New Approach for Evaluating the Bioaccumulation Potential of Metals in the New Chemicals Division

EPA Publication Number: 747S25002

New Approach for Evaluating the
Bioaccumulation Potential of Metals in the
New Chemicals Division

January 13, 2025

1. Summary

The bioaccumulation mechanism for metals differs from that for typical organic chemicals (i.e.,
lipophilic partitioning). Therefore, when evaluating metals in the New Chemicals Division
(NCD), the standard lipid-based approach for assessing bioaccumulation potential based on
bioconcentration factor (BCF) or bioaccumulation factor (BAF) measurements or estimations is
not appropriate. Here, we present a new approach to evaluate the bioaccumulation potential of
metals that can be applied in the new chemicals program. This approach does not quantitatively
predict the metal's bioaccumulation potential but rather, it considers multiple lines of evidence
and endpoints to determine whether the metal is likely to accumulate in a way that presents a
concern for unreasonable risk. The weight of evidence (WoE) approach considers:

1. An assessment that integrates a literature review of available field measurements of metal
concentrations in fish and shellfish with critical concentrations based on human chronic
oral toxicity values criteria (i.e., non-cancer screening level, NCSL, and/or cancer slope
factor, CSF).

2. A critical review of the test data, conclusions, and rationales related to bioaccumulation
utilized by other programs, agencies, and organizations in their risk assessments for
metals.

3. A critical review of relevant metal-specific information on bioconcentration,
bioaccumulation, and trophic transfer available in peer-reviewed scientific literature.

2. NCD Assessment of Bioaccumulation

As part of the new chemical evaluation under TSCA, NCD's fate team typically assigns both a
persistence (P) and a bioaccumulation (B) rating, consistent with EPA's 1999 PBT policy as
outlined in the Federal Register.1 The 1999 framework focused on standard organic chemicals
and provides a bioaccumulation rating scheme based on BCF/BAF cutoffs. Accordingly, NCD
rates standard organic chemicals as B1 (BCF/BAF < 1000), B2 (1000 < BCF/BAF < 5000), or
B3 (BCF/BAF > 5000), with a B2 or B3 rating being sufficient for the "B" portion of a PBT
designation.

For chemicals that do not bioaccumulate via typical lipophilic partitioning, NCD fate assessors

1 Category for Persistent, Bioaccumulative, and Toxic New Chemical Substances, 64 F.R. 60,194 (November 4,
1999). https://www. gpo. gov/fdsvs/pkg/FR-1999-11 -04/pdf/99-28888.pdf.

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EPA Publication Number: 747S25002

may deviate from B1-3 to assign a rating of either B How or B*high to indicate their predicted
bioaccumulation potential. "B*" denotes that the rating is for a chemical that does not
bioaccumulate via lipophilic partitioning. The "low" or "high" designation indicates whether the
chemical is expected to have low or high potential to bioaccumulate via other mechanisms. NCD
does not have a formal cutoff between the "low" and "high" designation. However, a measured
or estimated BCF or BAF > 1000, in combination with any additional information, has been
considered as a screening level indication that a B*high rating may be warranted. The final
designation is ultimately based on professional judgement.

In the TSCA new chemicals program, the bioaccumulation rating (i.e., B rating) serves two
primary purposes: 1) to determine, in combination with the persistence (P) and toxicity (T)
ratings, whether a new chemical substance is PBT and therefore subject to risk management, and
2) to assess potential risks to humans via fish and shellfish consumption. A rating of B*high is
considered sufficient to contribute to a PBT designation.

Previously, NCD fate assessors have relied on available, measured BCF and/or BAF values to
assign either a B*low or B*high rating when assessing a metal. However, the latest scientific
data on bioaccumulation do not support the use of BCF or BAF when applied as generic
threshold criteria for the hazard potential of inorganic metals in human and ecological risk
assessment, as explained in Section 3.2'3 Single-value BCF/BAFs and mechanistic
bioaccumulation models for metals offer the most value for site-specific risk assessments when
extrapolation across different exposure and environmental conditions is minimized.2'3 Their
utility is limited for national-scale risk assessments such as those performed within NCD.

3. Bioaccumulation Considerations for Metals

Metals are naturally occurring in the environment and vary in concentrations across geographic
regions. Some metals are essential for maintaining the proper health of humans, animals, plants,
and microorganisms. As a result, many species have evolved physiological or anatomical
mechanisms to regulate accumulation and/or storage of certain metals, particularly essential
metals and those that may mimic essential metals within the organism. In these species,
homeostatic mechanisms can maintain optimal tissue levels over a range of exposures, even
when exposure concentrations (e.g., in water, air, and/or food) exceed those normally
encountered by the organism.2 In contrast, certain metals can bioaccumulate to high levels in
some aquatic organisms (for example zinc in barnacles and copper in crayfish) by active
regulation due to species-specific physiological requirements, regardless of exposure level.2
BCFs and BAFs cannot not distinguish between metals that are elevated to meet physiological
requirements and those by which adverse effects may result when elevated.4

Unlike hydrophobic, nonionic organic chemicals, which generally cross biological membranes
via passive diffusion, metals are taken up by a number of specific transport mechanisms. Some

2 U.S. EPA. 2007. Framework for Metals Risk Assessment. EPA 120/R-07/001 March 2007. 172pp.
www.epa.gov/sites/defauit/files/2013-09/documents/metals-risk-assessment-final.pdf

3 McGeer JC et al. 2004. Issue paper on the bioavailability and bioaccumulation of metals. Submitted by Eastern
Research Group (ERG) to the U.S. EPA on August 10, 2004. 126pp. www.epa.gov/sites/default/files/2014-

.1.1/doeiiments/bio finai.pdf.

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of these transport mechanisms involve binding with membrane carrier proteins, transport through
hydrophilic membrane channels, and endocytosis. Passive diffusion is thought to be reserved for
certain lipid soluble forms of metals, such as alky-metal compounds and neutral, inorganically
complexed metal species (e.g., HgChO). The implication of these specific transport mechanisms
is that metal bioaccumulation can involve saturable uptake kinetics, such that BCFs and BAFs
depend on exposure concentration.2 The existence of saturable uptake mechanisms, the presence
of significant amounts of stored metal in organisms, and the ability of some organisms to
regulate bioaccumulated metal within certain ranges are primarily responsible for the inverse
relationship that has frequently been reported between BCFs/BAFs and metal exposure
concentrations.4'5 In these cases, higher BCFs or BAFs are associated with lower exposure
concentrations and may be associated with lower tissue concentrations within a given aquatic
BCF or BAF study. This is counter to the implicit assumption that higher BCFs or BAFs indicate
higher metal hazard.2 Using BCF and BAF data can lead to conclusions that are inconsistent with
the toxicological data, as these values are sometimes highest (indicating hazard) at low exposure
concentrations and are lowest (indicating low/no hazard) at high exposure concentrations, where
impacts are likely.4

In addition, other biotic and abiotic factors influence metal bioavailability and bioaccumulation.
Assimilation efficiencies can vary widely depending on the metal, its form and distribution in
prey, species digestive physiology (e.g., gut residence time), environmental conditions, food
ingestion rate, and metal concentration in the diet.2 Considerable uncertainty can be associated
with the application of literature-derived BCFs and BAFs for assessing the risks of metals, as
variability in BCFs and BAFs for metals is known to be high. Much of this uncertainty results
from bioavailability differences among the studies in which the BCFs or BAFs are measured
(e.g., differences in water quality characteristics, metal speciation, and exposure pathways).2

Quantitative measures of trophic transfer (i.e., biomagnification factors [BMF] and trophic
magnification factors [TMF]) are susceptible to many of the same complications discussed for
BCF/BAF that lead to high variability and reduced utility beyond site-specific assessments. The
availability of these measures is also limited for many metals.

Bioaccumulation and trophic transfer of metals can occur despite the fact that the movement of
metals through the food web is complicated by factors of bioaccessibility, bioavailability,
essentiality, regulation (uptake and internal distribution), detoxification, storage, and the natural
adaptive capacity of organisms.2 However, biomagnification (i.e., increases in concentration
through multiple levels of the food web) is rare, with the exception of certain organometallic
compounds, such as methylmercury, that can biomagnify many orders of magnitude in the
aquatic food chain.2'3 But lack of biomagnification cannot be interpreted as lack of exposure or
concern via trophic transfer 2 Even in the absence of biomagnification, organisms can
bioaccumulate relatively large amounts of metals and become a significant source of dietary

4	McGeer, JC el al. 2003. Inverse relationship between bioconcentration factor and exposure concentration for
metals: implications for hazard assessment of metals in the aquatic environment. Environ Toxicol Chem 22(5):
1017-1037.

5	Borgmann, U el al. 2004. Re-evaluation of metal bioaccumulation and chronic toxicity in Hyalella azteca using
saturation curves and the biotic ligand model. Environ Pollut 131(3): 469-484.

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EPA Publication Number: 747S25002

metal to their predators.6

As a result of these numerous uncertainties, the application of measured BCF, BAF, or BMF
values for metals is not appropriate beyond individual well characterized site- and food web-
specific scenarios. The current science does not support the use of a single, generic threshold
BCF/BAF/BMF value for a given metal as an indicator of that metal's hazard potential. For
national-scale risk assessments, use of a single BCF/BAF/BMF value holds little utility due to
high uncertainty that results from differences in bioavailability, exposure conditions, and
species-specific factors that influence metal bioaccumulation by aquatic organisms.2

There are no simple metrics available that allow for the quantification of the potential for metal
bioaccumulation.2 Existing regulatory and scientific guidances do not provide a single
quantitative approach for metals that is appropriately suited to the national-scale, screening-level
assessment of bioaccumulation that is required for risk assessments in NCD. As described below,
NCD has developed a WoE approach to assess the bioaccumulation potential of metals.
Considering multiple lines of evidence will reduce uncertainty and allow for a more robust and
scientifically supported assessment.

4. New Approach for Determining the Bioaccumulation Potential of
Metals

This memorandum presents a WoE approach to consider the bioaccumulation potential of metals
as part of the screening-level risk assessment of new chemicals under TSCA. This approach
considers the following lines of evidence:

1. An assessment that integrates a literature review of available field measurements of metal
concentrations in fish and shellfish with critical concentrations based on human chronic
oral toxicity values (i.e., non-cancer screening level, NCSL, and/or cancer slope factor,
CSF).

2. A critical review of the data, conclusions, and rationales related to bioaccumulation
utilized by other programs, agencies, and organizations in their risk assessments for
metals.

3. A critical review of relevant metal-specific information on bioconcentration,
bioaccumulation, and trophic transfer available in peer-reviewed scientific literature.

NCD will review the complete body of evidence and assign a B rating of either B*low or B*high
to each individual metal of interest. This approach does not quantitatively predict the metal's
bioaccumulation potential; rather, it considers multiple lines of evidence and endpoints to
determine whether the metal is likely to accumulate in a way that presents a concern for
unreasonable risk.

4.1. Line of Evidence #1: Fish/Shellfish Tissue Concentrations Relative to Human Intake
Criteria

The first line of evidence uses human chronic dietary toxicity values (i.e., RfD or CSF) to

6 Reinfelder, JR el al. 1998. Trace element trophic transfer in aquatic organisms: a critique of the kinetic model
approach. Sci Total Environ 219(213): 117-35.

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determine whether individual metals pose a risk to human consumers based on literature-derived
fish/shellfish tissue metal concentrations. Despite the uncertainties associated with
bioavailability, bioaccumulation, and trophic transfer of dietary metals, the use of whole-body
inorganic metal concentrations in prey species has utility to risk assessors for conservatively
screening for exposure and risk to consumers.2 Such an analysis can discriminate between metals
that have the potential to cause effects via trophic transfer and metals that do not. Metals that
bioaccumulate to levels in prey organisms (i.e., fish/shellfish) that cause impacts in predatory
organisms (i.e., humans) are critical to identify and address through risk management.

This methodology does not provide a pure prediction of bioaccumulation, but rather integrates
bioaccumulation and chronic toxicity data to examine metal accumulation in fish relative to
human toxicity values. The methodology is analogous to the peer-reviewed methodology
employed by EPA's Office of Water to generate their list of contaminants of concern to be
included in fish tissue monitoring programs.7 The scope of this line of evidence is limited to
assessing risks to human consumers from the adult general population.

Metals have an abundance of published fish and shellfish tissue data available. These data span
environmental exposure scenarios, including sites with wide ranging water chemistries, across
geographical ranges with variations in background metal concentrations, and a variety of fish and
shellfish species commonly consumed by humans. Thus, the data integrate many of the variables
that affect organism tissue concentrations. The range and maximum tissue concentrations can be
used as indicators of possible human exposure scenarios via fish and shellfish consumption for a
given metal. Comparing this range to human dietary intake criteria (i.e., non-cancer and cancer
screening levels and CSL) allows the assessor to consider both bioaccumulation and toxicity
concurrently to answer the question of whether a given metal can accumulate in fish and
shellfish to concentrations that pose a risk to human consumers within the bounds of reasonably
anticipated environmental conditions. While not every possible environmental scenario can be
represented by the available data, the approach provides a conservative, screening-level line of
evidence that is an improvement over the previous reliance on BCFs/BAFs alone.

4.1.1. Step 1: Literature search for metal concentrations in fish and shellfish tissue
The NCD fate assessor first performs a literature search for available field measurements of fish
and shellfish tissue concentrations for the metal of interest. To be considered in the analysis,
studies must include the identity of the metal tested, fish/shellfish tissue metal concentrations,
the identity of the tissue in which the metals were measured, the study species, and the collection
site's general location and description. Studies not including all these criteria are excluded from
the analysis. Studies without an indication of whether reported concentrations were on a wet or
dry weight basis may still be included. The conservative assumption that the measurements are
on a wet weight basis will be made and the uncertainty will be noted in the database.

Measurements in fish muscle (i.e., fillet) or whole soft bodies of shellfish will be preferred as
these are the most common tissues consumed by the general population. In the absence of

7 U.S. EPA Office of Water. 2024. Contaminants to Monitor in Fish and Shellfish Advisory Programs: Compilation
of Peer Review-Related Information. EPA 823-R-24-001. July 2024. www.epa.gov/svstem/fites/doeuments/2024-
06/conta mi nants-monitor-fish-peer-review-package.pdf.

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sufficient data in preferred tissues, whole body fish metal concentrations may be included in the
assessment. Whole body metal concentrations are often higher than those in the edible portion of
fish, so their inclusion represents a more conservative assessment of bioaccumulation potential.
Studies included are limited to field studies to best reflect ambient conditions (i.e., laboratory
studies excluded) and should include only fish and shellfish species representative of those
consumed by humans. Tissue concentration should be considered on a wet-weight basis. If the
tissue concentration is reported on a dry-weight basis (Cdw), it can be converted to an equivalent
wet-weight concentration (Cww) according to the equation:

Cww = Cdw * 0.2 [Equation 1]

The calculation assumes 80% moisture content (20% dry mass) in fish and shellfish tissues. A
moisture content of 80% was chosen as a reasonable estimate appropriate for screening-level
purposes based on measured raw fish and shellfish values (Table 1).

Table 1: Percent moisture measured in raw fish and shellfish8

Organism Type

Number of Species
Measured

Percent Moisture (%)

Mean

Median

Range

Fish

75.7

76.4

63.6- 83.2

Shellfish

80.0

80.3

74.1 - 86.2

When tissue concentrations are very near the human toxicity concentrations, species-specific
moisture content information can be applied for a more precise estimate. These values are
available for a variety of commonly consumed by humans in the U.S.8

Ideally, the waters from which the organisms were collected should span a broad geographical
range to capture populations that are adapted to a variety of background metal concentrations.
Field studies should represent environmental contamination scenarios ranging from background
metal concentrations to sites anticipated to be highly contaminated by the metal of interest. As
many water body types as possible should be included to cover a wide range of water
chemistries. If the available data for a metal are narrower in scope, this will be noted in the
assessment and considered when interpreting the conclusions of the line of evidence.

All data extracted from the literature will be put into a database that includes relevant
information such as:

• Identity of metals measured

• Fish or shellfish tissue mean metal concentration

• Fish or shellfish tissue metal concentration range

• Concentration basis: wet weight or dry weight

• Tissue type (e.g., muscle, whole body)

• Test organism

• Water type: freshwater vs. marine

• Geographic location/further site details

8 U.S. Environmental Protection Agency. 2011. Exposure Factors Handbook: 2011 Version. EPA/600/R-09/052F.
https://www.epa.gov/expobox/exposure-factors-handbook-2011-edition

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•	Study literature reference

•	Additional notes to assist in the interpretation of the study results

Each database entry represents the mean concentration in one species at one location within a
given study. Thus, if there are multiple species or sampling locations in a single study, there may
be multiple database entries related to that study.

4.1.2.	Step 2: Determination of the human chronic oral reference dose (RfD) and cancer slope
factor (CSF)

NCD will identify a reference dose (RfD, mg/kg/day) for each metal for use in the methodology.
The RfD will be based on a no observed adverse effect level (NOAEL), lowest observed adverse
effect level (LOAEL), or benchmark dose for non-cancer endpoints and the associated safety
factor (SF), as detailed in existing EPA guidances.9

For cancer endpoints for which a linear low dose extrapolation is appropriate, NCD will utilize
an oral cancer slope factor (CSF), when available. The CSF is an estimate of the increased cancer
risk from oral exposure to a dose of 1 mg/kg-day for a lifetime and can be multiplied by an
estimate of lifetime exposure (in mg/kg-day) to estimate the lifetime cancer risk.

4.1.3.	Step 3: Translation of the RfD and CSF into acceptable fish/shellfish tissue concentrations
for human consumption (CNSL and CSL)

The fish tissue concentration corresponding to the RfD for non-cancer endpoints (i.e., the non-
cancer screening level, NCSL) can be calculated using the RfD (mg/kg/day) for a given metal
along with the consumer body weight (BW, kg) and fish consumption rate (FCR, g/day).

NCSL = [(RfD * BW) / FCR] * 1000 [Equation 2]

The NCSL (jug/g) thus represents the predicted maximum fish tissue (e.g., muscle) concentration
of the metal of interest that can be consumed over a lifetime by a human consumer of a given
BW and FCR with no expected adverse health impact.

For each contaminant with a cancer slope factor (CSF), NCD will also calculate a cancer
screening level (CSL) using the following equation:

CSL = (CRL * BW) / (CSF * FCR)	[Equation 3]

CRL is the cancer risk level and represents the increased lifetime risk of developing cancer from
exposure to a substance. Thus, the CSL represents the fish tissue concentration of a given
substance that will result in an increased cancer risk (e.g, 1 in 1,000,000 increase if a CRL of 10"6
is utilized) from a lifetime of fish consumption by a human consumer of a given BW and FCR.

The BW and FCR utilized will be selected to be consistent with the current methodologies of

9 U.S. EPA. A Review of the Reference Dose and Reference Concentration Processes. U.S. Environmental
Protection Agency, Risk Assessment Forum, Washington, DC, EPA/630/P-02/002F, 2002.
https://www.epa.gov/sites/default/files/2014-12/documents/rfd-final.pdf.

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NCD at the time of the assessment. These values can easily be adapted within the methodology
as needed, such as if NCD updates the FCR applied to the adult general population or if there is a
need to apply the approach to other populations, such as potentially exposed or susceptible
subpopulations (PESS). At this time, NCD Exposure assessors consider the adult general
population when assessing risks due to fish/shellfish consumption and employ a BW of 80 kg
and a FCR of 7.5 g/day. These values come from the 2014 EFAST Manual,10 and were derived
from 2011 Exposure Handbook.8 The fish consumption rate includes consumption of both fish
and shellfish.

NCD may also run a sensitivity analysis of the results by additionally calculating NCSL and CSL
using an alternate FCR, such as the 22 g/day rate utilized for the general population by EPA's
Office of Water (OW) in their assessments.11 This will provide information on whether the
conclusions of the assessment would be impacted if NCD chooses to align the NCD fish
consumption rate with that of OW in the future.

4.1.4. Step 4: Comparison of the NCSL and CSL to the literature-derived fish/shellfish tissue
concentrations

The NCSL and CSL are next compared to the literature-derived fish/shellfish tissue
concentrations to ascertain whether there is a likelihood that a human fish/shellfish consumer
may consistently be exposed to fish concentrations exceeding the NCSL/CSL over a lifetime of
fish consumption. Consistent exceedances of the either value (i.e., fish/shellfish tissue
concentrations are greater than the NCSL or CSL for a significant portion of the measured values
extracted from the peer-reviewed literature) may indicate that a human consumer has the
potential to exceed the metal RfD via dietary exposure. Factors to consider in making this
determination include:

• What proportion of the fish tissue concentrations found in the literature exceed the NCSL
and/or CSL?

• Are the exceedances mean concentrations or single tissue concentrations measured in the
study?

• What were the environmental and site conditions under which the NCSL/CSL
exceedances were measured?

• Are the species with measured NCSL/CSL exceedances widely consumed or only rarely,
or are they only local to a geographic region outside the U.S.?

• Are there any other factors that make the data less representative, valid, or applicable?

Additional information to assist in the interpretation of the results may include whether there are
any reports in the literature of human exposures to the metal via fish consumption leading to
adverse health outcomes or human body burdens of the metal above levels of concern, as well as
whether any regulatory actions (e.g., advisories) have been issued for the metal with regards to
fish consumption.

10 U.S. Environmental Protection Agency. 2018. Exposure and Fate Assessment Screening Tool (E-FAST) 2014
Version Documentation Manual. Prepared by Versar, Inc. February 2018 under EPA Contract No. EP-W-16-009.
187 pp.

11 U.S. Environmental Protection Agency, Office of Water. Human Health Ambient Water Quality Criteria: 2015
Update. EPA 820-F-15-001. June 2015. https://www.epa.gov/sites/defanit/files/2015-10/docnments/hnman-heaith-
2015-npdate-factsheet. pdf

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4.2.	Line of Evidence #2: Benchmarking Against Other Agencies/Organizations
The second line of evidence uses existing risk assessments for metals to inform NCD's
assessment of bioaccumulation potential. For each metal assessed, existing risk assessments that
have been performed by other agencies or other EPA offices will be located and reviewed. This
may include:

•	Agency for Toxic Substances and Disease Registry (ATSDR) Toxicological Profiles

•	Priority Substances List Assessment Reports under the Canadian Environmental
Protection Act (CEPA)

•	Environment and Climate Change Canada/Health Canada (ECCC/HC) Screening
Assessments

•	European Union (EU) Risk Assessment Reports

•	Organization for Economic Cooperation and Development (OECD) Assessment Profiles

•	EPA Water Quality Criteria documents (human health and/or aquatic life)

•	European Chemicals Agency (ECHA) dossiers

•	Environmental Health Criteria Monographs from the World Health Organization's
International Programme for Chemical Safety (WHO/IPCS)

•	Risk assessments from states

For each available assessment identified, any information pertinent to bioaccumulation will be
extracted and summarized including any conclusions about bioaccumulation potential and the
accompanying rationale.

4.3.	Line of Evidence #3: Literature Review of Pertinent Metal-Specific Bioaccumulation
Information

The third line of evidence uses studies from the peer-reviewed literature to compile information
on the bioaccumulation characteristics of each metal, such as whether they are subject to
homeostatic regulation and/or show consistent evidence of biomagnification or biodilution in
aquatic food webs.

As described above, individual BCF, BAF, and BMF values found in the literature have little
utility for the purposes of NCD's broad-scale risk assessments. However, more general
information about the bioaccumulation behavior of specific metals can contribute to the WoE
used to determine a B rating. For example, if an inverse relationship between BCF and exposure
concentration in the water is consistently observed for a given metal, this suggests that the metal
undergoes homeostatic regulation, lending support to a conclusion that the metal has low
bioaccumulation potential.4 Similarly, a body of literature demonstrating no significant
relationship between the organism metal concentration and trophic level within food webs could
suggest that the metal is unlikely to undergo trophic transfer in similar food webs, lending
support to a low bioaccumulation potential rating.

A literature search will be conducted for each metal and relevant information for aquatic food
webs will be extracted and summarized. The focus will be to derive general information on the
bioaccumulation process and its controls (e.g., homeostatic regulation) for a given metal, rather
than to identify quantitative BCF and BAF values.

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5. Situations in Which There is Incomplete or Conflicting Information

EPA has a long history of using WoE approaches to support scientific conclusions for risk
assessment.12"15 This document provides a reasonable, scientifically supportable approach using
three lines of evidence:

2. A critical review of the test data, conclusions, and rationales related to bioaccumulation
utilized by other programs, agencies, and organizations in their risk assessments for
metals.

3. A critical review of relevant metal-specific information on bioconcentration,
bioaccumulation, and trophic transfer available in peer-reviewed scientific literature.

EPA recognizes that there may be metals for which data from one or more lines of evidence are
unavailable, of reduced quantity or quality and/or provide conflicting evidence. The quality and
adequacy of the data will be considered, and any data gaps or uncertainties will be considered
and described transparently. Where possible and appropriate, NCD will fill data gaps with
information available for similar metals (i.e., chemically similar, close neighbors in the periodic
table). In some cases, NCD may need to rate the metal as "BU", indicating the bioaccumulation
potential is unknown (U), to ensure its risks can be conservatively managed.

6. Example of the Application of the WoE Approach to Cobalt

Cobalt is highlighted here to demonstrate the application of the WoE approach to a metal of
interest to NCD.

6.1. Cobalt Line of Evidence #1: Fish/Shellfish Tissue Concentrations Relative to Human
Intake Criteria

NCD conducted an extensive literature review and extracted data for cobalt studies with reported
fish and shellfish tissue concentration data meeting the criteria outlined above from 38 peer-
reviewed studies, providing 264 reported values representing cobalt tissue concentrations in 246
species/location combinations (see Appendix).

12 U.S. EPA (Office of Pesticide Programs). Guidance on Use of Weight of Evidence When evaluating the Human
Carcinogenic Potential of Pesticides. June 2023. https://www.epa.gov/svstem/files/documents/2023-
06/2023%20CARC%20WoE%20Guidance.pdf.

13 U.S. EPA (Office of Research and Development). Application of Weight-of-Evidence Methods for Transparent
and Defensible Numeric Nutrient Criteria. May 2024. https://www.epa.gov/svstem/files/documents/2024-

05/woe nne 508 final.pdf.

14 U.S. EPA. Weight of Evidence in Ecological Assessments. December 2016. EPA/100/R-16/001.

https://www.regiiiations.gOv/docnment/EPA-HO-OPPT-20.l.6-0654-0100.

15 OECD (2019), Guiding Principles and Key Elements for Establishing a Weight of Evidence for Chemical
Assessment, OECD Series on Testing and Assessment, No. 311, OECD Publishing, Paris,
https://doi.org/10.1787/fll597f6-en.

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NCD identified an RfD of 0.003 mg/kg BW/day based on polycythemia.16 This translates to a
NCSL of 36 |ig/g using Equation #3 for a consumer with a body weight of 80kg at a fish
consumption rate of 7.5g/day, the values currently utilized in NCD risk assessments. An oral
CSF could not be established due to inadequate evidence for carcinogenicity of cobalt and cobalt
compounds by the oral route of exposure.17 Therefore, a CSL was not calculated for comparison
to fish/shellfish tissue concentrations. Figure 1 compares the fish/shellfish tissue concentrations
from the literature to the NCSL, with the blue bars representing fish tissue concentrations for
each of the 264 species/location combination measurements and the yellow line indicating the
NCSL for cobalt at a FCR of 7.5 g/day.

NCSL = 12 ng/g
@ FCR = 22 g/day

NCSL = 36 ng/g
@ FCR = 7.5 g/day

38 Studies
264 Data Points

0 5 10 15 20 25 30 35

Fish/Shellfish Literature Co Concentrations (ng/g wet weight)

Figure 1: Literature values of cobalt fish tissue concentrations relative to the non-cancer
screening level (NCSL) at a FCR of 7.5 g/day (yellow line; value currently utilized in NCD
assessments) and 22 g/day (red line; value currently utilized in EPA Office of Water
assessments)

All fish and shellfish tissue concentrations across species and environmental and contamination
conditions were approximately an order of magnitude or more below the NCSL. Over 75% of
reported values were at least two orders of magnitude below the NCSL. Evidence from a wide
variety of field conditions indicates that cobalt is unlikely to bioaccumulate in fish or shellfish
species representative of those consumed by humans to a concentration exceeding the NCSL.

In addition, no current or historical U.S. fish consumption advisories based on cobalt were
found.18 EPA's database of U.S. fish tissue data collected by States and Tribes for fish

16 Davis JE, Fields JP. 1958. Experimental production of polycythemia in humans by administration of cobalt
chloride. Proc Soc Exp Biol Med 99(2): 493-495.

17 California Enviromnental Protection Agency. 2010. Cobalt and Cobalt Compounds Cancer Inhalation Unit Risk
Factors: Technical Support Document for Cancer Potency Factors Appendix B.
https://oelilia.ca.gov/media/downloads/crnr/cobaltcpfl00220.pdf.

18 https://fisliadvisorvonline.epa.gov/Advisories.aspx. Accessed 10/23/2024.

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EPA Publication Number: 747S25002

consumption advisories contains 43 measurements of cobalt in fish tissue.19 The highest reported
concentration is 1.95 |ig/g wet weight, well below the NCSL of 36 |ig/g. No reports of adverse
health impacts in humans linked to consuming cobalt-contaminated fish were found. Further,
cobalt is not included in the EPA Office of Water's list of contaminants to monitor in fish and
shellfish advisory programs720 This information supports the conclusion that there is a low
likelihood that human fish/shellfish consumers are consistently exposed to cobalt via fish
consumption at levels exceeding the NCSL.

A sensitivity analysis was conducted and led to the same overall conclusion. When OW's current
FCR of 22 g/day is considered, rather than NCD's 7.5 g/day, the NCSL decreases from 36 |ig/g
to 12 |ig/g (red line in Figure 1). All fish and shellfish tissue concentrations extracted from the
peer-reviewed literature were below this more conservative NCSL. Measurements available in
the EPA's database of U.S. fish tissue data collected by States and Tribes for fish consumption
advisories were also all below 12 |ig/g.

6.2. Cobalt Line of Evidence #2: Benchmarking Against Other Agencies/Organizations
NCD searched programs/agencies to identify existing risk assessments containing information on
cobalt bioaccumulation. The search results are shown in Table 2, including if information was
available for cobalt and which contained a discussion of cobalt bioaccumulation. NCD found
four existing risk assessments that included a discussion of bioaccumulation, all of which
concluded that the bioaccumulation and biomagnification potential for cobalt is low. Details of
each risk assessment are summarized below.

Table 2: Availability of Existing Risk Assessments

'or Cobalt

Risk Assessment

Available?

Contains Bioaccumulation
Discussion?

ATSDR Toxicological Profile

Priority Substances List Assessment Report under CEPA

N/A

ECCC/HC Screening Assessment

EU Risk Assessment Report

N/A

OECD Assessment Profile

EPA Water Quality Criteria Document

N/A

ECHA dossier

WHO/IPCS Enviromnental Health Criteria Monograph

N/A

Risk assessments from states

N/A

The Agency for Toxic Substances and Disease Registry (ATSDR) Toxicological Profile for
Cobalt states that mollusks accumulate little cobalt in their edible parts (BCF = 1 to 300 in soft
tissue), citing two references.21 The ATSDR profile also reports cobalt concentrations from
literature in some species. In the studies cited by ATSDR, fish cobalt concentrations were <1
|ig/g wet weight from three studies in freshwater fish and from two studies on marine fish,
consistent with the conclusion from Line of Evidence #1 that fish tissue concentrations are not

19 https://fishadvisorvonline.epa.gov/FishTissue .aspx. Accessed 10/4/2024.

211 U.S. EPA Office of Water. 2024. Fact Sheet: Contaminants to Monitor in Fish and Shellfish Advisory Programs.
EPA-823-F-24-011. https://www.epa.gov/svstem/files/documents/2024-06/contaminants-monitor-fish-factsheet-
iulv2024.pdf

21 ATSDR. 2023. Toxicological Profile for Cobalt: Draft for Public Comment.
https://www.atsdr.cdc.gov/ToxProfiles/tp33.pdf.

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EPA Publication Number: 747S25002

likely to exceed the NCSL of 36 jug/g. The ATSDR profile also described a study in an
amphipod-fish-seabird food web in Antarctica that showed that cobalt concentrations did not
increase with trophic level, indicating no biomagnification. ATSDR concluded that cobalt does
not biomagnify.

The Environment and Climate Change Canada (ECCC) and Health Canada (HC) Screening
Assessment states that cobalt and soluble cobalt compounds do not meet the bioaccumulation
criteria as set out in the Persistence and Bioaccumulation Regulations of CEPA (i.e., BAF >
5000).22 The ECCC/HC assessment's discussion of bioaccumulation in aquatic systems begins
by acknowledging that BCF and BAF are considered to have "little usefulness in predicting
metal hazards." Despite this, ECCC/HC do report literature BAF values from 20 references for
various species of algae, invertebrates, fish, and zooplankton for marine and fresh water ranging
from 7.4 to 3110, with a mean value of 878 and a median value of 720. The raw data were not
presented in the assessment, so the BAF ranges for specific taxa could not be determined.
ECCC/HC did note that no groups of organisms seemed to have higher BCF/BAF than others.
The assessment also cites four studies reporting zooplankton-fish BMFs (marine and freshwater)
ranging from 0.004-0.087. ECCC/HC also cite four TMF studies that showed no statistically
significant relationship between cobalt concentration and nitrogen stable isotopes in food webs.
Based on these results, ECCC/HC concluded that the bioaccumulation potential of cobalt
in natural ecosystems is relatively low and that cobalt does not present a risk for
biomagnification.

The Organization for Economic Cooperation and Development (OECD) Assessment Profile
provides a brief discussion on bioaccumulation, beginning with a statement that cobalt uptake is
expected to be regulated to some extent by many organisms through mechanisms of homeostasis
and detoxification because it is an essential micronutrient for bacteria, plants and animals.23
OECD cites the same BAF and BMF studies and values reported in the ECCC/HC Screening
Assessment.22 No additional information is provided. Considering these values and regulation
mechanisms for cobalt in most organisms, OECD expects the bioaccumulation and
biomagnification potentials of cobalt in aquatic ecosystems to be low.

The European Chemicals Agency (ECHA) Dossier for cobalt discusses the essentiality and
active regulation of cobalt by homeostatic mechanisms in plants and animals. According to the
Dossier, existing information suggests that cobalt does not biomagnify, as with most metals;
rather, cobalt exhibits biodilution, particularly in upper levels of both aquatic and terrestrial food
chains 24 From a review of 54 studies, ECHA found that cobalt accumulates from water to plants
in aquatic systems (BCF >100 to 5000); however, higher trophic levels show reduced
accumulation: BCF < 515 for invertebrates, with both freshwater and marine fish showing
BCF/BAF <10. ECHA also cites a marine trophic transfer study that reported trophic transfer
values <1 based on cobalt tissue concentrations across a number of trophic pathways,

22 ECCC/HC. 2017. Screening Assessment Cobalt and Cobalt-Containing Substances.

https://www.canada.ca/content/dam/eccc/migration/ese-ees/dceb359c-245f-4a06-b2e5-62887d47c806/en cobalt-
20fsar-20fi na 1-20 ma i -2025-2020.1.7-20. pdf.

23 OECD. 2010. Initial Targeted Assessment Profile (Human Health and Environment).
https://hpvchemicals.oecd.org/ui/handler.axd?id=e6f30459-3de7-402f-a7b6-9b394f34efel.

24 https://efaem.eefaa.eiiropa.eu/100.028.325/overyiew?searefaText=eobatt. Accessed 10/25/2024.

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EPA Publication Number: 747S25002

incorporating phytoplankton, zooplankton, sea bream and sea bass. The ECHA Dossier
concludes that cobalt does not biomagnify through either freshwater or marine trophic
food webs.

6.3. Cobalt Line of Evidence #3: Literature Review of Pertinent Metal-Specific
Bioaccumulation Information

NCD identified ten studies in the peer-reviewed literature that measured trophic transfer of
cobalt and found no evidence of biomagnification in aquatic food webs (Table 3). Five studies,
three in marine and two in freshwater food webs, observed trophic dilution of cobalt, indicated
by an inverse correlation between organismal cobalt concentrations and trophic level, as
indicated by stable isotope measurements (i.e., 815N).25-29 Chouvelon eial. (2019) did not
measure S15N directly, but found continuously decreasing cobalt concentrations and BAFs from
phytoplankton to zooplankton to fish in a marine food web, also indicating trophic dilution of
cobalt.30 The remaining four studies, three in marine food webs and one in a freshwater food
web, found no correlation between organismal cobalt concentrations and S15N, indicating a lack
of cobalt biomagnification.31"34 None of the studies identified observed evidence of
biomagnification in aquatic food webs.

25 Asante, K.A., et al. 2008. Trace elements and stable isotopes (513C and 515N) in shallow and deep-water
organisms from the East China Sea. Environmental pollution, 156(3), pp.862-873.

26 Balzani, P., et al. 2021. Combining metal and stable isotope analyses to disentangle contaminant transfer in a
freshwater community dominated by alien species. Environmental Pollution, 268, p. 115781.

27 Briand, M.J., et al. 2018. Tracking trace elements into complex coral reef trophic networks. Science of the Total
Environment, 612, pp.1091-1104.

28 Fey, P., et al. 2019. Does trophic level drive organic and metallic contamination in coral reef organisms?. Science
of the Total Environment, 667, pp.208-221.

29 Revenga, J.E., et al. 2012. Arsenic, cobalt and chromium food web biodilution in a Patagonia mountain lake.
Ecotoxicology and Environmental Safety, 81, pp.1-10.

30 Chouvelon, T., et al. 2019. Patterns of trace metal bioaccumulation and trophic transfer in a phytoplankton-
zooplankton-small pelagic fish marine food web. Marine Pollution Bulletin, 146, pp. 1013-1030.

31 Campbell, L.M., et al. 2005. Mercury and other trace elements in a pelagic Arctic marine food web (Northwater
Polynya, Baffin Bay). Science of the Total Environment, 351, pp.247-263.

32 Erasmus, A., et al. 2020. Trophic transfer of pollutants within two intertidal rocky shore ecosystems in different
biogeographic regions of South Africa. Marine Pollution Bulletin, 157, p. 111309.

33 Ikemoto, T., et al. 2008. Biomagnification of trace elements in the aquatic food web in the Mekong Delta, South
Vietnam using stable carbon and nitrogen isotope analysis. Archives of environmental contamination and
toxicology, 54, pp.504-515.

34 Nfon, E., et al. 2009. Trophodynamics of mercury and other trace elements in a pelagic food chain from the Baltic
Sea. Science of the Total Environment, 407(24), pp.6267-6274.

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EPA Publication Number: 747S25002

Table 3: Summary of Studies that Examine the Trophic Transfer of Cobalt (Co)

Organism Description

Location

Freshwater/
Marine

Reference

Evidence

Conclusion

35 fish species and 15 invertebrate species of diverse
feeding strategies

East China Sea

Marine

Asante et al.
(2008)25

Significant
negative
correlation
between trophic
level (515N) and
organismal Co
concentrations.

Co undergoes
trophic dilution
in the food web
studied.

Three crustacean and eight fish species of various trophic
levels

Arno River (central
Italy)

Freshwater

Balzani et al.
(2021)26

Primary producers, consumers (herbivorous, omnivorous
and carnivorous invertebrates) and high-level predators
(anguilliform fish)

Lagoon of New
Caldonia (South
Pacific)

Marine

Briand et al.
(2018)27

Ice algae, three species of

zooplankton, Arctic cod (Boreogadus saida), ringed seals

(Phoca hispida) and eight species of

seabirds

Northwater Polynya,
Baffin Bay (arctic
Ocean)

Marine

Fey et al.
(2019)28

Plankton, benthic invertebrates, forage fish, and, plants

LakeMoreno,
Patagonia, Argentina

Freshwater

Revenga et
al. (2012)29

Phytoplankton, zooplankton, and pelagic fish (European
sardine and anchovy)

Gulf of Lions, NW
Mediterranean Sea

Marine

Chouvelon et
al. (2019)30

Decreasing Co
BAF with
increasing general
trophic level.

Ice algae, three species of zooplankton, Arctic cod
(Boreogadus saida), ringed seals (Phoca hispida) and eight
species of
seabirds

Northwater Polynya,
Baffin Bay (Arctic
Ocean)

Marine

Campbell et

al. (2005)31

No significant
correlation
between trophic
level (515N) and
organismal Co
concentrations.

Co does not
biomagnify in
the food web
studied.

37 species of algae, invertebrates, and (i.e. primary
producers, primary consumers, secondary consumers and
tertiary consumers)

South African coast
(Indian Ocean)

Marine

Erasmus et
al. (2020)32

Particulate organic matter (POM), phytoplankton,
gastropod (1 species), crustaceans (5 species), and fish (15
species)

Mekong Delta, South
Vietnam

Freshwater

Ikemoto et al.
(2008)33

Phytoplankton, zooplankton, mysis and herring

Baltic Sea

Marine

Nfon et al.
(2009)34

15

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EPA Publication Number: 747S25002

Jeffree etal. (2014) measured metal concentrations, including cobalt, in two fish species along a
contamination gradient within a mining impacted river in Australia.35 Populations of both fish
species exposed to the highest concentrations of mine-related metals (cobalt, copper, lead,
manganese, nickel, uranium, and zinc) in surface water and sediment had the lowest tissue (bone,
liver, and muscle) concentrations of these metals. The authors explored several hypotheses for
the observation and concluded that the most plausible interpretation is that populations of both
fish species have modified kinetics within their metal bioaccumulation physiology, via
adaptation or tolerance responses, to reduce their body burdens of metals.

6.4. Cobalt Conclusions from the WoE Approach

EPA used a WoE approach to determine the bioaccumulation potential of cobalt for the purposes
of TSCA new chemicals risk assessments. All three lines of evidence support a low concern for
cobalt bioaccumulation in aquatic food webs.

• LoE 1 (Fish/shellfish tissue concentrations relative to human intake criteria): There is a
low likelihood that human consumers are consistently exposed to cobalt via fish/shellfish
consumption at levels exceeding the non-cancer screening level (NCSL) over a lifetime
of consumption.

• LoE 2 (Benchmarking asainst other asencies/orsanizations): Four out of four available
risk assessments concluded that the bioaccumulation and biomagnification potential for
cobalt is low.

• LoE 3 (Literature review of pertinent metal-syecific bioaccumulation information): Ten
EPA-reviewed studies indicated either trophic dilution in aquatic food webs or no
relationship between cobalt concentrations in organisms and 815N (an indicator of
trophic level); none found evidence of biomagnification. Another study documented an
inverse relationship between cobalt concentrations in fish and environmental
contamination levels, suggesting active homeostatic regulation of the metal in these
species.

Based on the weight of evidence, cobalt has a low potential to bioaccumulate and is therefore
assessed as B*low rating in NCD risk assessments.

35 Jeffree, R. A., S. J. Markich and J. R. Twining (2014). "Diminished Metal Accumulation in Riverine Fishes
Exposed to Acid Mine Drainage over Five Decades." PLOS ONE 9(3): e91371.

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EPA Publication Number: 747S25002

Appendix

Literature with Cobalt Fish or Shellfish Tissue Concentration Data Used for Line of
Evidence #1 (Fish and Shellfish Tissue Concentrations Relative to Human Intake Criteria)

1.	Anandkumar, A., R. Nagarajan, K. Prabakaran, C. H. Bing and R. Rajaram (2018).
"Human health risk assessment and bioaccumulation of trace metals in fish species
collected from the Miri coast, Sarawak, Borneo." Mar Pollut Bull 133: 655-663.

2.	Andreji, J., Stranai, I., Massanyi, P., & Valent, M. (2005). "Concentration of selected
metals in muscle of various fish species." Journal of environmental science and health
40(4): 899-912.

3.	Asante, K. A., T. Agusa, H. Mochizuki, K. Ramu, S. Inoue, T. Kubodera, S. Takahashi,
A. Subramanian and S. Tanabe (2008). "Trace elements and stable isotopes (deltal3C
and deltal5N) in shallow and deep-water organisms from the East China Sea." Environ
Pollut 156(3V 862-873.

4.	Badsha, K. S. and C. R. Goldspink (1988). "Heavy metal levels in three species of fish in
Tjeukemeer, a Dutch polder lake." Chemosphere 17(2): 459-463.

5.	Balzani, P., P. J. Haubrock, F. Russo, A. Kouba, P. Haase, L. Vesely, A. Masoni and E.
Tricarico (2021). "Combining metal and stable isotope analyses to disentangle
contaminant transfer in a freshwater community dominated by alien species." Environ
Pollut 268(Pt B): 115781.

6.	Barlas, N. (1999). "A pilot study of heavy metal concentration in various environments
and fishes in the Upper Sakarya River Basin, Turkey." Environmental Toxicology 14(3):
367-373.

7.	Bouchoucha, M., R. Chekri, A. Leufroy, P. Jitaru, S. Millour, N. Marchond, C. Chafey,
C. Testu, J. Zinck, P. Cresson, F. Miralles, A. Mahe, N. Arnich, M. Sanaa, N. Bemrah
and T. Guerin (2019). "Trace element contamination in fish impacted by bauxite red mud
disposal in the Cassidaigne canyon (NW French Mediterranean)." Sci Total Environ 690:
16-26.

8.	Brotheridge, R., Newton, K., Evans, S., Taggart, M., & McCormick, P. (1998). "Nickel,
cobalt, zinc and copper levels in brown trout (Salmo trutta) from the river Otra, southern
Norway." Analyst 123(1): 69-72.

9.	Chouvelon, T., E. Strady, M. Harmelin-Vivien, O. Radakovitch, C. Brach-Papa, S.
Crochet, J. Knoery, E. Rozuel, B. Thomas, J. Tronczynski and J. F. Chiffoleau (2019).
"Patterns of trace metal bioaccumulation and trophic transfer in a phytoplankton-
zooplankton-small pelagic fish marine food web." Mar Pollut Bull 146: 1013-1030.

10.	Erasmus, A., Y. Ikenaka, S. M. M. Nakayama, M. Ishizuka, N. J. Smit and V. Wepener
(2020). "Trophic transfer of pollutants within two intertidal rocky shore ecosystems in
different biogeographic regions of South Africa." Mar Pollut Bull 157: 111309.

11.	Gashkina, N. A. and T. I. Moiseenko (2020). "Influence of Thermal Pollution on the
Physiological Conditions and Bioaccumulation of Metals, Metalloids, and Trace Metals
in Whitefish (Coregonus lavaretus L.)." Int J Mol Sci 21(12).

12.	Gebrekidan Asgedom, A., M. Berhe Desta and Y. Weldegebriel Gebremedh (2013).
"Bioaccumulation of Heavy Metals in Fishes of Hashenge Lake, Tigray, Northern
Highlands of Ethiopia." American Journal of Chemistry 2(6): 326-334.

17

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EPA Publication Number: 747S25002

13.	Greig, R. A. and J. Jones (1976). "Nondestructive neutron activation analysis of marine
organisms collected from ocean dump sites of the middle eastern United States." Arch
Environ Contam Toxicol 4(4): 420-434.

14.	Hantoush, A., G. Al-Najare, A. Amteghy, H. Al-Saad and K. Ali (2012). "Seasonal
variations of some trace elements concentrations in Silver Carp Hypophthalmichthys
molitrix Consolidated from farms in central Iraq." Marsh Bulletin 7: 126-136.

15.	Hellou, J., L. L. Fancey and J. F. Payne (1992). "Concentrations of twenty-four elements
in bluefin tuna, Thunnus thynnus from the Northwest Atlantic." Chemosphere 24(2): 211-
218.

16.	Ikemoto, T., N. P. Tu, N. Okuda, A. Iwata, K. Omori, S. Tanabe, B. C. Tuyen and I.
Takeuchi (2008). "Biomagnification of trace elements in the aquatic food web in the
Mekong Delta, South Vietnam using stable carbon and nitrogen isotope analysis." Arch
Environ Contam Toxicol 54(3): 504-515.

17.	Jayaprakash, M., R. S. Kumar, L. Giridharan, S. B. Sujitha, S. K. Sarkar and M. P.
Jonathan (2015). "Bioaccumulation of metals in fish species from water and sediments in
macrotidal Ennore creek, Chennai, SE coast of India: A metropolitan city effect."
Ecotoxicol Environ Saf 120: 243-255.

18.	Jeffree, R. A., S. J. Markich and J. R. Twining (2014). "Diminished Metal Accumulation
in Riverine Fishes Exposed to Acid Mine Drainage over Five Decades." PLOS ONE
9(3): e91371.

19.	Jelodar, H., H. Fazli and A. Salman Mahiny (2016). "Study on heavy metals (Chromium,
Cadmium, Cobalt and Lead) concentration in three pelagic species of Kilka (Genus
Clupeonella) in the southern Caspian Sea." Iranian Journal of Fisheries Sciences 15: 567-
574.

20.	Kumar, N., Chandan, N. K., Bhushan, S., Singh, D. K., & Kumar, S. (2023). "Health risk
assessment and metal contamination in fish, water and soil sediments in the East Kolkata
Wetlands, India, Ramsar site." Scientific Reports 13(1): 1546.

21.	Mannzhi, M. P., J. N. Edokpayi, O. S. Durowoju, J. Gumbo and J. O. Odiyo (2021).
"Assessment of selected trace metals in fish feeds, pond water and edible muscles of
Oreochromis mossambicus and the evaluation of human health risk associated with its
consumption in Vhembe district of Limpopo Province, South Africa." Toxicol Rep 8:
705-717.

22.	Nfon, E., I. T. Cousins, O. Jarvinen, A. B. Mukherjee, M. Verta and D. Broman (2009).
"Trophodynamics of mercury and other trace elements in a pelagic food chain from the
Baltic Sea." Sci Total Environ 407(24): 6267-6274.

23.	Owhonda, N. K., R. E. Ogali and S. E. Ofodile (2016). "Assessment of chromium, nickel,
cobalt and zinc in edible flesh of two tilapia fish species found in Bodo River, Rivers
State, Nigeria." Journal of Applied Sciences and Environmental Management 20(3).

24.	Raja, P., et al., (2009). Heavy Metals Concentration in Four Commercially Valuable
Marine Edible Fish Species from Parangipettai Coast, South East Coast of India."
International Journal of Animal and Veterinary Advances 1(1): 10-14.

25.	Rashed, M. N. (2001). "Monitoring of environmental heavy metals in fish from Nasser
Lake." Environment International 27(1): 27-33.

26.	Revenga, J. E., L. M. Campbell, M. A. Arribere and S. Ribeiro Guevara (2012). "Arsenic,
cobalt and chromium food web biodilution in a Patagonia mountain lake." Ecotoxicol
Environ Saf 81: 1-10.

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EPA Publication Number: 747S25002

27.	Sani, U. (2011). "Determination of some heavy metals concentration in the tissues of
Tilapia and Catfishes." Biokemistri 23(2).

28.	Sapozhnikova, Y., N. Zubcov, S. Hungerford, L. A. Roy, N. Boicenco, E. Zubcov and D.
Schlenk (2005). "Evaluation of pesticides and metals in fish of the Dniester River,
Moldova." Chemosphere 60(2): 196-205.

29.	Schmitt, C. J., Brumbaugh, W. G., & May, T. W. (2009). "Concentrations of cadmium,
cobalt, lead, nickel, and zinc in blood and fillets of northern hog sucker (Hypentelium
nigricans) from streams contaminated by lead-zinc mining: implications for monitoring."
Archives of environmental contamination and toxicology 56: 509-524.

30.	Shaqiri, L., & Mavromati, J. (2019). "Concentration of the Cobalt (Co) in Wild Fish
Squalius Cephalus and Barbus Barbus Tissues in Vardar River of North Macedonia."
Journal of Multidisciplinary Engineering Science and Technology (JMEST), 6(9): 2458-
9403.

31.	Squadrone, S., E. Burioli, G. Monaco, M. K. Koya, M. Prearo, S. Gennero, A. Dominici
and M. C. Abete (2016). "Human exposure to metals due to consumption of fish from an
artificial lake basin close to an active mining area in Katanga (D.R. Congo)." Science of
The Total Environment 568: 679-684.

32.	Swaibuh Lwanga, M., Kansiime, F., Denny, P., & Scullion, J. (2003). "Heavy metals in
Lake George, Uganda, with relation to metal concentrations in tissues of common fish
species." Hydrobiologia 499: 83-93.

33.	Szefer, P., J. Pempkowiak, B. Skwarzec, R. Bojanowski and E. Holm (1993).
"Concentration of selected metals in penguins and other representative fauna of the
Antarctica." Science of The Total Environment 138(1): 281-288.

34.	Turkmen, M. and C. Ciminli (2007). "Determination of metals in fish and mussel species
by inductively coupled plasma-atomic emission spectrometry." Food Chemistry 103(2):
670-675.

35.	Turkmen, M., A. Turkmen and Y. Tepe (2008). "Metal concentrations in five fish species
from Black, Marmara, Aegean and Mediterranean Seas, Turkey." Journal of the Chilean
Chemical Society 53: 1424-1428.

36.	Turkmen, M., A. Turkmen, Y. Tepe, Y. Tore and A. Ate§ (2009). "Determination of
metals in fish species from Aegean and Mediterranean seas." Food Chemistry 113(1):
233-237.

37.	Winger, P. V., D. P. Schultz and W. W. Johnson (1990). "Environmental contaminant
concentrations in biota from the lower Savannah River, Georgia and South Carolina."
Arch Environ Contam Toxicol 19(1): 101-117.

38.	Yilmaz, A. B., M. K. Sangiin, D. Yaglioglu and C. Turan (2010). "Metals (major,
essential to non-essential) composition of the different tissues of three demersal fish
species from iskenderun Bay, Turkey." Food Chemistry 123(2): 410-415.

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