g/person/day.
This Guidance document represented current agency thinking in regards to the available science at the time it was issued. It no longer represents the current state of science and is presented here for the historical record only.
United States Food and Drug Administration
200 C St., S.W.
Washington, D.C. 20204
August 1993
CONTENTS
i. AUTHORS AND CONTRIBUTERS ii. EXECUTIVE SUMMARY iii. FORWARD I. STATUTORY AUTHORITY II. PUBLIC HEALTH STATEMENT III. SAMPLING OF AND TRACE ELEMENT ANALYSIS IN SHELLFISH 1. Sampling 1.1 Field Sampling Procedure 1.2 Laboratory Sample Preparation 2. Analytical Methodology 2.1 Mineralization 2.2 Determinative Techniques 3. Quality Assurance/Quality control (QA/QC) IV. CONSUMPTION AND EXPOSURE ASSESSMENT 1. Shellfish Intake 2. Lead Concentrations in Shellfish 3. Lead Exposure form Shellfish 4. Background and Relative Source Contribution to Lead Exposure 4. Select Subpopulations V. HAZARD ASSESSMENT 1. Adverse Health Effects of Lead 1.1 Children, Infants, and Fetuses 1.2 Adults 2. Lead Ingestion and Blood Lead Levels 2.1 Infants, Young Children, and Adults 2.2 Older Children 2.3 Pregnant Women and Fetal Exposure 2.4 Women of Childbearing Age and Fetal Exposure 2.5 Lactating Women and Infant Exposure 3. Provisional Tolerable Total Intake Levels VI. LEVELS OF CONCERN VII. REFERENCES
Michael A. Adams, Ph.D
Chemistry Review Branch, HFS-247
Division of Product Manufacture and Use
Office of Pre-Market Approval
Michael Bolger, Ph.D., D.A.B.T.
Contaminants Standards Monitoring and Programs Branch, HFS-308
Division of Programs and Enforcement Policy
Office of Plant and Dairy Foods and Beverages
Clark D. Carrington, Ph.D., D.A.B.T.
Contaminants Standards Monitoring and Programs Branch, HFS-308
Division of Programs and Enforcement Policy
Office of Plant and Dairy Foods and Beverages
Curtis E. Coker
Special Assistant to the Director, HFS-301
Office of Plant and Dairy Foods and Beverages
Gregory M. Cramer, Ph.D.
Policy and Guidance Branch, HFS-416
Division of Programs and Enforcement Policy
Office of Seafood
Michael J. DiNovi, Ph.D.
Chemistry Review Branch, HFS-247
Division of Product Manufacture and Use
Office of Pre-Market Approval
Scott Dolan
Elemental Research Branch, HFS-338
Division of Pesticides and Industrial Chemicals
Office of Plant and Dairy Foods and Beverages
This document has been developed to satisfy requests from local and state officials for federal guidance regarding the public health significance of lead in shellfish. (The term "shellfish" is used in a general sense throughout this document and is meant to include both molluscan bivalves and crustacea.) This document is designed to assist local and state health officials in their deliberations concerning the possible need to either issue consumption advisories or to close waters for fishing because of excessive lead contamination. The contents of this document include sections on FDA's statutory authority, a public health statement, sampling techniques and trace element analysis, consumption and exposure information, hazard assessment, and a discussion of estimating levels of concern for local consumption advisories or water closures.
Lead is a widely distributed and ubiquitous element found in a
variety of minerals. Environmental lead contamination arises
from various sources including manufacturing processes, paints
and pigments, and atmospheric emissions from motor vehicles,
incineration of municipal solid wastes, and combustion of coal.
The primary sources of lead intake in the general population are
food (including water), soil, paint and dust. The levels of lead
exposure from different sources and routes of exposure indicates
that the levels of risk to children and adults varies widely
between the sources and routes of exposure. Data from FDA's
Total Diet Study (which includes water but does not include
shellfish) suggest that the average dietary intake of lead for
the population is around 5 to 10 g/person/day.
Adults absorb approximately 5-15% of ingested lead and retain less than 5%. Children absorb about 50% of ingested lead. Lead distributes in the blood, soft tissues and bones with blood and soft tissues comprising the active pool and bones representing the storage pool. The half-life of lead in blood is about 35 days and in bones it is 5 years to decades depending on the types and location of the bones. Total body burden for children under age 10 is estimated to be less than 0.1 mg/kg but it will increase with a person's age to levels ranging from 1.4 - 5.7 mg/kg in an adult who has reached the later decades of life.
Acute lead toxicity, which occurs at high levels of lead
exposure, generally in occupational settings, rarely occurs in
part because lead is a cumulative poison with relatively low
solubility. Chronic toxicity which is associated with low to
intermediate levels of exposure, is manifested in a number of
ways including loss of appetite, anemia, headaches, nervous
irritability, muscle and joint pains, and fine tremors. Lead
affects the nervous system and at higher level exposure causes
peripheral neuropathy in adults and encephalopathy in children.
In fetuses, levels as low as 10 g/dl in umbilical cord blood
have been reported to adversely affect neurological development.
The provisional tolerable total intake levels (PTTIL) in this
document were developed using information on the lowest levels of
lead exposures associated with adverse effects (i.e.,
neurobehavioral and cognitive development). The recommended
PTTILs are 6 g/day for children up to the age of 6 years, 15
g/day for children 7 years and older, 25 g/day for pregnant
women and 75 g/day for adults.
Surveys of contaminants in shellfish conducted by FDA and the
National Marine Fisheries Service have found mean lead levels
ranging from 0.1 ppm up to 0.8 ppm. FDA has combined these
survey results with nationally representative shellfish
consumption information to estimate the range of lead exposures
that is possible among shellfish consumers. For individuals who
chronically consume an average of either 15 g/day of molluscan
bivalves or 17 g/day of crustaceans (90th percentile average
intake over 14-days for consumers among survey population) that
have mean lead levels of 0.3 ppm and 0.6 ppm, respectively, lead
intake will average 4 g/person/day and 10 g/person/day.
Local patterns of shellfish consumption and/or levels of lead contamination may vary from national averages. Hence, lead exposures from shellfish in particular regions may also vary from values estimated using national figures. In order to decide whether local lead exposure levels are of concern, it is suggested that the PTTIL figures for lead be used to calculate Levels of Concern, either maximum permitted levels of chronic shellfish consumption or maximum permitted levels of lead contamination.
Although local figures for shellfish consumption or shellfish contamination would be most appropriate for evaluating local situations, reference to national figures may also prove useful. As an example, if it is assumed that total lead exposure for pregnant women is derived solely from shellfish, it is calculated that the lead level of concern for pregnant women consuming molluscan bivalves on a chronic basis at the 90th percentile level would be 1.7 ppm. The corresponding consumption level of concern for individuals consuming molluscan bivalves with lead levels equivalent to the highest average level (0.3 ppm) found in FDA's national surveys is 83 g/person/day.
The PTTIL figures consider only the amount of lead intake, not the source of the lead. It is imperative when assessing the hazard and risk of lead from a particular source that this be done in the context of total lead exposure. This means that the assessment must consider the relative risk of the source, how it contributes to the total risk of lead, as well as the avoidability of the lead exposure. This way, appropriate measures are considered that are not disproportionate to the risk from that one source. In addition, due to the ubiquitous nature of lead in the environment and the difficulties in obtaining reproducible and accurate lead analyses, it is essential that the analytical methods used to support any assessment of lead hazard and risk be of the highest quality.
Purpose
Over the years, and particularly of late, concern has been raised regarding the public health significance of the presence of contaminants in shellfish in both fresh water and marine species. Local and state officials have repeatedly sought guidance at the federal level on these matters; in particular, the advice and position of the U.S. Food and Drug Administration (FDA) has often been requested. FDA is developing a series of guidance documents in an attempt to satisfy the requests for federal guidance. They are designed to assist local and state health officials in their deliberations concerning contaminants in shellfish and the possible need to either issue fish consumption advisories or to close waters for fishing. The term "shellfish" is used in a general sense throughout these documents and is meant to include both molluscan bivalves and crustacea.
Standards have been developed for those contaminants which have been identified as national problems (e.g. methylmercury). Guidance documents have been and will continue to be developed for contaminants which could cause health effects, based on the toxicity of the contaminant and potential exposure. These health effects generally have a low probability of occurring. In some localities, however, adverse health effects may occur under extreme conditions of exposure. Should such conditions exist or occur the information in this guidance document indicates how tolerable levels of shellfish consumption or contamination might be determined; however, it does not dictate an approach or decision regarding a particular contaminant in shellfish. These guidance documents have no statutory authority. They merely present the relevant scientific information on each contaminant. How this information is used is entirely up to public health officials who consult them and may in large part be determined by the particular circumstances of each case.
Based on conclusions reached from a meeting with representatives of state public health organizations, the decision was made not to develop a single advisory level. Instead, a broad guidance approach was developed to address the relevant information on a contaminant so that the information can be used as part of the overall determination of the public health significance of contaminants in shellfish.
Selection of Contaminants
The first guidance documents (for arsenic, cadmium, chromium, lead, and nickel) address contaminants chosen because they are most likely to occur and frequent concerns have been raised regarding their presence in shellfish. Concern about these elements is to be expected, since shellfish tend to accumulate elemental contaminants present in the environment.
Relevant Statutory Authority
The guidance documents were developed with a clear understanding of the statutory responsibilities of the Environmental Protection Agency (EPA) (e.g., Clean Water Act), the FDA, and the states. There is no intent to circumvent the statutory authority of any state or federal authority.
Scope
These guidance documents have been designed for use by public health officials at the federal, state and local level, other interested parties and members of the general public. These documents are being issued to present FDA's assessment of the current state of knowledge on specific contaminants and they will be revised when important new information becomes available. The agency requests and strongly encourages anyone with relevant information to add to these documents to contact the Office of Seafood at the address shown on the cover of this document.
Each guidance document begins with a public health statement much like that given in the toxicological profiles published by the Agency for Toxic Substances and Disease Registry (ATSDR). The sections that follow present information about sampling shellfish, analytical procedures for analysis of elemental contaminants in shellfish, consumption and exposure assessments, and conclude with a summary of toxicological and adverse effects of these contaminants. Each document identifies and summarizes the key literature describing the information, including the work of other health organizations (e.g toxicological profiles - ATSDR and environmental health criteria -International Program on Chemical Safety/World Health Organization).
The purpose of the Federal Food, Drug, and Cosmetic Act (FD&C Act) is to ensure a safe and wholesome food supply. The FD&C Act and other related statutes, including the Public Health Service Act, provide the regulatory framework under which the Food and Drug Administration assesses the effects of environmental contaminants on the safety of consumption of fish and shellfish (molluscan bivalves and crustacea). FDA has jurisdiction over foods that are intended for introduction into, or have been shipped in interstate commerce.
The basic provision of the Act dealing with poisonous or deleterious substances in food is section 402(a)(1). This section states:
(a)(1) if it bears or contains any poisonous or deleterious substance
which may render it injurious to health; but in case the substance is not
an added substance such foodshall not be considered adulterated under
this clause if the quantity of such substance in such food does not
ordinarily render it injurious to health;
This section applies to all poisonous or deleterious substances, whether they are naturally occurring or added.
Section 402(a) (1) of the Act distinguishes between naturally occurring and "added" substances and sets different standards for each. Naturally occurring substances would adulterate a food only if they render the food "ordinarily" injurious to health. Added substances would adulterate the food if the food "may" be injurious to health.
The term "added" in this legal context is important. An added poisonous or deleterious substance is a substance that does not occur naturally in foods. A "naturally occurring poisonous and deleterious substance" is a substance that is an inherent, natural constituent of a food and is not the result of environmental, agricultural, industrial, or other types of contamination. When a naturally occurring, poisonous, or deleterious substance is increased to abnormal levels through mishandling or other intervening acts, it is regarded as an added substance.
Under conditions where an unavoidable contaminant occurs at a level that is of concern to FDA, FDA may establish a formal tolerance limiting the extent of allowable contamination of a food. Compliance with a tolerance is strictly determined on the basis of the results of analysis of commingled representative subsamples of edible portions taken from a fish shipment, not on the basis of a single fish. However, when toxicological data are scanty or conflicting, when additional data are being developed, or when other conditions are rapidly changing, FDA may choose not to establish a tolerance. Nevertheless, it may still be appropriate to take some regulatory action to control exposure to a contaminant. In such circumstances, FDA may consider developing guidelines or regulatory limits.
The authority of the FD&C Act extends only to fish and shellfish that are moving in interstate commerce. Accordingly, tolerances and guidelines are typically established on a national basis when it is judged that a national problem exists for a particular contaminant. Thus, guidelines are generally, but not always, tailored to national needs and national patterns of consumption. For example, consumption levels of fish on a national per capita basis are generally considerably less than those typical of sports fishermen or of individuals living near lake shores or coastal regions of the U.S. Nonetheless, the toxicity information {Reference Dose, (RFD) or Acceptable Daily Intake (ADI)} that is considered in the setting of national guidelines, may be useful to the states in establishing local controls or advisories on local shellfish consumption. If a potential local health threat exists, a state or locality may wish to issue warnings or provide guidance on the quantity of contaminated shellfish that may be safely consumed. Local authorities can utilize the available federal toxicity information and compare it with the level of the contaminant found locally and local shellfish consumption patterns to establish acceptable levels.
Lead is a widely distributed and ubiquitous element found in a variety of minerals. In most inorganic compounds lead is in the +2 oxidation state. Most salts are not readily soluble in water. The production of lead from lead ores involves mining, processing, roasting and sintering, reduction and refining. Commercially, it is used primarily in the production of lead-acid batteries, in paint pigments, as gasoline additives (tetramethyl- and tetraethyl lead), for soldering and plumbing, in cable sheathing, in metal products, and in ammunition.
Environmental lead contamination arises from various sources
including mining, smelting, refining, manufacturing processes
(battery plants), paints and pigments, atmospheric emissions from
motor vehicles, combustion of coal, recycling of batteries,
incineration of municipal solid wastes and hazardous wastes. The
atmospheric concentration of lead ranges between 0.3 - 1.1 g/m3 in
urban areas and 0.15 - 0.3 g/m3 in rural areas.
Nonoccupational sources of lead exposure include: hobbies, home distilled whisky, and acidic foods contacting lead soldered containers and improperly fired lead-glazed ceramics. Additional exposure in some children results from practices of pica (old lead-based paints, dusts or soil) or treatment with Hispanic, Hmong, and Indian folk medicine. The frequent hand-to-mouth activity of infants and children is an important consideration in their exposure to lead from soil and dust. Children under 3 years have been shown to ingest approximately 30-100 mg of soil and dust per day.
In the general population the primary sources of lead intake are food, soil, paint and dust. Lead in the diet can be attributed to (a) natural sources of lead in the soil; (b) deposition of lead particles onto crops, forage, feed, soils, and water; and (c) harvesting, processing, transporting, and preparing and storing food products. Lead-containing solder used for cans, though now greatly reduced, still contributes to overall dietary lead. Leaching of lead from ceramic ware and crystal may also contribute to lead in the diet.
The primary factor for determining absorption of lead from dietary sources including drinking water is the efficiency of the gastrointestinal (GI) absorption. GI absorption varies with one's age, diet, and nutritional status. Deficiencies of iron, copper, zinc, calcium, and vitamin D are associated with increased GI absorption of lead in animals. Evidence suggests that transport of lead from the GI tract to the blood is a saturable process and thus the intake of large doses may not indicate proportionally larger uptake into the blood.
Data from FDA's Total Diet Study (which includes water but does not
include shellfish) suggests that the average dietary intake of lead
for the population is around 5 to 10 g/person/day. Adults absorb
approximately 5-15 % of the ingested lead and retain less than 5%.
Children absorb about 50 % of ingested lead. Increased absorption
combined with decreased excretion may account for the increase in
susceptibility to lead toxicity in children. Children, infants and
fetuses are at particularly high risk for lead toxicity. Women of
child-bearing age and pregnant women are considered to be at an
increased risk due to the increased susceptibility to lead of a
fetus, especially during the period of neuronal development.
Lead serves no beneficial purpose in the body. It distributes in the blood, soft tissues, and bones, with blood and soft tissues comprising the active pool and bones representing the storage pool. More than 95% of the lead found in human blood is bound to erythrocytes under steady-state conditions. Most of the erythrocyte lead is bound within the cell primarily to hemoglobin. In plasma and extracellular fluids, nearly all of the lead is bound to proteins (mainly albumin). Ninety percent of the body burden of lead is found in the bones at steady state. The half-life of lead in bones is 5 years to decades depending on the type and location of the bone. In blood, the half-life of lead is estimated to be 35 days, and in soft tissues it is intermediate between these two extremes. Lead accumulates in the skeletal system. Total body burden for children under age 10 is estimated to be less than 0.1 mg/kg but it will increase with a person's age to levels ranging from 1.4 - 5.7 mg/kg in an adult who has reached the later decades of life.
Acute lead toxicity, which may occur at high levels of lead exposure (generally in occupational settings), rarely occurs since lead is a cumulative poison with relatively low solubility. Chronic toxicity, which is associated with low to intermediate levels of exposure, is manifested by loss of appetite, a metallic taste in the mouth, constipation, anemia, pallor, malaise, weakness, insomnia, headaches, nervous irritability, muscle and joint pains, fine tremors, encephalopathy, and colic. At the cellular level lead exerts its toxicity by complexing ligands and inhibiting enzymes. The most critical target sensitive to subtle increases in lead levels for adults is the hematopoietic system. At very low lead concentrations heme synthesis is inhibited, resulting in anemia. Lead interferes with several enzymes that participate in the heme synthesis pathway including delta-aminolevulinic acid synthetase (ALA-S), delta-aminolevulinic acid dehydratase (ALA-D) and ferrochelatase (heme synthetase). Erythrocyte ALA-D activity is the most sensitive biological indicator of lead toxicity.
Lead intoxication affects the nervous system causing peripheral
neuropathy in adults and encephalopathy in children. In fetuses,
levels as low as 10 g/dl in umbilical cord blood have been
reported to adversely effect neurobehavioral development. Similar
adverse effects on intelligence are seen in children postnatally
exposed to lead that resulted in blood lead levels of 10/dl. The
most important symptoms of pediatric lead poisoning (in descending
order of frequency) are: drowsiness, irritability, vomiting,
gastrointestinal symptoms, ataxia, stupor and fatigue. Lead has
toxic effects in the kidney producing either reversible renal
dysfunction, predominantly in children with acute poisoning, or
irreversible chronic nephropathy usually from chronic occupational
exposures.
Other pathological findings due to lead toxicity include gout,
hypertension, sterility, spontaneous abortions, neonatal mortality
and morbidity, and suppression of the immune system. A number of
studies show that lead poisoning is associated with cardiotoxic
effects (electro-myocardiographic abnormalities) in both children
and adults as well as suggestions of significant association
between moderately increased blood lead levels (>30 g/100 ml) and
a small increase in blood pressure in males. Case reports of
clinically overt lead poisoning and controlled studies of male
workers have shown that exposure to lead is associated with
depressed sperm counts and abnormal mobility and morphology and
also that moderate exposure to lead was associated with both
primary and secondary effects of lead on the testes.
In rodent studies lead acetate, lead subacetate and lead phosphate are carcinogenic to rats and lead acetate is carcinogenic to mice following oral or parenteral administration producing both benign and malignant tumors of the kidney. Lead arsenate and lead chromate have also been classified as carcinogenic. No final conclusions as to the carcinogenicity of lead in humans have been reached by IARC and EPA although several studies have shown increased cancer mortality among lead smelter and battery plant workers, particularly with respect to cancers of the GI tract and respiratory system. Evidence for the genotoxicity of lead is conflicting in regard to the induction of chromosomal aberrations in peripheral lymphocytes of lead-exposed populations. Soluble lead acetate and nitrate are mutagenic in V79 cells but do not include sister chromatid exchange and DNA single strand breaks.
Levels of lead exposure from different sources and routes of exposure (e.g., dust, soil, paint, lead solder, etc.) indicate that the level of risk to children and adults varies widely between the sources and routes of exposure. For many individuals, their total lead exposure from an individual source (e.g., paint) is significant. This is evidenced by the fact that known, subtle, adverse effects occur in humans at levels of lead exposure that are observed in the general population.
The provisional tolerable total intake levels (PTTIL) discussed in
this document were developed using information on the lowest levels
of lead exposure associated with adverse effects (i.e.,
neurobehavioral and cognitive development). The PTTIL figures
reflect the rapid developments in identifying adverse effects
associated with low levels of lead exposure. The recommended
PTTILs are 6 g/day for children up to the age of 6 years, 15
g/day for children 7 years of age and older, 25 g/day for
pregnant women and 75 g/day for adults. These intake levels were
developed to be applied to short term and chronic exposures and
were not meant to be applied to incidents involving acute
exposures.
It is imperative to keep in mind when assessing the hazard and risk of lead from any source that this analysis be done with the understanding that lead exposure for any population occurs from a number of sources which vary in degree and level of risk. Further, given the ubiquitous nature of lead in the environment and the difficulties in obtaining reproducible and accurate lead analyses, it is essential that the analytical methods used to support any assessment of lead hazard and risk be of the highest quality.
In this section methods for the determination of elemental contaminants in shellfish are examined. Necessary steps for shellfish analysis are outlined without repeating published methodologies. Specific analytical procedures are cited.
1. Sampling
Collecting, processing, preserving, and shipping primary samples are the critical steps required for generating valid analytical data. A detailed sampling plan for proposed specific sample collection sites should be developed before actual collection is started. The plan should include site locations, target species, target contaminants, sampling times, types of samples, and sample replication. Samples must be representative of the site or lot being studied. In addition, these steps must be conducted in a manner that preserves the integrity of the analyte(s) of interest and precludes inadvertent contamination (EPA, 1991). If sampling of processed shellfish products is required, specific sampling procedures can be found in the FDA Inspection Operations Manual (FDA, 1990).
1.1 Field Sampling Procedure
A minimum of 3 replicate composite samples from each sampling location is recommended for a representative site or lot. The shellfish collected should satisfy any legal requirements for harvestable size or weight or be of consumable or "market" size when no legal harvestable requirements are in effect. Selection of the largest animals is recommended if a worst-case exposure sampling is desired (EPA, 1991).
Animals used in composited samples should be of similar size with the total length (size) of the smallest animal having no less than 75 percent of the total length (size) of the largest animal. Composites should be composed of equal numbers of from 10 to 50 animals (depending on the species and size class being sampled) for a total minimum weight of 500 g of tissue homogenate. The relative difference (in percent) between the overall mean length (size) of replicate composites and the mean length (size) of any individual composite should be no greater than 10 percent (EPA In progress).
1.2 Laboratory Sample Preparation
Shellfish should be preserved with ice if they are not processed immediately. If shellfish are shipped or stored more than 24 h before processing, dry ice must be used. All laboratory samples should be prepared and analyzed, or frozen, as soon as possible after collection, preferably within 48 h. All instruments, work surfaces, and containers used in processing laboratory samples must be composed of materials that can be easily cleaned and that are not themselves potential sources of contamination. To avoid cross-contamination, all equipment used in sample handling should be cleaned before each test sample (EPA, 1991). Cleaning plastic equipment for trace element analysis has been studied and guidelines are given by Moody and Lindstrom (1977).
Equipment should be chosen to minimize contamination. Contamination may be minimized by using titanium, aluminum, quartz, TFE (tetraflouroethylene), polypropylene, or polyethylene utensils, and a glass blender or food processor with titanium or stainless steel blades and Teflon, polyethylene, or polystyrene gaskets (FDA, 1975). Stainless steel that is resistant to corrosion may be used if necessary. Stainless steel scalpels have been found not to contaminate mussel samples (Stephenson et al., 1979). However, other biological tissues (e.g., fish muscle) containing low concentrations of heavy metals may be contaminated significantly by any exposure to stainless steel (EPA, 1991). The predominant metal contaminants from stainless steel are chromium and nickel. Titanium utensils have been used successfully and are available commercially (Zeisler et al.,1988; Private Communication 1). Aluminum is also an option to consider, especially if it is not an element being studied. Titanium and aluminum each have the advantage of contaminating the sample with only one element. Quartz utensils are ideal but expensive. The analyst must know the elements of interest and their approximate levels in the species to be analyzed in order to make an informed decision about utensil selection.
Before shellfish are "shucked" (opened), the external shell surface should be thoroughly cleaned with brush and water to remove all sand, dirt, etc., adhering to the shell to prevent contamination of composite samples. After shellfish are shucked, the meat and liquor are carefully removed from the shell and collected to minimize incorporation of shell fragments and sand into the test sample. The presence of shell fragments or sand will complicate mineralization. Separation can be accomplished by transferring the meat and decanting the liquor, leaving sand and shell fragments behind. For analysis of canned, frozen, or otherwise processed products, the test sample should include meats, liquor, and any breading. Thaw frozen products prior to homogenization (McMahon and Hardin, 1968). Once shellfish are frozen, thawing of shellfish samples should be kept at a minimum during tissue removal procedures to avoid loss of liquids.
Comminute entire test sample in a glass blender or food processor to obtain a homogeneous mixture. When a food processor is used, adding dry ice successfully freezes the suspension and minimizes water separation. Precaution: If dry ice is used, check dry ice for interferences and store frozen test samples loosely covered overnight to allow for dissipation of carbon dioxide. Composite test samples should be homogenized to a paste-like consistency before portions are taken for analysis. Store prepared test samples frozen in glass jars with Teflon-lined lids or in Teflon containers. If analysis is not started immediately after homogenization, appropriate analytical portions may be weighed and stored frozen. This will eliminate the need to rehomogenize a thawed test sample.
2. Analytical Methodology
Review of the literature reveals that many inorganic analytical studies of shellfish have been prompted largely by a concern that abnormally high levels of toxic elements contaminate some of the world's shellfish growing areas (Marcus and Thompson 1986; Lytle and Lytle, 1982; Watling and Watling, 1982; May and McKinney, 1981; Micallef and Tyler,1989; Balogh, 1988; Eisenberg and Topping, 1984; Gault et al., 1983; Meeus-Verdinne, 1983; Favretto et al., 1981; Cooper et al., 1982).
This concern has focused mainly on elements of biological interest including antimony, arsenic, cadmium, chromium, copper, lead, manganese, mercury, molybdenum, nickel, selenium, tin, and zinc. Various techniques have been applied to analysis of shellfish; however, in the laboratories surveyed (Private Communications 2-5) routine shellfish analysis is conducted usually by using only a few methods. Most methods of shellfish analysis require mineralization of test portions. The digestion procedures can then be coupled with the determinative techniques presented in 2.2 of this section to form complete analytical methods.
2.1 Mineralization
Digestion procedures fall into one of two general categories, dry ash or wet ash. Dry ash digestions use a long, slow ashing step, usually performed overnight in a muffle furnace. The ashing process is completed by the addition of a small quantity of inorganic acid to the residue and evaporation on a hot plate. The residue is redissolved in acid and the solution is brought to volume with distilled or deionized water. Dry ash procedures can be found in the Association of Official Analytical Chemists (AOAC) method 972.23 (Helrich, 1990). Element loss during the ashing process is greater for dry ash procedures than for wet ash procedures. "Ashing aids" are often employed to prevent loss of the more volatile elements like arsenic and selenium (Maher, 1983; May, 1982).
Wet ash digestions are characterized by short ashing times (normally 3 to 4 h) and the use of acids (HCl, HNO3, H2SO4, HClO4). Wet ashing is performed by adding acid or an acid mixture to the test portion and boiling until digestion is complete. A variety of procedures have been developed using different types of digestion apparatus to retain volatile elements. The most common procedure is performed in beakers on a hot plate. The analytical portion is boiled with HNO3 or a mixture of HNO3 and HCl until digestion is complete (FDA, 1975; ASTM, 1986). Combinations of HNO3, HClO4, and H2SO4 have also been used for more efficient oxidation. Perchloric acid requires constant operator attention, specialized equipment (HClO4 hoods, fume traps, etc.), and safety precautions (Michie et al., 1978).
"Hot-block" digesters have been used for improved temperature control and their reflux capabilities (Agemian et al., 1980). "PTFE (polytetrafluoroethylene) bomb" digesters have been used because digestion can take place in a sealed vessel, thereby retaining volatile elements (Welz and Melcher, 1985).
Another wet ash approach that has been applied to fish analysis is microwave digestion (Schnitzer et al., 1988; Nadkarni, 1984; McCarthy and Ellis, 1991). This technique has been subject to extensive research and development since microwave technology was first applied to digestions (Abu-Samra et al., 1975; Barrett et al., 1978; Nadkarni, 1984; White and Douthit,1985; Kingston and Jassie, 1986, 1988; Burguera et al., 1986; Fernando et al., 1986). Current systems incorporate many features to improve digestion time, element recoveries, contamination, and safety. Improved recoveries can be achieved with systems operating under power-regulated control of pressure because these systems allow digestions to be completed without venting volatile elements. Sealed digestion vessels also reduce acid consumption and, in combination with Teflon construction, reduce contamination problems. Microwave digestion methods developed for shellfish do not require HClO4 to complete mineralization. Microwave digestion has been compared to some of the other digestion procedures (Blust et al., 1988).
Regardless of the digestion procedure selected, appropriate quality assurance and quality control guidelines need to be followed. These include the use of contamination control, digestion blanks, spiked test portions, replicate analyses, an appropriate standard reference material (e.g., National Institute of Standards and Technology: Oyster or Bovine Liver), and recovery calculations (Boyer and Horwitz, 1986; Jones, 1988).
2.2 Determinative Techniques
Selection of a determinative technique is often dictated by available equipment and quantitation requirements. Ease of operation, number of analyses, and familiarity of personnel with techniques are also influential factors. For these reasons only a few methods developed for shellfish analysis are used for routine testing. Spectrometric techniques are most commonly used and include flame atomic absorption spectrometry (FAAS), graphite furnace atomic absorption spectrometry (GFAAS), and inductively coupled plasma-atomic emission spectrometry (ICP-AES) (see Table 1).
FAAS is often used to determine many elements in shellfish analysis because it is easy and affordable (Private Communications 2-5). The American Society for Testing and Materials (ASTM) method D3559 (ASTM, 1986) and the American Public Health Association (APHA) method 303 (APHA, 1986) both use FAAS. FAAS has been combined with high-performance liquid chromatography (HPLC) for the study of metal ion speciation in biological systems. Information about the distribution of metal ions in biological extracts can be used to estimate exposure of an animal to chemical toxins, and to estimate the relationship between chemical form and bioavailability (Guy, 1985).
FAAS has been applied to two methods to improve detection of some elements. First, measurement of arsenic and lead has been improved by on-line generation of volatile hydrides with hydride generation-AAS (HG-AAS) (Holak, 1969). In applications of hydride generation to fish analysis, (Agemian and Thompson, 1980; Julshamn et al., 1982; Cutter, 1985; Maher, 1982, 1986; Madrid et al., 1988) the enhancement of detection limits ranged from a factor of 5 to a factor of over 500 (see Table 1). Hydride generation (HG) has also been coupled with GFAAS (Willie et al., 1986; Sturgeon et al., 1986), direct current plasma-AES (DCP-AES) (Panaro and Krull, 1984), and ICP-AES (de Oliveira et al., 1983; Montaser and Golightly, 1987). HG-AAS has been coupled with HPLC for determination of inorganic and methylated arsenic species in marine samples (Maher, 1981).
GFAAS has been used for measurement of selected elements when FAAS determination limits were insufficient for shellfish analysis (Cruz et al., 1980;Serra and Serrano, 1984). Clearly, GFAAS is more sensitive than FAAS although GFAAS analysis time is longer. The main problems with GFAAS shellfish analysis are matrix and spectral interferences. Deuterium arc and Zeeman-effect background corrections have been applied to reduce spectral interference in shellfish analysis. While deuterium arc background correction is plagued by spectral interferences, Zeeman-effect background correction appears to be satisfactory for routine analysis of marine biological tissue (Welz and Schlemmer, 1986; McMahon et al., 1985). Various "matrix modifiers" (organic and inorganic reagents) have been used to reduce matrix effects. Molybdenum, lanthanum, and ammonium dihydrogen phosphate have been used as matrix modifiers for shellfish analysis (Poldoski, 1980; Mckie, 1987). Solvent extraction is another approach taken to minimize matrix effects (Stephenson and Smith, 1988; Harms, 1985).
Solid sampling has been developed for GFAAS to increase relative sensitivity by avoiding the large dilution factors required in the sample dissolution. Solid sampling also eliminates contamination and/or loss of analyte resulting from dissolution. Solid sampling has not been widely used for shellfish analysis because of possible interference by very large background matrix absorbance (Chakrabarti, 1979; May and Brumbaugh, 1982). A complete analysis by GFAAS can be found in ASTM method D3559 (ASTM, 1986).
ICP-AES has also been used for routine elemental analysis of shellfish (Grant and Ellis, 1988). Sequential or simultaneous mode ICP-AES allows rapid analysis, dramatically improving throughput. Detection limits in ICP-AES are generally the same as those in FAAS except for elements that are difficult to atomize. Such elements have lower ICP-AES detection limits because of the higher temperature of the plasma (see Table 1). A complete analysis by ICP-AES can be found in the EPA's water method 200.7 (EPA, 1979).
Although not widely used for routine shellfish analysis, techniques have been developed using voltammetry for trace element determination (ASTM, 1986; Adeloju and Bond, 1983; Breyer and Gilbert, 1987), polarography (Lemly, 1982), ICP-MS (Munro et al., 1986; Beauchemin et al., 1988; Park et al., 1987), HPLC with electrochemical and spectrophotometric detection (Bond and Nagaosa, 1985), and neutron activation analysis (NAA) (Zeisler et al., 1988; LaTouche et al., 1981; DeSilva and Chatt, 1982; Chisela and Bratter, 1986).
Regardless of the analytical method chosen, sound scientific analytical principles must be followed. All methods used by a laboratory must be validated by the laboratory prior to routine sample analysis. The detection and quantitation limits, and accuracy and precision of each method must be assessed and documented to be sufficient for reliable quantitation at or below the level of interest for each metal. All analytical methods used routinely for the analysis of shellfish should be documented thoroughly; formal standard operating procedures (SOP's) are preferred. A published method may serve as an analytical SOP only if the analysis is performed exactly as described. Analytical SOP's should be followed as written (EPA, 1991).
3. Quality Assurance/Quality Control (QA/QC)
Initial demonstration of laboratory capability and routine analysis of appropriate QA/QC samples to document data quality and to demonstrate continued acceptable performance is essential. QA/QC requirements should be based on specific performance criteria, or control limits, for data quality indicators, such as accuracy and precision. Typically, control limits for accuracy are based on the historical mean recovery plus or minus three standard deviation units, and control limits for precision are based on the historical standard deviation or coefficient of variation (or mean relative percent difference for duplicate samples) plus three standard deviation units. Some recommended QA/QC procedures include: instrument calibration and calibration checks, assessment of method detection and quantitation limits, assessment of method accuracy and precision, routine monitoring of interferences and contamination, regular external QA assessment of analytical performanceinterlaboratory comparison programs, appropriate documentation, and reporting of data (including QA/QC data) (EPA, 1991).
Table 1. Quantitation Limits (
ICP-AES FAAS GFAAS(f) Solution Hydride Solution Hydride Element neb.(b) gen.(c) neb.(d) gen.(e)
As 0.5 0.008 10.0 0.03 0.01 Cd 0.05 0.007 0.0005 Cr 0.3 0.20 0.003 Ni 0.2 0.3 0.02 Pb 0.8 0.03 0.3 0.003 0.005 (a) Quantitation limits (10 sigma) were estimated from instrumental detection limits (3 sigma), and a 5 g test portion in 50 ml of test solution was assumed. (b) Values for pneumatic nebulizer/ICP-AES are taken from Applied Research Laboratories literature on Model 3580 OES. (c) Values are reported in Montaser and Golightly (1987). (d) Values are taken from Varian literature on Models AA-1275 and AA-1475 best value was selected for either C2H2-air or C2H2-N2O flame. (e) Value for arsenic are reported in Robbins and Caruso (1979); value for lead in Ikeda et al. (1982). (f) Values are taken from Varian literature on Model Spectra AA-300 (nominal 20 uL injection).
The following sections provide estimates of chronic shellfish intake as well as estimates of lead exposures resulting from chronic shellfish consumption. In addition, estimates of lead exposure are provided for background sources, both dietary (i.e. non-seafood) and non-dietary sources.
1. Shellfish Intake
The frequency of shellfish eating occasions has been tabulated in the Market Research Corporation of America (MRCA) 14-day survey (5-Year Menu Census, 1982-87) (MRCA, 1988). The MRCA reports that only 13% of the surveyed population consumed crustaceans and only 4.8% of the surveyed population (25,726 individuals, 2+ years) consumed molluscan bivalves. Using standard portion sizes from the USDA's 3-day National Food Consumption Survey (NFCS, 1977-78) (Pao et al., 1982) we estimate the 14-day-average mean and 90th percentile daily intakes of molluscan bivalves. These are presented in Table 2. The intakes for crustacean shellfish are presented in Table 3.
Table 2. 14-Day average intake of molluscan bivalves,
grams/person/day, for eaters-only.
Age Group Mean 90th
Percentile
2+ years (all ages) 10 15
2-5 years 4 8*
(male/female)
18-44 years** 12 18
(male/female)
* Estimated value. Reliable data are not available in the MRCA
survey. The 90th percentile value is estimated to be twice the
mean (WHO, 1983).
** USDA portion size for 33-44 year age group used in the
calculation. This age subgroup has the highest consumption of
any subgroup in the 18-44 year range (Pao et al., 1982).
Table 3. 14-Day average intake of crustacean shellfish, grams/person/day, for eaters-only.
Age Group Mean 90th Percentile
2+ years (all ages) 9 17 2-5 years 5 10 (male/female) 18-44 years* 9 19 (male/female) * USDA portion size for 33-44 year age group used in the calculation. This age subgroup has the highest consumption of any subgroup in the 18-44 year range (Pao et al.,1982).
2. Lead Concentrations in Shellfish
In 1985-86 the Agency surveyed the levels of lead in softshell clams (Mya arenaria), hardshell clams (Mercenaria), Eastern oysters (Crassostrea virginica), and Pacific oysters
(C. gigas) (S. Capar, FDA, Division of Contaminants Chemistry, unpublished data). The shellfish samples were harvested from approved waters in 20 coastal states (all coastal states except Alaska and New Hampshire). The results of that survey are tabulated in Table 4.
Table 4. Results of the 1985-86 FDA shellfish survey for lead.
Mean lead level* Range* Species (
Clam, hardshell 0.24 0.054-0.340 Clam, softshell 0.29 0.069-0.430 Oyster, Eastern 0.11 0.027-0.260 Oyster, Pacific 0.06 0.029-0.070 Clams, overall 0.26 Oysters, overall 0.09 * Wet weight basis.
In 1978, the National Marine Fisheries Service (NMFS) published the results of a survey on trace elements in fish (Hall et al., 1978). The lead levels in several species of shellfish were reported. The results are presented in Table 5.
Table 5. Results of the NMFS survey of trace elements in the
fishery resource for selected shellfish.
Range of mean lead levels(a)
Species: (g/g)
Clam, hardshell 0.7-0.8
Clam, softshell 0.4-0.5
Oyster, Eastern 0.6-0.7
Oyster, Pacific 0.6-0.7
Crab, blue, body/claw 0.6-0.7
Crab, dungeness, body/claw 0.7-0.8
Lobster, American, claw/tail 0.5-0.6
Lobster, spiny, Atlantic, tail 0.6-0.7
Lobster, spiny, Pacific, tail 0.5-0.6
Shrimp, several species, tail 0.6-0.7
Range of mean lead levels for 0.4-0.8
Molluscs and Crustaceans
a) Shellfish from a single survey site formed a sample. Lead
content was averaged for each sample. Each species was sampled
at many sites. This table reports the range of the mean lead
levels that encompassed all samples.
Lead levels in molluscan bivalves reported in the NMFS survey are consistently higher than in the FDA survey. Although no clear explanation for this difference is available, it is worth noting that the FDA survey data were gathered seven years or more after the NMFS survey. Perhaps the levels of available lead in the environment had dropped as the result of abatement actions to such an extent that marine organisms sampled in 1985 had lower lead levels than in 1978. Another explanation may be that analytical procedures for lead underwent considerable improvement during this interval. The differences might only reflect differences in analytical procedures. We believe that FDA's data provides more up to date information for estimating possible lead exposures from molluscan bivalves. In the case of crustacea, the NMFS data from 1978 is the most complete data set available. Lacking a more up to date set of data for lead in crustacea, we have used the NMFS data for estimating possible lead exposure from crustacea recognizing that lower estimates might result if more current data were available.
3. Lead Exposure from Shellfish
Using information on chronic shellfish consumption and typical
levels of lead in shellfish, it is possible to estimate lead
intake on a chronic basis for consumers of shellfish. According
to the MRCA survey, only 13% of the population eat crustacean
shellfish, and only 4.8% of the population eat molluscan
shellfish. Assuming that mean lead levels in molluscan shellfish
equal 0.3 g/g (ppm) and 0.6 g/g in crustacean shellfish,
consumer exposures (for eaters only) are as follows:
Table 6. Molluscan bivalves - chronic lead intake for
specified level of contamination, g/person/day.
Contamination at 0.3 ppm
Mean 90th
Percentile
2+ (all ages) 3 5
(male/female)
2-5 1 2*
(male/)
18-44 4 5
(male/female)
* Estimated value. Reliable data are not available in the MRCA
survey. The 90th percentile value is estimated to be twice the
mean (WHO, 1983).
Table 7. Crustacean shellfish - chronic lead intake for
specified levels of contamination, g/person/day.
Contamination at 0.6 ppm
Age Group, Yrs. Mean 90th
Percentile
2+ (all ages) 5 10
(male/female)
2-5 3 6
(male/female)
18-44 5 11
(male female)
4. Background and Relative Source Contributions to Lead Exposure
Intakes of lead from all sources (food, air, soil, and dust) are presented in Table 8. Estimates of dietary exposure to lead are derived from the FDA Total Diet Study (TDS) (which does not include shellfish) (E. Gunderson, FDA, Division of Contaminants Chemistry, unpublished data). These are representative mean values and do not account for any special lead exposure scenarios that a consumer might encounter (e.g., consumption of lead-contaminated shellfish, consumption of wine containing large amounts of lead, and preparation or consumption of food using lead-glazed ceramics).
Table 8. Relative source contributions of lead.
Source (
0-6 months 2(c) 0.07/0.1 - -/- 6-11 months 4.1 0.07/0.1 0.29 1.5/20 26 2 years 5.3 0.07/0.1 0.29 1.5/20 27 14-16 years 7.0 0.17/0.55 0.02 0.11/5.3 13 female 14-16 years 9.1 0.17/0.55 0.02 0.11/5.3 15 male 25-30 years 8.8 0.17/0.55 0.02 0.11/5.3 15 female 25-30 years 9.8 0.17/0.55 0.02 0.11/5.3 16 male 60-65 years 8.6 0.17/0.55 0.02 0.11/5.3 15 female 60-65 years 9.0 0.17/0.55 0.02 0.11/5.3 15 male (a) All values are mean values. Except where noted, values are from EPA (1989). (b) Other than the 0-6 months age group, values are taken from the FDA Total Diet Study, July, 1986 - April 1988 reporting period (E. Gunderson, FDA, Division of Contaminants Chemistry, unpublished data). Values include contributions from solid and liquid foods including water. Shellfish are not included in the FDA Total Diet Study. (c) Infants are assumed to consume ca. 750 mL of formula/day as their sole source of nutrition. A lead level of 2.5 ppb in formula was assumed, resulting in a 5 kg infant consuming about 2
Comparison of the lead exposures from different sources (Table 8) with the exposures estimated for consumers of shellfish (Tables 6 and 7) reveals that chronic shellfish consumption can result in lead exposures that are comparable to cumulative lead exposure from all other foods surveyed in FDA's Total Diet Study (E. Gunderson, FDA, Division of Contaminants Chemistry, unpublished data). It is noted that only a small percentage of the population consumes crustacean shellfish (13%) and molluscan shellfish (4.8%) on a chronic basis. Thus, the total food lead intake for regular and frequent consumers of shellfish might be expected to be significantly higher than those individuals who consume shellfish infrequently or not at all.
5. Select Subpopulations
The preceding exposure estimates for lead intake from shellfish consumption are based on nationally representative food consumption surveys. These surveys may not be suitable for estimating exposures to particular sub-populations or individuals residing in specific regions of the country. State or local organizations will want to tailor estimates to their specific needs.
In addition to the nationwide food consumption surveys mentioned previously {(MRCA, 1988) 14-day Survey; USDA 3-Day National Food Consumption Survey (USDA-NFCS) (Pao et al, 1982)}, the National Purchase Diary (NPD) survey (TRF, 1975) presents a 14-day survey covering the continental United States.
Several other food consumption databases are also available for use in preparing estimates of exposure. Although none have the national character of the MRCA survey, NPD survey, or USDA-NFCS, they do address either the consumption of specific types of food (e.g. fish, shellfish), or they reflect the consumption patterns and habits of regional areas or of specific groups (e.g., fishermen). Some available databases, along with selected shellfish consumption values, are listed in Table 9, below.
TABLE 9.Shellfish Consumption
Seafood Consumption Level Population Source of the Category (g/day) Data
All 5 (availability(a)) Entire U.S. NMFS "Fisheries of Shellfish the U.S."Series 1986)(b) 5 (average) Persons Earning NMFS (1981)(c) More than $15,000 per Year 9 (average) Shellfish-Eaters 1977-1978 Menu Census(d) 16 (90th percentile) Shellfish-Eaters 1977-1978 Menu Census(d) 30 (availability(a)) Shellfish-Eaters NMFS "Fisheries of U.S." Series (1986)(b) Clams 1 (average) New Englanders NMFS (1981)(c) 22 (95th percentile) Texans 1973-1974 Tuna Institute Survey(e) Oysters 0.5 (average) Persons Earning NMFS (1981)(c) More than $15,000 per Year 21 (95th percentile) Californians 1973-1974 Tuna Institute Survey(e) (a) Total availability of edible shellfish "meat" (excluding bones, viscera, shells), fresh, frozen and canned. (b) C. Lewis, FDA, in memo to I. Boyer, FDA, "Estimating a Level of Concern for Cadmium in Shellfish," 10-26-88. (c) Hu (1985). (d) Market Research Corporation of America (MRCA) as reported by C. Lewis, FDA, in memo to I. Boyer, FDA, "Estimating a Level of Concern for Cadmium in Shellfish," 10-26-88. (e) Tuna Research Foundation (1975), Seafood Consumption Study, National Purchase Diary Panel, Inc.,Schaumburg, IL.
It is important to note that the level of consumption, and thus the level of contaminant intake, varies from survey to survey. The variation is controlled by several factors, among which are:
Finally, we note that in some circumstances it may be difficult or impossible to find a survey that adequately addresses a particular public health problem. In this case, the best choice may be to initiate a survey that will be specifically designed to address the question.
1. Adverse Health Effects of Lead
The primary targets for lead toxicity are the nervous system, red blood cells, and the kidney. The effects of concern that appear at the lowest levels of exposure are impaired neuorbehavioral development in children, and increased blood pressure in adults. Presently, there are no levels of lead exposure for children or adults at which it may be considered that no adverse effects occur. Consequently, the provisional tolerable total intake levels proposed in this document are based on the lowest observed effect levels instead of no-observed effect levels.
1.1 Children, Infants, and Fetuses.
Fetuses, infants, and children are at particularly high risk from
lead exposure. Due to the sensitivity of the fetus, particularly
during development of the fetal nervous system, women of
child-bearing age and particularly pregnant women are at the
greatest risk among the adult population. Umbilical cord levels
as low as 10 g Pb/dL in the fetus have been reported to
adversely affect neurobehavioral development (Bellinger et
al.,1987). This level served as the basis for the FDA
proposal to
limit exposure of infants and children to lead from ceramic
pitchers (FDA, 1989). While there is a tendency for cord blood
levels to be, on the average, about 20% lower in the cord than in
the maternal blood supply (EPA, 1989), there is no clear evidence
that the placenta represents a significant barrier to lead. We
therefore consider this effect level of 10 g Pb/dL to be
suitable for limiting intake by pregnant women as well.
1.2 Adults.
Levels of 30 g Pb/dL or less have been associated with
peripheral nerve dysfunction, red blood cell protoporphyrin
elevation, and elevated blood pressure in adults (EPA, 1986;
WHO, 1987). Higher blood levels (50 to 100 g Pb/dL) have been
associated with adverse effects on central nervous system
function, chronic renal failure, reproductive dysfunction in
males and females, and anemia. Several studies have found
correlations between elevated blood pressure and blood lead
levels below 30 g/dl (Pirkle et al.., 1985; Weiss et
al., 1986; Sharp et al.., 1989).
However, in a large study of adult males, a relationship between
blood lead and hypertension could only be observed at levels of
37 g Pb/dl or higher (Pocock et al.., 1984). The higher
incidence of hypertensive disease in workers with occupational
exposure to lead (Selevan et al.., 1985) and elevated blood
pressure in laboratory animals exposed to lead
(Victery, 1988), indicates that the hypertension results from
high blood lead levels, rather than the converse. In conclusion,
the weight of the evidence indicates that a blood lead level of
30 g/dl and greater may result in a significant elevation in
blood pressure which may be expected to increase the incidence of
hypertension related diseases.
2.1 Infants, Young Children, and Adults.
The relationship between lead ingestion and blood lead levels in
children and adults has been estimated to be 0.16 and 0.04 g
Pb/dl blood per g Pb/day ingested, respectively (EPA, 1986).
These conversion factors are appropriate for predicted blood
levels for relatively low level exposures resulting in blood
levels of up to 10 g Pb/dl in children and 30 g Pb/dl in
adults. Higher exposures result in considerably lower increments
in blood lead concentration (EPA, 1989). However, since the FDA
is primarily concerned with relatively low exposures, the
conversion factors for low level exposures are appropriate.
These values which relate daily dietary exposure to lead with
internal blood levels are empirically derived and do not take
into account any specific variation in lead absorption due to sex
or diet (Mahaffey, 1985). Using a conversion factor of 0.16
and a blood level of 10 g Pb/dl, a dietary effect level of 60
(rounded from 62.5) g/day is estimated for children. Using a
conversion factor of 0.04 and a blood level of 10 g Pb/dl, a
dietary effect level of 250 g/day is estimated for pregnant
women. Using a conversion factor of 0.04 and a blood level of 30
g Pb/dl, a dietary effect level of 750 g/day is estimated for
adults.
2.2 Older Children.
The 0.16 conversion factor for children described above, and most
of the other data relating lead intake to lead blood levels, has
been derived from studies on infants. However, due to changes
in body weight and other pharmacokinetic parameters, the
quantitative relationship between lead intake and blood lead
levels may be expected to change with the age of the individual,
with the appropriate conversion factor eventually decreasing to
the adult value of 0.04. Table 10 contains lead ingestion levels
that are predicted by a pharmacokinetic model developed under
contract (EPA, 1989) to result in 10 g Pb/dl in children aged 1
through 10. The dietary intakes were calculated using two
different estimates for the rate of lead absorption. The first
estimate (column 2) assumes that a rate of 48% uptake from the
gastrointestinal tract, an average of values taken from studies
in infants, is also applicable to older children. The second
estimate assumes that there is a progressive decrease in
absorption with age in which the rate of absorption decreases
with decreasing growth rate until it reaches the average of 11%
that has been reported for adults.
Table 10. Predicted Ingestion Levels (
Ingestion Level Age (years) No Growth(a) Growth Empirical
1 71 (48%) 71 (48%) 60 2 48 (48%) 48 (48%) 3 52 (48%) 71 (35%) 4 52 (48%) 71 (35%) 5 54 (48%) 74 (35%) 6 54 (48%) 74 (35%) 7 58 (48%) 133 (21%) 8 77 (48%) 176 (21%) 9 94 (48%) 214 (21%) 10 79 (48%) 181 (21%) 250 (a) All ingestion levels are in
The intake values predicted with the use of a 48% absorption rate
for all ages is close to the value obtained by using the 0.16
conversion factor. A slightly lower intake value is predicted
for children ages 2-7 and the values are slightly higher for
infants and for children aged 8 or older. Even if adjustments
are made for changes in the rate of absorption that may be
expected with development, the figure of 60 g/day is still
reasonably close for children aged 6 or less. For older children
however, the available physiological data indicates that a much
larger ingestion rate (150 to 200 g Pb/day) would be required to
produce the same blood level. Unfortunately, there are no
empirical data to support this prediction.
2.3 Pregnant Women and Fetal Exposure.
A number of physiological changes occur during pregnancy. Since lead is known to be disposed through the same mechanisms which operate to govern the distribution of calcium, the gestational changes which affect calcium may also be expected to affect lead. For instance, the fractional absorption of calcium from the GI tract has been reported to increase by about 70% during pregnancy (Heaney and Skilman, 1971). Some, but not all, studies also indicate that there is increased mobilization of calcium from bone during pregnancy, which may result in a higher blood lead level even with a constant lead uptake (Pitkin, 1985). This increase may be due to a pregnancy-induced calcium deficiency. We have previously considered that an adjustment of the factor for converting dietary exposure to blood lead levels as a result of increased absorption from the GI tract is necessary (memorandum from Clark Carrington, dated 6/12/90). However, in view of empirical evidence that indicates that blood lead levels in pregnant women are not significantly different from those of nonpregnant women and do not change appreciably during pregnancy (Gershanik et al., 1974; Alexander and Delves, 1981), it appears that the physiological changes that occur during pregnancy do not result in a net increase in maternal blood lead levels. One possible explanation is that any increases in calcium and lead uptake from bone or the GI tract are compensated by removal to fetal bone stores. While large releases of lead from bone or increases in GI absorption may be important in particular individuals, they do not appear to be a general phenomenon. Therefore, the conversion factor derived from normal adults (0.04) may also be considered to be suitable for pregnant women for the purpose of limiting fetal exposure to lead.
2.4 Women of Childbearing Age and Fetal Exposure.
While there is no compelling evidence which indicates that there
is an increase in the mobilization of calcium, and therefore
lead, from bone due to pregnancy (Pitkin, 1985), some release is
normal. Under steady-state conditions, the release of lead from
bone and other soft tissue will be equal to the rate of uptake
from blood. The half-lives for the dissemination of lead from
internal stores (40 days for soft tissue, several years for bone)
are long enough that the release of lead from internal stores
during at least the first two months following cessation of
intake from a contaminated source will reflect prior exposure,
rather than current exposure. Using data obtained from adult
males, Rabinowitz, et al. (1976) estimated that the amount of
lead released from bone and soft tissue under steady-state
conditions is equal to about 20% of the daily lead uptake. If it
is assumed that these values also apply to women both before and
during pregnancy, a steady-state blood lead level of 50 g Pb/dl
(10 g Pb/dl / 20 %) would need to be maintained in order to
result in bone lead levels that will result in the release of
lead of an amount sufficient to give a blood lead level of
10 g Pb/dl after exposure is terminated. For a hypothetical
single-source pre-gestational lead exposure that is terminated
upon the onset of pregnancy (e.g., wine), an exposure of 1250
g/day (50 g Pb/dl / 0.04 g Pb/dl per g/day)
would be
necessary to generate blood lead levels causing fetal effects.
Since this is higher than the effect level for adults (750 g
Pb/day), a limit of lead intake which will protect the woman
herself will also protect a developing fetus in a subsequent
pregnancy as long as lead intake is reduced to the appropriate
level (25 g/day) during pregnancy. Therefore, nonpregnant women
of child-bearing age may be considered along with other adults
for the purpose of limiting lead intake.
2.5 Lactating Women and Infant Exposure.
While there has been some concern of the possibility of high lead
exposure occurring through breast feeding, the available evidence
concerning lead levels in human milk indicates that levels are
not significantly higher than those encountered in infant formula
(Ryu et al.., 1983), and that blood lead levels in nursing
infants are somewhat lower than maternal blood lead levels
(Wolff, 1983). In one study which attempted to relate maternal
blood lead levels to levels of lead in breast milk, mean lead levels
less than 3 ppb were encountered in breast milk in a population with
a mean maternal blood level of over 11 g/dl (Rockway et al..,
1984), and there was no significant correlation between maternal
blood levels and milk lead concentrations. We therefore conclude
that there is not sufficient evidence to suggest that exposure to
lead through nursing is a more direct source of exposure to maternal
lead than when the fetus and the mother share a common blood
supply. However, in the absence of data concerning the effects
of high maternal lead intakes on nursing infants, a conservative
approach would dictate that a standard set for pregnant women
should also apply while the mother is nursing.
3. Provisional Tolerable Total Intake Levels
Provisional total tolerable intake levels (PTTIL) are presented in Table 11. These values were obtained by dividing the lowest levels of lead ingestion causing observed effects (as estimated from blood lead levels) by a factor of 10. The PTTIL figures are provisional because safe levels of lead exposure have not been identified. It is recognized that these intake levels are not presently achievable for most children and a significant portion of the adult population. Nonetheless, they represent levels where some margin of safety would be reached.
Table 11. Provisional Tolerable Total Intake Levels (PTTIL)
for Various Population Groups.
Effect Level
Blood Diet PTTIL
Population (g Pb/dl) (g Pb/day) (g Pb/day)
Children Ages 0-6 10 60 6
Children Over 7 10 150 15
Pregnant Women 10 250 25
Adults 30 750 75
The PTTIL figures consider only the amount of lead intake, not
the source of the lead. Even without the contribution from the
diet, lead exposure from dust may be expected to exceed the PTTIL
in a significant fraction of the population, particularly young
children (EPA, 1989). It is imperative when assessing the hazard
and risk of lead from a particular source that this be done in
the context of total lead exposure. This means that the
assessment must consider the relative risk of the source, how it
contributes to the total risk of lead, as well as the
avoidability of the lead exposure. This way, appropriate
measures are considered that are not disproportionate to the risk
from that one source.
The following section illustrates how Levels of Concern, either permitted amounts of chronic shellfish consumption or permitted levels of contamination, can be determined using information on the provisional tolerable total intake levels (PTTIL) for lead. For the purpose of this illustration, it is assumed that total lead exposure is derived solely from shellfish. This approach leads to estimates of the maximum permitted levels of either shellfish intake or levels of lead contamination in the shellfish. As other sources of lead exposure are considered (e.g., relative source contribution including other dietary sources of lead), then corresponding adjustments in the levels of concern will need to be made.
Due to the ubiquitous nature of lead and the difficulties in obtaining reproducible and accurate lead analyses, it is cautioned that any effort to determine total lead exposure, Levels of Concern, or to limit lead exposure should be based on the highest quality analytical data.
The following equations illustrate how the PTTIL for lead can be combined with information on either shellfish consumption or lead levels to estimate the corresponding levels of concern of either shellfish contamination or shellfish intake.
Total Lead Level of Concern: = {PTTIL of Lead} / {Daily Intake of Shellfish}
Consumption Level of Concern = {PTTIL of Lead} / {Lead Concentration}
As an example, the Total Lead Level of Concern is estimated for pregnant women consuming molluscan bivalves on a chronic basis at the 90th percentile level (15 g/person/day, see Table 2).
Total Lead Level of Concern = {25 g/p/d} / {15 g/p/d} = 1.7g/gFor molluscan bivalves with lead levels corresponding to the highest mean concentration found in FDA's survey (see Table 4), the corresponding molluscan bivalve consumption level of concern is calculated as follows:
Consumption Level of Concern = {25 g/p/d} / {0.3 g/g} = 83 g/p/dThe following tables (12-15) present a number of plausible levels of concern, either consumption or concentration, that could be used in the assessment of the potential impact of lead on human health through exposure from shellfish. For reference, the following values should be compared with shellfish intake and lead contamination figures presented in Section IV.
Table 12. Molluscan Bivalve Consumption Levels of Concern(g/p/d)*
Age Group Levels of Concern
Children 0-6 years 20 Children Over 7 years 50 Pregnant Women 83 Adults 250 * Derived from the PTTIL for lead and the highest mean concentration for total lead (see Table 4) found in FDA's 1985-86 survey. These consumption levels do not consider other sources of lead exposure.
Table 13. Crustacean Shellfish Consumption Levels of Concern (g/p/d)*
Age Group Levels of Concern
Children 0-6 years 10 Children Over 7 years 25 Pregnant Women 42 Adults 120 * Derived from the PTTIL for lead and the mean concentration of lead in crustacea (Table 5) reported in the 1978 NMFS survey. These consumption levels do not consider other sources of lead exposure.
Table 14. Lead Levels of Concern in Molluscan Bivalves (
Age Group Mean 90th Percentile Children 2-5 years 1.5 0.8 Pregnant Women 2.1 1.4 Adults 18-44 years 6.3 4.2 * Derived from the PTTIL for lead for each age group and intake figures for molluscan bivalves presented in Table 2. These concentration levels do not consider other sources of lead exposure.
Table 15. Lead Levels of Concern for Crustacean Shellfish (
Age Group Mean 90th Percentile
Children 2-5 years 1.2 0.6 Pregnant women 2.8 1.3 18-44 years 8.3 3.9(male/female) * Derived from the PTTIL for lead for each group and intake figures for crustacean shellfish presented in Table 3. These concentration levels do not consider other sources of lead exposure.
The Levels of Concern presented in the above tables do not take into account other sources of lead exposure. As Tables 8 and 11 illustrate, nonshellfish sources of lead exposure may be equivalent to a major portion of the PTTIL for particular population subgroups. Therefore, it is imperative when assessing the hazard and risk of lead from a particular source that this be done in the context of total lead exposure. This means that the assessment must consider the relative risk of the source, how it contributes to the total risk of lead, as well as the avoidability of the lead exposure. This way, appropriate measures are considered that are not disproportionate to the risk from that one source.
Abu-Samra, A. Morris, J. S. and Koirtyohann, S.R. (1975) Anal. Chem. 47:1475.
Adeloju, A.B. and Bond A.M. (1983) Anal. Chim. Acta 148:59.
Agemian, H., Sturtevant, D.P. and Austen, K.D. (1980) Analyst 105:125.
Agemian, H. and Thomson R. (1980) Analyst 105:902.
Alexander F.W. and Delves H.T. (1981). Int. Arch. Occup. Environ. Health 48:35.
APHA (American Public Health Association) (1986) APHA Standard Methods for the Examination of Water and Waste Water, 16th Ed., APHA, Washington.
ASTM (American Society for Testing and Materials) Annual Book of ASTM Standards, (1986) 11.01, D3559-85, ASTM, Philadelphia, PA.
Balogh, K.V. (1988) Bull. Environ. Contam. Toxicol. 41(6):910.
Barrett, P., Davidowski, L.J., Penaro K.W. and Copeland, T.R. (1978) Anal. Chem. 50:1021.
Beauchemin, D., McLaren, J.W. and Berman, S.S. (1988) J. Anal. At. Spectrom. 3:775.
Bellinger, D., Leviton, A., Waternaux, C., Needleman, H. and Rabinowitz, M. (1987) New Eng. J. Med. 316:1037-1043.
Blust, R., Van der Linden, A., Verheyen, E. and Decleir, W.J. (1988) J. Anal. At. Spectrom. 3:387.
Bond, A.M. and Nagaosa, Y. (1985) Anal. Chim. Acta 178:197.
Boyer, I.W. and Horwitz, W. (1986) Special Considerations in Trace Element Analysis of Foods and Biological Materials. In Environmental Carcinogens-Selected Methods of Analysis O'Neil, I.K., Schuller, P. and Fishbein, L., Eds., International Agency for Research on Cancer, Lyon, 8, France, 191.
Breyer, P.H. and Gilbert, B.P. (1987) Anal. Chim. Acta 201:23.
Burguera, M., Burguera, J.L. and Alarcon, O.M. (1986) Anal. Chim. Acta. 179:351.
Chakrabarti, C.L., Wan, C.C. and Li, W.C. (1979) Spectrochim. Acta 35B:547.
Chisela, F. and Bratter, P. (1986) Anal. Chim. Acta 188:85.
Cooper, R.J., Langlois, D. and Olley, J. (1982) J. Appl. Toxicol. 2(2):99.
Cruz, R.B., Lorouso, C., George, S., Thomassen, Y., Kinrade, J.D., Butler, L.R.P., Lye, J. and Van Loon, J.C. (1980) Spectrochim. Acta 35B:775.
Cutter, G.A. (1985) Anal. Chem. 57:2951.
de Oliveira, E., McLaren, J.W. and Berman, S.S. (1983) Anal. Chem. 55:2047.
DeSilva, K.N. and Chatt, A. (1982) J. Trace Microprobe Tech. 1(3):307.
Eisenberg, M. and Topping, J.J. (1984) J. Environ. Sci. Health Part B. 19(7):649.
EPA (U.S. Environmental Protection Agency) (1979) Methods for Chemical Analysis of Water and Waste, EPA-600/47902, Cincinnati, OH.
EPA (U.S. Environmental Protection Agency) (1986) Air Quality Criteria for Lead EPA-600/8-83/028aF, Research Triangle Park, N.C.
EPA (U.S. Environmental Protection Agency) (1989) Review of the National Ambient Air Quality Standards for Lead: Exposure Analysis Methodology and Validation OAQPS Staff Report, Research Triangle Park. EPA-450/2-89-011.
EPA (U.S. Environmental Protection Agency) (1991) Draft Fish Sampling and Analysis: A Guidance Document for Issuing Fish Advisories Washington, DC, 20460.
Favretto, L.G., Marletta, G.P. and Favretto, L. (1981) Recent Developments in Food Analysis: Proceedings of the First European Conference on Food Chemistry (EURO FOOD CHEM I),Vienna, Austria, 372.
Federal Register (1989). Lead from Ceramic Pitchers 54:23485.
Fernando, L.A., Heavner, W.D. and Gabrielli, C.C.(1986) Anal. Chem. 58:511.
FDA (U.S. Food and Drug Administration) (1990) Inspection Operations Manual, Rockville, MD.
Gault, N.F. S., Tolland, E.L.C. and Parker, J.G. (1983) Mar. Biol. 77(3):307.
Gershanik, J.J., Brooks, G.G. and Little, J.A. (1974) Am. J. Obstet. Gynecol. 119:508.
Grant, W.A. and Ellis, P.C. (1988) J. Anal. At. Spectrom. 3:815.
Guy, R.D. (1985) Anal. Chim. Acta 174:269.
Hall, R.A., Zook, E.G. and Meaburn, G.M. (March, 1978) National Marine Fisheries Service Survey of Trace Elements in the Fishery
Resource. NOAA Technical Report NMFS SSRF-721, U.S. Department of Commerce, National Oceanic and Atmospheric Administration, Washington, D.C.
Harley, N.H. and Kneip, T.H. (1985) An Integrated Metabolic Model for Lead in Humans of All Ages. Final report U.S. EPA, Contract No. B44899 with New York University School of Medicine.
Harms, U. (1985) Fresenius Z. Anal. Chem. 322:53.
Heaney, R.P. and Skillman, T.G. (1971) J. Clin. Endocrinol. Metab. 33:661.
Helrich, K., Ed., (1990) Official Methods of Analysis of the Association of Official Analytical Chemists 15th Ed. Vol. I, Chap. 9, Method 972.23, AOAC, Arlington, VA.
Holak, W. (1969) Anal. Chem. 41:1712.
Hu, T.-W. (1985), Analysis of Seafood Consumption in the U.S.: 1970, 1974, 1978, 1981. NTIS PB86-135043.
Ikeda, M., Nishibe, J., Hamada, S. and Tujino, R. (1982) Anal. Chim. Acta. 125:109.
Jones, J.W. (1988) In Quantitative Trace Analysis of Biological Materials, H. A. McKenzie and L. E. Smythe, Eds., Elsevier Science, New York, N.Y., 353.
Julshamn, K., Ringdal, O., Slinning, K.E. and Braekkan, O.R. (1982) Spectrochim. Acta 37B(6):473.
Kingston, H.M. and Jassie, L.B. (1986) Anal. Chem. 58:2534.
Kingston, H.M. and Jassie, L.B. (1988) Introduction to Microwave Sample Preparation, ACS, Washington, DC.
LaTouche, Y.D., Bennett, C.W. and Mix, M.C. (1981) Bull. Environ. Contam. Toxicol. 26:224.
Lemly, A.D. (1982) Environ. Technol. Lett. 3:497.
Lytle, T.F. and Lytle, J.S. (1982) Bull. Environ. Contam. Toxicol. 29:50.
Madrid, Y., Bonilla, M. and Camara, C. (1988) J. Anal. At. Spectrom. 3:1097.
Mahaffey, K.R. (1985) Factors modifying susceptibility to lead. In Dietary and Environmental Lead: Human Health Effects, K.R. Mahaffey, Ed., Elsevier, Amsterdam, 373-419.
Maher, W.A. (1981) Anal. Chim. Acta 126:157.
Maher, W. (1982) Anal. Chim. Acta 138:365.
Maher, W.A.(1983) Talanta 30:534.
Maher, W. (1986) Anal. Lett. 19:295.
Marcus, J. M. and Thompson, A.M. (1986) Bull. Environ. Contam. Toxicol. 36:587.
May, T.W. and McKinney, G. L. (1981) Pestic. Monit. J. 15(1):14.
May, T.W. (1982) J. Assoc. Off. Anal. Chem. 65:1140.
May, T.W. and Brumbaugh, W.G. (1982) Anal. Chem. 54:1032.
McCarthy, H. and Ellis, P.C. (1991) J. Assoc. Off. Anal. Chem. 74:566.
Mckie, J.C. (1985) Anal. Chim. Acta 197:303.
McMahon, B.M. and Hardin, B.F., Eds., (1968) Pesticide Analytical Manual, Vol. I, 2nd Ed., and updates, U.S. Food and Drug Admin istration, Rockville, MD.
McMahon, J.W., Docherty, A.E., Judd, J.M.A. and Gentner, S-R (1985) Int. J. Environ. Anal. Chem. 24:297.
Meeus-Verdinne, K. MPNBA, Van Cauter, R. and De Borger, R. (1983) Mar. Pollut. Bull. 14(5):198.
Micallef, S. and Tyler, P.A. (1989) Bull. Environ. Contam. Toxicol. 42(3):344.
Michie, N.D., Dixon, E.J. and Bunton, N.G. (1978) J. Assoc. Off. Anal. Chem. 61:48.
Montaser, A. and Golightly, D.W., Eds. (1987) Inductively Coupled Plasmas in Analytical Atomic Spectrometry, VCH, New York, 487-511.
Moody, J.R. and Lindstrom, R.M. (1977) Anal. Chem. 49:2264.
MRCA (Market Research Corporation of America) (1988) 14 Day Survey (5 Year Menu Census, 1982-87), MRCA, Northbrook, IL.
Munro, S., Ebdon, L. and McWeeny, D.J. (1986) J. Anal. At. Spectrom. 1:211.
Nadkarni, R.A. (1984) Anal. Chem. 56:2233.
Panaro, K.W. and Krull, I.S. (1984) Anal. Lett. 17:157.
Pao, E.M., Fleming, K.H., Guenther, P.M., et al. (1982) Foods Commonly Eaten by Individuals: Amount Per Day and Per Eating Occasion U.S. Department of Agriculture. Home Economics Report No. 44.
Park, C.J., Van Loon, J.C, Arrowsmith, P. and French, J.B. (1987) Anal. Chem. 59:2191.
Pirkle, J.L., Schwartz J., Landis, J.R. and Harlan, W.R. (1985) Am. J. Epidemiol. 121:246.
Pitkin, R.M. (1985) Am. J. Obstet. Gynecol. 151:99-109.
Pocock, S.J., Shaper, A.G., Ashby, D., Delves T. and Whitehead, T.P. (1984) Brit. Med. J. 289:872.
Poldoski, J.E. (1980) Anal. Chem. 52:1147.
Private Communication 1, Hans Kurner, Post FACH 171, D-8200 Rosenheim, Federal Republic of Germany, Phone: 08031/88088.
Private Communication 2, Commonwealth of Virginia, Department of General Services, Division of Consolidated Laboratory Services.
Private Communication 3, P. C. Ellis, Rhode Island Dept. of Health, Division of Laboratories, 50 Orms St., Providence, RI 02904.
Private Communication 4, J. Anderson, Bureau of Chemistry, Division of Consolidated Laboratories, Commonwealth of Virginia, 1 North 14th St., Richmond, VA 23219.
Private Communication 5, K. Wiles, Texas Department of Health, Shellfish Sanitation Branch, 1100 W. 49th Street, Austin, Texas 78756.
Private Communication 6, E. Feerst, State of New Jersey, Department of Environmental Protection, Marine Water Classification and Analysis, P.O. Box 405, Stoney Hill Road, Leeds Point, NJ 08220.
Rabinowitz, M.B., Wetherill, G.W. and Kopple, J.D. (1976) J. Clin. Invest. 58:260.
Robbins, W.B. and Caruso, J.A. (1979) Anal. Chem. 51:889A.
Rockway, S.W., Weber, C.W., Lei, K.Y. and Kemberling, S.R. (1984) Int. Arch. Occup. Environ. Health 53:181.
Ryu, J.E., Ziegler, E.E., Nelson S.E. and Fomon, S.J. (1983) Am. J. Dis. Child 137:886.
Schnitzer, G., Pellerin, C. and Clouet, C. (1988) Lab. Pract. 37(1):63.
Selevan, S.G., Landrigan, P.J., Stern, F.B. and Jones, J.H. (1985) Am. J. Epidemiol. 122:673.
Serra, T.M. and Serrano, J.F.B. (1984) J. Assoc. Off. Anal. Chem. 67(1):186.
Sharp, D.S., Smith, A.H., Holman, B.L. and Fisher, J.M. (1989) Arch. Environ. Health 44:18.
Stephenson, M.D., Martin, M., Lange, S.E., Flegal, A.R. and Martin, J.H. (1979) California mussel watch 1977-78. Volume II: Trace metals concentrations in the California mussel, Mytilus californianus, State Water Resources Control Board, Water Quality Monitoring Report, No. 79-22, Sacramento, CA.
Stephenson, M.D. and Smith, D.R. (1988) Anal. Chem. 60:696.
Sturgeon, R.E., Willie, S.N. and Berman, S.S. (1986) J. Anal. At. Spectrom. 1:115.
TRF (Tuna Research Foundation) (1975) Seafood Consumption Study, National Purchase Diary Panel, Inc., Schaumburg, IL.
Victery, W. (1988) Environ. Health Perspect. 78:71.
Watling, H.R. and Watling, R.J. (1982) Bull. Environ. Contam. Toxicol. 28:460.
Weiss, S.T., Munoz, A., Stein, A., Sparrow, D. and Speizer, F. E. (1986) Am. J. Epidemiol. 123:800.
Welz, B. and Melcher, M. (1985) Anal. Chem. 57:427.
Welz, B. and Schlemmer, G. (1986) J. Anal. At. Spectrom. 1:119.
Willie, S.N., Sturgeon, R.E. and Berman, S.S. (1986) Anal. Chem. 58:1140.
White, R.T., Jr. and Douthit, G.E. (1985) J. Assoc. Off. Anal. Chem. 68:766.
Wolff, M.S. (1983) Am. J. Indust. Med. 4:259.
WHO (World Health Organization) (1987) Air Quality Guidelines for Europe, WHO Regional Publications, European Series No. 23. Copenhagen, D.K., 242-261.
WHO (World Health Organization) (1983) "Guidelines for the Study of Dietary Intakes of Chemical Contaminants" in Global Environmental Monitoring System, WHO, Geneva, Switzerland, 49-50.
Zeisler, R., Stone, S.F. and Sanders, R.W. (1988) Anal. Chem. 60:2760.
This Guidance document represented current agency thinking in regards to the available science at the time it was issued. It no longer represents the current state of science and is presented here for the historical record only.