elevated postmortem liver copper con- 
centrations that ranged from 1,236 to 2,179 mg/kg DM. All 
cows that died were in late pregnancy. The addition of am- 
monium molybdate to the diet for 18 days and removal of 
the copper-containing mineral supplement arrested the out- 
break of copper toxicosis (Bradley, 1993). Lactating dairy 
cows (Holstein) tolerated 40 (Engle et al., 2001) or 80 mg 
(Du et al., 1996) supplemental Cu/kg DM for 60 days. How- 
ever, over the 60-day periods, liver copper concentrations 
increased by over 7 mg Cu/kg DM/day in both studies. If 
this rate of increase in liver copper had continued, cows may 
have eventually showed signs of copper toxicosis. 

Goats can also tolerate much higher dietary copper con- 
centrations than sheep. Zervas et al. (1990) compared the 
susceptibility of lambs and goats to chronic copper toxico- 
sis. Lambs supplemented with 30 or 60 mg Cu/kg DM had 
elevated serum asparate aminotransferase after 50 days, and 
some lambs died from copper toxicosis beginning as early as 
67 days. Goats fed the same diets (30 or 60 mg Cu/kg) had 
normal serum asparate aminotransferase activity and exhib- 
ited no signs of toxicosis during the 137-day study. Lambs 
stored 6 to 9 times more copper in their livers than goats. 
Goats fed a total dietary copper of 36 mg/kg DM for 88 days 
had increased liver copper but no signs of toxicosis or liver 
damage (Luginbuhl et al., 2000). Goats administered 600 
mg Cu/d (gelatin capsule) for 4 weeks showed clinical signs 
of toxicosis and one of two goats receiving 1,200 mg Cu/d 
died (Solaiman et al., 2001). 

Copper is relatively nontoxic to nonruminant animals. 
Supplementation of swine diets with 250 mg Cu/kg diet in- 
creased liver copper concentration, but generally has produced 
no negative effects on animal growth or health. In fact, 250 
mg/kg of added copper normally stimulates growth rates of 
weanling pigs (Hill et al., 2000). Addition of 250 mg Cu/kg to 
sow diets for up to 775 days had no adverse effects on animal 
health and actually increased piglet birth and weaning weights 
(Cromwell et al., 1993). In growing pigs, 500 mg Cu/kg diet 
has reduced growth and hemoglobin concentrations (Bunch et 
al., 1965; Kline et al., 1971) and resulted in increased mortal- 
ity in one study (DeGoey et al., 1971). 

Poultry are similar to swine in their ability to tolerate ex- 
cess dietary copper. In long-term studies with laying hens, 
400 mg Cu/kg diet or higher reduced feed intake and egg 
production, and increased gizzard weight (Jackson et al.. 



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MINERAL TOLERANCE OF ANIMALS 



1979; Jackson and Stevenson, 1981). Some studies with 
broilers have indicated that supplementation with 250 mg 
Cu/kg diet can cause slight gizzard lining erosion (Robbins 
and Baker, 1980), proventriculitis (Wideman et al., 1996), 
and lesions in the oral cavity, tongue, and pharynx (Chiou et 
al., 1999). In other studies, no adverse effects were noted in 
broilers supplemented with 375 (Pesti and Bakalli, 1996) or 
450 (Ledoux et al., 1991) mg Cu/kg diet. Supplementation 
with 500 mg Cu/kg diet reduced body weights and feed in- 
take, and caused gizzard erosion in turkey poults (Christmas 
and Harms, 1979). In older turkeys, addition of up to 500 mg 
Cu/kg diet did not affect performance (Leeson et al., 1997). 
Ducklings accumulated more copper in their livers than 
chicks when fed high dietary copper concentrations (Wood 
and Worden, 1973) and thus, may be more sensitive to cop- 
per toxicosis. Addition of 500 mg Cu/kg to duckling diets 
caused myopathy characteristic of selenium-vitamin E defi- 
ciency (Van VIeet, 1982). High mortality and severe necro- 
sis in the gizzard, skeletal muscle, and intestine were ob- 
served in ducklings fed 1,000 mg Cu/kg diet. 

Horses and rabbits appear to be more resistant to copper 
toxicosis than swine or poultry. Ponies fed diets containing 
up to 79 1 mg Cu/kg for 6 months showed no signs of toxico- 
sis, including liver damage despite final liver copper con- 
centrations over 3,000 mg/kg DM (Smith et al., 1975). Some 
of the ponies were pregnant during the study and foaled nor- 
mally 3 to 4 months after the study ended. Rabbits can toler- 
ate 500 mg Cu/kg diet for up to 32 days without adverse 
effects (Patton et al., 1982; Grobner et al., 1986). In some 
studies, high dietary copper has increased gain and reduced 
mortality from enteritis in rabbits (Patton et al., 1982). Addi- 
tion of 1,000 mg Cu/kg diet reduced gain, feed intake, and 
feed efficiency in rabbits (Grobner et al., 1986). 

Rats are very tolerant to dietary copper. Diets supple- 
mented with 1,000 mg Cu/kg produced no adverse effects in 
rats (Hebert et al., 1993; Aburto et al., 2001a). Aburto et al. 
(2001a) reported reduced body weight and slight liver dam- 
age in rats fed 1,250 mg Cu/kg diet for 3 months, and more 
extensive liver damage in animals fed 1,500 mg Cu/kg. Rats 
fed 2,000 to 8,000 mg Cu/kg diet for 13 weeks exhibited 
hyperplasia and hyperkeratosis of the squamous mucosa on 
the limiting ridge separating the forestomach from the glan- 
dular stomach, inflammation in the liver, with indications of 
cellular damage, and an increase in the size and number or 
cytoplasmic protein droplets present in the epithelium of the 
proximal convoluted tubules of kidney (Hebert et al., 1993). 
Severity of stomach, liver, and kidney lesions increased with 
increasing dietary copper. Some research indicates that rats 
are able to adapt to prolonged exposure to high copper, with 
elevated liver and kidney concentrations eventually decreas- 
ing, with subsequent recovery from copper-induced liver and 
kidney damage (Haywood, 1985; Fuentealba et al., 1993). 
Mice are even less sensitive to copper than rats (Hebert et 
al., 1993). Chronic copper toxicosis did not affect reproduc- 
tion in either rats or mice (Hebert et al., 1993). 



Copper toxicosis resulting from a genetic disorder in cop- 
per metabolism is a problem in certain breeds of dogs, espe- 
cially in Bedlington terriers (Hyun and Filippich, 2004). 
Dogs with this disorder absorb normal amounts of copper, 
but biliary excretion of copper is reduced. Progressive accu- 
mulation of copper within hepatic lysosomes results in liver 
damage that can be fatal. Occurrence of a hemolytic crisis is 
uncommon in dogs with inherited copper toxicosis, but can 
be seen in the terminal stages. Once diagnosed, inherited 
copper toxicosis is treated by restricting copper intake, add- 
ing an antagonist, such as zinc, to the diet to reduce copper 
absorption and/or use of chelators that increase urinary cop- 
per excretion (Hyun and Filippich, 2004). Limited research 
has examined the level of copper needed to cause toxicosis 
in normal dogs. During a 12-month study, 2 of 12 Beagle 
dogs fed 8.4 mg Cu/kg BW developed elevated serum 
alanine aminotransferase (WHO, 1998). 

The amount of dietborne copper needed to cause toxicosis 
differs among fish species (Clearwater et al., 2002). Channel 
catfish may be most sensitive to dietary copper. Mural et al. 
(1981)found thatonly 8mg Cu/kg diet reduced gain:feed, and 
that 16 to 32 mg Cu/kg diet reduced gain and gain:feed during 
a 16-week study with catfish. In contrast, Gatlin and Wilson 
(1986) reported that copper concentrations up to 40 mg/kg 
diet caused no adverse effects in channel catfish during a 13- 
week study. In Atlantic salmon fry, copper concentrations of 
500 to 1,750 mg/kg diet reduced weight gain (Berntssen et al., 
1999). Atlantic salmon parr are more sensitive to lower di- 
etary concentrations of copper than fry (Clearwater et al., 
2002). Lanno et al. (1985) concluded that 665 mg Cu/kg diet 
was the maximum tolerable level in rainbow trout fry, when 
copper was supplied from CUSO4. Reduced gain and increased 
feed:gain were observed in trout fed diets containing 730 mg/ 
kg and increased mortality occurred when diets supplied 1 ,5 85 
to 3,088 mg Cu/kg (Lanno et al., 1985). Dietary copper con- 
centrations as low as 660 mg/kg increased mortality in rain- 
bow trout fry when supplemental copper was provided from 
shrimp, enriched with copper (Mount et al., 1994). Water- 
borne copper released from shrimp may have contributed to 
the higher toxicosis of copper in this study (Mount et al., 1994). 
Rainbow trout exposed to water containing 144 |ig Cu/L for 
12 weeks had reduced growth and increased mortality when 
fed high carbohydrate diets and maintained at 10°C (Dixon 
and Hilton, 1985). This concentration of copper in water had 
no effect when fish were fed low carbohydrate diets and/or 
maintained at a water temperature of 15°C. 

Factors Influencing Toxicity 

A number of dietary, physiological, and genetic factors 
affect the occurrence of copper toxicosis, especially in rumi- 
nants. Length of exposure will affect copper toxicity because 
copper generally accumulates in the liver over time when 
animals are fed high dietary copper. In ruminants, young 
animals are more susceptible to copper toxicosis than older 



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animals during relatively short-term exposure periods (Todd, 
1969). 

Molybdenum and sulfur are potent copper antagonists that 
greatly affect the level of copper needed to cause toxicosis in 
ruminants. Addition of 0.2 percent sulfur and 2 to 16 mg 
Mo/kg diet reduced liver copper and liver damage in sheep 
fed a high copper diet (45 mg Cu/kg) for 18 weeks (Suttle, 
1977). In sheep previously exposed to high dietary copper, 
supplementation with 0.3 percent sulfur and 14 or 30 mg 
Mo/day reduced liver copper concentrations by 34 to 47 per- 
cent over a 79-day period (Van Ryssen, 1994). Deaths from 
outbreaks of copper toxicosis can also be alleviated by 
supplementation of molybdenum and sulfur (Hidiroglou et 
al., 1984). However, sheep consuming diets low in molyb- 
denum and sulfur are susceptible to toxicosis at relatively 
low (10 to 14 mg/kg) dietary copper concentrations. High 
dietary sulfur intake via feed or water will reduce copper 
toxicity even if dietary molybdenum is low. Consumption of 
high sulfate (1,500 mg SO4/L) water versus normal water 
greatly reduced liver copper accumulation in cattle fed 100 
mg Cu/kg diet (Wright et al., 2000). 

Dietary iron and zinc can also influence copper toxicity. 
High dietary zinc can reduce liver copper and clinical signs 
of copper toxicosis. In lambs fed diets containing 29 mg Cu/kg, 
increasing dietary zinc from 43 to 220 or 420 mg/kg reduced 
liver copper concentrations and liver damage, and prevented 
onset of a hemolytic crisis (Bremner et al., 1976). Providing 
high dietary zinc to sheep that had previously been loaded 
with copper was ineffective in reducing liver copper concen- 
trations over an 88-day period (Van Ryssen, 1994). How- 
ever, administration of 200 mg Zn/day for 2 years to dogs 
(Bedlington terriers and West Highland White terriers) with 
inherited copper toxicosis reduced liver copper concentra- 
tions and liver damage (Brewer et al., 1992). 

Factors that reduce or eliminate ruminal protozoa increase 
copper bioavailability and, thus, increase the susceptibility 
of ruminants to chronic copper toxicosis (Ivan et al., 1986). 
Liver copper concentrations increased to a much greater ex- 
tent in fauna-free than in faunated sheep when diets contain- 
ing 13 mg Cu/kg were fed (Ivan et al., 1986). Lower copper 
bioavailability in sheep with ruminal protozoa may relate to 
the role of protozoa in degradation of dietary protein. In- 
creased degradation of dietary protein by ruminal microor- 
ganisms increases ruminal sulfide, which subsequently can 
reduce copper absorption. Feeding monensin, a carboxylic 
ionophore, increased liver copper in sheep (Van Ryssen and 
Barrowman, 1987; Ivan et al., 1992). Higher liver copper in 
sheep fed monensin may relate to the ability of the iono- 
phore to reduce ruminal protozoa numbers. 

Plants containing toxins that compromise liver function 
decrease tolerance for copper in ruminants. Plants belonging 
to the Heliotropium echium and Senecio genera contain 
pyrrolizidine alkaloids that cause liver damage. Concurrent 
exposure to high copper and Heliotropium europaeum in 
sheep enhanced the toxicity of both substances and caused 



greater accumulation of copper in liver than high copper 
alone (Howell et al. 1991). Mycotoxins that cause liver dam- 
age can also increase liver copper concentrations and may 
lower the level of copper needed to produce toxicosis (White 
et al., 1994). 

High dietary copper may increase the requirement for 
certain antioxidants because of its ability to cause oxidative 
damage. Addition of 2 mg Se/kg or 200 lU vitamin E/kg to 
diets adequate in these nutrients prevented mortality and 
muscle necrosis in ducklings fed 1,500 mg Cu/kg diet (Van 
Vleet et al., 1981). Dietary selenium did not affect hepatic 
damage in rats fed 2,000 mg Cu/kg diet (Aburto et al., 
2001b). Ascorbic acid supplementation reduced liver copper 
accumulation, but did not alleviate the growth depression 
observed in chicks fed 1,000 mg Cu/kg diet (Persia et al., 
2003). 

Copper toxicosis in poultry is affected by dietary sulfur 
amino acid level. Addition of 0.4 percent methionine or 0.33 
percent L-cysteine to diets adequate in sulfur amino acids 
prevented growth depression and reduced liver copper in 
chicks fed 500 mg Cu/kg diet (Jensen and Maurice, 1979). 
Cysteine supplementation at 0.5 percent also alleviated 
growth depression and reduced elevated liver copper con- 
centrations in chicks receiving 1,000 mg Cu/kg diet (Persia 
et al., 2003). In turkey poults, addition of 0.4 percent me- 
thionine partially prevented growth depression and gizzard 
erosion seen in birds fed 500 or 750 mg Cu/kg diet (Christ- 
mas and Harms, 1979). 

Susceptibility to copper toxicosis, especially in sheep and 
dogs, is greatly affected by genetics. Breeds of sheep differ 
in their ability to accumulate copper in their livers when fed 
low (Littledike and Young, 1993; Suttle et al., 2002) or mod- 
erate to high (Woolliams et al., 1982) copper levels. When 
fed moderately high dietary copper, Texel-sired lambs were 
most susceptible to copper toxicosis followed by Suffolk 
lambs (Woolliams et al., 1982). Finnish landrace were inter- 
mediate and Scottish Blackface appeared to be least suscep- 
tible to copper toxicosis. In crossbred lambs fed diets low in 
copper (4 to 5 mg/kg), Texel-sired lambs had the highest 
liver copper followed by Dorset- and Montadale-sired lambs 
with lambs from Finnsheep or Romanov rams being lowest 
(Littledike and Young, 1993). 

Genetic differences among breeds in copper metabolism 
also occur in cattle. Simmental and Charolais cows and their 
calves had lower plasma copper concentrations than Angus 
when fed diets low in copper (Ward et al., 1995). Simmental 
cattle also have lower liver copper concentrations than An- 
gus (Mullis et al., 2003). Biliary copper excretion is higher 
in Simmental compared to Angus (Gooneratne et al., 1994). 
Copper accumulated more rapidly in the liver of Jerseys than 
in Holsteins when copper was supplemented at 80 mg/kg 
diet (Du et al., 1996). During an outbreak of copper toxicosis 
in nursing beef calves, 32 percent of Angus calves were af- 
fected, but only 5.5 percent of Charolais calves (Sargison 
and Scott, 1996). 



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MINERAL TOLERANCE OF ANIMALS 



Liver copper concentrations vary greatly among and 
witliin breeds in dogs (Thiornburg et al., 1990). Inherited 
copper toxicosis affects approximately 70 percent of 
Bedlington terriers in the United States (Hyun and Filippich, 
2004). The condition is caused by an autosomal recessive 
trait that reduces biliary excretion of copper. Inherited cop- 
per toxicosis has also been seen in Doberman pinschers. 
West Highland White terriers, Skye terriers, Dalmatians, and 
in some mixed breeds (Hyun and Filippich, 2004). 

Acute toxicity of waterborne copper in fish is affected by 
water hardness, pH, and temperature (WHO, 1998). The 96-hr 
LCjQ for rainbow trout increased with increasing water hard- 
ness and was higher at alkaline pH (Howarth and Sprague, 
1978). The effect of water temperature varies depending on 
fish species (Smith and Heath, 1979). 

TISSUE LEVELS 

Representative copper concentrations in tissues from vari- 
ous animals fed normal or high levels of copper are shown in 
Table 13-2. Copper concentrations are highest in the liver, 
and liver copper increases in animals fed high dietary cop- 
per. Normal liver copper concentrations are higher in rumi- 
nants than in pigs and chickens, and relatively small amounts 
of dietary copper greatly increase liver copper concentra- 
tions in ruminants. In nonruminants and fish, liver copper 
increases to a much smaller extent with increasing dietary 
copper unless high levels of copper (>100 mg Cu/kg) are 
fed. Kidney copper concentrations also can increase in ani- 
mals fed high dietary copper, but to a lesser extent than liver. 
Muscle contains less than 4 mg Cu/kg DM in most animals 
and does not increase in animals fed high copper diets. Cop- 
per concentration in eggs is also not affected by level of 
copper in diets of laying hens. The copper content of milk is 
low and varies with species and stage of lactation 
(Underwood, 1977). Copper addition to diets already ad- 
equate in copper has little effect on milk copper concentra- 
tion in cows (Underwood, 1977). 

MAXIMUM TOLERABLE LEVELS 

The maximum tolerable level for copper is defined as the 
dietary level that, when fed for a defined period of time, will 
not impair animal health and/or performance. Nonruminant 
animals are tolerant to high dietary copper concentrations rela- 
tive to copper requirements. Based on published literature in 
nonruminants, maximum tolerable levels for copper in mg 
Cu/kg diet were set by the NRC committee at chicken and 
turkey, 250; duck, 100; swine, 250; horse, 250; rabbit, 500; 
rat, 1,000; and mouse, 2,000. Insufficient data are available 
to set a maximum tolerable level for copper for dogs and cats. 

In broilers, addition of 250 mg Cu/kg to diets may in- 
crease the incidence of proventriculitis (Wideman et al., 
1996) and lesions in the oral cavity, tongue, and pharynx 
(Chiou et al., 1999), but negative effects on performance are 



generally not observed. Ponies tolerated up to 791 mg Cu/kg 
diet for 6 months without showing clinical signs of toxicosis 
(Smith et al., 1975). However, liver copper concentrations 
were elevated to concentrations that could cause toxicosis 
problems in stressful situations that affect liver function. 
Because of the limited research data, and possible genetic 
differences in susceptibility to copper toxicity, a maximum 
tolerable level of 250 mg Cu/kg is recommended in horses. 

The maximum tolerable level of dietborne copper for fish 
is affected by species and fish size or life stage (Clearwater 
et al., 2002). The upper limit for copper intake from diets in 
Atlantic salmon and rainbow trout is approximately 100 and 
500 mg/kg diet, respectively. The maximum tolerable cop- 
per level may be lower for channel catfish, but sufficient 
data are not available to arrive at a reliable upper limit. 
Waterborne copper is generally more toxic to fish than 
dietborne copper. However, it is impossible to set a maxi- 
mum tolerable water copper concentration for fish because 
of the major impact of water hardness and pH on copper 
toxicity from water. 

In sheep, the maximum tolerable level was set at 15 mg 
Cu/kg diet DM. Some sheep breeds may be susceptible to 
copper toxicosis at lower dietary concentrations if dietary 
molybdenum and/or sulfur are below normal levels (1 to 2 
mg Mo/kg diet; 0.15 to 0.25 percent S) (Hogan et al., 1968; 
MacPherson et al., 1997). This upper limit for copper is simi- 
lar to the current European Commission (EC, 2003) limit of 
17 mg Cu/kg DM in sheep diets. A recent study (Suttle et al., 
2002) suggested that the EC limit of 17 mg Cu/kg diet was 
too high for some sheep breeds when fed high concentrate 
diets (contained 0.27 percent sulfur and unspecified molyb- 
denum concentration). 

The level of dietary copper that causes toxicosis in rumi- 
nants is greatly affected by dietary sulfur and molybdenum, 
and genetics. Other factors that may affect the maximum 
level of copper that ruminants can tolerate include: (1) length 
of exposure, (2) age, (3) initial liver copper concentration at 
the onset of high copper exposure, (4) dietary toxins that 
impair liver function and trigger a hemolytic crisis, and 
(5) dietary concentrations of zinc and iron. Maximum toler- 
able copper levels for ruminants were set based on available 
literature, and assuming normal dietary sulfur (0.15 to 0.25 
percent DM) and molybdenum (1 to 2 mg/kg DM). 

The maximum tolerable level for copper in cattle was set at 
40 mg Cu/kg diet DM. Higher concentrations of copper can 
often be tolerated by cattle for several weeks or even months. 
However, long-term feeding of diets slightly less than 40 mg 
Cu/kg can result in copper toxicosis (Bradley, 1993). Feeding 
high dietary copper will greatly increase liver copper concen- 
trations. Factors that regulate liver copper in cattle are poorly 
understood, and the minimum liver copper concentration that 
may result in clinical signs of toxicosis is not known. How- 
ever, cattle not showing clinical signs of copper toxicosis, but 
with high liver copper concentrations, would be expected to 
be more susceptible to a hemolytic crisis when exposed to 



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COPPER 



143 



toxins that cause liver damage or other stressors. Increased 
incidence of copper toxicosis in cattle has been reported re- 
cently by the Veterinary Laboratories Agency (VLA) in En- 
gland (Bidewell et al., 2000; VLA, 2001). A few years ago a 
considerable amount of the supplemental copper provided in 
cattle supplements was in the form of copper oxide (primarily 
cupric oxide) powder. Based on research indicating that 
bioavailability of copper from copper oxide was extremely 
low, more bioavailable sources of copper have now replaced 
copper oxide in most ruminant supplements. Supplementation 
of more bioavailable copper sources to cattle diets may have 
contributed to increased incidence of copper toxicosis. Recent 
studies with goats suggest that their tolerance to copper is simi- 
lar to cattle. 

HUMAN HEALTH 

Copper in edible tissue is not likely to present a human 
health concern, when copper is provided in animal diets at 
the maximum tolerable levels indicated. Muscle copper is 
low and increasing dietary copper does not increase muscle 
copper concentration. Copper intake from animal products 
could possibly be of concern in humans that consume large 
quantities of liver. Livers from animals fed dietary copper 
concentrations within the maximum tolerable levels sug- 
gested can contain from approximately 20 to 900 mg Cu/kg 
DM (Table 13-2). 

FUTURE RESEARCH NEEDS 

Maximum tolerable levels of copper are fairly well defined 
for poultry and swine, and copper toxicosis is not a common 
problem in these species. Copper toxicosis in ruminants is 
primarily due to poor copper homeostasis, resulting in liver 
copper accumulation. Research is needed to better understand 
biliary copper excretion in ruminants and factors that affect it. 
Under certain conditions, which are not well defined, cattle 
appear to regulate liver copper concentrations once concen- 
trations reach a certain level in spite of high copper intakes. 
However, in other situations, liver copper continues to increase 
until ruminants succumb to copper toxicosis. Additional re- 
search is also needed to identify factors (dietary toxins, stress, 
etc.) that may trigger the occurrence of hemolytic crisis, and 
thus clinical signs of copper toxicosis in ruminants with el- 
evated liver copper concentrations. 

SUMMARY 

Copper occurs primarily in the cuprous (+1) and cupric 
(h-2) oxidation states. Because of its high electrical and ther- 
mal conductivity, and resistance to corrosion, copper is a 
widely used industrial metal. Copper compounds are also 
used in nutritional supplements for animals, fungicides, and 
as algicides in streams and reservoirs. Copper is an essential 
trace mineral, and functions as a component of a number of 



enzymes that are involved in maturation and stability of col- 
lagen and elastin, energy metabolism, the antioxidant de- 
fense system, and pigmentation, as well as other processes. 
Homeostatic control mechanisms for copper are usually very 
efficient in preventing copper toxicosis in nonruminants. 
Concentrations of copper needed to cause toxicosis in 
nonruminants exceed requirements by at least 25-fold. Ru- 
minants, particularly sheep, are very susceptible to copper 
toxicity. Copper requirements of sheep generally range from 
3 to 8 mg Cu/kg diet DM, while 15 mg Cu/kg diet or even 
less can cause toxicosis under certain conditions. Cattle and 
goats can generally tolerate 40 mg Cu/kg diet DM for sev- 
eral weeks or months. Maximum tolerable concentrations of 
copper for ruminants are greatly affected by dietary concen- 
trations of sulfur and molybdenum as well as dietary toxins 
that compromise liver function. Susceptibility of sheep and 
dogs to copper toxicity is greatly affected by genetics. Cer- 
tain fish species are also quite susceptible to toxicosis from 
waterborne copper. In all species, liver and kidney are the 
major organs affected by copper toxicosis. 



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inemi Tnlemnce nf Animals- Sennnd Revised Edition 



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COPPER 



153 



TABLE 13-2 Copper Concentrations in Fluids and Tissues of Animals (mg/kg) 



Animal 



Quantity 



Source 



Duration 



Route Liver Kidney Muscle 



Heart Egg Reference 



Rabbits 



Chicks 



Laying hens 


Control 


CUSO4 


48 wk 


Diet 


12" 


3.4° 


Jackson and Stevenson, 




+ 150 mg/kg 








16 


3.4 


1981 




+ 300 mg/kg 








32 


3.3 






+ 450 mg/kg 








61 


3.0 





Fish, 
rainbow 
trout 



Control 

+ 250 mg/kg 

+ 500 mg/kg 

Control 

+ 250 mg/kg 

Control 
+ 150 n 
+ 300n 
+ 450n 

Control 

+ 120 mg/kg 

+ 240 mg/kg 

Control 
+ 20 mg/kg 
+ 40 mg/kg 

Control 
+ 30 mg/kg 

Control 

+ 300 mg/kg 

+ 1,000 mg/kg 



CuSO, 



CuSO, 



28 d 



42 d 



Diet 



Diet 



12° 
195 
890 

3.1'' 
5 



10 
11 
15 



2.6 
3.6 
3.4 

0.4* 
0.4 



2.8" 
3.0 



Grobner et al., 1986 



Pesti and Bakalli, 1996 



CuSO, 



132 d 



Bradley et al., 1983 




CuSO 



CuSO, 



350° 14° 

2,400 50 



7.0° 
26.0 



28 d 



Diet 



38* 

45 

100 



1.7* 

1.7 

10.0 



0.3* 

0.3 

0.3 



Zervas et al., 1990 



Kamunde et al., 2001 



°Dry tissue basis. 
*Wet tissue basis. 



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14 



Fluorine 



INTRODUCTION 

Fluorine (F) is tiie most electronegative and reactive of 
all elements and can form compounds with all elements ex- 
cept helium, neon, and argon. It has an atomic number of 9 
and an atomic weight of 18.9984. Fluorine is a pale green- 
ish-yellow, highly toxic gas with a characteristic pungent 
odor. In nature, it does not occur in the elemental state but 
forms organic or inorganic compounds as fluorides. Fluo- 
rine occurs mainly as salts containing the halide anion, fluo- 
ride. It is found in both igneous and sedimentary rock and 
constitutes about 0.06-0.09 percent of the upper layers of 
the Earth's crust. It ranks 13th in abundance among elements 
in the Earth's crust. The most important inorganic fluorine- 
bearing minerals are fluorspar (CaFj), fluorapatite 
(Caj(P04)3F), and cryolite (NajAlFg). The largest known 
deposits of fluoride are located in the United States, United 
Kingdom, and Germany. 

Inorganic fluorides are used in steel, glass, brick, ceramic, 
and adhesive manufacture. Hydrogen fluoride (HF), a major 
industrial compound, is widely used in the production of 
cryolite, aluminium fluoride (AIF3), gasoline alkylates, and 
chlorofluorocarbons. It is also used for cleaning or etching 
compounds for semiconductors, glass, aluminium, and tan- 
ning leather, and it is also used in rust removers. HF is highly 
soluble in water in which it forms hydrofluoric acid. Cal- 
cium fluoride (CaFj), with a fluorine content of 48.7 per- 
cent, is relatively insoluble in water and dilute acids and 
bases. It is the principal fluoride-containing mineral pro- 
duced for industrial use as a flux in steel, glass, and enamel 
production, and as the raw material for production of hy- 
drofluoric acid and anhydrous HF. Sodium fluoride (NaF), 
fluorosilic acid (HjSiFg), and sodium hexafluorosilicate 
(NajSiFg) are used for controlled fluoridation of drinking 
water. 

Fluorides are ubiquitous in the environment and are re- 
leased naturally through the weathering and dissolution of 
minerals, as emissions from volcanoes, and as marine aero- 



sols (Symonds et al., 1988; ATSDR, 2003). Inorganic fluo- 
rides are released in the environment from phosphate fertil- 
izer production and use; aluminium smelting; steel produc- 
tion; glass, enamel, brick, and ceramic manufacturing; glue 
and adhesive production; pesticides; and drinking water fluo- 
ridation (Burns and AUcroft, 1964; Neumiiller, 1981; Fuge, 
1988; Fuge and Andrews, 1988). Transformation and trans- 
port of inorganic fluorides in the aquatic environment is in- 
fluenced by pH, hardness, and the concentration of ion-ex- 
change materials (e.g., bentonite clay and humic acid) in 
water. In the environment, vaporization, aerosol, and hy- 
drolysis are the main factors that determine the fate of fluo- 
rides. Generally, the formation of aluminium and calcium 
complexes and pH affect the fate of fluorides in soils. In 
areas of extreme acidity and alkalinity, inorganic fluorides 
leach into surface or ground water, and their solubilization 
in water may be further enhanced by the presence of ion- 
exchange materials (Pickering et al., 1988). During weather- 
ing under acidic conditions, certain fluorine-bearing miner- 
als (e.g., cryolite) dissolve rapidly; however, the solubility 
of fluorapatite and calcium fluoride is relatively slow (Fuge 
and Andrews, 1988). Soils rich in calcium carbonate or 
amorphous aluminium hydroxides may bind inorganic fluo- 
ride by forming insoluble calcium fluoride or aluminium- 
fluoro-hydroxide complexes, thus limiting leaching from the 
soil and uptake by plants (Fluhler et al., 1982). Other cations 
(e.g., iron) also contribute to the fixation of fluoride (Murray, 
1983, 1984); however, soil phosphate may contribute to the 
mobility of inorganic fluoride (Kabata-Pendias and Pendias, 
1984). The fate of inorganic fluorides released to soil also 
depends on their chemical form, rate of deposition, soil 
chemistry, and climate (Davison, 1983). 

ESSENTIALITY 

Fluorine is considered an essential element, although spe- 
cific deficiency signs have not been observed in experimen- 
tal animals. No one has yet produced an environment suffi- 



154 



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FLUORINE 



155 



ciently devoid of this element tliat survival of an animal was 
virtually threatened. Fluorine was identified as a constituent 
of bones and teeth as early as 1905. Attempts to demonstrate 
the essential role of fluorine in rat experiments with other 
than dental criteria have not been successful. McClendon 
and Gershon-Cohen (1953) fed weaning rats for 66 days 
upon materials grown hydroponically in water considered to 
be "fluorine free." The rats weighed 5 1 g and had 10 carious 
molars per animal compared to fluoride-supplemented rats 
that weighed 128 g and had 0.5 carious molars per animal. 
Maurer and Day (1957) purified dietary ingredients and pro- 
duced a diet that contained about 0.007 |jg/g fluoride on 
which four generations of rats were raised without evidence 
of impaired general health, dental health, or weight gain as 
compared to rats raised on the same diet plus 2 |jg/mL of 
fluoride in their drinking water. In the early 1970s, it was 
reported that mice fed low fluoride (0.005 pg/g diet) exhib- 
ited anemia and infertility compared to mice supplemented 
with fluoride (50 |Jg/mL drinking water) (Messer et al., 1972, 
1973). Subsequently, it was determined that the diets of these 
mice were low in iron and that high dietary fluoride, similar 
to that fed in supplemented controls, improved iron absorp- 
tion or use (Tao and Suttie, 1976). Mice fed low fluoride 
diets containing sufficient iron neither exhibited anemia nor 
infertility. Relatively high fluoride supplementation (2.5- 
7.5 |Jg/g diet) to rats fed low fluoride (0.04 |ig/g diet) slightly 
improved the growth of suboptimally growing rats. Weber 
(1966) reported that in six generations of study on mice, no 
definite differences in reproduction were observed between 
the low (0.25 |Jg/g) and high (65 |Jg/g) fluoride groups. 
Schwarz and Milne (1972), working in a filtered-air envi- 
ronment, reported a favorable growth response when small 
increments (1-2.5 pg/g) of fluoride were added to a low 
fluoride diet for rats. Studies on high fluoride supplements 
indicate that these growth-promoting effects were probably 
pharmacological. High or pharmacological amounts of fluo- 
ride have been also found to depress lipid absorption and to 
alleviate nephrocalcinosis induced by feeding phosphorus 
and to alter soft tissue calcification by magnesium deprivation. 
Several decades of research on humans have strength- 
ened the view that fluorine is essential to reduce the preva- 
lence of dental caries, and it should be considered as an es- 
sential element on this basis (NRC, 1993). A fluoride 
supplement of 80 pg/g diet enhanced the growth of broiler 
chickens (Gutierrez et al., 1993). To date, sufficient evidence 
of the essentiality of fluorine in farms animals has been pro- 
vided only by Anke et al. (1997). Female goats developed 
skeletal abnormalities and poor growth in their offspring for 
10 generations on a diet containing < 0.3 mg F/kg DM. These 
findings need to be confirmed in other animal species before 
being accepted as evidence for essentiality in higher ani- 
mals. Fluoride may be needed for proper functioning of some 
enzymes, given that in vitro its activities, including histidine 
methyl transferase, stabilize the interaction between gua- 



nosine triphosphatase (GTPase) and GTPase-activitating 
proteins, and affect the posttranslational assembly of 
glycosaminoglycan chains in mineralizing bone cells (Kirk, 
1991). 

DIFFICULTIES IN METHODS OF ANALYSIS AND 
EVALUATION 

Fluorine is too reactive to be analyzed directly in biologi- 
cal samples and environmental media, and its measurement 
is restricted to the detection of the free anion (F"). The most 
widely used analytical method is a potentiometric technique 
that measures free anion using the fluoride ion-selective elec- 
trode (F-ISE) (Neumiiller, 1981; Harzdorf et al., 1986; 
ATSDR, 2003). This method has been used for the quantifi- 
cation of fluoride in biological tissues and fluids (e.g., urine, 
serum, plasma, organs, bone, and teeth), foodstuffs, and en- 
vironmental media (e.g., air, water, and soil). The variations 
in the sample preparation procedures and the recovery of 
this element may affect the detection limits using F-ISE. The 
values reported by this method range from 0. 1 to 300 ng/m^ in 
air, from 1 to 1,000 |ig/L in water, and from 0.05 to 20 mg/kg 
in nonskeletal tissues (Harzdorf et al., 1986; ATSDR, 2003). 

Fluoride ions form a stable, colorless complex such as 
(AlFg)-'", (FeFg) ^", and (ZrFg) ^^ with certain trivalent ions. 
Colorimetric methods are based on the bleaching of these 
metal complexes with organic dyes in the presence of fluo- 
ride (WHO, 1984). Other methods for the quantification of 
fluorine include gas chromatography (GC), ion chromatog- 
raphy, capillary electrophoresis, atomic absorption, and pho- 
ton activation (Neumiiller, 1981; Harzdorf et al., 1986; Wen 
et al., 1996; ATSDR, 2003). GC methods have been used to 
measure fluoride concentrations in urine and plasma but require 
extraction and derivatization using trimethylchlorosilane in 
toluene to produce trimethylfluorosilane (Ikenishi et al., 
1988). Analytical methods based on neutron or proton acti- 
vation of fluorine- 19 have been also developed that measure 
emitted gamma rays or x-rays using lithium-drifted germa- 
nium detectors (Shroy et al., 1982; Knight et al., 1988). 
These techniques do not depend on the specific sample 
matrix or chemical form. 

Appropriate sample preparation is the critical step in the 
accurate quantification of fluoride, especially where only the 
free fluoride ion is measured. For analysis involving bio- 
logical materials, the most accurate method is microdiffusion 
techniques such as the acid-hexamethyldisioxane (HMDS) 
diffusion method described by Taves (1968). Methods in- 
volving acid or alkali digestion may not convert all complex 
organic fluorine into an ionic form that can be conveniently 
measured (Venkateswarlu, 1983). Open-ashing methods 
may result in the loss of volatile fluoride compounds or of 
fluoride itself at temperatures in excess of 550°C, or they 
may result in contamination with extraneous fluoride 
(Venkateswarlu, 1983; Campbell, 1987). 



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MINERAL TOLERANCE OF ANIMALS 



REGULATION AND METABOLISM 

Approximately 75 to 90 percent of ingested fluoride is 
absorbed in the stomach and small intestine as well as rumen 
of animals (Messer, 1984; WHO, 1994; Cerklewski, 1997). 
The efficiency of fluoride absorption depends on the solubil- 
ity of the fluoride compound and the presence of other di- 
etary components. In the stomach, low pH conditions favor 
the formation of highly diffusible hydrogen fluoride (pK^ = 
3.4). Therefore, the conditions that promote gastric acidity 
increase the rate of fluoride absorption, and it is impaired by 
alkalinity. The absorption from the small intestine occurs as 
the fluoride ion by non-pH dependent diffusion (Nopakun 
and Messer, 1990). In the fasted state, fluoride absorption 
from either fluoridated water or sodium fluoride is almost 
100 percent (Rao, 1984; Trautner and Seibert, 1986). Soluble 
fluorides, such as sodium fluoride, are almost completely 
absorbed. Less soluble sources, such as bone meal, are rela- 
tively poorly absorbed (<50 percent). Calcium, magnesium, 
aluminum, sodium chloride, and high lipid levels are the 
main dietary factors that depress fluoride absorption (NRC, 
1980; Cerklewski, 1997). 

The respiratory tract is the major route of absorption of 
both gas and particulate fluoride from industrial emissions. 
Depending on their aerodynamic characteristics and solubil- 
ity, fluoride-containing particles are deposited in the bron- 
chioles and nasopharynx and absorbed either immediately or 
gradually released from lungs (Mclvor, 1990). Dermal ab- 
sorption of fluoride has been reported only in the case of 
burns resulting from exposure to hydrofluoric acid (Burke et 
al., 1973). 

Removal of fluoride from circulation occurs principally 
through two mechanisms: renal excretion and calcified tis- 
sue deposition. Following absorption, the concentration of 
ionic fluoride (F") increases in plasma where it reacts with 
calcium to form calcium fluoride. Some ionic fluoride may 
become non-ionic by coordinating with macromolecules 
such as proteins (Singer and Armstrong, 1964; Kirk, 1991). 
About 75 percent of fluoride in the blood is in the plasma 
(Carlson et al., 1960), with 15 to 70 percent (0.01-0.04 mg/L) 
in the ionic form (Singer and Armstrong, 1964). Nearly 5 
percent of plasma fluoride is bound to protein, but most of 
the bound fluoride is associated with compounds having 
molecular weights less than albumin. However, HF, not ionic 
fluoride, apparently is the form that is in diffusion equilib- 
rium across cell membranes. It is removed from circulation 
by mineralized tissues in exchange for other anions such as 
hydroxyl ion, citrate, and carbonate and subsequently enters 
bone crystal lattice. Soft tissue fluoride levels are maintained 
within narrow limits and are marginally affected by long- 
term fluoride intake or by short-term fluctuations in plasma 
levels. However, soft tissue fluoride is readily exchangeable 
with extracellular fluid fluoride, as demonstrated by the rapid 
distribution of '^F following intravenous injection (Wallace, 
1953; Carlson et al., 1960). The rate of uptake of fluoride 



into bones and teeth is most efficient during early develop- 
ment stages. Approximately 50 percent of fluoride absorbed 
each day is deposited in the calcified tissues (bone and de- 
veloping teeth), which results in approximately 99 percent 
of body burden of fluoride being associated with these tis- 
sues (NRC, 1980). Excretion of fluoride occurs mainly via 
the urine, which accounts for approximately 90 percent of 
total excretion. Urinary excretion of fluoride is directly re- 
lated to urinary pH; thus factors that affect urinary pH, such 
as diet, drugs, metabolic and respiratory disorders, and alti- 
tude residence, can affect how much absorbed fluoride is 
excreted. Generally, > 20 percent of ingested fluoride is ex- 
creted in feces. 

Milk and saliva have fluoride concentrations similar to 
plasma ionic fluoride levels, and variations in plasma fluo- 
ride levels are reflected in these secretions. Milk fluoride 
concentrations are affected only minimally by dietary fluo- 
ride (Greenwood et al., 1964). Fluoride crosses the placental 
barrier of cows, and fluoride levels in the bones of the off- 
spring are correlated with those of maternal blood (NRC, 
1974). However, bone fluoride concentration of calves born 
to cows consuming as much as 108 mg F/kg diet (from so- 
dium fluoride) were low (Hobbs and Merriman, 1962), and 
it appeared that neither placental fluoride transfer nor milk 
fluoride concentration were sufficient to adversely affect the 
health of these calves. 

Homeostatic regulation of plasma fluoride concentration 
involves the kidneys and skeleton (Smith et al., 1950; Singer 
and Armstrong, 1964). Bone has a great affinity to incorpo- 
rate fluoride into hydroxyapatitite to form fluorapatite. This 
results in larger, less soluble, more stable apatite crystals 
(Zipkin et al., 1964). The fluoride cannot be removed with- 
out resorption of this mineral unit. Even low levels of fluo- 
ride intake will result in appreciable accumulation of fluo- 
ride in the skeleton and teeth. These accumulations can 
increase, within limits, over a period of time without mor- 
phological evidence of pathology. However, in some cases 
of high-fluoride intake, structural bone changes develop 
(Shupe et al., 1963b). Most soft tissues do not accumulate 
much fluoride, even during high intakes, although tendon 
(Armstrong and Singer, 1970), aorta (Ericsson and Ullberg, 
1958), and placenta (Gardner et al., 1952) have a higher 
fluoride concentration than other soft tissues, possibly asso- 
ciated with their relatively high levels of calcium and mag- 
nesium. Kidney will usually exhibit a high fluoride concen- 
tration during high fluoride ingestion due to urine retained in 
the tubules and collecting ducts. 

In laboratory animals and humans, approximately 99 per- 
cent of fluoride is retained in bones and teeth (Kaminsky et 
al., 1990; Hamilton, 1992), with the remainder distributed in 
vascularized soft tissues and the blood (Mclvor, 1990). In 
calcified tissues, the highest concentration is found in bone, 
dentine, and enamel, and the concentration varies with age, 
sex, and in different parts of the bone. During the rapid 
growth phase at the early stages of development, a high por- 



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FLUORINE 



157 



tion of fluorine is deposited in skeletal tissues. Retention of 
fluorine in skeletal tissues is related to the turnover of miner- 
als and previous exposure to this element (Carracio et al., 
1983). The selective affinity of fluoride for mineralized tis- 
sues is, in the short term, due to uptake on the surface of 
bone crystallites by the processes of isoionic and heteroionic 
exchange. In the long run, fluoride is incorporated into the 
crystal lattice structure of teeth and skeletal tissues by re- 
placing some hydro xyl ions within the unit cells of hydroxya- 
patite (Ca|g(P04)g(OH)2), producing partially fluoridated 
hydroxyapatite (Ca|Q(P04)g(0H)^ ^-^F^^)- 

Metabolic Interactions and Mechanism of Toxicity 

The mechanism of fluorine toxicity is partially under- 
stood, and the clinical manifestations of the toxicosis de- 
pend on the age and species of animal, dosage, fonn and 
duration of exposure to fluorine, and nutritional status. The 
biochemical and clinical basis of acute fluorine toxicity has 
been classified into four major categories: (1) enzyme inhibi- 
tion, (2) calcium complex formation, (3) shock, and (4) spe- 
cific organ injury (Hodge and Smith, 1981). Fluoride inhibits a 
large number of metalloenzymes containing copper, manga- 
nese, zinc, nickel, and iron (heme and non-heme) by binding 
to the metal on the active site (reviewed by Messer, 1984; 
Kirk, 1991). Enzymes requiring divalent cations (e.g., magne- 
sium) are also inhibited, and in some instances inhibition of 
fluoride ion is enhanced by inorganic phosphate (e.g., enolase, 
succinic dehydogenase). Although the inhibition of enzyme 
activities by fluoride toxicosis interrupts metabolic processes 
such as glycolysis and synthesis of proteins (Kessabi, 1984), 
the molecular mechanisms involved in these biochemical 
processes are mainly speculative. Fluoride concentrations of 
blood and tissues increase rapidly, and there is a severe hypo- 
calcemia probably from inhibition of lactate required for 
extrusion of Ca^"^ from bone (Hodge and Smith, 1981). The 
mechanism of sudden death during acute toxicity may also 
involve hyperkalemia or diminished Na+ZK-H-ATPase activity 
affecting ATP production and causing glycolysis inhibition 
(Messer, 1984). 

Many of the biochemical actions of fluoride toxicity have 
been explained in terms of its substitution for hydro xyl ions, 
which the fluoride ions closely resemble in their physical 
properties (hydration number, ion radius, and charge), par- 
ticularly in mineralized tissues where the substitution results 
in dissolution of minerals in bones. For enolase and pyro- 
phosphatase, which have been examined in considerable de- 
tail, the substitution of the fluoride ion for the hydroxyl ion 
(that normally participates in this enzymatic reaction) results 
in the formation of stable, less reactive complexes. Chronic 
toxicosis occurs in extracellular fluid when the fluoride con- 
centration is in the low micromolar range and includes both 
inhibitory and stimulatory effects on cells that cannot readily 
be explained in terms of biochemical effects (Messer, 1984; 
Kirk, 1991). 



SOURCES AND BIOAVAILABILITY 

Fluoride is present in varying amounts in air, water, soil, 
and in plant and animal tissues. Active volcanoes and fuma- 
roles, as well as certain industrial processes, may contribute 
significantly to local concentrations of fluoride. Fluoride 
levels in surface water vary according to local geology and 
proximity to emission sources. Fluoride concentrations in 
surface freshwater range from 0.01 to 0.03 mg F~/L and the 
range in seawater is 1.2 to 1.5 mg F-/L (EPA 1980; IPCS, 
2002). Higher concentrations are found in areas where there 
is geothermic and volcanic activity. In rivers, fluoride con- 
centrations range from < 0.001 to 6.5 mg F~/L; the average 
fluoride concentration is approximately 0.2 mg F~/L 
(Fleischer et al., 1974). Fluoride levels may be higher in 
lakes, especially in saline lakes and lakes in closed basins in 
areas of high evaporation. The Great Salt Lake in Utah has a 
fluoride content of 14 mg F~/L (Fleischer et al., 1974). The 
fluoride content of ground water generally ranges from 0.02 
to 1.5 mg/L (Fleischer, 1962; EPA, 1980). Highest fluoride 
levels in U.S. ground water are generally found in the South- 
west, and maximum ground water levels in Nevada, south- 
ern California, Utah, New Mexico, and western Texas ex- 
ceed 1.5 mg/L. In endemic fluorosis areas, deep-well water 
may percolate through fluorapatite and frequently contains 3 
to 5 mg F~/L, and sometimes 10 to 15 mg F~/L (Harvey, 1952; 
Cholak, 1959). The amount of fluoride in water is influenced 
by pH, water hardness, and the presence of clay that has ion- 
exchange properties. In the Rift Valley of Kenya and Tanza- 
nia, high fluoride levels in water and a high incidence of 
fluorosis have been correlated with low levels of calcium 
and magnesium in the water (Gaciri and Davies, 1993). 

Industrial pollution furnishes airborne fluorine in one of 
three principal forms: hydrofluoric acid, silicon tetrafluo- 
ride, or fluoride-containing particulate matter. The average 
fluorine concentrations in air are generally less than 0.1 pg/m-' 
(IPCS, 2002) with higher concentrations in urban than rural 
areas. Generally, the airborne fluorine contamination in ar- 
eas located in the vicinity of industrial emissions does not 
exceed 2 to 3 [ig/va^. Direct inhalation of fluoride does not 
contribute significantly to fluoride accumulation in animals; 
however, these emissions contaminate plants, soil, and wa- 
ter. Gaseous fluorine may be absorbed and incorporated in 
plant tissues. Particulate fluorides accumulate on plant sur- 
faces and may be ingested by animals as the plant is eaten. 
Rain may wash off some of these inert particles, but their 
toxicity is related largely to their solubility in water. 

Although soil is undoubtedly the principal source of fluo- 
ride in plants, there is no consistent relationship between 
total fluoride in soil and plants (NRC, 1974). Fluorine con- 
tent of pastures and forages are particularly low unless they 
have been contaminated by a deposition of dust and fumes 
from volcanic and industrial origin or by fluoride-rich 
geothermic and well water used for irrigation (Shupe, 1980). 
Animals ingest soil during grazing, which may contribute to 



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158 



MINERAL TOLERANCE OF ANIMALS 



more than 50 percent of the dietary fluorine in grazing sheep 
and cattle, and this amount may increase to > 80 percent in 
winter months (Cronin et al., 2000). There is some indica- 
tion that acid soils promote fluoride uptake in plants, and 
liming of these soils may reduce it. Fluoride content of soils 
ranges from 20 to 1,000 mg total F7kg in areas without natu- 
ral phosphate and fluoride deposits. Some soils unusually 
high in fluoride have been found in Idaho (3,870 mg/kg) and 
in Tennessee (8,300 mg/kg). The natural fluoride content of 
the soil increases with increasing depth. Only 5 to 10 percent 
of the total fluoride content in soil is soluble (Noemmik, 
1953). The fluoride content increase with depth is in the 
usual range in the United States, as 20 to 500 mg/kg (aver- 
age, 190 mg/kg) from to 8 cm deep and 20 to 1,620 mg/kg 
(average, 292 mg/kg) from to 30 cm deep (Robinson and 
Edgington, 1946). The extent of fluoride retention depends 
upon the amount of clay and pH, and the distribution at vari- 
ous depths follows the clay pattern of soil. Several anthropo- 
genic sources of fluoride can enrich soil, especially phos- 
phate fertilizers, insecticides containing fluorides, and 
emissions from industry. Soils may contain fluoride in sev- 
eral different minerals. 

Natural forage normally contains 2 to 20 mg F/kg dry 
weight (NRC, 1974). Cereal grains and cereal by-products 
usually contain 1 to 3 mg/kg (Underwood and Suttle, 1999), 
which mainly accumulates in the outer layer and embryo 
(Kumpulainen and Koivistoinen, 1977). The fluorine con- 
tent of both leaf and root vegetables do not differ apprecia- 
bly from those of cereals, with the exception of spinach, 
which is rich in this element. The tea plant and camellia are 
exceptions, and fluoride concentrations of 100 mg/kg or 
more have been reported (Underwood and Suttle, 1999). 
Fluorine is a normal component of calcified animal and fish 
tissues. Animal by-products containing bone may contribute 
significant quantities of fluoride to animal diets, depending 
upon the amount of by-product used (and bone contained) 
and the dietary history of the animals from which the by- 
products were derived. Bone ash normally contains less than 
1,500 mg F/kg and would contribute only minor amounts. 
However, cattle grazing fluoride-contaminated pastures can 
have bone ash containing over 10,000 mg/kg F. Aquatic or- 
ganisms absorb fluoride to a limited extent from food sources 
and directly from water where that uptake depends on fluo- 
ride concentration, exposure time, and temperature (Hemens 
and Warwick, 1972; Milhaud et al., 1981; Nell and Livanos, 
1988). In natural environments, the proximity of anthropo- 
genic sources, geology, physiochemical conditions, and food 
source also influence fluoride content in fish, invertebrates, 
and aquatic plants. 

The primary sources of dietary fluorides for farmed ani- 
mals are phosphorus supplements and feed ingredients of 
animal origin. Phosphorus supplements greatly vary in their 
fluoride content, depending on origin and manufacturing 
processes. The majority of U.S. feed phosphate originates 
from rock phosphate deposits, which contain 2 to 5 percent 



(average, 3.5 percent) fluorine (vanWazer, 1961). When pro- 
cessed sufficiently to qualify as defluorinated, feed-grade 
phosphates must contain no more than 1 part of fluoride to 
100 parts phosphorus (AAFCO, 2004). Processed low-fluo- 
ride, feed phosphates include mono-, di-, and tricalcium 
phosphates, mono- and diammonium phosphates, mono- and 
disodium phosphates, ammonium and sodium poly- 
phosphates, feed-grade phosphoric acid, and defluorinated 
phosphate. Unprocessed feed phosphates, supplying substan- 
tial amounts of fluoride, include soft rock phosphate, ground 
rock phosphate, and ground low-fluoride rock phosphate. 
More readily absorbed sources of fluoride, when incorpo- 
rated in animal diets, are undefluorinated, fertilizer-grade 
phosphates. 

Fluoride levels in blood, urine, and feces are commonly 
used to assess bioavailability and toxic levels of fluorine for 
humans and animals from dietary sources; however, skeletal 
tissue uptake has been used for experimental animal investi- 
gations (Rao, 1984). Fluoride is readily absorbed from wa- 
ter, but the absorption from diet may depend on the follow- 
ing factors: concentration, source, chemical form (inorganic 
or organic), other elements (Al, Ca, Mg, P, CI, S04^"), fluo- 
rine exposure from other sources (water, air, etc.), and physi- 
ological status (age, acid-base balance, disease, etc.). Soluble 
forms of fluorine, such as sodium fluoride, are readily ab- 
sorbed compared with bone meal, rock phosphate, and 
defluorinated rock phosphate. Fluorine from dicalcium phos- 
phate and raw rock phosphate was 50 percent as available as 
fluorine from NaF (Clay and Suttle, 1985). The fluorine from 
hay is available as NaF (Shupe et al., 1962). 

Some reduction in fluorine absorption from diet may be 
associated with insoluble complex formation between the 
fluoride anion and multivalent cations in the alkaline envi- 
ronment of the small intestine. Calcium and magnesium form 
insoluble complexes with fluoride, which significantly de- 
creases fluoride absorption (Cerklewski, 1997). Aluminium 
also forms an insoluble complex with fluoride. Among the 
anions, only chloride significantly influences fluoride 
bioavailability. Diets low in chloride reduce fluorine excre- 
tion and increase uptake in bone and teeth (Cerklewski, 
1997). Fluoride absorption in laboratory animals increased 
when either fat or protein were increased (McGown et al., 
1976; Boyde and Cerklewski, 1987). 

TOXICOSIS 

The toxicity of fluorine in animals via excessive fluoride 
ingestion from water or from industrial exposure is referred 
to as fluorosis, chronic fluorine toxicity, or fluorine toxico- 
sis. The toxicosis is initially manifested as dental fluorosis 
(mottled enamel) and, at higher levels of intake, skeletal fluo- 
rosis. Several reviews have been published on fluoride tox- 
icity in animals and humans (Krishnamachari, 1987; 
Whitford, 1996; Kirk, 1991; Underwood and Suttle, 1999; 
Cronin et al., 2000; ATSDR, 2003; Camargo, 2003; IPCS, 



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2002; McDowell, 2003). Table 14-1 summarizes some stud- 
ies on the effects of fluorine exposure in animals. 

Single Dose 

The oral LDjg for fluorine varies among animal species 
and also by gender. In rats, the following LD^q values or their 
range (mg F"/kg body weight) have been reported for three 
common fluoride supplements (DeLopez et al., 1976; Lim et 
al., 1978; Whitford, 1990; ATSDR, 2003): sodium fluoride, 
31 to 101; sodium monofluorophosphate, 75 to 102; and stan- 
nous fluoride, 45.7. For mice, LD^q values reported were 44.3; 
58, 54, and 94; and 25.5 and 3 1 .2 for sodium fluoride, sodium 
monofluorophosphate, and stannous fluoride, respectively 
(Lim et al., 1978; ATSDR, 2003). The predominant signs of 
toxicosis in these animals upon administration of a single large 
oral dose of fluoride were nausea, salivation, lacrimation, 
vomiting, diarrhea, respiratory arrest, tetany, and coma. Intra- 
peritoneal injection (13.6 and 21.8 mg F"/kg BW) of sodium 
fluoride into female rats caused proximal tubular necrosis and 
marked changes in kidney weight, urine osmolarity and pH, 
and chloride excretion; however, these effects were less severe 
in the younger animals (Daston et al., 1985). When sodium 
fluoride solutions (1 to 50 mmol ¥^fL dissolved in 0. 1 N HCl) 
were introduced in the stomach of rats, several histopathologi- 
cal changes in the gastrointestinal tract (hemorrhage, disrup- 
tion of epithelial integrity and glandular structure, lysis, and 
loss of epithelial cells) were observed (Easmann et al., 1984, 
1985). 

The inhalation exposure of rats, mice, and guinea pigs to 
fluorine gas (F,) and HF causes severe ocular and nasal irri- 
tation, pulmonary congestion, edema, respiratory distress, 
and erythraemia of exposed skin (ATSDR, 2003). Fluorine 
is more toxic than HF (Stokinger, 1949; Keplinger and 
Suissa, 1968). For rats, LC^q values (mg F"/m-^) ranging from 
4,065 to 14,400 have been reported for 5-minute exposure 
(WHO, 1984; ATSDR, 2003) and 1,084 for 60-minute expo- 
sure to HF (Rosenholtz et al., 1963). The LC^q for mice were 
approximately 5,000 and 280 when exposed to HF for 5 and 
60 minutes, respectively (WHO, 1984; ATSDR, 2003). 
Direct exposure of the skin to hydrofluoric acid produces 
severe burns and tissue damage depending upon the concen- 
tration and the length of the exposure (Derelanko et al., 
1985). In rats, direct application of aqueous solution of so- 
dium fluoride (0.5 and 1.0 percent) to the abraded skin 
caused necrosis, edema, and inflammation (Essman et al., 
1981). 

Acute 

Acute fluoride toxicosis is rare. It usually results from the 
accidental ingestion of compounds such as sodium 
fluorosilicate (NaiSiFg), used as a rodenticide, or sodium 
fluoride, used as an ascaricide in swine. The rapidity with 
which toxic signs appear depends on the amount of fluoride 



ingested (Cass, 1961). Toxic signs include high fluoride con- 
tent of blood and urine, restlessness, stiffness, anorexia, re- 
duced milk production, excessive salivation, nausea, vomit- 
ing, urinary and fecal incontinence, chronic convulsions, 
necrosis of gastrointestinal mucosa, weakness, severe de- 
pression, and cardiac failure (Shupe et al., 1963a,b). Death 
occurred within 12 to 14 hours in dairy cows after ingesting 
sodium fluorosilicate (Krug, 1927). Acute fluoride toxicosis 
also occurred in cattle after exposure to wood treatment com- 
pounds used on utility poles (Bischoff et al., 1999). The pre- 
dominant systematic effects of acute oral exposure to so- 
dium fluoride in humans and laboratory animals include 
hypocalcemia, hyperkalemia, and gastrointestinal pain 
(ATSDR, 2003). 

Chronic 

Chronic skeletal fluorosis is prevalent in cattle, sheep, 
goats, horses, and humans in parts of India, Argentina, Aus- 
tralia, Turkey, Africa, the United States, and other regions of 
the world (Underwood, 1981; WHO, 1994), often due to 
consumption of water high in fluoride. The degree to which 
inorganic fluoride induces skeletal changes varies consider- 
ably in farm animals. Cattle are most sensitive to fluorosis, 
followed by sheep, horses, pigs, rabbits, rats, guinea pigs, 
and poultry (Franke, 1989). Young animals are most affected 
by fluorosis. The dietary concentration at which fluoride in- 
gestion becomes harmful in farm animals is not clearly de- 
fined. Diagnosis of fluoride toxicosis at low levels of inges- 
tion is difficult because there is an extended interval of time 
between ingestion of elevated levels and the appearance of 
toxic signs (Shupe, 1970). Low-level toxicosis also depends 
upon solubility of the fluoride source, general nutritional sta- 
tus, species of animal, age when ingested, and dietary com- 
ponents that modify toxicity. 

Both domestic and wild grazing animals also develop 
chronic fluorosis due to contamination with volcanic gas and 
ash (Roholm, 1937; Araya et al., 1990; Shanks, 1997), inges- 
tion of commercial phosphorus supplements (Shupe et al., 
1992; Jubb et al., 1993; Singh and Swarup, 1995), phosphate 
fertilizer residues (Clark and Stewart, 1983), forage or water 
polluted with industrial emissions (Kay et al., 1975; Singh and 
Swarup, 1995; Kierdorf et al., 1996), and drinking water from 
ground and geothermal sources that are rich in fluoride 
(Harvey, 1952; Shupe et al., 1984; Botha et al., 1993). 

The effect of fluorine toxicosis generally takes weeks or 
months to become manifest and some of the excess fluorine 
is excreted in urine (Underwood and Suttle, 1999). Plasma 
fluoride concentrations reflect short-term changes in fluo- 
rine uptake with levels of < 0. l|Jg g"' in normal animals and 
1 \ig g"' indicating a high fluorine uptake (Suttie et al., 1972). 
The skeleton of animals normally contains the greatest pro- 
portion of fluorine within the animal and the normal whole 
bone fluorine concentration ranges between 300 and 600 |ig 
g"' (dry fat-free basis), the highest concentration being 



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within the cancellous bones such as rib and vertebrae 
(Underwood and Suttle, 1999). In ruminants, teeth contain 
approximately half the fluorine concentration of bone 
(Underwood and Suttle, 1999). The period during which 
developing teeth in cattle are sensitive to excess fluoride is 
from approximately 6 months to 4 years of age. Teeth that 
have erupted are not influenced adversely by subsequent 
fluoride ingestion (Garlick, 1955), and cattle that are more 
than 3 years old will not develop typical dental lesions. Den- 
tal fluorosis is generally diagnosed by examining the inci- 
sors. Gross fluoride lesions of the incisor enamel begin with 
inside mottling (white, chalky patches or striations) and 
progresses to defined mottling, hyperplasia, and 
hypocalcification. A scoring system for classification of den- 
tal fluorosis has been proposed (NRC, 1974). 

The amount of fluoride stored in bone may increase over 
time with no apparent change in bone structure or function. 
However, if excess fluoride ingestion is sufficiently high, 
and occurs over a sufficiently long period of time, morpho- 
logical abnormalities will develop. In livestock, clinically 
palpable (bilateral) lesions usually develop first on the medi- 
cal surface of the proximal third of the metatarsals. Subse- 
quent lesions are seen on the mandible, metacarpals, and ribs. 
Osteofluorotic lesions tend to be more severe in those bones, 
and parts of the bones, that are subject to greatest physical 
stress. Radiographic evidence of osteoporosis, osteosclero- 
sis, osteomalacia, hyperostosis, and osteophytosis or any 
combination of these lesions has been described (Johnson, 
1965; Shupe and Alther, 1966; Shupe, 1969). Grossly, se- 
verely affected bones appear chalky white, are larger in di- 
ameter, and heavier than normal, and they have a roughened, 
irregular periosteal surface. In cattle poisoned by industrial 
fluoride emissions, Krook and Maylin (1979) suggested that 
the primary target of fluoride was the resorbing osteocyte. 
Morphological signs of osteolysis were absent, and the fail- 
ure of resorption caused osteopetrosis with retention of 
lamellar bone in cortices. 

Animal movement may be impaired by intermittent peri- 
ods of stiffness and lameness, associated with advanced ar- 
eas with calcification of particular structures and tendon in- 
sertions. In animals with marked periosteal hyperostosis, 
spurring and bridging of the joints may lead to rigidity of the 
spine and limbs. Anorexia, unthriftiness, dry hair, and thick, 
nonpliable skin have been noted in fluorotic animals 
(Roholm, 1937; Shupe et al., 1963b). 

Primary adverse effects on reproduction and lactogenesis 
have not been demonstrated although milk production may 
decrease with high fluoride intake, secondary to dental and 
skeletal damage and consequent reductions in feed and wa- 
ter intake (Stoddard et al., 1963). Suttle et al. (1957b) have 
demonstrated that cows first exposed to fluoride at 4 months 
of age can consume 40 to 50 mg/kg of fluorine as sodium 
fluoride in their diet for two or three lactations without a 
measurable effect on milk production. Milk production was 
reduced in the fourth and subsequent lactations. Higher di- 



etary fluoride levels (93 mg/kg) affected the milk production 
in the second lactation slightly and definitely reduced milk 
yield in a subsequent lactation (Stoddard et al., 1963). Irre- 
spective of level or duration of fluoride intake, clinical signs 
of toxicosis will normally precede impaired milk produc- 
tion. No characteristic, unequivocal histological or func- 
tional changes in blood or soft tissues have been correlated 
with fluoride intakes sufficient to induce chronic fluorosis of 
bones and teeth. 

Natural surface waters are often contaminated with fluoride 
from discharges of fluoridated municipal waters, fertilizers, and 
other industrial pollutants that cause ecological risk to aquatic 
organisms and plants. In certain areas, high concentrations ( 10 
mg F^VL) have been measured in rivers (CEPA, 1993; 
Camargo, 1996). Aquatic organisms accumulate soluble inor- 
ganic fluorides. When sodium fluoride was released into an 
experimental pond, within 24 hours the concentration of fluo- 
ride in aquatic vascular plants increased 35 -fold and a signifi- 
cant increase in its uptake was observed in algae (14-fold), 
molluscs (12-fold), and fish (7-fold) (Kudo and Garrec, 1983). 
Limited data on the toxicity of fluorine in freshwater to algae, 
aquatic plants, invertebrates, and fish are available (reviewed 
by Camargo, 2003). Aquatic plants can effectively remove fluo- 
ride from contaminated water, and the concentration increases 
with exposure time. The toxicity is linked to fluoride ions that 
affect nucleotide and nucleic acid metabolism that influences 
algal cell division (Antia and Klut, 1981). Some algae can toler- 
ate inorganic fluoride levels as high as 200 mg/L (Antia and 
Klut, 1981; Camargo, 2003). Fluoride toxicity in aquatic inver- 
tebrates and fish increases with increasing fluoride concentra- 
tions, exposure time, and water temperature. Marine inverte- 
brates appear to be more tolerant to toxicity than freshwater 
species (reviewed by Camargo, 2003). Fluorine accumulates in 
the exoskeleton of invertebrates and bones of fish. The LCjq 
values (mg F"/L) of several fish at different exposure times 
range from 5 1 to 460 (Camargo, 2003). In soft water with low 
ionic content, a fluoride concentration as low as 0.5 mg F"/L 
produces toxicosis in invertebrates and fish. Among fishes, rain- 
bow trout have been used widely to study fluoride toxicity. In 
addition to water temperature, calcium and chloride content of 
water and body size also affect the fluorine toxicity. 

In humans, the major toxic effects range from dental fluo- 
rosis (Fejerskov et al., 1977; DenBesten and Thariani, 1992), 
reversible gastric disturbances (Jowsey et al., 1979), and 
lower urinary concentrating ability (Goldemberg, 1931; 
Whitford and Taves, 1973) to skeletal fluorosis (Singh and 
Jolly, 1970) and death (Hodge and Smith, 1965; Church, 
1976; Dukes, 1980;Eichleretal., 1982; Gessner et al., 1994). 

Reproduction 

The adverse effects of high levels of fluoride intakes on 
reproduction of several laboratory animal species have been 
reported (IPCS, 2002). Reproductive function was severely 
affected in female mice orally administered >5.2 mg F/kg 



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BW/day after mating (Pillai et al., 1989), and in male rabbits 
orally administered 9.1 mg F/kg BW/day for 30 days 
(Chinoy et al., 1991). Histopatliological changes have also 
been observed in testes of male rabbits fed 4.5 mg F/kg 
BW/day for 18 to 29 months (Susheela and Kumar, 1991) 
and male mice fed 4.5 mg F/kg BW/day for 30 days (Chinoy 
and Sequeira, 1989a,b) and in ovaries of female rabbits in- 
jected subcutaneously with 10 mg F/kg BW for 100 days 
(Shashi, 1990). Exposure of dams to 150 mg/L fluorine in 
drinking water prior to breeding and during pregnancy and 
lactation had no adverse effects on the bones of developing 
rats (Ream et al., 1983a), although effects on the bones of 
the dams were observed (Ream et al., 1983b). The lowest- 
observable-effect-level (LOEL) of dams was considered to 
be 21.4 mg/kg BW/day. In most studies, the fluoride con- 
centrations associated with adverse effects were much higher 
than those measured in drinking water or animal feeds. The 
apparent threshold concentration for inducing reproductive 
effects in animals in drinking water has been listed by NRC 
(1993): 100 mg/L for mice, rat, foxes, and cattle; 100-200 
mg/L for mink, owls, and kestrels; and >500 mg/L for 
hens. 

Genotoxicity and Carcinogenicity 

The genotoxicity of fluoride has been examined in a large 
number of in vitro and in vivo assays (WHO, 1984; Li et al., 
1987; Tong et al., 1988; Zeigler et al., 1993; IPCS, 2002). 
Generally, fluoride is not mutagenic in microbial cells, but 
increases the frequency of gene-locus mutations in cultured 
mammalian cells and induces the morphogenic transforma- 
tion of Syrian hamster embryo cells at cytotoxic concentra- 
tion in vitro. Although the results of some studies have indi- 
cated that sodium fluoride increases unscheduled DNA 
synthesis in mammalian cells, these results were not con- 
firmed when steps were taken to eliminate potential artifacts 
(i.e., formation of precipitable complexes of magnesium 
fluoride and pH] thymidine). Sodium fluoride has been con- 
sidered capable of inducing chromosomal aberrations, mi- 
cronuclei, and sister-chromatid exchanges in vitro in mam- 
malian cells; however, the results from these studies were 
not consistent. Chromosal aberrations have not been detected 
in cells exposed in vitro to levels of fluoride less than 10 [igl 
mL, which is considered as a threshold for the clastogenic 
activity of fluoride. 

The results of early carcinogenicity bioassays conducted 
with NaF administered in water were largely negative. How- 
ever, the documentation and protocols of these studies were 
inadequate according to current bioassay techniques and thus 
of limited value. Although several carcinogenicity bioassays 
have been conducted in recent years, the results of two stud- 
ies were selected to be reliable by NRC (1993). The National 
Toxicological Program (NTP, 1990) administered 175 mg 
F/L in drinking water to male and female mice. Although 
results were negative, there was some evidence of a dose- 



related increase in the incidence of osteosarcomas in male 
rats. These results were not confirmed by another study con- 
ducted by Procter and Gamble (Maurer et al., 1990) in which 
fluorine in the diets was at doses higher than those in the 
NTP study. The later study did produce significant dose- 
related incidence of osteomas (benign bone tumors) in male 
and female mice. The available laboratory animal experi- 
mental data are insufficient to demonstrate a carcinogenic 
effect of fluorine (NRC, 1993). 

Factors Influencing Toxicity 

Toxicity of fluoride, at a given level of exposure, depends 
on animal species, source, concentration, chemical form (in- 
organic or organic), other elements, and physiological status 
(age, acid-base balance, disease, etc.) of the animal. Fluo- 
ride is readily absorbed from water; therefore, the toxicity of 
soluble fluoride compounds will depend on their water solu- 
bility. Based on the skeletal accumulation of fluoride by rats, 
Hobbs et al. (1954) ranked the toxicity of fluoride com- 
pounds in the order from highest to lowest as follows: rock 
phosphate, natural and synthetic cryolite, calcium and mag- 
nesium fluosilicates, and calcium fluoride. Fluoride in rock 
phosphate was considerably less toxic to beef heifers than 
that in sodium fluoride (Hobbs and Merriman, 1962). Natu- 
rally fluorinated water produced toxicity comparable to equal 
amounts of fluoride from sodium fluoride added to water 
(Wagner and Muhler, 1957) or to a dry diet (Harvey, 1952; 
Wuthier and Phillips, 1959). Variations in fluoride intake, 
with alternate periods of high and low exposure, were more 
damaging to young cattle than were constant intakes of the 
same amount (Suttie et al., 1972). 

Some dietary components that have been shown to re- 
duce fluoride toxicosis include aluminum, calcium, magne- 
sium, phosphorus, sodium chloride, boron, and sulfates 
(NRC, 1980; Krishnamachari, 1987). Calcium given orally 
or intravenously counteracts the effects of fluoride, particu- 
larly toxicity of an acute nature (Krishnamachari, 1987). 
Feeding of calcium carbonate, aluminum oxide, or alumi- 
num sulfate reduces absorption of fluoride and may control 
chronic fluorosis under some conditions. However, the con- 
sumption of a combination of aluminum sulfate with cal- 
cium carbonate in a mineral mixture was not effective in 
reducing bone fluoride accumulation in cattle grazing in a 
fluoride-contaminated pasture (Allcroft and Burns, 1968). 
Aluminum compounds may also adversely affect dietary 
phosphorus retention (Street, 1942; Hobbs et al., 1954; 
Alsmeyer et al., 1963; Storer and Nelson, 1968); if they are 
used to alleviate fluoride toxicosis, increased levels of phos- 
phorus must be fed. Aluminum chloride and aluminum ac- 
etate also appear to be effective in reducing fluorosis in 
cattle, but aluminum oxide produces only slight alleviation 
(Hobbs et al., 1954). For chickens, free-choice access to di- 
etary aluminum sulfate at 800 mg Al/kg completely pre- 
vented toxicity of 1,000 mg F/kg; the oxide form was not 



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MINERAL TOLERANCE OF ANIMALS 



effective (Cakir et al., 1978). Aluminum seems to reduce the 
gastrointestinal absorption of fluoride. 

High intakes of boron have been shown to be an effective 
antidote to fluoride toxicosis in rabbits (Baer et al., 1977; 
Elsair et al., 1980), pigs (Seffner and Teubener, 1983), and 
sheep (Wheeler et al., 1988). Boron may induce formation of 
a complex, BF^, which is excreted in the urine. Magnesium 
metasilicate has been tested in chronic fluorosis patients, who 
responded with partial clinical amelioration of their signs (Rao 
et al., 1975). Toxicity of fluorine is also affected by dietary fat 
level, probably due to its effects on absorption of this element. 
Increase in the level of dietary fat from 5 to 15 or 20 percent 
enhanced the growth-retardation effect of high fluorine intake 
in rats (Miller and Phillips, 1953; Buttner and Muhler, 1958) 
and chickens (Bixler and Muhler, 1960). Fluorine, in the pres- 
ence of fat, causes delayed gastric emptying, which may ac- 
count for the increased toxicity of fluorine in rats fed high-fat 
diets (McGown et al., 1976). 

TISSUE LEVELS 

The concentrations of fluorine in bone, tissues, blood, 
eggs, and milk vary with fluorine intakes from water and 
diet (NRC, 1980). Mineralized tissues contain approximately 
99 percent of total body fluorine, with a major proportion 
concentrated in bone. The skeleton of animals normally con- 
tains the highest proportion of fluorine, with normal bone 
having ranges from 0.3 to 0.6 mg F/g of dry tissue on a fat- 
free basis (Underwood and Suttle, 1999). The fluorine con- 
centration in the soft tissues is relatively low and changes 
little with age. Kidneys contain higher levels of fluorine than 
other tissues partly due to urine retained in this organ. Nor- 
mal fluorine levels (|Jg/g, dry matter basis) of cow and sheep 
in the following soft tissues have been reported (Harvey, 
1952; Suttie et al., 1958): liver, 2.3 and 3.5; kidney, 3.5 and 
4.2; thyroid, 2.1 and 3.0; and heart, 2.3 and 3.0. Dairy cows 
fed a ration supplemented with 50 pg F/g as NaF for 5.5 
years only increased the levels in heart, liver, thyroid, and 
pancreas 2- to 3-fold above 3 |Jg/g found in tissues of normal 
cows (Suttie et al., 1958). In normal cow's milk, fluorine 
levels range from 0.01 to 0.4 (ig/g (Suttie et al., 1957b; 
Bergmann, 1995), whereas fluorosis can occur in cattle when 
levels reach 0.64 |jg/g (Suttie et al., 1957b). 

Plasma fluoride concentrations are maintained within nar- 
row limits by regulatory mechanisms involving skeletal tis- 
sues. In cattle, the plasma fluorine concentration is less than 
0.1 [Jg/g in normal animals and 1 pg/g indicates a high fluo- 
rine uptake from diet (Suttie et al., 1972). The fluorine con- 
centration in plasma of healthy humans ranges from 7.6 to 
28.5 pg/L (Guy, 1979) and values as high as 106 ± 76 mg/L 
in adults exposed to 5.03 mg F/L in drinking water have 
been reported (Li et al., 1995). Elevated intakes of fluoride 
also result in an increased concentration of fluoride in urine 
and bone. In several long-term experiments with beef and 
dairy cattle, the skeletal concentration of fluorine was ap- 



proximately proportional to the concentration of NaF (NRC, 
1974). Urine fluoride levels are approximately correlated 
with dietary intake, although the duration of fluoride inges- 
tion, sampling time, and total urinary output will introduce 
variation. With excess ingestion of fluorine, urinary levels 
increased from 15 to 30 mg/L to an upper limit of 70 to 80 
mg/L (Suttie and Kolstad, 1977). The following urine con- 
centrations indicate fluorine status of cattle: normal, <5 mg 
F/L; borderline toxicity, 20 to 30 mg F/L; systematic toxic- 
ity, >35 mg F/L (McDowell, 2003). 

Birds fed high-fluoride diets accumulate fluorine in their 
eggs, particularly in yolk. The fluorine content of normal 
chicken eggs increased from 0.8 to 0.9 |Jg/g to as high as 3 
|jg/g with an increase in dietary rock phosphate concentra- 
tion (Phillips et al., 1935). Generally the fluorine in deboned 
poultry meat is low (0.2 mg/kg), but higher values in the 
range of 0.3 to 2.7 mg/kg in chicken and turkey meat have 
been reported (Jedra et al., 2001). Fluoride is mainly con- 
centrated in skeletal tissues of fish and exoskeletons of in- 
vertebrates. In freshwater and marine fish muscle, it ranges 
from 0.6 to 26 mg/kg wet weight (Lall, 1994; Camargo, 
2003) and in whole fish, from 10 to 60 mg/kg (Soevik and 
Braekkan, 1981). The average amount in the edible part of 
muscle is 0.8 mg F/kg, but small bones retained in fish 
muscle can affect the fluorine content (Lall, 1994). Fluorine 
in the muscle of crab, shrimps, and prawn from the North 
Sea varies from 1 to 4 mg/kg (Soevik and Braekkan, 1981). 
Antarctic krill contains exceptionally large amounts of fluo- 
rine (mg/kg dry weight): whole animal, 1,000; muscle, 70; 
and exoskeleton, 2,000. The concentrations of fluoride in 
the body of wild invertebrates consumed by fish have ranged 
from 7 to 3,500 mg/kg (Camargo, 2003). The consumption 
of these organisms could have a significant effect on the 
fluorine content of fish tissues. 

HUMAN HEALTH 

Fluorine is considered a hazardous substance with inges- 
tion of large doses of this element in either food or water or 
both. Fluoride consumed from water and beverages accounts 
for more than 70 percent of total fluoride intake, because 
most animal meat, milk, and eggs contain less than 30 |ig F/ 
100 g (Table 14-2). Regular consumption of marine fish and 
crustaceans can significantly increase fluoride intake. The 
drinking water in many parts of the world is naturally rich in 
fluoride, which causes local inhabitants to develop endemic 
fluorosis. Several reviews related to the risk of chronic fluo- 
ride toxicity to humans have been published (Kaminsky et 
al., 1990; USPHS Ad Hoc Subcommittee on Fluoride, 1991; 
NRC, 1993; Whitford, 1996; IPCS, 2002; ATSDR, 2003). 

MAXIMUM TOLERABLE LEVELS 

The tolerance levels for domestic animals are based on 
clinical signs of fluoride toxicosis, and most of the values 



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were obtained before 1980. While small intakes of fluoride 
may be beneficial or even essential, prolonged intakes of 
oral fluoride concentrations above these maximum tolerable 
levels may result in reduced performance. The tolerable lev- 
els are based on tolerances to sodium fluoride or other fluo- 
rides of similar toxicity (fluoride in certain phosphorus 
sources appears to be less toxic) and assume that the diet is 
essentially the sole source of fluoride. When water also con- 
tains appreciable fluoride (> 3 mg/L), these dietary levels 
should be reduced. The maximum fluoride levels for young 
beef or dairy calves and heifers are 35 and 40 mg/kg of diet, 
respectively; for animals kept for breeding, 30 to 40 mg/kg 
of diet for heifers; and for mature animals, 40 to 50 mg/kg of 
diet. Long-term studies (approximately 7 years) conducted 
with young calves showed that 30 mg F/kg diet caused ex- 
cessive staining of teeth (Hobbs and Merriman, 1959; Shupe 
et al., 1963b). Minor morphological lesions may be seen in 
cattle teeth when dietary fluoride during tooth development 
exceeds 20 mg/kg, but a relationship between these lesions 
and animal performance has not been established. 

Mature dairy cattle consume more feed in relation to body 
weight than mature beef cattle, so maximum recommended 
concentrations of dietary fluoride are 40 mg/kg for dairy 
cattle and 50 mg/kg for beef cattle. Lifetime fluoride expo- 
sure for finishing cattle is less than for breeding cattle, so the 
maximum tolerable level for this productive class is esti- 
mated at 100 mg/kg. Excessive exposure during tooth devel- 
opment in cattle may result in exaggerated tooth wear, im- 
paired mastication, and sensitivity to cold drinking water. 
Maximum tolerable levels for other species are based on rela- 
tively limited published data in some cases, and the level of 
40 mg F/kg for horses and rabbits represents an estimate. 
Levels for lifetime exposure for sheep, swine, and poultry 
are less than that for cattle and horses. Poultry are relatively 
less sensitive to fluoride. The maximum tolerable levels are 
150, 150, and 200 mg F/kg for swine, turkeys, and chicken, 
respectively. Sheep raised for lamb or wool can tolerate 60 
mg F/kg diet, and finishing lamb can tolerate up to 150 mg 
F/kg diet (NRC, 1980). 

Studies on laboratory species under a wide range of ex- 
perimental design have been summarized by CEP A (1993) 
and IPCS (2002), and the NOAEL and the LOAEL are as 
follows (mg of F/kg of BW/day): rats, 1.8-12.7, 3.2-12.8; 
and mice, 2.7-9.1,4.5-5.7. The estimated average safe con- 
centrations (infinite hour LCg Q^ values) of fluoride for fry 
(8-10 cm) of rainbow trout and brown trout in soft water 
(21.2-22.4 mg CaCOj/L) were 5.14 (range 3.10-7.53) and 
7.49 (range 4.42-10.96) mg F-/L, respectively (EPA, 1986; 
Lee et al., 1995). A fluoride concentration of 0.5 mg F"/L is 
considered safe for freshwater upstream migrating salmon 
inhabiting soft water of low ionic content and 1-1.5 mg F"/L 
in hard water with high ionic content (Camargo and La 
Point, 1995; Camargo, 1996). 

The LCjQ values of fluoride for freshwater, brackish, and 
marine invertebrates at different stages of development, ex- 



posure time, and temperature have ranged from 10.5 to 308 
F"/L. However, the safe concentration (infinite hour LCqqj 
values) of fluoride for many species in soft freshwater is in 
the range of 0.5 mg F"/L (Camargo, 2003). 

The lethal dose (LDjqq) of sodium fluoride in an average 
human adult has been estimated between 5 and 10 g (32 to 
64 mg of F/kg BW) (WHO, 1984; Whitford, 1990). The tol- 
erable upper intake level of fluorine ranges from 0.7 to 2.2 
mg/day for children to 10 mg/day for adult men and women 
(NRC, 1993), approximately 2- to 3-fold higher than the rec- 
ommended dietary allowance. The EPA derived an oral ref- 
erence dose (RFD) of 0.06 mg/kg/day for fluorine (ATSDR, 
2003) that was based on a NOAEL of 0.06 mg of F/kg of 
BW/day and the LOAEL of 0.12 mg of F/kg of BW/day 
from data on fluorosis in children. The maximum amount of 
fluoride allowed by EPA in drinking water is 4 mg/L. 

FUTURE RESEARCH NEEDS 

The beneficial effects of fluoride on dental health of 
farmed and laboratory animals and humans have been known 
for more than 60 years; however, the exact biochemical 
mechanisms to qualify fluorine as an essential trace element 
remain to be established. Numerous publications describe 
the effects of fluorine supplementation (in some cases, phar- 
macological dose) that include prevention of dental caries or 
osteoporosis. As well, the amelioration of anemia and fertil- 
ity in experimental animals has been reported; however, the 
biochemical mechanisms remain unclear. The potential haz- 
ard of fluoride on farm animals by ingestion of phosphate 
fertilizers, volcanic ash, and industrial wastes is likely to 
continue and it will affect terrestrial and aquatic animal pro- 
duction. Research is needed to establish the risk of fluorine 
toxicity in pastoral systems and aquaculture, as well as to 
develop sustainable strategies for the future. The impact of 
human activities — such as aluminum smelters, discharges of 
municipal waters, and industries manufacturing brick, ce- 
ramics, glass, and chemicals containing fluorides — are caus- 
ing significant increases in the fluoride concentration of sur- 
face waters. Despite detennining the safe levels of fluoride 
for certain aquatic organisms, there are limited data on 
bioaccumulation in the edible portion of fish and shellfish. 

SUMMARY 

Fluorine is present as fluoride in igneous and sedimentary 
rock. Most plants have a limited capacity to absorb fluoride 
from the soil; however, animals may consume significant 
quantities from contaminated surface waters, deep-well 
water percolating through fluorapatite; forages contaminated 
by fluoride-bearing dusts, fumes, or water; animal by-prod- 
ucts containing bone high in fluoride; and a variety of inor- 
ganic phosphate supplements. Animals normally ingest low 
levels without harm, and small amounts of fluoride may be 
beneficial and perhaps even essential. Ingestion of excessive 



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fluoride chronically induces characteristic lesions of the skel- 
eton and teeth, resulting in intermittent lameness, excessive 
tooth wear, reduced feed and water intake, and decreased 
weight gain and milk production. Developing teeth and bone 
are particularly sensitive, and excessive exposure in early 
postnatal life is especially damaging. Other disorders caused 
by acute fluorine toxicosis in animals and/or humans include 
gastrointestinal irritation, severe cardiac effects (e.g., tetany, 
cardiovascular collapse, ventricular fibrillation), and paren- 
chymal liver degeneration. In general, those fluoride com- 
pounds that are most soluble are most toxic. Aquatic organ- 
isms absorb fluorine from their diet and surrounding water 
and accumulate it in skeletal tissues. Toxicity data are avail- 
able for terrestrial and aquatic animals; however, oral maxi- 
mum tolerable levels for fish are not established. 

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180 



T3 

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FLUORINE 



181 



TABLE 14-2 Fluorine Concentrations in Fluids and Tissues of Animals (mg/kg or |Jg/L) 





Level and 






Liver or 






Egg 




Animal 


Source of F 


Plasma 


Bone 


Soft Tissues 


Muscle 


Milk 


Yolk 


Reference 


Humans 


Diet 


0.003- 
0.080 


1.5-2.2 


19.6-159 


J 






Guy et at., 1976; Zipkin 

et at., 1960; Taves et at., 1983 


Chickens, 


16 


0.001 


538 


5.2 


4.0 




4.3 


Hahn and Guenter, 1986 


laying 


too 


0.003 


2,247 


5.5 


4.0 




3.3 




hens 


1,300 


0.010 


2,600 


19.2 


6.7 




18.4 





Meat and 
poultry 



Cattle, 
fluorosis 

Cows 



Cows 



Cows 



Cows 



Sheep 



17 varieties 
of muscle 
and poultry 
in Canada 



Meat and 


7 varieties of 


poultry 


meat and 




poultry in 




Hungary 



12 varieties 
of diary 
products in 
Canada 

13 varieties 
of dairy 
products in 

[ Hungaiy _ 



Milk and 

milk products 
sampled 
between 
1981-1989 in 
Gennany 

50 Mg/g 
fluorine in 
feed ration 
for 5.5 yr 

10 mg/g liter 
fluorine in 
water for 
2 yr 



0.04-1.2 




Dabeka and McKenzie, 1995 



0.01-1.7 



885-6,918 




0.072-0.64 



0.01-0.8 



0.045-0.51 



Schamschula et al., 1988 



Hillman et al., 1979 



Dabeka and McKenzie, 1995 



Schamschula et al., IS 



0.019-0.16 



Bergmann, 1995 




Liver: 3.6 
Kidney: 19.3 
Heart: 4.6 
Thyroid: 7.3 

Liver: 2.2 
Kidney: 16.8 
Thyroid: 7.2 
Heart: 2.2 




Harvey, 1952 



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15 



Iodine 



INTRODUCTION 

The name iodine (I) is derived from thie Greek word iodes, 
meaning violet. Iodine (atomic weighit 126.9045), a nonme- 
tallic element of the halogen group, is volatile at ambient 
temperature and pressure. It can exist in various oxidation 
states with valences of "1, """l, ^3, """S, or "'"7, but the main 
oxidation state is "1 as iodide. The term iodide refers to its 
ionic form I". lodate refers to lO-^". Elemental iodine occurs 
as a purple-black solid with rhombic crystalline structure that 
sublimes into the gaseous form (Ij), giving off an intense 
violet vapor and a characteristic odor. It is also a component 
of fallout produced by nuclear explosions. Iodine is less re- 
active than other halogens (fluorine [F], chlorine [CI], and 
bromine [Br]) but readily forms compounds with other ele- 
ments. It dissolves in alcohol, carbon disulfide, carbon tetra- 
chloride, and chloroform, but it is only slightly soluble in 
water. In water, hydrogen and iodine form hydrogen iodide. 
Iodine forms compounds with certain nonmetals (e.g., car- 
bon, nitrogen, phosphorus, and oxygen), and it can be dis- 
placed by the other halogens. Among 36 isotopes of iodine 
with masses between 108-143 (Chu et al., 1999), 14 yield 
significant radiation. The naturally occurring isotopes of io- 
dine, '^^I and '^^I, are stable and radioactive. The other iso- 
topes of iodine are instable isotopes. Iodine"'^' is a radioac- 
tive isotope with a half-life of 8 days. It is widely used to 
diagnose abnormalities of the thyroid gland in humans and 
experimental animals. 

Iodine, the 64th most abundant element (10"^ percent of 
Earth's crust), is widely distributed in nature and present in 
both organic and inorganic substances in very small amounts. 
The ocean is the primary source of iodine, containing be- 
tween 50-60 l-ig/L. Only a few substances, such as the salt- 
petre deposits of Chile and some marine products, have con- 
centrations of up to 1,000-2,000 mg/kg. Iodine is present in 
soil, air, and water and becomes a constituent of plants and 
animals used for food. The average iodine concentration 
in air, soil, freshwater, and animal body are 0.7 |ig/m-^. 



300 Mg/kg, 5 Mg/1, and 0.4 mg/kg (WHO, 1998; ATSDR, 
2004). The iodine content of water reflects the iodine con- 
tent of the rocks and soils of the region. Thus, crops grown 
in certain areas, such as the Ganges plains of India, are io- 
dine deficient, and animals and humans in this area suffer 
from a deficiency of this element. An important source of 
iodine is caliche, a nitrate rock that contains up to 0.2 per- 
cent iodine in the form of iodate salts. Iodine is also found as 
an iodide in certain seaweeds. 

Iodine was first isolated in 1811 by a French chemist, 
Bernard Courtois, from seaweed ash during the making of 
gunpowder. In 1895, Baumann discovered iodine in the hu- 
man thyroid gland and the relationship of iodine deficiency 
to enlargement of the thyroid gland was first clinically shown 
by Marine and Kimball in 1922 (Hetzel and Dunn, 1989). A 
comprehensive review of the historical aspects of goiter has 
been published (Matovinovic, 1983). Iodine and its com- 
pounds are used as supplements in feeds and foods (e.g., 
iodized salt), agricultural chemicals (e.g., herbicides and fun- 
gicides), animal drugs (e.g., ethylenediamine dihydriodide 
[EDDI]) and sanitizers (e.g., iodophors). EDDI is used at 
relatively high levels to prevent or treat foot rot and soft 
tissue lumpy jaw in cattle (Miller and Tillapaugh, 1966). 
Iodophors are widely used in the dairy industry as teat dips 
and udder washes. lodophor solutions are also used as sani- 
tizing agents for cleansing equipment. In industry iodine is 
used mainly as organoiodine compounds in pharmaceuticals, 
photography, pigments, sterilization, dyestuffs, and rubber 
manufacture. 

ESSENTIALITY 

Iodine is an essential element for animal species, includ- 
ing humans, mainly because it is an integral component of 
the thyroid hormones, 3,3',5-triiodothyronine (T3) and 
3,3',5,5'-tetraiodothyronine (thyroxine, T^). Thyroid hor- 
mones regulate cell activity and growth in virtually all tis- 
sues and therefore are essential in intermediary metabolism. 



182 



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IODINE 



183 



reproduction, growth and development, hematopoiesis, cir- 
culation, neuromuscular functioning, and thermoregulation. 
The concentration of iodine in the environment, as well as in 
plant material and water, is extremely variable throughout 
the world and iodine deficiency disorders (IDD) can be a 
major problem in both domestic animals and humans (Hetzel 
and Dunn, 1989; Delange, 1994; Underwood and Suttle, 
1999). 

In all farm animals, deficiency is accompanied by thyroid 
hyperplasia or "goiter" and a decrease in thyroglobulin con- 
centration within the follicles of the thyroid gland. The 
goitrous condition is a hyperplastic response of the thyroid 
glands to an increased stimulation of thyroid growth by thy- 
roid-stimulating hormones produced in the pituitary gland. 
With mild iodine deficiency, the hyperplastic thyroid gland 
can compensate for the reduced absorption of iodine (Hetzel 
and Welby, 1997). Deficiency signs vary depending upon 
the animal species and the severity of the deficiency. Calves 
may be born hairless, weak, or dead. Fetal death can occur at 
any stage of gestation, but cows often appear normal 
(Hemken, 1960). Reproductive failures have been reported 
in both male and female cattle, sheep, and pigs suffering 
from goiter (Underwood and Suttle, 1999). Chickens may 
show thyroid hyperplasia without a significant effect on 
growth and reproductive performance. Laying hens fed diets 
containing 0.01 to 0.02 mg I/kg, produced eggs of low hatch- 
ability and poor embryonic development. While 0.35 mg 
I/kg was adequate to maintain good hatchability, 0.75 mg 
I/kg was not sufficient to prevent thyroid hyperplasia in em- 
bryos (Rogler et al., 1961). 

Goitrogenic substances in feed may increase iodine re- 
quirement substantially (2- to 4-fold) depending on the 
amount and type of this natural toxicant (Bell, 1984). Cy- 
anogenetic goitrogens include thiocyanate derived from cya- 
nide in white clover and the glucosinolates found in Bras- 
sica seeds and forages (e.g., kale, turnips, and rapeseeds). 
These thiocyanate compounds impair iodine uptake by the 
thyroid, but their effect can be overcome by increasing di- 
etary iodine intake. Several cases of fetal death, abortion, or 
the birth of weak lambs or calves associated with hypothy- 
roidism have been reported where plants containing goitro- 
gens constituted an appreciable proportion of the diet during 
pregnancy (Sinclair and Andrews, 1958). Goitrogens, par- 
ticularly the thiooxazolidone type, readily pass from the 
bloodstream to the milk of lactating animals and conse- 
quently the milk possesses goitrogenic potency (Arstila et 
al., 1969; Laurberg et al., 2002). 

Recent estimates of iodine requirements (mg/kg diet) pub- 
lished in NRC publications where diet does not contain 
goitrogens are as follows: chicken and cats, 0.35; turkey, 
0.40; beef cattle, 0.50; dairy cattle, 0.25 (growing) and 0.5 
(lactating); sheep, 0.1-0.8; horses, 0.10; swine, 0.14; rats 
and mice, 0.15; nonhuman primates, 2 (reviewed by 
McDowell, 2003). Lactating animals require more dietary 
iodine because approximately 10 percent or more of the io- 



dine intake may be excreted in milk, depending upon the rate 
of milk production (Miller et al., 1975). Iodine requirements 
may also be influenced by genetic differences, climate, and 
environment. The thyroid hormone secretion in certain ani- 
mals has been shown to be inversely related to environmen- 
tal temperature. Cattle, sheep, and goats show a significant 
decrease in thyroid hormone production during the summer 
(ARC, 1980). 

DIFFICULTIES IN METHODS OF ANALYSIS AND 
EVALUATION 

Iodine is found in the organic forms in plants and animals 
and in the inorganic form in natural water samples. Methods 
for the identification and quantitative determination of io- 
dine depend upon the type of compound and the matrices in 
which it occurs. Iodine normally exists in nature as iodide 
(L); however, other common forms include iodate (lO^) and 
molecular iodine (Ij). For analysis of iodine in most biologi- 
cal samples, a dissolution step (wet or dry ashing) is required 
prior to analysis in order to convert and isolate it to a chemi- 
cal or physical form suitable for detection. Spectrophotom- 
etry was initially used for the determination of iodine based 
on the iodized catalyzed reaction of cerium (IV) by arsenic 
(III) in food and feeds. 

Several analytical methods — including chromatography 
(ion and gas), spectrometry, and neutron activation analy- 
sis — are used to determine iodine in feeds and foods. The 
ionic forms of iodine, iodide or iodate, can be determined 
using ion chromatography where detection limits with con- 
ductivity detection generally range between 0.1-lmg/L. 
Other analytical techniques, particularly those based on ra- 
dioiodine measurements, require highly specialized equip- 
ment and multi-step sample preparation techniques, which 
are prone to iodine loss or contamination. Due to the high 
selectivity and sensitivity obtained by ICP-MS, it is com- 
monly used for the quantitative determination of iodine in 
biological tissues (Larsen and Ludwigsen, 1997; Julshamn 
etal., 2001). 

Some iodine species, such as hydrogen iodide, are volatile 
at room temperature and therefore must be stabilized in sample 
solutions at alkaline pH prior to ICP-MS determination. Vola- 
tilization of iodine may lead to memory and adhesion effects 
in ICP instruments, which may cause problems in sample in- 
troduction systems. Several methods such as digestion using 
perchloric acid in combination with nitric acid have been used 
successfully to digest samples in polytetrafluoroethylene-lined 
steel bombs. The acid mixture oxidizes potentially volatile 
iodine species to nonvolatile species such as iodate. Other 
analytical approaches using ICP-MS are based on alkali ex- 
traction combining potassium hydroxide and tetramethylam- 
monium hydroxide (TMAH) to determine iodine in milk or 
serum by flow injection ICP-MS or use TMAH as a strong 
alkali to measure iodine in diets supplemented with the ele- 
ment. The microwave digestion system using 0.5 percent 



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184 



MINERAL TOLERANCE OF ANIMALS 



ammonium solution lias also been successfully used for rapid 
and sensitive determination of iodine (Baumann, 1990; 
Vanhoe et al., 1993). Volatile and semi-volatile forms of or- 
ganic iodine may be determined by gas chromatography and 
gas chromatography-mass spectrometry. 

REGULATION AND METABOLISM 
Absorption and Metabolism 

Ingested inorganic iodine and iodate are reduced to iodide 
and absorbed almost completely from the gastrointestinal tract 
(Hetzel and Maberly, 1986). In the ruminant, the rumen is the 
major site of absorption of iodine and the abomasum is the 
major site of endogenous secretion (Barua et al., 1964). After 
absorption, the iodide is rapidly distributed throughout the 
body. The major sites of iodine concentration are the thyroid 
and the kidney. In addition, iodine is concentrated by the sali- 
vary glands, mammary glands, gastric mucosa, placenta, 
ovary, skin, and hair (Gross, 1962). In thyroid follicular cells, 
iodine is transformed through a series of metabolic steps into 
the thyroid hormones, T4 and T3. These two hormones ex- 
press almost all of the biological effects of iodide. Other forms 
of iodine in the thyroid include inorganic iodine, 3- 
monoiodotyrosine (MIT), 3, 5 -diiodotyrosine (DIT), polypep- 
tides containing thyroxine, and thyroglobulin. 

Iodide trapped by the thyroid is oxidized by thyroid peroxi- 
dase to active iodine, which iodinates the tyrosyl residues in 
thyroglobulin. This large glycoprotein (MW 660,000), the 
storage form of thyroid hormones, serves as the substrate for 
iodination and coupling of peptide-linked iodotyrosines to T^ 
and T3. This coupling reaction is also catalyzed by thyroid 
peroxidase and results in conversion of an alanine side chain 
to dehydroalanine. Thyroglobulin consists of two polypeptide 
subunits that undergo post-translational glycosylation and io- 
dination prior to secretion into the lumen of the thyroid fol- 
licle. The iodoprotein is reabsorbed into the thyroid cells and 
hydrolyzed to release free thyroid hormones for secretion into 
the circulation sytem and distribution to the peripheral tissues. 
This process also occurs in lactating mammary glands and, to 
a limited extent, in the ovum within the ovary. In other sites, 
the element remains in the form of iodide. 

Thyroglobulin in the colloid contains the greatest part of 
iodine in a normal thyroid. Approximately 70 percent of the 
dry weight of a thyroid gland is thyroglobulin. One mole of 
thyroglobulin containing 1 percent iodine consists of 10-12 
residues each of MIT and DIT, 3-4 residues of T4, and less 
than one residue of T3. The percent iodine varies and is a 
function of iodine in the diet. The colloid can store a sufficient 
amount of thyroid hormones to supply the body needs of hu- 
mans for several months (Taurog, 2000); however, the storage 
time may vary among different animals. The iodide pool is 
replenished continuously, exogenously from the diet and en- 
dogenously from the saliva, gastric juice, and breakdown of 
thyroid hormones and iodothyronines by deiodination. The 



pool is in a dynamic equilibrium with the thyroid gland and 
kidneys. Approximately 80-90 percent of iodine intake is ex- 
creted via the kidneys in the urine (Vought and London, 1967; 
Nath et al., 1992); other routes include saliva, bile, sweat, and 
feces. In lactating animals, milk is also a major route of iodine 
excretion. Undigested organic iodine is excreted in feces. 

Thyroid honnones are primarily transported in blood mainly 
(>99.7 percent) loosely bound to plasma proteins (thyroxine 
binding protein, transthyretin, albumin, and lipoproteins). The 
regulation of thyroid metabolism is a complex process, which 
involves the thyroid, anterior pituitary, hypothalmus, and pe- 
ripheral tissues (Hetzel and Welby, 1997). Thyroid-stimulating 
hormones (TSH) from the pituitary gland stimulate thyroid 
metabolism via cyclic AMP and the synthesis of thyroid hor- 
mones. The TSH released from the pituitary is inhibited through 
a feedback control by the thyroid hormones, and its release is 
stimulated by thyrotropin-releasing hormones (TRH), a tripep- 
tide (pyroglutamyl histidyl-prolinamide). Synthesis of TRH 
occurs in the hypothalamus, and they are transported to the pi- 
tuitary through blood vessels. The secretion of TRH from the 
hypothalamus is in turn inhibited by T3. These interacting fac- 
tors of the thyroid-pituitary-hypothalamus axis maintain ho- 
meostasis with regard to thyroid hormones. Under normal con- 
ditions most of the thyroid hormones released from the thyroid 
gland are in the form of T^ and only a small amount as T3. Three 
selenium-dependent enzymes, deiodinase (Type I, II, and III) 
convert T4 into T3, the biological active form of thyroid hor- 
mone (Arthur, 1999). Thiouracil only inhibits Type I deiodinase 
(IDl). A major difference exists between ruminants and 
nonruminants with respect to ID 1 ; this enzyme is particularly 
important in brown tissue metabolism of newborn ruminants 
and serves as source of T3 for other tissues (Nicol et al., 1994). 
Free MIT and DIT are deiodinated by tissue deiodinases. Nor- 
mal concentrations of thyroid hormones are necessary to main- 
tain a wide range of essential physiological processes including 
metabolic rate, protein and enzyme synthesis, thermoregula- 
tion, and growth and development of most vital organs. 

Fish and other aquatic organisms obtain iodine primarily 
from gills and, to a lesser extent, from food sources. Rain- 
bow trout derive 80 percent of their iodine from water, 19 
percent from diet, and less than 1 percent from recycling 
iodine originating from thyroid hormone degradation (Hunt 
and Bales, 1979). When dietary uptake is low or absent, fish 
are able to maintain their plasma iodine levels by uptake of 
iodide from water and mobilization of this element from tis- 
sue stores. Major differences exist between fish and mam- 
mals in the handling of iodine and extrathyroidal metabo- 
hsm of T4 and T3 (Higgs et al., 1982). 

IVIetabolic Interactions and IVIechanism of Toxicity 

The cellular functions of thyroid hormone are mediated 
through triiodothyronine in a complex molecular mechanism 
involving steroid and thyroid hormones and nuclear recep- 
tors (Hetzel and Welby, 1997). Retinoic acid (vitamin A) 



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IODINE 



185 



stimulates, synergistically with triiodothyronine, the produc- 
tion of growth hormones in cultured cells. Goitrogenic sub- 
stances in a wide range of vegetables and plant products are 
capable of producing thyroid hyperplasia by interfering with 
thyroid hormone synthesis. The pituitary responds by in- 
creasing TSH output, thus causing thyroid gland hypertro- 
phy in an effort to increase thyroid hormone production. In- 
organic elements in diets that interfere with iodine 
metabolism include: (1) high intake of fluorine, arsenic, and 
calcium, (2) cobalt deficiency or excess, and (3) low manga- 
nese intake (Underwood and Suttle, 1999). Higher amounts 
of dietary potassium increase urinary loss of iodine in cattle 
(Dennis and Hemken, 1978). Synthetic flavonoids have been 
shown to interfere with iodine metabolism in a rat fetus by 
crossing the placenta and reducing the availability of T^. 

Iodine toxicosis in animals and humans markedly reduces 
thyroidal iodine uptake, and the response is dependent on 
dose and duration of iodine intake (Hetzel and Welby, 1997). 
Excess iodine may result in either hypothyroidism, with or 
without goiter, or hyperthyroidism (thyrotoxicosis), but the 
mechanisms involved in these responses are not completely 
understood. Numerous studies conducted on the mechanisms 
of iodine toxicity show the following direct effects on thy- 
roid glands: (1) inhibition of iodide transport and uptake by 
the thyroid; (2) accumulation of iodotyrosines; (3) inflam- 
mation and degeneration of follicular cells; and (4) damage 
to follicular cells DNA. Indirect effects of iodine toxicosis 
include: (1) changes in thyroid hormones transport in 
plasma; (2) interference in TSH or transthyrectin metabo- 
lism; (3) higher hepatic microsomal enzyme activities caus- 
ing an increase in iodotyrosine excretion; and (4) poor ab- 
sorption of thyroid hormones and their excretion in feces. 

The adverse effects of iodine toxicosis may not be related 
exclusively to the source of the iodine. Large doses of iodine 
temporarily inhibit either organic iodine synthesis (Wolff- 
Chaikoff effect), possibly by binding the active form of io- 
dine as I3", or inhibit thyroglobulin proteolysis with reduc- 
tion in hormone secretion. Both effects may contribute to the 
stimulation of release of TSH from the pituitary gland and to 
an increase in serum concentration of TSH and hypertrophy 
of the thyroid gland. However, the thyroid gland adapts to 
excess iodine by decreasing its transport into the cell. The 
mechanisms for the Wolff-Chaikoff effect involve both io- 
dide transport and iodination reactions, possibly through an 
inhibition of the expression of sodium-iodine symport (NIS) 
and thyroid peroxidase. This reaction is mediated by iodide 
or an iodinated metabolic intermediate (Eng et al., 1999). 
Iodine may escape when iodide transport into the thyroid 
gland is either depressed to release it from inhibition of thy- 
roid peroxidase or by other biochemical processes in the pro- 
duction of iodothyronines (Sailer et al., 1998). Several 
chemical inhibitors of iodine thyroid metabolism have been 
identified (ATSDR, 2004). 

The mechanism by which the iodide suppresses iodina- 
tion and thyroid hormone release appears to involve inhibi- 



tion of adenyl cylase. The stimulatory actions of TSH on the 
thyroid gland, which include increased iodide transport and 
increased iodination of thyroglobulin production and release 
of T4 and T3, occur in response to an increase in intracellular 
cAMP levels. Iodide inhibits adenylate cyclase in thyroid 
follicle cells and decreases the TSH-induced rise in intracel- 
lular cAMP. However, inhibitors of the iodination reaction, 
such as prophylthiouracil, can prevent the effects of iodide 
on adenylate cyclase. This indicates that the active inhibitor 
may be an endogenous iodinated species, which is produced 
in the reaction involving thyroid peroxidase. 

Excess iodide intake may also be a contributing factor in 
the development of lymphocytic thyroiditis in rats and mice 
and autoimmune thyroiditis in humans. Hyperthyroidism has 
been observed after iodine supplementation of iodine-defi- 
cient populations. Chronic iodine deficiency results in thy- 
roid gland proliferation and the development of autonomous 
nodules that do not respond to serum TSH levels. Under 
these conditions, excess amounts of iodine cause increased 
levels and nonregulated thyroid hormone production 
(Corvilain et al., 1998; Roti and Uberti, 2001). 

SOURCES AND BIOAVAILABILITY 

Iodine is distributed widely in nature in both organic and 
inorganic forms in very small amounts. Iodine is present in 
soil, air, and water and is a constituent of plants and animals 
used for food. It exists in several chemical forms such as mo- 
lecular iodine, iodide, iodate, and periodate. It is also subject 
to oxidation-reduction reactions to yield other forms, as well 
as microbial alkyation (e.g., methyl iodide). The highest con- 
centration of iodine (0.5 to 2 g/kg) is found in nitrate deposits 
(saltpetre) of Chile, where it was brought from Antarctic anti- 
cyclonic airflows from the Pacific Ocean. Several marine al- 
gae (brown, red, and green) cultured in Southeast Asia contain 
high concentrations of iodine (up to 3 g I/kg). The iodine con- 
tent of water reflects the iodine content of the rocks and soils 
of the region. Iodine in water is in the form of iodide and 
iodate. The iodine content in seawater averages 40- 60 |Jg/L 
and unpolluted surface water contains 2- 4 |Jg/L (NRC, 1974). 
In certain areas, where water is polluted with municipal waste- 
water or urban run-off, the concentration may be as high as 
8.7 |jg I/L (FDA, 1974; WHO, 1988). In Denmark, iodine 
concentrations ranging from 2 to 139 |Jg/L have been reported 
in drinking water (Pedersen et al., 1999; Rasmussen et al., 
2000). In surface water, the proportion of iodide to iodate de- 
pends on the microbial activity and release of iodine from 
terrestrial sources (De Luca Rebello et al., 1990). The iodine 
content of river water may range from 0.1 to 18 |Jg/L (NRC, 
1979), with a relative proportion of iodide to iodate of about 
55:45. The average atmospheric iodine concentration ranges 
between 2-52 ng/m^ (NRC, 1979; WHO, 1988), with gaseous 
iodine exceeding particulate iodine by a factor of 2-6 (White- 
head, 1984). The source of atmospheric iodine is mainly from 
the vaporization of seawater; in this process, iodide is 



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oxidized by solar liglit. Ttie concentration of atmosphieric io- 
dine is much higher over the ocean. Air pollution, particularly 
the combustion of gasoline and oil, increases the iodine con- 
centration in air. 

The concentration of iodine in common feedstuffs is 
highly variable. Protein sources of animal origin other than 
fishery by-products contain relatively high amounts of this 
element. Oilseed proteins or their concentrates contain 100 
to 300 |Jg/kg and common cereal grains 40 to 100 |Jg/kg. 
Concentrations of iodine in forage depend on the iodine con- 
tent of soil. Many soils of the world are low in iodine and 
goiter regions have been identified in many areas of all con- 
tinents. During the glaciation period, iodine was swept away 
and replaced by crystalline soil that lacks humus to retain 
sufficient amounts of iodine. Heavy rain and flooding may 
also leach iodine in certain areas. Soils near the oceans pro- 
vide iodine to plants. Iodine concentrations in forages from 
the Great Lakes region and the Northwest United States are 
generally low enough to cause iodine deficiency in cattle 
unless iodine is supplemented to diets. 

Iodine sources permitted as feed additives include cal- 
cium iodate, sodium iodate, potassium iodate, potassium 
iodide, sodium iodide, EDDI, calcium iodobehenate, 
cuprous iodide, 3,5-diiodosalicylic acid, pentacalcium 
orthoperiodate, and thymol iodide (AAFCO, 2004). Iodine 
occurs in foods largely as inorganic iodide. In this form, it is 
almost completely absorbed from the gastrointestinal tract 
(Underwood, 1977). The reported bioavailability of potas- 
sium iodide, sodium iodide, EDDI, and pentacalcium 
orthoperiodate for poultry, cattle, and rats is about 100 per- 
cent, and for calcium iodide between 90-95 percent 
(Ammerman and Miller, 1972; Miller and Ammerman, 
1990). Diiodosalicylic acid is a stable compound and well 
absorbed (80 percent) by rats, but it is not well utilized (~15 
percent) by ruminants. Pentacalcium orthoperiodate and 
EDDI are also more stable and less soluble and are added to 
mineral blocks for licking by cattle to supply dietary iodine. 
Limited information is available on the bioavailability of 
iodine from plant material and animal by-products (Miller 
and Ammerman, 1990). 

Potassium iodate used in salt iodization and as a maturing 
agent and dough conditioner in bread may appear in animal 
and human food sources. Some non-food sources also sup- 
ply iodine (e.g., iodine-containing drugs, antiseptics and dis- 
infectants, and iodized oil used for intramuscular injections), 
lodophors used as antiseptic agents for udder washes and 
milking machines — as well as disinfection of tanks used for 
handling, storing, and transport of milk — can increase the 
iodine content of dairy products. 

TOXICOSIS 

Iodine toxicosis can result from a single large dose of 
iodine or from repeated exposure to iodine concentrations 
that exceed animal requirements. Several studies evaluating 



oral ingestion of high concentrations of iodine are summa- 
rized in Table 15-1. Several reviews are available on iodine 
toxicity in mammals (NRC, 1980; SCF, 2002; McDowell, 
2003; ATSDR, 2004); however, reports on iodine toxicity to 
fish from diet and the aquatic environment are sparse. More 
than a century ago, in practically all of the areas of Europe 
affected by goiter, there was a wave of enthusiasm for the 
use of some form of inorganic iodine to prevent and treat 
goiter. It appears that indiscriminate use of various iodine 
preparations was practiced with many cases of toxicosis. The 
adverse effects of iodine in humans and animals have re- 
sulted from the consumption of food supplements (e.g., io- 
dized salt, bread or water), use of iodine-containing pharma- 
ceuticals (e.g., oral, intramuscular injection, applications to 
skin and mucus membrane), iodine in water supplies and 
consumption of seaweed or animal meat containing thyroid 
tissues. Iodine toxicity has been studied in many laboratory 
animals, dogs, poultry, pigs, cattle, and goats. Significant 
species differences exist in the tolerance to high levels of 
iodine because of the differences in basal metabolic rate and 
iodine metabolism. All species appear to have a wide margin 
of safety for this element. 

Single Dose 

The reported oral LD^q in rats fed Nal was 3,320 mg I /kg 
BW and the oral LDjqq for mice fed KI was 1,425 mg I /kg 
BW (Clayton and Clayton, 1981). The oral LDj^ of iodate 
and iodide in mice were 483-698 and 1,550-1,580 mg/kg 
BW, respectively (Webster et al., 1957). Webster et al. 
(1966) determined the minimum lethal dose and the maxi- 
mum allowable dose of potassium iodate for dogs. Oral ad- 
ministration of three single doses of potassium iodate (100, 
200, or 250 mg/I kg BW) to dogs caused anorexia and occa- 
sional vomiting at the low level; however, the higher levels 
(200 and 250 mg/kg) caused death preceded by anorexia, 
prostration, and coma (Webster et al., 1966). Severe retinal 
degenerative changes have been reported in laboratory ani- 
mals intravenously administered sodium iodate above 10 mg/ 
kg (Biirgi et al., 2001), but no such retinal changes were 
observed in guinea pigs given potassium iodate in the drink- 
ing water (Highman et al., 1955). 

Acute and Chronic 

The prolonged administration of large doses of iodine 
markedly reduces iodine uptake by the thyroid, thus causing 
antithyroidal or goitrogenic effects in many domestic and 
experimental animals (Radostits et al., 2000). High levels of 
iodide inhibit organic-iodine formation and saturate the ac- 
tive-transport mechanism of this ion causing iodide goiter. 
Significant species differences exist in tolerance to high di- 
etary iodine intakes. In comparison to other trace element 
toxicities, the dietary level of iodine necessary to cause toxi- 
cosis is high relative to the minimal requirement. For ex- 



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IODINE 



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ample, the margin of safety is 1 ,000 times ttie minimal re- 
quirement of chickens (NRC, 1994) and pigs (Newton and 
Clawson, 1974; NRC, 1998). A significant amount of data is 
available on iodine toxicity in humans (Hetzel and Welby, 
1997; ATSDR, 2004); however, limited research has been 
conducted on acute and chronic toxicosis in domestic and 
laboratory animals since the NRC last report (NRC, 1980). 
Iodine toxicity data on laboratory animals is considered of 
limited use for humans (ATSDR, 2004) because of the higher 
sensitivity of this element to animals and some differences 
in their dietary requirements and energy metabolism. 

Although early work on the minimum toxic iodine intake 
(as calcium iodate) for calves (80-112 kg) was close to 50 
mg/kg diet, some animals showed adverse effects at lower 
levels (Newton and Clawson, 1974; Newton et al., 1974). 
Elevated levels of dietary iodine depressed growth rate and 
feed intake, with the depression being significant for diets 
containing 50, 100, or 200 mg/kg added iodine. The feeding 
of either 100 or 200 mg I/kg, and in some cases lower levels, 
produced toxic signs that included coughing and nasal dis- 
charge. All levels of added iodine increased serum iodine, 
and calves fed 200 mg/kg had significantly lower hemoglo- 
bin and serum calcium levels. Calves fed diets with added 
iodine had heavier adrenal glands, but there was no consis- 
tent effect on the weight of the thyroid glands. Based on 
trends in growth rate and adrenal weights, Newton et al. 
(1974) concluded that 50 mg/kg diet appeared to be the mini- 
mum toxic level for calves. Another study showed that calves 
(100 kg) tolerated 20 and 40 mg I/kg from EDDI, but daily 
gains were slightly depressed at 86 and 174 mg/kg diet (Fish 
and Swanson, 1977). Iodine levels of 71, 140, and 283 mg/kg 
had no effect, but a level of 435 mg/kg diet depressed daily 
gains in yearling (320 kg) heifers (Fish and Swanson, 1977). 
Mild and severe signs of iodine toxicosis were observed 
in cattle fed 2.2 and 20 mg I/animal for 6 months 
(Mangkoewidjojo et al., 1980). 

Iodine toxicosis has been reported in adult dairy cows 
with dietary intakes of 50 mg/day (NRC, 1980, 2001). High 
concentrations of iodine in the diet increase iodine concen- 
tration in milk. Humans are more susceptible to iodine thy- 
ro toxicity than cows; therefore, the excess iodine in the dairy 
cow must be limited due to public health concerns (Hetzel 
and Welby, 1997). Feeding 170 mg I/day to high-yielding 
cows for 30 days has resulted in abortions within a 68-day 
period following treatment (Morrow and Edwards, 1981). 
Other clinical signs of iodine toxicosis in dairy cows and 
cattle include excessive nasal and ocular discharge, cough- 
ing, nervousness, tachycardia, decrease in appetite, dermati- 
tis and alopecia, exopthalmos, weight loss, decreased milk 
production, susceptibility to infectious and respiratory dis- 
eases, and increased mortality of dams (Olson et al., 1984; 
Radostits et al., 2000). 

Sheep were able to tolerate daily diets of 102 mg I/kg (as 
KI) and 214 mg I/kg (as EDDI) for 22 days without any 
adverse effects on growth and feed intake (McCauley et al.. 



1973). Body weight gains were depressed by daily intakes of 
393 mg potassium iodide (300 mg iodine) or 562 mg EDDI 
(450 mg iodine) per lamb per day. Other signs of iodine toxi- 
cosis in sheep include cough, anorexia, hyperthermia, respi- 
ratory diseases, and sometimes death (McCauley et al., 1973; 
Paulikova et al., 2002). 

A high incidence of goiter and leg weakness was reported 
in foals born to mares fed 48-432 mg I/day (Baker and 
Lindsey, 1968; Drew et al., 1975), although an upper tolerable 
limit for horses has not been established. Excess iodine caused 
osteoporosis and increase in serum phosphate and alkaline 
phophatase levels in thoroughbred horses (Silva et al., 1987). 

Pigs and chickens are more tolerant of excess iodine than 
are cattle. Newton and Clawson (1974) fed levels of iodine 
ranging from 10 to 1,600 mg/kg diet to growing-finishing 
pigs and found that the minimum toxic level was between 
400 and 800 mg/kg. Growth rate, feed intake, and hemoglo- 
bin levels were depressed at 800 and 1,600 mg I/kg, and 
liver iron levels were significantly depressed at 400 mg/kg 
diet. A decrease in serum Tj concentrations was observed in 
finishing pigs fed 10 mg I/kg diet (Schone and Leiterer, 
1999). During lactation and the last 30 days of gestation, 
neither 1,500 nor 2,500 mg I/kg diet affected reproduction in 
sows (Arrington et al., 1965). 

Early work on iodine toxicity in chickens showed that 
their performance was not affected by feeding 500 mg I/kg 
in the form of potassium iodide for up to 6 weeks (Wilgus et 
al., 1953). More recent studies on oral iodine toxicity of 
potassium iodide in chickens fed >900 mg I/kg showed 
depressed growth and neurological clinical signs (Baker et 
al., 2003); however, severe growth depression in chickens 
fed 1,000-1,500 mg I/kg was totally reversed by dietary 
supplementation of 50-100 mg Br/kg as sodium bromide. In 
chicken and turkey laying hens, typical iodine toxicosis signs 
observed with high intake of dietary iodine include decreased 
egg production, egg size, and hatchability; low fertility; 
enlarged thyroids in hatching chicks; and wiry down 
(Perdomo et al., 1966; Arrington et al., 1967; Marcilese et 
al., 1968; Christensen et al., 1991; Christensen and Ort, 
1991). Turkey breeder hens are more sensitive to high dietary 
iodine than chickens (Christensen and Ort, 1991). When lay- 
ing chickens were fed 625 to 5,000 mg I/kg, egg production 
varied inversely with the iodine level and ceased with dietary 
levels of 5,000 mg/kg (Arrington et al., 1967). Early 
embryonic death, reduced hatchability, and delayed hatch- 
ing were also observed; however, egg production com- 
menced within 1 week after cessation of feeding iodine. Diets 
containing 5,000 mg I/kg caused an increase in serum 
calcium level, as well as a marked reduction in egg produc- 
tion and in the size of ovaries and oviducts of chickens 
(Roland et al., 1977). 

Marked differences exist between rabbits, hamsters, 
and rats in their tolerance to high intakes of iodine. Mor- 
tality was high in the offspring of rabbits fed 250 mg I/kg 
in late gestation. However, the feeding of diets containing 



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MINERAL TOLERANCE OF ANIMALS 



2,500 mg/kg iodine to liamsters during gestation did not 
affect deatii loss in ttie offspring (Arrington et al., 1965). 
The survival of the offspring of rats was not affected by 
feeding gestating female rats 500 mg I/kg, but high mor- 
tality of the young was found when the gestation diets 
contained 1,000 mg I/kg (Ammerman et al., 1964). No 
gross lesions or abnormalities in mice or guinea pigs that 
received 5,000 mg/L of potassium iodate in their drinking 
water for several weeks were observed (Webster et al., 
1959). However, mice showed hemosiderin deposits in 
the renal convoluted tubules. 

High concentrations of dietary iodine (up to 14 mg/kg of 
diet) in commercial and experimental feed have been shown 
to cause a transient decrease in serum thyroid hormone lev- 
els in cats (Tarttelin et al., 1992; Kyle et al., 1994; TartteUn 
and Ford, 1994). Excessive dietary iodine intake may be 
linked to feline hyperthyroidism; however, epidemiologic 
studies have not identified a clear relationship between di- 
etary iodine and adenomas or adenocarcinomas of the thy- 
roid gland in cat (Scarlett, 1994). The minimum lethal dose 
for potassium iodate administered orally to dogs was esti- 
mated to be 200 to 250 mg/kg (Webster et al., 1966). In 
puppies, high levels of iodine (5.6 mg/kg of diet) caused a 
decrease in uptake of radioiodine by the thyroid gland 
(Castillo et al., 2001a), and an excessive amount of iodine 
present in some commercial feed caused impairment of thy- 
roid function and hypothyroidism (Castillo et al., 2001b). 

In humans, an iodine intake of 2 mg/day is considered 
harmful (Hetzel and Welby, 1997). Oral iodine toxicosis has 
occurred in certain areas of China and Japan in populations 
exposed to chronic high intakes (50-80 mg/day) of iodine 
(Wolff, 1969; NRC, 2001). Inhabitants of coastal regions 
that consume large amounts of seaweed developed goiter 
from high iodine intake (Hetzel and Welby, 1997). The re- 
lease of at least 15 different radioactive isotopes of iodine 
occurs as gases when uranium or plutonium atoms are split 
resulting in typical signs of iodine toxicosis in animals and 
humans (ATSDR, 2004). In 1986, the Chernobyl nuclear 
accident in the Soviet Union caused excess iodine levels in 
milk from dairy cows grazing on contaminated pastureland. 
Children drinking contaminated milk showed iodine toxicity 
(Kazakov et al., 1992; Nauman and Wolff, 1993). 

Oral toxicity of iodine in fish has not been studied, but 
high amount of iodine in water is toxic. An exposure of 8 mg 
I/L was lethal to mullet (Gozlan, 1968). Acute toxicity of 
iodine to channel fish varied with the exposure period 
(Le Valley, 1982). Concentrations as low as 0.72 mg I/L re- 
sulted in 100 percent mortality during a 24-hour exposure 
period and 7.2 mg I/L within an hour. Mortality was caused 
due to gill damage and asphyxiation. 

Factors Influencing Toxicity 

Natural or synthetic chemicals distributed in plants and 
plant products used for animal feeds or for forage possess 



goitrogenic properties and they affect iodine bioavailability 
and metabolism (Talbot et al., 1976). Although the effects of 
goitrogens on iodine toxicity are not clearly defined, changes 
in thyroid metabolism associated with high intake of these 
compounds may influence iodine toxicity as well as concen- 
tration of iodine in tissue and animal products. There are two 
types of goitrogens: thiocyanates and goitrin (L-5-vinyl-2- 
thiooxazolidone). Among the naturally occurring goitrogens, 
the best characterized are the glucosinolate derivatives iso- 
lated from the Brassica species. Rapeseed, kale, cabbage, and 
turnip contain high concentrations of goitrogens. Thiocyan- 
ates inhibit the uptake of iodine in the thyroid, but their action 
is reversible by iodine supplementation. However, goitrin in- 
hibits thyroid hormone synthesis, probably through inhibition 
of thyroid peroxidase, and this effect is irreversible by iodine 
supplements (Talbot et al., 1976). Iodine supplementation al- 
leviates the hypothyroidism and the adverse effects on perfor- 
mance caused by feeding rapeseed meal to swine, and the ef- 
ficacy of iodine can be improved by dietary copper 
supplementation (Liidke and Schone, 1988; Schone et al., 
1988). Glucosinolates are inactivated by treatment with cop- 
per sulphate solution. Moreover, extrusion of rapeseed with 
barley eliminates its hypothyroidism effects (Maskell et al., 
1988). Other plant products such as soybean, cottonseed, lin- 
seed meals, lentils, and peanuts also contain goitrogenic sub- 
stances (Matovinovic, 1983; Fenwick et al., 2000). 

Thiocyanates, perchlorates, and rubidium salts are known 
to interfere with iodine uptake by the thyroid, and high lev- 
els of arsenic can induce goiter in rats (Underwood, 1977). 
Bromide, fluoride, cobalt, manganese, and nitrate may also 
inhibit normal iodine uptake (Talbot et al., 1976). Excess 
calcium intake has been shown to have an antithyroid effect 
(Taylor, 1954). Adequate amounts of both iodine and sele- 
nium are necessary for optimum thyroid metabolism (Hotz 
et al., 1997). Selenium deficiency can influence iodine 
bioavailability. High iodine intake when selenium is defi- 
cient in diets of rats causes thyroid tissue damage as a result 
of low thyroidal GSH-Px activity during thyroid stimulation 
(Hotz et al., 1997). 

However, a moderately low selenium intake normalized 
the circulating T4 concentration in the presence of iodine 
deficiency. Protein-calorie malnutrition may reduce iodine 
absorption and thyroid iodine clearance and radiodide up- 
take (Ingenbleek and Beckers, 1973, 1978). Excess dietary 
iodine intake has been also shown to inhibit thyroid activity, 
presumably by blocking the uptake of iodine by the thyroid 
(Baker and Lindsey, 1968; Nagasaki, 1974; Newton and 
Clawson, 1974; Fish and Swanson, 1983; Kaneko, 1989). 
Certain food coloring agents (e.g., erythrosine), pharmaceu- 
tical products, water purification tablets, and disinfectants 
contain high amounts of iodine and are likely to increase 
total intake of this element by the animal. 

Few studies have been conducted to compare the relative 
toxicity of various iodine compounds. No differences in toxic- 
ity of sodium and potassium iodide to rats have been reported 



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(Arrington et al., 1965). lodate salts were more toxic than io- 
dide salts in mice when administered orally, intraperitoneally, 
or intravenously (Webster et al., 1957). Although absorption of 
EDDI and sodium or potassium iodide does not differ widely in 
dairy cows, EDDI was retained in most tissues longer than the 
iodide salts (Miller and Swanson, 1973). The presence of food 
in the stomach greatly decreased the acute toxic effects of orally 
administered iodine to mice and guinea pigs, and small or inter- 
mittent doses of potassium iodate in drinking water were better 
tolerated by these animals than a single dose given by stomach 
tube (Webster et al., 1959). 



contain 4-10 |ig I/kg, which is located mainly in the yolk. 
Feeding large amounts of iodine supplements and seaweed 
to laying hens increases the amount in eggs by 100-fold or 
more (Underwood and Suttle, 1999). When high con- 
centrations of iodine (100-150 |Jg/day) were fed to laying 
hens, the iodine concentration reached 50-120 |Jg/kg 
(Marcilese et al., 1968). Iodine concentrations in fish muscle 
vary widely (30 to 3,500 pg/kg) between species as well as 
within species, and the concentration is generally higher in 
marine fish (Varo et al., 1982; Lall, 1994; Julshamn et al., 
2001; Karl etal., 2001). 



TISSUE LEVELS 

Iodine concentration in animal products, particularly milk 
and eggs, is directly influenced by iodine intake from feed 
and water. In mammals, approximately 80 percent of total 
iodine is distributed in the thyroid gland, and the remaining 
amount is found in other soft tissues including muscle, liver, 
kidney, and heart (Downer et al., 1981; Kaufmann and 
Rambeck, 1998). The highest iodine concentrations are in 
fish, shellfish, marine algae, and seaweed. Representative 
iodine concentrations in tissues from various animals fed 
normal or high levels of iodine are shown in Table 15-2. 

Although iodine is a natural constituent of milk, the con- 
centration is influenced by iodine intake, stage of lactation, 
season, level of milk production, and the use of 
iodine-containing disinfectants to maintain udder and dairy 
equipment. Average iodine concentrations in milk of cows 
fed 0, 40, 81, 162, 405, and 810 mg I/kg diet as EDDI was 8, 
362, 895, 1,559, 2,036, and 2,383 pg/L (Miller et al., 1975). 
The iodine content of milk varies seasonally, with higher 
concentrations present in winter (80- 930 |Jg/L) than in sum- 
mer (40-200 |Jg/L) (Varo et al., 1982; Dellavalle and 
Barbano, 1984; Pennington, 1990; Lee et al., 1994; Larsen et 
al., 1999; MAFF, 2000). The colostrum of dairy cows is 
much higher in iodine than milk, and in late lactation there is 
a decrease in concentration. A mean value of 264 ± 100 |Jg/L 
for colostrom compared to 98 ± 82 |Jg/L for milk was re- 
ported (Kirchgessner, 1959). Iodine content of milk is re- 
duced below normal in goiter regions (Feng et al., 1999). 
Dipping of teats in iodophor after milking increased the io- 
dine levels from 89 and 94 |Jg/L to 127 and 152 |ig/L (Funke 
et al., 1975). Average iodine concentration reported in sheep 
and goat milk were 105.5 and 63.0 |Jg/L, respectively; how- 
ever, the concentrations were much higher when these ani- 
mals had access to mineral licks containing the element 
(Travnicek and Kursa, 2001). 

The early work of Fisher and Carr (1974) showed that the 
iodine content of beef, pork, and mutton was low (27 to 45 
|jg/kg). A wide range of iodine concentration ranging from 2 
to 580 |Jg/kg has been reported in animal meat products from 
Europe and the United States (Hemken et al., 1972; Hemken, 
1979; Varo et al., 1982; Wenlock et al., 1982; Dunn, 1993; 
Lee et al., 1994; Jahreis et al., 2001). Generally, hen eggs 



MAXIMUM TOLERABLE LEVELS 

The tolerance levels of domestic animals are based on clini- 
cal signs of iodine toxicosis, and most animals tolerate high 
dietary iodine concentrations relative to iodine requirements. 
Iodine toxicity in domestic animals has not been properly 
quantified. Based on available literature, maximum tolerable 
levels (mg I/kg diet) suggested for iodine are cattle, 50; sheep, 
50; swine, 400; chicken and turkey, 300. These values are 
unchanged from those reported by NRC (1980) since recent 
data do not support a change in these recommendations. Al- 
though cattle can tolerate 50 mg/kg iodine, this level in the 
diet may result in undesirably high levels of iodine in the milk. 
Among the species studied, horses are most susceptible to io- 
dine toxicity. High levels of iodine, for example of kelp con- 
sumption by horses, cause goiter in the offspring of mares. 
Assuming that mares consume 10 kg air dry diet daily, the 
maximum tolerable level for iodine in horse diets is 5 mg/kg. 

Laboratory animal models have been useful to study the 
mechanism of iodine toxicity. However, the major 
toxicokinetics data on oral exposure have been obtained in 
experimental and clinical studies of humans using 
biomarkers (e.g., urinary excretion, serum TSH, thyroid scin- 
tillation scan, and antibodies) (ATSDR, 2004). Thus, unlike 
other minerals, most toxicological studies conducted with 
laboratory animals lack toxicokinetic data, and results do 
not distinguish specific effects of an overdose of iodine or 
iodate supplements (Biirgi et al., 2001). Mice showed 
marked toxic effects of iodate administered in water for 4 
weeks such as hemolysis and renal damages, which occurred 
from 300 mg I/kg BW upward with a no-observable-effect 
level (NOEL) at approximately 120 mg/kg BW. However, 
guinea pigs exposed via the same route tolerated 300 mg/kg 
BW without apparent effect. When given intravenously to 
rats, doses above 10 mg I/kg BW were highly toxic to retina 
(Biirgi etal., 2001). 

HUMAN HEALTH 

Iodine toxicity in humans is well documented and the 
subject of several comprehensive reviews by national, re- 
gional, and international organizations. Estimates of di- 
etary intake of iodine have ranged from <50 |Jg/day in 



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MINERAL TOLERANCE OF ANIMALS 



iodine-deficient regions to >10 mg/day in populations that 
regularly consume seaweeds high in iodine. The NRC's 
Recommended Dietary Allowance (RDA) for iodine is 
150 |ig/day (2.1 |jg/kg/day for a 70-kg adult), with addi- 
tional allowances for 70 and 140 pg/day during pregnancy 
and lactation, respectively (NRC, 2001). An upper toler- 
able level of iodine intake for an adult human is 1,100 |Jg/ 
day (NRC, 2001). A provisional maximum tolerable daily 
intake of 1 mg/day (equivalent to 17 |Jg/kg BW) from all 
sources has been recommended by WHO (1988). In coun- 
tries with long-standing IDD, the intake should not ex- 
ceed 500 |Jg I/day to prevent hyperthyroidism (AFSSA, 
2001). A minimal risk level (MRL) of 0.01 mg/kg/day 
has been derived for acute duration of oral exposure (1- 
14 days) to iodine, which is based on a NOAEL of 0.024 
mg/kg/day in healthy adult males (ATSDR, 2004). This 
NOAEL value derived from human adults may also be 
applicable to children and the elderly. Genotoxicity and 
carcinogenicity data for iodine are limited and are nonex- 
istent for iodate (Biirgi et al., 2001). 

FUTURE RESEARCH NEEDS 

The toxicity of iodine in most domestic and aquatic ani- 
mals should be better defined. There is a need to determine 
the mechanisms involved in iodine metabolism, particularly 
the interaction of iodine with other nutrients such as sele- 
nium, bromine, and iron. Although the toxicity of iodine for 
humans has been extensively studied, there is a need for more 
research to better establish the basis for beneficial or essen- 
tial action of iodine, and the intake below which this action 
is compromised. This would be helpful in setting the lower 
limits of toxicity standards for iodine and would allow better 
use of animal by-products in human nutrition. 

SUMMARY 

Iodine is an essential element for all animals. Its only 
known function in the body is in the synthesis of the thy- 
roid hormones. Sodium iodide, potassium iodide, and eth- 
ylenediamine dihydroiodide are well utilized as a source of 
iodine. Limited information is available on the 
bioavailability of this element from plant and animal by- 
products. Species differ widely in their susceptibility to 
iodine toxicity, but all animals can tolerate iodine levels far 
in excess of their requirements for this element. Feeding 
excessive levels of iodine has resulted in decreased egg 
production in hens, inhibition of lactation in rats, decreased 
hemoglobin levels in pigs, and goiter and reduced thyroid 
hormone synthesis in several species. Increasing the iodine 
intake of lactating cows and laying hens increases the lev- 
els of iodine in milk and eggs. 



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J. Pharmacol. Exp. Ther. 120:171-192. 

Webster, S. H., M. E. Rice, B. Highman, and E. F. Stohlman. 1959. The 
toxicology of potassium and sodium iodates. II. Subacute toxicity of 
potassium iodate in mice and guinea pigs. Toxicol. Appl. Pharmacol. 
1:87-96. 

Webster, S. H., E. F. Stohlman, and B. Highman. 1966. The toxicology of 
potassium and sodium iodates. III. Acute and subacute oral toxicology 
of potassium iodate in dogs. Toxicol. Appl. Pharmacol. 8:185-192. 

Wenlock, R. W., D. H. Buss, R. H. Moxon, and N. G. Bunton, 1982. Trace 
nutrient. 4. Iodine in British food. Br. J. Nutr. 47:381-390. 

Whitehead, D. C. 1984. The distribution and transformations of iodine in 
the environment. Environ. Int. 10:321-339. 

WHO (World Health Organization). 1988. Toxicological evaluation of cer- 
tain food additives and contaminants, FAS 24: 267—294. Geneva: World 
Health Organization. 

WHO. 1998. Iodine. Environmental Health Criteria 200. Geneva: World 
Health Organization. 

Wilgus, H. F., F. X. Gassner, A. P. Patton, and G. S. Harshfield. 1953. The 
iodine requirements of chickens. Ft. Collins: Colo. Agric. Exp. Stn. 
Tech. Bull. 49. 

Wolff, J. 1969. Iodide goiter and the pharmacological effects of excess 
iodide. Am. J. Med. 47(1):101-124. 



Copyright © National Academy of Sciences. All rights reserved. 



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194 



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ttp7/www nap edi]/ratalng/1 1 rina htmll 



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Copyright © National Academy of Sciences. All rights reserved. 



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inemi Tnlemnce nf Animals- Sennnd Revised Edition 



ttp7/www nap edi]/ratalng/1 1 rina htmll 



196 



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Copyright © National Academy of Sciences. All rights reserved. 



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ineral Tolerance nf Animals- Sennnd Revised Edition 



ttp7/www nap edi]/ratalog/1 1 rina htmll 







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Copyright © National Academy of Sciences. All rights reserved. 



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198 



MINERAL TOLERANCE OF ANIMALS 



TABLE 15-2 Iodine Concentrations in Fluids and Tissues of Animals 



Animal 



Quantity 



Source 



Duration 



Route 



Milk Egg Yolk 

(Mg/100 ml) (|jg/g) 



Reference 



Chickens 


Control (0.5 mg I/kg) 
+ 2.0 mg I/kg 
+ 4.4 mg I/kg 
+ 2.7 mg I/kg 
+ 7.2 mg I/kg 


Kelp 
Kelp 
Calj 
Cal^ 


45 d 


Diet 


0.18 
0.87 
2.00 
1.36 
0.93 


Rys'etaf, 1997 




Control 




5 d after 


Diet 


0.13 






+ 10 mg I/kg live wt 


KI 


single dose 


Diet 


12.86 






+ 10 mg I/kg live wt 


Iodized oil 




Diet 


14.04 






+ 10 mg I/kg live wt 


Iodized oil 




Intramuscular 
injection 


0.44 






Control 




21 d after 


Diet 


0.05 






+ 10 mg I/kg live wt 


KI 


single dose 


Diet 


0.09 






+ 10 mg I/kg live wt 


Iodized oil 




Diet 


0.01 






+ 1 mg I/kg live wt 


Iodized oil 




Intramuscular 
injection 


0.99 





Cows, dairy 



Control 
+ 10 mg I/kg 
+ 20 mg I/kg 
+ 30 mg I/kg 
+ 40 mg I/kg 



KI 



5 wk 



Diet 



1.8 

15.7 

23.6 

30.1 

36.2 



Feng et al., 1999 



Cows, dairy 


Control (5.8 mg I/cow/d) 


EDDI 


42 d 


Diet 


20.6 


Brzoska et at., 2000 


Spring 


+ 28.3 mg I/cow/d 
+ 43.3 mg I/cow/d 
+ 58.3 mg I/cow/d 










^^H 


Cows, dairy 


Control (5.8 mg I/cow/d) 








■ 


^^^^^^^^^H 


Summer 


+ 28.3 mg I/cow/d 
+ 43.3 mg I/cow/d 
+ 58.3 mg I/cow/d 


EDDI 


47 d 


Diet 




^^H 


Cows, dairy 


Control (5.8 mg I/cow/d) 








^H 


^^^^^^^^^H 


Autumn 


+ 28.3 mg I/cow/d 








^H 


^^^^^^^^^^^^^^1 




+ 43.3 mg I/cow/d 


EDDI 


32 d 


Diet 


^H 


^^^^^^^^^^^^^^1 




+ 58.3 mg I/cow/d 








239.2 


^^^ 



Cows, 


dairy 


Control 
+ 3.8 mg I/kg 
+ 3.8 mg I/kg 
+ 7.6 mg I/kg 
+ 7.6 mg I/kg 
+ 1 1.5 mg I/kg 
+ 1 1.5 mg I/kg 




KI 
EDDI 

KI 

EDDI 
KI 
EDDI 


Goats 




20.5-162.4 |jg/kg 
+ 3.05 ^ig I/kg 


DM 


Hay 

Hay + iodized 
salt 


Sheep 




20.5-162.4 |jg/kg 
+ 3.05 Mg/kg 


DM 


Hay 

Hay + mineral 
lick 



5d 



Diet 



Herzigetal., 2001 



Diet 



Diet 



20 ± 5.2 
50 ±15.2 
80 ± 3.0 
64 ±13.9 
173 ±39.3 
121 ±52.8 

19.3 
142.2 

105.5 
243.3 



Travnicek and Kursa, 
2001 



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16 



Iron 



INTRODUCTION 

The atomic symbol for iron is Fe, wliicti is derived from 
the halin ferrum. Iron (atomic number, 26; atomic weight, 
55.847) is a silvery, lustrous metal. There are 14 known iso- 
topes of iron. Common iron consists of a mixture of four 
isotopes. 

The melting point of iron is 1,535°C, boiling point is 
2,750°C, specific gravity is 7.874 (20°C), with a valence of 
2, 3, 4, or 6. Pure iron is chemically reactive and corrodes 
rapidly, especially in moist air or at elevated temperatures. 
Four allotropic forms, or ferrites, are known: a, (3, y, and 5, 
with transition points at 770, 928, and 1,530°C. The a form 
is magnetic, but when iron is transformed into the p form, 
the magnetism disappears, although the lattice remains un- 
changed. 

Iron is relatively abundant in the universe. It is found in 
the sun and many types of stars in considerable quantity. The 
core of the earth is thought to be largely composed of iron 
with about 10 percent occluded hydrogen. On a weight ba- 
sis, the metal makes up 5 percent of the Earth's crust and is 
the fourth most abundant element. Pure iron is rarely en- 
countered in commerce; it is usually alloyed with carbon or 
other metals. Pig iron is an alloy containing about 3 percent 
carbon with various amounts of sulfur, silicon, manganese, 
and phosphorus. Iron is hard, brittle, and fairly fusible and is 
used to produce other alloys, including steel. Wrought iron, 
which contains only a few tenths of a percentage of carbon, 
is tough, malleable, and less fusible. The pure metal is very 
reactive chemically and rapidly corrodes, especially in moist 
air or at elevated temperatures. 

Iron is one of the most useful metals in both technology 
and biology. Iron compounds are involved in numerous oxi- 
dation-reduction reactions, beginning with the reduction of 
hydrogen and its incorporation into carbohydrates during 
photosynthesis in the presence of ferredoxins (Fairbanks, 



1994). Iron is a vital constituent of plant and animal life and 
works as an oxygen carrier in hemoglobin. 

The ancient Greeks, Egyptians, and Hindus prescribed iron 
as a treatment for general weakness, diarrhea, and constipa- 
tion. The role of iron in blood formation became apparent in 
the 17th century when it was shown that iron salts were of 
value in treating chlorosis, now known as iron-deficiency ane- 
mia, in young women. The first clinical description of iron 
overload was reported in 1871 (Fairbanks, 1994). 

ESSENTIALITY 

Iron has been recognized as an essential nutrient for more 
than 100 years when it was found to be present in all body 
cells. The largest portion is found as a necessary component 
of the protein molecules hemoglobin and myoglobin. The 
major role of iron in both hemoglobin and myoglobin are 
similar. Hemoglobin is found in red blood cells (erythro- 
cytes) and transports oxygen from the lungs to the tissues, 
whereas myoglobin binds oxygen for immediate use by 
muscle cells (Morris, 1987). 

Iron is also found in plasma (transferrin), milk 
(lactoferrin), placenta (uteroferrin), and liver (ferritin and 
hemosiderin) (Underwood, 1977; Underwood and Suttle, 
1999). Species differences in total body iron concentrations 
occur in the newborn, but become much less pronounced in 
the adult. For example, the pig has relatively little iron in its 
body at birth (29 mg/kg) because it is born with low liver 
iron stores and has no polycythemia (venous hematocrit of 
greater than 65 percent) of the newborn as does the human 
infant. The newborn rabbit, by contrast, has an exceptionally 
high total body iron concentration (135 mg/kg) because of 
its high liver stores (Underwood, 1977). The iron concentra- 
tion of skeletal muscle (on a fresh tissue basis) in adult dogs, 
rabbits, hens, pigs, and cattle ranges from 12 to 16 mg/kg 
(Georgievskii et al., 1979). 



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Iron plays an important role not only in oxygen delivery 
to the tissues, but also as a cofactor with several enzymes 
involved in energy metabolism and thermoregulation. Mito- 
chondrial iron enzymes are essential for oxidative produc- 
tion of cellular energy. Aerobic metabolism depends on iron 
because of its role in the functional groups of most of the 
enzymes of the Krebs cycle, as an electron carrier in cyto- 
chromes, and as a means of oxygen and carbon dioxide trans- 
port in hemoglobin (Fairbanks, 1994). 

Iron deficiency is the most commonly known potential 
mineral deficiency in humans (Baynes, 1994). It is estimated 
that about half of the world's population suffers from iron 
deficiency. Iron deficiency is of limited practical significance 
in most livestock, but examples of situations in which animals 
are vulnerable to iron deficiency are newborn pigs, calves 
raised for veal, copper-supplemented pigs, and animals with 
parasitic infestations (Underwood and Suttle, 1999). Iron is 
required in growing, laying, and lactating animals at between 
50 and 100 mg/kg, depending on age and species. 

DIFFICULTIES IN METHODS OF ANALYSIS AND 
EVALUATION 

The most common methods currently used for the analy- 
sis of iron in biological samples are flame AAS, graphite 
furnace AAS, ICP-AES, ICP-MS, and x-ray fluorescence 
spectroscopy. The method of choice for the analysis of iron 
at very low concentrations is graphite furnace AAS, but 
AAS and ICP-AES are acceptable for measuring usual or 
toxic levels. Limits of detection for iron using AAS are on 
the order of pg/mL (mg/kg) and for graphite furnace AAS 
are generally in the low ng/mL (ppb) range. Sample prepara- 
tion usually consists of wet ashing the specimen with strong 
acid and heat, and redissolving the residue in dilute acid prior 
to analysis so that all iron species are converted quantita- 
tively to the same iron compound. 

To ensure adequate quality control, a reference material 
should be used that closely matches the matrix type and con- 
centration of the experimental samples to be analyzed. Iron 
contamination during sample collecting and processing must 
be avoided. All glass equipment involved in blood collec- 
tion and storage should be made of iron-free silicate glass 
and acid washed. Because of the high levels of iron in the 
atmosphere, samples can become contaminated during the 
analytical process. It is essential, therefore, to complete all 
analyses keeping the sample covered or in a hood. Blanks 
should be included in all analyses. 

REGULATION AND METABOLISM 
Absorption 

Gastrointestinal absorption of iron occurs primarily in the 
duodenum, and the proportion of the dose that is absorbed 
decreases as the dose increases. It is likely that iron absorp- 



tion occurs by the divalent metal transporter- 1, which prima- 
rily transports non-heme iron (Bressler et al., 2004). The rate- 
limiting processes in iron absorption are associated with 
transfer of iron from enterocytes to the blood rather than 
transport across the apical membrane of the enterocytes. 
Because of the limited capacity of the body to excrete iron, 
iron homeostasis is maintained primarily by adjusting iron 
absorption to bodily needs. The absorption of iron is affected 
by (1) the age, iron status, and state of health of the animal; 
(2) the conditions within the gastrointestinal tract; (3) the 
amount and chemical form of the iron ingested; and (4) the 
amounts and proportions of various other components of the 
diet, both organic and inorganic (Underwood, 1977). 

Soluble forms of iron are absorbed better than insoluble 
forms (Skikne and Baynes, 1994). The proportion of a dose 
of highly soluble iron absorbed by adults may vary from 2 to 
20 percent when ingested (Layrisse and Martinez-Torres, 
1971). Phytate, milk proteins, and soy proteins reduce iron 
absorption. Young animals absorb iron more efficiently than 
do older animals. Pregnancy, lactation, or a deficiency of 
iron also increases the efficiency of absorption. 

Metabolic Interactions 

Once absorbed, iron enters the blood where it is taken up 
by transferrin (Baker and Morgan, 1994). Iron in circulation 
is primarily bound to transferrin. Transferrin distributes iron 
throughout the body to wherever it is needed, mostly to 
erythrocyte precursors in the bone marrow for new hemo- 
globin synthesis. Cell membranes contain a protein called 
transferrin receptor. On the cell membrane, diferric transfer- 
rin binds to transferrin receptors, and then the iron-transfer- 
rin-transferrin receptor complex is internalized by endocyto- 
sis. The cellular regulation of iron is dependent on the effect 
of iron, or iron lack, in stimulating the synthesis of ferritin or 
of transferrin receptors, respectively (Fairbanks, 1994). This 
regulatory mechanism involves the effect of iron on iron- 
responsive elements (IRE) in the untranslated regions of 
transferrin receptor mRNA and ferritin mRNA. An iron-re- 
sponsive element binding protein (IRE-BP) appears to be 
involved in the activation or repression of ferritin and also of 
the transferrin receptor gene. 

Much of the absorbed iron is transferred to the bone mar- 
row where the iron-transferrin receptor complex enters eryth- 
roblasts by endocytosis. Iron is released into the cytosol and 
apotransferrin is returned to circulation. Within the cytosol, 
iron either is transported to mitochondria to be incorporated 
into heme or taken up by ferritin within siderosomes 
(Fairbanks, 1994). Within the mitochondria, iron is inserted 
into protoporphyrin, forming heme, a reaction catalyzed by 
heme synthetase (ferrochelatase). Heme inhibits the release 
of iron from transferrin, an important feedback mechanism 
for adjusting the iron supply to the rate of hemoglobin syn- 
thesis. Approximately 20 to 25 mg of iron is used daily for 
hemoglobin synthesis in humans. 



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In addition to the synthiesis of lieme, iron is also required 
for tfie synthesis of iron-containing enzymes (Fairbanks, 
1994). Examples of iron-containing enzymes are cytochrome 
c, aconitase, cytochrome oxidase, phenylalanine hydroxy- 
lase, and a-glycerophosphate dehydrogenase. These en- 
zymes are sensitive to iron depletion; the degree of loss var- 
ies from enzyme to enzyme and from tissue to tissue. 
Cytochrome c and aconitase are readily depleted during iron 
deficiency, as are the iron-sulfur enzymes in muscle mito- 
chondria. 

Iron in excess of need is stored intracellularly as ferritin 
and hemosiderin, principally in the macrophage system of 
the liver, spleen, and bone marrow (Fairbanks, 1994). Fer- 
ritin is the basic storage form; hemosiderin appears to be 
aggregated ferritin stripped of its protein component. Mobi- 
lization of iron from iron stores requires the presence of the 
Cu-containing enzyme of the plasma ceruloplasmin 
(ferroxidase I) (Chung et al., 2004). 

Daily excretion of iron is limited, and losses from the 
body are relatively small except during hemorrhage or men- 
struation. Although iron is released from breakdown of 
erythrocytes and is secreted in bile, most of this iron is reab- 
sorbed and used to form new hemoglobin. The primary 
routes of iron excretion are via feces and urine, and there are 
additional losses in sweat, hair, and nails (Georgievskii et 
al., 1979; McDowell, 2003). 

Mechanism of Toxicity 

Organ damage arising from iron overload is remarkable 
for the range of tissues affected and for the slow and insidi- 
ous onset of organ dysfunction with chronic toxicity (Eaton 
and Qian, 2002). The organs affected most by iron overload 
are the liver, heart, and pancreatic beta cells; all are tissues 
with highly active mitochondria, which generate activated 
oxygen species capable of causing synergistic toxicity with 
intracellular iron. 

One hypothesis concerning the etiology of cell and organ 
damage arising from iron overload is that excess iron selec- 
tively targets mitochondria and, perhaps, the mitochondrial 
genome (Eaton and Qian, 2002). The leak of electrons from 
mitochondria accounts for about 90 percent of the activated 
oxygen generated by most cells. Experimental acute iron 
loading of cultured (beating) rat myocardial cells caused a 
decrease in membrane polyunsaturated fatty acid (PUFA) 
content, loss of thiol-dependent enzyme activities, decreased 
ATP, and increased lysosomal fragility (Link et al., 1989). 
Similar changes in mitochondrial energy production with 
iron loading have been observed in the livers of rats with 
chronic iron overload (Bacon et al., 1993). It is also known 
that abnormal accumulation of iron within mitochondria is 
associated with extensive and irreversible damage. Affected 
mitochondria show not only iron accumulation but also in- 
activation of iron-sulfur enzymes — complexes I, II, and III 
and aconitase (Rotig et al., 1997). One of the pathological 



consequences of iron overload might be damage to mito- 
chondrial DNA (Eaton and Qian, 2002). There are several 
reasons for the apparent fragility of mitochondrial DNA 
(mtDNA): (1) mitochondria generate reactive oxygen spe- 
cies; (2) mitochondria are intrinsically rich in iron; (3) 
MtDNA is deficient in histones that may normally provide 
partial protection against oxidant damage; and (4) repair of 
damage to mtDNA is slow and less effective. 

An alternative, but not mutually exclusive, mechanism 
for the toxic effects of iron on cells and organs involves the 
proposition that an accumulation of iron within the cellular 
lysosomal compartment sensitizes the lysosomes to damage 
and rupture, releasing damaging lysosomal digestive en- 
zymes into the cytoplasm of the cell (Eaton and Qian, 2002). 
Minimal release of lysosomal enzymes may induce transient 
reparative autophagocytosis, while moderate lysosomal rup- 
ture is followed by apoptosis. Severe oxidative stress, caus- 
ing massive lysosomal breakdown, causes necrosis. 

In summary, iron overload or toxicosis causes a number 
of serious and life-threatening pathologies. The molecular 
bases of these changes are not known, but pathologies in- 
volving an interaction between iron and mitochondrial res- 
piration seem to be at the core. 

SOURCES AND BIOAVAILABILITY 
Sources 

The iron content of animal feeds is highly variable. The 
level of iron in herbage plants is determined by the species 
and type of soil in which the plants grow. Values as high as 
700 to 800 mg/kg iron have been recorded for uncontami- 
nated alfalfa and as low as 40 mg/kg for some grasses grown 
on sandy soils (Underwood, 1977). Most cereal grains con- 
tain 30 to 60 mg/kg, and species differences are small. The 
leguminous and oil seeds may contain 100 to 200 mg/kg 
iron. 

Feeds containing animal products are rich sources of 
iron. Meat meals and fish meals contain 400 to 600 mg/kg, 
and blood meals contain more than 3,000 mg/kg iron. 
Ground limestone, oyster shell, and many forms of calcium 
phosphate used as mineral supplements contain 2,000 to 
5,000 mg/kg iron (Underwood, 1977). Iron is commonly 
added to animal diets in the form of iron sulfate, iron chlo- 
ride, iron proteinates, and blood meal. Red iron oxide is 
added to some pet foods and trace mineral supplements as 
a coloring agent, but the iron in this source is essentially 
unavailable to animals. 



Water 

The EPA has established National Primary Drinking 
Water Regulations that set mandatory water quality stan- 
dards for drinking water contaminants (EPA, 1992). These 
are enforceable standards called "maximum contaminant 



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MINERAL TOLERANCE OF ANIMALS 



levels" (MCL), which are established to protect the public 
against consumption of drinking water contaminants that 
present a risk to human health. An MCL is the maximum 
allowable amount of a contaminant in drinking water that is 
delivered to the consumer. The MCL set for iron is 0.3 mg/ 
L. Excessive amounts of iron in water cause a rusty color, 
sediment, metallic taste, and reddish or orange staining of 
containers and cooking utensils. Iron is not generally listed 
in tables with safe upper limits of concentrations of minerals 
in water for livestock and poultry (NRC, 1974). Iron content 
of surface waters in the United States ranges from 0.10 to 
4,600 Mg/L with a mean of 43.9 pg/L (NRC, 1974). Well 
water with high iron content may cause bacteria to prolifer- 
ate, forming a red, slimy mass. 

Bioavailability 

Henry and Miller (1995) published a comprehensive re- 
port on the bioavailability of iron in a wide variety of 
feedstuff s and feed supplements. The proportion of dietary 
iron absorbed is largely determined by the iron requirement 
of the individual or animal. Absorption is regulated by the 
body iron stores; the percentage absorbed is inversely pro- 
portional to serum ferritin concentrations (Bothwell et al., 
1979). Iron bioavailability is also influenced by the compo- 
sition of the diet. 

A variety of factors in feeds can have an enhancing or 
inhibiting effect on iron bioavailability. Ascorbic acid 
strongly enhances the absorption of non-heme iron. In the 
presence of ascorbic acid, dietary ferric iron is reduced to 
ferrous iron, which forms a soluble iron-ascorbic acid com- 
plex in the stomach. Animal tissues enhance the uptake of 
non-heme iron. It is thought that low molecular weight pep- 
tides released during digestion bind iron and improve its 
bioavailability. 

Inhibitors of non-heme iron absorption include phytate, 
polyphenols, and calcium. Phytic acid (inositol 
hexaphosphate) is present in legumes, rice, and grains. There 
appears to be a dose-response relationship between the level 
of phytate in a food and iron absorption (Hallberg and 
Hulthen, 2000). Because phytate and iron are concentrated 
in the aleurone layer and germ of grains, milling to white 
flour and white rice reduces the content of phytate and iron, 
thereby increasing the bioavailability of the remaining iron 
(Larsson et al., 1996). Polyphenols markedly inhibit the ab- 
sorption of non-heme iron. Iron binds to tannic acid in the 
intestinal lumen forming an insoluble complex that reduces 
absorption. The inhibitory effects of tannic acid are dose 
dependent and reduced by the addition of ascorbic acid 
(lOM, 2000). Calcium appears to inhibit the absorption of 
both heme and non-heme iron (Gleerup et al., 1995). The 
mechanism is not fully understood, but it may involve inter- 
ference with the degradation of phytic acid and/or inhibition 
of iron absorption during the transfer through the mucosal 
cell. 



Trace mineral interactions may also alter iron 
bioavailability. An excess of one trace mineral may impair 
the absorption or transportation of another mineral with simi- 
lar divalent form, possibly by competing for intestinal bind- 
ing sites on the mucosa; conversely, a deficiency of one min- 
eral may be associated with an enhanced absorption of 
another (Hill and Matrone, 1970). Interactions affecting iron 
transport that have been described in animals include zinc- 
copper-iron, calcium-phosphorus-iron, manganese-iron, co- 
balt-iron, nickel-iron, and iron-cadmium. For example. 
Baker andHalpin (1991) reported that when young chickens 
are fed excessive dietary concentrations of manganese, the 
blood hemoglobin concentrations decrease. 

Additional examples of interactions that affect 
bioavailability of iron for animals, along with a discussion 
of the effects of feed processing, are provided in the review 
by Henry and Miller (1995). 

TOXICOSIS 

Iron toxicosis is not a common problem in most domestic 
animals, probably because of the limited absorption and up- 
take of iron when intakes are high. Nevertheless, if intakes 
are sufficiently high, signs of iron toxicosis occur. When 
animals consume large amounts of iron over sustained peri- 
ods, tissue overload occurs, iron binding capacity is ex- 
ceeded, and reactive (free) iron levels become sufficient to 
cause peroxidative damage, especially in liver. According to 
Underwood and Suttle (1999), the underlying pathogenic 
mechanism is peroxidative damage of lipid membranes and 
therefore the extent of the damage will depend to some ex- 
tent on the antioxidant status of the animal. Rats, and prob- 
ably other animals, become more susceptible to iron over- 
load with age (Wu et al., 1990). 

Single Dose 

Very large doses of soluble iron sources can be fatal. 
Accidental fatal toxicoses have occurred in children, and 
there have been fatalities in adults due to suicides (Abdel- 
Mageed and Oehme, 1990). The lethal dose in adults is 200 
to 250 mg Fe/kg BW. Three to 10 grams of ferrous sulfate 
are usually fatal in young children (Mullaney and Brown, 
1988). Autopsy findings include periportal hepatic necrosis 
and congestion along with necrosis of gastric and duodenal 
mucosa. Iron toxicosis in most animals (dogs may be an ex- 
ception) requires much higher iron levels along with high 
intakes of cobalt, zinc, manganese, or copper (Abdel- 
Mageed and Oehme, 1990). A few studies have examined 
the effect of single doses of iron in animals (Table 16-1). In 
piglets given 200 mg iron from ferric ammonium citrate 
within six hours of birth, only 33 percent survived for 21 
days. Lesions in piglets dosed with ferrous sulfate include 
hydropericardium, hydrothorax, and coagulative necrosis of 
skeletal muscle. Ferrous sulfate given at 200 mg/kg BW to 



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rabbits caused death within a few hours of administration 
(NRC, 1980). A lethal dose of iron (200 mg/kg as ferrous 
sulfate) was administered into the duodenum of seven rab- 
bits as heart rate, right atrial pressure, arterial pressure, car- 
diac output, left ventricular pressure, right ventricular devel- 
oped force, and arterial pH were monitored (Artman et al., 
1982). The acute lethal dose increased systemic vascular re- 
sistance and depressed stroke volume, cardiac output, and 
ventricular force. In these rabbits, acute iron toxicosis sig- 
nificantly depressed myocardial contractility. 

Single dose iron toxicity has also been studied in swine. 
Eight pigs (mean weight 25 kg) were given 60 mg of iron as 
either solid iron tablets or chewable multivitamins with iron 
per kg body weight (Nordt et al., 1999). After 10 hours, gross 
examination of the esophagus, liver, small and large intes- 
tines, and stomach of all the animals was performed by a 
surgical pathologist. All of the animals survived for the 10- 
hour study. The group receiving the solid iron tablets had 
severe esophageal inflammation and focal erosion, but no 
significant changes were identified in the liver, small intes- 
tine, or large intestine of either group. 

A newly born pig has very low iron reserves, rapid 
growth, limited iron intake from the sow's milk, and is gen- 
erally raised in an environment (away from soil) where ex- 
ogenous iron is not available; therefore, pigs are routinely 
supplied with an oral or injectable dose of iron (usually dex- 
trin, dextran, or gleptoferrin). In some cases, after a single 
dose injection of iron in a newborn pig, toxicosis has oc- 
curred. Death may result as quickly as 30 minutes to 6 hours 
postinjection or even be delayed for 2 to 4 days. A toxic oral 
single dose of iron from ferrous sulfate is approximately 600 
mg/kg BW for 3- to 10-day-old pigs (Campbell, 1961). A 
single injection of 200 mg/kg BW of iron dextran to new- 
born pigs from sows fed adequate vitamin E will result in 
normal hemoglobin concentrations and growth performance 
(Hill et al., 1999). However, toxicosis signs were observed 
when 100 mg/kg BW of iron dextran was injected into new- 
born pigs from vitamin E deficient sows (Lannek et al., 
1962). 

The severity of iron toxicosis in young pigs from oral 
doses of 200 mg iron administered within 6 hours of birth 
depended on the iron source given. Dosing with ferric am- 
monium citrate resulted in 66 percent mortality by 21 days 
of age (Cornelius and Harmon, 1976). 

Within 8 hours of birth, five foals were given 16 mg/kg 
BW of ferrous fumarate orally (Mullaney and Brown, 
1988). Two of the five foals died 50 hours after dosing, 
one died at 7 days, and the other two survived. Necropsies 
showed that the foals had liver disease. The dose of iron 
causing acute toxicosis in foals (about 16.5 mg/kg BW) is 
strikingly low when compared with the dose rates of other 
species. This may be related to the high serum iron and 
percentage saturation of transferrin levels that foals have 
at birth. Also, since iron absorption is elevated at birth, a 
small amount of orally administered iron could be well 



absorbed and exceed the iron binding capacity of serum 
and result in free or unbound iron reaching tissues, espe- 
cially the liver. 

Iron toxicity is sometimes encountered in dogs and cats, 
usually as the result of consumption of large amounts of 
readily ionizable iron such as multivitamins, dietary min- 
eral supplements, or human pregnancy supplements. Acute 
doses from 200 to 600 mg of iron per kilogram of body 
weight are fatal in dogs (Reissman and Coleman, 1955; 
Bronson and Sisson, 1960; D' Arcy and Howard, 1962). For 
cats, the LD^g (median lethal dose) for an acute dose of 
ferrous sulfate has been estimated to be 500 mg/kg BW 
(Hoppe et al., 1955). 

A study using rat hepatocytes showed that glutathione 
and vitamin E can be protective against the lipid peroxidation 
associated with iron toxicity (Milchak and Bricker, 2002). In 
piglets, iron toxicosis is often associated with vitamin E de- 
ficiency (Lannek et al., 1962). 

Acute 

Rats injected intraperitoneally with iron dextran at doses 
of 250, 500, or 1,000 mg/kg BW developed oxidative stress 
and im