A rriA United States
Environmental Protection
Agency September 2016
Technical Support for Fish Tissue Monitoring for
Implementation of EPA's 2016 Selenium Criterion
Draft
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EPA Draft for Public Comment
Technical Support for Fish Tissue Monitoring for Implementation of
EPA's 2016 Selenium Criterion
Draft
This document provides an overview on how to establish or modify existing fish tissue monitoring
programs to facilitate the collection and analysis offish tissue for the implementation of the fish tissue-
based criterion elements in the 2016 selenium water quality criterion, including waterbody assessment
and listing as well as development of water column-based site-specific criteria. The document does not
address the development of fish-tissue-based site-specific criteria. The document does not impose legally
binding requirements on EPA, states, authorized tribes, other regulatory authorities, or the regulated
community, and may not apply to a particular situation based upon the circumstances. EPA, state, tribal
and other decision makers retain the discretion to adopt approaches on a case-by-case basis that differ
from those provided in this technical support document where appropriate and consistent with statutory
and regulatory requirements. EPA could update this document as new information becomes available. In
addition to this document, EPA has other documents which provide considerations and recommendations
on implementing the selenium criterion and can be found at EPA's selenium website:
h(lm:,//www.eixt.eov/wac/aaualic-life-criterion-selenium
Table of Contents
Document Overview 4
Criteria Overview 4
Monitoring Strategy 6
Tissue Type 6
Egg-ovary Tissue Sample 8
Whole-body and Muscle Tissue Samples 9
Sample Type 11
Composite Samples 11
Individual Sample 13
Target Species 13
Leveraging Existing Fish Tissue Monitoring Programs and Sample Designs. 16
Considerations for Augmenting Existing Fish Tissue Monitoring Programs 16
Consistency with Existing Programs 17
Temporal Considerations 17
Spatial Considerations 18
Selenium Differences in Lentic and Lotic Environments 19
Existing Resources and Information 20
Available Expertise 20
Existing Guidance 21
Using Existing Data to Enhance Selenium Monitoring 23
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Sample Assessment: Analytical Chemistry 24
Sample Assessment: Statistical Analysis 27
Literature Cited 27
Appendix A Egg and Ovary Sample Preparation 33
Appendix B Spawning Seasons for Example Fish Assemblages from Select U.S. Watersheds. 36
Appendix C Conversion of Wet to Dry Tissue Weight 45
Tables
Table 1: Summary of the Recommended Freshwater Selenium Ambient Chronic Water Quality Criterion
for Protection of Aquatic Life 5
Table 2: Sampling Considerations Associated with Different Types of Fish Tissue 8
Table 3: Target Species for Implementation of Selenium Criterion 14
Table 4: Recommended Documents for Additional Guidance 23
Table 5: List of Test Procedures for Total Selenium in Tissue 25
List of Acronyms
USFWS United States Fish and Wildlife Service
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Definitions
Anadromous fish
Types of fish whose life cycle is divided between fresh and saltwater, including migrating to spawn in
freshwater. Migrations should be cyclical and predictable and cover more than 100 km. (FishBase, 2016)
Asynchronous spawners
Eggs are released in batches over a period that can last days or even months. (Murua and Saborido-Rey,
2003)
Exogenous feeding
Nutrient acquisition in which the food source is orally ingested and digested in the intestines. (Balon,
2013)
Gravid
Having the body distended with ripe eggs. (FishBase, 2016)
Indeterminate fecundity
Potential annual fecundity is not fixed before the onset of spawning and eggs can develop at any time
during the spawning season. (FishBase, 2016)
Iteroparous
Producing offspring in successive, e.g., annual or seasonal batches, as is the case in most fishes.
(FishBase, 2016)
Potamodromous
Fish species that spend their whole life in fresh water, but generally migrate for spawning purposes,
typically back to a natal upstream tributary from a mainstream river or between connected lake and river
systems. Migrations should be cyclical and predictable and cover more than 100 km. (FishBase, 2016)
Synchronous spawners
Eggs are released in a single episode in each breeding season. (Murua and Saborido-Rey, 2003)
Vitellogenesis
The process by which the yolk is formed and accumulated in the ovum. This is also the period when
nutrients stored in the liver are transferred to the developing oocytes in the ovary or ovaries. (FishBase,
2016)
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Document Overview
This document is part of a series of documents prepared by the U.S. Environmental Protection Agency
(EPA) Office of Water to provide an overview to states, authorized tribes, and other agencies on EPA's
2016 CWA section 304(a) recommendations for Aquatic Life Water Quality Criterion for Selenium -
Freshwater (USEPA 2016a). This document is intended to be used in conjunction with three companion
documents:
1) Technical Support for Adopting and Implementing EPA's Selenium 2016 Criterion in Water
Quality Standards
2) Frequently Asked Questions (FAQs): Implementing WQS that Include Elements Similar or
Identical to EPA's 2016 Selenium Criterion in Clean Water Act Section 402 NPDES Programs
3) Frequently Asked Questions (FAQs): Implementing the 2016 Selenium Criterion in Clean Water
Act Sections 303(d) and 305(b) Assessment, Listing, and Total Maximum Daily Load (TMDL)
Programs
Collectively, these four documents comprise the Technical Support Materials (TSM) for EPA's Aquatic
Life Ambient Water Quality Criterion for Selenium-Freshwater 2016 (USEPA 2016a). This document
provides an overview on how to establish or modify existing fish tissue monitoring programs to facilitate
implementation of the fish tissue-based criterion elements in the 2016 selenium water quality criterion.
This includes monitoring for waterbody assessment and listing as well as development of water column-
based site-specific criteria. The document does not specifically address monitoring for the development of
fish-tissue-based site-specific criteria. States and authorized tribes who wish to develop fish-tissue-based
site-specific criteria should engage their EPA Regional office early in the process to ensure the
development of sound scientific analyses.1
Criteria Overview
The EPA updated its national recommended chronic aquatic life criterion for selenium in freshwater to
reflect the latest scientific information, which indicates that toxicity to aquatic life is driven by dietary
exposures. The criterion has four elements: (1) a fish egg-ovary element, (2) a fish whole-body and/or
muscle element, (3) a water column element (one value for lentic and one value for lotic aquatic systems),
and (4) a water column intermittent element to account for potential chronic effects from short-term
exposures (one value for lentic and one value for lotic aquatic systems). EPA's Aquatic Life Ambient
Water Quality Criterion for Selenium-Freshwater 2016 contains a recommendation that states and
authorized tribes adopt into their water quality standards (WQS) a selenium criterion that includes all four
elements (USEPA 2016a). The criterion document also recommends that—because the fish tissue-based
concentration is a more direct measure of selenium toxicity to aquatic life than water column
concentrations—fish tissue elements supersede the water column elements when both types of data are
available (Table 1). All tissue elements have primacy over water element(s), except where there are no
fish, or for water bodies with new discharges where tissue concentrations in fish might not have
stabilized. EPA did not develop an acute criterion for selenium when it updated the chronic criterion. In
the case of bioaccumulative compounds like selenium, acute toxicity studies do not address risks that
result from exposure to chemicals via the diet (through the food web). Such studies also do not account
1 Throughout this document and in the CW A. the term "states" means the fifty states, the District of Columbia, the
Commonwealth of Puerto Rico, the United States Virgin Islands, Guam, American Samoa, and the Commonwealth
of the Northern Mariana Islands. The term "authorized tribe" means those federally recognized Indian tribes with
authority to administer a CWA WQS program.
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for the slow accumulation kinetics of many bioaccumulative compounds such as selenium and may
underestimate effects from long-term accumulation in different types of aquatic systems. Because
exposure to selenium toxicity is primarily driven by organisms eating selenium-contaminated food rather
than being exposed only to selenium dissolved in water, chronic exposure is a more relevant concern for
aquatic life. However, as described in the criterion document, EPA included an intermittent criterion
element. Application of the intermittent exposure criterion element will provide protection from the most
important selenium toxicity effect, reproductive toxicity, by protecting against selenium bioaccumulation
in the aquatic ecosystem resulting from short-term, high exposure events (USEPA 2016a).
The selenium aquatic life chronic criterion is unique, in part, because it is the first aquatic life criterion
based on fish tissue. EPA has previously published fish tissue-based criteria for methyl-mercury, but
those criteria are for protecting human health. Therefore, states and authorized tribes have experience
sampling fish tissue for the purposes of issuing fish consumption advisories, thus collection of fish tissue
for water quality assessment is common.
Table 1: Summary of the Recommended Freshwater Selenium Ambient Chronic Water Quality
Criterion for Protection of Aquatic Life.
Media Type
Fish Tissue1
Water Column4
Criterion
Element
Egg-ovary 2
Fish Whole-body
or Muscle3
Monthly
Average
Exposure
Intermittent Exposure5
Magnitude
15.1 mg/kg dry
weight
8.5 mg/kg dry
weight whole-
body
or
11.3 mg/kg dry
weight muscle
(skinless, boneless
fillet)
1.5 (ig/L in lentic
aquatic systems
3.1 (ig/L in lotic
aquatic systems
WQCint =
WQC30-day Cbkgrndd f int)
f int
Duration
Instantaneous
measurement6
Instantaneous
measurement6
30 days
Number of days/month with an
elevated concentration
Frequency
Not to be
exceeded
Not to be exceeded
Not more than
once in three
years on average
Not more than once in three
years on average
1. Fish tissue elements are expressed as steady-state.
2. Egg-ovary supersedes any whole-body, muscle, or water column element when fish egg-ovary concentrations are
measured.
3. Fish whole-body or muscle tissue supersedes water column element when both fish tissue and water
concentrations are measured.
4. Water column values are based on dissolved total selenium in water and are derived from fish tissue values via
bioaccumulation modeling. Water column values are the applicable criterion element in the absence of steady-
state condition fish tissue data.
5. Where WQC3o-day is the water column monthly element for either lentic or lotic waters; Cbkgmd is the average
background selenium concentration; and fmt is the fraction of any 30-day period during which elevated selenium
concentrations occur, with fmt assigned a value >0.033 (corresponding to 1 day).
6. Fish tissue data provide instantaneous point measurements that reflect integrative accumulation of selenium over
time and space in fish population(s) at a given site.
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EPA derived fish tissue and water column elements from the underlying scientific studies on selenium
reproductive effects in fish taking into consideration the implementation of criteria for Clean Water Act
purposes (e.g., permitting, monitoring, and assessment). Available toxicity data indicate the selenium
concentration in fish eggs and ovaries is the most robust and consistent measurement endpoint directly
tied to adverse aquatic effects. Toxicity in developing embryos and larvae is directly linked to egg
selenium concentration (USEPA 2016a). EPA derived the whole-body and muscle tissue elements from
the egg-ovary element so that states and authorized tribes could more readily implement EPA's selenium
criterion.
EPA recommends that states and authorized tribes adopt into their water quality standards a selenium
criterion that expresses the four elements as a single criterion composed of multiple parts in a manner that
explicitly affirms the primacy of the whole-body or muscle element over the water column elements, and
the egg-ovary element over any other element. Adopting the fish whole-body and muscle tissue element
into water quality standards ensures the protection of aquatic life when measurements from fish eggs or
ovaries are not available. Adopting the water column element ensures protection when fish tissue
measurements are not available. For approaches for translating between fish tissue and water column
selenium concentrations, see Appendix K of Aquatic Life Ambient Water Quality Criterion for Selenium-
Freshwater 2016 (USEPA 2016a). For information on how to use the four-part criterion for the purposes
of National Pollutant Discharge Elimination System (NPDES) permitting and waterbody assessment,
listing, and TMDL development, see Frequently Asked Questions (FAQs): Implementing WQS that
Include Elements Similar or Identical to EPA's 2016 Selenium Criterion in Clean Water Act Section 402
NPDES Programs (USEPA 2016b) and I'AOs: Implementing the 2016 Selenium Criterion in Clean Water
Act Sections 303(d) and 305(b) Assessment, Listing and Total Maximum Daily Load (TMDL) Programs
(USEPA 2016c), respectively.
Monitoring Strategy
The following sections review study design and sampling considerations regarding fish tissue types,
sample types, target species and sizes, and spatial and temporal concerns. Additional information
regarding adoption of, implementation of, and compliance with the criteria can be found in the three
companion documents (USEPA 2016b, USEPA 2016c, and USEPA 2016d).
When considering monitoring strategies, agencies should first review their existing fish tissue monitoring
programs, if any exist, and determine how best to incorporate fish tissue sampling for selenium. The
relationship between fish tissue sampling locations, species habits and natural history, and selenium
sources should be understood and taken into account during sampling for implementation of the criterion.
Detailed field collection procedures can be found in EPA's 2000 Fish Advisory Guidance (USEPA
2000a) and the Field Sampling Plan for the National Study of Chemical Residues in Lake Fish Tissue
(USEPA 2002). Appendix A of this document presents egg and ovary collection and sample preparation
methods.
Tissue Type
From the toxicology standpoint, the most relevant measure of exposure to a toxic substance is its
concentration at the site of toxic action. Because of selenium's mode of action in fish, the most
ecologically relevant sites of toxic action are the developing tissues during early life stages. This was a
major point of consensus of the 2009 SETAC Pellston workshop on selenium risk assessment (Chapman
et al. 2009). The 304(a) selenium aquatic life criterion is based on reproductive impacts in fish. Egg
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and/or ovary tissue is the closest surrogate for measuring actual reproductive effects from maternal
exposure to selenium. Therefore, selenium concentrations in egg-ovary tissue is the most useful exposure
measure for estimating ecological effects.
Egg-ovary tissue of adult female fish may be the best surrogate for assessment of reproductive toxicity in
fish, however some states or authorized tribes may instead sample muscle or whole-body tissue from
adult fish due to the following considerations:
• Temporal. Most fish species that are synchronous spawners do so in the spring; whereas fish
tissue collection for advisories typically occur in the late summer or early fall, when contaminant
loads in the edible portion of the fish are highest.
• Spatial. Some fish species (e.g., salmonids) migrate to upstream areas to spawn; areas may be
harder to access than larger order downstream segments that are inhabited during non-spawning
seasons.
• Size: It is difficult to collect egg-ovary (or muscle) tissue samples from small fish species (e.g.,
certain species in the family Cyprinidae or Cyprinodontidae) because the amount of tissue
available for analysis is small, and many of these species are asynchronous spawners that do not
have a large number or biomass of eggs at any one time.
Due to these various concerns, states or authorized tribes have considerable discretion when selecting the
fish tissue type to be used in their sampling protocols. The flexibility provided by having multiple fish
tissue types for water quality monitoring and assessment purposes also leverages existing monitoring
capacity since a number of the species that are good target species for selenium sampling may also be
commonly collected as muscle (fillet) samples in state and tribal fish tissue monitoring programs (e.g.,
trout/salmon, bass/sunfish). The whole-body tissue criterion element also simplifies the collection and
processing of small fish species that may be the dominant trophic level in smaller order stream networks.
When developing a new or modifying an existing fish tissue monitoring strategy, states or authorized
tribes should consider the available resources, existing information on the spawning habits and size of
target species, and potential population level effects associated with lethal sampling techniques. They
should also consider where the relevant exposure is, and understand where fish are feeding and obtaining
their selenium body burdens. Sampling considerations associated with different types of fish tissue are
presented in Table 2.
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Table 2: Sampling Considerations Associated with Different Types of Fish Tissue
Issue
Egg-ovary*
Whole-body*
Muscle/Fillet*
Comments
Ease of
collection
Difficult
Easy
Easy - except
on small fish
Egg-ovary samples are only
collected from gravid females;
there are seasonal and logistical
considerations, and species-
specific sampling windows. See
Appendices A and B.
Consistency
with existing
state & tribal
methods
Not typically
collected
Sometimes
collected
Primary tissue
collected
Whole-body might be collected
in special cases, for certain
populations that consume whole
fish, or for eco-risk assessments.
Sample
availability
Limited - only
from gravid
females
Always
Always
For water bodies with small
species at top trophic levels,
whole-body may be the only
option due to issues collecting
sufficient muscle tissue.
Ability to
make
composite
sample
Yes
Yes
Yes
Composite samples are the most
cost-effective way to represent
average selenium tissue
concentrations. However,
information on elevated levels of
chemical contamination in
individual organisms is likely
attenuated.
Ability to test
individual
sample
Yes on larger
species; smaller
species or
asynchronous
spawners may
require
composited tissue
Yes on larger
species, may
be difficult on
small species
Yes on larger
species, may be
difficult on
small species
Individual samples are more
resource intensive to prepare and
more expensive to analyze, but
are valuable when sampling
from waters known or suspected
to be impacted by selenium
discharges.
*See Appendix C for methods to convert from wet weight to dry weight and vice versa.
Egg-ovary Tissue Sample
Egg-ovary is the preferable tissue to collect because the egg-ovary tissue of pre-spawn, reproductively
mature (also called "gravid" or "vitellogenin') females will give the most accurate view of potential
selenium hazard to reproduction. Egg-ovary tissue (which refers to eggs, ovaries, or both) data provide
point measurements that reflect integrative dietary accumulation, transfer, and deposition of selenium
over time and space in female fish at a given site. Research has shown that selenium concentrations in
egg-ovary tissue is strongly correlated with selenium in the maternal diet, which is transferred from the
adult female during vitellogenesis. Buhl and Hamilton found concentrations 2-5 times higher in eggs than
that in the maternal muscle tissue, indicating that dietary selenium was transferred from the female in a
concentration-dependent manner (Buhl and Hamilton 2000). When using egg-ovary tissue for the
implementation of the selenium criterion, states and authorized tribes must be careful to consider the
difficulty in timing egg-ovary sampling with spawning periods. Timing errors related to fish reproduction
may result in data that falsely indicate the selenium criterion is being met.
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Monitoring programs should sample for reproductively mature females from iteroparous fish species (i.e.,
fish that have multiple reproductive life cycles over the course of its lifetime) that are single batch
(synchronous) or multiple batch (asynchronous) spawners. Fish species that spawn multiple times per
season (asynchronous; e.g., species in the family Cyprinidae) have variable cycles of oogenesis and thus
special care should be taken when using these for egg-ovary monitoring as the pre-spawn window can be
hard to predict. Egg maturation may occur well before, immediately prior to, or during the spawning
season. For example, Lepomis cyanellus (Green Sunfish) can spawn multiple times per season
(Osmundson and Skorupa 2011, Chapman et al. 2010). For many fish species, vitellogenesis can occur
over several months prior to spawning, with a relatively large amount of yolk deposited into eggs
(Osmundson and Skorupa 2011). It is also possible that species with relatively large eggs and yolks
deposit more selenium in their eggs than species with smaller eggs and yolks (Osmundson and Skorupa
2011). Selenium concentrations in the eggs and ovarian tissues are expected to be at their maximum level
when eggs have maximum levels of vitellogenin prior to spawning; therefore, egg-ovary tissue samples
collected outside of the pre-spawn window are not suitable for assessment in comparison to the national
egg-ovary fish tissue criterion element. Reproductively mature females of most fish species, except
indeterminate spawning species and viviparous species (i.e., live bearing), will produce eggs that can be
sampled for selenium. Appendix A of this document presents egg and ovary collection and sample
preparation methods.
An egg-ovary tissue sample from a female that is not gravid will not be representative for monitoring and
assessment when compared with gravid egg-ovary results, since the egg-ovary tissues represent the
potential selenium load available to eggs and larvae through maternal transfer. Larger game species such
as Rainbow Trout (Oncorhynchus mykiss) and Walleye (Stizostedion vitreum) will be logistically simpler
to sample because they spawn once per year, which allows for easier collection of egg-ovary tissue since
the reproductive timing and habits of these species in freshwater tend to be well understood in most areas.
Species should be sampled when females are expected to be gravid. This will depend on the species and
geography, and for most species this will happen in spring but may happen later at higher latitudes. For
example, different species of trout begin releasing eggs and sperm (spawning) during different times of
the year. Rainbow Trout (Oncorhynchus mykiss) spawn in the late spring and early summer as water
temperatures rise. Brown Trout (Salmo trutta) spawn in the fall, typically from late September to early
November, and Lake Trout (Salvelinus namaycush) also spawn during the autumn months. See Appendix
B of this document for spawning windows of different species in various regions across the US.
The egg-ovary tissue element has primacy over all other elements, thus, when available, it is the ultimate
arbiter for compliance with the selenium water quality criterion. Most states and authorized tribes do not
currently collect egg-ovary tissue as part of their regular monitoring programs. EPA recognizes that many
states and authorized tribes may not have the resources to augment their existing monitoring programs to
include egg-ovary tissue collection. While egg-ovary remains the preferable tissue type, whole-body or
muscle samples can be used as an alternative.
Whole-body and Muscle Tissue Samples
The whole-body and muscle tissue elements of EPA's selenium criterion were derived from the egg-ovary
element. Whole-body and muscle tissue samples are acceptable alternatives because selenium
concentrations in fish collected at any time of the year (except pre-spawn windows for females) will
provide sufficient information on selenium bioaccumulation, although there will likely be some variation
across seasons, due to prey availability, temperature, depuration of selenium from tissue during
vitellogenesis prior to spawning, and other factors. Summer and fall may be prime periods for whole-
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body and muscle tissue collection due to the engorgement of populations to replenish fat and energy
reserves post-spawn and for over-wintering. Winter tissue collection is discouraged, except for
subtropical regions. Whole-body and muscle fish tissue data provide point measurements that reflect
integrative dietary accumulation and deposition of selenium in fish tissues over time and space in fish
population(s) at a given site. The whole-body tissue element is intended to be used for whole fish for
small fish species or small individuals of larger fish species. Whole-body and muscle tissue are equally
preferred in the absence of egg-ovary tissue.
Whole-body and muscle tissue samples are relatively easy to collect, and do not have the same spatial
considerations and temporal restrictions as egg-ovary tissue. Muscle tissue is the most common type of
sample collected and analyzed by monitoring programs, and whole-body samples are sometimes
submitted by states and authorized tribes for analysis. A portion of these samples already collected can be
submitted for selenium analysis. States or authorized tribes will realize cost efficiencies by choosing to
use whole bodies or fillets that are already being collected for an existing monitoring program. EPA is
aware that some states and authorized tribes make use of muscle plugs in their monitoring programs.
However, it is important to remember that contaminant concentrations can vary considerably depending
on where the plug is collected. Plugs provide very small tissue quantities (about a gram of tissue per fish)
and therefore not enough biomass for possible reanalysis or quality assurance/quality control
considerations. In addition, relatively small individuals may not recover from a muscle plug biopsy
punch. Care should be taken to ensure that the sampling protocols involving plugs have a sound scientific
basis and that there is enough tissue for the analytical method.
States or authorized tribes might choose to use whole-body or muscle tissue samples because seasonal
restrictions on fish sampling may prevent sampling for egg-ovary tissue, or because existing monitoring
programs can incorporate selenium analysis into their existing fish tissue monitoring strategies. States or
authorized tribes might also choose to use whole-body samples because juvenile or small-bodied species
are the most appropriate to sample in a particular situation (Beatty and Russo 2014). In small streams and
watersheds that are dominated by lower trophic level fish, it may be difficult to collect egg-ovary tissue
from small fish species (e.g., species in the family Cyprinidae or Cyprinodontidae), due to the small
amount of egg-ovary tissue available for analysis. In addition, most small bodied fish (i.e., minnows -
cyprinids, cyprinodonts and Killifish [Fundulus spp.]) are asynchronous spawners, and produce eggs
sporadically over the spawning season such that there is no one "best" time to collect mature eggs.
Furthermore, the small body mass (even at adult stage) for many of these fish necessitates the collection
of multiple individuals to ensure a sufficient tissue sample for processing and analytical chemistry
analyses.
Another case where whole-body or muscle samples might be used is for Pacific anadromous juvenile
(smolt) salmonids. Anadromous fish species are those spawned in freshwater, then migrate to the ocean as
juveniles (e.g., smolts), where they grow into adults before migrating back into freshwater to
spawn. Notable among these species are the coho, chum, and Chinook salmon, as well as marine adapted
rainbow trout (steelhead). Adult anadromous females (in the genus Oncorhynchus) stop eating prior to re-
entering freshwater environments as part of the physiological modifications required for the migratory
spawning process, and thus, lack exposure to freshwater selenium sources. They are also semelparous
(except steelhead), meaning they die after spawning so there is no post-spawn residual exposure. Since
adults of these species are not residents of the waterbody, the selenium concentrations will not be
representative of localized freshwater selenium sources (see Section 6.4.1 of the criterion document)
(USEPA 2016a). An exception are landlocked variants of striped bass that cannot migrate out to sea, or
hybrids (e.g., "wipers" which are striped bass-white bass crosses) in the Midwest. Adult fish in these
landlocked populations may be representative of localized freshwater selenium concentrations, and thus
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appropriate for sampling. Although more uncertain, some studies indicate that selenium might affect
endpoints such as juvenile growth and survival (Hamilton et al. 1990, DeForest and Adams 2011), so
monitoring of selenium in the whole body of Pacific anadromous salmon smolt is the most appropriate
tissue to assess selenium hazard to these fish species.
Seasonal considerations are less stringent for whole-body and muscle tissue sampling. Seasonal collection
of whole-body or muscle fish tissue samples should be timed to avoid the pre-spawning influence on
selenium tissue concentrations, particularly for females, since enhanced depuration of selenium from
tissue stores may occur during vitellogenesis prior to spawning (USEPA 2016a).
Sample Type
For fish tissue monitoring of selenium for implementing EPA's recommended selenium criterion, EPA
recommends using composite samples. This is based on current EPA guidance on fish tissue monitoring
which recommends using composite samples (USEPA 2000a).
Composite Samples
Composite samples are homogeneous mixtures of one type of tissue (e.g., egg-ovary sample, whole-body,
or muscle) from two or more individual organisms of the same species collected at a particular site and
analyzed as a single sample. Composite samples of fish tissue are recommended for selenium analysis to
help identify those sites where selenium concentrations are elevated. They are also best for small fish
species where they become a logistical necessity due to small amounts of tissue per individual
fish. Because the costs of individual chemical analyses are usually higher than field costs, EPA
recommends using composite samples as the most cost-effective way to represent average selenium tissue
concentrations in target species populations (see Table 3). Since composites represent a physical
averaging of the samples, they also avoid the issue of how non-detections will be factored into averaging
(USEPA 2010a). Additionally, composite samples ensure adequate sample mass to allow analyses for any
additional target analytes. A disadvantage of using composite samples, however, is that elevated/extreme
contaminant concentration values for individual organisms are attenuated.
Current EPA guidance on fish tissue monitoring recommends using composite samples and recommends
using 3 to 10 individuals for a composite sample for each target species as availability allows (USEPA
2000a). In Section 6.1.2.7.1 of the Fish Advisory Guidance ("Guidelines for Determining Sample Sizes"),
the guidance maintains that it is not possible to recommend a single set of sample size requirements for all
fish contaminant monitoring studies (USEPA 2000a). Rather, EPA presents a more general approach to
sample size determination that is both scientifically defensible and cost-effective. EPA provides a table in
this section of the guidance that shows the varying precision achieved by using additional numbers of
individuals per composite, and additional replicate composite samples. The data suggest that greater
precision in the estimated standard error is gained by increasing the number of replicate samples than by
increasing the number of fish per composite.
At each site, states and authorized tribes should determine the appropriate number of individuals per
composite sample and number of replicate composite samples. This should be based on site-specific
estimations of the population variance of the target analyte concentration, fisheries management
considerations, and statistical power consideration. For example, fewer replicate composite samples
and/or fewer individuals per composite sample may be required if the population variance of the selenium
concentration at a site is small. In this case, it would not be cost-effective to use sample sizes that are
larger than required to achieve the desired statistical power. Additionally, fish tissue monitoring for
criteria implementation may be conducted on much smaller streams than those sampled for fish
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consumption purposes, and there may be limited numbers of fish available in these smaller tributaries.
In EPA's National Lake Fish Tissue Study, composites were generally required to include five fish
(USEPA 2002a). This composite size represented a reasonable number of fish that also satisfied statistical
requirements. Based on this precedent and EPA's Fish Advisory Guidance (USEPA 2000a), EPA
recommends that in most waters composites of five fish be used for fish tissue monitoring for selenium
criteria implementation. However, EPA recognizes that sometimes it might not be possible to collect a
five-fish composite (or, as described above, five fish might not be needed to have statistical power). In
these limited cases, EPA encourages the state or tribe to use as many fish as possible in the composite.
Organisms used in a composite sample should meet the following recommendations (USEPA 2000a):
• All the same species.2
• Of similar size so that the smallest individual in a composite is no less than 75% of the total
length (size) of the largest individual (the "75% rule"; does not apply to egg-ovary samples).
• Collected at the same time (i.e., collected as close to the same time as possible but no more than 1
week apart).
• Collected in sufficient numbers to provide at least 20 grams composite homogenate sample of
tissue for analysis of selenium.
EPA's 2000 Fish Advisory Guidance (USEPA 2000a) provides recommendations on the number of
composite samples to collect. It recommends collecting at least two composite samples at each site, and
encourages a third, in order to properly estimate the site variance. For the purposes of sampling fish in
potential selenium impacted waters, the number of composite replicates may be determined on a case-by-
case basis. This decision would primarily be based on the presence of target species and the numbers of
individuals present at the site in question.
Individual organisms used in composite samples must be of the same species, in part because of the
differences in selenium bioaccumulation potential between species (USEPA 2016a). Accurate taxonomic
identification is essential to prevent the mixing of species in a sample. EPA recognizes that, in contrast to
other bioaccumulative contaminants in fish, selenium concentrations are generally conserved or increase
incrementally at each trophic level in a food web. This is because there is relatively little variation across
all trophic levels of fish since the trophic transfer factors from prey to fish are small, with some
exceptions (e.g., molluscivorous fish) (USEPA 2016a). However, EPA still recommends following the
"75% rule" for whole body or muscle tissue (does not apply to egg-ovary samples) for the sizes of
individual specimens within a composite.
The tissue mass recommendation is based on EPA Method 200.8 for solid samples, which states that a 20
gram sample is sufficient if the sample is <35% moisture; a 50-100 gram sample is recommended if the
moisture content is >35% (USEPA 1994a). Since many fish tissue samples are 70-80% moisture,
monitoring agencies should consider the tissue mass as they develop their sampling and analysis plans.
Monitoring agencies typically collect composite samples for other analytes in addition to selenium;
additional biomass should be collected to accommodate selenium as well as standard contaminant
analyses, if necessary. If agencies currently discard or archive the composite homogenates in excess of
their current analytical needs, it may be easy to save the excess tissue to use an additional 20 grams (or
2 Ensuring that a composite sample consists of the same species is particularly important for selenium as different
species can have different sensitivity to selenium and have different bioaccumulation potential (see "Target Species"
discussion below).
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more if needed) for selenium analysis. Agencies that submit composite tissue samples for their advisory
analyses could take advantage of the opportunity to add selenium as an analyte to their sampling protocol.
Individual Sample
An individual sample is a discrete sample from a single fish, and can be an egg-ovary sample, a whole
body, or a muscle (fillet) sample. Although EPA recommends states or authorized tribes use composite
samples for selenium fish tissue monitoring, there are some instances where collecting individual fish
may be desirable.
Analysis of individual fish samples may be of interest to evaluate spatial and temporal differences among
individuals of a species of similar size or across the population of a species residing in a specific water
body. For water bodies or segments that are known to be impacted by selenium, individual samples may
better estimate the magnitude (i.e., extreme values) of the impact and may provide information about
selenium source-exposure relationships in large water bodies. Individual samples may also allow for the
identification of fish that are migrant or transient in a population, since that fish may have a higher or
lower concentration of selenium than other fish in the area. EPA recommends 20 grams as a minimum
tissue mass required per individual fish for analysis and QA/QC (USEPA 1994a).
If using individual samples for the purposes of selenium criteria implementation, all fish should be the
same species and from the same waterbody (or site for large waterbodies) within the same sampling
period. Where the monitoring agency plans to arithmetically composite such individual samples or
calculate an average concentration, the fish should be of similar size (within the 75% rule) and the
samples should be of the same tissue type. When using individual fish tissue samples for selenium
monitoring, EPA recommends targeting at least 5 individuals for analysis to achieve measurements of a
reasonable statistical power (see discussion of statistical power in the "Composite Sample" discussion
above). In the event that collecting at least 5 individuals of one species is not possible, fewer specimens
may be sufficient to provide adequate biomass for both selenium analysis and quality assurance/quality
control (QA/QC), but the statistical power of the analysis may be affected. EPA recommends 20 grams as
a minimum tissue mass required per individual fish for analysis and QA/QC.
Target Species
Different species have varying sensitivity to selenium and as such, states or authorized tribes should
consider selenium sensitivity, along with bioaccumulation potential, when designing fish tissue
monitoring plans. EPA recommends that states or authorized tribes target species that have higher
selenium sensitivity, but if this is not possible, the selenium criterion is designed to be used for any fish
species (with the exception of anadromous fish species). Migratory and highly mobile fish species should
be avoided for selenium sampling, if possible. Recently stocked fish should also be avoided, regardless of
species, since their residence time before sampling may be too short to provide a representative sample.
Since the selenium criterion applies to ecological risk and not human health, monitoring agencies could
evaluate their target species list and decide if they are including appropriate species for assessing
selenium risk in their regions (see Table 3). When selecting target fish species for selenium criterion
monitoring, monitoring agencies should focus on species that are sensitive to selenium, that may
potentially accumulate high concentrations of selenium, and that are easy to identify (USEPA 2000a).
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Table 3: Target Species for Implementation of Selenium Criterion
F amily
Scientific Name
Common Name
Acipenseridae
Scaphirhynchus platorynchus
Shovelnose Sturgeon
Acipenseridae
Acipenser fulvescens
Lake Sturgeon
Catostomidae
Ictiobus bubalus
Smallmouth Buffalo
Catostomidae
Ictiobus cyprinellus
Bigmouth Buffalo
Catostomidae
Catostomus commersonii
White Sucker
Catostomidae
Catostomus catostomus
Longnose Sucker
Catostomidae
Catostomus macrocheilus
Large scale Sucker
Catostomidae
Minytrema melanops
Spotted Sucker
Catostomidae
Moxostoma anisurum
Silver Redhorse
Catostomidae
Moxostoma congestum
Grey Redhorse
Catostomidae
Moxostoma duquesnei
Black Redhorse
Catostomidae
Moxostoma erythrurum
Golden Redhorse
Catostomidae
Moxostoma macrolepidotum
Shorthead Redhorse
Catostomidae
Moxostoma poecilurum
Blacktail Redhorse
Catostomidae
Carpiodes cyprinus
Quillback
Centrarchidae
Micropterus salmoides
Largemouth Bass
Centrarchidae
Micropterus dolomieu
Smallmouth Bass
Centrarchidae
Pomoxis annularis
White Crappie
Centrarchidae
Pomoxis nigromaculatus
Black Crappie
Centrarchidae
Lepomis macrochirus
Bluegill
Centrarchidae
Lepomis cyanellus
Green sunfish
Centrarchidae
Ambloplites rupestris
Rock Bass
Cyprinidae
Cyprinus carpio
Common Carp
Cyprinidae
Campostoma anomalum
Central Stoneroller
Cyprinidae
Rhinichthys cataractae
Longnose Dace
Cyprinidae
Rhinichthys atratulus
Blacknose Dace
Cyprinidae
Semotilus atromaculatus
Creek Chub
Cyprinidae
Semotilus corporalis
Fallfish
Cyprinidae
Pimephales promelas
Fathead Minnow
Cyprinidae
Pimephales notatus
Bluntnose Minnow
Cyprinidae
Notemigonus crysoleucas
Golden Shiner
Cyprinidae
Notropis atherinoides
Emerald Shiner
Cyprinidae
Notropis hudsonius
Spottail Shiner
Cyprinidae
Nocomis micropogon
River Chub
Esocidae
Esox lucius
Northern Pike
Esocidae
Esox masquinongy
Muskellunge
Ictaluridae
Ictalurus catus
White Catfish
Ictaluridae
Ictalurus punctatus
Channel Catfish
Ictaluridae
Ictalurus melas
Black Bullhead
Ictaluridae
Ictalurus nebulosus
Brown Bullhead
Ictaluridae
Ictalurus natalis
Yellow Bullhead
Ictaluridae
Pylodictis olivaris
Flathead Catfish
Moronidae
Moron e chrysops
White Bass
Moronidae
Morone saxatilis1
Striped Bass1
Moronidae
Morone americana
White perch
Percidae
Sander vitreus
Walleye
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F amily
Scientific Name
Common Name
Percidae
Sander canadensis
Sauger
Percidae
Perca flavescens
Yellow Perch
Salmonidae
Coregonus clupeaformis
Lake Whitefish
Salmonidae
Oncorhynchus kisutch2,3
Coho Salmon2,3
Salmonidae
Oncorhynchus mykiss
Rainbow Trout
Salmonidae
Oncorhynchus tschawytscha2,4
Chinook Salmon2,4
Salmonidae
Salvelinus namaycush
Lake Trout
Salmonidae
Salmo trutta
Brown Trout
Salmonidae
Salvelinus fontinalis
Brook Trout
Sciaenidae
Aplodinotus grunniens
Freshwater Drum
Common molluscivorous fish species are indicated in bold. Molluscivorous fish species have a higher potential to
bioaccumulate selenium, since the available data indicate that mollusks generally have a higher trophic transfer
factor than other invertebrate taxa (USEPA 2016a).
1 Adult specimens are acceptable if the population is landlocked
2 Where Pacific anadromous fish are listed, the target species only includes juveniles (smolt stage)
3 Endangered in Central California Coast; Threatened in Lower Columbia River, Oregon Coast, and Southern
Oregon - Northern California Coast (USFWS 2016)
4 Endangered in Sacramento River and Upper Columbia River; Threatened in California Coastal, Central Valley,
Lower Columbia River, Puget Sound, Snake River, and Upper Willamette River (USFWS 2016)
Bioaccumulation of selenium by higher trophic level fish is highly influenced by diet. For example, fish
that primarily consume freshwater mollusks (e.g., Lepomis microlophus, or redear sunfish) will exhibit
greater selenium bioaccumulation than fish that consume primarily insects or crustaceans from waters
with the same concentration of dissolved selenium because mollusks tend to accumulate selenium at
higher concentrations than other trophic level 2 organisms (Luoma and Presser 2009; Stewart et al. 2004).
Because of this, diet is an important factor to consider when selecting species to monitor. For example, in
the San Francisco estuary, sturgeon are monitored not only because they are sensitive to the toxic effects
of selenium, but also because their primary prey accumulates selenium very efficiently. As a result, the
sturgeon receive large doses of selenium.
Based on the best available and acceptable reproductive-effect studies as well as extensive analyses, EPA
developed a species sensitivity distribution (SSD) to support the derivation of the national selenium
criterion (USEPA 2016a). This SSD presents the four most sensitive genera for fish reproductive effects
(in decreasing order) to be Acipenser, Lepomis, Salmo, and Oncorhynchus. These genera have known
sensitivity to selenium and should be targeted for selenium monitoring, but care should be taken to avoid
sampling threatened or endangered species and anadromous species. For example, Acipenser, although
the most sensitive, is a genus of sturgeon; many species are threatened or endangered and thus are not
suitable for sampling. When selecting species from these genera, it is important to consider the diet of
certain species compared to others, and select the species that best represent the potential accumulation in
the waterbody. As mentioned, fish that primarily consume freshwater mollusks will exhibit greater
selenium bioaccumulation than fish that consume primarily insects or crustaceans form the same waters
(Luoma and Presser 2009; Stewart et al. 2004). Fish that consume primarily benthic insects will tend to
exhibit greater selenium bioaccumulation than fish that feed higher in the water column (Schneider et al.,
2015; Simmons and Wallschlager, 2005).
Species that are sensitive to selenium are commonly present but if they are not available in sufficient
numbers, then other species that are available in sufficient numbers can be used for fish tissue monitoring.
In smaller streams, cyprinids may be the only species available. Species known to be tolerant to selenium
may also be appropriate to use, since their selenium tissue concentration will be compared to the tissue
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element threshold (see Table 1) which is designed to be protective of the entire aquatic community. A
waterbody with selenium impacts and only tolerant species should still show selenium impacts, since
even tolerant fish bioaccumulate selenium. For example, there are data from West Virginia and Colorado
that show some native cyprinids including blacknose dace and central stoneroller with tissue
concentrations over 40 mg/kg dry weight. (USEPA, 2016a). {Note: Research is needed to determine
whether certain species are resistant to bioaccumulation of selenium versus other species.)
When selecting target species, it is important to consider all of the organisms and trophic levels that are
potentially at risk in the study area. For example, certain species will have habitat preferences that expose
them to higher levels of accumulated selenium. If possible, migratory species and highly mobile species
should be avoided. Highly mobile fish species such as potamodromous and anadromous species could
travel back and forth between areas with low and elevated selenium concentrations, resulting in variable
tissue selenium concentration data (Beatty and Russo 2014). It is possible that typical adult selenium
exposure concentrations would be lower than concentrations at rearing grounds, and for these reasons
resident species should be the first choice for selecting target species.
If migratory or highly mobile species must be sampled, then sampling plans should account for the life
history of these species so that the correct locations for sampling within a watershed are selected.
Potamodromous species vary in the extent to which they migrate for spawning. Most simply migrate from
a lake or reservoir to a nearby river or stream, or from a larger downstream section of the river to a
smaller upstream tributary. For example, some Walleye {Sander vitreus) spawn in lakes with suitable
habitat, and some return to river systems or streams that connect with the lake. However, some
Pikeminnows (genus Ptychocheilus) migrate over 100 miles to spawn. In riverine systems, some
individuals migrate short distances to suitable habitat, while others migrate longer distances. The
proximity of the selenium source sampling locations should also be considered; the nearest source of
selenium may be located some distance upstream, or it may be located at or near a sampling site. If
Pacific anadromous species are selected as target species to be used for sampling, EPA recommends that
states and authorized tribes use the whole-body criterion element for juvenile (smolt) as the primary
criterion element over the other elements. This recommendation is due to the unique life history of these
species, specifically, the lack of exposure to adult salmonids from selenium in freshwater prior to
reproduction (see Section 6.4.1.1 in USEPA 2016).
The use of a limited number of target species allows comparison of fish contaminant data among sites
over a broad geographic area. It is difficult to compare contaminant monitoring results within a state or
among states unless the data are from the same species because of differences in habitat, food preferences,
and rate of contaminant uptake among various fish species. However, it is impracticable to sample the
same species at every site. Limiting the number of species allows for collection and comparison of
contaminant data from across a state, region, or nationally. Table 3 lists EPA's recommended target
species for implementation of the selenium criteria (adapted from existing EPA guidance on fish tissue
monitoring (USEPA 2000a). Common molluscivorous fish species are indicated in bold. Molluscivorous
fish species have a higher potential to bioaccumulate selenium, since the available data indicate that
mollusks generally have a higher trophic transfer factor than other invertebrate taxa (USEPA 2016a).
Leveraging Existing Fish Tissue Monitoring Programs and Sample Designs
Considerations for Augmenting Existing Fish Tissue Monitoring Programs
In 2010, forty-five states monitored chemical contaminants in fish tissue for assessing human health risks.
The design of an agency's existing fish tissue monitoring program will likely drive its approach to
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selenium monitoring. Twenty-eight states identify selenium as a contaminant in their monitoring program
(USEPA 2010a). Many states already have monitoring programs and sample designs that can be
leveraged for the new selenium criterion. Several case studies are provided in the following sections as
examples of programs that might have the capacity and framework to augment their existing monitoring
strategies to include fish tissue monitoring for the selenium criteria.
Consistency with Existing Programs
To the extent possible within a state or tribal program, EPA recommends that fish tissue monitoring for
the assessment of the selenium aquatic life criterion should be consistent with state's current practices
regarding spatial and temporal considerations of the program, species collected, and sample type
collected. In this way, logistical modifications to a state's fish tissue monitoring program can be
minimized. However, care should be taken when utilizing existing sampling programs that are designed
for human health protection, as existing sampling designs and methods for human health may need to be
amended before being used for selenium sampling. States should take into consideration the information
presented in this document when amending their programs. Where deviation from existing state or tribal
programs is necessary because of spatial or temporal considerations, or species/sample type due to
concerns regarding specific waterbodies with selenium inputs, these can potentially be accommodated by
leveraging expertise and logistical assistance from other agencies. Various state (e.g., Department of
Natural Resources) or federal (i.e., National Oceanic and Atmospheric Administration - National Marine
Fisheries Service, United States Fish and Wildlife Service [USFWS], United States Geological Survey
[USGS]) agencies have the expertise to provide such assistance. Alternatively, in the absence of an
existing program, additional monitoring may need to be planned for criteria implementation.
Temporal Considerations
Various temporal factors will influence fish tissue monitoring strategies for selenium. For example, as
described earlier in this document, most fish species that are synchronous spawners do so in the spring,
whereas fish tissue collection for advisories typically occurs in the late summer or early fall, when
contaminant loads in the edible portion of the fish are highest. If an agency is limited to sampling outside
of the pre-spawning period due to resource constraints, that will need to be considered when incorporating
selenium fish tissue monitoring into the existing programs, or when developing a new program (e.g.,
sampling whole body or muscle tissue instead of egg-ovary tissue).
The only appropriate time to collect egg-ovary tissue from suitable species is when the female is gravid in
the pre-spawn stage, just prior to mating and spawning. This is typically a very small window (see
Appendix B) of time for most synchronous species, and may occur in the spring or early summer, or in
the fall to early winter. In northern latitudes, spawning may occur slightly later than in southern latitudes.
It is the selenium concentration in eggs that drives early life stage toxicity, so adult female fish must be
collected during the late vitellogenic or pre-ovulatory periods of oogenesis for this criterion to be
scientifically and toxicologically meaningful. Measuring selenium concentration in ovarian tissue during
other periods of oogenesis will be much less informative. Summer and fall may be prime periods for
whole-body and muscle tissue collection due to the engorgement of populations to replenish fat and
energy reserves post-spawn.
For egg-ovary tissue sampling, agencies with fish tissue monitoring responsibilities should consult with a
state fisheries biologist to determine the appropriate time for sampling specific species in their region in
order to capture the specimens in their pre-spawning phase. These regional experts will be familiar with
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the local species, and able to use their best professional judgment to determine which are appropriate for
selenium sampling, and the appropriate sampling time frame based on spawning season. If agency
resources limit fish tissue collection to times outside of these species-specific windows, then the only
appropriate samples to collect are whole-body and muscle tissue. Target fish species collected in the fall
may be common to selenium monitoring and human health risk assessment. In this case, muscle tissue
can be composited and evaluated for selenium in addition to contaminants of interest for fish consumption
advisories. Seasonal restrictions (e.g., due to spawning seasons, high flows) on fish sampling may also
prevent sampling for egg-ovary tissue in specific areas.
Spatial Considerations
Spatial factors will need to be considered when augmenting existing programs, or when developing a new
program. For example, as described earlier in this document, some fish species migrate to upstream areas
to spawn; these areas may be harder to access than larger order downstream segments that are inhabited
during non-spawning seasons. However it may still be possible to sample such species on their way up
stream. It may be necessary to monitor smaller order stream segments of a larger stream network than is
traditionally monitored (e.g., downstream river segment) to get closer to the selenium input. This may
require some adjustment to monitoring plans that would consider the species of fish available in the small
stream segment, temporal issues (e.g., spring flood/safety, low flow availability of fish), and the types of
appropriate sampling gear. Agencies should consider a species' home range in relation to the location of a
known selenium source (e.g. the migratory patterns of a certain species versus the location of a power
plant on a reservoir). It is also important to consider the relationship of an upstream source to downstream
habitats.
States currently use a number of different methods for selecting sites for sampling fish tissue. Monitoring
agencies generally will target high-use fishing areas, areas of special concern, and areas of suspected
contamination, such as water bodies where fish advisories have been issued in the past (USEPA 2010a).
States using this survey design should consider possible selenium prevalence and potential areas of
contamination when targeting areas for sampling. If problem areas are identified through best professional
judgment or through screening studies to determine the magnitude of chemical contamination in sensitive
fish species, these areas can then continue to be targeted to monitor trends. Additional information
regarding screening studies and intensive studies can be found in the "Existing Guidance" section of this
document.
Geology may cause certain areas to be prone to selenium bioaccumulation, resulting in elevated
concentrations. This should be kept in mind when selecting sites, and when analyzing data from these
areas (Beatty and Russo 2014). In many areas, selenium sources have been well characterized; in these
areas an intensive study designed to capture the magnitude and geographical extent of the selenium
contamination in fish tissue (rather than following the results of a screening study) is recommended to
ensure protection of aquatic life from reproductive impacts and aquatic community balance. Results of
these intensive studies could be used to help identify the geographic extent of the selenium contamination,
either downstream in a lotic environment, or by area in a lentic environment.
Forty agencies monitor fish sampling areas at regular intervals, and several conduct statewide, rotating
basin sampling programs over a multi-year period (USEPA 2010a). Agencies can monitor state- or basin-
wide, and track progress in individual basins relative to other areas. Regular yearly sampling could be
conducted, with intensified sampling in the targeted basins as indicated (see Table 4 for several
documents that provide guidance for sampling and survey designs). Several states use a probabilistic
survey design to select sampling sites. This type of sampling design can produce estimates that represent
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the condition of the whole watershed, and an estimate of random spatial variability (USEPA 2000a).
Probability sampling provides the basis for estimating resource (i.e., fish population(s)) extent and
condition, for characterizing trends in resource extent or condition, and for representing spatial patterns,
all with known certainty (USEPA 2009). The case study below presents the Kansas Department of Health
and the Environment's (KDHE) fish tissue monitoring program, which uses several designs for selecting
sites. Based on the information available, it is likely that a state or authorized tribe with a similar program
could take advantage of their current sampling strategy to perform screening level selenium analysis
throughout their state or tribe. Where selenium is already a primary parameter of interest, the state or tribe
may have the data to support more intensive studies in certain water bodies.
CASE STUDY: The Kansas Department of Health and the Environment
The Kansas Department of Health and the Environment (KDHE) currently collects fish samples
annually from 50 or more fixed and rotating stations. The KDHE selects sites based on targeted,
census, and probability based study designs. Specific sub-program objectives determine the
numbers, species, and sizes of fish collected from a particular water body, and the tissues and
parameters of interest.
Highlights (KDHE 2013):
• Whole fish, muscle, muscle plugs, or other specific tissues are collected for different programs.
• Selenium is a primary parameter of interest.
• Specific tissues (such as egg-ovary) are analyzed for specific chemicals of concern known to
accumulate in certain organs.
The KDHE maintains a comprehensive fish tissue sampling program that routinely collects various
tissue types.
http://www.kdheks.gov/environment/qmp/download/Fish Tissue Part III.pdf
Selenium Differences in Lentic and Lotic Environments
Selenium concentrations and bioaccumulation patterns are different in lotic (flowing water) versus lentic
(very slow moving or still water) environments. It is of greatest concern in lentic water bodies, where
reducing conditions create an environment where selenium accumulates in sediment more readily.
Benthic organisms are therefore exposed to higher concentrations of selenium in the sediment, leading to
increased bioaccumulation potential in other organisms feeding on the benthic organisms (Simmons and
Wallschlager 2005; Orr et al. 2006). Several studies have concluded that fish feeding on benthic
organisms are expected to have higher selenium concentrations than fish feeding from the water column
(Schneider et al., 2015; Simmons and Wallschlager, 2005). This suggests that bottom feeding fish may
have higher selenium levels, at least for the lifecycle that ties their energy needs to food webs with
benthic insects. Other studies (Saiki et al. 1993; Saiki and Lowe 1987) have shown that detritivores may
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be exposed to high levels of dietary selenium, as high concentrations of selenium were measured in
detritus. Reducing conditions may also lead to higher bioavailability in the water column (Luoma and
Rainbow 2008).
Hillwalker et al. (2006) found that the body burden concentrations of selenium in insects within similar
taxa were up to 7 times greater in lentic systems than lotic systems within the same watershed.
Additionally, they concluded that selenium bioaccumulation in insects gave a more accurate measurement
of accumulation risk throughout the food chain than surface water selenium concentrations (Beatty and
Russo 2014).
Mollusks such as mussels and clams accumulate selenium to a much greater extent than planktonic
crustaceans and insects due to higher ingestion rates of both particulate-bound (algae) and dissolved
selenium from the water column through filter feeding. These organisms also have a lower selenium
elimination rate (Johns et al. 2008; Reinfelder et al. 1997). Certain ecosystems with mollusk-based food-
webs may create a pathway for more selenium to bioaccumulate, particularly in molluscivorous fish, since
the available data indicate that mollusks generally have a higher trophic transfer factor than other
invertebrate taxa (USEPA 2016a). Common molluscivorous fish species are indicated in Table 3.
Existing Resources and Information
Available Expertise
The fish tissue sampling infrastructure (experience, equipment, etc.) for the purposes of implementing the
selenium fish tissue criterion typically resides in the agency charged with protection of natural resources
(e.g., a natural resources department or a fish and game department). EPA recommends that states or
authorized tribes leverage the appropriate expertise and logistical knowledge for compiling the necessary
information and data to implement sampling.
All states, in addition to most authorized tribes and interstate commissions, have established biological
assessment programs. This means that there should be capacity to establish or modify existing fish tissue
monitoring programs to facilitate implementation of the new fish tissue-based criteria elements in the new
selenium water quality criterion. In addition to individual state and tribal agencies and local expertise,
federal (e.g., USFWS) and state resource agency collaborations could be used as necessary to fill in data
gaps and provide supporting data. By using all available resources for information and expertise,
monitoring agencies should be able to:
• Identify potential sites/locations, water bodies, and watersheds for selenium sampling beyond the
coverage of current monitoring program
• Design an appropriate monitoring strategy
• Select target species
• Identify pre-spawning periods
• Procure analytical support
The case study below presents Minnesota's Fish Contaminant Monitoring Program, which is
implemented through a collaborative partnership of four state agencies to maximize available expertise.
Based on the available information, a state or authorized tribe with a similar collaborative program could
take advantage of their joint resources to devise the most efficient approach for adding selenium to their
current monitoring strategy. They could also use their extensive database to determine where to conduct
more intensive studies in certain water bodies.
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CASE STUDY: Minnesota's Fish Contaminant Monitoring Program
Minnesota's Fish Contaminant Monitoring Program is implemented through a partnership of
Minnesota Departments of Natural Resources (DNR), Health (MDH), and Agriculture (MDA)
and the Minnesota Pollution Control Agency (MPCA). The data are used to issue fish
consumption advisories, identify impaired waters, research mercury cycling, and document long
term trends for PCBs and mercury.
Highlights (MPCA 2008):
• Approximately 130 lakes and river sites are sampled annually.
• The Fish Contaminant Monitoring Program database contains over 31,000 data records.
• As of 2008, the program has sampled 22% of the estimated 5,500 fishing lakes in the state
(15% of the lakes <2,000 acres and 80% of the lakes >2000 acres).
This program is a robust example of how interagency cooperation can maximize available
expertise, resources, and cost effectiveness.
https://www.pca.state.mn.us/sites/default/files/p-p2s4-05.pdf
Existing Guidance
Existing EPA guidance related to monitoring of contaminants in fish was published in Guidance for
Assessing Chemical Contaminant Data for Use in Fish Advisories, Vol 1: Fish Sampling and Analysis
(USEPA 2000a). This Guidance was developed specifically for assessing human health risks associated
with consumption of fish and shellfish, so there are aspects of the aquatic life selenium fish tissue-based
criterion that are not covered by the 2000 Guidance (e.g., fish egg-ovary sampling). The 2000 Guidance
recommends selenium as a target analyte based on its relevance to human health and focuses on fish
consumption advisories.
The monitoring strategy in the 2000 Guidance document discusses two tiers of studies with the goal of
identifying locations where fish consumption advisories may be needed. Tier 1 studies are screening
studies that cover a large number of sites for chemical contamination with few samples per site. These are
most useful in water bodies, regions, or states where there are no known or expected selenium problems.
Screening studies help states identify those sites where selenium concentrations are elevated relative to
other water bodies in the state and prioritize water bodies for future monitoring, thus enabling resources
to be used more efficiently. For example, water bodies with fish having low selenium may be monitored
less frequently in the future, while water bodies with fish having elevated selenium at or near the tissue
elements may be prioritized for more frequent or more intensive monitoring. Other information (e.g.,
location of sources), can also be used to prioritize sites for screening and prioritization.
Tier 2 studies are intensive studies of problem areas identified in screening studies to determine the
magnitude of chemical contamination in sensitive fish species, and to assess the geographic extent of the
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contamination. Agencies will typically use Tier 2 studies to determine the overall magnitude and
variability of a specific contaminant that was found at elevated levels during a Tier 1 study.
For the purposes of implementing the aquatic life selenium criterion recommendations, the process is
different. In the waterbody assessment context, once a criterion element threshold is exceeded, the
waterbody is considered impaired (and placed on the state's or tribe's CWA section 303(d) list), and the
next step would be additional monitoring for a TMDL or site specific criterion. Data from intensive
studies might help to support TMDL development for those waters where fish tissue criteria elements are
exceeded by identifying the magnitude of selenium in fish tissue ("worst case scenario"). Monitoring at
points downstream in a lotic water body may define the area of impact for an impairment based on
selenium in tissues of sensitive resident fish species. In lentic systems, intensive monitoring in a large
lake or reservoir, for example, might demonstrate that selenium contamination in fish is limited to a
certain area such as an embayment or a tributary arm of a reservoir.
Although the focus of the 2000 Guidance document is different, it still provides information that is useful
to state and tribal programs monitoring for implementation of the fish tissue components of EPA's
aquatic life selenium criterion recommendations. In particular, the 2000 Guidance document discusses the
importance of selecting target species for tissue samples, and provides lists of species for various feeding
habits and habitats (bottom feeder, predators) that are recommended by EPA, USFWS, and USGS as
targets for monitoring. The 2000 Guidance also discusses study design considerations and the major
parameters that must be specified for field collection activities, such as site selection, analyte screening
values, sampling times, sampling type, and quality assurance/quality control (QA/QC) samples such as
replicate samples.
Additionally, numerous documents on bioassessment techniques have been produced by EPA and other
stakeholders. Specific sections of these documents contain information that may be helpful for developing
guidelines for sampling fish (particularly for species like cyprinids not typically targeted by state
monitoring programs) for the purposes of selenium fish tissue analysis. For example, Rapid
Bioassessment Protocols for Use in Streams and Wadeable Rivers: Periphyton, Benthic
Macroinvertebrates, and Fish - Second Edition Chapter 3 (Barbour et al. 1999) provides guidance and
information on the elements of biomonitoring including seasonality for fish collections and fish collection
methodologies. A selection of recommended documents for additional guidance is presented in Table 4.
22
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EPA Draft for Public Comment
Table 4: Recommended Documents for Additional Guidance
Title
Author
Link
Guidance for Assessing Chemical
Contaminant Data for Use in Fish
Advisories, Vol 1: Fish Sampling and
Analysis
USEPA
2000a
https ://www.epa. aov/sites/production/files/2015-
06/documents/volume 1 .pdf
Rapid Bioassessment Protocols for Use
in Streams and Wadeable Rivers:
Periphyton, Benthic Macroinvertebrates,
and Fish - Second Edition
Barbour et
al. 1999
https://nepis.epa.aov/Exe/ZvPDF.cai/2000400K.P
DF?Dockev=2000400K.PDF
Field Sampling Plan for the National
Study of Chemical Residues in Lake Fish
Tissue
USEPA
2002a
http ://www.epa. aov/sites/production/files/2015-
07/documents/fish-studv-fieldplan.pdf
The National Study of Chemical Residues
in Lake Fish Tissue (Final Report)
USEPA
2009
https://nepis.epa.aov/Exe/ZvPDF.cai/P1005P2Z.P
DF?Dockev=P1005P2Z.PDF
Concepts and Approaches for the
Bioassessment ofNon- Wadeable
Streams and Rivers
Flotemersch
et al. 2006
https://nepis.epa. e;ov/Exe/ZvPDF.ce;i/600006KV.P
DF?Dockev=600006KV.PDF
Guidance on Choosing a Sampling
Design for Environmental Data
Collection
USEPA
2002b
http ://www.epa. aov/sites/production/files/2015-
06/documents/a5 s-final.pdf
Spatially Balanced Survey Designs for
Natural Resources. Design and Analysis
of Long-Term Ecological Monitoring
Studies
Olsen et al.
2012
https://www.cambridae.ora/core/books/desian-and-
analvsis-of-lona-term-ecoloaical-monitorina-
studies/508A10FEE39E7E93EF07B005D06952F5
Spatially Balanced Sampling of Natural
Resources
Stevens and
Olsen, 2004
https://archive.epa.aov/nheerl/ann/web/pdf/arts as
a.pdf
Application of Global Grids in
Environmental Sampling
Olsen et al.
1998
https://archive.epa.aov/nheerl/ann/web/litail/abolse
n98.html
Using Existing Data to Enhance Selenium Monitoring
All available existing data should be considered and utilized as necessary to inform and enhance selenium
monitoring. According to the EPA's 2010 Fish Advisory Survey Report, 28 states identify selenium as a
contaminant in their monitoring program (USEPA 2010a). Several states have conducted extensive
statewide assessments, and could have existing state selenium data. The Ohio River Valley Water
Sanitation Commission (ORSANCO) collects samples for selenium analysis as part of their Fish
Consumption Advisory Program, and has data available online (http://www .orsanco .org/fish-tissue).
National scale data sources for selenium in fish tissue samples include EPA's 2008-2009 National Rivers
and Streams Assessment; the data are publicly available at http://www.epa.gov/fish-tech/fish-tissue-data-
collected-epa. One hundred paired mercury and selenium fish fillet concentration data from samples
collected in 2007 are available at http://www.epa.gov/sites/production/files/2015-07/mercurv-
finaldata2012.xlsx. Sample sites are randomly selected U.S. locations where existing mercury advisories
were in place at the time of sampling. The USGS has also conducted numerous state surveys of selenium
in fish tissue. The USGS National Water Quality Assessment (NAWQA) database
(http://cida.usgs.gov/nawqa www/nawqa data redirect.html) contains analytical results for fillet and
whole-body fish tissue samples from across the country.
23
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EPA Draft for Public Comment
Sample Assessment: Analytical Chemistry
Fish tissue sampling for the selenium criterion will involve many of the same types of analytical concerns
as with any tissue monitoring and assessment program. Various researchers have shown that analytical
results on the same population of fish can differ between studies and even within studies. These inherent
uncertainties are minimized through a rigorous study design, clear data quality objectives, meticulous
QA/QC protocols, and careful execution of the monitoring and assessment program in the field.
Standardized methods should be followed in the field to ensure the appropriate samples (that have been
handled, preserved, and shipped according to protocol) are analyzed in the laboratory (Beatty and Russo
2014). Consistent analytical procedures should be used across implementation programs, (e.g., ambient
monitoring, NPDES compliance monitoring).
Quality assurance in the laboratory should be closely monitored, and laboratories should be selected
carefully based on lab accreditations, strong QA/QC protocols, and experience with using analytical
methods for selenium and the fish tissue matrix. Samples should be prepared in accordance with the tissue
type. (Section 7.2.2 of EPA's 2000 Fish Advisory Guidance (USEPA 2000a) includes detailed direction
for preparing muscle and whole body samples. Please refer to Appendix A of this document for egg and
ovary sample preparation.) EPA does not have approved methods under 40 CFR Section 136 for
measuring selenium in fish tissue. However, states and authorized tribes are not required to use EPA-
approved methods for monitoring and assessment of criteria attainment or other activities not related to
permit applications or permit compliance reports (USEPA 2016a). Several methods for selenium analysis
in animal tissue are presented in Table 5. Four methods have a method detection limit (MDL) that is ten
times lower than the range expected given the criteria limits for tissue (the exception is EPA Method
6010C).
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EPA Draft for Public Comment
Table 5: List of Test Procedures for Total Selenium in Tissue
Method
Digestion /
Preparation in
reference method?
Example
MDL1
Links to Methods
EPA Method 6010C -
Inductively Coupled Plasma -
Atomic Emission
Spectroscopy
No-
Recommended: 3052
(total), or 3 05 0B (total
recoverable)
5 mg/kg
httr>://www.et>a.eov/sites/t>roduction/file
s/2015-07/do c u l lie n t s/e oa-6 010 c. od f
httt>s://www.et>a.eov/sites/t>roduction/fil
cs/2015-12/documcnts/3052.Ddr
httt>s://www.et>a.eov/sites/t>roduction/fil
es/2015 -06/documents/et>a-3050b .odf
EPA Method 6020A -
Inductively Coupled Plasma -
Mass Spectrometry (ICP -
MS)
No-
Recommended: 3052
(total), or 3 05 0B (total
recoverable)
0.2 mg/kg
httt>s://www.et>a.eov/sites/t>roduction/fil
es/2015-07/documents/et>a-6020a.t>df
httt>s://www.et>a.eov/sites/t>roduction/fil
cs/2015-12/documcnts/3052.Ddr
httt>s://www.et>a.eov/sites/t>roduction/fil
es/2015 -06/documents/et>a-3050b .odf
EPA Method 7742 -
Atomic Absorption,
Borohydride Reduction
No-
References 301 OA for
water (total)
Recommended: 3052
(total), or 3 05 0B (total
recoverable)
0.05
mg/kg
httt>s://www.et>a.eov/sites/t>roduction/fil
es/2015-12/do c u l lie n t s/7742. od f
lUtDs://\Yww.cDa.aov/sitcs/Droduction/ril
cs/2015-12/documcnts/3052.Ddf
lUtDs://\Yww.cDa.aov/sitcs/Droduction/ril
es/2015-06/documents/et>a-3050b.t>df
USGS 1-9020-05 -
Determination of Elements in
Natural-water, Biota,
Sediment, and Soil Samples
using Collision /Reaction Cell
ICP - MS
No-
References 3052 (total)
Recommended: 3052
(total), or 3 05 0B (total
recoverable)
0.008 (xg/g
httt>s://t>ubs.uses. sov/tm/2006/tm5b 1/P
DF/TM5-Bl.r>df
lUtDs://\Yww.cDa.aov/sitcs/Droduction/ril
cs/2015-12/documcnts/3052.Ddf
lUtDs://\Yww.cDa.aov/sitcs/Droduction/ril
es/2015 -06/documents/et>a-3050b .odf
NOAA 140.1-
Graphite Furnace-Atomic
Absorption for the Analysis
of Trace Metals in Marine
Animal Tissues
Yes -
Teflon Bomb
0.1 ng/g
httr)s://www.nemi.sov/methods/method
summarv/7185/
1 MDL - Establish empirically; MDLs will be laboratory, and potentially instrument, or analyst-specific. To
determine MDLs, commercial laboratories generally follow the procedures described in 40 CFR Part 136
Appendix B using analyte free reference material for spiking.
States can also use methods for analyzing selenium in water to measure selenium in fish tissue, as long as
the samples are made soluble. Tissue samples are homogenized and digested prior to analysis using strong
acid or dry-ashing digestion. The suitability for a given technique should be determined by the individual
lab and its capabilities and preference. Care should be taken to use a process that will minimize the loss of
volatile selenium. For example, fluorometric techniques require sample digestion and sample reduction;
loss of volatile selenium compounds is possible because several steps are required (ATSDR 2003).
Standard reference materials, analytical duplicates, and matrix spike samples are recommended to
determine the applicability of a selected digestion procedure. EPA recommends three specific EPA-
approved analytical methods for aqueous selenium; these methods are presented in Table 6 (USEPA
25
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EPA Draft for Public Comment
2016a). All three methods have an MDL that is ten times lower than the range expected given the criteria
limits for tissue.
Table 6. List of Test Procedures for Total Selenium in Water
Method
Digestion /
Preparation in
reference method?
Example
MDL1
Links to Methods
American Public Health Standard
Method 3114 B-
Arsenic and Selenium by Manual
Hydride Generation/Atomic
Absorption Spectrometry (2009)
or 3114 C -
Arsenic and Selenium by Continuous
Hydride Generation/Atomic
Absorption Spectrometry (2009)
Yes -
3114 B includes
digestions (Section
4), but references SM
3 03 OF for sample
preparation
2 ng/L
httos ://www. nemi. eov/methods/meth
od summarv/9703/
httt>s://www. scribd.com/doc/1771889
O/Standard-Methods-21 st-ed-Part-
3000-Metals
EPA Method 200.8, Rev 5.4 -
Determinations of Trace Elements in
Waters by ICP- MS (1994a)
Yes -
Section 11.2 (total
recoverable)
Alternative digestion
301 OA (total)
7.9 ng/L
htft>s://www.et>a.eov/sites/t>roduction
/files/2015-06/documcnts/cDa-
200.8.odf
htft>s://www.et>a.eov/sites/t>roduction
/files/2015-12/documents/30 lOa.odf
EPA Method 200.9, Rev.2.2-
Detennination of Trace Elements by
Stabilized Temperature Graphite
Furnace Atomic Absorption (1994b)
Yes -
Section 11.2, (total
recoverable)
Alternative digestion
301 OA (total)
0.6 \xg!h
htft>s://www.et>a.eov/sites/t>roduction
/files/2015-
08/documents/method 200-9 rev 2-
2 1994.odf
httDs://\\\\\\.CDa.ao\/sitcs/Droduction
/files/2015-12/documents/30 lOa.odf
1 MDL - Establish empirically; laboratory- and potentially instrument- or analyst-specific. To determine MDLs,
commercial laboratories generally follow the procedures described in 40 CFR Part 136 Appendix B using "analyte
free" reference material for spiking.
The North American Metals Council (NAMC) has published a comprehensive discussion of analytical
concerns relevant to selenium, contained in Ohlendorf et al. 2008 and 2011. An additional NAMC
document (Ralston et al. 2008) presents guidance on analytical methods and considerations for selenium
and its chemical species. Inductively coupled plasma mass spectrometry is the typical method used for
analyzing selenium in tissue and other matrices; however, this method is sensitive to interferences.
Alternative methods for analyzing selenium are discussed in D'Ulivo (1997), Ohlendorf et al. (2008), and
Ralston et al. (2008). States and authorized tribes should choose an analytical method that is sufficiently
sensitive to implement its water quality standard for selenium.
If a state or authorized tribe is using a data set that includes several values below the detection level, it
must decide how it will evaluate these values. EPA's Guidance for Assessing Chemical Contaminant
Data for Use in Fish Advisories (USEPA 2000a), recommends using one-half of the MDL for non-detects
in calculating mean values (Section 9.1.2). Measurements between the MDL and the method quantitation
limit are assigned a value of the detection limit plus one-half the difference between the detection limit
26
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EPA Draft for Public Comment
and the quantitation limit. Other statistical methods could also be used to calculate the average of data that
includes values below the detection limit. States or authorized tribes could conduct a sensitivity analysis
to determine how best to quantify samples below the detection limit (USEPA 2010b). For further
discussion on handling non-detects, see USEPA 2000a and USEPA 2010b.
Additional information regarding analysis can be found in Appendix L of the Criteria Document (USEPA
2016a). Complete descriptions of analytical methods appropriate for analyzing selenium in different
media can be found in the National Environmental Methods Index at http://www.nemi.gov.
Sample Assessment: Statistical Analysis
EPA guidance related to recommended statistical approaches for comparing contaminant measurements
measured at different locations or over time is outlined in Appendix N of Guidance for Assessing
Chemical Contaminant Data for Use in Fish Advisories, Vol 1: Fish Sampling and Analysis (USEPA
2000a). The guidance recommends using the t-test to statistically compare the mean of all fish tissue data
for a single species to the criterion. States and authorized tribes can evaluate whether the t-test statistic of
the mean exceeds the water quality standards. Intensive studies may include the collection of fish
contaminant data from several locations within a region of interest or for multiple time periods (e.g.,
seasons or years) from a single location, or a combination of both. Data from intensive studies such as
these may be used to perform spatial (i.e., between stations) or temporal (i.e., overtime) analyses. Spatial
and temporal comparisons of contaminant data may yield important information about the variability of
target analyte concentrations in specific populations of a particular target species. EPA recommends that
states and authorized tribes consult a statistician to determine the specific statistical tests needed for a
particular data set, and choose a method best suited to how they express their water quality standards.
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APHA. 2009b. Method 3114C. Continuous hydride generation/atomic absorption spectrometric method.
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Stewart R.A., S.N. Luoma, C.E. Schlekat, M.A. Doblin, K.A. Hieb. 2004. Food web pathway determines
how selenium affects aquatic ecosystems: a San Francisco Bay case study. Environ. Sci. Technol. 38:
4519-4526. http://pubs.acs.org/doi/pdf/10.1021/es0499647
Tyler, C. R., and J. P. Sumpter. 1996. Oocyte growth and development in teleosts. Reviews in Fish
Biology and Fisheries. September 1996, Volume 6, Issue 3, pp 287-318.
http://link.springer.com/article/10.1007/BF0Q122584
USEPA. 1992. Method 3010A. Acid digestion of aqueous samples and extracts for total metals an
analysis by FLAA or ICP spectroscopy, Revision 1. U.S. Environmental Protection Agency, Office of
Research and Development, Environmental Monitoring Systems Laboratory, Cincinnati, OH.
https ://www.epa. gov/sites/production/files/2015-12/documents/3 01 Oa.pdf
USEPA. 1994a. Method 200.8. Determination of Trace Elements in Waters and Wastes by Inductively
Coupled Plasma-Mass Spectrometry, Revision 5.4. U.S. Environmental Protection Agency, Office of
Research and Development, Environmental Monitoring Systems Laboratory, Cincinnati, OH.
https://www.epa.gov/sites/production/files/2015-08/documents/method 200-8 rev 5-4 1994.pdf
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USEPA. 1994b. Method 200.9. Determination of Trace Elements by Stabilized Temperature Graphite
Furnace Atomic Absorption, Revision 2.2. U.S. Environmental Protection Agency, Office of
Research and Development, Environmental Monitoring Systems Laboratory, Cincinnati, OH.
https://www.epa.gov/sites/production/files/2015-08/documents/method 200-9 rev 2-2 1994.pdf
USEPA. 1994c. Method 7742. Atomic Absorption, Borohydride Reduction, Revision 0. U.S.
Environmental Protection Agency, Office of Research and Development, Environmental Monitoring
Systems Laboratory, Cincinnati, OH. https://www.epa.gov/sites/production/files/2015 -
12/documents/7742 .pdf
USEPA. 1996a. Method 3052. Microwave assisted acid digestion of siliceous and organically based
matrices, Revision 0. U.S. Environmental Protection Agency, Office of Research and Development,
Environmental Monitoring Systems Laboratory, Cincinnati, OH.
https://www.epa.gov/sites/production/files/2015-12/documents/3052.pdf
USEPA. 1996b. Method 3050B. Acid digestion of sediments, sludges, and soil, Revision 2. U.S.
Environmental Protection Agency, Office of Research and Development, Environmental Monitoring
Systems Laboratory, Cincinnati, OH. https: //www .epa. gov/site s/production/file s/2015 -
06/documents/epa-3 05 Ob .pdf
USEPA. 1998. Method 6020A. Inductively Coupled Plasma - Mass Spectrometry, Revision 1. U.S.
Environmental Protection Agency, Office of Research and Development, Environmental Monitoring
Systems Laboratory, Cincinnati, OH. https: //www .epa. gov/site s/production/file s/2015 -
07/documents/epa-6020a.pdf
USEPA. 2000a. Guidance for Assessing Chemical Contaminant Data for Use in Fish Advisories. Vol 1
Fish Sampling and Analysis. EPA 823-B-00-007. U.S. Environmental Protection Agency, Office of
Water, Washington, DC. https://www.epa.gov/sites/production/files/2015-06/documents/volumel.pdf
USEPA. 2000b. Method 6010C. Inductively Coupled Plasma - Atomic Emission Spectrometry, Revision
3. U.S. Environmental Protection Agency, Office of Research and Development, Environmental
Monitoring Systems Laboratory, Cincinnati, OH. https: //www .epa. gov/site s/production/file s/2015 -
07/documents/epa-601 Oc .pdf
USEPA. 2002a. Field Sampling Plan for the National Study of Chemical Residues in Lake Fish Tissue.
EPA-823-R-02-004. U.S. Environmental Protection Agency, Office of Water, Washington, DC.
http://www.epa.gOv/sites/production/files/2015-07/documents/fish-studv-fieldplan.pdf
USEPA. 2002b. Guidance on Choosing a Sampling Design for Environmental Data Collection. EPA-240-
R-02-005. U.S. Environmental Protection Agency, Office of Environmental Information,
Washington, DC. http://www.epa.gov/sites/production/files/2015-06/documents/g5s-final.pdf
USEPA. 2009. The National Study of Chemical Residues in Lake Fish Tissue. EPA-823-R-09-006. U.S.
Environmental Protection Agency, Office of Water, Washington, DC.
https://nepis.epa.gOv/Exe/ZvPDF.cgi/P 1005P2Z.PDF?Dockev=P1005P2Z.PDF
USEPA. 2010a. Summary of Responses to the 2010 National Survey of Fish Advisory Programs. EPA-
820-R-12-001. U.S. Environmental Protection Agency, Office of Water, Washington, DC. Accessed
on-line at https://nepis.epa.gOv/Exe/ZvPDF.cgi/P 100LIPR.PDF?Dockev=P 100LIPR.PDF
USEPA. 2010b. Guidance for Implementing the January 2001 Methylmercury Water Quality Criterion.
EPA 823-R-10-001. U.S. Environmental Protection Agency, Office of Water, Washington, DC.
https://nepis.epa.gov/Exe/ZvPDF.cgi/P1007BKQ.PDF?Dockev=P1007BKQ.PDF
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USEPA. 2016a. Aquatic Life Ambient Water Quality Criterion for Selenium-Freshwater 2016. EPA 822-
R-16-006. U.S. Environmental Protection Agency, Office of Water, Office of Science and
Technology, Washington, DC. https://www.epa.gov/wqc/aquatic-life-criterion-selenium-documents.
USEPA. 2016b. Frequently Asked Questions (FAQs): Implementing WQS that Include Elements Similar
or Identical to EPA's 2016 Selenium Criterion in Clean Water Act Section 402 NPDES Programs.
U.S. Environmental Protection Agency, Office of Water, Washington, DC.
USEPA. 2016c. Frequently Asked Questions (FAQs): Implementing the 2016 Selenium Criterion in
Clean Water Act Sections 303(d) and 305(b) Assessment, Listing, and Total Maximum Daily Load
(TMDL) Programs . U.S. Environmental Protection Agency, Office of Water, Washington, DC.
USEPA. 2016d. Technical Support for Adopting and Implementing EPA's Selenium 2016 Criterion in
Water Quality Standards. U.S. Environmental Protection Agency, Office of Water, Washington, DC.
USFWS. 2016. ECOS Environmental Conservation Online System. Species Search.
http://ecos.fws.gov/ecpO/reports/ad-hoc-species-report-input
USGS. 2006. Determination of elements in natural-water, biota, sediment, and soil samples using
collision/reaction cell inductively coupled plasma-mass spectrometry: U.S. Geological Survey
Techniques and Methods, book 5, sec. B, chap. 1, 88 p.
https://www.epa.gov/sites/production/files/2015 -06/documents/epa-3 05 Ob .pdf
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Appendix A
Egg and Ovary Sample Preparation
Scope
This guidance is for egg and ovary collection from freshwater fish. The egg extraction method is
excerpted and adapted from a more comprehensive guidance that includes gamete collection,
embryo incubations and evaluation of selenium-induced deformities in freshwater fish, and the
ovary dissection method was compiled from peer-reviewed literature.
1. Field collection and handling of adult fish
Spawning adults can be collected in the field using a wide variety of techniques, including fish traps (e.g.,
hoop or trap nets), electrofishing or angling in areas close to spawning areas. Gillnets are also effective in
capturing fish during spawning migrations, but it is essential to monitor these nets constantly to remove
fish immediately after capture. If possible, the use of passive capture methods (e.g., hoop or trap nets) is
recommended since this is the least stressful capture technique of those listed above. Trap nets are usually
set up in creeks, streams or narrows in lakes, although successful fish capture can also occur when these
nets are set perpendicular to shore in lentic habitats. Trap or hoop nets can be purchased from fisheries
suppliers, or even constructed in creeks and streams using chicken wire, baling wire and reinforcing bar
(Janz and Muscatello, 2008). Fish should be held in livewells until adult female fish are selected for egg
collection.
2. Egg collection procedures
Fish should be carefully observed for signs of physical damage, mortality or other sources of stress. Since
any handling of the fish will remove the protective body layer of slime, fish should be handled as little as
possible using dip nets and soft material gloves. Adult fish for egg collection should be randomly selected
from livewells. Eggs should not be in contact with water; thus, it is imperative to dry the area surrounding
the urogenital opening with paper towels. All the material used for egg collection should be carefully
cleaned and dried. Precautions to avoid fecal, blood or urine contamination should be taken. Eggs must be
kept covered to avoid direct sun exposure.
Egg collection should proceed after recording weight and length. Gentle pressure from behind the
pectoral fins towards the anus is applied to express the eggs. This process needs to be repeated several
times. Check that eggs are released "clean" (e.g., without feces) before starting collection to avoid
contamination of the entire egg batch. Eggs are individually collected into pre-cleaned stainless steel
bowls and kept covered in a cool place until use. Collected eggs should be closely inspected and eggs
with adhered feces, urine or blood discarded by using a clean plastic pipette (Janz and Muscatello, 2008).
Eggs are then weighed to the nearest gram using a top-loading digital scale, frozen for storage, and
shipped for laboratory analysis when appropriate. A composite homogenate sample of 20 grams of tissue
should be collected for analysis of selenium (USEPA 1994a).
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3. Ovary dissection procedures
Fish designated for ovary collection should be humanely euthanized, and necropsy procedures should
commence immediately following euthanasia (Wolf et al. 2004). The fish should be placed in right lateral
recumbency on a piece of acetone-washed and baked aluminum foil. The left body wall should be
removed by using fine dissecting instruments. To identify female specimens for ovary collection, sex is
determined by macroscopic inspection when the body cavity is opened. The ovaries are paired organs
suspended from the dorsal wall, with color ranging from clear to white to yellow-orange. A yellow-
orange color is indicative of a ripening or ripe adult specimen. Further, increased blood flow during the
reproductive season causes the ovaries to become highly vascularized and appear reddish. In cross-
section, the ovaries are round to elliptical and contain a central cavity (lumen). In young fish, the texture
of the ovaries varies from smooth to slightly granular. The ovarian texture in a ripe fish will be highly
granular (FIN 2006). If inspection of the ovaries reveals that the specimen is immature or developing, it is
not recommended that the eggs/ovarian tissue be used for tissue monitoring for selenium.
After confirmation that the specimen is a ripe female, the ovaries should be excised by severing the
oviducts and mesenteric attachments. All gonads are dissected in a caudal to cranial direction (Wolf et al.
2004). Ovaries are then weighed to the nearest gram using a top-loading digital scale, frozen for storage,
and shipped for laboratory analysis when appropriate (Orr et al. 2012). A composite homogenate sample
of 20 grams of tissue should be collected for analysis of selenium (USEPA 1994a).
4. Storing fish eggs and ovaries
Eggs and ovaries should be kept frozen until analysis. After collection, samples should be kept in a
container with ice or freezer packs until transfer to a freezer (-20°C) for storage. It is recommended to
transfer the samples collected from each individual female into sealed Ziploc® bags to prevent water
(from ice melting) entering the sample. Storage time is 6 months to 2 years at -20°C for the majority of
trace metals, including selenium (Janz and Muscatello, 2008).
5. Laboratory Preparation of egg and tissue samples for metal analysis
Egg and tissue samples should be thawed, and wet weight recorded for each individual sample. To
prevent cross contamination between samples, a plastic foil (e.g., parafilm®) should be placed on the
scale and replaced after each weighing. Samples are oven dried at 60°C until constant weight is recorded.
It is required to record the moisture content for each individual sample in order to express analytical data
on a dry weight basis. Trace element (e.g., selenium) analysis is routinely performed using hydride
generation atomic absorption spectrophotometry (HG-AAS) or inductively coupled plasma-mass
spectrometry (ICP-MS) and reported on a dry-weight basis (Janz and Muscatello, 2008).
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References
FIN. 2006. Biological Sampling Manual. Gulf States Marine Fisheries Commission.
http://www.gsmfc.org/pubs/FIN/Biological%20Sampling%20Manual.pdf
Janz, D.M. and J.R. Muscatello. 2008. Standard operating procedure for evaluating selenium-induced
deformities in early life stages of freshwater fish. Appendix A in Selenium tissue thresholds: Tissue
selection criteria, threshold development endpoints, and potential to predict population or community
effects in the field. Washington (DC, USA): North America Metals Council - Selenium Working
Group. http://www.namc.org/docs/00043675.PDF
Orr, P. L., C.I.E. Wiramanaden, M.D. Paine, W. Franklin, and C. Fraser. 2012. Food chain model based
on field data to predict westslope cutthroat trout (Oncorhynchus clarkii lewisi) ovary selenium
concentrations from water selenium concentrations in the Elk Valley, British Columbia.
Environmental Toxicology and Chemistry 31.3: 672-680.
http://onlinelibrarv.wilev.com/doi/10.1002/etc. 1730/abstract
USEPA. 1994a. Method 200.8: Determination of Trace Elements in Waters and Wastes by Inductively
Coupled Plasma-Mass Spectrometry, Revision 5.4. U.S. Environmental Protection Agency, Office of
Research and Development, Environmental Monitoring Systems Laboratory, Cincinnati, OH.
https://www.epa.gov/sites/production/files/2015-08/documents/method 200-8 rev 5-4 1994.pdf
Wolf, J.C., D.R. Dietrich, U. Friederich, J. Caunter, and A.R. Brown. 2004. Qualitative and quantitative
histomorphologic assessment of fathead minnow Pimephales promelas gonads as an endpoint for
evaluating endocrine-active compounds: a pilot methodology study. Toxicologic pathology 32.5: 600-
612. http://tpx.sagepub.com/content/32/5/600.full.pdf+html
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Appendix B
Spawning Seasons for Example Fish Assemblages from Select U.S.
Watersheds
This appendix contains spawning season calendars for fish assemblages from selected watersheds in
six different areas of the United States. The calendars are intended to provide examples of spawning
periods for fish species commonly collected in those areas. Monitoring agencies should use all
available locally relevant resources to determine the appropriate time to collect fish for the purpose
of implementing the selenium criteria.
References
Auer, N.A. (editor). 1982. Identification of Larval Fishes of the Great Lakes Basin with Emphasis on the
Lake Michigan Drainage. Great Lakes Fishery Commission, Ann Arbor, MI. Special Pub. 82 - 3:744
pp.
Boschung, H.T., and Mayden, R.L. 2004. Fishes of Alabama. Washington, D.C: Smithsonian Books.
Nevada Division of Environmental Protection. 2006. Fact Sheet. Temperature Criteria for Various Fish
Species as Recommended to NDEP during the 1980s.
https://ndep.nv.gov/bwqp/file/recommended temp criteria06.pdf
Page, L.M. and B.M. Burr. 1991. Peterson Field Guides: Freshwater Fishes. Boston: Houghton Mifflin
Company.
Scarola, J.F. 1973. Freshwater Fishes of New Hampshire. New Hampshire Fish and Game Department,
Division of Inland and Marine Fisheries.
Wang, J.C.S. and R.J. Kernehan. 1979. Fishes of the Delaware Estuaries: A Guide to the Early Life
Histories. Towson, MD: EA Communications.
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Spawning Seasons for Example Fish Assemblages in the Merrimack River, MA and NH Watershed
F amily
Scientific Name
Common Name
Spawning Season
Atherinopsidae
Menidia menidia
Atlantic Silverside
April through August
Catostomidae
Catostomus commersonii
White Sucker
March through July
Centrarchidae
Ambloplites rupestris
Rock Bass
April through July
Centrarchidae
Enneacanthus obesus
Banded Sunfish
April through July
Centrarchidae
Lepomis auritus
Redbreast Sunfish
April through July
Centrarchidae
Lepomis gibbosus
Pumpkinseed
June through August
Centrarchidae
Lepomis macrochirus
Bluegill
May through August
Centrarchidae
Micropterus dolomieu
Smallmouth Bass
April through June
Centrarchidae
Micropterus salmoides
Largemouth Bass
April through June
Centrarchidae
Pomoxis nigromaculatus
Black Crappie
April through July
Clupeidae
Dorosoma cepedianum
Gizzard Shad
March through August
Cyprinidae
Carassius auratus
Goldfish
March through August
Cyprinidae
Cyprinus carpio
Common Carp
April through August
Cyprinidae
Luxilus cornutus
Common Shiner
May through July
Cyprinidae
Notemigonus crysoleucas
Golden Shiner
May through July
Cyprinidae
Notropis atherinoides
Emerald Shiner
May through June
Cyprinidae
Notropis bifrenatus
Bridle Shiner
May through August
Cyprinidae
Notropis hudsonius
Spottail Shiner
May through September
Cyprinidae
Rhinichthys atratulus
Blacknose Dace
April through July
Cyprinidae
Rhinichthys cataractae
Longnose Dace
April through June
Cyprinidae
Semotilus atromaculatus
Creek Chub
March through June
Cyprinidae
Semotilus corporalis
Fallfish
April through May
Esocidae
Esox lucius
Northern Pike
March through May
Esocidae
Esox niger
Chain Pickerel
March through May
Fundulidae
Fundulus diaphanus
Banded Killifish
April through August
Fundulidae
Fundulus heteroclitus
Mummichog
June through July
Gadidae
Lota lota
Burbot
January through April
Gasterosteidae
Apeltes quadracus
Fourspine Stickleback
April through May
Gasterosteidae
Gasterosteus aculeatus
Threespine Stickleback
March through June
Gasterosteidae
Pungitius pungitius
Ninespine Stickleback
April through August
Ictaluridae
Ameiurus catus
White Catfish
May through July
Ictaluridae
Ameiurus natalis
Yellow Bullhead
May through June
Ictaluridae
Ameiurus nebulosus
Brown Bullhead
April through June
Ictaluridae
Ictalurus punctatus
Channel Catfish
April through September
Ictaluridae
Noturus gyrinus
Tadpole Madtom
May through July
Ictaluridae
Noturus insignis
Margined Madtom
June through July
Moronidae
Morone americana
White Perch
May through June
Percidae
Etheostoma fusiforme
Swamp Darter
April through May
Percidae
Etheostoma olmstedi
Tessellated Darter
March through May
Percidae
Percaflavescens
Yellow Perch
May through July
Percidae
Sander vitreus
Walleye
April through May
Salmonidae
Oncorhynchus mykiss
Rainbow Trout
April through June
Salmonidae
Salmo trutta
Brown Trout
October through February
Salmonidae
Salvelinus fontinalis
Brook Trout
September through November
(Scarola 1973, Page and Burr 1991)
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Spawning Seasons for Example Fish Assemblages in the Delaware River, DE Watershed
F amily
Scientific Name
Common Name
Spawning Season
Aphredoderidae
Aphredoderus sayanus
Pirate Perch
April through May
Atherinopsidae
Membras martinica
Rough Silverside
May through August
Atherinopsidae
Menidia peninsulae
Tidewater Silverside
May through August
Atherinopsidae
Menidia menidia
Atlantic Silverside
April through August
Catostomidae
Catostomus commersonii
White Sucker
March through May
Catostomidae
Erimyzon oblongus
Creek Chubsucker
March through May
Centrarchidae
Acantharchus pomotis
Mud Sunfish
May through June
Centrarchidae
Enneacanthus chaetodon
Blackbanded Sunfish
May through July
Centrarchidae
Enneacanthus gloriosus
Bluespotted Sunfish
May through September
Centrarchidae
Enneacanthus obesus
Banded Sunfish
June through September
Centrarchidae
Lepomis auritus
Redbreast Sunfish
May through June
Centrarchidae
Lepomis gibbosus
Pumpkinseed
May through August
Centrarchidae
Lepomis macrochirus
Bluegill
May through August
Centrarchidae
Micropterus salmoides
Largemouth Bass
April through June
Centrarchidae
Pomoxis annularis
White Crappie
April through June
Centrarchidae
Pomoxis nigromaculatus
Black Crappie
May through June
Clupeidae
Dorosoma cepedianum
Gizzard Shad
April through June
Cyprinidae
Carassius auratus
Goldfish
June through July
Cyprinidae
Cyprinus carpio
Common Carp
May through July
Cyprinidae
Hybognathus nuchalis
Mississippi Silvery Minnow
April through May
Cyprinidae
Notemigonus crysoleucas
Golden Shiner
April through July
Cyprinidae
Cyprinella analostana
Satinfin Shiner
March through July
Cyprinidae
Notropis bifrenatus
Bridle Shiner
March through August
Cyprinidae
Notropis chalybaeus
Ironcolor Shiner
April through May
Cyprinidae
Notropis hudsonius
Spottail Shiner
April through July
Cyprinidae
Rhinichthys atratulus
Blacknose Dace
May through June
Esocidae
Esox americanus americanus
Redfin Pickerel
February through March
Fundulidae
Fundulus diaphanus
Banded Killifish
April through August
Fundulidae
Fundulus heteroclitus
Mummichog
April through September
Fundulidae
Fundulus majalis
Striped Killifish
April through September
Fundulidae
Lucania parva
Rainwater Killifish
May through July
Ictaluridae
Ameiurus catus
White Catfish
April through July
Ictaluridae
Ameiurus nebulosus
Brown Bullhead
May through July
Ictaluridae
Ictalurus punctatus
Channel Catfish
May through July
Ictaluridae
Noturus gyrinus
Tadpole Madtom
May through July
Moronidae
Morone americana
White Perch
April through June
Percidae
Etheostoma fusiforme
Swamp Darter
April through May
Percidae
Etheostoma olmstedi
Tessellated Darter
March through May
Percidae
Percaflavescens
Yellow Perch
March through April
Poeciliidae
Gambusia affmis
Mosquitofish
May through August
Umbridae
Umbra pygmaea
Eastern Mudminnow
April through June
(Wang and Kernehan 1979, Page and Burr 1991)
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Spawning Seasons for Example Fish Assemblages in the Cahaba River, AL Watershed
F amily
Scientific Name
Common Name
Spawning Season
Amiidae
Amia calva
Bowfin
March through June
Atherinopsidae
Labidesthes sicculus
Brook Silverside
June through August
Catostomidae
Carpiodes cyprinus
Quillback
March through September
Catostomidae
Carpiodes velifer
Highfin Carpsucker
May through July
Catostomidae
Erimyzon oblongus
Creek Chubsucker
March through May
Catostomidae
Erimyzon sucetta
Lake Chubsucker
March through April
Catostomidae
Erimyzon tenuis
Sharpfin Chubsucker
March through April
Catostomidae
Hypentelium etowanum
Alabama Hog Sucker
April through June
Catostomidae
Ictiobus bubalus
Smallmouth Buffalo
March through April
Catostomidae
Minytrema melanops
Spotted Sucker
April through May
Catostomidae
Moxostoma carinatum
River Redhorse
April
Catostomidae
Moxostoma duquesnii
Black Redhorse
April through May
Catostomidae
Moxostoma erythrurum
Golden Redhorse
April through June
Catostomidae
Moxostoma poecilurum
Blacktail Redhorse
April
Centrarchidae
Ambloplites ariommus
Shadow Bass
May through October
Centrarchidae
Centrarchus macropterus
Flier
February through May
Centrarchidae
Lepomis macrochirus
Bluegill
March through May
Centrarchidae
Lepomis marginatus
Dollar Sunfish
May through August
Centrarchidae
Lepomis megalotis
Longear Sunfish
May through August
March through May;
Centrarchidae
Lepomis microlophus
Redear Sunfish
September through November
Centrarchidae
Lepomis miniatus
Redspotted Sunfish
March through September
Centrarchidae
Micropterus coosae
Redeye Bass
May through July
Centrarchidae
Micropterus dolomieu
Smallmouth Bass
March through May
Centrarchidae
Micropterus punctulatus
Spotted Bass
April through May
Centrarchidae
Micropterus salmoides
Largemouth Bass
April through June
Centrarchidae
Pomoxis annularis
White Crappie
April through June
Centrarchidae
Pomoxis nigromaculatus
Black Crappie
February through May
Clupeidae
Dorosoma cepedianum
Gizzard Shad
April through May
Clupeidae
Dorosoma petenense
Threadfin Shad
April through August
Cottidae
Cottus carolinae
Banded Sculpin
January through March
Cyprinidae
Campostoma oligolepis
Large scale Stoneroller
April through May
Cyprinidae
Cyprinella callistia
Alabama Shiner
March through May
Cyprinidae
Cyprinella trichroistia
Tricolor Shiner
June through July
Cyprinidae
Cyprinella venusta
Blacktail Shiner
March through October
Cyprinidae
Hybognathus nuchalis
Mississippi Silvery Minnow
March through April
Cyprinidae
Hybopsis winchelli
Clear Chub
February through April
Cyprinidae
Luxilus chrysocephalus
Striped Shiner
April through August
Cyprinidae
Lythrurus bellus
Pretty Shiner
April through June
Cyprinidae
Macrhybopsis storeriana
Silver Chub
May through August
Cyprinidae
Notemigonus crysoleucas
Golden Shiner
April through July
Cyprinidae
Notropis ammophilus
Orangefin Shiner
April through October
Cyprinidae
Notropis asperifrons
Burrhead Shiner
April through June
Cyprinidae
Notropis atherinoides
Emerald Shiner
May through July
Cyprinidae
Notropis baileyi
Rough Shiner
May through October
Cyprinidae
Notropis buccatus
Silveijaw Minnow
March through June
Cyprinidae
Notropis candidus
Silverside Shiner
June through September
Cyprinidae
Notropis chrosomus
Rainbow Shiner
May through June
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F amily
Scientific Name
Common Name
Spawning Season
Cyprinidae
Notropis edwardraneyi
Fluvial Shiner
May through June
Cyprinidae
Notropis stilbius
Silverstripe Shiner
March through August
Cyprinidae
Notropis texanus
Weed Shiner
February through October
Cyprinidae
Notropis uranoscopus
Skygazer Shiner
May through July
Cyprinidae
Notropis volucellus
Mimic Shiner
April through August
Cyprinidae
Opsopoeodus emiliae
Pugnose Minnow
April through September
Cyprinidae
Phenacobius catostomus
Riffle Minnow
April through May
Cyprinidae
Pimephales notatus
Bluntnose Minnow
April through August
Cyprinidae
Pimephales vigilax
Bullhead Minnow
May through August
Cyprinidae
Semotilus atromaculatus
Creek Chub
April through May
Cyprinidae
Semotilus thoreauianus
Dixie Chub
April through May
Elassomatidae
Elassoma zonatum
Banded Pygmy Sunfish
March through April
Esocidae
Esox americanus
Redfin Pickerel
April through May
Esocidae
Esox niger
Chain Pickerel
April through October
Fundulidae
Fundulus olivaceus
Blackspotted Topminnow
March through September
Hiodontidae
Hiodon tergisus
Mooneye
April through May
Ictaluridae
Ameiurus melas
Black Bullhead
May through August
Ictaluridae
Ameiurus natalis
Yellow Bullhead
April through June
Ictaluridae
Ameiurus nebulosus
Brown Bullhead
April through August
Ictaluridae
Ictalurus furcatus
Blue Catfish
April through June
Ictaluridae
Ictalurus punctatus
Channel Catfish
April through July
Ictaluridae
Noturus funebris
Black Madtom
May through June
Ictaluridae
Noturus gyrinus
Tadpole Madtom
May through September
Ictaluridae
Pylodictis olivaris
Flathead Catfish
June through July
Lepisosteidae
Lepisosteus oculatus
Spotted Gar
May through July
Lepisosteidae
Lepisosteus osseus
Longnose Gar
April through August
Moronidae
Morone chrysops
White Bass
February through March
Percidae
Ammocrypta beanii
Naked Sand Darter
March through October
Percidae
Etheostoma meridianum
Southern Sand Darter
April through June
Percidae
Etheostoma chlorosomum
Bluntnose Darter
April
Percidae
Etheostoma jordani
Greenbreast Darter
April through May
Percidae
Etheostoma nigrum
Johnny Darter
March through May
Percidae
Etheostoma parvipinne
Goldstripe Darter
March through April
Percidae
Etheostoma ramseyi
Alabama Darter
March through May
Percidae
Etheostoma rupestre
Rock Darter
March through April
Percidae
Etheostoma stigmaeum
Speckled Darter
March through May
Percidae
Etheostoma swaini
Gulf Darter
March through April
Percidae
Percina kathae
Mobile Logperch
April through June
Percidae
Percina maculata
Blackside Darter
March through June
Percidae
Percina nigrofasciata
Blackbanded Darter
May through June
Percidae
Percina vigil
Saddleback Darter
February through April
Percidae
Sander vitreus
Walleye
March through April
Sciaenidae
Aplodinotus grunniens
Freshwater Drum
May through June
(Boschung and Mayden 2004)
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Spawning Seasons for Example Fish Assemblages in the Chicago River, IL Watershed
F amily
Scientific Name
Common Name
Spawning Season
Amiidae
Amia calva
Bowfin
March through June
Catostomidae
Catostomus commersonii
White Sucker
April through May
Centrarchidae
Ambloplites rupestris
Rock Bass
May through July
Centrarchidae
Lepomis cyanellus
Green Sunfish
June through August
Centrarchidae
Lepomis humilis
Orangespotted Sunfish
May through July
Centrarchidae
Lepomis gibbosus
Pumpkinseed
May through July
Centrarchidae
Lepomis gulosus
Warmouth
May through August
Centrarchidae
Lepomis macrochirus
Bluegill
May through August
Centrarchidae
Micropterus dolomieu
Smallmouth Bass
April through June
Centrarchidae
Micropterus salmoides
Largemouth Bass
April through June
Centrarchidae
Pomoxis nigromaculatus
Black Crappie
May through July
Clupeidae
Dorosoma cepedianum
Gizzard Shad
May through July
Cyprinidae
Campostoma anomalum
Central Stoneroller
April through July
Cyprinidae
Carassius auratus
Goldfish
May through June
Cyprinidae
Cyprinella spiloptera
Spotfin Shiner
May through August
Cyprinidae
Cyprinus carpio
Common Carp
May through August
Cyprinidae
Hybopsis dorsalis
Bigmouth Shiner
May through June
Cyprinidae
Notemigonus crysoleucas
Golden Shiner
May through August
Cyprinidae
Notropis atherinoides
Emerald Shiner
April through August
Cyprinidae
Notropis hudsonius
Spottail Shiner
June through July
Cyprinidae
Notropis stramineus
Sand Shiner
May through July
Cyprinidae
Pimephales notatus
Bluntnose Minnow
May through August
Cyprinidae
Pimephales promelas
Fathead Minnow
May through August
Cyprinidae
Semotilus atromaculatus
Creek Chub
April through June
Cyprinodontidae
Fundulus notatus
Blackstripe Topminnow
May through August
Esocidae
Esox americanus
Grass Pickerel
May through June; November
Esocidae
Esox lucius
Northern Pike
March through May
Gobiidae
Neogobius melanostomus
Round Goby
April through May
Ictaluridae
Ameiurus melas
Black Bullhead
May through June
Ictaluridae
Ameiurus natalis
Yellow Bullhead
May through June
Ictaluridae
Ictalurus punctatus
Channel Catfish
April through August
Moronidae
Morone americana
White Perch
May through June
Moronidae
Morone chrysops
White Bass
April through June
Moronidae
Morone mississippiensis
Yellow Bass
April through May
Percidae
Etheostoma nigrum
Johnny Darter
April through June
Percidae
Sander vitreus
Walleye
April through May
Percidae
Percaflavescens
Yellow Perch
May through July
Umbridae
Umbra limi
Central Mudminnow
April through May
(Auer, NA. 1982, Page and Burr 1991)
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EPA Draft for Public Comment
Spawning Seasons for Example Fish Assemblages in the Truckee and Carson River, NV
Watersheds
F amily
Scientific Name
Common Name
Spawning Season
Centrarchidae
Micropterus dolomieu
Smallmouth Bass
April through July
Centrarchidae
Micropterus salmoides
Largemouth Bass
April through July
Centrarchidae
Lepomis macrochirus
Bluegill
May through August
Centrarchidae
Pomoxis nigromaculatus
Black Crappie
May through July
Ictaluridae
Ictaluridae
Catfish species
June through July
Moronidae
Morone saxatilis
Striped Bass*
April through June
Moronidae
Morone chrysops
White Bass
April through June
Percidae
Sander vitreus
Walleye
January through April
Salmonidae
Oncorhynchus mykiss
Rainbow Trout
March through May
Salmonidae
Salmo trutta
Brown Trout
January through March
Salmonidae
Pros opium williamsoni
Mountain Whitefish
October through December
* This population of striped bass is landlocked, and cannot migrate out to sea.
(Nevada Division of Environmental Protection 2006)
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EPA Draft for Public Comment
Spawning Seasons for Example Fish Assemblages in the Rio Grande and Colorado River, TX
Watersheds
F amily
Scientific Name
Common Name
Spawning Season
Amiidae
Amia calva
Bowfin
March through June
Anguillidae
Anguilla rostrata
American Eel
February through June
Catostomidae
Ictiobus bubalus
Smallmouth Buffalo
March through September
Catostomidae
Ictiobus cyprinellus
Bigmouth Buffalo
April through May
Catostomidae
Ictiobus niger
Black Buffalo
April through May
Centrarchidae
Lepomis macrochirus
Bluegill
April through September
Centrarchidae
Lepomis cyanellus
Green Sunfish
April through August
Centrarchidae
Lepomis megalotis
Longear Sunfish
May through June
Centrarchidae
Lepomis auritus
Redbreast Sunfish
April through October
Centrarchidae
Lepomis microlophus
Redear Sunfish
May through July
Centrarchidae
Lepomis gulosus
Warmouth
March through October
Centrarchidae
Micropterus salmoides
Largemouth Bass
February through May
Centrarchidae
Micropterus dolomieu
Smallmouth Bass
April through May
Centrarchidae
Micropterus punctulatus
Spotted Bass
April through June
Centrarchidae
Micropterus treculii
Guadalupe Bass
March through June
Centrarchidae
Pomoxis nigromaculatus
Black Crappie
March through May
Centrarchidae
Pomoxis annularis
White Crappie
March through May
Cichlidae
Herichthys cyanoguttatus
Rio Grande Cichlid
March through August
Clupeidae
Dorosoma cepedianum
Gizzard Shad
April through June
Clupeidae
Dorosoma petenense
Threadfin Shad
April through September
Cyprinidae
Ctenopharyngodon idella
Grass Carp
April through July
Cyprinidae
Cyprinus carpio
Common Carp
March through June
Cyprinidae
Cyprinella lutrensis
Red Shiner
April through September
Cyprinidae
Cyprinella venusta
Blacktail Shiner
April through September
Cyprinidae
Notropis amabilis
Texas Shiner
February through September
Cyprinidae
Notemigonus crysoleucas
Golden Shiner
April through July
Cyprinidae
Pimephales promelas
Fathead Minnow
May through September
Esocidae
Esox niger
Chain Pickerel
December through February
Ictaluridae
Ictalurus furcatus
Blue Catfish
April through May
Ictaluridae
Ictalurus punctatus
Channel Catfish
April through June
Ictaluridae
Pylodictis olivaris
Flathead Catfish
June through July
Ictaluridae
Ameiurus melas
Black Bullhead
April through June
Ictaluridae
Ameiurus natalis
Yellow Bullhead
May through July
Lepisosteidae
Atractosteus spatula
Alligator Gar
April through May
Lepisosteidae
Lepisosteus oculatus
Spotted Gar
April through June
Lepisosteidae
Lepisosteus osseus
Longnose Gar
April through July
Lepisosteidae
Lepisosteus platostomus
Shortnose Gar
May through July
Moronidae
Morone chrysops
White Bass
March through May
Moronidae
Morone mississippiensis
Yellow Bass
April through June
Moronidae
Morone saxatilis
Striped Bass*
February through April
Percidae
Sander vitreus
Walleye
February through April
Polyodontidae
Polyodon spathula
Paddlefish
February through June
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EPA Draft for Public Comment
Family Scientific Name Common Name Spawning Season
Salmonidae Oncorhynchus mykiss Rainbow Trout November through February
Sciaenidae Aplodinotus grunniens Freshwater Drum April through June
Sciaenidae Sciaenops ocellatus Red Drum August through October
* This population of striped bass is landlocked, and cannot migrate out to sea.
(Hendrickson and Cohen, 2015; Texas Parks and Wildlife Department, 2016)
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EPA Draft for Public Comment
1 Appendix C
2 Conversion of Wet to Dry Tissue Weight
3 Conversion of Wet to Dry Tissue Weight
4 Selenium data in fish tissues can be reported in either dry weight or wet weight concentrations. It is
5 essential that exposure assessors be aware of this difference so that they may ensure consistency between
6 units. If the contaminant concentration is measured in wet weight of fish, then the concentration must be
7 converted to dry weight units to compare against the selenium criterion, which is expressed in dry weight
8 (USEPA 2008). Wet weight may be converted to dry weight using the following equation:
9 WW = DW x [1 - (percent moisture/100)] (Lusk et al. 2005)
10 Measurements reported as wet weight can be converted to equivalent dry weights using available percent
11 moisture data for the relevant species and tissue type. If percent moisture data is unavailable for a fish
12 species, percent moisture data for a similar species (i.e., same genus or, if unavailable, same family)
13 should be used. Table C-l lists percent moisture targeted species by tissue type (USEPA 2016). Percent
14 moisture can vary within species; therefore, these data should generally be used when dealing with
15 historical data. Field collected samples can be analyzed for % moisture, thus giving more accurate
16 conversions between dry weight and wet weight data.
17 Table C-l. Percent moisture, by species and tissue type
Average
% Moisture by Tissue
%
Whole
Egg-
Scientific Name
Common Name
Moisture
body
Muscle
ovary
Reference
Cyprinus carpio
Common Carp
75.64a
75.8 lb
aUSEPA 2014;
bChatakondi et al.
1995
Rhinichthys cataractae
Longnose Dace
73.25
USEPA 2014
Rhinichthys atratulus
Blacknose Dace
73.75
USEPA 2014
Semotilus
Creek Chub
76.71
USEPA 2014
atromaculatus
Pimephales promelas
Fathead Minnow
76.64a
75.3b
USEPA 2014;
bUSEPA 2015
Pimephales notatus
Bluntnose Minnow
74.8
USEPA 2014
Nocomis micropogon
River Chub
75.2
USEPA 2014
Ictalurus punctatus
Channel Catfish
81.223
78.43b
aPinkney 2003;
bMay et al. 2009
Ictalurus melas
Black Bullhead
76.82
USEPA 2014
Pylodictis olivaris
Flathead Catfish
75.97
May et al. 2009
Catostomus
White Sucker
77.37
USEPA 2014
commersonii
Coregonus
clupeaformis
Lake Whitefish
80
Rieberger 1992
Oncorhynchus kisutch
Coho Salmon
80
Rieberger 1992
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EPA Draft for Public Comment
Scientific Name
Common Name
Average
%
Moisture
% Moisture by Tissue
Reference
Whole
body
Muscle
Egg-
ovary
Oncorhynchus mykiss
Rainbow Trout
77.54
61.2
USEPA 2016
Sander canadensis
Sauger
77
USEPA 2014
Percaflavescens
Yellow Perch
73.98
USEPA 2014
Micropterus salmoides
Largemouth Bass
75.74a
79.06b
78.53c
a USEPA 2014; b
Pinkney 2003,c May
et al. 2009
Micropterus dolomieu
Smallmouth Bass
74.22
USEPA 2014
Pomoxis annularis
White Crappie
80.57
May et al. 2009
Pomoxis
nigromaculatus
Black Crappie
79.75
May et al. 2009
Lepomis macrochirus
Bluegill
74.8
80.09
76
USEPA 2016
Ambloplites rupestris
Rock Bass
74.95
USEPA 2014
Esox lucius
Northern Pike
78
Rieberger 1992
Pylodictis olivaris
Flathead Catfish
58.97
May et al. 2009
Scaphirhynchus
platorynchus
Shovelnose
Sturgeon
77.13
47.18
May et al. 2009
18
19 References
20 Chatakondi, N., R.T. Lovell, P.L. Duncan, M. Hayat, T.T. Chen, D.A. Powers, J.D. Weete, K. Cummins,
21 R.A. Dunham. 1995. Body composition of transgenic common carp, Cyprinus carpio, containing
22 rainbow growth hormone gene. Aquaculture 138: 99-109.
23 http://www.sciencedirect.com/science/article/pii/004484869501Q785
24 Lusk, J.D., E. Rich, and R.S. Bristol. 2005. Methylmercury and Other Environmental Contaminants in
25 Water and Fish Collected from Four Recreational Fishing Lakes on the Navajo Nation, 2004.
26 Prepared for the Navajo Nation Environmental Protection Agency.
27 https://www.fws.gov/southwest/es/newmexico/documents/final nnlfwqi report.pdf
28 May, T.W., Walther, M.J., Brumbaugh, W.G., McKee, M., 2009. Concentrations of elements in whole-
29 body fish, fish contaminant monitoring program: U.S. Geological Survey Open-File Report 2009-
30 1278. 11 p. https://pubs.usgs.gov/of/2009/1278/pdf/QF2009 1278.pdf
31 Pinkney, A.E. 2003. Investigation of Fish Tissue Contaminant Concentrations at Painted Turtle Pond,
32 Occoquan Bay National Wildlife Refuge, Woodbridge, Virginia. Annapolis, MD: US Fish and
33 Wildlife Service, https://www.fws.gov/chesapeakebav/pdf/cbfo-c0305.pdf
34 Rieberger, K. 1992. Metal Concentrations in Fish Tissue from Uncontaminated B.C Lakes. Ministry of
35 Environment, Lands and Parks, Province of British Columbia.
36 http://www.env.gov.bc.ca/wat/wq/reference/metalinfish.pdf
37 USEPA. 2008. Child-Specific Exposure Factors Handbook. EPA/600/R-06/096F. National Center for
38 Environmental Assessment, Office of Research and Development, Washington, DC.
39 https://cfpub.epa.gov/ncea/risk/recordisplav.cfm?deid=199243
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41
42
43
44
45
46
47
48
49
50
51
52
53
EPA Draft for Public Comment
USEPA. 2008. Child-Specific Exposure Factors Handbook. EPA/600/R-06/096F. National Center for
Environmental Assessment, Office of Research and Development, Washington, DC.
https://cfpub.cpa.gov/ncca/risk/rccordisplay.cfin7dcidH 99243
USEPA. 2014. External Peer Review Draft Aquatic Life Ambient Water Quality Criterion for Selenium-
Freshwater 2014. EPA 822-P-14-001. U.S. Environmental Protection Agency, Office of Water,
Office of Science and Technology, Washington, DC.
https ://www.epa.gov/sites/production/files/2016-
07/documents/2014 draft document external peer review draft aquatic life ambient wqc for se
freshwater.pdf
USEPA. 2016. Aquatic Life Ambient Water Quality Criterion for Selenium-Freshwater 2016. EPA 822-
R-16-006. U.S. Environmental Protection Agency, Office of Water, Office of Science and
Technology, Washington, DC. https://www.epa.gov/wqc/aquatic-life-criterion-selenium-documents
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