Heavy Metals in the Environment - Chapter 1 pot

Heavy
Metals
in
the
Environment
edited
by
Bibudhendra
Sarkar
The
Hospital
for
Sick Children
and
University
of
Toronto
Toronto,
Ontario,
Canada
MARCEL
n
DECKER
MARCEL DEKKER,
INC.
NEW
YORK
•
BASEL
Copyright © 2002 Marcel Dekker, Inc.
ISBN: 0-8247-0630-7

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

The publisher offers discounts on this book when ordered in bulk quantities. For more
information, write to Special Sales/Professional Marketing at the headquarters address
above.
Copyright  2002 by Marcel Dekker, Inc. All Rights Reserved.
Neither this book nor any part may be reproduced or transmitted in any form or by any
means, electronic or mechanical, including photocopying, microfilming, and recording,
or by any information storage and retrieval system, without permission in writing from
the publisher.
Current printing (last digit):
10987654321
PRINTED IN THE UNITED STATES OF AMERICA
Copyright © 2002 Marcel Dekker, Inc.
Preface
Our global environment now consists of numerous natural and artificial metals.
Metals have played a critical role in industrial development and technological
advances. Most metals are not destroyed; indeed, they are accumulating at an
accelerated pace, due to the ever-growing demands of modern society. A fine
balance must be maintained between metals in the environment and human
health. It is with this view in mind that this book has been written to address

diverse issues surrounding heavy metals in the environment. Nineteen chapters
have been contributed by 50 experts from around the world, known for their
expertise and outstanding research. The book provides a critical review and analy-
sis of the current state of knowledge of heavy metals in the environment.
The volume begins with a chapter on the essentiality and toxicity of metals.
The widespread distribution of metals in the environment is of great concern
because of their toxic properties; however, some metals are also essential for
normal growth and development. This chapter provides a critical assessment of
nutritional and toxicological information based on available data on humans. The
evaluation has used information available on speciation and bioavailability to
identify the critical effects and clinical manifestations of metal deficiency and
toxicity. New principles and basic concepts are presented to define the acceptable
range of oral intake (AROI) at which no adverse effects occur and the correspond-
ing safe range of population mean intake (SRPMI) of essential trace metals such
as selenium, iron, manganese, zinc, and copper. The interdependence of various
Copyright © 2002 Marcel Dekker, Inc.
elements is discussed with regard to metabolic and functional interactions involv-
ing storage and metabolism.
Analytical measurements of heavy metals in the environment are an integral
component of monitoring and assessing their toxic effects. They are required for
regulatory purposes and routine monitoring to ensure compliance with allowed
levels to determine hazardous conditions. Clean-ups of contaminated locations
are commenced on the basis of measurements indicating the site and extent of
contamination. Chapter 2, on analytical methods for quantitative determination
of heavy metals discusses various analytical tools and speciation analyses of
heavy metals as well as their microscopic analyses. Techniques used for specia-
tion analyses are discussed for individual metals such as chromium, arsenic, mer-
cury, lead, and cadmium. This chapter also describes recent developments in the
use of microprobe beamline to monitor intracellular distribution of elements in
a single cell.

The need to develop and establish new toxicological approaches to assess
the potential cytotoxic and genotoxic effects of heavy metals in the environment
is addressed in Chapter 3, which focuses on a variety of in vitro toxicological
screening methods for the biomonitoring of heavy metals. These methods take
advantage of intracellular effects of metals to induce the expression of detoxify-
ing proteins, other protective proteins, and proteins involved in cell cycles and
proliferation and apoptosis. Suggestions are made as to the future of heavy metal
biomarker research and how it can be more carefully monitored in the human
environment.
A large spectrum of radionuclides was produced after the creation of the
cosmos. Their radioactive half-lives are very long, and they remain ubiquitous
components of the environment. Additionally, as a result of the development of
nuclear weapons and nuclear technology, a number of artificial radionuclides
have become a part of the human environment. Chapter 4 discusses the distribu-
tion and concentration of both natural and manmade radionuclides and the mecha-
nism of their transfer to plants, animals, and humans. Possible long-term effects
of their distribution in human tissue in terms of health implications are discussed.
Metallic agents, as a class, make up a substantial portion of known human carcin-
ogens. Chapter 5 reviews the topic of metal carcinogenesis, following the Interna-
tional Agency for Research on Cancer (IARC) classification system, with particu-
lar emphasis on known human carcinogens.
In recent years, both carcinogenic and noncarcinogenic potential of arsenic
have been intensely studied. Chapters 6 and 7 review the global perspective on
arsenic in the environment and aspects of arsenic toxicity. Chapter 6 explores
the environmental behavior of arsenic with special reference to the abundance
and distribution of arsenic in the lithosphere, sediments, soil environment, and
groundwater. It also discusses various pathways of arsenic emission into the envi-
ronment, methods for arsenic determination in drinking water, and techniques
Copyright © 2002 Marcel Dekker, Inc.
for remediation of arsenic-contaminated soil and groundwater systems. Chapter

7 discusses the sources of human exposure and aspects of human toxicology with
special emphasis on chronic arsenic poisoning and its general effects related to
dermatological manifestations, cardiovascular diseases, neurological impair-
ments, and cancer effects.
Individual chapters are devoted to selected metals in the environment, in-
cluding cadmium, chromium, aluminum, nickel, lead, mercury, and molybdenum.
Chapter 8 reviews the pertinent literature of cadmium toxicology, with discus-
sions of the health effects in humans of cadmium exposure and the molecular
mechanisms underlying these effects. The connection between inhalation of chro-
mium (VI) compounds and the causation of cancers of the airways and lungs
is well established. Chapter 9 describes epidemiological studies along with the
toxicokinetics and molecular mechanisms underlying the carcinogenicity of chro-
mium (VI). It is followed by an in-depth consideration of approaches to the bio-
logical monitoring of chromium (VI)–exposed subjects. Chapter 10 presents an
assessment of the hazards of aluminum exposure to humans, animals, and plants.
Chapter 11, on nickel, reviews its distributions in the environment, human expo-
sure, metabolism, systemic and molecular toxicology, and carcinogenesis. This
chapter also includes a discussion on the interaction of nickel with other essential
metals such as magnesium, calcium, iron, zinc, and manganese. Chapter 12 dis-
cusses the release of lead in the environment, human body burdens, and the popu-
lation at risk. Special emphasis is given to analytical methods for the assessment
of lead exposure and its metabolism, treatment of lead poisoning, in vitro and
animal studies, molecular mechanisms, reproductive outcome, risk assessment
and human epidemiological studies.
It is believed that the global cycling of mercury of natural and anthropo-
genic sources is responsible for the transport and deposition of mercury in areas
remote from the original source. Chapter 13 takes a detailed look at mercury in
the environment and its toxic actions, including a discussion on epidemiological
studies of prenatal exposure. Molybdenum is essential to a variety of organisms,
and is distributed widely in the environment owing to its diverse chemistry and

its technological and agricultural applications. Chapter 14 provides a balanced
picture of the complex environmental chemistry of molybdenum, including its
interactions with copper, which can be either antagonistic or beneficial from the
interplay of individual components in the biogeosphere.
The intracellular concentration of heavy metals is kept in balance by a
variety of metal-transporters. Many of the metals are toxic in excess. Bacterial
metal resistance probably arose early in evolution owing to widespread geochem-
ical sources of metals. Chapter 15, devoted to the microbial resistance mechanism
of heavy metals, discusses the mechanisms of resistance to zinc, cadmium, lead,
copper, arsenic, and antimony in bacteria. The exposure to metal that is harmless
to some bacteria may be destructive to others with specific genetic changes. Chap-
Copyright © 2002 Marcel Dekker, Inc.
ter 16 examines genetic susceptibility to heavy metals in the environment, noting
how each metal is expected to have its own series of transporters. Transport of
several metals is highly dependent upon the concentration of the other metals.
This balance can be disrupted when any gene within the balanced system is non-
functional. The interaction between genes and environment—considered critical
for avoiding metal toxicity not only for humans but also for a wide variety of
animal species—is described in detail. Selenium has multiple biological actions
as an essential trace element, a modifier of other toxic elements, an anticarcino-
genic agent, and a toxicant. These are all discussed in Chapter 17, which provides
an overview of the entire profile of biological actions of selenium in nutrition
and toxicology.
Over the past three decades, elements such as arsenic, antimony, gallium,
and indium have been used in the manufacture of semiconductors for computer
chips, cellular telephones, and light-emitting diodes. Many tons of these elements
have been incorporated into these devices, either as dopants for silicon-based
computer chips or in higher-speed semiconductors, such as gallium arsenide and
indium arsenide. With the increased demand for higher-speed devices, older de-
vices have been discarded, generating a large stockpile of electronic equipment

containing these elements known collectively as ‘‘e-waste.’’ This is a new phe-
nomenon, and the magnitude of this growing problem has been recognized only
recently, since there are no well-established recycling programs for such item.
Chapter 18, on semiconductors, provides an assessment of the present state of
knowledge of the role and biological effects of metal/metalloids utilized in the
semiconductor industry. The potential human health and environmental effects
of these elements, either alone or as mixtures, are discussed in relation to areas
of future studies.
There is a growing need for methods of assessing the amount of heavy
metals pollution in our natural and industrial environments. While it is relatively
straightforward to use the techniques of analytical chemistry to detect heavy
metal concentrations in a particular location, they do not indicate how much of
this metal is a ‘‘biological hazard.’’ Chapter 19 describes biosensors for monitor-
ing heavy metals, and how researchers are exploiting various biological mecha-
nisms to determine the amount of ‘‘bioavailable’’ heavy metal in the natural and
industrial environments. These methods are still in their infancy compared with
the techniques of analytical chemistry, but they clearly offer advantages in terms
of ease of use, and biological relevance. The recent progress made in the develop-
ment of whole-cell and protein-based biosensors is encouraging and holds much
promise for the future.
The book was written by contributors in close collaboration with me. I
visited some of their laboratories, intensively discussed their work with them,
and made a few field trips to environmentally affected areas to obtain first-hand
knowledge. Despite conscientious efforts by all concerned, the chapter authors,
Copyright © 2002 Marcel Dekker, Inc.
the editor, and the publisher cannot assume any liability for errors that this book
may contain. Every effort has been made to keep the error rate as low as possible.
Heavy Metals in the Environment will be an invaluable resource for toxicol-
ogists; biochemists; bioinorganic, inorganic, environmental, and medicinal chem-
ists; immunologists; oncologists; physiologists; pharmacologists; geneticists;

bacteriologists; molecular biologists; environmental scientists; and upper-level
undergraduate and graduate students in these disciplines.
I thank many of my international colleagues who provided valuable sugges-
tions in the selection of topics and other advice. Special thanks are due to Loretta
LeBlanc for preparing the manuscript and to Suree Narindrasorasak, Ping Yao,
and Negah Fatemi for their assistance in the preparation of the index.
Bibudhendra Sarkar
Copyright © 2002 Marcel Dekker, Inc.
Contents
Preface
Contributors
1.EssentialityandToxicityofMetals
Gunnar F. Nordberg, Brittmarie Sandstro
¨
m, George Becking,
Robert A. Goyer
2. Analytical Methods for Heavy Metals in the Environment:
Quantitative Determination, Speciation, and Microscopic
Analysis
Richard Ortega
3. In Vitro Toxicological Assessment of Heavy Metals and
IntracellularMechanismsofToxicity
Wendy E. Parris and Khosrow Adeli
4.RadionuclidesintheEnvironment
David M. Taylor
5.MetalCarcinogenesis
Michael P. Waalkes
Copyright © 2002 Marcel Dekker, Inc.
6.ArsenicintheEnvironment:AGlobalPerspective
Prosun Bhattacharya, Gunnar Jacks, Seth H. Frisbie,

Euan Smith, Ravendra Naidu, and Bibudhendra Sarkar
7.EnvironmentalAspectsofArsenicToxicity
J. Thomas Hindmarsh, Charles O. Abernathy,
Gregory R. Peters, and Ross F. McCurdy
8.Cadmium
Monica Nordberg and Gunnar F. Nordberg
9.ChromiumandCancer
Montserrat Casadevall and Andreas Kortenkamp
10.Aluminum
John Savory, R. Bruce Martin, Othman Ghribi, and
Mary M. Herman
11.Nickel
Jessica E. Sutherland and Max Costa
12.Lead
Emily F. Madden, Mary J. Sexton, Donald R. Smith, and
Bruce A. Fowler
13.Mercury
Thomas W. Clarkson
14.Molybdenum
Edward I. Stiefel and Henry H. Murray
15. Microbial Resistance Mechanisms for Heavy Metals and
Metalloids
Mallika Ghosh and Barry P. Rosen
16.GeneticSusceptibilitytoHeavyMetalsintheEnvironment
Diane W. Cox, Lara M. Cullen, and John R. Forbes
17.SeleniuminNutritionandToxicology
Seiichiro Himeno and Nobumasa Imura
Copyright © 2002 Marcel Dekker, Inc.
18.Semiconductors
Bruce A. Fowler and Mary J. Sexton

19. Bacterial Metal-Responsive Elements and Their Use in
BiosensorsforMonitoringofHeavyMetals
Ibolya Bontidean, Elisabeth Cso
¨
regi, Philippe Corbisier,
Jonathan R. Lloyd, and Nigel L. Brown
Copyright © 2002 Marcel Dekker, Inc.
Contributors
Charles O. Abernathy Office of Water Quality, U.S. Environmental Protec-
tion Agency, Washington D.C.
Khosrow Adeli Division of Clinical Biochemistry, Department of Laboratory
Medicine and Pathology, and Program in Structural Biology and Biochemistry,
The Hospital for Sick Children and University of Toronto, Toronto, Ontario,
Canada
George Becking World Health Organization, Research Triangle Park, North
Carolina
Prosun Bhattacharya Department of Land and Water Resources Engineering,
Royal Institute of Technology, Stockholm, Sweden
Ibolya Bontidean Department of Biotechnology, Lund University, Lund,
Sweden
Nigel L. Brown School of Biosciences, The University of Birmingham, Edg-
baston, Birmingham, United Kingdom
Copyright © 2002 Marcel Dekker, Inc.
Montserrat Casadevall Centre for Toxicology, The School of Pharmacy, Lon-
don, England
Thomas W. Clarkson Department of Environmental Medicine, University of
Rochester School of Medicine, Rochester, New York
Philippe Corbisier Joint Research Centre of the European Commission, Insti-
tute for Reference Materials and Measurements, Geel, Belgium
Max Costa Department of Environmental Medicine, New York University

School of Medicine, Tuxedo, New York
Diane W. Cox Department of Medical Genetics, University of Alberta, Edmon-
ton, Alberta, Canada
Elisabeth Cso
¨
regi Department of Biotechnology, Lund University, Lund,
Sweden
Lara M. Cullen Department of Medical Genetics, University of Alberta, Ed-
monton, Alberta, Canada
John R. Forbes Department of Biochemistry, McGill University, Montreal,
Quebec, Canada
Bruce A. Fowler Department of Epidemiology, Laboratory of Cellular and
Molecular Toxicology, University of Maryland, Baltimore, Maryland
Seth H. Frisbie Better Life Laboratories, Inc., Plainfield, Vermont
Mallika Ghosh Department of Biochemistry and Molecular Biology, Wayne
State University School of Medicine, Detroit, Michigan
Othman Ghribi Department of Pathology, University of Virginia, Charlottes-
ville, Virginia
Robert A. Goyer University of Western Ontario, London, Ontario, Canada
Mary M. Herman National Institute of Mental Health, National Institutes of
Health, Bethesda, Maryland
Seiichiro Himeno School of Pharmaceutical Sciences, Kitasato University,
Tokyo, Japan
Copyright © 2002 Marcel Dekker, Inc.
J. Thomas Hindmarsh Department of Pathology and Laboratory Medicine,
The University of Ottawa and The Ottawa Hospital, Ottawa, Ontario, Canada
Nobumasa Imura School of Pharmaceutical Sciences, Kitasato University,
Tokyo, Japan
Gunnar Jacks Department of Land and Water Resources Engineering, Royal
Institute of Technology, Stockholm, Sweden

Andreas Kortenkamp Centre for Toxicology, The School of Pharmacy, Lon-
don, England
Jonathan R. Lloyd School of Biosciences, The University of Birmingham,
Edgbaston, Birmingham, United Kingdom
Emily F. Madden Center for Devices and Radiological Health, U.S. Food &
Drug Administration, Rockville, Maryland
R. Bruce Martin Department of Chemistry, University of Virginia, Charlottes-
ville, Virginia
Ross F. McCurdy InNOVAcorp., Dartmouth, Nova Scotia, Canada
Henry H. Murray Corporate Strategic Research, ExxonMobil Research and
Engineering Co., Annandale, New Jersey
Ravendra Naidu CSIRO Land and Water, Commonwealth Scientific & Indus-
trial Research Organization, Glen Osmond, South Australia, Australia
Gunnar F. Nordberg Department of Environmental Medicine, Umea
˚
Univer-
sity, Umea
˚
, Sweden
Monica Nordberg Institute of Environmental Medicine, Karolinska Institute,
Stockholm, Sweden
Richard Ortega Chimie Nucle
´
aire Analytique Bioenvironnementale, Univer-
sity of Bordeaux, Gradignan, France
Wendy E. Parris Division of Clinical Biochemistry, Department of Laboratory
Medicine and Pathology, and Program in Structural Biology and Biochemistry,
The Hospital for Sick Children and University of Toronto, Toronto, Ontario,
Canada
Copyright © 2002 Marcel Dekker, Inc.

Gregory R. Peters Philip Analytical Services Inc., Bedford, Nova Scotia,
Canada
Barry P. Rosen Department of Biochemistry and Molecular Biology, Wayne
State University School of Medicine, Detroit, Michigan
Brittmarie Sandstro
¨
m Research Department of Human Nutrition, The Royal
Veterinary and Agricultural University, Copenhagen, Denmark
Bibudhendra Sarkar Program in Structural Biology and Biochemistry, The
Hospital for Sick Children and University of Toronto, Toronto, Ontario, Canada
John Savory Department of Pathology, Biochemistry and Molecular Genetics,
University of Virginia, Charlottesville, Virginia
Mary J. Sexton Department of Epidemiology and Preventive Medicine, School
of Medicine, University of Maryland, Baltimore, Maryland
Donald R. Smith Department of Environmental Toxicology, University of Cal-
ifornia, Santa Cruz, California
Euan Smith CSIRO Land and Water, Commonwealth Scientific & Industrial
Research Organization, Glen Osmond, South Australia, Australia
Edward I. Stiefel Department of Chemistry, Princeton University, Princeton,
New Jersey
Jessica E. Sutherland Department of Environmental Medicine, New York
University School of Medicine, Tuxedo, New York
David M. Taylor Department of Chemistry, Cardiff University, Cardiff, Wales
Michael P. Waalkes Laboratory of Comparative Carcinogenesis, National
Cancer Institute and National Institute of Environmental Health Sciences, Na-
tional Institutes of Health, Research Triangle Park, North Carolina
Copyright © 2002 Marcel Dekker, Inc.
1
EssentialityandToxicityofMetals
Gunnar F. Nordberg

Umea
˚
University, Umea
˚
, Sweden
Brittmarie Sandstro
¨
m
The Royal Veterinary and Agricultural University, Copenhagen, Denmark
George Becking
World Health Organization, Research Triangle Park, North Carolina
Robert A. Goyer
University of Western Ontario, London, Ontario, Canada
1. INTRODUCTION
Many metals are of concern because of their toxic properties and some metals
are also essential for survival and health of animals and humans.
In risk assessments concerning toxicity of essential metals, their essentiality
should also be taken into account to avoid too low intakes. This has not always
been done in a proper way and authorities responsible for protecting the public
from adverse health effects from toxicity have issued recommendations that have
1
Copyright © 2002 Marcel Dekker, Inc.
been partly in conflict with those issued with an aim of protecting from deficiency.
There is an obvious need for an approach including a balanced consideration of
nutritional as well as toxicological data for these metals.
These new principles of evaluation take into account some basic concepts
of interindividual variability in sensitivity to deficiency and toxicity. Such varia-
tion translated into one interval of (low) daily intake, at which there is a risk of
developing deficiency, and another interval of (high) dietary intake, at which
toxicity may occur. In between there is a set of intakes that represent the accept-

able range of oral intakes (AROI), at which no adverse effects occur. While it
is possible to define such a range that will protect most people, a range will
not usually be found that protects all persons from adverse effects. Those with
genetically determined sensitivity may require higher intakes to avoid deficiency
or lower intakes to avoid toxicity than those defined by the acceptable range.
AROI is defined as protecting 95–98% of healthy individuals in specified gender
and life stage population groups from even minimal adverse effects of deficiency
or toxicity. While AROI is defined for intakes by individuals in a population
group, the corresponding range for mean intakes is the safe range of population
mean intakes (SRPMI).
This chapter reviews principles and methodologies that may be applied in
defining limits of safety for nutritionally essential metals. Excessive intake of
these metals can give rise to toxicity. There is increasing use of various standards
worldwide that express the maximum acceptable limits for human exposures for
various substances present in the environment including nutritionally essential
trace elements. In some instances, the methodology applied to standard setting
for these nutritionally essential substances has been the same as applied to toxic
metals. In the case of zinc, RDA (recommended daily allowances set by the U.S.
National Research Council) and RfD (reference dose, set by the U.S. Environ-
mental Protection Agency) were found to be almost identical and, for certain age
groups, the RDA was higher than the Rfd (1,2). It is becoming apparent that
standard setting for nutritionally essential trace elements requires consideration
beyond approaches traditionally applied to metals that have no biological require-
ment for good health.
Recognition of this problem has prompted a number of conferences on this
topic that have resulted in publications and discussions of potential approaches
as well as potential problems. These activities include a workshop sponsored by
the U.S. Environmental Protection Agency, the Agency for Toxic Substances and
Disease Registry, and the International Life Sciences Institute’s Risk Sciences
Institute held in March 1992 in Herndon, Virginia, which reviewed the problem

and identified a number of topics that had been inadequately considered to date
(1). Similarly, a Nordic Working Group on Food and Nutrition and the Nordic
Group on Food Toxicology have prepared a report (3), and a conference on
‘‘Trace Elements in Human Health’’ held in Stockholm in May 1992 has also
Copyright © 2002 Marcel Dekker, Inc.
been reported (2). The conceptual framework for the preparation of a WHO/
IPCS methodology document was based on discussions from a WHO planning
meeting held in Washington in April 1996 (4) and on IPCS Workshop held in
Santiago de Chile in 1998. The methodology [reviewed in part also by Nordberg
et al. (5)] proposed in the present chapter has evolved from the activities.
Metals have been classified as essential, beneficial, or detrimental. Trace
elements recognized as essential for human health include iron, zinc, copper,
chromium, iodine, cobalt, molybdenum, and selenium (6). For the purpose of
this chapter all metallic elements in this listing will be considered in discussions
of methodology. Later in the chapter, the methodology described will be applied
to a few of these as examples. There is also a second group of elements thought
to be beneficial to life (e.g., silicon, manganese, nickel, boron, and vanadium).
Some of these elements may be essential to vegetative life and perhaps beneficial
to human health but, generally, they are not yet accepted as essential for human
health. If any of these or other elements become accepted as essential for humans
and quantitative nutritional requirements are established, the approaches outlined
in this chapter should be applicable for setting an acceptable range of oral intakes
(AROI).
The methodology described for determining the AROI is not intended to
be applied to detrimental metals or metals that are regarded as purely toxic metals
such as lead, cadmium, and mercury, which are not known to provide any essen-
tial or potentially beneficial health effect at any level of exposure. Also this meth-
odology, in its present form, is not intended to assess risk for carcinogenicity,
although at present this is only of concern for one essential element, chromium,
and is probably limited to inhalation and not oral exposure. While the essential

elements are not by themselves known to be carcinogenic by the oral route, sev-
eral play important roles as modulators of carcinogens by promoting or protecting
from oxidative damage. For example, selenium deficiency and excess iron intake
may act synergistically to enhance oxidative damage of macromolecules, nucleic
acids, and lipid membranes.
Presently recommended dietary intakes of essential trace metals are based
on estimates of amounts needed to prevent clinical or biochemical deficiency. It
is increasingly recognized that higher intakes of some of the trace elements may
have beneficial health effects in relation to risk reduction of degenerative diseases
such as cardiovascular disease and cancer. These suggested higher levels are
usually not met by ordinary foods but require supplements, often administered
in a more available form than dietary minerals. Long-term intake of high but not
immediately toxic doses of trace elements may lead to interaction with other
trace elements and/or other changes not identified with a classical toxicological
approach. Thus the safety of essential trace elements is a subtle issue and as new
criteria for estimates of requirements are emerging there may be a need to redefine
also the criteria used to estimate adverse-effect levels.
Copyright © 2002 Marcel Dekker, Inc.
For essential metals health risk assessment requires consideration of both
toxicity from excess exposures and health effects as a consequence of deficiencies
from severe restriction of intake. Such an approach involves principles from nutri-
tion as well as toxicology. The objective is to make recommendations that result
in a range of recommended intakes that recognizes consequences of both nutri-
tional deficiency and toxicity.
The approach described in this chapter outlines the principles that support
the concept of AROI, or a ‘‘homeostatic model’’ for determining the distribution
of intakes for essential trace metals (ETM) that meet nutritional requirements of
a healthy population as well as preventing toxicity. The methodology presented
in this document recognizes the importance of variability in exposure and bioki-
netics arising from age, gender, physiological conditions, and nutritional status.

In addition it should be noted that dietary/food intake is only part of oral intake.
Oral intake also includes intake from water, dietary supplements, and a fraction
of inhalation exposures that are subsequently ingested after coughing and swal-
lowing.
1.1 Nutritionally Essential Trace Metals (ETM)
The traditional criteria for human health are that absence or deficiency of the
element from the diet produces either functional or structural abnormalities and
that the abnormalities are related to, or a consequence of, specific biochemical
changes that can be reversed by the presence of the essential metal (6).
The criteria for identifying ETM have evolved over the past 50 years and
may be expected to expand as the result of future research. New end points re-
flecting effects of deficiency have been considered in recent investigations of
essentiality of ETMs in experimental animals (7). These have included mild re-
ductions in growth rate, impairment of reproductive performance, decreased life
span, sudden unexpected death, and some anatomical lesions.
1.2 Homeostatic Mechanisms
A defining characteristic of ETMs is that there are homeostatic mechanisms that
maintain optimum tissue levels over a range of exposures for the performance
of essential functions (8). Homeostatic mechanisms involve regulation of absorp-
tion, tissue retention, and excretion of ETMs. There are specific homeostatic
mechanisms for each ETM (6). For some ETMs, namely those that are handled as
cations (zinc, iron, copper, manganese, chromium), the homeostatic mechanisms
operate predominantly via the gastrointestinal tract and the liver. They may regu-
late uptake and transfer by the gut (Fe, Zn) or by biliary excretion (Cu). For each
of these ETMs there may be a specific chain of protein carriers and receptors to
effect uptake into cells. Anionic ETMs, like molybdenum and selenium, are more
Copyright © 2002 Marcel Dekker, Inc.
soluble and systemic absorption is less regulated than for cationic ETMs. They
are absorbed very efficiently and subsequent control, tissue deposition, and excre-
tion are managed by oxidation state. Total body burden is regulated by renal

excretion. A third category of homeostatic mechanism is illustrated by cobalt,
which is a highly reactive element with several oxidation states. The physiologi-
cal role for this metal is as one of highly regulated form in cobalamin and there
is no evidence that humans require inorganic cobalt. Whether or not an analogous
situation applies to chromium and the ‘‘glucose tolerance factor’’ is unclear par-
ticularly since inorganic chromium has been effective in alleviating chromium
deficiency in patients on total parenteral nutrition. However, there is a wide diver-
sity within populations and individuals as to the efficiency of these mechanisms.
2. CONCEPTS OF EVALUATION—CONSIDERATION
OF TOXICITY AND ESSENTIALITY
Interindividual variation that occurs in human populations is considerable. This
applies to the expression of toxicity from higher doses of an ETM as well as for
the expression of deficiency symptoms as a result of too low intakes of the same
essential element.
2.1 Acceptable Range of Oral Intake (AROI)
InFigure1theinterindividualdistributionofsensitivityisshownfornutritional
requirements and for expression of toxicity. For a specific adverse effect of defi-
ciency, individuals exist in a population who display average sensitivity to devel-
oping symptoms (mean nutrient requirement, Fig. 1) as well as more sensitive
individuals, i.e., those developing deficiency symptoms at somewhat higher in-
takes (ϩ1.5–2.5 D nutrient requirement), and individuals with less sensitivity,
i.e., those that develop deficiency symptoms first when intakes are lower (Ϫ1.5–
2.5 D nutrient requirement). A similar situation applies for a specific toxic effect;
i.e., some individuals display symptoms at doses lower than those giving rise to
symptoms in the individuals of average sensitivity and there are also individuals
who are less than average sensitive and they require higher doses to develop
symptoms. The situation can be depicted as two bell-shaped curves describing
the distribution of sensitivity to deficiency and toxicity. In most cases an interval
between these curves describes the AROI in which no adverse effects occur in
the large majority (95–98%) of subjects (cf. Fig. 1). If these conditions are instead

depicted with curves in cumulative forms, a U-shaped curve is formed and the
AROIappearsatthebottomoftheU(Fig.2).Furtheraspectsandexamplesof
how AROI for specific essential metals can be derived will be given in later
sections.
Copyright © 2002 Marcel Dekker, Inc.
F
IGURE
1 Theoretical model describing distribution of intakes to meet nutri-
tional requirements (left) and distribution of intakes giving rise to toxicity
(right), with the acceptable range of oral intakes (AROI) in between. Lower
limit to the AROI should cover the requirements of most (97.5%) of the popu-
lation; the higher limit if AROI should protect a similar proportion of the popu-
lation from toxic effects.
2.2 Groups with Special Sensitivity/Resistance
2.2.1 Genetically Determined
InFigure3,thesamemodelfordistributionofintakestomeetnutritionalrequire-
ments and to prevent toxicity is displayed as in Figure 1. The low limit of the
AROI covers the requirement of most of the population and the high limit of the
AROI should protect most of the population from toxic effects. Special popula-
tion subgroups, such as persons with Wilson’s disease, may exhibit toxicity at
relatively low intakes of copper, lower than the acceptable range for normal per-
sons. On the other hand, some population subgroups such as B may have require-
ments higher than the upper limit of acceptable range (for example, zinc intake
in subjects with acrodermatitis enteropatica). Further aspects on genetically deter-
mined variation in sensitivity have been given by WHO 1996 (6) and will be
given in a future document on ‘‘Principles of Risk Assessment for Essential Trace
Elements’’ by IPCS/WHO.
Copyright © 2002 Marcel Dekker, Inc.
F
IGURE

2 Percent of population subjected to deficiency or toxicity to oral
exposure/intake.ThisisthesamemodelasinFigure1,butinacumulative
form. As intake drops below A (lower limit of AROI), risk for deficiency in-
creases; at extremely low intakes all subjects will manifest deficiency. As in-
takes increase beyond B, a progressively larger proportion of subjects will
exhibit effects of toxicity.
2.2.2 Nongenetically Determined
Many diseases are genetically determined but this is not considered here if the
disease is not known to involve a specific metabolic defect related to essential
elements. In celiac disease, there is a deficient uptake of several nutrients in-
cluding essential elements such as iron (9) and zinc (10). In addition, gastrointes-
tinal losses of trace elements can be increased due to diarrhea. If the disease is
not well controlled by exclusion of gluten from the diet, and/or the decreased
uptake is not compensated by an increased intake of these elements, iron and/
or zinc deficiency may develop. Increased urinary losses of zinc are observed in
patients with alcoholic cirrhosis (11) and diabetes (12). It has been shown that
iron deficiency gives rise to an increased uptake of manganese from the diet (13).
It can therefore be assumed that there would be an increased risk of manganese
toxicity if persons with iron deficiency were exposed to high oral intakes of man-
ganese.
Sensitivity to manifestations of zinc deficiency is known to be dependent
on certain metabolic situations. When discussing effects of deficiency or toxicity
of an essential element it is therefore of fundamental importance to specify the
Copyright © 2002 Marcel Dekker, Inc.
F
IGURE
3 Theoretical model for distribution of intakes to meet nutritional re-
quirements of healthy population and prevent toxicity. The lower limit to
AROI should cover the requirements of most (97.5%) of the population; the
higher limit should protect most of the population from toxic effects. Special

population subgroups, such as A, may exhibit toxicity at intakes lower than
the acceptable range (e.g., Wilson’s disease and copper intake), or in contrast,
some population subgroups, such as B, may have requirements higher than
the upper limit of acceptable range (e.g., zinc intakes in subjects with acroder-
matitis enteropathica).
background conditions of the group of persons under consideration. Such back-
ground conditions can be all-determining for the dose-response relationships. For
example, at a certain low zinc intake (e.g., under conditions of total parenteral
nutrition) clinical symptoms of zinc deficiency may not develop in a group of
individuals who are in metabolic balance, but may be clinically manifest in per-
sons who undergo growth or who are in an anabolic phase (14). Dose-response
relationships for zinc deficiency thus can be quite different depending on meta-
bolic state.
2.3 Nutritional Requirement and Safe Range of Population
Mean Intake (SRPMI)
Public health aspects concerning adverse effects as a result of deficiency from
an essential element have been discussed in many documents. Definitions relating
Copyright © 2002 Marcel Dekker, Inc.
to the needs of individuals were defined by the Joint FAO/IAEA/WHO Expert
Consultation on Trace Elements in Human Nutrition (6).
Requirement. This is the lowest continuing level of nutrient intake that,
at a specified efficiency of utilization, will maintain the defined level of
nutriture in the individual.
Basal requirement. This refers to the intake needed to prevent
pathologically relevant and clinically detectable signs of impaired func-
tion attributable to inadequacy of the nutrient.
Normative requirement. This refers to the level of intake that
serves to maintain a level of tissue storage or other reserve that is judged
[by the Expert Consultation] to be desirable.
Ideally the establishment of estimates of requirement of essential element

should be based on functional criteria for adequacy. For many trace elements
sensitive and specific criteria are lacking and other approaches have to be
adopted. The factorial technique, i.e., the sum of the obligatory endogenous
losses of the element via skin, kidney, and intestine with the addition of require-
ments for synthesis of new tissue in periods of growth, has been the basis for
the estimates of zinc requirement in the WHO 1996 report (6). This approach
was originally used for estimates of protein requirements. The obligatory losses
are usually determined in balance studies at different intakes. The principal prob-
lem in relation to zinc has been to account for the ability to adapt to different
intakes by changes in endogenous losses.
Most early reports on recommended intakes of essential elements have pro-
vided estimates of the requirements of individuals and the ‘‘recommended’’ or
‘‘safe’’ level of intake has been defined as the average requirement ϩ 2SDin
requirement. Thus for an individual consuming this amount of element there
would be a very low probability that the individual’s requirement was not met.
The WHO 1996 report is concerned with population (group) mean intakes rather
than intakes of individuals. The lower limit of population mean intake is set so
that very few individuals in the population (group) would be expected to have
intakes below their requirement; i.e., the population mean intake corresponds to
the estimates of average individual requirement ϩ 2 SD in intakes. The term
‘‘population’’ in the WHO 1996 report refers to a group that is homogeneous in
terms of age, sex, and other characteristics believed to affect requirement and
not, for example, to demographically or culturally defined groups. The variability
in usual intakes within a population group is usually larger than estimates of
variability of requirements and it has empirically been demonstrated that the eval-
uation of the prevalence of inadequate intakes is relatively insensitive to the vari-
ability in requirements.
WHO/FAO/IAEA Expert Consultation (6) identified the need to work with
recommendations of mean population intakes, since it is difficult in practice to
Copyright © 2002 Marcel Dekker, Inc.

give a precise description of upper and lower ends of intake distributions. They
defined the mean normative requirement so as to protect 98–99% of the popula-
tion from minimal adverse effects (including undesirably low tissue stores). The
upper limit of population mean intake to avoid (minimal) toxicity in 98–99% of
the population was also discussed (6). If 98–99% of the population is protected
from deficiency and the same percentage is protected from toxicity, this means
that 96–98% of the population is protected from both deficiency and toxicity.
Based on these considerations, SRPMI was derived (6) thus protecting 96–98%
of healthy persons in specific gender and life stages from even minimal effects
of deficiency or toxicity.
3. FACTORS MODIFYING DOSE-RESPONSE
RELATIONSHIPS
As mentioned earlier, homeostatic mechanisms regulating ETMs are dependent
on basic properties of the essential metals, i.e., their belonging to one of three
groups, namely those that are handled as cations (zinc, iron, and copper), anions
(molybdenum and selenium), or bioinorganic complexes (e.g., cobalt as cobala-
min). These properties in terms of speciation of the elements are of fundamental
importance to their behavior in the human body including bioavailability and
also influence interactions. There is a considerable body of literature available
on factors influencing dose-response relationships of metals. Early literature on
this subject was reviewed by Nordberg (15) and a recent review is included in
the 1996 WHO document (6). The following are a few aspects.
3.1 Bioavailability, Uptake, and Utilization
Bioavailability of trace metals is influenced by chemical form or species but also
by such factors as food source, or dietary media, nutritional state, age, pathologi-
cal conditions, and interactions with other nutrients or toxic substances.
These factors are important and should be considered both when estimating
the nutritional requirement and when assessing toxicity. A number of physiologi-
cal and dietary variables influence trace element utilization (6). Intrinsic (or phys-
iological) variables that influence the absorptive process, such as the poorly regu-

lated situation during infancy for the absorption of a number of elements such
as chromium, iron, and zinc and the probable decline in absorptive efficiency
during senility (copper and zinc). There may be an adaptation to low trace element
status or high demand by modifying activity/concentration of receptors involved
in uptake from gastrointestinal tract (chromium, copper, manganese, zinc).
With regard to metabolic/functional interactions there is an interdepen-
dence of elements in processes involved in storage and metabolism, e.g., in rela-
tion to catecholamine metabolism (copper and iron) and in relation to iodine
Copyright © 2002 Marcel Dekker, Inc.
utilization (selenium) and protein synthesis (zinc). With regard to extrinsic (di-
etary) variables, mucosal uptake may be influenced by the solubility and molecu-
lar dimensions of trace-element-bearing species within food, digesta, and gut
lumen (iron oxalates, zinc, and iron phytates associated with calcium). Dietary
components may also increase the mobility of an ETM; e.g., citrate enhances
zinc absorption.
Antagonism involving, for example, competition with receptors regulating
absorption transport and storage have been shown to limit ETM uptake and stor-
age (e.g., in cadmium/zinc/copper antagonism).
3.2 Age, Gender, Pregnancy, and Lactation
Growth and development of the fetus is dependent on availability of essential trace
metals, for example zinc. Developmental abnormalities of the nervous system have
also been shown in experimental animals as reviewed by Sandstead (16). In in-
fancy, gastrointestinal absorption may be higher, that is, less well regulated, than
in later stages of life. In the elderly, intestinal uptake of trace metals may decline
even in those with normal health (17). There are also differences in nutritional
requirements depending on differences in metabolic handling of trace metals be-
tween the two sexes. For example, men are larger of stature than females. Skeletal
size is related to height as is body calcium. Protein and energy requirements are
considerably lower for females than for males. For these reasons it is important
to define specific nutritional requirements and toxic levels separately for men and

women and sometimes in relation to age. Increased demand for ETM, particularly
iron, zinc, and copper, occurs during pregnancy and lactation (6,18). Specific rec-
ommendations for pregnant and lactating women are therefore warranted.
3.3 Interactions
Interactions between ETMs are important in determining bioavailability and, in
turn, the AORI for metals involved. For example, copper can interact with a
number of other nutrients (19) leading to reduced availability.
Copper uptake in the gut is inhibited by high zinc intakes. Both a direct
interaction and an interaction mediated by metallothionein induction may explain
this phenomenon. It is well known that zinc can induce metallothionein synthesis
in intestinal cells. Such metallothionein may sequester copper, since copper has
higher affinity to SH groups on metallothionein than zinc. By this mechanism
copper may be trapped in the intestinal mucosa, not reaching the systemic circula-
tion to the same extent as in persons with normal zinc intake and therefore in-
creasing susceptibility to copper deficiency. The critical effects of zinc toxicity
are considered to be related to induced copper deficiency (20).
There are some studies in animals indicating a decrease of copper absorp-
tion when intake of iron is high. In human preterm neonates the usual increase
Copyright © 2002 Marcel Dekker, Inc.