£EPA
United States
Environmental Protection
Agency
Office of Water
EPA-822-R-24-008
September 2024
Standard Operating Procedures
for Systematic Review of Ecological Toxicity Data
in Support of
Ambient Water Quality Criteria, Benchmark and Screening
Value Derivation
for Aquatic Life and Aquatic Dependent Wildlife
September 2024
United States Environmental Protection Agency
Office of Water
Office of Science and Technology
Health and Ecological Criteria Division
Ecological Risk Assessment Branch
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Table of Contents
1 Introduction 3
1.1 Purpose 3
1.2 Background 3
1.3 Organization of the Document 4
2 Screening the Open Literature 6
2.1 Studies Accepted by ECOTOX and OW 8
2.2 Studies Accepted by ECOTOX but not by OW 8
2.3 Studies Rejected by either ECOTOX or OW 9
2.4 Consideration of Papers in the "Other" Category 9
3 Reviewing the Open Literature 10
3.1 Study Classifications 11
3.2 Guidance for Open Literature Data Review 12
3.2.1 Studies Classified as Unacceptable 19
3.2.2 Studies for Quantitative and Qualitative Use 19
4 Documentation of Open Literature Used in AWQC Development 20
4.1 Documenting Study Screening Outcomes 20
4.2 Documenting Study Review Outcomes 21
4.2.1 Completion of DERs for Endpoints Used Quantitatively 22
4.2.2 Completion of DERs for Endpoints Used Qualitatively 23
4.2.3 Completion of DERs for Unacceptable Open Literature Studies 23
4.3 Open Literature Review Documentation in AWQC Documents 23
4.3.1 Documentation for Endpoints Used Quantitatively 23
4.3.2 Documentation for Endpoints Used Qualitatively 24
4.3.3 Documentation for Unacceptable Endpoints 24
5 References 25
Attachments
Attachment A 1985 Guidelines Minimum Data Requirements for Acute and Chronic
AWQC 27
Attachment B Acceptability Criteria for Aquatic Effects Data 29
Attachment C ECOTOX Exclusion Reasons 32
Attachment D Instructions for Completing Data Evaluation Records (DERs) for Toxicity
Studies in the Open Literature (September 2024) 35
Attachment E Fish Data Evaluation Record (DER) Template (September 2024) 42
Attachment F Aquatic Invertebrate Data Evaluation Record (DER) Template
(September 2024) 66
Attachment G Aquatic Nonvascular Plant Data Evaluation Record (DER) Template
(September 2024) 92
Attachment H Aquatic Vascular Plant Data Evaluation Record (DER) Template
(September 2024) 113
Attachment I Amphibian Data Evaluation Record (DER) Template (September 2024).... 135
Attachment J Avian Data Evaluation Record (DER) Template (September 2024) 159
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1 Introduction
1.1 Purpose
The purpose of this Standard Operating Procedures (SOP) document is to provide information on
the long-standing process used to assist the EPA Office of Water (OW) scientists and the EPA
contractors in systematic review of the quality of ecological effects studies in the development of
numeric Ambient Water Quality Criteria (AWQC) for the protection of aquatic life and aquatic-
dependent wildlife. In addition, EPA is making these materials publicly available to offer
information to criteria developers outside of the EPA, including states and authorized Tribes, that
could consider conducting systematic review of ecological effects data following OW's process
described in this SOP. This document is only intended to be informative and descriptive and does
not provide or make recommendations about what other entities should do to conduct systematic
review in the development of AWQC. This document supports the screening, review,
documentation, and use of data from various sources and can assist criteria developers in
determining if and in what manner (e.g., quantitatively or qualitatively) a study could be used in
an effects assessment to support development of aquatic life criteria and other protective values
(e.g., aquatic life benchmarks and screening values). The SOP is generally consistent with
evaluation guidelines developed by the EPA Office of Chemical Safety and Pollution Prevention
(OCSPP) (e.g., U.S. EPA 2011) to support a common approach to data evaluation for chemicals.
1.2 Background
One of the objectives of the Clean Water Act (CWA) (33 U.S.C. Sections 125 l-1387is to protect
and restore the biological, chemical, and physical integrity of waters in the United States.
Pursuant to CWA Section 304(a), the EPA is required to publish, and from time to time
thereafter revise, criteria for water quality to accurately reflect the latest scientific knowledge.
AWQC are levels of individual pollutants, water quality characteristics, or descriptions of
conditions of a water body that, if met, are expected to protect the aquatic community. AWQC
for the protection of aquatic life and aquatic-dependent wildlife, developed under Section 304(a),
reflect current scientific knowledge and are based on data and scientific determinations of the
relationship between concentrations of a pollutant and its effects on aquatic life. The EPA's
national recommended AWQC are not rules, nor do they automatically become part of a state's
water quality standards. The EPA develops recommended AWQC based on the best available
science, a scientific literature review, established procedures for risk assessment, EPA policy,
and external scientific peer review and public input.
States and authorized Tribes may adopt criteria that the EPA publishes under CWA Section
304(a), modify the CWA 304(a) criteria to reflect site-specific conditions, or adopt criteria based
on other scientifically defensible methods (40 C.F.R. § 131.11(b)(1)). Under CWA Section 303
and the EPA's implementing regulations at 40 C.F.R. Part 131, the EPA must review and
approve new or revised state water quality standards, including criteria. A state must adopt
criteria that protect the designated use and are based on a sound scientific rationale (40 C.F.R. §
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131.11(a)(1)).
Since 1985, the EPA's AWQC for the protection of aquatic life have been derived based on
methods outlined in the EPA's Guidelines for Deriving Numerical National Water Quality
Criteria for the Protection of Aquatic Organisms and Their Uses (hereafter referred to as
"GuidelinesUS EPA 1985). The Guidelines have provided consistency and transparency in the
derivation methodology of AWQC for pollutants. The Guidelines include detailed direction on
acceptability of toxicity test results and minimum data requirements (see Attachment A) to
ensure the quality and sufficiency of data used in the derivation of nationally recommended
AWQC. This SOP was developed by synthesizing the principles in the 1985 Guidelines and
other EPA data quality guidance to transparently and consistently document the systematic
review of data used in the development of AWQC.
AWQC are based on an evaluation of available toxicological effects data for a pollutant for
aquatic life and/or aquatic-dependent wildlife [taxa that depend on aquatic prey (e.g., fish and
emergent aquatic insects) as their major food source]. Most ecological effects data for pollutants
are obtained from the U.S. EPA ECOTOXicology Knowledgebase 1 (ECOTOX, Olker et al.
2022), maintained by the EPA's Office of Research and Development. Another important source
of information used in the development of AWQC for pesticides is toxicity data acquired through
U.S. EPA's Office of Pesticide Programs (OPP) (under the Federal Insecticide, Fungicide, and
Rodenticide Act, 40 CFR Part 158). Applicable toxicity data from EPA researchers and sources
external to the EPA, such as other federal agencies, academic researchers, and other international
environmental regulatory authorities are also considered during the development of AWQC.
1.3 Organization of the Document
This document is divided into the following three sections that describe a systematic review
approach:
• Screening the Open Literature: Discusses the general screening of papers and
categorization of acceptability (i.e., applicable, non-applicable, and "others").
• Reviewing the Open Literature: Provides technical support for reviewing studies that
pass the screening process to assess the quality of the study and determine its usability in
AWQC development.
• Documenting the Open Literature: Provides technical support for the efficient and
consistent process for documenting reviews of the open literature and avoiding
duplicative and possibly conflicting efforts associated with study reviews across
reviewers.
1 ECOTOX is a publicly available database that curates the ecological effects of single chemicals to aquatic and
terrestrial plants and animals. For more information on ECOTOX literature review and data curation processes,
database documentation, controlled vocabularies, and guides for users, see the overview in Olker et al. 2022 and
visit EPA's ECOTOX website at https://cfpub.epa.gov/ecotox/.
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The flowchart depicted in Figure 1 summarizes the Systematic Review Process for open
literature studies.
Study obtained from open
literature (e.g., ECOTOX).
Screen
Acceptable to OW based
on ECOTOX Applicability
Criteria?
No
Studies classified as
"rejected."
Yes
Data should be considered
potentially relevant for
inclusion in the AWQC
effects assessment and
should be obtained.
No
Pass additional OW screen
for relevant taxa or
endpoints?
Yes
Review
Are data considered
scientifically rigorous and
provide reliable and/or
useful information?
No
Studies classified as
"unacceptable."
Yes
Are data considered
appropriate for use in
AWQC derivation or other
quantitative measures of
effect?
No
Studies classified as
"qualitative," data can be
used as additional
lines/weight of evidence
to support the effects
assessment.
Yes
Studies classified as
"quantitative."
Documentation
Document rejected
studies, including rejection
rationale, and review if
appropriate. DERs not
required.
Provide citations (in
AWQC appendix) including
rejection rationale.
Abbreviated DERs
required.
Provide citations (in
AWQC appendix). Full
DERs required.
Provide citations (in
AWQC appendix). Full
DERs and verification of
calculations and statistics
required.
Figure 1. The EPA Office of Water's screening, review, and documentation steps in
overarching systematic review process of open literature studies.
ECOTOX = US EPA Ecotoxicology Database; AWQC = Ambient Water Quality Criteria: DER = Data Evaluation
Record.
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2 Screening the Open Literature
The purpose of this section is to discuss the screening process applied to identify potentially
useful open literature studies. OW uses ECOTOX as its primary search engine to obtain relevant
acute and chronic toxicity information for AWQC development.
ECOTOX is a source for locating single chemical toxicity data for aquatic life, terrestrial plants,
and wildlife. ECOTOX was created and is maintained by the U.S. EPA's Office of Research and
Development/Center for Computational Toxicology and Exposure's Great Lakes Toxicology and
Ecology Division, and has well-established systematic review and data curation processes used
for both Program offices and database chemical searches procedures. The screening process
begins with a comprehensive chemical-specific literature search of the open literature conducted
according to ECOTOX Standard Operating Procedures. The search terms in ECOTOX are
comprised of chemical terms, synonyms, degradates, and verified Chemical Abstracts Service
(CAS) numbers. For example, the literature search terms for Phosphoric acid, triphenyl ester are
as follows: "Antioxidant TTP" OR "BRN1888236" OR "Celluflex TPP" OR "DHPF 005" OR
"Disflamoll TP" OR "NSC 57868" OR "0,0,0-Triphenyl phosphate" OR "Phenyl phosphate"
OR "Phoscon FR 903N" OR "Phosflex TPP" OR "Phosphoric acid, triphenyl ester" OR
"Phosphoric acid, triphenyl ester radical ion(l+)" OR "Reofos TPP" OR "Sumilizer TPP" OR
"Triphenolphosphate " OR "Triphenoxyphosphine oxide " OR "Triphenylphosphate " OR
"Triphenylphosphat" OR "Triphenylphosphate" OR "UN3077" OR "UNII-YZE19Z66EA" OR
"Wako TPP" OR "WSFR-TPPOnce the references are generated, they are tagged with
exclusion keywords (see Figure 2 and Attachment C).
After developing the literature search strategy, the ECOTOX literature review process includes
conducting the searches, identifying potentially applicable studies based on title and abstract,
acquiring potentially applicable studies, applying the ECOTOX applicability criteria (see
Section 2.1), and abstracting the data, with respect to the acceptability of the study report for
inclusion in the knowledgebase, into ECOTOX (see Figure 2). Studies that meet the
applicability criteria are coded to reflect information on the chemical, species, habitat, test
location, exposure route, control type, endpoint and effect. At each step, search terms and results,
screening decisions, and respective tags are recorded and stored electronically in the ECOTOX
Knowledgebase for both applicable and non-applicable studies. This process is documented in
U.S. EPA (2023) and described in Olker et al. (2022).
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Figure 2. Overview of the literature searches, citation identification, and applicability criteria used
by the ECOTOX Knowledgebase as described in U.S. EPA (2023).
Although this section of this SOP focuses on the evaluation of toxicological studies obtained
through ECOTOX, many of the approaches applied to the evaluation of these studies are also
applicable to the evaluation of studies from other sources. All papers identified in the ECOTOX
database search as having data that are potentially relevant to AWQC derivation are obtained and
screened for data quality. When requesting the data pull from ECOTOX, OW obtains all
applicable studies (with pdfs), as well as the non-applicable studies (with reason for exclusion),
and the studies in the "other" category. To ensure data quality and verifiability, these studies
should meet certain applicability criteria described to be considered for use in the development
of AWQC. Studies are assigned to one of three categories based on the applicability evaluation:
(1) Studies accepted by ECOTOX and OW (Section 2.1);
(2) Studies accepted by ECOTOX but not by OW (Section 2.2);
(3) Studies rejected by either ECOTOX or OW (Section 2.3); and
(4) "Other" studies (Section 2.4).
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2.1 Studies Accepted by ECOTOX and OW
Traditionally, toxic effects of a chemical should be relevant to a whole organism (including
aquatic animal, aquatic-dependent wildlife or aquatic plant species) to be considered for use in
the development of AWQC. However, increasingly in vitro methods are used to assess toxicity
for priority setting and screening assessments, and as quantitative linkages between these in vitro
endpoints and apical endpoints of regulatory concern (survival, growth, and reproduction) are
established, they may be considered for quantitative risk assessment. To be accepted by OW,
regardless of whether the study is identified through the ECOTOX database or outside literature
sources, papers should meet the following minimum criteria based on the ECOTOX applicability
criteria:
(1) The toxic effects are related to single chemical exposure (unless the study is being
considered as part of a mixture effects assessment);
(2) There is a biological effect on live, whole organisms or in vitro preparation including
gene chips or omics data on adverse outcome pathways potentially of interest;
(3) Chemical test concentrations are reported;
(4) There is an explicit duration of exposure;
(5) Toxicology information that is relevant to OW is reported for the chemical of
concern;
(6) The paper is published in the English language;
(7) The paper is available as a full article (not an abstract);
(8) The paper is publicly available;
(9) The paper is the primary source of the data;
(10) A calculated endpoint is reported or can be calculated using reported or available
information;
(11) Treatment(s) are compared to an acceptable control;
(12) The location of the study (e.g., laboratory vs. field) is reported; and
(13) The tested species is reported (with recognized nomenclature).
Attachment B discusses the ECOTOX and OW applicability criteria in detail, as well as the
criteria for studies that are rejected from the ECOTOX database and thus tagged as non-
applicable.
"Acceptable" studies from the open literature papers that pass the ECOTOX and OW screens of
applicability should be considered potentially relevant for inclusion in the AWQC effects
assessment and should be obtained for further review.
2.2 Studies Accepted by ECOTOX but not by OW
After studies from the open literature that pass the ECOTOX applicability criteria are obtained,
an additional [title and abstract] scan of the "acceptable" studies should be conducted to identify
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studies with taxa that are not relevant to AWQC (e.g., strictly terrestrial species). If non-relevant
studies are identified, the studies will follow the process outlined in Section 2.3. All remaining
acceptable studies will be further reviewed. Additional discussion of the review of studies and
documentation for use in AWQC development is provided in Sections 3 and 4, respectively.
2.3 Studies Rejected by either ECOTOX or OW
Open literature studies found not acceptable for ECOTOX or OW, based on the factors
summarized in Section 2.1 and 2.2, are not typically used to derive AWQC; however, they may
provide some useful information for AWQC development. Studies that do not meet the criteria
for ECOTOX are designated as "non-applicable," and are given rejection keywords (see
Attachment C), which can be cited to explain the reason for excluding a study from
consideration. The EPA, may review these rejected studies, if appropriate, to determine whether
the study includes information useful to the effects assessment or effects characterization. For
example, studies may be rejected by ECOTOX because they include data on mixtures of
chemicals. Studies that are coded with the rejection code "MIXTURE" during the initial
evaluation may still be considered as a "line of evidence" during AWQC development. Other
studies that are likely to be rejected by ECOTOX, but that may be useful to the EPA, as a "line
of evidence" include those with data related to modeling, monitoring, incident reports, and
review articles.
2.4 Consideration of Papers in the "Other" Category
In addition to providing citations for "acceptable" (i.e., acceptable for ECOTOX and OW) and
"rejected" (i.e., not acceptable for ECOTOX or OW) studies, a file of citations called "Other" is
provided to the EPA as part of the ECOTOX search. The "Other" category documents citations
for studies into one of the following four categories:
(1) Target (for pesticides): toxicity of chemical on intended pest including efficacy
studies;
(2) On Order: potentially acceptable but publication has not been received;
(3) To Abstract: applicable but not coded; and
(4) To Apply Criteria: potentially acceptable but not evaluated.
Papers included in the "Other" category are not coded into ECOTOX and do not appear as
"applicable" data. Depending on the chemical, citations for "target" data are routinely included,
whereas citations from the other three categories of "on order," "to abstract" and "to apply
criteria" are encountered less frequently. In an effort to address the omission of information
included in the "Other" category, all citations listed in the ECOTOX file name "Other" should be
reviewed for potential inclusion in acceptable studies category.
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3 Reviewing the Open Literature
All studies identified through ECOTOX or other relevant sources that are identified as
potentially useful based on the screening process discussed in Section 2 should be reviewed and
classified as described in this section.
When reviewing ecotoxicity data, it is important to have a systematic method for reviewing and
evaluating the quality of a study. This applies to all types of ecotoxicity studies that have the
potential to be included in AWQC development (e.g., single-species toxicity data, multispecies
laboratory studies, mesocosm studies, and field tests). Information that should be considered
when evaluating the test data quality includes:
• species and test properties (e.g., age, weight, lifestage)
• test compound and dosing (nominal and measured doses)
• dosing methodology (static, static renewal, or flow-through)
• exposure duration (acute or chronic, or subchronic)
• water quality data during the test (dissolved oxygen levels, pH, temperature, etc.)
• ecotoxicological endpoint data for both control and treatment groups
• statistical methods used to analyze the test results
• any unexpected observations or unusual circumstances that would be important in the
interpretation of the test (e.g., tank overflow, malfunction of aeration system)
High quality data should be both relevant and reliable for AWQC development. The Information
Quality Act of 2001 (Data Quality Act) charged the Office of Management and Budget (OMB)
with issuing government-wide guidelines that "provide policy and procedural guidance to
Federal agencies for ensuring and maximizing the quality, objectivity, utility, and integrity of
information (including statistical information) disseminated by Federal agencies." In 2002, the
OMB issued Information Quality Guidelines and the EPA then developed the policy and
procedural guidance, Guidelines for Ensuring and Maximizing the Quality, Objectivity, Utility,
and Integrity of Information Disseminated by the Environmental Protection Agency (EPA/206R-
02-008, October 2002).
Data quality, as defined by OMB and in the Informational Quality Guidelines, is the overarching
data condition that must be determined by evaluating each of the following:
1. Utility refers to the usefulness of the literature to its intended purpose and use. Studies,
data, information and methods must only be relied upon to the extent their use is
scientifically justified.
2. Objectivity refers to whether the information in the study is presented in an accurate,
clear, complete, appropriate, and unbiased manner.
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3. Transparency refers to the clarity of the process. For example, uncertainties and error
sources, assumptions, statistical methods, and justifications must be identified, as this
high degree of transparency facilitates reproducibility by third parties.
4. Integrity refers to ensuring the information is not compromised through alteration or
improper interpretation.
In the derivation of AWQC, ecotoxicity data need to be particularly evaluated based on three
major points: 1) relevance to criteria derivation; 2) level of documentation; and 3) acceptability.
The level of documentation and acceptability together define the reliability of a study. The
ECOTOX system for documentation of aquatic and terrestrial toxicity data from laboratory and
field studies is widely accepted. International jurisdictions use similar processes (e.g., Dutch
National Institute for Public Health and the Environment (RIVM) 2001) and acceptance criteria
(e.g., Klimisch et al. 1997) in the data evaluation process.
To minimize uncertainty in AWQC, only data that meet stated data quality guidelines should be
used for criteria derivation (Section 3.2.2), Toxicity and physical-chemical data should be from
studies conducted according to accepted protocols that are appropriate for the chemical and
organism being tested. As in vitro methods develop, technical support information on accepted
protocols will be incorporated. Some international jurisdictions simply state that tests must have
been conducted according to accepted, standardized protocols or according to principles of good
laboratory practices (GLP) while the U.S. EPA (U.S. EPA 1985, U.S. EPA OSCPP Section 850
Guidelines for aquatic species) and European Commission (2011) list very specific data
requirements.
3.1 Study Classifications
Studies identified in ECOTOX and other sources that may provide data relevant to criteria
derivation should be reviewed and classified. Data should be classified into one of three general
categories:
• Quantitative: Appropriate for use in AWQC derivation or other quantitative measures of
effect (e.g., Sensitivity Distribution development, acute-to-chronic ratio calculation);
• Qualitative: Not appropriate for quantitative use, but of sufficient quality to be used
descriptively; for example, as a line of evidence in the effects assessment; and
• Unacceptable: Inappropriate for quantitative and qualitative use in AWQC development
due to insufficient quality and/or lack of scientific defensibility.
General guidelines for reviewing open literature studies and identifying data usability from a
study as "quantitative," "qualitative," or "unacceptable" are provided in the following section
(Section 3.2).
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3.2 Guidance for Open Literature Data Review
The 1985 Guidelines provides guidance to enable classification of data from open literature
studies into one of three categories, as discussed in the preceding section. OW has developed this
SOP document as an enhancement to the 1985 Guidelines, to support transparency and
consistency in evaluations. The approach outlined in this SOP document is based on the 1985
Guidelines and additionally reflects information available in the EPA OCSPP Series 850
Ecological Effects Test Guidelines for aquatic and aquatic-dependent species.
Prior to development of this SOP, OW a conducted a review comparing OW's data quality and
test acceptability guidelines for the review of open literature studies on aquatic and aquatic-
dependent species, as presented in the 1985 Guidelines, with OPP's 40 CFRPart 158
requirements and guidance on reviewing open literature studies (U.S. EPA 2011) for the
evaluation of data quality and test acceptability under the Federal Insecticide, Fungicide, and
Rodenticide Act (7 U.S.C. § 136 et seq., 1996). The results of this review indicate that test
acceptability and data quality requirements do not differ substantively between the two
programs, and that data would generally be excluded or included based on similar data quality
requirements. The approach outlined in this SOP document is also generally consistent with data
quality and test acceptability criteria applied in data quality review for aquatic and aquatic-
dependent species' data by the EPA's Office of Pollution Prevention and Toxics under the
Toxics Substances Control Act (15 U.S.C. §§ 2601 et seq., 1976).
EPA reviewers also apply their best professional judgment to determine the appropriate
classification for aquatic toxicity studies. The templates for taxa-specific Data Evaluation
Records (DERs) found in Attachment D of this document include detailed information for the
study reviewer on specific attributes necessary to consider a study acceptable for use in criteria
development. The information in the DER templates on specific attributes of a study reflect the
EPA (1985 Guidelines, 1995 Addendum, and EPA OCSPP's Series 850 Ecological Effects Test
Guidelines), ASTM (American Society for Testing and Materials), and OECD (Organisation for
Economic Co-operation and Development) standardized test guidelines. The sources for each
specific attribute of the study are cited on the DER.
While a single factor may result in a study being considered unacceptable (e.g., excessive control
mortality), more typically, several factors in combination render a study unacceptable. The
following should be considered when evaluating open literature studies:
• Data used in derivation of AWQC. The data should be from a primary source published in
English and available either as a publication or in the form of a dated and signed document
(e.g., manuscript, memo, letter; U.S. EPA 1985). Reports should include enough supporting
information to evaluate the acceptability of test procedures and reliability of study results.
Unpublished studies deemed useful for criteria derivation should undergo focused the EPA
review, and in some cases external peer review, prior to usage in criteria derivation. The
1985 Guidelines (U.S. EPA 1985) and associated Technical Support Document (U.S. EPA
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1987) provide specific data quality guidance and information on quality criteria for
acceptance/rejection of toxicity tests. This SOP document summarizes and updates previous
data quality information found in the 1985 Guidelines (U.S. EPA 1985) and the Technical
Support Document (U.S. EPA 1987).
• Nature of the test substance (percent active ingredient; source/manufacturer). The study
should indicate the exact nature and source of the chemical toxicant being tested, including
the grade and purity. Data for test substances less than 80% pure and chemical mixtures are
typically deemed unacceptable (e.g., drilling muds, effluents, sludges), but may be
considered on a case-by-case basis. For most metals, acceptable data for only a few salts
(chloride, nitrate, and sulfate) are used quantitatively. Data for other salts are typically
classified as either qualitative or unacceptable but may be used on a case-by-case basis. If the
chemical is a pesticide, the percent active ingredient and/or the purity of the test compound
should be reported. If a vehicle is used for solubilization of the chemical in the test dilution
water, the vehicle should be identified and should be known not interfere with the absorption,
distribution, metabolism, or the elimination (ADME) of the test substance, nor alter the
behavior/response of the test organisms. Toxicity studies which rely on solvents should
include solvent controls to document that the solvent did not affect the organism response.
• Species, age, sex, size, and life stage and source of the test species. The test organism
should be identified by scientific name and the health of the test organism should be reported.
The test organisms, to the extent possible, should be of uniform weight, size and age, and life
stage, and have no history of pre-exposure to other chemicals. Any acclimation of test
organisms prior to testing should be reported and adhere to established protocols (e.g., U.S.
EPA or ASTM). Observed diseases and treatment should be reported and may disqualify test
animals or a specific study from quantitative use. Data obtained with species non-resident in
North American were previously generally categorized as unacceptable and rejected without
further review based on discussions in the 1985 Guidelines. Current scientific approaches
reflect the interest in considering all quality toxicity tests on aquatic species as potential
surrogate data for the thousands of untested species in the environment and thus may be
included on a case-by-case basis in the systematic review process, so that data needed to fill
information gaps is not unnecessarily excluded without appropriate consideration of their
utility. Species native to temperate regions are considered more representative surrogates for
potential untested species in the U.S. than tropical species.
• Method of chemical addition and exposure. The test material is typically dosed to the
dilution water by either static, static-renewal or flow-through procedures. Acute tests can use
any of these three methods, if the chemical is known to be sufficiently stable in water over
the test duration, but chronic tests should use either static-renewal or flow-through
procedures. Data from tests in which the test organisms were exposed to the test material by
injection or gavage are typically classified as unacceptable, but may be considered as part of
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the weight of the evidence. Data from tests with dietary exposure will be considered for
bioaccumulative chemicals.
• Number of organisms tested per concentration/dosage level and number of
concentrations/dosage levels evaluated, as well as the number of replicates for each
concentration/dosage level. This type of information should be reported and be sufficient to
yield statistically-sound results. An inadequate number of test organisms per test level or not
randomly assigning test organisms to the different treatments, can produce unreliable results.
U.S. EPA (e.g., Office of Chemical Safety and Pollution Prevention 850 Draft or Final Test
Guidelines2) or ASTM standardized test guidelines should be consulted for further
information on the adequate number of test organisms per test level and on assigning test
organisms. Data from tests in which enzymes, excised or homogenized tissue or cell cultures
can be considered in examining adverse outcome pathways or used if a quantitative
relationship between the effect and an apical endpoint (e.g., survival, growth, and
reproduction) has been determined.
• Quantification of exposure. The concentration (and total volume of test material
administered, if available) in water or sediment should be reported, as well as the type of test
(static, renewal, flowthrough) and duration of exposure. For all studies, the exposure
conditions should be clearly described and documented. Measured concentrations are
strongly preferred over nominal concentrations for acute tests, while measured
concentrations are required for chronic tests and field studies. Measured and nominal
concentrations should be reported. Measured concentrations should be analyzed according to
procedures set forth in standardized analytical chemistry test guidelines, such as USEPA,
ASTM standard test guidelines or other analytical methods determined to be of high
performance (e.g., documented research method). Measured concentrations should not vary
excessively during the study period. Studies should not be used quantitatively where
measured concentrations deviate more than identified as acceptable in the published
analytical method. Studies should also not be used quantitatively if variability in
concentration between the different treatment groups is sufficiently high (e.g., results deviate
by more than 20%) to render mean exposure concentrations between the different treatment
groups statistically indistinguishable and/or overlapping concentrations occur between
different treatments. The following factors should be considered when reviewing a study
with high variability between treatment group measured concentrations: a) the treatment
group with high variability is not an endpoint of concern (e.g., different than the no observed
effect concentration [NOEC] or lowest observed effect concentration [LOEC] value); b) the
dose-response is strong despite the +/- 20% variance; c) the frequency and duration of
occurrence (i.e., variance at a low frequency vs. all the time); d) the study authors have
provided justification for variability in measured concentrations and identified all measures
2 https://www.epa.gov/test-guidelines-pesticides-and-toxic-substances/series-850-ecological-effects-test-guidelines
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taken to mitigate the problem; and e) the duration of the study (i.e., <20% variability is rarely
achieved in fish full lifecycle studies). Where only nominal concentrations are reported, the
reviewer should consider whether the test compound is subject to degradation, volatilization,
partitioning and/or a combination of these properties such that exposure levels may be
considerably different than any reported nominal values (discussed under "Test chemical
properties"). Additionally, the reviewer should consider whether test conditions could allow
exposure to other chemicals that could potentially confound the study outcomes.
• Control conditions and performance. A suitable number of controls should be run
concurrently with treatments, and control performance should be used as an indicator of
whether study conditions and animal health are acceptable. Negative controls should be run
concurrent with the study and failure to do so would typically invalidate the study;
exceptions can be made under special circumstances (e.g., lab with a strong history of
known, non-variable control responses and outcomes with toxicants consistent with other
studies). Positive controls should also be run with a reference toxicant if organisms are field
collected or if sublethal biochemical endpoints are assessed to ensure responsiveness of the
organisms tested. Studies which rely on co-solvents should also report concurrent solvent
control performance. As an indicator of study conditions, control performance in terms of
mortality and disease should be carefully evaluated and reported to determine the adequacy
of the study. For most species, mortality of greater than 10% in controls for acute tests and
greater than 20% in controls for chronic studies is sufficient to invalidate studies. Ideally,
studies should also report the measured concentrations of test chemical in the controls.
Studies reporting test chemical residues in the controls greater than the limit of detection
(LOD) should be invalidated, as the ability of the study to discriminate a treatment effect
may be compromised. Exceptions may occur for naturally occurring test materials (e.g.,
copper, other metals, various salts) and in situations where the presence of the test material in
controls can be discounted given acceptable performance in the controls and where effect
concentrations in the treatment groups are orders of magnitude higher than the control.
Normal growth/development times (where available) for the tested species should be
compared to those reported for the study controls. Where the growth/development of the
control organisms differs substantially from normal reported values, the reviewer must
determine whether study conditions have impaired the organisms' ability to thrive. In cases
where growth or development in the controls is substantially different than typically observed
for the test organisms, the study results should not be used because the ability to distinguish
treatment effects is uncertain.
• Wild-caught animals. Tests using wild-caught animals with unknown previous exposure
histories are acceptable provided their source is not an area where prior exposure to
pollutants is likely. Test organisms that are reported as having been previously exposed to the
test material or other contaminants are invalid. Likewise, wild caught test organisms
collected from water with high natural concentrations of potential environmentally-derived
toxicants (e.g., metals) should not be used. Also, wild caught animals used in tests that are
not quarantined and sufficiently acclimated should be invalidated. Documentation of
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quarantine and acclimation should be available for all wild caught test organisms used in
toxicity tests. As an exception, wild caught organisms collected from high and low exposure
sites may be used to generate an exposure gradient for maternal transfer studies for certain
chemicals (e.g., selenium maternal transfer studies where effects were observed in offspring).
• Macroscopic observations of the test animals. During a study, a detailed description of the
nature, incidence, time of occurrence, severity, and duration of all observed effects, including
death and any other abnormal or unusual signs and symptoms (i.e., sublethal effects) should
be reported for controls and treatment groups.
• Husbandry and test conditions. Standardized conditions (U.S. EPA OSCPP 850
Guidelines, ASTM studies) have been established in part to minimize the potential for
husbandry conditions to confound the study outcome. Reviewers should be cognizant of
husbandry conditions and verify that the study conditions are adequately described and
acceptable. This description should include:
o number of animals per cage or test container (i.e., biological loading rate)
o test organism health (treatment or observation of disease/stress should be reported)
o ambient temperature (use of water temperature outside the range of values that are
acceptable for the test species is invalid, unless temperature-related responses are an
experimental factor)
o humidity (as applicable)
o photoperiod
o dietary composition and feeding rate
o source of food
o dimensions of the test container
o source of the dilution water and description of its chemical characteristics (use of
distilled or deionized water without reconstitution is invalid)
o description of the toxicant delivery system and flow rate (expressed as the average
water volume of test solution passing through each test chamber per unit time).
Reviewers should consider whether the water exchange (static, static renewal, or flow-
through conditions) is adequate to support the number of test animals in the selected test
chambers and is appropriate given the test chemical's stability. Control performance and
variability should be used as an indicator of the test environment suitability. Results from
tests using nonstandard protocols are acceptable for use if the conditions described above are
adequately documented and no unexplained irregularities are observed.
• Feeding during acute tests. Results of acute tests during which test organisms were fed
should not be used (except for tests using certain species such as saltwater annelids and
mysids) unless data indicate that food did not affect the toxicity of the test material and/or the
test material has a low Kow value (< 2). For compounds with a log Kow of less than two, the
presence of food is assumed to be not likely to significantly alter the dissolved concentration
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or bioavailability of the test material. For pesticides with log Kow values between 5 and 7,
laboratory toxicity data should be carefully reviewed to ensure that feeding regimes are
eliminated to minimize any effects from interaction of the pesticide with food particles (e.g.,
reduction of test solution concentration as a result of partitioning into the food particles, or
introduction of a dietary exposure route if animals ingest food that has sorbed to the
pesticide, if the test is intended to capture only water column-based effects).
• Test chamber material. Tests conducted with organic materials in plastic test chambers (test
vessels constructed from materials other than glass) without measurement of test material
should be considered invalid unless it can be confirmed that the test material is stable and/or
has a low Kow value. Tests conducted in plastic test chambers should be documented in the
study evaluation because there is the potential for plasticizers to leach into the dilution water,
which could compromise the test results.
• Test chemical properties (e.g., solubility, Kd and Koc, vapor pressure/Henry's law
constant). This information is important in determining if actual exposure concentrations
could differ substantially from nominal concentrations and where it might be critical to have
measured values throughout the study. The U.S. EPA OCSPP Guideline 850.1000 (Draft;
U.S. EPA 1996) provides useful guidance on the design and conduct of aquatic studies with
difficult to test substances and should be considered when determining the acceptability of a
toxicity study. The solubility and stability of the test material should be known for the
conditions under which the test is being conducted to provide scientifically defensible
information. This representative analysis of the material should be conducted under the same
conditions as those used for the test. The limit of detection (LOD) and limit of quantification
(LOQ) for the chemical being analyzed should be identified. Incomplete dissolution of
materials with low water solubility, such as evidenced by the presence of precipitates or films
in or on the water could affect actual exposure levels, and the solubility of the chemical in
relation to the reported test concentrations should be considered when evaluating such a test
outcome. The use of aerated treatment units when testing a chemical that is volatile is likely
to overestimate exposure concentrations, and should be considered invalid, unless exposure
concentrations are regularly measured. For any chemicals where stability, solubility,
volatility, and/or sorption may be issues, chemical measurements at the study initiation and
termination are considered important and should be considered when determining the validity
of the test.
• Water quality. All relevant water quality parameters (e.g., dissolved oxygen, temperature,
pH) should be reported. Hardness, Dissolved Organic Carbon (DOC), and pH are crucial for
the evaluation of metals toxicity, particularly for those where bioavailability is affected by
these parameters. However, results of acute tests conducted in dilution water with total
organic carbon or particulate matter exceeding 5 mg/L should not be used, unless a
relationship between acute toxicity and organic carbon or particulate matter has been
established and/or data show that organic carbon, particulate matter, or similar substances do
not affect exposure/toxicity. In addition, biological loading rates should be suitable for the
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test container and not compromise water quality during the study. For water column tests,
mean dissolved oxygen concentrations should not drop below 60% saturation for prolonged
periods, unless justification is provided indicating the dissolved oxygen suppression did not
interfere with the test outcomes.
• Negative and solvent control performance. The concentration of organic solvent in test
solution should not exceed 0.1 mL/L for acute and/or chronic invertebrate studies or acute
fish flow-through studies; the solvent should not exceed 0.5 mL/L for acute fish static or
static-renewal studies (see the appropriate comparable U.S. EPA or ASTM study guideline).
OPP has developed guidance (U.S. EPA 2008) for aquatic studies with pesticides for
determining whether negative and solvent control performance is adequate. This guidance
should be considered when determining whether an aquatic toxicity study conducted with
high Kow organic compounds is valid.
• Endpoint selection. Measured toxicity outcomes should be representative and applicable to
the assessment endpoint being evaluated (e.g., risks to species, populations, communities).
For acute tests, these endpoints would typically include acute median effective concentration
(ECso) or median lethal concentration (LC50) values. For chronic studies, endpoints may
include NOEC and LOEC, and/or preferably ECx (e.g., EC 10 or EC20) values. Where toxicity
data are available for multiple life stages of the same species (e.g., eggs, juveniles, adults),
OW uses the data from the most sensitive life stage to develop AWQC. This helps to ensure
that a given species can survive an exposure during the most sensitive stages of its life cycle,
and thus maintain a viable population. Studies reporting sublethal endpoints other than those
traditionally used in AWQC development may be used qualitatively as additional lines of
evidence to support tests that report direct effects on survival, growth, and reproduction.
Sublethal effects can include hormonal, biochemical, cellular, osmoregulatory, and
behavioral measurement endpoints, among others. Some of these endpoints, such as
adaptational behaviors (e.g., predator avoidance, feeding behaviors), reproductive behaviors
(e.g., mating behavior, nest guarding behavior), and morphological effects are important to
the overall fitness of both the individual and the population. Consideration should be given to
whether there is a quantitative relationship between the observed sublethal effect and an
effect that is relevant to organism and/or population viability and is of regulatory concern
(e.g., survival, growth, reproduction). These types of endpoints (Kramer et al. 2011) may also
be linked via Adverse Outcome Pathways (AOP) for survival and reproduction to population-
level effects in aquatic animals (Ankley et al. 2010).
• Statistical methods used to analyze the test outcome. Verification of the statistical analysis
is an integral part of the data evaluation process. As such, studies should report the specific
measures of central tendency (e.g., means, medians) and dispersion (e.g., standard deviations,
standard errors) that were used, along with associated sample sizes (N values). The report
should state which methods of statistical comparison (e.g., t-test, ANOVA, chi square) were
used and the assumed data distribution (parametric versus nonparametric). Tests using
parametric statistics should indicate whether the conditions for such tests (i.e., normal
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distribution and homogeneity of variance) have been met. Specific statistical software used
should be identified.
• Information necessary to provide a complete and accurate evaluation of test outcomes.
Each report should include a summary of the data, a description of the statistical analysis of
the data, and a statement of conclusions drawn from the analysis that allows the reader to
independently evaluate the conclusions of the author. The availability of raw data is
particularly important when needed to recalculate an endpoint for a study that could
substantively affect the criteria value (e.g., studies indicating effects near the 5th percentile),
and efforts should be made to obtain the raw data from the study author if these data are not
reported in the study.
• Important information to determine study reliability. Inconsistencies or deviations with
recommended methodologies should be reported, as discussed in the applicable guidelines
(U.S. EPA guidelines, ASTM test methods, and/or OSCPP guidelines Standard Evaluation
Procedure [SEP]), for each of the respective studies. The U.S. EPA specific test guidelines
can provide additional measures to gauge the reliability of study conditions.
3.2.1 Studies Classified as Unacceptable
Open literature studies classified as unacceptable are those that are not considered sufficiently
scientifically rigorous for use in criteria derivation and do not provide useful and/or reliable
information. Studies classified as unacceptable can include those performed under conditions
that deviated so significantly from the recommended protocols that they bring into question the
validity of the toxicity test results.
Aquatic studies commonly placed in this category include those with improper test conditions
(e.g., static exposures with volatile chemicals in aerated test chambers), test vessels constructed
of materials other than glass coupled with a test substance that is expected to sorb to the test
chamber walls, excessive mortality of control animals, substantial amounts of missing test
information, a study where the test material was not properly identified, and/or environmental
conditions or results that cannot be readily interpreted from the information provided. A detailed
list of factors that could result in invalidation of open literature data is provided above in Section
3.2.
3.2.2 Studies for Quantitative and Qualitative Use
If a study is determined to be acceptable based on the guidelines described above, a
determination is made regarding whether the information provided in the study is adequate for
"quantitative" or " qualitative" use in AWQC derivation. For OW's purposes, 'quantitative'
means the data from the study can be used to derive a numeric AWQC. " Qualitative" refers to
data that are not adequate for the derivation of numeric aquatic life criteria, but that can be used
as additional lines/weight of evidence to support the effects assessment as described in the
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effects characterization.
As previously discussed, to be used quantitatively, the endpoint(s) reported in the open literature
should meet all the following general guidelines:
• The endpoint is reported in (or can be converted to) acceptable units (e.g., |ig/L)
• The endpoint reported can be used to derive AWQC (e.g., LC50 for acute exposure in
fish) for apical endpoints of concern: survival, growth and reproduction; and
• Sufficient information is provided in the study to substantiate or independently evaluate
whether the reported study conclusions (i.e., dose-response) and endpoints are accurate.
Depending on the measured endpoint, study evaluation criteria similar to those in OCSPP 850
Test Guidelines or ASTM methods, should be used to gauge the utility of the study. If a study
does not contain sufficient information to meet the key acceptance criteria including the general
guidelines summarized above in this SOP, the data from the study should be classified as
"qualitative."
OW recognizes that the third criterion of "sufficient information" listed above requires best
professional judgment. The most reliable means of determining whether study conclusions can
be verified is through accessing the raw data for a study; however, it is recognized that many
open literature papers, particularly older ones, may not provide this type of information.
Therefore, the quantitative use of open literature requires that the study provide a relatively
comprehensive description of the conditions under which the study was conducted, and the data
generated by the study. The study should report detailed measures of the variability associated
with the data and the methods used to analyze the data. Reviewers should note whether the
statistical tests used in the study are appropriate for the study design, the nature of the measured
endpoints, and the data generated by the study.
Where raw data are not available to verify the study endpoints, the reviewer should discuss the
uncertainties associated with quantitative use of the data. Consideration should be given to the
extent to which the measured test endpoints align with other lines of evidence.
4 Documentation of Open Literature Used in AWQC
Development
This section discusses the process of tracking and documenting studies obtained from the open
literature through the screening (Section 4.1) and reviewing process (Section 4.2), and how this
information is presented in AWQC documents (Section 4.3).
4.1 Documenting Study Screening Outcomes
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All studies identified through an ECOTOX search or other relevant searches should be
documented throughout the screening process. The files provided with the results of the
ECOTOX database search and screen can include the studies accepted by ECOTOX as well as
studies considered non-applicable with defined rejection reasons and "other" studies with
identification terms. These files should be retained to document which studies require further
review and which studies do not. The files should also be updated if studies are found from
sources other than the ECOTOX database and in cases when a study is accepted by ECOTOX
but not by OW.
4.2 Documenting Study Review Outcomes
Studies identified in the open literature (ECOTOX and other sources) that may provide data
relevant to criteria derivation should be reviewed and classified (i.e., quantitative, qualitative,
unacceptable), and the evaluation should be documented as described in this section.
Data Evaluation Record (DER) templates have been developed by OW to describe specific
recommendations for toxicity test aspects and to document review outcomes for studies
considered for use in AWQC development. The purpose of completing the DER for each study
as outlined below is to ensure a transparent, efficient, and consistent process for completing and
documenting reviews of studies and avoiding duplicative and possibly conflicting conclusions
associated with study reviews by different reviewers. The EPA is using an electronic format of
the DERs housed under the EPA's ECOTOX database. The information captured in the DER is
identical to those included below. In the future, the information captured in the DER could be
captured in another electronic format. DER templates for fish, aquatic and terrestrial
invertebrates, plants, amphibians, and avian species are included in Attachment E through
Attachment J.
Data Evaluation Records (or an equivalent documentation approach) should be completed:
• For open literature studies that pass the initial screening phase as described in
Section 2.
• For all studies with acceptable endpoints that are classified as 'quantitative,'
'qualitative,' or 'unacceptable' as described in Section 3.
o 'Unacceptable' studies receive an abbreviated DER review, as noted
below.
• By a primary and secondary reviewer.
The DERs are separated into three parts:
• Part A includes a general overview (citation, summary of any deficiencies, study
classification [quantitative, qualitative, unacceptable]), abstract, summary of
relevant endpoints, and results.
• Part B includes information on materials and methods.
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• Part C includes verification of calculations and statistical results (if raw data are
provided or obtained from the study author, and statistical re-analyses are
deemed useful or important).
The DERs should be saved with a file name that includes:
• Chemical (e.g., "Hg" for mercury)
• Taxonomic group (e.g., "invert" for invertebrate)
• Species name or common name (e.g., "C. dubia" or "Cladoceran"). If data for
more than one species are provided in the paper, then use term that best conveys
the information (e.g., "multiple inverts")
• Abbreviated citation beginning with year published followed by first author (e.g.,
year and first author [et al.])
• Reviewer's initials and date
For example, BaCl_Fish_ D. rerio _2016_Kwon et al._ CB_05-09-19 would be the file name
of the study review completed by "Catherine Brown" of:
Kwon, B., N. Ha, J. Jung, P.G. Kim, Y. Kho, K. Choi and K. Ji. 2016. Effects of barium
chloride exposure on hormones and genes of the hypothalamic-pituitary-gonad axis, and
reproduction of zebrafish (Danio rerio). Bull. Environ. Contam. Toxicol. 96(3): 341-346.
The procedures for completion of the DERs for endpoints that are classified as "quantitative",
"qualitative", or "unacceptable" are described below.
4.2.1 Completion of DERs for Endpoints Used Quantitatively
DERs of open literature data and other studies that are used quantitatively to derive numeric
criteria should be completed in their entirety (i.e., Parts A, B, and C). The DER should include:
the basic study requirements, such as test organism source/acclimation, use of solvent and
negative controls, control mortality rates (or other issues with controls that could affect the study
validity), number of test concentrations, number of treatments (e.g., 3 applications, 7-day
application interval), water quality parameters, and verification of suitable replication. The
review should also document all statistically or biologically significant effects. In addition, the
duration of exposure, the magnitude of the effect, and the test concentration (nominal, measured,
and time-weighted average, if it can be determined) at which the effect was observed should be
documented. Each documented endpoint should specify the affected taxa and/or individual
species. In addition, the reviewer should include relevant figures and tables from the study that
include key findings (e.g., a screenshot from the publication); table and figure captions should
properly cite the relevant publication if the figure and/or table is copied from the publication.
Statistical software and methods (e.g., R, TRAP, BMDS, with associated version number/date
identified) used to verify the reported study or test results and calculate point estimates should be
completed when possible and deemed necessary, and reported in Part C. This step may occur
separately in time, after Parts A and B have been completed. All open literature studies that are
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classified as 'quantitative' and used to derive criteria should undergo two levels of internal
review, including a primary review of the study, typically by the EPA contractor, and a
secondary review by staff typically within OW's Ecological Risk Assessment Branch (ERAB).
In the future the information captured in a DER could be captured in another electronic format.
4.2.2 Completion of DERs for Endpoints Used Qualitatively
DERs should be completed for open literature studies that include endpoints to be used
qualitatively in criteria development. To the extent possible, DERs for qualitative endpoints
should include the same type of information and level of detail as reviews that are completed for
quantitative endpoints, but only through Parts A and B. In addition, DERs for qualitative
endpoints should include a description of the study limitations which preclude its quantitative
use.
4.2.3 Completion of DERs for Unacceptable Open Literature Studies
For those open literature studies that are classified as "unacceptable," DERs should be
completed, however, the length and level of detail relative to "quantitative" and "qualitative"
reviews should be significantly reduced. The abbreviated DER for unacceptable studies should
consist of Part A only and focus on the limitations of the study which preclude its use in criteria
development. Detailed description of the experimental design is not required for studies that are
classified as "unacceptable," however, screenshots of figures and tables of relevant results should
be included.
4.3 Open Literature Review Documentation in A WQC Documents
Studies identified that may provide data relevant to criteria derivation and are reviewed and
classified as quantitative, qualitative, or unacceptable are listed as appendices in AWQC
documents as described below.
4.3.1 Documentation for Endpoints Used Quantitatively
Numeric AWQC development by OW is detailed in the Effects Analysis section of aquatic life
AWQC documents, along with the final acute and chronic criteria (with allowed durations and
frequencies). Numerous quantitative data are provided in this section, as is an overview of key
drivers of the criteria magnitude. The final main section of the aquatic life AWQC document, the
Effects Characterization, describes confidence and uncertainties in those studies and includes
relevant qualitative studies as other lines of evidence.
A table listing the studies used quantitatively can also be found in appendices labeled
"quantitative toxicity data" (previously referred to as "acceptable toxicity data" in aquatic life
AWQC developed prior to 2017). This table includes relevant study information including, but
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not limited to, the species tested, notes on the test method (e.g., static versus flow-through), test
material, water quality, acute or chronic effect value, and the study reference.
4.3.2 Documentation for Endpoints Used Qualitatively
Although endpoints from studies that are classified as 'qualitative' are not appropriate for
quantitative use (i.e., numeric AWQC derivation), they should be discussed in the Effects
Characterization section of the AWQC document as additional lines of evidence to support
conclusions. A clear rationale should be provided in an appendix. The Effects Characterization
section of the AWQC document should focus on how these studies provide supporting lines of
evidence and briefly reference why the endpoints were not used quantitatively. These reasons
might include test duration, limitations in the study design, lack of sufficient information to
substantiate the test results/conclusions, or other uncertainties that confound the ability to
discriminate a quantitative treatment-related effect. As previously stated, best professional
judgment should be used to determine the appropriate use of studies in criteria development.
A table listing the studies categorized as qualitative can also be found in appendices labeled
"qualitative toxicity data" (previously referred to as "other toxicity data"). This table includes
relevant study information including, but not limited to, the species tested, test material, test
duration, water quality, acute or chronic effect value, species mean acute or chronic value
(SMAV or SMCV), the study reference, and notes on the reason it is categorized for qualitative
use.
4.3.3 Documentation for Unacceptable Endpoints
To create and maintain a record of all studies reviewed and considered in criteria development, a
table listing the studies determined as unacceptable for use is also included in appendices labeled
"unacceptable toxicity data" (previously referred to as "unused toxicity data") in each AWQC
document. This table includes the citation and rationale for considering the study unacceptable
for criteria development.
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5 References
Ankley, G.T., R.S. Bennett, R.J. Erickson, D.J. Hoff, M.W. Hornung, R.D. Johnson, D.R.
Mount, J.W. Nichols, C.L. Russom, P.K. Schmieder, J.A. Serrrano, J.E. Tietge andD.L.
Villeneuve. 2010. Adverse outcome pathways: A conceptual framework to support
ecotoxicology research and risk assessment. Environ. Toxicol. Chem. 29(3): 730-741.
European Commission. 2011. Common implementation strategy for the Water Framework
Directive (2000/60/EC), Guidance Document No. 27, Technical Report - 2011 - 055.
Brussels, Belgium.
Klimisch, H.-J., M. Andreae and U. Tillmann. 1997. A Systematic Approach for Evaluating the
Quality of Experimental Toxicological and Ecotoxicological Data. Reg. Toxicol. Chem.
25: 1-5.
Kramer, V.J., M.A. Etterson, M. Hecker, C.A. Murphy, G. Roesijadi, D.J. Spade, J.A.
Spromberg, M. Wang and G.T. Ankley. 2011. Adverse outcome pathways and ecological
risk assessment: Bridging to population-level effects. Environ. Toxicol. Chem. 30: 64-
76.
Olker, J. H., Elonen, C. M., Pilli, A., Anderson, A., Kinziger, B., Erickson, S., Skopinski, M.,
Pomplun, A., LaLone, C. A., Russom, C. L., & Hoff, D. 2022. The ECOTOXicology
Knowledgebase: A Curated Database of Ecologically Relevant Toxicity Tests to Support
Environmental Research and Risk Assessment. Environmental Toxicology and
Chemistry. 41(6), 1520-1539. https://doi.org/10.1002/etc.5324
U.S. EPA. 1985. Guidelines for Deriving Numerical Water Quality Criteria for Protection of
Aquatic Organisms and Their Uses. U.S. Environmental Protection Agency. Office of
Research and Development. Environmental Research Laboratories. PB85-227049.
U.S. EPA. 1987. Manual of instructions for preparing aquatic life water quality criteria
documents. U.S. EPA, Environmental Research Laboratory. Duluth, MN.
U.S. EPA. 1996. Ecological Effects Test Guidelines. OPPTS 850.1000. Special Considerations
for Conducting Aquatic Laboratory Studies. Office of Prevention, Pesticides and Toxic
Substances. EPA 712-C-96-113. https://www.regulations.gov/document/EPA-HQ-OPPT-
2014-0908-0091
U.S. EPA. 2008. Memorandum on Guidance for Use of Dilution-Water (Negative) and Solvent
Controls in Statistical Data Analysis for Guideline Aquatic Toxicity Studies. From Mary
Frankenberry, Carolyn Hammer, and Keith Sappington to Donald Brady (EFED Division
Director). September 25, 2008.
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U.S. EPA. 2011. Evaluation Guidelines for Ecological Toxicity Data in the Open Literature:
Procedures for Screening, Reviewing, and Using Published Open Literature Toxicity
Data in Ecological Risk Assessments. Office of Pesticide Programs. May 9, 2011.
U.S. EPA. 2013. Aquatic Life Ambient Water Quality Criteria for Ammonia - Freshwater. Office
of Water. Washington, DC. EPA-822-R-13-001.
U.S. EPA. 2022. ECOTOX ECOTOXicology Knowledgebase System User Guide - Version 5.5.
August 2022.
U.S. EPA. 2023. ECOTOX ECOTOXicology Knowledgebase System SOP: ECOTOX Literature
Searches, Citation Identification and Applicability Criteria. September 2023.
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Attachment A 1985 Guidelines Minimum Data Requirements
for Acute and Chronic AWQC
• The acute freshwater toxicity testing requirement is fulfilled with the following eight
minimum data requirements:
o the family Salmonidae in the class Osteichthyes;
o a second family in the class Osteichthyes, preferably a commercially or
recreationally important warmwater species (e.g., bluegill, channel catfish);
o a third family in the phylum Chordata (may be in the class Osteichthyes or may
be an amphibian, etc.);
o a planktonic crustacean (e.g., cladoceran, copepod);
o a benthic crustacean (e.g., ostracod, isopod, amphipod, crayfish);
o an insect (e.g., mayfly, dragonfly, damselfly, stonefly, caddisfly, mosquito,
midge);
o a family in a phylum other than Arthropoda or Chordata (e.g., Rotifera, Annelida,
Mollusca); and
o a family in any order of insect or any phylum not already represented.
• The acute estuarine/marine requirement is fulfilled with the following eight minimum
data requirements:
o two families in the phylum Chordata;
o a family in a phylum other than Arthropoda or Chordata;
o either the Mysidae or Penaeidae family;
o three other families not in the phylum Chordata (may include Mysidae or
Penaeidae, whichever was not used above); and
o any other family.
• Chronic toxicity test data (longer-term survival, growth, or reproduction) are required
for a minimum of three taxa, with at least one chronic test being from an acutely-
sensitive species. Acute-chronic ratios can be calculated with data from species of
aquatic animals from at least three different families if the following data requirements
are met:
o at least one is a fish;
o at least one is an invertebrate; and
o for freshwater chronic criterion: at least one is an acutely sensitive freshwater
species (the other two may be estuarine/marine species) or for estuarine/marine
chronic criterion: at least one is an acutely sensitive estuarine/marine species (the
other two may be freshwater species).
• At least one acceptable test with a freshwater alga or vascular plant is required. If plants
are among the aquatic organisms most sensitive to the material, results of a plant in
another phylum should also be available.
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• At least one acceptable bioconcentration factor determined with an appropriate
freshwater species is required.
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Attachment B Acceptability Criteria for Aquatic Effects Data
ECOTOX Reference No.:
General instructions: If more than one experimental design is used in the study, multiple
Literature Acceptance Criteria Checklist forms may be required, but the acceptability of the paper
is based on at least one experimental design meeting all the Acceptability Criteria.
No.
Criteria / Instructions
Yes / No
1
The paper reports effects associated with a single chemical exposure.
However, OWwill consider studies examining additivity, synergism, or
antagonism of two or more chemicals where pertinent to the derivation of an
aquatic life criterion. Effluents, leachates, drilling muds, fly ashes, natural-
sediments, and sludges are not considered single chemicals. In addition, the
single chemical cannot be introduced as a component of an effluent, etc.
Formulated products, such as emulsifiable concentrates and wettable powders
while considered single chemicals, may not be used for quantitative criteria
derivation unless this is the only material that has valid data. The data can be
used qualitatively in the effects characterization section.
2
The paper reports a biological effect on live, whole organisms or in vitro
preparation.
The authors clearly identify an observed effect response related to the exposure
ofa live organism to the chemical of concern. In vitro studies may be considered
in the criteria derivation process. "Positive" effects (e.g., increased
reproduction) will be recorded and considered in the effects characterization
section.
3
The paper reports a concurrent environmental chemical concentration/dose or
application rate.
Authors clearly report a concentration/dose or application rate associated with
the observed effect response. If the study results are only available in a
graphical format, add a comment to the remarks field.
4
The paper reports an explicit duration of exposure.
Authors must explicitly report the duration of the exposure related to the
observed effect.
Durations can include qualitative terms (e.g., at hatch, at harvest).
5
The paper reports toxicology information for the pollutant of concern to
OST/OW.
6
The article is published in the English language.
The full article is published in the English language. Translations will not be
conducted.
7
The study is presented as a full article.
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No.
Criteria / Instructions
Yes / No
Abstracts from journal publications where the full article is published in non-
English, the abstract is published in English and conference proceedings
published as brief abstracts will not be considered.
8
The paper is a publicly available document.
Publications that are not publicly available (e.g., internal memoranda,
government reports not readily available from NTIS) may be considered.
However, the documents must be made available to the public via the Federal
docket when the assessment goes out in the Federal Register. In addition,
certain key studies that may not be in press may be used if the reviewer is
provided the opportunity to conduct an internal/external review and the
author's permission to publicly disclose the information. If a registrant-
sponsored study for a pesticide, all information needed for criteria derivation
would need to be made available to OW through a data evaluation record (DER)
or similar vehicle.
9
The paper is the primary source of the data.
A document is considered a primary source if at least one of the investigators
who conducted the toxicity test is an author, and the authors do not cite another
publication as the original source of the data.
10
The paper reports a calculated endpoint.
For the purposes of this evaluation, an endpoint is defined as the quantification
of an observed effect obtained through statistics or other means of calculation
for the expressed purpose of comparing equivalent effects (e.g., IC50, BCF,
NOEC). If within a single experiment, the authors report the same endpoint at
multiple durations, the duration most relevant to the OW's standard acceptable
test durations for acute and chronic studies (e.g., 96-hr IC/ECsos will be used
and note the other endpoints in the remarks field). If NOEC and IOEC
endpoints are not explicitly reported by the authors, reviewers should interpret
endpoints based on levels of significance reported in the paper. If more than
one measurement (e.g., juveniles per litter, juveniles per females) is observed
for a particular effect (e.g., reproduction), then only the most sensitive
measurement will be used, and the remaining measurements are noted in the
remarks field.
11
The paper reports that treatment(s) were compared to an acceptable control.
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No.
Criteria / Instructions
Yes / No
The control treatment must be comparable to the other treatments and must be
free of the chemical stressor. Appropriate controls include: baseline or
background control - parameters of actual or representative test species
measured before and after administration of test chemical though not as part
of the same test scenario; negative control - organisms maintained under
conditions identical to exposed organisms except for the absence of the test
substance; positive controls - organisms maintained under conditions identical
to the exposed organisms except the test substance is replaced with a
substance known to elicit a consistent toxic response; and solvent controls -
organisms exposed to carrier or solvent that is used as a vehicle for
administrating the test substance to exposed organisms.
The number of treatments (other than controls) should be reported in the data
summan> table.
12
The paper reports the location of the study (e.g., laboratory vs. field).
Authors clearly state the locations of the study, either in a controlled laboratory
setting or in the field. Field studies are not typically used in a quantitative
manner in the criteria derivation process, but may be used qualitatively to
support quantitative laboratory studies. Field studies can include natural or
artificial settings (e.g.. microcosms, mesocosms).
13
The paper reports the species that was tested; and this species can be verified
The authors clearly identify the test species and the organism's scientific name
can be verified in a reliable reference. The preferred scientific name should be
reported. If a specific genus/species is not reported, reviewers report the species
information at the lowest taxonomic level.
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U.S. EPA OW
Attachment C ECOTOX Exclusion Reasons
General Instructions: The following is a list of ECOTOX exclusion keywords and definitions utilized under the
ECOTOX database efforts.
Exclusion keywords
Description
ABSTRACT
Study published as an abstract only.
ARCHIVED CHEMICAL
When all chemical name(s) in a publication are unable to be verified. Also used
for individual chemicals on the second page of the screening module.
BACTERIA
Bacteria; includes microbes and Microtox tests.
Only use when COC is affecting/effecting bacteria.
*If bacteria is creating the effect, use NO TOXICANT.
BENEFICIAL EFFECT
Study reports only a positive effect (improving the health of the organism). Also
used for individual chemicals on the second page of the screening module.
BIOLOGICAL
TOXICANT
Biological toxicants including venoms, fungal toxins, and plant, animal or
microbial extracts or toxins not purified.
This is used only when the toxicant (COC) is in a biological toxicant form. For
example, if acetylsalicylic acid is derived from birch bark rather than prepared in
lab.
*This is rarelv used in TIAB.
CHEM METHODS
The description of chemical analysis procedures and measurements in a
laboratory setting. No organism effects are reported in the paper.
EFFICACY
A secondary positive benefit to one organism; for example insect species are
reduced and agricultural yield is improved.
FATE
Chemical distribution in natural media (water, soil, air, and tissue if no
biological effect).
HUMAN HEALTH
Studies with human subjects or with surrogate animal subjects. Also includes
human or human surrogate species DNA injected into non-human cells and
studies on food.
INCIDENT
Reports of accidental or intended animal deaths by exposure to a toxicant or
poison; not a controlled experiment.
METHODS
Publication only provides documentation for toxicology test methods,
experimental design, statistical methods, standard terminology, recently
developed test methods.
*must include COC
MIXTURE
No single chemical effects reported. This includes ambient toxicants in lakes,
rivers, soil, air and co-exposure with other chemicals, including microplastics.
Howevenn 'Chemical 1' mixed with 'Chemical 2' based on abstract alone is not
sufficient enough to exclude as there may be single exposures as well within the
full test.
*Not commonly used in TIABing, except for 'effluent,' indication an unknown
mixture.
MODELING
Modeling or QSAR papers.
*must include COC
NO CONC
Not including search chemical, no usable dose or concentration reported after
examination of the entire paper; includes lead shot studies lacking dose
information (i.e., report only the number of pellets) as well as endpoint
concentrations reported in log units only.
NO DURATION
No duration reported (entire publication examined).
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U.S. EPA OW
Exclusion keywords
Description
NO TOXICANT
No chemical toxicant added as stressor; ambient air components not included in
ECOTOX.
- ambient air component chemicals (ozone, C02, S02) and pollution - ambient
conditions, including radioactivity, ultraviolet light (UV), temperature, pH,
salinity, dissolved oxygen (DO), or other water, air or soil parameters
NUTRIENT
In situ chemical of interest used as nutrient.
PEST MANUAL
More than one insect species is tested; and some are in the PEST group and
some are not; Unify does not make this distinction. Used with a second
inclusion/exclusion keyword, e.g., OK.
REVIEW
All toxicity tests reported in other primary publications; REVIEW bibliography
may be Full Text Screened to identify relevant citations
SEDIMENT CONC
Chemical concentration reported in sediment only (if pore or overlying water
concentrations reported, then applicable).
SURVEY
Effects observed in field collected organisms and/or brought to laboratory for
residue measurement.
TARGET MANUAL
Used when more than one species group is Full Text Screened and different
categories are needed, e.g., and one is a target species and the other has
beneficial effect associated with the chemical application. For example, a plant is
tested (with positive/efficacy effects) and the insect is a target species. Often
accompanied by the exclusion keyword EFFICACY.
VIRUS
Virus used as a test organism.
*If virus is causing the effect, use NO TOXICANT
YEAST
Yeast used as test organism.
WEEDS MANUAL
Used when more than one plant species is attached to the paper and there is only
endpoint data for the weed species. For example, a pine tree has no endpoint
data, but knapweed has endpoint data.
ADDENDUM
[Bibliographic]
Publication is a supplement to another publication. The Addendum citation is
cross-referenced to the original publication and the PDFs are merged (erratum or
addendum). Erratums and Addendums are ordered and attached to the back of
the corresponding publication.
DATASET*
[Bibliographicl
Author linked repository dataset.*
DUPLICATE
[Bibliographicl
Publication duplicated in different journal or source.
ECOCHEM
VERIFICATION
SOURCE
[Bibliographicl
Publication used to verify chemical CAS or physical/chemical properties.
NO SOURCE
[Bibliographic]
Source of publication undetermined, publication unavailable; order status
ARCHIVE (includes internal chemical company document and personal
communication citations).
NON-ENGLISH
[Bibliographicl
Paper's full text language other than English
PUBL AS [Bibliographic]
Entire study was published in another source; only data from one source is
abstracted. The second source is linked by exclusion keyword.
REFS CHECKED
[Bibliographicl
References in a REVIEW have been checked.
RETRACTED
[Bibliographicl
Retracted article from publication by journal.
SCREENED
[Bibliographicl
Identifies a book or journal where applicability criteria have been applied to all
chapters or publications.
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U.S. EPA OW
Exclusion keywords
Description
SPECIES
VERIFICATION
SOURCE
[Bibliographic!
Publication used to verify species.
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Attachment D
Instructions for Completing Data Evaluation Records (DERs) for Toxicity Studies in the
Open Literature
Attachment D Instructions for Completing Data Evaluation Records
(DERs) for Toxicity Studies in the Open Literature
(September 2024)
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Attachment D
Instructions for Completing Data Evaluation Records (DERs) for Toxicity Studies in the
Open Literature
The purpose of Attachment D is to provide instructions for completing DERs while reviewing toxicity
studies from the open literature and to ensure that DERs are completed in a consistent manner across
reviewers.
All DERs should be:
• Completed in their entirety (except for noted exemptions) following the instructions provided
below
o Do not leave sections/tables blank unless noted otherwise. Use not applicable (NA), not
calculable, not provided, not verified, or not measured as appropriate
• Completed for each chemical and species combination (i.e.. if a study focused on 2 separate
chemicals and 2 separate species, 4 individual DERs should be completed for the one study)
• Saved with a file name that follows:
o DER_ Chemical_Taxa_ Abbreviated Citation (First author [et al.])_ Year of Publication_ Species
Name _ Reviewers Initials - Example: DERBariumFishKwon et al._2016_D. rerio CB
Part A: Overview Complete an abbreviated DER of Part A only for studies marked "Not Acceptable for
Use"
I. Test Information
Chemical Information: Complete information as provided.
Test Type: Place Xby one classification. These test type classifications should be applied when
completing Part B: Detailed Review
¦ Controlled Experiments are defined here as studies where the chemical exposure and test
community is manipulated (e.g.. laboratory, microcosm, and mesocosm tests regardless if
conducted indoors or outdoors)
¦ Field Study/Observations are defined here as studies where the either the chemical exposure
and/or test community is not manipulated and the exposure observations occur in a natural
waterbody (e.g., observed effects of a chemical at a contaminated stream, lake, pond, and in-situ
stream or a whole lake experiments)
Reviewer Information: Complete reviewer information. Primary and secondary reviewer should
be designated by the EPA project lead and will typically consist of a contractor as the primary
reviewer and EPA staff as the secondary reviewer.
Citation: Complete citation as noted in SOP and on DER, being sure to indicate author(s), year,
study title, journal, volume and pages.
Companion Papers: Provide a list of any companion papers, including other publications, reports,
or theses associated with the current publication being reviewed using the same citation format
noted in the SOP and on the DER. If companion papers are listed, identify if separate DERs were
completed for companion papers and list file names for each of the DERs.
¦ Companion papers include separate publications reporting other aspects of the experimental design
and/or results or other papers used in the development of the experimental design of the publication
being reviewed
EPA OWDER INSTRUCTIONS
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Attachment D
Instructions for Completing Data Evaluation Records (DERs) for Toxicity Studies in the
Open Literature
Study Classification for Aquatic Life Criteria Development: Place Xby one classification based
on the study's highest use (e.g., mark "Acceptable for Quantitative Use " only if a study has both
Quantitative and Qualitative Use endpoints).
¦ This overall Study Classification for Aquatic Life Criteria Development should take the Study
Design/Methods Classification (in Materials and Methods section of Part B) and Response-Curve
Classification (in Statistical Verification of Results section of Part C) into consideration.
¦ Provide any necessary details related to the study's use classification for all pertinent endpoints,
including non-apical endpoints (i.e., differences in the study's use classification for certain
endpoints).
Major Deficiencies: Check all that apply, paying attention to any noted exceptions. Checking any
of these items may make the study "Not Acceptable for Use". If occurrence of mixtures is identified
as a major deficiency, describe potential chemical mixtures in areas provided. Identify any other
notable concerns that may classify the study as "Not Acceptable for Use" under General Notes.
Minor Deficiencies: List and describe any minor deficiencies or other concerns with test. Listing
any items in this section may make the study "Acceptable for Qualitative Use" (exceptions apply to
field studies as noted on the DER) and listing one (particularly chemical solubility issues.
anomalous or inconsistent results, previous or variable exposure, dosing via gavagc) or several
items may make the study "Not Acceptable for Use".
The EPA project lead will typically make this distinction in use classification. A study may be
considered "Acceptable for Quantitative Use " even if one or more minor deficiencies are identified
and the study results are consistent with similar studies focused on a related taxon (i.e.. up to a
family level) and measured the similar endpoints. For field studies/observations check mixtures if
observed effects are not justifiably contributed to single chemical exposure and uncharacterized
reference sites/conditions if appropriate. If either of these are checked, that may make the study
"Not Acceptable for Use". Describe potential chemical mixtures present at the site and exposure
variability across the study site(s). Identify any other notable concerns that may classify the study
as "Acceptable for Qualitative Use" or "Not Acceptable for Use" under General Notes.
* Minor Deficiencies may include: analytical or chemical solubility issues, problems encountered
with the test organisms or treatments (minor variability in concentrations, loss of replicate(s),
small sample sizes, only one test concentrations), description of dilution water not provided (e.g.,
uncharacterized stream water or potential presence of unknown containments, high organic
content, extreme hardness, pH), too few exposure concentrations, anomalous or inconsistent
results, unmeasured test concentrations, previous or variable exposure, dosing via gavage,
insufficient details regarding methods or analyses.
¦ For field studies/observations: Only publications with a range of exposure concentrations (i.e.,
study design incorporates both low and high exposure concentrations) and those where observed
effects are justifiably contributed to a single chemical exposure (within a mixture of
concentrations) should be considered "Acceptable for Quantitative Use."
Reviewer's Comments: Add comments not captured elsewhere on DER, including pertinent
information for drafting study summaries for the Effects Analysis and/or Effects Characterization
sections of the AWQC.
Abstract: Copy and paste abstract from publication.
Summary Tables: Complete acute and chronic tables for "Acceptable for Quantitative Use" and
"Acceptable for Qualitative Use" studies with information on the most sensitive apical and/or non-
EPA OWDER INSTRUCTIONS
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Attachment D
Instructions for Completing Data Evaluation Records (DERs) for Toxicity Studies in the
Open Literature
apical endpoint measured (i.e.. those usually captured in the data appendices/tables of the criteria
document). Modify tables as needed under the direction of the EPA project lead. DO NOT
complete tables for studies classified as "Not Acceptable for Use. "
* To fill in Reported Effect Concentration see Part A Section II: Results below.
¦ To fill in Verified Effect Concentration see Part C: Statistical Verification below. If Statistical
verification not preformed (i.e., paper marked "Not Acceptable for Use"), indicate not verified.
II. Results: Provide results as reported in the publication (including supplemental materials). Add
pertinent information for drafting study summaries that is otherwise not captured in the result
section of the DER under General Notes for the relevant section. Complete each results section as
follows:
• For all studies, paste screen shots of tables and/or figures reporting results from the article,
including those tables and/or figures found in the supporting materials, in the respective
subsections and after the associated pre-tabulated results table. If a particular result (i. e..
mortality, growth, reproduction, and/or sublethal effects) was not part of the study design note
this under the General Notes area of the associated results section.
• For studies marked "Acceptable for Quantitative Use" or "Acceptable for Qualitative Use"
complete all pre-tabulated tables (including any needed modifications to supplied tables) as
follows:
¦ The table headers, particularly the supplied entries in brackets (Example: Mean percent mortality [or
number of immobilized] of [test organism] exposed to [test substance] for [test duration])
* The number and titles of treatments, including controls (including negative and solvent controls if
any) by adding rows as needed and changing the treatment name in brackets to a consistent
nomenclature in publication (e.g., categorical names used by study authors (low, medium, high),
nominal, or measured concentrations). Ensure all treatment group labels are consistent throughout
the results section.
¦ The observed effect(s) in Tables A.II.3 through Table A.II.6 to be consistent with the observed
effects reported in the study (e.g., mean percent mortality, growth, or reproductive effects). Common
observed effects are supplied in each pre-tabulated table. Include units when appropriate. Add
columns for additional effects (and standard deviation or standard error), if needed.
¦ Edit Standard Deviation or Standard Error Column headers based on which is reported.
¦ Copy and paste additional reproductive effects tables for each generation of a multi-generational
study (remembering to label each generation) and the sublethal effect table for each observed
sublethal effect.
¦ Identify the values that are reported to be significantly different from control with a superscript.
¦ Provide the toxicity values (e.g., LCX, ECX, NOEC and LOEC) identified in the study, whether stated
by the study authors or not. Edit toxicity values (e.g., LCX and ECX) provided in brackets as needed.
If values for LC50, LT50, NOEC are greater than the highest treatment concentration, use > symbol.
Water Quality Parameters: Summarize water quality parameters measurements made in test
solutions. If only general summary data of water quality parameters are provided by study authors
(i.e.. specific details of water quality parameters on a treatment level is not provided), summarize
any information regarding water quality parameters under General Notes.
EPA OWDER INSTRUCTIONS
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Attachment D
Instructions for Completing Data Evaluation Records (DERs) for Toxicity Studies in the
Open Literature
Chemical Concentrations: Summarize the concentration verification data from the test
solutions/media and discuss the acceptability for deriving AWQC under General Notes. Expand
table to include measured concentration data for each media type (i.e., muscle, liver, blood, etc.).
Mortality Effects: Summarize mortality results (if any). Comment on concentration response
relationship and slope of response if provided under General Notes.
Growth Effects: Summarize growth results (if any). Comment on concentration response
relationship and slope of response if provided under General Notes.
Reproductive Effects: Summarize reproductive endpoint results (if any). For multi-generational
studies, copy and paste Table A.II.5 for each generation with reproductive endpoint data. Comment
on concentration response relationship and slope of response, if provided, under General Notes.
Other Sublethal Toxicity Effects: Summarize any other reported sublethal effect(s), including
behavioral abnormalities or other signs of toxicity. Copy Table A.II.6 for each additional sublethal
effect observed. Comment on concentration response relationship and slope of response if provided
under General Notes.
Reported Statistics: Briefly summarize statistical analysis conducted by study authors or copy and
paste statistical section from article.
Part B: Detailed Review Do not complete for studies marked "Not Acceptable for Use"
I. Materials and Methods: In text citations for test guidance and recommendations provided brackets.
Protocol/Guidance Followed: Indicate Protocol/Guidance (e.g., U.S. EPA, ASTM, Environment
Canada, European Union) followed if identified by study authors, otherwise complete with relative
information (e.g., not provided).
Deviation from Protocol/Guidance: Indicate deviations from protocol/guidance as described by
study authors, otherwise complete with relative information (e.g., not provided).
Study Design and Methods: Briefly describe the experimental design or copy and paste related
information from appropriate section of the article.
Test Organism Matrix: Complete for both Controlled Experiments and Field
Studies/Observations. Complete information under Details column as noted in the study and add
any pertinent notes under the Remarks column.
Study Parameters Matrix: Complete for both Controlled Experiments and Field
Studies/Observations. Complete designated information in the Details column, paying particular
attention to any relevant guidance information in the bulleted lists under the Parameter column.
Summarize any pertinent information or deficiencies in the Remarks column.
Controlled Experiment Study Parameters Matrix: Complete designated information in the
Details column, paying particular attention to any relevant guidance information in the bulleted lists
under the Parameter column. Summarize any pertinent information or deficiencies in the Remarks
column. Complete this Controlled Experiment Study Parameters Matrix for Controlled Experiments
only. Leave blank for field studies/observations.
EPA OWDER INSTRUCTIONS
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Attachment D
Instructions for Completing Data Evaluation Records (DERs) for Toxicity Studies in the
Open Literature
Study Design/Methods Classification: Place Xby one classification. Provide details of major and
minor deficiencies/concerns with study design under the associated sections of Part A of the DER
paying attention to designations of study use classification noted on the DER (/'. e., items indicated
under the Major Deficiencies section classify the study as "Not Acceptable for Use" and items
indicated under the Minor Deficiencies section may make the study classification as "Acceptable
for Qualitative Use"). This study design/methods classification should be taken into consideration
for the overall study classification for aquatic life criteria development in Part A (e.g., if the study
design classification is "Not Acceptable for Use" the study classification in Part A should be
consistent and not'Acceptable for Quantitative Use").
Additional Notes: Provide additional considerations related to the study design/methods, including
details of particular study design parameters that may influence the use of measured study results or
treatment groups.
II. Observations
Observations Matrix: Complete designated information in the Details column, paying particular
attention to any relevant guidance information in the bulleted lists under the Parameter column.
Summarize any relevant information or deficiencies in the Remarks column. Complete this
Observations Matrix for both Controlled Experiments and Field Observations. This information
should be consistent with the Results section in Part A.
Available Concentration-Response Data: Answer all questions related to data availability. The
EPA project lead should stipulate who is responsible for contacting study authors and will identify
the software that should be used to estimate concentration-response data from graphs.
Part C. Statistical Verification of Results Complete for all studies marked "Acceptable for Quantitative
Use" and only for the five most sensitive genera and sensitive apical endpoint. If multiple sensitive apical
endpoints were measured copy Sections I and II of Part C for each endpoint as needed. Completion of
Part C should be designated by the EPA project lead.
I. Statistical Verification Information: Complete all information as provided.
Statistical Reviewer Information: Complete reviewer information. Primary and secondary reviewer
should be designated by the EPA project lead and will typically consist of a secondary reviewer for
studies marked for "Quantitative Use" and that are used to derive the criteria (e.g., five most sensitive
genera).
Endpoint(s) Verified: List all endpoints verified. Verification of endpoints should be designated by the
EPA project lead and will typically focus on apical endpoints only.
Additional Calculated Endpoint(s): List all endpoints calculated. Statistical verification of additional
calculated endpoints should be designated by the EPA project lead.
Statistical Method: Report statistical methods (e.g., R, EPA TRAP, BMDS or other statistical packages)
used to verify the test results and/or those used to calculate toxicity value point estimates, including for
tests where toxicity values were not provided.
EPA OWDER INSTRUCTIONS
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Attachment D
Instructions for Completing Data Evaluation Records (DERs) for Toxicity Studies in the
Open Literature
II. Toxicity Values: Provide the statistically verified toxicity values identified on the DER. If the values
for the LC50, LT50, and/or NOEC are greater than the highest test concentration, use the " > " symbol.
Include confidence intervals if applicable.
Response-Curve Classification: Place Xby one classification. This response-curve classification
should be taken into consideration for the overall study classification for aquatic life criteria
development in Part A (e.g., if the response-curve classification is "Not Acceptable for Use" the
study classification in Part A should be consistent and not "Acceptable for Quantitative Use"). The
response-curve classifications should be based on model performance as follows:
• Acceptable for Deriving Criterion - Model performs well on all statistical metrics.
• Acceptable for Supporting Information - Model presents some metrics that may call estimates into
question.
• Not Acceptable for Deriving Criterion - Model does not perform well to fit data and should not be
used.
Summary of Statistical Verification: Provide summary of methods used in statistical verification,
including pertinent information for drafting study summaries.
Additional Notes: Add notes related to the statistical verification not captured elsewhere in Part C,
including pertinent information for drafting study summaries.
Attachments: Provide the attachments listed below as follows:
• Concentration-Response Data: Provide attachments to ensure that all data used in Part C is
captured. This includes:
¦ Study results reported in the publication (including supplemental materials), which are
captured in Results section of Part A of DER above.
¦ Additional data requested and provided by study authors, which are captured in Table C.II. 1
below and include the original correspondence with study authors as an attachment to the
DER.
• Model Assessment: Include all model figures and tables associated with the statistical
verification.
• Statistical Code: Provide statistical code used to fit dose-response curve. This code can be saved
in one file and referenced by file name in DER.
Additional Data Used in Response-Curve: Provide all data used to fit response-curve not already
captured in the Results section of Part A of the DER in Table C.II.l. This data would typically involve
replicate and/or raw data provided by study authors. Add rows as needed. First row in italicized text is an
example. Edit pre-tabulated information designated by brackets and/or add columns for additional
parameters (e.g., non-detect concentrations, DOC, pH, hardness) to be consistent with the data used to fit
the dose-response curve. Note: It should be anticipated that this data table will be copied and pasted into
broader database with all studies and endpoints used in the criterion derivation.
EPA OWDER INSTRUCTIONS
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Attachment E Fish Data Evaluation Record (DER) Template
(September 2024)
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Data Evaluation Record on the Effects of [Chemical] on Fish [Species]
Part A: Overview
I. Test Information
Chemical name:
CAS name: CAS Number:
Purity: Storage conditions:
Solubility in Water (units):
Controlled Experiment Field Study/Observation {Place X by One)
(imanipulated) {not manipulated)
Primary Reviewer: Date: EPA Contractor {PlaceXby One)
Secondary Reviewer: Date: EPA Contractor {PlaceXby One)
{At least one reviewer should be from EPA for sensitive taxa)
Citation: Indicate: author(s), year, study title, journal, volume, and pages.
(e.g., Slonim, A.R. 1973. Acute toxicity of beryllium sulfate to the common guppy. J. Wat. Pollut. Contr. Fed. 45(10): 2110-2122)
Companion Papers: Identify any companion papers associated with this paper using the citation format above.
{Ifyes, list file names of
Were other DERs completed for Companion Papers? Yes No DERs below)
Study Classification for Aquatic Life Criteria Development: Place X by One Based on Highest Use
Acceptable for Quantitative Use
Acceptable for Qualitative Use
Not Acceptable for Use/Unused
General Notes: Provide any necessary details regarding the study's use classification for all pertinent endpoints,
including non-apical endpoints within the study (e.g., note all study classifications for each endpoint if the use varies)
Major Deficiencies (note any stated exclusions): Check all that apply. Checking any of these items make the study "Not
Acceptable for Use"
.. . ^ , No Controls (for controlled experiments
Mixture (for controlled experiments only) only)
Excessive Control Mortality (> 10% for acute and > 20% for chronic)
Bioaccumulation: steady state not reached
Dermal or Injection Exposure Pathway
Review paper or previously published without modification
Other: (if any list here, e.g., use of distilled water)
POTENTIAL CHEMICAL MIXTURES: Describe any potential chemicals mixtures as characterized by study authors
(including any confirmation of chemical mixtures).
EPA OWDER INSTRUCTIONS
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Data Evaluation Record on the Effects of [Chemical] on Fish [Species]
General Notes:
Minor Deficiencies: List and describe any minor deficiencies or other concerns with test. These items may make the study
"Acceptablefor Qualitative Use" (exceptions may apply as noted)
DESCRIPTION OF UNMEASURED TEST CONCENTRATIONS: Describe concerns with unmeasured test
concentrations and the influence of the study classification.
DESCRIPTION OF CONCERNS WITH DILUTION WATER: Describe concerns with characterization of and/or
deficiencies with dilution water (e.g., uncharacterized stream or lake water, potential presence of unknown containments,
high organic content, extreme hardness, pH, etc).
For Field Studies/Observations: A field study/observation may be considered "Acceptable for Quantitative Use" if it
consisted of a range of exposure concentrations and the observed effects are justifiably contributed to a single chemical
exposure
Mixture (observed effects not justifiably contributed to single chemical exposure)
Uncharacterized Reference Sites/Conditions
POTENTIAL CHEMICAL MIXTURES PRESENT AT SITE: Describe any potential chemicals mixtures present at
the site as characterized by study authors (including any confirmation of chemicals present at study site).
EXPOSURE VARIABILITY ACROSS STUDY SITE(S): Describe any exposure variability across study site(s) as
characterized by study authors (i.e., description of study design with reference and contaminated sites).
General Notes:
Reviewer's Comments: Provide additional comments that do not appear under other sections of the DER.
EPA OWDER INSTRUCTIONS
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Data Evaluation Record on the Effects of [Chemical] on Fish [Species]
ABSTRACT: Copy and paste abstract from publication.
SUMMARY: Fill out for the most sensitive endpoint (apical and/or non-apical) and modify as needed. If study is classified as "Not Acceptable for Use " DO
NOT complete summary tables.
Acute:
Species (lifestage)
Method"
Test
Duration
Chemical
/ Purity
|)H
Temp.
(°C)
Hardness
(mg/L as
CaCOi)
or
Salinity
(ppt)
DOC
(mg/L)
Effect
Reported
Effect
Concentration
(mg/L)
Verified Effect
Concentration1"
(mg/L)
Classification
Quantitative /
Qualitative
a S=static, R=renewal, F=flow-through, U=unmeasured, M=measured, T=total, D=dissolved, Diet=dietary, MT=maternal transfer
b Verification following completion of Part C of the DER
Chronic:
Species (lifestage)
Method3
Test
Duration
Chemical
/ Purity
pH
Temp.
(°C)
Hardness
(mg/L as
CaCOi)
or
Salinity
(ppt)
DOC
(mg/L)
Chronic
Limits
Reported
Chronic
Value
(mg/L or
ng/g)
Verified
Chronic
Valueb
(mg/L or
ng/g)
Chronic
Value
Endpoint
Classification
Quantitative /
Qualitative
a S=static, R=renewal, F=flow-through, U=unmeasured, M=measured, T=total, D=dissolved, Diet=dietary, MT=maternal transfer
b Verification following completion of Part C of the DER
EPA OWDER INSTRUCTIONS
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Data Evaluation Record on the Effects of [Chemical] on Fish [Species]
II. Results Provide results as reported in the publication (including supplemental materials). Include screen shots of tables and/or
figures reporting results from the article following tabulated data table in each associated results section for all studies. Complete
tabulated data tables for all studies for studies marked "Acceptable for Quantitative Use" and "Acceptable for Qualitative Use".
Water Quality Parameters: If only general summary data of water quality parameters is provided by study authors (i.e., no
specific details of water quality parameters on a treatment level is provided), summarize any information regarding water quality
parameters under General Notes below and indicate data not provided in Table A. II. 1.
General Notes: For aquatic life criteria development, measured water quality parameters in the treatments nearest the toxicity
test endpoint(s), e.g., LC50, EC20, etc., are most relevant.
Table A.II.1. Measured Water Quality Parameters in Test Solutions.
Dissolved oxygen, temperature, pH and [other parameters (hardness, salinity, DOC)] in test solutions during the /117-day
exposure of [test organism] to [concentration of treatment(s)] of [test substance] under [static renewal/flow-through]
conditions.
Parameter
Treatment
Mean
Range
Dissolved
Oxygen
(% saturation or
mg/L)
[1]
[2]
j
j
Temperature (C)
[1]
[2]
j
j
pH
[I]
[2]
j
j
Other (e.g.,
hardness,
salinity, DOC)
[I]
[2]
j
j
EPA OWDER INSTRUCTIONS
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Data Evaluation Record on the Effects of [Chemical] on Fish [Species]
Chemical Concentrations: Summarize the concentration verification data from test solutions/media. Expand table to include
measured concentration data for each media type (i.e., water, diet, muscle, liver, blood, etc.).
General Notes: Provide any necessary detail regarding the measured concentrations, including any identified cause for
substantial differences between nominal and measured concentrations, if samples were collected on separate days (and if so provide
details), and any potential cross contamination.
Table A.II.2. Measured and Nominal Chemical Concentrations in Test Solutions/Media.
[Analytical Method] verification of test and control concentrations during an [X]-day exposure of [test organism] to [test
substance] under [static renewal/flow-through] conditions.
[Mean]
Number of
[Standard
Nominal
Measured
Samples
Deviation or
Concentration
Concentration
Number of
Non-
Below Non-
Standard
Treatment
(units)
(units)
Samples
Detect3
Detect
Error]
Range
Control
[1]
[2]
[3]
[4]
[5]
[6]
./'
aNon-Detect: 0 = measured and detected; 1= measured and not detected; if not measured or reported enter as such
EPA OWDER INSTRUCTIONS
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Data Evaluation Record on the Effects of [Chemical] on Fish [Species]
Mortality: Briefly summarize mortality results (if any).
General Notes: Comment on concentrations response relationship and slope of response ifprovided. Compare mortality in
treatments with control group and/or the reference chemical.
Table A.II.3. Mean Percent [Mortality or Survival].
Mean percent mortality [or number of immobilized, survival] of [test organism] exposed to [test substance] for [test duration]
under [static/renewal/flow-through] conditions. Superscript(s) used to identify the values reported to be significantly different
from control as p value of [0.05/ or any other provided by authors].
Treatment
[Standard Deviation
(units)
[Mean % Mortality]
Sample Size
or Standard Error]
Control
[1]
[2]
[3]
[4]
[5]
[6]
ILC.xl
NOEC
LOEC
a Use superscript(s) to identify the values reported to be significantly different from control.
EPA OWDER INSTRUCTIONS
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Data Evaluation Record on the Effects of [Chemical] on Fish [Species]
Growth: Briefly summarize growth results (if any).
General Notes: Comment on concentrations response relationship and slope of response ifprovided. Compare growth endpoints
in treatments with control group and/or the reference chemical.
Table A.II.4. Mean [Growth].
Mean growth [length and/or weight] of [test organism] exposed to [test substance] for [test duration] under
[static/renewal/flow-through] conditions. Superscript(s) used to identify the values reported to be significantly different from
control as p value of [0.05/ or any other provided by authors].
Treatment
Mean Growth
[Length/Weight]
(units)
Sample Size
[Standard
Deviation or
Standard Error]
Mean Percent
Change in
[Length/
Biomass]
Sample Size
[Standard
Deviation or
Standard Error]
Control
[1]
[2]
[3]
[4]
[5]
[6]
¦/'
[ECx]
NOEC
LOEC
a Use superscript(s) to identify the values reported to be significantly different from control.
EPA OWDER INSTRUCTIONS
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Data Evaluation Record on the Effects of [Chemical] on Fish [Species]
Reproductive: Briefly summarize reproduction endpoint results (if any). For multi-eenerational studies, copy and vaste Table
A.II.5 below for each generation with reproductive effects data.
General Notes: Comment on concentrations response relationship and slope of response ifprovided. Compare reproductive
endpoints in treatments with control group and/or the reference chemical.
Table A.II.5. Mean [Reproductive] Effect.
Mean [reproductive] effects for [generation] of [test organism] exposed to [test substance] for [test duration] under
[static/renewal/flow-through] conditions. Superscript(s) used to identify the values reported to be significantly different from
control as p value of [0.05/ or any other provided by authors].
[Mean
Number
[Standard
Deviation
[Mean
[Standard
Deviation
[Mean
[Standard
Deviation
[Mean
Percent
Survival
[Standard
Deviation
Treatment
of
Sample
or Standard
Number
Sample
or Standard
Percent
Sample
or Standard
Post
Sample
or Standard
(units)
Spawns!
Size
Error!
ofEggsl
Size
Error!
Hatchl
Size
Error!
Hatchl
Size
Error!
Control
rn
\2\
[31
T41
rsi
T61
J
[ECxl
NOEC
LOEC
a Use superscript(s) to identify the values reported to be significantly different from control.
EPA OWDER INSTRUCTIONS
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Data Evaluation Record on the Effects of [Chemical] on Fish [Species]
Sublethal Toxicity Endpoints: Include other sublethal effect(s), including behavioral abnormalities or other signs of toxicity,
if any. Copy Table A.II.6 as needed to provide details for each sublethal effect obser\>ed.
General Notes: Briefly summarize obser\>ed sublethal effects otherwise not captured in the results table(s) below.
Table A.II.6. Mean [Sublethal] Effect.
Mean /Sublethal effect, (e.g., beha\>ioral abnormalities, etc.)] in [test organism] during [test duration (acute/chronic)]
exposure to [test substance] under [static/renewal/flow-through] conditions. Superscript(s) used to identify the values
reported to be significantly different from control as p value of [0.05/ or any other provided by authors].
[Mean Sublethal Response]
[Standard Deviation or
Treatment
(units)
Sample Size
Standard Error]
Control
[1]
[2]
[3]
[4]
[5]
[6]
¦/'
[ECxl
NOEC
LOEC
a Use superscript(s) to identify the values reported to be significantly different from control
EPA OWDER INSTRUCTIONS
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Data Evaluation Record on the Effects of [Chemical] on Fish [Species]
Reported Statistics: Copy and paste statistical section from publication.
EPA OWDER INSTRUCTIONS
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Data Evaluation Record on the Effects of [Chemical] on Fish [Species]
Part B: Detailed Review
I. Materials and Methods
Protocol/Guidance Followed: Indicate ifprovided by authors.
Deviations from Protocol: If authors report any deviations from the protocol noted above indicate here.
Study Design and Methods: Copy and paste methods section from publication.
TEST ORGANISM: Provide information under Details and any relevant or related information or clarifications in Remarks.
Parameter
Details
Remarks
Species:
Useful sites include:
• httDs://www.itis.eov/
• httt>s://www.fws.eov/endaneered/
• httt>s://www.fisheries.noaa.eov/fmd-st>ecies
Common Name:
Scientific Name:
Order Name:
Family Name:
North American species?
Surrogate for North American
Taxon?
Is this species Threatened or
Endangered?
(Place A' if applicable)
Strain/Source:
• Wild caught from unpolluted areas [4]
o Quarantine for at least 14 days or until they are
disease free, before acclimation [2,4]
o Quarantine at least 7 days before holding, which
should be at least 12 days [1]
• Must originate from same source and population
[1-2.4] "
• Salmon and trout should be obtained from a hatchery
certified disease free [1]
• Should not be used:
o If appeared stressed, diseased, have physical
abnormalities, or show unusual behavior [2,4]
o If more than 5% die or show signs of stress during
the 48 hours before test initiation [1,4]
o If they were used in previous test treatments or
controls [1,5]
o If collected by electroshocking, chemical
treatments, or gill netting [1,2]
• No treatments of diseases may be administered:
o Within 16 hours of field collection [4]
o Within 48 hours of testing or during testing [1]
o Within 10 days of testing or during testing [4]
o Embryos should not be obtained from fish treated
for disease within past 14 days [2]
o Embryos should not be treated for diseases during
testing [2]
Age at Study Initiation:
Acute:
• Juvenile stages preferred [1,4]
o Should be less than 3 g weight and actively
feeding [1]
Chronic:
• Life-cycle test:
o Embryos or newly hatched young < 48 hours old
[5]
• Partial life-cycle test:
o Immature juveniles at least 2 months prior to
active gonad development [5]
• Early life-stage test:
o Shortly after fertilization [2,5]
¦ <24 hours post fertilization preferred, < hours
encouraged [21
U.SEPA OWFISHDER
Part B: Detailed Review
Page 53 of 178
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Data Evaluation Record on the Effects of [Chemical] on Fish [Species]
Parameter
Details
Remarks
Was body weight or length recorded at
test initiation?
Yes No
Was body weight or length recorded at
regular intervals?
Yes No
If yes, describe regular inter\>als:
U.SEPA OWFISHDER
Part B: Detailed Review
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Data Evaluation Record on the Effects of [Chemical] on Fish [Species]
STUDY PARAMETERS: Provide information under Details and any relevant information of deficiencies in Remarks.
Complete for both Controlled Experiments and Field Studies/Obser\>ations.
For Both Controlled Experiments and Field Obsen'ations
Parameter
Details
Remarks
Number of Replicates per Treatment Group:
• Generally, at least 2 replicates/treatment
recommended for acute [1,4] and chronic [6] tests.
• At least 4 replicates/treatment recommended for
early life stage (ELS) test [21
Control(s):
Treatment(s):
Number of Organisms per Replicate/
Treatment Group:
• At least 10 organisms/treatment recommended [1,6]
• At least 7 organisms/treatment acceptable [1,7]
• At least 20 organisms/replicate (80
organisms/concentration) recommended for ELS test
m
Control(s):
Treatment(s):
Exposure Pathway:
(i.e., water, sediment, gavage, or diet).
Note: all other pathways (e.g., dermal, single dose via
gavage, and injection) are unacceptable.
Exposure Duration:
Acute
• Should be at least 96 hours [1]
• Should be 96 hours [5]
Chronic
• Life-cycle tests:
o Ensure that all life stages and life processes are
exposed [5]
o Begin with embryos (or newly hatched young),
continue through maturation and reproduction, and
should end not less than 24 days (90 days for
salmonids) after the hatching of the next
generation [5]
• Partial life-cycle tests:
o Allowed with species that require > 1 year to reach
sexual maturity, so that all major life stages can be
exposed to the test material in <15 months [5]
o Begin with immature juveniles at least 2 months
prior to active gonad development, continue
through maturation and reproduction, and end not
less than 24 days (90 days for salmonids) after the
hatching of the next generation [5]
• Early life-cycle tests:
o 28 to 32 days (60 day post hatch for salmonids)
exposures from shortly after fertilization through
embryonic, larval, and early juvenile development
[2.51'
Acute
Partial Life Cycle
Early Life Stage
Full Life Cycle
Other (please remark):
Observation Intervals:
Should be an appropriate number of observations over
the study to ensure water quality is being properly
maintained [71
Test Concentrations (remember units):
Recommended test concentrations include at least three
concentrations other than the control; four or more will
provide a better statistical analysis [61
Nominal:
Measured:
Media measured in:
What analytic methods were used to
measure test concentrations?
What was the recovery of the test material?
What was the reporting limit of the
analytical method used to measure the test
concentrations?
Were standards used as part of the analytical
method?
U.SEPA OWFISHDER
Part B: Detailed Review
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Data Evaluation Record on the Effects of [Chemical] on Fish [Species]
CONTROLLED EXPERIMENT STUDY PARAMETERS: Provide information under Details and any relevant
information of deficiencies in Remarks. Complete for Controlled Experiments only.
Parameter
Details
Remarks
Acclimation/Holding:
• Should be placed in a tank along with the water in
which they were transported
o If culture water (or other source water, e.g. wild
caught organisms) differs from test water, should
be changed gradually to 100% test dilution water
Duration:
Identify number of individuals excluded from testing and/or
analysis (if any):
Feeding:
(usually 2 or more days) [1,2,4]
o For wild-caught animals, test water temperature
should be within 5°C of collection water
Water type:
temperature [4]
o Temperature change rate should not exceed 3°C
within 72 hours [4]
Temperature (°C):
• To avoid unnecessary stress and promote good
health:
o Organisms should not be crowded [4]
¦ See "Biomass/Loading Rate for guidance on
holding densities
o Water temperature variation should be limited [4]
o Dissolved oxygen:
¦ Maintain between 60 - 100% saturation [4]
¦ Continuous gentle aeration if needed [4]
o Unionized ammonia concentration in holding and
acclimation waters should be < 35 (.ig/L [4]
o Mortality during the week preceding the test
(following a 48 hour adjustment period) must be <
10%, or the batch should be rejected [1]
¦ If between 5-10%, holding should be extended
an additional 7 days 111
Dissolved Oxygen (mg/L):
£:
O
•*2
s:
£
Health {any mortality obser\>ed?)\
s:
Acclimation followed published guidance?
Describe, if any
Yes No
If yes, indicate which guidance:
Test Vessel:
• Test chambers should be loosely covered [4]
• Test chamber material:
o Should minimize sorption of test chemical from
Material:
Briefly describe the test vessel:
water [4]
o Should not contain substances that can be leached
or dissolved in solution and are free of substances
that could react with exposure chemical [4]
o Glass, No. 316 stainless steel, nylon screen and
Size:
perfluorocarbon (e.g. Teflon) are acceptable for
most chemicals [3,4]
¦ Other materials recommended for specific
chemicals and should be used when appropriate
(e.g., polyethylene for PFAS chemicals [8]
o Rubber, copper, brass, galvanized metal, epoxy
glues, lead and flexible tubing should not come
into contact with test solution, dil. water, or stock
[3.4]
• Size/volume should maintain acceptable biomass
loading rates (see Biomass Loading Rate below) [4]
Fill Volume:
U.SEPA OWFISHDER
Part B: Detailed Review
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Data Evaluation Record on the Effects of [Chemical] on Fish [Species]
Parameter
Details
Remarks
For Controlled Experiments Only
Test Solution Delivery System/Method:
• Flow-through preferred for some highly volatile,
hydrolyzable or degradable materials [5]
o Concentrations should be measured often enough
using acceptable analytical methods [5]
• Chronic exposures:
o Flow-through, measured tests required for tests
with fish [5]
Test Concentrations Measured
Yes No
Test Solution Delivery System:
Static
Renewal
Indicate Inten'al:
Flow-through
Indicate Type of DiInter:
Dilution Water Source & Characteristics:
• Dilution water must be characterized (natural surface
water, well water, etc.) [6]
o Clean surface water, ground water, reconstituted
water, or natural or artificial seawater (for
saltwater species) are acceptable [1,2]
o Dechlorinated tap water should not be used as
some forms of chlorination difficult to adequately
remove [1,2]
o Distilled/deionized water without the addition of
appropriate salts should not be used [5]
• Freshwater hardness range should be < 5 mg/L or <
10% of the average (whichever is greater) [4]
o Recommended hardness <250 mg/L (preferably
<180 mg/L); or 40-50 mg/L for metals [1,2]
o LTnless study is examining effects of hardness on
toxicity.
• Saltwater salinity range should be < 2 g/kg or < 20%
of the average (whichever is greater) [4]
o Recommended salinity 15-25 %o [1,2]
o LTnless study is examining effects of salinity on
toxicity.
• Dissolved oxygen in dilution water at start of test
recommended to be 90-100% of saturation [1,2]
• pH should be between 6-8.5 for freshwater species
and 7.5-8.5 for saltwater species [1,2]
• Dilution water in which total organic carbon (TOC) >
2 mg/L [OCSPP Guidance - 1,2] should not be used
(LIS. EPA Guidelines recommends limit of >5 mg/L
-5)
o LTnless data show that TOC or particulate matter
do not affect toxicity [5], or the study is examining
effects of TOC on toxicity
Dilution Series (e.g., 0.5x, 0.6x, etc.):
Dilution Water Parameters:
Measured at the beginning of the experiment or
averaged over the duration of the experiment (details of
water quality parameters measured in test solutions
should be included under the results section)
Dissolved Oxygen (mg/L):
pH:
Temperature (°C):
Hardness (mg/L as CaCCb):
Salinity (ppt):
Total Organic Carbon (mg/L):
Dissolved Organic Carbon (mg/L):
U.SEPA OWFISHDER
Part B: Detailed Review
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Data Evaluation Record on the Effects of [Chemical] on Fish [Species]
Parameter
Details
Remarks
Aeration:
• Acceptable to maintain dissolved oxygen at 60 -
100% saturation at all times [1,2,4]
• Avoid aeration when testing highly oxidizable,
reducible and volatile materials [4]
• Turbulence should be minimized to prevent stress on
test organisms and/or re-suspend fecal matter [1,2,4]
• Aeration should be the same in all test chambers at all
times [4]
• Generally not recommended. Only permitted when
D.O. levels are in danger of falling below 60%
saturation [1,2]
Yes
No
Describe Preparation of Test
Concentrations (e.g., water exposure,
diet):
Test Chemical Solubility in Water:
List units and conditions (e.g., 0.01% at 20X1)
Were concentrations in water or diet
verified by chemical analysis?
Measured test concentrations should be reported in
Table A.II.2 above.
Yes
Indicate media:
No
s:
O
•*2
s:
£
"3
Were test concentrations verified by
chemical analysis in tissue?
Measured test concentrations can be verified in test
organism tissue (e.g., blood, liver, muscle) alone if a
dose-response relationship is observed.
Measured test concentrations should be reported in
Table A.II.2 above.
Yes
Indicate tissue type:
No
If test concentrations were verified in test organism
tissue, was a dose-response relationship observed?
Were stability and homogeneity of test
material in water/diet determined?
Yes
No
s:
Was test material regurgitated/avoided?
Yes
No
£
Solvent/Vehicle Type (Water or Dietary):
• When used, a carrier solvent should be kept to a
minimum concentration [4]
o Should be restricted to situations where no other
acceptable method of media preparation is
available [3]
• Should not affect either survival or growth of test
organisms [4]
• Should be reagent grade or better [4]
• Should not exceed 0.5 ml/L (static) or 0.1 ml/L (flow
through) unless it was shown that higher
concentrations do not affect toxicity [6]
• Should not exceed 0.1 mL/L [1-3]
o Solvent concentration as low as 0.02 mL/L
recommended [1-3]
• Examples of preferred solvents include
dimethylformamide, Methylene glycol, methanol,
acetone, and ethanol [31.
Negative Control:
Yes
No
Reference Toxicant Testing:
Yes
No
If Yes, identify substance:
U.SEPA OWFISHDER
Part B: Detailed Review
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Data Evaluation Record on the Effects of [Chemical] on Fish [Species]
Parameter
Details
Remarks
For Controlled Experiments Only
Other Control: If any (e.g. solvent control)
Biomass Loading Rate:
• Loading should be limited so as not to affect test
results. Loading will vary depending on temperature,
type of test (static vs. flow-through), species,
food/feeding regime, chamber size, test solution
volume, etc. [4]
• This maximum loading would be determined for the
species, test duration, temperature, flow rate, test
solution volume, chamber size, food, feeding regime,
etc.
• Loading should be sufficiently low to ensure:
o Dissolved oxygen is at least 60% of saturation
(40% for warm-water species) [4,9]
o Unionized ammonia does not exceed 35 (.ig/L [4]
o LTptake by test organisms does not lower test
material concentration by > 20% [4]
o Growth of organisms is not reduced by crowding
• Generally, at the end of the test, the loading (grams of
organisms; wet weight; blotted dry) in each test
chamber should not exceed the following:
o Static tests: >0.8 g/L (lower temperatures); >0.5
g/L (higher temperatures) [1,4]
o Flow through tests: > 1 g/L/day or > 10 g/L at any
time (lower temperatures); >0.5 g/L/day or > 5
g/L at any time (higher temperatures) [4]
• >0.5 g/L/day or > 5 g/L at any time (all
temperatures) [1,2]
• Lower temperatures are defined as the lower of 17°C
or the optimal test temperature for that species [6]
U.SEPA OWFISHDER
Part B: Detailed Review
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Data Evaluation Record on the Effects of [Chemical] on Fish [Species]
Parameter
Details
Remarks
For Controlled Experiments Only
Feeding:
• Unacceptable for acute tests [1,5]
o Should not be fed for 24-48 hours before test
initiation [1]
o Exceptions:
¦ Data indicate that the food did not affect the
toxicity of the test material [5]
¦ Test material is very soluble and does not sorb
or complex readily (e.g., ammonia) [5]
• Feeding during chronic tests should be appropriate to
the species and size of the test organisms [2]
o Should be adjusted during the test to account for
size and number of individuals per chamber [2]
o Feeding levels should be identical across treatment
levels [2]
o Should observe food consumption and any
bacterial development, which should be avoided
[2]
o Fish should not be fed during the final 24 hours of
a test 121
Yes No
Lighting:
• Depends on the type of test (acute or chronic) and
endpoint (e.g., reproduction) of interest.
• Light levels between 540-1080 lux (50-100 foot
candles) that are constant throughout the test are
recommended [1,2]
• Constant photoperiod between 12 light: 12 dark and
16 light: 8 dark recommended [1,2]
• Newly hatched larvae should be kept in the dark
(except for inspection) for one week [2]
• Artificial light cycles should have a 15 - 30-minute
transition period to avoid stress due to rapid increases
in light intensity [1.2,41
Study Design/Methods Classification: (Place Xbv One Based on Ch>erall Study Design/Methods Classification)
Provide details of Major or Minor Deficiencies/Concerns with Study Design in Associated Sections of Part A: Overview
This classification should be taken into consideration for the overall study classification for aquatic life criteria development in Part A.
Study Design Acceptable for Quantitative Use
Study Design Acceptable for Qualitative Use
Study Design Not Acceptable for Use
Additional Notes: Provide additional considerations for the classification of study use based on the study design.
Clarifying Questions for Study Authors and the Other Pertinent Information/Notes from Discussion: Provide clarifying
questions for study authors.
U.SEPA OWFISHDER
Part B: Detailed Review
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Data Evaluation Record on the Effects of [Chemical] on Fish [Species]
OBSERVATIONS: Provide information under Details and any relevant information in Remarks. This information should be
consistent with the Results Section in Part A.
Parameter
Details
Remarks
Parameters measured including sublethal
effects/toxicity symptoms:
Common Apical Parameters Include:
Acute
• EC50 based on percentage of organisms exhibiting
loss of equilibrium plus the percentage of organisms
immobilized plus percentage of organisms killed [5]
0 If not available, the 96-hr LC50 should be used [5]
Chronic
• Life-cycle/Partial Life-cycle test:
0 Survival and growth of adults and young,
maturation of males and females, eggs spawned
per female, embryo viability (salmonids only), and
hatchability [5]
• Early life-cycle test:
0 Survival and growth 151
List parameters:
Was control survival acceptable?
Acute
• > 90% control survival at test termination [5]
Chronic
• > 80% control survival at test termination [51
Yes No
Control survival (%):
Were individuals excluded from the
analysis?
Yes No
If yes, describe justification provided:
Was water quality in test chambers
acceptable?
• If appropriate, describe any water quality issues
(e.g., dissolved oxygen level below 60% of
saturation)
Yes No
Availability of concentration-response
data:
• Were treatment level concentration-response
data included in study publication (can be from
tables, graphs, or supplemental materials)?
specify endpoints in remarks
• Were replicate level concentration-response
data included in study publication (can be from
tables, graphs, or supplemental materials)?
specify endpoints in remarks
Yes No
Yes No
• If treatment and/or replicate level
concentration-response data were included, how
was data presented? (check all that apply)
Tables
Graphs
Supplemental Files
• Were concentration-response data estimated
from graphs study publication or supplemental
materials?
Yes No
If yes, indicate software used:
Yes No
• Should additional concentration-response data
be requested from study authors?
If concentration-response data are available, complete
Verification of Statistical Results (Part C) for sensitive
Requested by:
Request date:
Date additional data received:
U.SEPA OWFISHDER
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Data Evaluation Record on the Effects of [Chemical] on Fish [Species]
Parameter
Details
Remarks
species.
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Data Evaluation Record on the Effects of [Chemical] on Fish [Species]
Part C: Statistical Verification of Results
I. Statistical Verification Information: Report the statistical methods (e.g., R, EPA TRAP, BMDS, other) used to verify the
reported study or test results for the five (5) most sensitive genera and sensitive apical endpoints (including for tests where such
estimates were not provided). If values for the LC50, LT50 and NOEC are greater than the highest test concentration, use the "> "
symbol.
Primary Reviewer: Date: EPA Contractor {PlaceXby One)
Secondary Reviewer: Date: EPA Contractor {PlaceXby One)
{At least one reviewer should be from EPA for sensitive taxa)
Endpoint(s) Verified:
Additional Calculated Endpoint(s):
Statistical Method (e.g., TRAP, BMDS, R, other):
II. Toxicity Values: Include confidence intervals if applicable
NOEC:
LOEC:
MATC:
ECs:
EC10:
EC20:
ECso or LCso
Dose-Response Curve Classification: (PlaceXby One)
This classification should be taken into consideration for the overall study classification for aquatic life criteria development in Part A
Dose-Response Curve Acceptable for Quantitative Use
Dose-Response Curve Acceptable for Qualitative Use
Dose-Response Curve Not Acceptable for Use
Summary of Statistical Verification: Provide summary of methods used in statistical verification.
Additional Notes:
Attachments:
1. Provide attachments to ensure all data used in Part C are captured, whether from study results reported in the publication
and/or from additional data requested from study authors
• Data from study results of the publication should be reported in Results section of Part A
• Additional data provided upon request from study authors should be reported in Table C.II.l below and original
correspondence with study authors should be included as attachments
2. Model assessment output (including all model figures, tables, and fit metrics)
3. Statistical code used for curve fitting
U.SEPA OWFISHDER
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Data Evaluation Record on the Effects of [Chemical] on Fish [Species]
III. Attachments: Include all attachments listed above after the table below.
Additional Data Used in Response-Curve: Provide all data used to fit dose-response cur\'e not captured in Results section of PER above in Part A. Add rows as needed.
First row in italicized text is an example.
Table C.II.1 Additional Data Used in Dose-Response Curve.
Curve ID
Species
Endpoint
Treatment
Replicate
[Standard
Deviation
or Standard
Error!
# of
Survivors
Na
ka
na
Response
Response
Unit
Cone
Cone units
Alchronicl
Ceriodaphnia dubia
#of
voimg/female
0
6
10
10
1
18
count
0.03
nig/L
a N = number of individuals per treatment; k = number of replicates per treatment level; n = number of individuals per replicate
U.SEPA OWFISHDER
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Data Evaluation Record on the Effects of [Chemical] on Fish [Species]
Part I): References to Test Guidance
1. U.S. EPA. 2016a. OCSPP 850.1075: Freshwater and saltwater fish acute toxicity test.
Ecological effects test guidelines. Office of Chemical Safety and Pollution Prevention.
EPA 712-C-16-007. October 2016.
2. U.S. EPA. 2016b. OCSPP 850.1400: Fish early life stage toxicity test. Ecological effects
test guidelines. Office of Chemical Safety and Pollution Prevention. EPA 712-C-16-008.
October 2016.
3. U.S. EPA. 2016c. OCSPP 850.1000: Background and special consideration-tests with
aquatic and sediment-dwelling fauna and aquatic microcosms. Ecological effects test
guidelines. Office of Chemical Safety and Pollution Prevention. EPA 712-C-16-014.
October 2016.
4. ASTM Standard E 729, 1980. 2002. Standard guide for conducting acute toxicity tests on
test materials with fishes, macroinvertebrates, and amphibians. ASTM International,
West Conshohocken, PA.
5. Stephan, C.E., D.I. Mount, D J. Hansen, I.H. Gentile, G.A. Chapman and W.A. Brungs. 1985.
Guidelines for Deriving Numerical National Water Quality Criteria for the Protection of Aquatic
Organisms and their Uses. PB85-227049. National Technical Information Service, Springfield,
VA.
6. Stephan, C.E. 1995. Review of results of toxicity tests with aquatic organisms. Draft.
U.S. EPA, MED. Duluth, MN. 13 pp.
7. OECD 203. 1992. Test No. 203: Fish, Acute Toxicity Test. OECD Guidelines for the
Testing of Chemicals, Section 2, OECD Publishing, Paris,
https://doi. org/10.1787/9789264069961 -en.
8. Boudreau, T.M., Sibley, P.K., Mabury, S.A., Muir, D.G.C., and Solomon, K.R. 2003.
Laboratory Evaluation of the Toxicity of Perfluorooctane Sulfonate (PFOS) on
Selenastrum capricornutum, Chlorella vulgaris, Lemna gibba, Daphnia magna, and
Daphnia pulicaria. Archives of Environmental Contamination and Toxicology. 44: 307-
313.
9. American Public Health Association (APHA). 2012. Standard methods for the
examination of water and wastewater. Part 8000 - Toxicity. APHA. Washington, DC.
U.SEPA OWFISHDER
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Data Evaluation Record on the Effects of [Chemical] on Aquatic Invertebrate [Species]
Attachment F Aquatic Invertebrate Data Evaluation Record (DER)
Template (September 2024)
U.SEPA OW FISH DER
Part D: References to Test Guidance
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Data Evaluation Record on the Effects of [Chemical] on Aquatic Invertebrate [Species]
Part A: Overview
I. Test Information
Chemical name:
CAS name: CAS Number:
Purity: Storage conditions:
Solubility in Water (units):
Controlled Experiment Field Study/Observation {Place X by One)
(imanipulated) {not manipulated)
Primary Reviewer: Date: EPA Contractor {PlaceXby One)
Secondary Reviewer: Date: EPA Contractor {PlaceXby One)
{At least one reviewer should be from EPA for sensitive taxa)
Citation: Indicate: author (s), year, study title, journal, volume, and pages.
(e.g., Keller, A.E and S.G. Zam. 1991. The acute toxicity of selected metals to the freshwater mussel, Anodonta imbecilis. Environ. Toxicol. Chem. 10(4): 539-546.)
Companion Papers: Identify any companion papers associated with this paper using the citation format above.
{Ifyes, list file names of
Were other DERs completed for Companion Papers? Yes No DERs below)
Study Classification for Aquatic Life Criteria Development:
Acceptable for Quantitative Use
Acceptable for Qualitative Use
Not Acceptable for Use/Unused
General N otes: Provide any necessary details regarding the study's use classification for all pertinent endpoints, including
non-apical endpoints within the study (e.g., note all study classifications for each endpoint if the use varies)
Major Deficiencies (note any stated exclusions): Check all that apply. Checking any of these items make the study "Not
Acceptable for Use"
.. . ^ , No Controls (for controlled experiments
Mixture (for controlled experiments only) only)
Excessive Control Mortality (> 10% for acute and > 20% for chronic)
Bioaccumulation: steady state not reached
Dermal or Injection Exposure Pathway
Review paper or previously published without modification
Other: (if any, list here, e.g., use of distilled water)
POTENTIAL CHEMICAL MIXTURES: Describe any potential chemicals mixtures as characterized by study authors
(including any confirmation of chemical mixtures).
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Data Evaluation Record on the Effects of [Chemical] on Aquatic Invertebrate [Species]
General Notes:
Minor Deficiencies: List and describe any minor deficiencies or other concerns with test. These items may make the study
"Acceptablefor Qualitative Use" (exceptions may apply as noted)
DESCRIPTION OF UNMEASURED TEST CONCENTRATIONS: Describe concerns with unmeasured test
concentrations and the influence of the study classification.
DESCRIPTION OF CONCERNS WITH DILUTION WATER: Describe concerns with characterization of and/or
deficiencies with dilution water (e.g., uncharacterized stream or lake water, potential presence of unknown containments,
high organic content, extreme hardness, pH, etc).
For Field Studies/Observations: A field study/observation may be considered "Acceptable for Quantitative Use" if it
consisted of a range of exposure concentrations and the observed effects are justifiably contributed to a single chemical
exposure
Mixture (observed effects not justifiably contributed to single chemical exposure)
Uncharacterized Reference Sites/Conditions
POTENTIAL CHEMICAL MIXTURES PRESENT AT SITE: Describe any potential chemicals mixtures present at
the site as characterized by study authors (including any confirmation of chemicals present at study site).
EXPOSURE VARIABILITY ACROSS STUDY SITE(S): Describe any exposure variability across study site(s) as
characterized by study authors (i.e., description of study design with reference and contaminated sites).
General Notes:
Reviewer's Comments: Provide additional comments that do not appear under other sections of the template.
U.SEPA OWFISHDER
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Data Evaluation Record on the Effects of [Chemical] on Aquatic Invertebrate [Species]
ABSTRACT: Copy and paste abstract from publication.
SUMMARY: Fill out for the most sensitive endpoint (apical and or non-apical) and modify as needed. If study is classified as ''Not Acceptable for
Use " DO NOT complete summary tables.
Acute:
Species (lifestage)
Method3
Test
duration
Chemical
/ Purity
|)H
Temp.
(°C)
Hardness
(mg/L as
CaCOi)
or
Salinity
(ppt)
DOC
(mg/L)
Effect
Reported
Effect
Concentration
(mg/L)
Verified Effect
Concentration1"
(mg/L)
Classification
Quantitative / Qualitative
a S=static, R=renewal, F=flow-through, U=unmeasured, M=measured, T=total, D=dissolved, Diet=dietary, MT=maternal transfer
b Verification following completion of Part C of the DER
Chronic:
Species (lifestage)
Method"
Test
duration
Chemical
/ Purity
pH
Temp.
(°C)
Hardness
(mg/L as
CaCOi)
or
Salinity
(ppt)
DOC
(mg/L)
Chronic
Limits
Reported
Chronic
Value
(mg/L or
ug/g)
Verified
Chronic
Valueb
(mg/L or
ug/g)
Chronic
Value
Endpoint
Classification
Quantitative /
Qualitative
a S=static, R=renewal, F=flow-through, U=unmeasured, M=measured, T=total, D=dissolved, Diet=dietary, MT=maternal transfer
b Verification following completion of Part C of the DER
U.SEPA OW FISH DER
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Data Evaluation Record on the Effects of [Chemical] on Aquatic Invertebrate [Species]
II. Results Provide results as reported in the publication (including supplemental materials). Include screen shots of tables and/or
figures reporting results from the article following tabulated data table in each associated results section for all studies. Complete
tabulated data tables for all studies for studies marked "Acceptable for Quantitative Use" and "Acceptable for Qualitative Use".
Water Quality Parameters: If only general summary data of water quality parameters is provided by study authors (i.e., no
specific details of water quality parameters on a treatment level is provided), summarize any information regarding water quality
parameters under General Notes below and include data not provided in Table A.II.l.
General Notes: For aquatic life criteria development, measured water quality parameters in the treatments nearest the toxicity
test endpoint(s), e.g., LC50, EC20, etc., are most relevant.
Table A.II.1. Measured Water Quality Parameters in Test Solutions.
Dissolved oxygen, temperature, pH and [other parameters (hardness, salinity, DOC)] in test solutions during the /117-day
exposure of [test organism] to [concentration of treatment(s)] of [test substance] under [static renewal/flow-through]
conditions.
Parameter
Treatment
Sample Size
Mean
Range
Dissolved
oxygen
(% saturation or
mg/L)
[1]
[2]
J
J
Temperature (C)
[11
[2]
J
J
pH
[11
[2]
J
J
Other (e.g.,
hardness,
salinity, DOC)
[1]
[2]
J
./'
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Data Evaluation Record on the Effects of [Chemical] on Aquatic Invertebrate [Species]
Chemical Concentrations: Summarize the concentration verification data from test solutions/media. Expand table to include
each measured concentration data for each media type (i.e., muscle, liver, blood, etc.).
General Notes: Provide any necessary detail regarding the measured concentrations, including any identified cause for
substantial differences between nominal and measured concentrations, if samples were collected on separate days (and if so provide
details), and any potential cross contamination.
Table A.II.2. Measured and Nominal Chemical Concentrations in Test Solutions/Media.
[Analytical Method] verification of test and control concentrations during an [X]-day exposure of [test organism] to [test
substance] under [static renewal/flow-through] conditions.
[Mean]
Number of
[Standard
Nominal
Measured
Samples
Deviation or
Concentration
Concentration
Number of
Non-
Below Non-
Standard
Treatment
(units)
(units)
Samples
Detect3
Detect
Error]
Range
Control
[1]
[2]
[3]
[4]
[5]
[6]
./'
aNon-Detect: 0 = measured and detected; l=measured and not detected; if not measured or reported enter as such
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Data Evaluation Record on the Effects of [Chemical] on Aquatic Invertebrate [Species]
Mortality: Briefly summarize mortality results (if any).
General Notes: Comment on concentrations response relations and slope of response ifprovided. Compare mortality with control
treatment and/or the reference chemical.
Table A.II.3. Mean Percent [Mortality or Survival].
Mean percent mortality [or number of immobilized] or survival of [test organism] exposed to [test substance] for [test
duration] under [static/renewal/flow-through] conditions. Superscript(s) used to identify the values reported to be
significantly different from control as p value of [0.05/ or any other provided by authors].
Treatment
[Mean % Mortality]
Sample Size
[Standard Deviation
or Standard Error]
Control
[1]
[2]
[3]
[4]
[5]
[6]
[LCxl
NOEC
LOEC
a Use superscript(s) to identify the values reported to be significantly different from control.
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Data Evaluation Record on the Effects of [Chemical] on Aquatic Invertebrate [Species]
Growth: Briefly summarize growth results (if any).
General Notes: Comment on concentrations response relations and slope of response ifprovided. Compare growth endpoints with
control treatment and/or the reference chemical.
Table A.II.4. Mean [Growth].
Mean growth [length and/or weight] of [test organism] exposed to [test substance] for [test duration] under
[static/renewal/flow-through] conditions. Superscript(s) used to identify the values reported to be significantly different from
control as p value of [0.05/ or any other provided by authors].
Mean Percent
Mean Growth
[Standard
Change in
[Standard
[Length/Weight]
Deviation or
[Length/
Deviation or
Treatment
(units)
Sample Size
Standard Error]
Biomass]
Sample Size
Standard Error]
Control
[1]
[2]
[3]
[4]
[5]
[6]
./'
[ECX]
NOEC
LOEC
a Use superscript(s) to identify the values reported to be significantly different from control.
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Data Evaluation Record on the Effects of [Chemical] on Aquatic Invertebrate [Species]
Reproductive: Briefly summarize reproduction endpoint results (if any). For multi-eenerational studies, copy and vaste Table
A.II.5 below for each generation with reproductive effects data.
General Notes: Comment on concentrations response relations and slope of response ifprovided. Compare reproduction
endpoints with control treatment and/or the reference chemical.
Table A.II.5. Mean [Reproductive] Effect.
Mean [reproductive] effects for [generation] of [test organism] exposed to [test substance] for [test duration] under
[static/renewal/flow-through] conditions. Superscript(s) used to identify the values reported to be significantly different from
control as p value of [0.05/ or any other provided by authors].
[Standard
[Standard
Deviation
[Standard
Deviation
Treatment
[Mean
Number of
Sample
Deviation or
Standard
[Mean
Number of
Sample
or
Standard
[Mean
Number of
Sample
or
Standard
(units)
Spawns]
Size
Error]
Eggsl
Size
Error]
Offspring]
Size
Error]
Control
[1]
[2]
[3]
[4]
[5]
[6]
./'
[ECx]
NOEC
LOEC
a Use superscript(s) to identify the values reported to be significantly different from control.
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Data Evaluation Record on the Effects of [Chemical] on Aquatic Invertebrate [Species]
Sublethal Toxicity Endpoints: Include other sublethal effect(s), including behavioral abnormalities or other signs of toxicity,
if any. Copy Table A.II.6 as needed to provide details for each sublethal effect obser\>ed.
General Notes: Briefly summarize obser\>ed sublethal effects otherwise not captured in the results table(s) below.
Table A.II.6. Mean [Sublethal] Effect.
Mean /Sublethal effect, (e.g., beha\>ioral abnormalities, etc.)] in [test organism] during [test duration (acute/chronic)]
exposure to [test substance] under [static/renewal/flow-through] conditions. Superscript(s) used to identify the values
reported to be significantly different from control as p value of [0.05/ or any other provided by authors].
Treatment
[Mean Sublethal Response]
(units)
Sample Size
[Standard Deviation or
Standard Error]
Control
[1]
[2]
[3]
[4]
[5]
[6]
./'
[ECxl
NOEC
LOEC
a Use superscript(s) to identify the values reported to be significantly different from control
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Data Evaluation Record on the Effects of [Chemical] on Aquatic Invertebrate [Species]
Reported Statistics: Copy and paste statistical section from publication.
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Data Evaluation Record on the Effects of [Chemical] on Aquatic Invertebrate [Species]
Part B: Detailed Review
I. Materials and Methods
Protocol/Guidance Followed: Indicate ifprovided by authors.
Deviations from Protocol: If authors report any deviations from the protocol noted above indicate here.
Study Design and Methods: Copy and paste methods section from publication.
TEST ORGANISM: Provide information under Details and any relevant or related information or clarifications in Remarks.
Parameter
Details
Remarks
Species:
North American species?
Common Name:
Surrogate for North American
Useful sites include:
Scientific Name:
Taxon?
• httDs://www.itis.eov/
Order Name:
Is this species Threatened or
• httt>s://www.fws.eov/endaneered/
Family Name:
Endangered?
• httt>s://www.fisheries.noaa.eov/find-st>ecies
(Place A' if applicable)
Strain/Source:
• If wild caught, organisms should be from unpolluted
areas from the same source/population [9]
o Quarantine for at least 7 days or until they are
disease free, before acclimation [9]
o Wild caught Gammarids should be quarantined
at least 14 days [2]
o Wild caught shrimp should be quarantined at
least 10 days [5]
• Daphnids should be cultured at the test facility [1,7]
o Should originate from same
culture population [1,7]
• Should not be used:
o If appeared stressed, such as discoloration or
unusual behavior [9]
o If more than 5% die during the 48 hours before
test initiation [1,2,4,7,9]
o If they were used in previous test treatments or
controls [1,2,4,10]
o If culture contains ephippa, adults do not
produce at least 3 offspring/day in 7 days before
test, or animals are first brood progeny
(Daphnids) [1.7]
o If adult brood stock (bivalves) were injured
during handling, exhibit abnormal shell
development, or underwent unplanned
spawning [6]
o If >5% culture or brood stock (C. virginica) dies
or shows signs of stress [3]
• No treatments of diseases may be administered:
o Within 16 hours of field collection [9]
o Within 10 days of testing or during testing [9]
Age at Study Initiation:
Acute:
• Larval stages preferred [9]
• Mayflies and Stoneflies
o Early instar [9]
• Daphnids/cladocerans:
o < 24-hr old [1.7.9]
• Midges:
o 2ntl or 3ri instar larva [9]
• Gammarid amphipods
o <24-hr postrelease [21
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Data Evaluation Record on the Effects of [Chemical] on Aquatic Invertebrate [Species]
Parameter
Details
Remarks
• Hyalella azteca (chronic exposure)
o Generally, 7-8 days old [11]
• Freshwater mussels (chronic exposure)
o Generally, 2 month old juveniles [12]
• Oysters (C. virginica)
o 30-50mm valve height and as similar in size as
possible (<20% coefficient of variation) [3]
• Mysids
o < 24-hr post release [4,9]
• Penaeid shrimp
o Post-larval juveniles [51
Was body weight or length recorded at
test initiation and/or at regular intervals?
Yes No
Was body weight or length recorded at
regular intervals?
Yes No
If yes, describe regular inter\>als:
U.S EPA OWAQUATIC INVERTEBRATE DER
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STUDY PARAMETERS: Provide information under Details and any relevant information of deficiencies in Remarks.
Complete for both Controlled Experiments and Field Studies/Obser\>ations.
Parameter
Details
Remarks
Number of Replicates per Treatment Group:
• At least 2 replicates/treatment recommended for
most tests [1-6,9,14]
• 4 replicates/treatment recommended for C. virginica
acute limit test [3]
• 4 replicates/treatment (flow-through) or 10
replicates/treatment (renewal) recommended for
Daphnia chronic tests 171
Control(s):
Treatment(s):
Number of Organisms per Replicate/
Treatment Group:
• Unless otherwise specified, at least 10
organisms/replicate recommended for most tests [1-
6.9]
Control(s):
• 8 organisms/replicate recommended for C. virginica
limit test [3]
• 15-30 embryos/replicate for Bivalves [6]
• 5 organisms/replicate (flow-through) or 1
organism/replicate (renewal) recommended for
Daphnia chronic tests [7]
Treatment(s):
Exposure Pathway:
(i.e., water, sediment, or diet). Note: all other pathways
(e.g., dermal, injection) are unacceptable.
For Both Controlled Experiments and Field Obsen'ations
Exposure Duration:
Acute
• Cladocerans and midges should be 48 hours [1.10]
o Longer durations acceptable if test species not fed
and had acceptable controls [10]
• Freshwater mussel glochidia should be a maximum
of 24 hours [12]
o Shorter durations (6. 12. 18 hours) acceptable so
long as 90% survival of control animals achieved
(see below) [12]
• Embryo/larva (bivalve mollusks. sea urchins,
lobsters, crabs, shrimp and abalones) should be 96
hours, but at least 48 hours [6.10]
• Other invertebrate species should be 96 hours [2-5.2]
Chronic
• Daphnids/cladocerans should be 21 days (3-brood
test) [7.10]
o Exception 7 days acceptable for Ceriodaphnia
dubia [10]
• Freshwater juvenile mussels should be at least 28
days [12]
• Hyalella azteca should be at least 42 days
o Beginning with 7-8 day old animals [11]
• Mysids should continue until 7 days past the median
time of first brood release in the controls [12]
Acute
Chronic
Other (please remark):
U.S EPA OWAQUATIC INVERTEBRATE DER
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Data Evaluation Record on the Effects of [Chemical] on Aquatic Invertebrate [Species]
Parameter
Details
Remarks
For Both Controlled Experiments and Field
1- - - .-t:
Observation Intervals:
Should be an appropriate number of observations over
the study to ensure water quality is being properly
maintained [91
Test Concentrations (remember units):
Recommended test concentrations include at least three
concentrations other than the control; four or more will
provide a better statistical analysis. Certain specific
test protocols require more (e.g., [1-6] require five
concentration plus a control)
Nominal:
Measured:
Media measured in:
• What analytic methods were used to
measure test concentrations?
What was the recovery of the test material?
What was the reporting limit of the
analytical method used to measure the test
concentrations?
Were standards used as part of the analytical
method?
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Data Evaluation Record on the Effects of [Chemical] on Aquatic Invertebrate [Species]
CONTROLLED EXPERIMENT STUDY PARAMETERS: Provide information under Details and any relevant
information of deficiencies in Remarks. Complete for Controlled Experiments only.
Parameter
Details
Remarks
Acclimation/Holding:
Identify number of individuals excluded from testing and/or
analysis (if any):
• Should be placed in a tank along with the water in
Duration:
which they were transported [9]
o Water should be changed gradually to 100%
dilution water (usually 2 or more days) [5,9]
o For wild-caught animals, test water temperature
Feeding:
should be within 5°C of collection water
temperature [9]
o Temperature change rate should not exceed 3°C
within 72 hours [9]
Water:
• Test specific recommendations:
u Mysids: should be maintained in dilution water 48
hours before test, with < l°C/day variation in water
temperature [4]
Temperature (°C):
o Gammarids: should be acclimated to dilution water
conditions over 48 hours, then held an additional 7
s:
O
days [2]
*3
o Bivalves: Brood adults should be held for at least
Dissolved Oxygen (mg/L):
S2
14 days in test dilution water prior to spawning [6]
.§
£
u C. Virginia: on arrival at test facility, brush shells
clean of fouling organisms [3]
Health {any mortality obser\>ed?)\
>
¦ Acclimate to dilution water and hold for 10-12
days, until demonstrated they are not stressed or
diseased [3]
"3
¦ Maintain in dilution water at test temperature
for 48 hours before test [3]
£
• To avoid unnecessary stress and promote good
health:
£
o Organisms should not be crowded [9]
¦ See "Biomass/Loading Rate" for guidance on
holding densities
o Water temperature variation should be limited
o Dissolved oxygen:
¦ Maintain between 60 - 100% saturation [9]
¦ Continuous gentle aeration if needed [9]
o Unionized ammonia concentration in holding and
acclimation waters should be < 35 (.ig/L [9]
o Mortality during the week preceding the test
(following a 48 hour adjustment period) must be <
10%, or the batch should be rejected [2,3,5,6]
o If between 5-10%, holding should be extended an
additional 7 days [2,3,5,61
Acclimation followed published guidance?
Describe, if any
Yes No
If yes, indicate which guidance:
U.S EPA OWAQUATIC INVERTEBRATE DER
Part B: Detailed Review
Page 81 of 178
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Data Evaluation Record on the Effects of [Chemical] on Aquatic Invertebrate [Species]
Parameter
Details
Remarks
Test Vessel:
Briefly describe the test vessel here
• Test chambers should be loosely covered [9]
• Test chamber material:
Material:
o Should minimize sorption of test chemical from
water [9]
o Should not contain substances that can be leached
or dissolved in solution and free of substances that
Size:
could react with exposure chemical [9]
o Glass, No. 316 stainless steel, nylon screen and
perfluorocarbon (e.g. Teflon) are acceptable [8,9]
¦ Other materials recommended for specific
chemicals should be used when appropriate
Fill Volume:
(e.g., polyethylene for PFAS chemicals [13]
n Rubber, copper, brass, galvanized metal, epoxy
glues, lead and flexible tubing should not come
into contact with test solution, dilution water or
stock [8,9]
¦ Stainless steel should not be used for saltwater
tests [8]
• Size/volume should maintain acceptable biomass
-&•
loading rates (see below) [9]
s
O
• Substrate:
o Required for some species (e.g., Hyalella aztecd)
[11]
o Common types: stainless steel screen, nylon
screen, quartz sand, cotton gauze and maple leaves
&
[11]
o More inert substances preferred over plant
material, since plants may break down during
Substrate Used (if applicable):
testing and promote bacterial growth [11]
"3
o Consideration should be given between substrate
-G
s:
and toxicant [11]
Cj
¦ Hydrophobic organic compounds in particular
can bind strongly to Nitex® screen, reducing
£
exposure concentrations, especially for studies
using static or intermittent renewal exposure
methods [11]
o Examples:
¦ Acid-washed sand, free of excess organic
matter, 2-3 cm depth (Penaeid shrimp) [5]
¦ Bent piece of stainless steel screen (Gammarid
amphipods) [21
Test Solution Delivery System/Method:
Test Concentrations Measured
• Flow-through preferred for some highly volatile,
Yes No
hydrolyzable or degradable materials [10]
o Concentrations should be measured often enough
Test Solution Delivery System:
using acceptable analytical methods [10]
• Acute C. virginica shell deposition test must be flow
Static
through [3]
Renewal
• Chronic exposures:
Indicate Inten'al:
o Flow-through, measured tests required [10]
o Exception: renewal is acceptable for Cladocerans
[7.10.14]
Flow-through
Indicate Type of DiInter:
U.S EPA OWAQUATIC INVERTEBRATE DER
Part B: Detailed Review
Page 82 of 178
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Data Evaluation Record on the Effects of [Chemical] on Aquatic Invertebrate [Species]
Parameter
Details
Remarks
For Controlled Experiments Only
Dilution Water Source & Characteristics:
• Dilution water must be characterized (natural surface
water, well water, etc.) [10]
o Clean surface water, ground water, reconstituted
water, or natural or artificial seawater (for
saltwater species) are acceptable [1-7]
o Dechlorinated tap water should not be used as
some forms of chlorination difficult to adequately
remove [1,2,7,8]
o Distilled/deionized water without the addition of
appropriate salts should not be used [10]
• Freshwater hardness range should be < 5 mg/L or <
10% of the average (whichever is greater) [9]
o Recommended hardness <250 mg/L (preferably
<180 mg/L); or 40-50 mg/L for metals [1,2,7]
o LTnless study is examining effects of hardness on
toxicity.
• Saltwater salinity range should be < 2 g/kg or < 20%
of the average (whichever is greater) [9]
o Recommended salinity 20±2 %o [3-6]
¦ For C. virginica test with unfiltered seawater,
recommended salinity >12±5%o [3]
o LTnless study is examining effects of salinity on
toxicity.
• Dilution water in which total organic carbon (TOC) >
2 mg/L [OCSPP Guidance - 1-7] should not be used
(LIS. EPA Guidelines recommends limit of >5 mg/L
-2)
o LTnless data show that TOC or particulate matter do
not affect toxicity [10], or the study is examining
effects of TOC on toxicity.
• Dissolved oxygen in dilution water at start of test
recommended to be 90-100% of saturation [1-7]
• pH should be between 6-8.5 for freshwater species
and 7.5-8.5 for saltwater species and should vary by
less than 1 pH unit during the test and between
concentrations [1-7]
• Dilution water for tests with Hyalella azteca
o Reconstituted waters should have at least 0.02 mg
bromide/L; natural ground or surface water
presumed to have sufficient bromide [11]
o Recommended that control/dilution waters have
chloride concentrations at or above 15 mg/L [11]
Dilution Series (e.g., 0.5x, 0.6x, etc.):
Dilution Water Parameters:
Measured at the beginning of the experiment or
averaged over the duration of the experiment (details of
water quality parameters measured in test solutions
should be included under the results section)
Dissolved Oxygen (mg/L):
pH:
Temperature (°C):
Hardness (mg/L as CaCCb):
Salinity (ppt):
Total Organic Carbon (mg/L):
Dissolved Organic Carbon (mg/L):
Aeration:
• Strongly discouraged unless dissolved oxygen (D.O.)
in danger of falling below 60% [1-7,9]
o Preferably performed prior to addition of test
substance [1-7]
• Avoid aeration when testing highly oxidizable,
reducible and volatile materials
• Assurances should be made to prevent stress on test
organisms [1-7,9]
• Aeration should be the same in all test chambers at all
times [1-7.9]
Yes No
U.S EPA OWAQUATIC INVERTEBRATE DER
Part B: Detailed Review
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Data Evaluation Record on the Effects of [Chemical] on Aquatic Invertebrate [Species]
Parameter
Details
Remarks
Describe Preparation of Test
Concentrations (e.g., water exposure,
diet):
Test Chemical Solubility in Water:
• List units and conditions (e.g.. 0.01% at 20°C)
Were concentrations in water or diet
verified by chemical analysis?
Measured test concentrations should be reported in
Table A.II.2 above.
Yes No
Indicate media:
Were test concentrations verified by
chemical analysis in tissue?
Measured test concentrations can be verified in test
organism tissue (e.g., blood, liver, muscle) alone if a
dose-response relationship is observed.
Measured test concentrations should be reported in
Table A.II.2 above.
Yes No
Indicate tissue type:
If test concentrations were verified in test organism
tissue, was a dose-response relationship observed?
Were stability and homogeneity of test
material in water/diet determined?
Yes No
For Controlled Experiments Only
Was test material regurgitated/avoided?
Yes No
Solvent/Vehicle Type:
• When used, a carrier solvent should be kept to a
minimum concentration [1]
o Should be restricted to situations where no other
acceptable method of media preparation is
available [8]
• Should not affect either survival or growth of test
organisms [9]
• Should be reagent grade or better [9]
• Should not exceed 0.5 ml/L (static), or 0.1 ml/L (flow
through) unless it was shown that higher
concentrations do not affect toxicity [14]
• Should not exceed 0.1 mL/L [1-8]
o Solvent concentration as low as 0.02 mL/L
recommended [1-8]
• Examples of preferred solvents include
dimethylformamide, Methylene glycol, methanol,
acetone, and ethanol [8],
Test Temperature
• Start between 18-22 °C. Maintain ±1°C of starting
temperature throughout test [1]
• 18°C recommended [2]
• 20 °C recommended [3]
• 25 °C recommended [4]
• 23 °C recommended [5]
• Between 16-25°C depending on test species [see ref.
6]
• Should be 20±2°C [71
Negative Control:
Yes No
Reference Toxicant Testing:
Yes No
If yes, identify substance:
Other Control: If any (e.g. solvent control)
U.S EPA OWAQUATIC INVERTEBRATE DER
Part B: Detailed Review
Page 84 of 178
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Data Evaluation Record on the Effects of [Chemical] on Aquatic Invertebrate [Species]
Parameter
Details
Remarks
For Controlled Experiments Only
Biomass Loading Rate:
• Loading should be limited so as not to affect test
results. Loading will vary depending on temperature,
type of test (static vs. flow-through), species,
food/feeding regime, chamber size, test solution
volume, etc. [9]
• This maximum number would have to be determined
for the species, test duration, temperature, flow rate,
test solution volume, chamber size, food, feeding
regime, etc.
• Loading should be sufficiently low to ensure:
o Dissolved oxygen is at least 60% of saturation
(40% for warm-water species) [9,15]
o Unionized ammonia does not exceed 35 (.ig/L [9]
o LTptake by test organisms does not lower test
material concentration by > 20% [9]
o Growth of organisms is not reduced by crowding
• Generally, at the end of the test, the loading (grams of
organisms; wet weight; blotted dry) in each test
chamber should not exceed the following:
o Static or renewal tests: > 0.8 g/L (lower
temperatures); > 0.5 g/L (higher temperatures) [9]
o >1 Daphnid / 20 mL [1]
o > 1 Daphnid / 40 mL for chronic renewal tests [7]
o >30 Mysids / L [4]
o >30 bivalve embryos/mL [6]
o >0.8 g/L [2.5]
o Flow through tests: > 1 g/L/day or > 10 g/L at any
time (lower temperatures); >0.5 g/L/day or > 5
g/L at any time (higher temperatures) [9]
o > 0.5 g/L/day or >5 g/L [1.2.4.5.7]
o C. virginica loading based on flow rate adequate to
promote shell growth [3]
¦ Flow rate of 1 L/hr/individual using unflltered
natural sea water [3]
o When loading based on temperature, lower
temperatures are defined as the lower of 17°C or
the optimal test temperature for that species. [91
Feeding:
• LTnacceptable for acute tests [1.2.5.6.10]
o Exceptions:
¦ Data indicate that the food did not affect the
toxicity of the test material [10]
¦ Test organisms will be severely stressed if they
are unfed for 96 hours [10]
• Mysids should be fed daily during testing
(same diet as during culturing and
acclimation) [4]
• C. virginica should be fed during testing [3]
¦ Test material is very soluble and does not sorb
or complex readily (e.g.. ammonia) [101
Yes No
Lighting:
• Generally, ambient laboratory levels (540-1080 lux or
50 - 100 foot candles) or natural lighting should be
acceptable, with a constant photoperiod between 12
light: 12 dark and 16 light: 8 dark [1-7]
• Artificial light cycles should have a 15 - 30 minute
transition period to avoid stress due to rapid increases
in light intensity [1-7.91
U.S EPA OWAQUATIC INVERTEBRATE DER
Part B: Detailed Review
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Data Evaluation Record on the Effects of [Chemical] on Aquatic Invertebrate [Species]
Study Design/Methods Classification: (Place Xby One Based on Overall Study Design/Methods Classification)
Provide details of Major or Minor Deficiencies/Concerns with Study Design in Associated Sections of Part A: Overview
This classification should be taken into consideration for the overall study classification for aquatic life criteria development in Part A.
Study Design Acceptable for Quantitative Use
Study Design Acceptable for Qualitative Use
Study Design Not Acceptable for Use
Additional Notes: Provide additional considerations for the classification of study use based on the study design.
Clarifying Questions for Study Authors and the Other Pertinent Information/Notes from Discussion:
Provide clarifying questions for study authors.
U.S EPA OWAQUATIC INVERTEBRATE DER
Part B: Detailed Review
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Data Evaluation Record on the Effects of [Chemical] on Aquatic Invertebrate [Species]
OBSERVATIONS: Provide information under Details and any relevant information in Remarks. This information should be
consistent with the Results Section in Part A.
Parameter
Details
Remarks
Parameters measured including sublethal
effects/toxicity symptoms:
Common Apical Parameters Include:
Acute
• Daphnids/cladocerans:
o EC50 based on percentage of organisms
immobilized plus percentage of organisms killed
[10]
• Embryo/larva (bivalve molluscs, sea urchins, lobsters,
crabs, shrimp, and abalones):
0 EC50 based on the percentage of organisms with
incompletely developed shells plus the percentage
of organisms killed [10]
¦ If available, the 96 hour EC50 based on the
percentage of organisms with incompletely
developed shells and the 96-hr LC50 should also
be reported separately [2]
• Freshwater mussel (glochidia and juveniles):
0 Glochidia: EC50 based on 100 x number closed
glochidia after adding NaCl solution - number
closed glochidia before adding NaCl solution) /
Total number open and closed glochidia after
adding NaCl solution [12]
0 Juvenile: EC50 based on percentage exhibiting foot
movement within a 5-min observation period [12]
• All other species and older life stages:
0 EC50 based on the percentage of organisms
exhibiting loss of equilibrium plus the percentage
of organisms immobilized plus the percentage of
organisms killed [10]
¦ If not available, the 96 hour LC50 should be
used [10]
Chronic
• Daphnid:
0 Survival and young per female [10]
• Mysids:
0 Survival, growth and young per female [10]
List parameters:
Were controls acceptable?
Acute
• > 90% control survival at test termination [10]
0 Glochidia 90% after 24 hours, or, the next longest
duration less than 24 hours that had at least 90%
survival [12]
0 >70% normal oyster embryos, or >60% normal
hard clam or mussel larvae at the end of the test [6]
0 >90% surviving and free of disease or stress with
an overall mean new shell growth of 2mm for C.
virginica [3]
Chronic
• > 80% control survival at test termination [10]
o >80% in 42 day test with Hyalella azteca, slightly
lower in tests substantially longer than 42 days [11]
o > 80% surviving and free of disease or stress that
did not produce ephippia and that produced at least
60 offspring in 21 days in Daphnid tests [7]
Yes No
Control survival (%):
U.S EPA OWAQUATIC INVERTEBRATE DER
Part B: Detailed Review
Page 87 of 178
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Data Evaluation Record on the Effects of [Chemical] on Aquatic Invertebrate [Species]
Parameter
Details
Remarks
Were individuals excluded from the
analysis?
Yes No
Ifves, describe justification provided:
Was water quality in test chambers
acceptable?
• If appropriate, describe any water quality issues
(e.g., dissolved oxygen level below 60% of
saturation)
Yes No
Availability of concentration-response
data:
• Were treatment level concentration-response
data included in study publication (can be from
tables, graphs, or supplemental materials)?
specify endpoints in remarks
Yes No
• Were replicate level concentration-response
data included in study publication (can be from
tables, graphs, or supplemental materials)?
specify endpoints in remarks
•
Yes No
• If treatment and/or replicate level
concentration-response data were included, how
was data presented? (check all that apply)
Tables
Graphs
Supplemental Files
• Were concentration-response data estimated
from graphs study publication or supplemental
materials?
Yes No
Ifves, indicate software used:
Yes No
Should additional concentration-response data be
requested from study authors?
If concentration-response data are available, complete
Verification of Statistical Results (Part C) for sensitive
species.
Requested by:
Request date:
Date additional data received:
U.S EPA OWAQUATIC INVERTEBRATE DER
Part B: Detailed Review
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Data Evaluation Record on the Effects of [Chemical] on Aquatic Invertebrate [Species]
Part C: Statistical Verification of Results
I. Statistical Verification Information: Report the statistical methods (e.g., R, EPA TRAP, BMDS„ other) used to verify the
reported study or test results for the five (5) most sensitive genera and sensitive apical endpoints (including for tests where such
estimates were not provided). If values for the LC50, LT50 and NOEC are greater than the highest test concentration, use the "> "
symbol.
Primary Reviewer: Date: EPA Contractor {PlaceXby One)
Secondary Reviewer: Date: EPA Contractor {PlaceXby One)
{At least one reviewer should be from EPA for sensitive taxa)
Endpoint(s) Verified:
Additional Calculated Endpoint(s):
Statistical Method (e.g., TRAP, BMDS, R, other):
II. Toxicity Values: Include confidence intervals if applicable
NOEC:
LOEC:
MATC:
ECs:
EC10:
EC20:
ECso or LCso
Dose-Response Curve Classification: (PlaceXby One)
This classification should be taken into consideration for the overall study classification for aquatic life criteria development in Part A
Dose-Response Curve Acceptable for Quantitative Use
Dose-Response Curve Acceptable for Qualitative Use
Dose-Response Curve Not Acceptable for Use
Summary of Statistical Verification: Provide summary of methods used in statistical verification.
Additional Notes:
Attachments:
1. Provide attachments to ensure all data used in Part C is captured, whether from study results reported in the publication
and/or from additional data requested from study authors
• Data from study results of the publication should be reported in Results section of Part A
• Additional data provided upon request from study authors should be reported in Table C.II.l below and original
correspondence with study authors should be included as attachments
2. Model assessment output (including all model figures, tables, and fit metrics)
3. Statistical code used for curve fitting
U.S EPA OWAQUATIC INVERTEBRATE DER
Part C: Statistical Verification of Results
Page 89 of 178
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Data Evaluation Record on the Effects of [Chemical] on Aquatic Invertebrate [Species]
III. Attachments: Include all attachments listed above after the table below.
Additional Data Used in Response-Curve: Provide all data used to fit dose-response cur\'e not captured in Results section of PER above in Part A, rows as needed. First
row in italicized text is an example.
Table C.II.1 Additional Data Used in Dose-Response Curve.
Curve ID
Species
Endpoint
Treatment
Replicate
[Standard
Deviation
or Standard
Error!
# of
Survivors
Na
ka
na
Response
Response
Unit
Cone
Cone units
Alchronicl
Ceriodaphnia dubia
#of
voimg/female
0
6
10
10
1
18
count
0.03
nig/L
a N = number of individuals per treatment; k = number of replicates per treatment level; n = number of individuals per replicate
U.S EPA OWAQUATIC INVERTEBRATE DER
Part C: Statistical Verification of Results
Page 90 of 178
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Data Evaluation Record on the Effects of [Chemical] on Aquatic Invertebrate [Species]
Part I): References to Test Guidance
10. U.S. EPA. 2016a. OCSPP 850.1010: Aquatic invertebrate acute toxicity test, freshwater
Daphnids. Ecological effects test guidelines. Office of Chemical Safety and Pollution Prevention.
EPA 712-C-16-013. October 2016.
11. U.S. EPA. 2016b. OCSPP 850.1020: Gammarid amphipod acute toxicity test. Ecological effects
test guidelines. Office of Chemical Safety and Pollution Prevention. EPA 712-C-16-012. October
2016.
12. U.S. EPA. 2016c. OCSPP 850.1025: Oyster acute toxicity test (Shell deposition). Ecological
effects test guidelines. Office of Chemical Safety and Pollution Prevention. EPA 712-C-16-010.
October 2016.
13. U.S. EPA. 2016d. OCSPP 850.1035: Mysid acute toxicity test. Ecological effects test guidelines.
Office of Chemical Safety and Pollution Prevention. EPA 712-C-l6-011. October 2016.
14. U.S. EPA. 2016e. OCSPP 850.1045: Penaeid acute toxicity test. Ecological effects test
guidelines. Office of Chemical Safety and Pollution Prevention. EPA 712-C-16-009. October
2016.
15. U.S. EPA. 2016f. OCSPP 850.1055: Bivalve acute toxicity test (Embryo-larval). Ecological
effects test guidelines. Office of Chemical Safety and Pollution Prevention. EPA 712-C-16-006.
October 2016.
16. U.S. EPA. 2016g. OCSPP 850.1300: Daphnid chronic toxicity test. Ecological effects test
guidelines. Office of Chemical Safety and Pollution Prevention. EPA 712-C-16-005. October
2016.
17. U.S. EPA. 2016h. OCSPP 850.1000: Background and special consideration-tests with aquatic and
sediment-dwelling fauna and aquatic microcosms. Ecological effects test guidelines. Office of
Chemical Safety and Pollution Prevention. EPA 712-C-16-014. October 2016.
18. ASTM Standard E 729, 1980. 2002. Standard guide for conducting acute toxicity tests on test
materials with fishes, macroinvertebrates, and amphibians. ASTM International, West
Conshohocken, PA.
19. Stephan, C.E., D.I. Mount, D.J. Hansen, J.H. Gentile, G.A. Chapman and W.A. Brungs. 1985.
Guidelines for Deriving Numerical National Water Quality Criteria for the Protection of Aquatic
Organisms and their Uses. PB85-227049. National Technical Information Service, Springfield,
VA.
20. Mount, D.R. and J.R. Hockett. 2015. Issue summary regarding test conditions and methods for
water only toxicity testing with Hyalella azteca. Memorandum to Kathryn Gallagher, U.S. EPA
Office of Water. U.S. EPA Office of Research and Development. MED. Duluth, MN. 9 pp.
21. Bringolf, R.B., M.C. Barnhart, and W.G. Cope. 2013. Determining the appropriate duration of
toxicity tests with glochidia of native freshwater mussels. Submitted to Edward Hammer. U.S.
EPA. Chicago, IL, May 8, 2013. 39 pp.
22. Boudreau, T.M., Sibley, P.K., Mabury, S.A., Muir, D.G.C., and Solomon, K.R. 2003. Laboratory
Evaluation of the Toxicity of Perfluorooctane Sulfonate (PFOS) on Selenastrum capricornuturn,
Chlorella vulgaris, Lemna gibba, Daphnia magna, and Daphniapulicaria. Archives of
Environmental Contamination and Toxicology. 44: 307-313.
23. Stephan, C.E. 1995. Review of results of toxicity tests with aquatic organisms. Draft. U.S. EPA,
MED. Duluth, MN. 13 pp.
24. American Public Health Association (APHA). 2012. Standard methods for the examination of
water and wastewater. Part 8000 - Toxicity. APHA. Washington, DC.
U.S. EPA OW AQUATIC INVERTEBRATE DER
Part D: References to Test Guidance
Page 91 of 178
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Data Evaluation Record on the Effects of [Chemical] on Aquatic Nonvascular Plant [Species or Grouping]
Attachment G Aquatic Nonvascular Plant Data Evaluation Record
(DER) Template (September 2024)
U.S. EPA OW AQUATIC NONVASCULAR PLANT DER
Part A: Overview
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Data Evaluation Record on the Effects of [Chemical] on Aquatic Nonvascular Plant [Species or Grouping]
Part A: Overview
I. Test Information
Chemical name:
CAS name:
Purity:
Solubility in Water (units):
Controlled Experiment
(,manipulated)
CAS Number:
Storage conditions:
Field Study/Observation
(inot manipulated)
Date:
{Place X by One)
Primary Reviewer:
Secondary Reviewer:
(At least one reviewer should be from EPA for sensitive taxa)
Date:
EPA
EPA
Contractor (Place X by One)
Contractor (Place X by One)
Citation: Indicate: author(s), year, study title, journal, volume, and pages.
(e.g., Levy, J.L., J.L. Stauber, and D.F. Jolley. 2007. Sensitivity of marine microalgae to copper: The effect of biotic factors on copper adsorption and toxicity. Sci.
Total Environ. 387(1-3): 141-154)
Companion Papers: Identify any companion papers associated with this paper using the citation format above.
Were other DERs completed for Companion Papers? Yes _
(Ifyes, list file names of
No DERs below)
Study Classification for Aquatic Life Criteria Development: Place X by One Based on Highest Use
Acceptable for Quantitative Use
Acceptable for Qualitative Use
Not Acceptable for Use/Unused
General Notes: Provide any necessary details regarding the study's use classification for all pertinent endpoints,
including non-apical endpoints within the study (e.g., note all study classifications for each endpoint if the use varies)
• Major Deficiencies (note any stated exclusions): Check all that apply. Checking any of these items make the
study "Not Acceptable for Use"
• No Controls (for controlled
experiments only)
Excessive Control Mortality
Bioaccumulation: steady state not reached
Mixture (for controlled experiments only)
Review paper or previously published without modification
Excessive EDTA or similar complexing agent
Other: (if any, list here, e.g. use of distilled water)
•
POTENTIAL CHEMICAL MIXTURES: Describe any potential chemicals mixtures as characterized by study
authors (including any confirmation of chemical mixtures).
U.S. EPA OW AQUATIC NONVASCULAR PLANT DER
Part A: Overview
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Data Evaluation Record on the Effects of [Chemical] on Aquatic Nonvascular Plant [Species or Grouping]
General Notes:
Minor Deficiencies: List and describe any minor deficiencies or other concerns with test. These items may make the study
"Acceptablefor Qualitative Use" (exceptions may apply as noted)
• DESCRIPTION OF UNMEASURED TEST CONCENTRATIONS: Describe concerns with unmeasured test
concentrations and the influence of the study classification.
• DESCRIPTION OF CONCERNS WITH DILUTION WATER: Describe concerns with characterization of
and/or deficiencies with dilution water (e.g., uncharacterized stream or lake water, potential presence of unknown
containments, high organic content, extreme hardness, pH, etc).
For Field Studies/Observations: A field study/observation may be considered "Acceptable for Quantitative Use" if it
consisted of a range of exposure concentrations and the observed effects are justifiably contributed to a single chemical
exposure
Mixture (observed effects not justifiably contributed to single chemical exposure)
Uncharacterized Reference Sites/Conditions
POTENTIAL CHEMICAL MIXTURES PRESENT AT SITE: Describe any potential chemicals mixtures present at
the site as characterized by study authors (including any confirmation of chemicals present at study site).
EXPOSURE VARIABILITY ACROSS STUDY SITE(S): Describe any exposure variability across study
site(s) as characterized by study authors (i.e., description of study design with reference and contaminated sites).
General Notes:
Reviewer's Comments: Provide additional comments that do not appear under other sections of the template.
U.S. EPA OW AQUATIC NONVASCULAR PLANT DER
Part A: Overview
Page 94 of 178
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Data Evaluation Record on the Effects of [Chemical] on Aquatic Nonvascular Plant [Species or Grouping]
ABSTRACT: Copy and paste abstract from publication.
SUMMARY: Fill out for the most sensitive endpoint (apical and or non-apical) and modify as needed. If study is classified as ''Not Acceptable for
Use " DO NOT complete summary tables.
Acute:
Species
(age of inoculum)
Method3
Test
duration
Chemical
/ Purity
|)H
Temp.
(°C)
Hardness
(mg/L as
CaCOi)
or
Salinity
(ppt)
DOC
(mg/L)
Effect
Reported
Effect
Concentration
(mg/L)
Verified Effect
Concentration1"
(mg/L)
Classification
Quantitative /
Qualitative
a S=static, R=renewal, F=flow-through, U=unmeasured, M=measured, T=total, D=dissolved
b Verification following completion of Part C of the DER
Chronic:
Species
(age of inoculum)
Method"
Test
duration
Chemical
/ Purity
pH
Temp.
(°C)
Hardness
(mg/L as
CaCOi)
or
Salinity
(ppt)
DOC
(mg/L)
Chronic
Limits
Reported
Chronic
Value
(mg/L)
Verified
Chronic
Valueb
(mg/L)
Chronic
Value
Endpoint
Classification
Quantitative /
Qualitative
a S=static, R=renewal, F=flow-through, U=unmeasured, M=measured, T=total, D=dissolved
b Verification following completion of Part C of the DER
U.S. EPA OW AQUATIC NONVASCULAR PLANT DER
Part A: Overview
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Data Evaluation Record on the Effects of [Chemical] on Aquatic Nonvascular Plant [Species or Grouping]
II. Results Provide results as reported in the publication (including supplemental materials). Include screen shots of tables and/or
figures reporting results from the article following tabulated data table in each associated results section for all studies. Complete
tabulated data tables for all studies for studies marked "Acceptable for Quantitative Use" and "Acceptable for Qualitative Use".
Water Quality Parameters: If only general summary data of water quality parameters is provided by study authors (i.e., no
specific details of water quality parameters on a treatment level is provided), summarize any information regarding water quality
parameters under General Notes below.
General Notes: For aquatic life criteria development, measured water quality parameters in the treatments nearest the toxicity
test endpoint(s), e.g., LC50, EC20, etc., are most relevant.
Table A.II.1. Measured Water Quality Parameters in Test Solutions.
Dissolved oxygen, temperature, pH and [other parameters (hardness, salinity, DOC)] in test solutions during the /117-day
exposure of [test organism] to [concentration of treatment(s)] of [test substance] under [static renewal/flow-through]
conditions.
Parameter
Treatment
Mean
Range
Dissolved
oxygen
(% saturation or
mg/L)
[1]
[2]
J
J
Temperature (C)
[11
[2]
J
J
pH
[11
[2]
J
J
Other (e.g.,
hardness,
salinity, DOC)
[1]
[2]
J
./'
U.S. EPA OW AQUATIC NONVASCULAR PLANT DER
Part A: Overview
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Data Evaluation Record on the Effects of [Chemical] on Aquatic Nonvascular Plant [Species or Grouping]
Chemical Concentrations: Summarize the concentration verification data from test solutions/media. Expand table to include
each measured concentration data for each media type (i.e., water, tissue, cells.).
General Notes: Provide any necessary detail regarding the measured concentrations, including any identified cause for
substantial differences between nominal and measured concentrations, if samples were collected on separate days (and if so provide
details), and any potential cross contamination.
Table A.II.2. Measured (and Nominal) Chemical Concentrations in Test Solutions/Tissues
[Analytical Method] verification of test and control concentrations during an [X]-day exposure of [test organism] to [test
substance] under [static renewal/flow-through] conditions.
[Mean]
Number of
[Standard
Nominal
Measured
Samples
Deviation or
Concentration
Concentration
Number of
Non-
Below Non-
Standard
Treatment
(units)
(units)
Samples
Detect3
Detect
Error]
Range
Control
[1]
[2]
[3]
[4]
[5]
[6]
./'
aNon-Detect: 0 = measured and detected; l=measured and not detected; if not measured or reported enter as such
U.S. EPA OW AQUATIC NONVASCULAR PLANT DER
Part A: Overview
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Data Evaluation Record on the Effects of [Chemical] on Aquatic Nonvascular Plant [Species or Grouping]
Mortality: Briefly summarize mortality results (if any).
General Notes: Comment on concentrations response relations and slope of response ifprovided. Compare mortality with control
treatment and/or the reference chemical.
Table A.II.3. Mean Percent [Mortality or Survival].
Mean percent [mortality or survival] of [test organism] exposed to [test substance] for [test duration] under
[static/renewal/flow-through] conditions. Superscript(s) used to identify the values reported to be significantly different from
control as p value of [0.05/ or any other provided by authors].
Treatment
[Mean % Mortality]
Sample Size
[Standard Deviation
or Standard Error]
Control
[1]
[2]
[3]
[4]
[5]
[6]
[LCxl
NOEC
LOEC
a Use superscript(s) to identify the values reported to be significantly different from control.
U.S. EPA OW AQUATIC NONVASCULAR PLANT DER
Part A: Overview
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Data Evaluation Record on the Effects of [Chemical] on Aquatic Nonvascular Plant [Species or Grouping]
Growth: Briefly summarize growth results (if any).
General Notes: Compare response on growth (such as cell density, biomass in dry weight, and growth rate) with control treatment
and/or the reference chemical. Also indicate if exponential growth in the control was obsen'ed.
Table A.II.4. Mean [Growth].
Mean growth [e.g., cell density, chlorophylls concentration, length and/or weight] of [test organism] exposed to [test
substance] for [test duration] under [static/renewal/flow-through] conditions. Superscript(s) used to identify the values
reported to be significantly different from control as p value of [0.05/ or any other provided by authors].
[Mean Cell
[Standard
Deviation or
[Mean Percent
Change in
[Standard
Deviation or
Treatment
Density]
Sample Size
Standard Error]
Biomass]
Sample Size
Standard Error]
Control
[1]
[2]
[3]
[4]
[5]
[6]
./'
[ECx]
NOEC
LOEC
a Use superscript(s) to identify the values reported to be significantly different from control.
U.S. EPA OW AQUATIC NONVASCULAR PLANT DER
Part A: Overview
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Data Evaluation Record on the Effects of [Chemical] on Aquatic Nonvascular Plant [Species or Grouping]
Reproductive: Briefly summarize reproduction endpoint results (if any). For multi-eenerational studies, copy and vaste Table
A.II.5 below for each generation with reproductive effects data.
General Notes: Comment on concentrations response relations and slope of response ifprovided. Compare reproduction
endpoints with control treatment and/or the reference chemical.
Table A.II.5. Mean [Reproductive] Effect.
Mean [reproductive] effects for [generation] of [test organism] exposed to [test substance] for [test duration] under
[static/renewal/flow-through] conditions. Superscript(s) used to identify the values reported to be significantly different from
control as p value of [0.05/ or any other provided by authors].
[Standard
[Standard
Deviation or
[Mean
Deviation or
Treatment
[Mean Number
Sample
Standard
Number of
Sample
Standard
(units)
of Spores]
Size
Error]
Cystocarps]
Size
Error]
Control
[1]
[2]
[3]
[4]
[5]
[6]
./'
[ECx]
NOEC
LOEC
a Use superscript(s) to identify the values reported to be significantly different from control.
U.S. EPA OW AQUATIC NONVASCULAR PLANT DER
Part A: Overview
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Data Evaluation Record on the Effects of [Chemical] on Aquatic Nonvascular Plant [Species or Grouping]
Sublethal Toxicity Endpoints: Include other sublethal effect(s), including unusual cell shape, color differences, flocculation,
adherence of algae to test vessels, aggregation of algal cells, precipitation in the test solution, or other signs of toxicity, if any. Cow
Table A.II.6 as needed to provide details for each sublethal effect obser\>ed.
General Notes: Briefly summarize obser\>ed sublethal effects otherwise not captured in the results table(s) below.
Table A.II.6. Mean [Sublethal] Effect.
Mean /Sublethal effect, (e.g., developmental abnormalities, loss of color, morphological changes, necrosis, and/or
floccing.)] in [test organism] during [test duration (acute/chronic)] exposure to [test substance] under [static/renewal/flow-
through] conditions. Superscript(s) used to identify the values reported to be significantly different from control as p value of
[0.05/ or any other provided by authors].
Treatment
[Mean Sublethal Response]
(units)
Sample Size
[Standard Deviation or
Standard Error]
Control
[1]
[2]
[3]
[4]
[5]
[6]
[ECx]
NOEC
LOEC
a Use superscript(s) to identify the values reported to be significantly different from control
U.S. EPA OW AQUATIC NONVASCULAR PLANT DER
Part A: Overview
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Data Evaluation Record on the Effects of [Chemical] on Aquatic Nonvascular Plant [Species or Grouping]
Reported Statistics: Copy and paste statistical section from publication.
U.S. EPA OW AQUATIC NONVASCULAR PLANT DER
Part A: Overview
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Data Evaluation Record on the Effects of [Chemical] on Aquatic Nonvascular Plant [Species or Grouping]
Part B: Detailed Review
I. Materials and Methods
Protocol/Guidance Followed: Indicate ifprovided by authors.
Deviations from Protocol: If authors report any deviations from the protocol noted above indicate here.
Study Design and Methods: Copy and paste methods section from publication.
TEST ORGANISM: Provide information under Details and any relevant or related information or clarifications in Remarks.
Parameter
Details
Remarks
Species:
Useful sites include:
• httDs://www.itis.eov/
• httt>s://www.fws.eov/endaneered/
• httt>s://www.fisheries.noaa.eov/find-st>ecies
Common or Grouping Name:
Scientific Name:
Order Name:
Family Name:
North American species?
Surrogate for North American
Taxon?
FIFRA 5 Species?
Is this species Threatened or
Endangered?
(Place A' if applicable)
Strain/Source:
• Obtained from laboratory or culture collection
o Specify clone if identified [1]
• Obtained from unpolluted areas in the wild
o Quarantine for at least 14 days or until they are
disease free, before acclimation [4]
• Must originate from same source and population [4]
• Should not be used:
o If mortality observed [1]
o If unusual shapes [1]
o If color differences [1]
o If there are differences in chloroplast morphology
[1]
o If clumping or flocculation [1]
o If used in a previous test, including as a control [11
Age of stock culture at Study Initiation
(micro algae):
• Generally 3-7 days old recommended (when
logarithmic growth is occurring) [1]
• 1985 Algal acute toxicity test (5-10 days) [51
Was growth (e.g., cell density, chlorophyll
concentration, length and/or weight)
recorded at test initiation?
Yes No
Was growth (e.g., cell density, chlorophyll
concentration, length and/or weight)
recorded at regular intervals?
Yes No
If yes, describe regular inten'als:
U.S. EPA OW AQUATIC NONVASCULAR PLANT DER
Part B: Detailed Review
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Data Evaluation Record on the Effects of [Chemical] on Aquatic Nonvascular Plant [Species or Grouping]
STUDY PARAMETERS: Provide information under Details and any relevant information of deficiencies in Remarks.
Complete for both Controlled Experiments and Field Studies/Obser\>ations.
For Both Controlled Experiments and Field Obsen'ations
Parameter
Details
Remarks
Number of Replicates per Treatment Group:
Control(s):
Treatment(s):
Cell Density or Biomass per Replicate/
Treatment Group:
Control(s):
Treatment(s):
Exposure Pathway:
(i.e., water, sediment, or mix).
Exposure Duration:
• Static algal tests are generally 72-120 hours
• OECD 201: recommends 72 hours for algal growth
inhibition test [6]
• 48 hour exposure (followed by 5-7 day development
period for reproduction tests (e.g., C. parvula test)
[7]
• Algistatic tests are generally 13-14 days (4-5 day
exposure plus up to 9 day recovery period) 111
Observation Intervals:
• Generally microalgal enumeration recommended
daily if possible using indirect methods [1]
Water quality (e.g., temperature, pH, light) should be
monitored throughout the test. Additional vessels can
be used for some measurements to avoid disturbing test
vessels [11
Test Concentrations (remember units):
Recommended test concentrations include at least three
concentrations other than the control; four or more will
provide a better statistical analysis.
Nominal:
Measured:
Media measured in:
What analytic methods were used to
measure test concentrations?
What was the recovery of the test material?
What was the reporting limit of the
analytical method used to measure the test
concentrations?
Were standards used as part of the analytical
method?
U.S. EPA OW AQUATIC NONVASCULAR PLANT DER
Part B: Detailed Review
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Data Evaluation Record on the Effects of [Chemical] on Aquatic Nonvascular Plant [Species or Grouping]
CONTROLLED EXPERIMENT STUDY PARAMETERS: Provide information under Details and any relevant
information of deficiencies in Remarks. Complete for Controlled Experiments only.
Parameter
Details
Remarks
Acclimation/Culturing:
• Should be incubated under test conditions [1]
• Should be used when still growing exponentially [1]
o Water should be changed gradually to 100%
dilution water (usually 2 or more days) [4]
o Temperature change rate should not exceed 2°C
during acclimation or testing [1]
• To avoid unnecessary stress and promote good
health:
Duration:
Identify number of individuals excluded from testing and/or
analysis (if any):
Standard Nutrient Medium used:
Yes No
If no, provide details of composition of
the nutrient medium under the remarks
section
o Organisms should not be crowded [1]
o Temperature should be maintained at optimal test
conditions for the test species, and temperature
Water type:
variation should not exceed 2°C during
acclimation or testing [1]
o Lighting should be maintained on a light:dark
cycle and at an intensity optimal for the test
species[1]
¦ Light intensity should be measured for each
culture vessel at the level of the culture solution
[1]
o Stock algal cultures should be shaken to prevent
clumping (at least daily) [1]
o pH of nutrient medium in which algae is cultured
should be maintained at optimal conditions for the
test species [1]
Temperature (°C):
£
o
Light:dark cycle:
+3
£
Salinity (for marine algae, ppt):
Cl
Chelator used:
"3
s:
¦ Should not be adjusted after adding test
organism [1]
o Growth medium chelators:
Carbon source:
O
£
¦ Not to be used if suspected to interact with test
chemical [1]
¦ Recommended growth media:
Dissolved Oxygen (mg/L):
• OECD TG 201: 0.0027 mM EDTA [6]
• EPA AAP: 0.00081 mM EDTA [6]
¦ Acceptable provided concentrations are not
excessive for test chemicals subject to
interferences by the chelator (e.g., > 200 (.ig/L
EDTA for metals) [81
Health {any mortality, abnormalities
obser\>ed?)\
Acclimation followed published guidance?
Describe, if any
Yes No
If yes, indicate which guidance:
Test Type:
Acute
Partial Life Cycle
Chronic
Spore Germination
Other (please remark):
U.S. EPA OW AQUATIC NONVASCULAR PLANT DER
Part B: Detailed Review
Page 105 of 178
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Data Evaluation Record on the Effects of [Chemical] on Aquatic Nonvascular Plant [Species or Grouping]
Parameter
Details
Remarks
Test Vessel:
• Test chambers should be covered [1]
o Acceptable covers include foam plugs, stainless
steel, glass, or plastic screw caps [1]
o Covers that contact test solution should 1)
minimize sorption of test chemical from water; 2)
Material:
Briefly describe the test vessel here
not contain substances that will leach or interact
with test solution or affect results [1]
• Test chamber material:
o Should minimize sorption of test chemical from
water [1,9]
o Should not contain substances that can be leached
Size:
£:
O
•*2
£
or dissolved in solution and free of substances that
could react with exposure chemical
o May not contain substances that inhibit the growth
of test organisms [1]
o Erlenmeyer flasks or culturing apparatus are
recommended for growth / growth inhibition tests
[1]
¦ Sizes between 125-500 mL are suggested. All
vessels should be the same size [1]
¦ Test solution volume should not exceed 50% of
test chamber volume [1]
o Erlenmeyer flasks or polystyrene cups are
acceptable for algal reproduction tests [7]
¦ Test chamber size and solution volume should
be appropriate for the species tested [7]
• Size/volume should maintain acceptable cell density
Fill Volume:
•1
£
Cl
"3
s:
o
Test Concentrations Measured
Yes No
Test Solution Delivery System:
Static
Test Solution Delivery System/Method:
Renewal
O
ro
Indicate Inten'al:
Flow-through
Indicate Type of DiInter:
Dilution Water Source & Characteristics:
• Freshwater hardness range should be < 5 mg/L or <
10% of the average (whichever is greater) [4]
• Saltwater salinity range should be < 2 g/kg or < 20%
of the average (whichever is greater) [4]
• Dilution water must be characterized (natural surface
water, well water, etc.) [10]
o Distilled/deionized water without the addition of
appropriate salts should not be used [8]
• Dilution water in which total organic carbon or
particulate matter exceed 5 mg/L should not be used
[8]
o Unless data show that organic carbon or particulate
matter do not affect toxicity [8]
Dilution Series (e.g., 0.5x, 0.6x, etc.):
• 0.667x or 0.5x recommended [2]
• < 0.25x not recommended [2]
Water Pretreatment
Yes No
U.S. EPA OW AQUATIC NONVASCULAR PLANT DER
Part B: Detailed Review
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Data Evaluation Record on the Effects of [Chemical] on Aquatic Nonvascular Plant [Species or Grouping]
Parameter
Details
Remarks
Intervals of water quality measurement:
Dilution Water Parameters:
Measured at the beginning of the experiment or
averaged over the duration of the experiment (details of
water quality parameters measured in test solutions
should be included under the results section)
Recommendations:
• pH
o ~ 7.5 for most freshwater algal species [1]
o ~8 for Skeletonema spp. [1]
o Recommend measuring at start and end of test [1]
• Temperature
o 24-25 °C for most species [1]
o 20°C fox Skeletonema spp. [1]
o 22-24 °C for Cliampia parvula reproduction test
[7]
• Salinity
o 28-32 %o for C. parvula reproduction test |71
Dissolved Oxygen (mg/L):
Temperature (°C):
Light:dark cycle:
pH (test initiation):
pH (test termination):
Hardness (mg/L as CaCCb):
Salinity (for marine algae, ppt):
Total Organic Carbon (mg/L):
Dissolved Organic Carbon (mg/L):
Chelator Used:
Carbon source:
s
O
5^
"o
£:
CJ
£
Aeration or Agitation:
• Aeration not recommended unless appropriate for the
test substance
• Rapliidocelis (formerly Selanastrum) and
Skeletonema should be shaken during test [2]
• C. parvula should be shaken or swirled 2x/day [7]
Yes
No
Describe Preparation of Test
Concentrations:
Test Chemical Solubility in Water:
List units and conditions (e.g.. 0.01% at 20°C)
Were concentrations in water or nutrient
medium verified by chemical analysis?
Measured test concentrations should be reported in
Table A.II.2 above.
Yes
No
Indicate media:
Were test concentrations verified by
chemical analysis in tissue?
Measured test concentrations can be verified in test
organism tissue alone if a dose-response relationship is
observed
Measured test concentrations should be reported in
Table A.II.2 above.
If test concentrations were verified in test organism
tissue, was a dose-response relationship observed?
Yes
No
Indicate tissue type:
Were stability and homogeneity of test
material in water/nutrient medium
determined?
Yes
No
Solvent/Vehicle Type:
• When used, a carrier solvent should be kept to a
minimum concentration [4]
• Should not affect either survival or growth of test
organisms [4]
• Should be reagent grade or better [4]
• Should not exceed 0.5 ml/L (static) or 0.1 ml/L (flow
through) unless it was shown that higher
concentrations do not affect toxicity [USEPA
Guidelines Addendum - 10]
• Should not exceed 0.1 mL/L [OCSPP - 1.3]
o Solvent concentration as low as 0.02 mL/L
recommended [3]
• Examples of preferred solvents include
dimethylformamide. Methylene glycol, methanol,
acetone, and ethanol ["31.
Negative Control:
Yes
No
Reference Toxicant Testing:
Yes
No
If Yes, identify substance:
U.S. EPA OW AQUATIC NONVASCULAR PLANT DER
Part B: Detailed Review
Page 107 of 178
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Data Evaluation Record on the Effects of [Chemical] on Aquatic Nonvascular Plant [Species or Grouping]
Other Control: If any (e.g. solvent control)
Parameter
Details
Remarks
For Controlled Experiments Only
Initial Cell Density:
• Recommended: ~1 x 104 cells/mL for R. subcapita
(formerly S. capricornatum), N. pelliciilosa, A. flos-
aquae; and 7.7 x 104 cells/mL for S. costatum [2]
• Recommended ~5 x 103 - 104 cells/mL for R.
subcapita , 2-5 x 10* >S'. subspicatus, 5 x 104 - 105 S.
leopoldensis, and comparable biomass for other
species [6]
• Biomass density should not exceed 0.5 mg/L dry
weight [6]
• This maximum number would have to be determined
for the species, test duration, temperature, flow rate,
test solution volume, chamber size, food, feeding
regime, etc.
Feeding:
¦ Nutrient medium added during renewal tests?
Yes No
Lighting:
• Continuous lighting for Rapliidocelis. Navicula.
Anabaena [2]
• 14:10 light: dark for Skeletonema [2]
• Fluorescent lights recommended [2]
• -4.3 K lx (4,306 lm/ni2) for Rapliidocelis. Navicula.
and Skeletonema [2]
• -2.2 K lx for Anabaena [2]
• Light should have PAR of-60-70 |iE/nr/s [2]
• Lighting conditions should be consistent with
conditions during culturing/acclimation 121
Study Design/Methods Classification (Place A' by One Based on Ch'erall Study Design/Methods Classification)
Provide details of Major or Minor Deficiencies/Concerns with Study Design in Associated Sections of Part A: Overview
This classification should be taken into consideration for the overall study classification for aquatic life criteria development in Part A.
Study Design Acceptable for Quantitative Use
Study Design Acceptable for Qualitative Use
Study Design Not Acceptable for Use
Additional Notes: Provide additional considerations for the classification of study use based on the study design.
Clarifying Questions for Study Authors and the Other Pertinent Information/Notes from Discussion:
Provide clarifying questions for study authors.
U.S. EPA OW AQUATIC NONVASCULAR PLANT DER
Part B: Detailed Review
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Data Evaluation Record on the Effects of [Chemical] on Aquatic Nonvascular Plant [Species or Grouping]
OBSERVATIONS: Provide information under Details and any relevant information in Remarks. This information should be
consistent with the Results Section in Part A.
Parameter
Details
Remarks
Parameters measured including sublethal
effects/toxicity symptoms:
Common Apical Parameters Include:
Growth
• ECX, ICX based on growth inhibition relative to
control [1]
• Algicidal or algistatic [1]
Other Endpoints
• Chlorophyll a, etc. [11
List parameters:
Was control cell density or biomass
acceptable?
• Did controls reach logarithmic growth by 96 hr?
• How was logarithmic growth determined?
Yes No
Were individuals excluded from the
analysis?
Yes No
Ifves, describe justification provided:
Was test chemical algicidal or algistatic?
• What method was used to make this determination?
o Evans stain, reincubation of subculture, etc.
Algicidal
Algistatic
Additional observations
• Changes in cell sizes or shapes (defonnations) [1]
• Unusual colors [1]
• Differences in chloroplast morphology [1]
• Flocculation, clumping, adhering to test containers
HI
Was water quality in test chambers
acceptable?
• If appropriate, describe any water quality issues
Yes No
Availability of concentration-response
data:
• Were treatment level concentration-response
data included in study publication (can be from
tables, graphs, or supplemental materials)?
specify endpoints in remarks
• Were replicate level concentration-response
data included in study publication (can be from
tables, graphs, or supplemental materials)?
specify endpoints in remarks
•
Yes No
Yes No
• If treatment and/or replicate level
concentration-response data were included, how
was data presented? (check all that apply)
Tables
Graphs
Supplemental Files
• Were concentration-response data estimated
from graphs study publication or supplemental
materials?
Should additional concentration-response data be
requested from study authors?
If concentration-response data are available, complete
Verification of Statistical Results (Part C) for sensitive
species.
Yes No
Ifves, indicate software used:
Yes No
Ifves, requested by:
Request date:
Date additional data received::
U.S. EPA OW AQUATIC NONVASCULAR PLANT DER
Part B: Detailed Review
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Data Evaluation Record on the Effects of [Chemical] on Aquatic Nonvascular Plant [Species or Grouping]
Part C: Statistical Verification of Results
I. Statistical Verification Information: Report the statistical methods (e.g., R,EPA TRAP, BMDS, other) used to verify the
reported study or test results for the five (5) most sensitive genera and sensitive apical endpoints (including for tests where such
estimates were not provided). If values for the LC50, LT50 and NOEC are greater than the highest test concentration, use the "> "
symbol.
Primary Reviewer: Date: EPA Contractor {PlaceXby One)
Secondary Reviewer: Date: EPA Contractor {PlaceXby One)
{At least one reviewer should be from EPA for sensitive taxa)
Endpoint(s) Verified:
Additional Calculated Endpoint(s):
Statistical Method (e.g., TRAP, BMDS, R, other):
II. Toxicity Values: Include confidence intervals if applicable
NOEC:
LOEC:
MATC:
ECs:
EC10:
EC20:
ECso or LCso
Dose-Response Curve Classification: (PlaceXby One)
This classification should be taken into consideration for the overall study classification for aquatic life criteria development in Part A
Dose-Response Curve Acceptable for Quantitative Use
Dose-Response Curve Acceptable for Qualitative Use
Dose-Response Curve Not Acceptable for Use
Summary of Statistical Verification: Provide summary of methods used in statistical verification.
Additional Notes:
Attachments:
1. Provide attachments to ensure all data used in Part C is captured, whether from study results reported in the publication
and/or from additional data requested from study authors
• Data from study results of the publication should be reported in Results section of Part A
• Additional data provided upon request from study authors should be reported in Table C.II.l below and original
correspondence with study authors should be included as attachments
2. Model assessment output (including all model figures, tables, and fit metrics)
3. Statistical code used for curve fitting
U.S. EPA OW AQUATIC NONVASCULAR PLANT DER
Part C: Statistical Verification of Results
Page 110 of 178
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Data Evaluation Record on the Effects of [Chemical] on Aquatic Nonvascular Plant [Species or Grouping]
III. Attachments: Include all attachments listed above after the table below.
Additional Data Used in Response-Curve: Provide all data used to fit dose-response cur\'e not captured in Results section of PER above in Part A, rows as needed. First
row in italicized text is an example.
Table C.II.1 Additional Data Used in Dose-Response Curve.
Curve ID
Species
Endpoint
Treatment
Replicate
[Standard
Deviation
or Standard
Error!
# of
Survivors
Na
ka
na
Response
Response
Unit
Cone
Cone units
Alchronicl
Ceriodaphnia dubia
#of
voimg/female
0
6
10
10
1
18
count
0.03
nig/L
a N = number of individuals per treatment; k = number of replicates per treatment level; n = number of individuals per replicate
U.S. EPA OW AQUATIC NONVASCULAR PLANT DER
Part C: Statistical Verification of Results
Page 111 of 178
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Data Evaluation Record on the Effects of [Chemical] on Aquatic Nonvascular Plant [Species or Grouping]
Part I): References to Test Guidance
25. U.S. EPA. 2012. OCSPP 850.4500: Algal toxicity. Ecological effects test guidelines. Office of Chemical
Safety and Pollution Prevention. EPA 712-C-006. January 2012.
26. U.S. EPA. 1996. OPPTS 850.5400 algal toxicity, tiers I and II. Ecological effects test guidelines.
Prevention, Pesticides and Toxic Substances. EPA 712-C-96-164. April 1996.
27. U.S. EPA. 2016. OCSPP 850.1000: Background and special consideration-tests with aquatic and
sediment-dwelling fauna and aquatic microcosms. Ecological effects test guidelines. Office of Chemical
Safety and Pollution Prevention. EPA 712-C-16-014. October 2016.
28. ASTM Standard E 739, 1980. 2002. Standard guide for conducting acute toxicity tests on test materials
with fishes, macroinvertebrates, and amphibians. ASTM International, West Conshohocken, PA.
29. U.S. EPA. 2002. 40 CFR 797.1050 - Algal acute toxicity test. Source: 50 FR 39321, Sept. 27, 1985, as
amended at 52 FR 19058, May 20, 1987. July 1, 2002 Edition, pp. 101:105.
30. OECD. 2011. Test No. 201: Freshwater Alga and Cyanobacteria, Growth Inhibition Test. OECD
Guidelines for the Testing of Chemicals, Section 2, OECD Publishing, Paris,
https://doi.org/10.1787/9789264069923-en.
31. ASTM Standard E 1498, 1992. 2012. Standard guide for conducting sexual reproduction tests with
seaweeds. ASTM International, West Conshohocken, PA.
32. Stephan, C.E., D.I. Mount, D.J. Hansen, J.H. Gentile, G.A. Chapman and W.A. Brungs. 1985.
Guidelines for Deriving Numerical National Water Quality Criteria for the Protection of Aquatic
Organisms and their Uses. PB85-227049. National Technical Information Service, Springfield, VA.
33. Boudreau, T.M., Sibley, P.K., Mabury, S.A., Muir, D.G.C., and Solomon, K.R. 2003. Laboratory
Evaluation of the Toxicity of Perfluorooctane Sulfonate (PFOS) on Selenastrum capricornutum,
Chlorella vulgaris, Lemna gibba, Daphnia magna, and Daphnia pulicaria. Archives of Environmental
Contamination and Toxicology. 44: 307-313.
34. Stephan, C.E. 1995. Review of results of toxicity tests with aquatic organisms. Draft. U.S. EPA, MED.
Duluth, MN. 13 pp.
U.S. EPA OW AQUATIC NONVASCULAR PLANT DER
Part I): References to Test Guidance
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Data Evaluation Record on the Effects of [Chemical] on Aquatic Vascular Plant [Species or Grouping]
Attachment H Aquatic Vascular Plant Data Evaluation Record (DER)
Template (September 2024)
-Page 113 of 178-
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Data Evaluation Record on the Effects of [Chemical] on Aquatic Vascular Plant [Species or Grouping]
Part A: Overview
I. Test Information
Chemical name:
CAS name: not provided
Purity: not provided
Solubility in Water (units):
CAS Number: not provided
Storage conditions: not provided
Controlled Experiment Field Study/Observation {Place X by One)
(imanipulated) {not manipulated)
Primary Reviewer: Date: EPA Contractor {PlaceXby One)
Secondary Reviewer: Date: EPA Contractor {PlaceXby One)
{At least one reviewer should be from EPA for sensitive taxa)
CITATION: Indicate: author (s), year, study title, journal, volume, and pages.
(e.g., Antunes, P.M.C., M.L. Scornaienchi, H.D. Roshon. 2012. Copper toxicity to Lemna minor modelled using humic acid as a surrogate for the plant root.
Chemosphere. 88 (4):389-394.)
Companion Papers: Identify any companion papers associated with this paper using the citation format above.
{Ifyes, list file names of
Were other DERs completed for Companion Papers? Yes No DERs below)
Study Classification for Aquatic Life Criteria Development: Place X by One Based on Highest Use
Acceptable for Quantitative Use
Acceptable for Qualitative Use
Not Acceptable for Use/Unused
General Notes: Provide any necessary details regarding the study's use classification for all pertinent endpoints, including
non-apical endpoints within the study (e.g., note all study classifications for each endpoint if the use varies)
• Major Deficiencies (note any stated exclusions): Check all that apply. Checking any of these items make the
study "Not Acceptable for Use"
# • No Controls (for controlled
experiments only)
Excessive Control Mortality
• Bioaccumulation: steady state not
reached
Mixture (for controlled experiments only)
Dilution water not adequately characterized
Review paper or previously published without modification
Excessive EDTA or similar complexing agent
Other: [Add Text if Applicable]
•
• POTENTIAL CHEMICAL MIXTURES: Describe any potential chemicals mixtures as characterized by study
authors (including any confirmation of chemical mixtures).
U.S. EPA OWAQUATIC VASCULAR PLANTDER
Part A: Overview
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Data Evaluation Record on the Effects of [Chemical] on Aquatic Vascular Plant [Species or Grouping]
• DESCRIPTION OF DILUTION WATER: Describe concerns with characterization of and/or major
deficiencies with dilution water.
General Notes:
Minor Deficiencies: List and describe any minor deficiencies or other concerns with test. These items may make the study
"Acceptablefor Qualitative Use" (exceptions may apply as noted)
For Field Studies/Observations: A field study/observation may be considered "Acceptable for Quantitative Use" if it
consisted of a range of exposure concentrations and the observed effects are justifiably contributed to a single chemical
exposure
Mixture (observed effects not justifiably contributed to single chemical exposure)
Uncharacterized Reference Sites/Conditions
POTENTIAL CHEMICAL MIXTURES PRESENT AT SITE: Describe any potential chemicals mixtures present at
the site as characterized by study authors (including any confirmation of chemicals present at study site).
•
EXPOSURE VARIABILITY ACROSS STUDY SITE(S): Describe any exposure variability across study
site(s) as characterized by study authors (i.e., description of study design with reference and contaminated sites).
General Notes:
Reviewer's Comments: Provide additional comments that do not appear under other sections of the template.
U.S. EPA OWAQUATIC VASCULAR PLANTDER
Part A: Overview
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Data Evaluation Record on the Effects of [Chemical] on Aquatic Vascular Plant [Species or Grouping]
ABSTRACT: Copy and paste abstract from publication.
SUMMARY: Fill out and modify as needed.
Acute:
Species
(life stage3)
Methodb
Category0
Test
duration
Chemical
/ Purity
|)H
Temp.
(°C)
Hardness
(mg/L as
CaCOi)
or
Salinity
(ppt)
DOC
(mg/L)
Effect
Reported
Effect
Concentration
(mg/L)
Verified
Effect
Concentration
(mg/L)
Classification
Quantitative /
Qualitative /
Unused
a e.g., seed, seedling, adult
b S=static, R=renewal, F=flow-through, U=unmeasured, M=measured, T=total, D=dissolved
0 B=bentliic, E=emergent, Sub=submerged
Chronic:
Species
(life stage3)
Methodb
Category0
Test
duration
Chemical
/ Purity
pH
Temp.
(°C)
Hardness
(mg/L as
CaCOi)
or Salinity
(ppt)
DOC
(mg/L)
Chronic
Limits
Reported
Chronic
Value
(mg/L)
Verified
Chronic
Value
(mg/L)
Chronic
Value
Endpoint
Classification
Quantitative /
Qualitative /
Unused
a e.g., seed, seedling, adult
b S=static, R=renewal, F=flow-tlirough, U=unineasured, M=measured, T=total, D=dissolved
0 B=bentliic, E=emergent, Sub=submerged
U.S. EPA OWAQUATIC VASCULAR PLANTDER
Part A: Overview
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Data Evaluation Record on the Effects of [Chemical] on Aquatic Vascular Plant [Species or Grouping]
II. Results Provide results as reported in the publication (including supplemental materials). Include screen shots of tables and/or
figures reporting results from the article following tabulated data table in each associated results section for all studies. Complete
tabulated data tables for all studies for studies marked "Acceptable for Quantitative Use" and "Acceptable for Qualitative Use".
Water Quality Parameters: If only general summary data of water quality parameters is provided by study authors (i.e., no
specific details of water quality parameters on a treatment level is provided), summarize any information regarding water quality
parameters under General Notes below.
General Notes: For aquatic life criteria development, measured water quality parameters in the treatments nearest the toxicity
test endpoint(s), e.g., LC50, EC20, etc., are most relevant.
Table A.II.1. Measured Water Quality Parameters in Test Solutions.
Dissolved oxygen, temperature, pH and [other parameters (hardness, salinity, DOC)] in test solutions during the /117-day
exposure of [test organism] to [concentration of treatment(s)] of [test substance] under [static renewal/flow-through]
conditions.
Parameter
Treatment
Mean
Range
Dissolved
oxygen
(% saturation or
mg/L)
[1]
[2]
j
j
Temperature (C)
[I]
[2]
j
j
pH
[I]
[2]
j
j
Other (e.g.,
hardness,
salinity, DOC)
[1]
[2]
j
j
U.S. EPA OWAQUATIC VASCULAR PLANTDER
Part A: Overview
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Data Evaluation Record on the Effects of [Chemical] on Aquatic Vascular Plant [Species or Grouping]
Chemical Concentrations: Summarize the concentration verification data from test solutions/media. Expand table to include
each measured concentration data for each media type (i.e., water, tissue, cells).
General Notes: Provide any necessary detail regarding the measured concentrations, including any identified cause for
substantial differences between nominal and measured concentrations, if samples were collected on separate days (and if so provide
details), and any potential cross contamination.
Table A.II.2. Measured (and Nominal) Chemical Concentrations in Test Solutions/Media.
[Analytical Method] verification of test and control concentrations during an [X]-day exposure of [test organism] to [test
substance] under [static renewal/flow-through] conditions.
[Mean]
Number of
[Standard
Nominal
Measured
Samples
Deviation or
Concentration
Concentration
Number of
Non-
Below Non-
Standard
Treatment
(units)
(units)
Samples
Detecf
Detect
Error]
Range
Control
[11
[21
[31
[41
[51
[61
i
aNon-Detect: 0 = measured and detected; l=measured and not detected; if not measured or reported enter as such
U.S. EPA OWAQUATIC VASCULAR PLANTDER
Part A: Overview
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Data Evaluation Record on the Effects of [Chemical] on Aquatic Vascular Plant [Species or Grouping]
Mortality: Briefly summarize mortality results (if any).
General Notes: Comment on concentrations response relations and slope of response ifprovided. Compare mortality with control
treatment and/or the reference chemical.
Table A.II.3. Mean Percent [Mortality or Survival].
Mean percent mortality or survival of [test organism] exposed to [test substance] for [test duration] under
[static/renewal/flow-through] conditions.
Treatment
[Mean % Mortality!
[Standard Deviation
or Standard Error]
Control
[11
[21
[31
[41
[51
[61
[LCx]
NOEC
LOEC
a Use superscript to identify the values reported to be significantly different from control.
U.S. EPA OWAQUATIC VASCULAR PLANTDER
Part A: Overview
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Data Evaluation Record on the Effects of [Chemical] on Aquatic Vascular Plant [Species or Grouping]
Growth: Briefly summarize growth results (if any).
General Notes: Comment on concentrations response relations and slope of response ifprovided. Compare growth endpoints with
control treatment and/or the reference chemical.
Table A.II.4. Mean [Growth].
Mean growth [e.g., length and/or weight, chlorophylls concentration] of [test organism] exposed to [test substance] for [test
duration] under [static/renewal/flow-through] conditions.
Mean Growth
Mean Percent
[Length/Weight]
[Standard Deviation
Change in [Length/
[Standard Deviation
Treatment
(units)
or Standard Error]
Biomassl
or Standard Error]
Control
[11
[21
[31
[41
[51
[61
i
[ECx]
NOEC
LOEC
a Use superscript to identify the values reported to be significantly different from control.
U.S. EPA OWAQUATIC VASCULAR PLANTDER
Part A: Overview
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Data Evaluation Record on the Effects of [Chemical] on Aquatic Vascular Plant [Species or Grouping]
Reproduction: Briefly summarize reproduction endpoint results (if any). For multi-eenerational studies, copy and vaste Table
A.II.5 below for each generation with reproductive effects data.
General Notes: Comment on concentrations response relations and slope of response ifprovided. Compare reproduction
endpoints with control treatment and/or the reference chemical.
Table A.II.5. Mean [Reproductive] Effect.
Mean [reproductive] effects for [generation] of [test organism] exposed to [test substance] for [test duration] under
[static/renewal/flow-through] conditions.
[Standard
[Standard
Deviation
Deviation
[Mean
or
[Mean
or
Treatment
Reproductive
Standard
Reproductive
Standard
(units)
Effectl
Error]
Effectl
Error]
Control
[11
[21
[31
[41
[51
[61
i
[ECxl
NOEC
LOEC
a Use superscript to identify the values reported to be significantly different from control.
U.S. EPA OWAQUATIC VASCULAR PLANTDER
Part A: Overview
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Data Evaluation Record on the Effects of [Chemical] on Aquatic Vascular Plant [Species or Grouping]
Sublethal Toxicity Endpoints: Include other sublethal effect(s), including unusual colors or shapes, or other signs of toxicity,
if any. Copy Table A.II.6 as needed to provide details for each sublethal effect obser\>ed.
General Notes: Briefly summarize obser\>ed sublethal effects otherwise not captured in the results table(s) below.
Table A.II.6. Mean [Sublethal] Effect.
Mean /Sublethal effect, (e.g., morphological changes, etc.)] in [test organism] during [test duration (acute/chronic)]
exposure to [test substance] under [static/renewal/flow-through] conditions.
[Mean Sublethal Response]
[Standard Deviation or
Treatment
(units)
Standard Error!
Control
rn
[21
[31
[41
[51
[61
i
[ECxl
NOEC
LOEC
a Use superscript to identify the values reported to be significantly different from control
U.S. EPA OWAQUATIC VASCULAR PLANTDER
Part A: Overview
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Data Evaluation Record on the Effects of [Chemical] on Aquatic Vascular Plant [Species or Grouping]
Reported Statistics: List and briefly summarize the statistical tests that were performed for each of the response parameters that
were analyzed (or, copy and paste statistical section from publication).
U.S. EPA OWAQUATIC VASCULAR PLANTDER
Part A: Overview
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Data Evaluation Record on the Effects of [Chemical] on Aquatic Vascular Plant [Species or Grouping]
Part B: Detailed Review
I. Materials and Methods
PROTOCOL/GUIDANCE FOLLOWED: If indicated by authors, provide protocol that was followed (e.g., U.S. EPA, ASTM,
OECD, Environment Canada, European Union, etc.).
DEVIATIONS FROM PROTOCOL: If authors report any deviations from the protocol noted above indicate here.
Study Design and Methods: Copy and paste methods section from publication.
U.S. EPA OWAQUATIC VASCULAR PLANTDER
Part B: Detailed Review
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Data Evaluation Record on the Effects of [Chemical] on Aquatic Vascular Plant [Species or Grouping]
TEST ORGANISM: Provide information in details and any relevant or related information or clarifications in remarks.
Parameter
Details
Remarks
Species:
Useful sites include:
• httDs://www.itis.eov/
• httt>s://www.fws.eov/endaneered/
• httt>s://www.fisheries.noaa.eov/find-st>ecies
Common or Group Name:
Scientific Name:
North American species?
Surrogate for North American
Taxon?
FIFRA 5 Species?
Is this species Threatened or
Endangered?
(Place A' if applicable)
Strain/Source:
• Obtained from laboratory culture or commercial
source. [1]
o Identification of clone is desirable [1]
• Obtained from unpolluted areas in the wild
o If collected from the field, plants should be
maintained in culture in the same medium as used
for testing for a minimum of eight weeks prior to
use.[2]
• Oryza sativa (rice) are obtained as seeds and can be
kept in a cool area for one year [5]
• Must originate from same source and population [4,
5]
• Should not be used:
o If contaminated by other organisms such as algae
and protozoa [2]
o If visible lesions or discoloration (chlorosis) [2]
o If large number of plants with single fronds [2]
Age of inoculum at Study Initiation:
• EPA recommends 7-12 day old cultures for Lemma
spp. [1]
• Oryza sativa (rice) approximately 8-10 cm tall 151
Was growth recorded at regular
intervals?
Yes No
Ifves, describe regular inten'als:
U.S. EPA OWAQUATIC VASCULAR PLANTDER
Part B: Detailed Review
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Data Evaluation Record on the Effects of [Chemical] on Aquatic Vascular Plant [Species or Grouping]
STUDY PARAMETERS: Provide information under Details and any relevant information of deficiencies in Remarks.
Complete for both Controlled Experiments and Field Studies/Obser\>ations.
For Both Controlled Experiments and Field Obsen'ations
Parameter
Details
Remarks
Number of Replicates per Treatment Group:
• EPA recommends 4 replicates for Lemna test [1]
• ASTM: 5 replicates for rice and other macroplrytes [51
Control(s):
Treatment(s):
Plants per Replicate/ Treatment Group:
• EPA recommends 3-5 plants/replicate for Lemna test
rn
Control(s):
Treatment(s):
Fronds per Plant (e.g., Lemna spp.)
• EPA recommends 3-4 fronds/plant for Lemna test [1]
Control(s):
Treatment(s):
Exposure Pathway:
(i.e., water, sediment, or mixed). Note: all other pathways
are unacceptable.
Exposure Duration:
• EPA recommends 7 days fox Lemna spp. Test [1, 2]
• ASTM: 2 weeks for rice and other macroplrytes [51
Observation Intervals:
• For Lemna test, EPA recommends every three days
during the test and at test termination [1]
• Should be an appropriate number of observations over
the exposure duration to establish the shape of the
toxicity curve
• Should allow for mathematical/statistical determination
of point estimates
Test Concentrations (remember units):
Recommended test concentrations include at least three
concentrations other than the control; four or more will
provide a better statistical analysis.
Nominal:
Measured:
Media measured in:
What analytic methods were used to measure
test concentrations?
What was the recovery of the test material?
What was the reporting limit of the analytical
method used to measure the test
concentrations?
Were standards used as part of the analytical
method?
U.S. EPA OWAQUATIC VASCULAR PLANTDER
Part B: Detailed Review
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Data Evaluation Record on the Effects of [Chemical] on Aquatic Vascular Plant [Species or Grouping]
CONTROLLED EXPERIMENT STUDY PARAMETERS: Provide information under Details and any relevant
information of deficiencies in Remarks. Complete for Controlled Experiments only.
Parameter
Details
Remarks
Acclimation/Culturing:
• Test plants should be from cultures maintained at test
conditions for an appropriate amount of time
o EPA recommends 8 weeks for Lemna. [1]
o Temperature change rate should not exceed 2°C
during acclimation or testing [1]
Duration:
Identify number of individuals excluded from testing and/or
analysis (if any):
Standard Nutrient Medium Used:
Yes No
• To avoid unnecessary stress and promote good
health:
o Organisms should not be crowded [1]
o Temperature should be maintained at optimal test
If no, provide details of composition of
the nutrient medium under the remarks
section
conditions for the test species, and temperature
variation should not exceed 2°C during
acclimation or testing (25 ±2°C for Lemna, 20-
30°C for other macrophytes) [1.5]
Water type:
o Lighting should be maintained on a light:dark
cycle and intensity at test conditions optimal for
the test species.
¦ Continuous light recommended for Lemna test
(4.200-6.700 lux) [1]
¦ Minimum of 16 hours light for other
macrophytes (30-40 W/nr) [5]
¦ Light intensity should be measured at test
initiation for each culture vessel at the level of
the culture solution. [1]
o pH of nutrient medium in which plant is cultured
should be maintained at optimal conditions for the
test species.
¦ EPA recommends pH of 6.5 for Lemna minor
tests. pH 7.5 fox Lemna gibba tests. [1]
n Growth medium (growth chelators):
¦ EPA recommends 20x-AAP growth medium for
L. gibba. and modified Swedish Standard (SIS)
growth medium for L. minor. [1]
¦ OECD recommends pH buffer addition for test
substances where pH stability is important. [2]
¦ OECD recommends less than 0.001 mmol/L
Temperature (°C):
s
O
+2
Light:dark cycle:
5
5
C
*
Salinity (for marine plants, ppt):
^3
-Si
"o
&
s
£
£
Chelator used:
Carbon source:
Dissolved Oxygen (mg/L):
chelator (if used). [2]
¦ EPA Guidelines note acceptable provided
concentrations not excessive for test chemicals
subject to interferences by the chelator (e.g.. >
200 jig/L EDTA for metals) [7]
¦ Were details provided if non-standard growth
medium used (yes/no)?
Health {any mortality, abnormalities
obser\>ed?)\
Acclimation followed published guidance?
Describe, if any
Yes No
If yes, indicate which guidance:
Test Type:
Acute
Partial Life Cycle
Chronic
Germination
Other (please remark):
U.S. EPA OWAQUATIC VASCULAR PLANTDER
Part B: Detailed Review
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Data Evaluation Record on the Effects of [Chemical] on Aquatic Vascular Plant [Species or Grouping]
Parameter
Details
Remarks
For Controlled Experiments Only
Test Vessel:
• Test chambers should be loosely covered [1]
o Test vessels/covers should not create shadows or
otherwise affect light levels. [1]
• Test chamber material:
o Should minimize sorption of test chemical from
water [1]
o Should not contain substances that can be leached
or dissolved in solution and free of substances that
could react with exposure chemical [1]
o May not contain substances that inhibit the growth
of test organisms. [1]
• Test chamber type:
n Erlenmeyer flasks, crystallizing dishes, glass petri
plates, or other container can be suitable.
¦ Sizes between 250-1,000 mL are suggested. All
vessels should be the same size. [1]
¦ Vessels for Lemna tests should be > 20 mm
depth and >100 mL volume. [1]
¦ Should be wide enough so that fronds in control
vessel do not overlap (Lemna tests). [1]
o Plastic pots with drainage holes in the bottom are
used for culturing and exposing other
macrophytes. [5]
• Size/volume should maintain acceptable biomass
loading rates (see below)
Material:
Briefly describe the test vessel here
Size:
Fill Volume:
Test Solution Delivery System/Method:
Static
Renewal; Interval:
Flow-through:
Delivered to water or sediment?
Test concentrations measured?
Sediment Used (For Rooted Plants):
• Origin (e.g., natural, artificial, field collected,
reference)
• Textural Classification (% sand, silt, clay)
• Organic Carbon (%)
• Geographic Location
• Chemical quality confirmed?
Source of Dilution Water:
• Freshwater hardness range should be <5 mg/L or
<10% of the average (whichever is greater) [4]
• Saltwater salinity range should be <2 g/kg or <20%
of the average (whichever is greater) [4]
• Dilution water must be characterized (natural surface
water, well water, etc.) [8]
o Distilled/deionized water without the addition of
appropriate salts should not be used. [7]
• Dilution water in which total organic carbon or
particulate matter exceed 5 mg/L should not be used
[7]
o LTnless data show that organic carbon or particulate
matter do not affect toxicity. [71
Dilution Series (e.g., 0.5x, 0.6x, etc.):
• 0.667x or 0.5x is recommended. [1]
• <0.25x not recommended. [1]
Water Pretreatment
Yes No
Intervals of water quality measurement:
U.S. EPA OWAQUATIC VASCULAR PLANTDER
Part B: Detailed Review
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Data Evaluation Record on the Effects of [Chemical] on Aquatic Vascular Plant [Species or Grouping]
Parameter
Details
Remarks
For Controlled Experiments Only
Dilution Water Parameters:
Measured at the beginning of the experiment or
averaged over the duration of the experiment (details of
water quality parameters measured in test solutions
should be included under the results section)
Recommendations:
• pH
o 6.5 for L. minor tests and 7.5 for L. gibba tests. [1]
o Recommend measuring at beginning and end of
test. [1]
• Temperature
o EPA and OECD recommend 24-25 °C (±2 °C) for
Lemna spp. tests
¦ Can be measured in growth medium of extra
chambers during test,
n ASTM recommends 20-30°C for other
macrophytes [5]
Dissolved Oxygen (mg/L):
Temperature (°C):
Light:dark cycle:
pH (test initiation):
pH (test termination):
Hardness (mg/L as CaCCb):
Salinity (for marine plants, ppt):
Total Organic Carbon (mg/L):
Dissolved Organic Carbon (mg/L):
Chelator used:
Carbon source:
Aeration or Agitation: (Describe if yes)
• Aeration not recommended unless appropriate for
the test substance [71
Yes No
Describe Preparation of Test
Concentrations:
(Indicate how test material was added to the growth
medium (e.g., added directly or used stock solution)
Test Chemical Solubility in Water:
List units and conditions (e.g., 0.01% at 20°C)
Were concentrations in water or nutrient
medium verified by chemical analysis?
Measured test concentrations should be reported in
Table A.II.2 above.
Yes No
Indicate media:
Were test concentrations verified by
chemical analysis in tissue?
Measured test concentrations can be verified in test
organism tissue alone if a dose-response relationship is
observed
Measured test concentrations should be reported in
Table A.II.2 above.
Yes No
Indicate tissue type:
If test concentrations were verified in test organism
tissue, was a dose-response relationship observed?
Were stability and homogeneity of test
material in water/nutrient medium
determined?
Yes No
U.S. EPA OWAQUATIC VASCULAR PLANTDER
Part B: Detailed Review
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Data Evaluation Record on the Effects of [Chemical] on Aquatic Vascular Plant [Species or Grouping]
Parameter
Details
Remarks
For Controlled Experiments Only
Solvent/Vehicle Type:
• When used, a carrier solvent should be kept to a
minimum concentration. [4]
• Should not affect either survival or growth of test
organisms (e.g., N, N-dimethyl-formamide
recommended for Lemma spp. instead of acetone). [1]
• Should be reagent grade or better [4]
• Should not exceed 0.1 ml/L, unless it was shown that
higher concentrations do not affect toxicity 111
Negative Control:
Yes No
Reference Toxicant Testing:
Yes No
If Yes, identify substance:
Other Control: If any (e.g. solvent control)
Biomass Loading Rate:
Feeding:
• Nutrient medium added during renewal tests?
Yes No
Lighting:
• Lighting conditions should be consistent with
conditions during culturing/acclimation [1, 2]
• Should be measured in the growth chamber at the
same distance from light source as test plants [1]
• Should be measured before, after, and at least once
during test [1]
• Day:night period should be appropriate to the test
species (e.g., continuous lighting recommended for
Lemna spp.) [1]
• Fluorescent lighting recommended [1, 2]
• OECD recommends 6,500-10,000 lux (85-125
|iE/nr/s) foe Lemna spp. test. [2]
• EPA recommends 4,200-6,700 lux (57-90 |iE/nr/s)
fox Lemna spp. test. [1]
• Should not change by more than 15% during test [2]
¦ Measure daily if suspected
Study Design/Methods Classification: (Place Xbv One Based on 0\>erall Use)
Provide details of Major or Minor Deficiencies/Concerns with Study Design in Associated Sections of Part A: Overview
This classification should be taken into consideration for the overall study classification for aquatic life criteria development in Part A.
Study Design Acceptable for Quantitative Use
Study Design Acceptable for Qualitative Use
Study Design Not Acceptable for Use
Additional Notes: Provide additional considerations for the classification of study use based on the study design.
U.S. EPA OWAQUATIC VASCULAR PLANTDER
Part B: Detailed Review
Page 130 of 178
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Data Evaluation Record on the Effects of [Chemical] on Aquatic Vascular Plant [Species or Grouping]
OBSERVATIONS: Provide information under Details and any relevant information in Remarks. This information should be
consistent with the Results Section in Part A.
Parameter
Details
Remarks
Parameters measured including sublethal
effects/toxicity symptoms:
Growth
• ECX, ICX based on growth inhibition (e.g., biomass,
frond reduction, etc.) relative to control. [1, 5]
Reproduction
• Seed germination, seedling production, etc. [5]
Other Endpoints
o Chlorophyll a, pigment content, etc. [51
List parameters:
Was control growth acceptable?
• Change in frond number, area, size (e.g., Lemna)!
• How was acceptable control growth determined?
Yes No
Were controls acceptable?
• Relative increase in frond number or size (e.g.,
Lemna) [1]
• Growth rate, etc.
Yes No
If yes, describe justification provided:
Were individuals excluded from the
analysis?
Yes No
Ifves, describe justification provided:
Additional observations
• Unusual colors [1]
• Differences in chloroplast morphology [1]
• Changes in test solution appearance (e.g., clarity,
films, precipitates, etc.) [1]
• Flocculation, clumping, adhering to test containers
[1]
• Was crowding observed? Ill
Was water quality in test chambers
acceptable?
• If appropriate, describe any water quality issues
(e.g., EPA and OECD recommend temperature of
24-25 °C (±2 °C) for Lemna spp. tests) [1. 21
Yes No
Availability of concentration-response
data:
• Were treatment level concentration-response
data included? (specify endpoints in remarks)
• Were replicate level concentration-response
data included? (specify endpoints in remarks)
• How was concentration-response data
presented? (check all that apply)
Yes No
Yes No
Tables
Graphs
Supplemental Files
• Were concentration-response data estimated
from graphs study publication or supplemental
materials?
Yes No
Ifves, indicate software used:
• Should additional concentration-response data
be requested from study authors?
If concentration-response data are available, complete
Verification of Statistical Results (Part C).
Yes No
Requested by:
Request date:
Date additional data received:
U.S. EPA OWAQUATIC VASCULAR PLANTDER
Part B: Detailed Review
Page 131 of 178
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Data Evaluation Record on the Effects of [Chemical] on Aquatic Vascular Plant [Species or Grouping]
Part C: Statistical Verification of Results
I. Statistical Verification Information: Report the statistical methods (e.g., EPA TRAP, BMDS, R, other) used to verify the
reported study or test results for the five (5) most sensitive genera and sensitive apical endpoints. Report the statistical methods (e.g.,
EPA TRAP, BMDS, R, other) used to verify the reported study or test results and calculate point estimates (including for tests where
such estimates were not provided). If concentration-response data were provided, include here along with all figures and tables
associated with the statistical verification of the results and toxicity values. If values for the LC50, LT50 and NOEC are greater than the
highest test concentration, use the "> " symbol.
Primary Statistical Reviewer: Date: EPA Contractor (Place X)
Secondary Statistical Reviewer: Date: EPA Contractor (Place X)
(At least one reviewer should be from EPA for sensitive taxa)
Endpoint(s) Verified:
Additional Calculated Endpoint(s):
Statistical Method (e.g., TRAP, BMDS, R, other):
II. Toxicity Values: Include confidence intervals if applicable
NOEC:
LOEC:
MATC:
ECs:
EC10:
EC20:
ECso or LCso
Dose-Response Curve Classification: (PlaceXby One)
This classification should be taken into consideration for the overall study classification for aquatic life criteria development in Part A
Dose-Response Curve Acceptable for Quantitative Use
Dose-Response Curve Acceptable for Qualitative Use
Dose-Response Curve Not Acceptable for Use
Summary of Statistical Verification: Provide summary of methods used in statistical verification.
Additional Notes:
Attachments:
• Provide attachments to ensure all data used in Part C is captured, whether from study results reported in the publication
and/or from additional data requested from study authors
o Data from study results of the publication should be reported in Results section of Part A
o Additional data provided upon request from study authors should be reported in Table C.II.l below and original
correspondence with study authors should be included as attachments
• Model assessment output (including all model figures, tables, and fit metrics)
• Statistical code used for curve fitting
U.S. EPA OWAQUATIC VASCULAR PLANTDER
Part C: Statistical Verification of Results
Page 132 of 178
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Data Evaluation Record on the Effects of [Chemical] on Aquatic Vascular Plant [Species or Grouping]
Additional Data Used in Response-Curve: Provide all data used to fit dose-response cur\'e not captured in Results section of PER above in Part A, rows as needed. First
row in italicized text is an example.
Table C.II.1 Additional Data Used in Dose-Response Curve.
Curve ID
Species
Endpoint
Treatment
Replicate
[Standard
Deviation
or Standard
Error!
# of
Survivors
Na
ka
na
Response
Response
Unit
Cone
Cone units
Alchronicl
Ceriodaphnia dubia
#of
voting/female
0
6
10
10
1
18
count
0.03
mg/L
a N = number of individuals per treatment; k = number of replicates per treatment level; n = number of individuals per replicate
U.S. EPA OWAQUATIC VASCULAR PLANTDER
Part C: Statistical Verification of Results
Page 133 of 178
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Data Evaluation Record on the Effects of [Chemical] on Aquatic Vascular Plant [Species or Grouping]
Part I): References to Test Guidance
35. U.S. EPA. 2012. OCSPP 850.4400: Aquatic plant toxicity test using Lemna spp. Ecological effects test
guidelines. Office of Chemical Safety and Pollution Prevention. EPA 712-C-008. January 2012.
36. OECD. 2002. Test No. 221. Lemna sp. Growth Inhibition Test. OECD Guidelines for the Testing of
Chemicals, Section 2, OECD Publishing, Paris. 22 pp.
37. U.S. EPA. 2016. OCSPP 850.1000: Background and special consideration-tests with aquatic and
sediment-dwelling fauna and aquatic microcosms. Ecological effects test guidelines. Office of Chemical
Safety and Pollution Prevention. EPA 712-C-16-014. October 2016.
38. ASTM Standard E 739, 1980. 2002. Standard guide for conducting acute toxicity tests on test materials
with fishes, macroinvertebrates, and amphibians. ASTM International, West Conshohocken, PA.
39. ASTM Standard E 1841-04. 2012. Standard Guide for Conducting Renewal Phytotoxicity Tests with
Freshwater Emergent Macrophytes. ASTM International, West Conshohocken, PA. 10 pp.
40. U.S. EPA. 2012. OCSPP 850.4600: Rhizobium-legume toxicity. Ecological effects test guidelines.
Office of Chemical Safety and Pollution Prevention. EPA 712-C-004. January 2012.
41. Stephan, C.E., D.I. Mount, D.J. Hansen, J.H. Gentile, G.A. Chapman and W.A. Brungs. 1985.
Guidelines for Deriving Numerical National Water Quality Criteria for the Protection of Aquatic
Organisms and their Uses. PB85-227049. National Technical Information Service, Springfield, VA.
42. Stephan, C.E. 1995. Review of results of toxicity tests with aquatic organisms. Draft. U.S. EPA, MED.
Duluth, MN. 13 pp.
U.S. EPA OWAQUATIC VASCULAR PLANTDER
Part I): References to Test Guidance
Page 134 of 178
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Data Evaluation Record on the Effects of [Chemical] on Amphibian [Species]
Attachment I Amphibian Data Evaluation Record (DER) Template
(September 2024)
-Page 135 of 178-
-------
Data Evaluation Record on the Effects of [Chemical] on Amphibian [Species]
Part A: Overview
I. Test Information
Chemical name:
CAS name:
Purity:
Solubility in Water (units):
CAS Number:
Storage conditions:
Controlled Experiment
(imanipulated)
Primary Reviewer:
Secondary Reviewer:
Field Study/Observation
(inot manipulated)
Date:
Date:
(At least one reviewer should be from EPA for sensitive taxa)
{Place X by One)
EPA
EPA
Contractor (Place X by One)
Contractor (Place X by One)
Citation: Indicate: author (s), year, study title, journal, volume, and pages.
(e.g., Fort, D. J., E.L. Stover, J. A. Bantle, J.N. Dumont and R. A. Finch. 2001. Evaluation of a reproductive toxicity assay using Xenopus laevis\ boric acid, cadmium and
ethylene glycol monomethyl ether. J. Appl. Toxicol. 2: 41-52.)
Companion Papers: Identify any companion papers associated with this paper using the citation format above.
Were other DERs completed for Companion Papers?
Yes
(Ifyes, list file names of
No DERs below)
Study Classification for Aquatic Life Criteria Development: Place X by One Based on Highest Use
Acceptable for Quantitative Use
Acceptable for Qualitative Use
Not Acceptable for Use/Unused
General Notes: Provide any necessary details regarding the study's use classification for all pertinent endpoints,
including non-apical endpoints within the study (e.g., note all study classifications for each endpoint if the use varies)
• Major Deficiencies (note any stated exclusions): Check all that apply. Checking any of these items make the
study "Not Acceptable for Use"
• No Controls (for controlled
experiments only)
Mixture (for controlled experiments only)
Excessive Control Mortality (> 10% for acute and > 20% for chronic)
^ ^ , i , • . • Bioaccumulation: steady state not
Diet not adequately characterized • , ,
reached
Dermal or Injection Exposure Pathway • •
Review paper or previously published without modification
Other: (if any, list here, e.g. use of distilled water)
U.S. EPA OWAMPHIBIANDER
Part A: Overview
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Data Evaluation Record on the Effects of [Chemical] on Amphibian [Species]
• POTENTIAL CHEMICAL MIXTURES: Describe any potential chemicals mixtures as characterized by study
authors (including any confirmation of chemical mixtures).
General Notes:
Minor Deficiencies: List and describe any minor deficiencies or other concerns with test. These items may make the study
"Acceptablefor Qualitative Use" (exceptions may apply as noted)
•
• DESCRIPTION OF UNMEASURED TEST CONCENTRATIONS: Describe concerns with unmeasured test
concentrations and the influence of the study classification.
• DESCRIPTION OF CONCERNS WITH DILUTION WATER: Describe concerns with characterization of
and/or deficiencies with dilution water (e.g., uncharacterized stream or lake water, potential presence of unknown
containments, high organic content, extreme hardness, pH, etc.).
For Field Studies/Observations: A field study/observation may be considered "Acceptable for Quantitative Use" if it
consisted of a range of exposure concentrations and the observed effects are justifiably contributed to a single chemical
exposure
Mixture (observed effects not justifiably contributed to single chemical exposure)
Uncharacterized Reference Sites/Conditions
POTENTIAL CHEMICAL MIXTURES PRESENT AT SITE: Describe any potential chemicals mixtures present at
the site as characterized by study authors (including any confirmation of chemicals present at study site).
EXPOSURE VARIABILITY ACROSS STUDY SITE(S): Describe any exposure variability across study
site(s) as characterized by study authors (i.e., description of study design with reference and contaminated sites).
General Notes:
Reviewer's Comments: Provide additional comments that do not appear under other sections of the template.
U.S. EPA OWAMPHIBIANDER
Part A: Overview
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Data Evaluation Record on the Effects of [Chemical] on Amphibian [Species]
ABSTRACT: Copy and paste abstract from publication.
SUMMARY: Fill out for the most sensitive endpoint (apical and or non-apical) and modify as needed. If study is classified as ''Not Acceptable for
Use " DO NOT complete summary tables.
Acute:
Species
(lifestage)
Exposure
Method3
Test
duration
Chemical
/ Purity
|)H
Temp.
(°C)
Hardness
(mg/L as
CaCOi)
or
Salinity
(ppt)
DOC
(mg/L)
Relative
Humidity
Effect
Reported
Effect
Concentration
(mg/L)
Verified Effect
Concentration1"
(mg/L)
Classification
Quantitative /
Qualitative
a S=static, R=renewal, F=flow-through, U=unmeasured, M=measured, T=total, D=dissolved, Diet=dietary, MT=maternal transfer, FETAX=Frog Embryo Teratogenesis Assay -
Xenopus
b Verification following completion of Part C of the DER
Chronic:
Species
(lifestage)
Exposure
Method"
Test
duration
Chemical
/ Purity
pH
Temp.
(°C)
Hardness
(mg/L as
CaCOi)
or
Salinity
(ppt)
DOC
(mg/L)
Relative
Humidity
Chronic
Limits
Reported
Chronic
Value
(mg/Lor
ug/g)
Verified
Chronic
Valueb
(mg/L or
ug/g)
Chronic
Value
Endpoint
Classification
Quantitative /
Qualitative
a S=static, R=renewal, F=flow-through, U=unmeasured, M=measured, T=total, D=dissolved, Diet=dietary, MT=maternal transfer, FETAX=Frog Embryo Teratogenesis Assay -
Xenopus
b Verification following completion of Part C of the DER
U.S. EPA OW AMPHIBIAN DER
Part A: Overview
Page 138 of 178
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Data Evaluation Record on the Effects of [Chemical] on Amphibian [Species]
II. Results
Provide results as reported in the publication (including supplemental materials). Include screen shots of tables and/or figures
reporting results from the article following tabulated data table in each associated results section for all studies. Complete tabulated
data tables for all studies for studies marked "Acceptable for Quantitative Use" and "Acceptable for Qualitative Use".
Water Quality Parameters: If only general summary data of water quality parameters is provided by study authors (i.e., no
specific details of water quality parameters on a treatment level is provided), summarize any information regarding water quality
parameters under General Notes below.
General Notes: For aquatic life criteria development, measured water quality parameters in the treatments nearest the toxicity
test endpoint(s), e.g., LC50, EC20, etc., are most relevant.
Table A.II.1. Measured Water Quality Parameters in Test Solutions.
Dissolved oxygen, temperature, pH and [other parameters (hardness, salinity, DOC)] in test solutions during the /117-day
exposure of [test organism] to [concentration of treatment(s)] of [test substance] under [static renewal/flow-through]
conditions.
Parameter
Treatment
Mean
Range
Dissolved
oxygen
(% saturation or
mg/L)
[11
[2]
J
J
Temperature (C)
[11
[21
J
J
pH
[11
[21
J
J
Other (e.g.,
hardness,
salinity, DOC)
[11
[21
J
i
U.S. EPA OWAMPHIBIANDER
Part A: Overview
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Data Evaluation Record on the Effects of [Chemical] on Amphibian [Species]
Chemical Concentrations: Summarize the concentration verification data from test solutions/media. Expand table to include
each measured concentration data for each media type (i.e., muscle, liver, blood, etc.).
General Notes: Provide any necessary detail regarding the measured concentrations, including any identified cause for
substantial differences between nominal and measured concentrations, if samples were collected on separate days (and if so provide
details), and any potential cross contamination.
Table A.II.2. Measured (and Nominal) Chemical Concentrations in Test Solutions/Media.
[Analytical Method] verification of test and control concentrations during an [X]-day exposure of [test organism] to [test
substance] under [static renewal/flow-through] conditions.
[Mean]
Number of
[Standard
Nominal
Measured
Samples
Deviation or
Concentration
Concentration
Number of
Non-
Below Non-
Standard
Treatment
(units)
(units)
Samples
Detect3
Detect
Error]
Range
Control
[1]
[2]
[3]
[4]
[5]
[6]
./'
aNon-Detect: 0 = measured and detected; l=measured and not detected; if not measured or reported enter as such
U.S. EPA OWAMPHIBIANDER
Part A: Overview
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Data Evaluation Record on the Effects of [Chemical] on Amphibian [Species]
Mortality: Briefly summarize mortality results (if any).
General Notes: Comment on concentrations response relations and slope of response ifprovided. Compare mortality with control
treatment and/or the reference chemical.
Table A.II.3. Mean Percent [Mortality or Survival].
Mean percent mortality [or number of immobilized] or survival of [test organism] exposed to [test substance] for [test
duration] under [static/renewal/flow-through] conditions. Superscript(s) used to identify the values reported to be
significantly different from control as p value of [0.05/ or any other provided by authors].
Treatment
[Mean % Mortality]
Sample Size
[Standard Deviation
or Standard Error]
Control
[1]
[2]
[3]
[4]
[5]
[6]
[LCx]
NOEC
LOEC
a Use superscript(s) to identify the values reported to be significantly different from control.
U.S. EPA OWAMPHIBIANDER
Part A: Overview
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Data Evaluation Record on the Effects of [Chemical] on Amphibian [Species]
Growth: Briefly summarize growth results (if any).
General Notes: Comment on concentrations response relations and slope of response ifprovided. Compare growth endpoints with
control treatment and/or the reference chemical.
Table A.II.4. Mean [Growth].
Mean growth [length and/or weight] of [test organism] exposed to [test substance] for [test duration] under
[static/renewal/flow-through] conditions. Superscript(s) used to identify the values reported to be significantly different from
control as p value of [0.05/ or any other provided by authors].
[Standard
[Standard
Mean
[Standard
Deviation
Mean Growth
Deviation
Percent
Deviation
Mean Time to
or
[Length/Weight]
Sample
or Standard
Change in
Sample
or Standard
[Developmental
Sample
Standard
Treatment
(units)
Size
Error!
[Biomassl
Size
Error!
Stagebl
Size
Error!
Control
[11
[21
[31
[41
[51
[61
./
[ECxl
NOEC
LOEC
a Use superscript(s) to identify the values reported to be significantly different from control.
b Developmental staging can be general (e.g., larval, metamorphosis, etc.) or it can be specific. Xenopus are staged
using the Nieuwkoop and Faber (1994) system, anurans are staged using the Gosner (1960) system, and salamanders
are staged using the Harrison (1969) system.
Nieuwkoop, P.D. and J. Faber. 1994. Normal table of Xenopus laevis (Daudin). Garland Publishing Inc,
New York.
Gosner, K.L. 1960. A simplified table for staging anuran embryos and larvae with notes on identification.
Herpetologica. 16(3): 183-190.
Harrison R. 1969. Harrison stages and description of the normal development of the spotted salamanders,
Ambvstoma punctatum (Limm.). Pages 44-66 in Harrison R, ed. Organization and Development of
the Embryo. New Haven, CT: Yale University Press.
U.S. EPA OWAMPHIBIANDER
Part A: Overview
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Data Evaluation Record on the Effects of [Chemical] on Amphibian [Species]
Reproductive: Briefly summarize reproduction endpoint results (if any). For multi-eenerational studies, copy and vaste Table
A.II.5 below for each generation with reproductive effects data.
General Notes: Comment on concentrations response relations and slope of response ifprovided. Compare reproduction
endpoints with control treatment and/or the reference chemical.
Table A.II.5. Mean [Reproductive] Effect.
Mean [reproductive] effects for [generation] of [test organism] exposed to [test substance] for [test duration] under
[static/renewal/flow-through] conditions. Superscript(s) used to identify the values reported to be significantly different from
control as p value of [0.05/ or any other provided by authors].
[Mean
[Standard
[Mean
[Standard
[Standard
Treatment
Number of
Sample
Deviation or
Percent
Sample
Deviation or
[Mean Number of
Sample
Deviation or
(units)
Eggsl
Size
Standard Error!
Hatchl
Size
Standard Error!
Larva/Metamorphosedl
Size
Standard Error!
Control
rn
T21
pi
K1
PI
rei
/
|ECxl
NOEC
LOEC
a Use superscript(s) to identify the values reported to be significantly different from control.
U.S. EPA OWAMPHIBIANDER
Part A: Overview
Page 143 of 178
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Data Evaluation Record on the Effects of [Chemical] on Amphibian [Species]
Sublethal Toxicity Endpoints: Include other sublethal effect(s), including behavioral abnormalities or other signs of toxicity,
if any. Copy Table A.II.6 as needed to provide details for each sublethal effect obser\>ed.
General Notes: Briefly summarize obser\>ed sublethal effects otherwise not captured in the results table(s) below.
Table A.II.6. Mean [Sublethal] Effect.
Mean /Sublethal effect, (e.g., beha\>ioral abnormalities, etc.)] in [test organism] during [test duration (acute/chronic)]
exposure to [test substance] under [static/renewal/flow-through] conditions. Superscript(s) used to identify the values
reported to be significantly different from control as p value of [0.05/ or any other provided by authors].
Treatment
[Mean Sublethal Response]
(units)
Sample Size
[Standard Deviation or
Standard Error]
Control
[1]
[2]
[3]
[4]
[5]
[6]
[ECx]
NOEC
LOEC
a Use superscript(s) to identify the values reported to be significantly different from control
U.S. EPA OWAMPHIBIANDER
Part A: Overview
Page 144 of 178
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Data Evaluation Record on the Effects of [Chemical] on Amphibian [Species]
Reported Statistics: Copy and paste statistical section from publication.
U.S. EPA OWAMPHIBIANDER
Part A: Overview
Page 145 of 178
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Data Evaluation Record on the Effects of [Chemical] on Amphibian [Species]
Part B: Detailed Review
I. Materials and Methods
Protocol/Guidance Followed: Indicate ifprovided by authors.
Deviations from Protocol: If authors report any deviations from the protocol noted above indicate here.
Study Design and Methods: Copy and paste methods section from publication.
TEST ORGANISM: Provide information in Details and any relevant or related information or clarifications in Remarks.
Parameter
Details
Remarks
Species:
Useful sites include:
• httDs://www.itis.eov/
• httt>s://www.fws.eov/endaneered/
• httt>s://www.fisheries.noaa.eov/fmd-st>ecies
Common Name:
Scientific Name:
Order Name:
Family Name:
North American species?
Surrogate for North American
Taxon?
Is this species Threatened or
Endangered?
(Place A' if applicable)
Strain/Source:
• Wild caught from unpolluted areas [2]
o Quarantine for at least 14 days or until they are
disease free, before acclimation [2]
• Must originate from same source and population [2]
• Should not be used:
o If appeared stressed, such as discoloration or
unusual behavior [2]
¦ Should avoid crowding or rapid changes in
temperature or water quality to avoid stress [3]
o If more than 5% die during the 48 hours before
test initiation [2]
o If they were used in previous test treatments or
controls [4]
• No treatments of diseases may be administered:
o Within 16-hr of field collection [2]
o Within 10 days or testing or during testing 121
Age at Study Initiation:
Acute:
• Young larvae should be used whenever possible [2]
FETAX:
• (Xenopus laevis)- embryos (cysteine-treated to
remove jelly coat) [5]
Chronic:
• Partial life-cycle test:
o Immature juveniles at least 2 months prior to
active gonad development [4]
• Xenopus LAGDA test: newly spawned embryos
(Nieuwkoop and Faber (NF) stage 8-10), also
cysteine-treated to remove jelly coat [6]
• Xenopus AMAtest: NF stage 51 [31
Embryonic
Larval
Juvenile
Adult
Specify stage if provided:
Was body weight or length recorded at
test initiation?
For field observations, was body weight measured in a
consistent manner (e.g., during blood sample collection)
detailed in methods?
Yes No
Was body weight or length recorded at
regular intervals?
Yes No
Ifves, describe regular inten'als:
U.S. EPA OWAMPHIBIANDER
Part B: Detailed Review
Page 146 of 178
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Data Evaluation Record on the Effects of [Chemical] on Amphibian [Species]
STUDY PARAMETERS: Provide information under Details and any relevant information of deficiencies in Remarks.
Complete for both Controlled Experiments and Field Studies/Obser\>ations.
Parameter
Details
Remarks
Number of Replicates per Treatment Group:
• FETAX: recommends 2 replicates per test
concentration and 4 replicates for controls [5]
• LAGDA: recommends 4 replicates per test
concentration and 8 replicated for controls [6]
• AMA: recommends at least 4 replicates per
treatment/control [3]
• At least 2 replicates/treatment recommended for
chronic tests [2]
• At least 2 replicates/treatment recommended for
chronic tests [71
Control(s):
Treatment(s):
Number of Organisms per Replicate/
Treatment Group:
• Unless otherwise specified, at least 10
organisms/treatment recommended [7]
• FETAX: 20 or 25 (A", laevis embryos) per replicate
[5]
• LAGDA: recommends 20 animals (A", laevis
embryos)/tank (replicate) at exposure initiation and
10 animals (juveniles)/tank (replicate) after NF stage
66 to exposure termination [6]
• AMA: 20 (A" laevis embryos) per replicate at test
initiation. 5 indiv/replicate randomly removed after
7d for growth & development measurements [31
Control(s):
Male:
Female:
Treatment(s):
Male:
Female:
Exposure Pathway:
(i.e., water, sediment, or diet). Note: all other pathways
(e.g., dermal, injection) are unacceptable.
Co
£
•2
c
Co
O
"55
s:
C3
-*2
s:
.§
£
Cl
t
Exposure Duration:
Acute
• Should be 96 hours [4]
FETAX
• Must be 96 hours [5]
Chronic
• Partial life-cycle tests:
o Begin with embryos or newly hatched tadpoles,
continue through completed metamorphosis
» Larval growth and development assay (LAGDA):
from NF stage 8-10 to ten weeks after the median
time to NF stage 62 in water and/or solvent control
group (maximum 17 weeks) [6]
» Amphibian metamorphosis assay (AMA): 21-day
exposure beginning with NF stage 51 tadpoles.
Final NF stage is one of the measured endpoints [3]
Acute
Partial Life Cycle
Larval Growth and
Development Assay (LAGDA)
Amphibian Metamorphosis
Assay (AMA)
Other (please remark):
Observation Intervals:
• No specific guidance on number of observation
intervals for changes in survival, deformities,
behavior, etc. of study organisms during a test.
Should be an appropriate number of observations over
the study to ensure water quality is being properly
maintained [21
o
s:
Cj
Observations:
Parental:
(e.g., mortality, body weight, mean feed consumption)
o
oq
£
Offspring:
(e.g., mortality, time to metamorphosis, snout-vent lent,
external abnormalities)
U.S. EPA OWAMPHIBIANDER
Part B: Detailed Review
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Data Evaluation Record on the Effects of [Chemical] on Amphibian [Species]
For Both Controlled Experiments and Field
Obsen'ations
Parameter
Details
Remarks
Test Concentrations (remember units):
Recommended test concentrations include at least three
concentrations other than the control; four or more will
provide a better statistical analysis.
Nominal:
Measured:
Media measured in:
What analytic methods were used to
measure test concentrations?
What was the recovery of the test material?
What was the reporting limit of the
analytical method used to measure the test
concentrations?
Were standards used as part of the analytical
method?
U.S. EPA OWAMPHIBIANDER
Part B: Detailed Review
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Data Evaluation Record on the Effects of [Chemical] on Amphibian [Species]
CONTROLLED EXPERIMENT STUDY PARAMETERS: Provide information under Details and any relevant
information of deficiencies in Remarks. Complete for Controlled Experiments only.
Parameter
Details
Remarks
Acclimation/Holding:
• If aquatic phase, should be placed in a tank along
Duration:
with the water in which they were transported [2]
Water should be changed gradually to 100% dilution
water (usually 2 or more days) [2]
o For wild-caught animals, test water temperature
should be within 5°C of collection water
Feeding:
Water type:
temperature [2]
o Temperature change rate should not exceed 3°C
Temperature (°C):
within 72 hours [2]
• To avoid unnecessary stress and promote good
health:
o Organisms should not be crowded [2]
o Water temperature variation should be limited
Dissolved Oxygen (mg/L):
Health {any mortality observed?):
£
o
*3
(e.g., <3°C in any 12 hour period) [2]
o Water dissolved oxygen:
¦ Maintain between 60-100% saturation [2]
¦ Continuous gentle aeration if needed [2]
o Un-ionized ammonia concentration in holding and
acclimation waters should be <35 |ig/L [21
Number of individuals excluded from
analysis:
Acclimation followed published guidance?
Describe, if any
Yes No
£
£
If yes, indicate which guidance:
Cl
"3
s:
£
Test Type:
Acute
Partial Life Cycle
Larval Growth and
Development Assay (LAGDA)
Amphibian Metamorphosis
Assay (AMA)
Other (please remark):
Test Vessel/Enclosure Size:
• Test chambers should be loosely covered [2]
• Test chamber material:
o Should minimize sorption of test chemical from
water [2]
Material:
Briefly describe the test vessel here
o Should not contain substances that can be leached
or dissolved in solution and free of substances that
could react with exposure chemical [2]
o Glass, No. 316 stainless steel, nylon screen and
perfluorocarbon (e.g. Teflon) are acceptable [1,2]
¦ Other materials recommended for specific
chemicals should be used when appropriate
(e.g., polyethylene for PFAS chemicals [8]
o Rubber, copper, brass, galvanized metal, epoxy
glues, lead and flexible tubing should not come
into contact with test solution, dilution water or
stock [1,2]
• Size/volume should maintain acceptable biomass
loading rates (see below) [2]
Size:
Fill Volume:
U.S. EPA OWAMPHIBIANDER
Part B: Detailed Review
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Data Evaluation Record on the Effects of [Chemical] on Amphibian [Species]
For Controlled Experiments Only
Test Solution Delivery System/Method:
• Flow-through preferred for some highly volatile,
hydrolysable or degradable materials [4]
o Concentrations should be measured often enough
using acceptable analytical methods [4]
• Chronic exposures:
o Flow-through, measured tests required [4]
• LAGDA: designed using a flow through system [6]
Test Concentrations Measured
Yes No
Test Solution Delivery System:
Static
Renewal
Indicate Inten'al:
Flow-through
Indicate Type of Diluter:
Dilution Water Source & Characteristics:
• Freshwater hardness range should be <5 mg/L or
<10% of the average (whichever is greater) [2]
• Saltwater salinity range should be <2 g/kg or <20%
of the average (whichever is greater) [2]
• Dilution water must be characterized (natural surface
water, well water, etc.) [4]
o Distilled/deionized water without the addition of
appropriate salts should not be used [4]
• Dilution water in which total organic carbon or
particulate matter exceed 5 mg/L should not be used
[4]
o Unless data show that organic carbon or particulate
matter do not affect toxicity [4]
• FETAX: FETAX solution preferred [5]
• LAGDA: any water that permists normal growth and
development of A". laevis (e.g., spring water or
charcoal filtered tap water) [61
Dilution Series (e.g., 0.5x, 0.6x, etc.):
Test Conditions/
Dilution Water Parameters:
Measured at the beginning of the experiment or
averaged over the duration of the experiment (details of
water quality parameters measured in test solutions
should be included under the results section)
• FETAX: 24 ± 2°C recommended [5]
• LAGDA: 21 ± 1°C recommended [6]
• FETAX: pFl should be between 6.5 and 9.0 [5]
• LAGDA: pFl should be between 6.5 and 8.5 [6]
• LAGDA: D.O. should be >40% of air saturation 161
Dissolved Oxygen (mg/L):
pH:
Temperature (°C):
Relative Humidity:
Hardness (mg/L as CaCCh):
Salinity (ppt):
Total Organic Carbon (mg/L):
Dissolved Organic Carbon (mg/L):
Aeration:
• Acceptable to maintain dissolved oxygen at 60-100%
saturation at all times [2]
• Avoid aeration when testing highly oxidizable,
reducible and volatile materials
• Turbulence should be minimized to prevent stress on
test organisms and/or re-suspend fecal matter [2]
• Aeration should be the same in all test chambers at all
times [21
Yes No
Describe Preparation of Test
Concentrations (e.g., water exposure,
diet):
Test Chemical Solubility in Water:
List units and conditions (e.g., 0.01% at 20X1)
U.S. EPA OWAMPHIBIANDER
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Data Evaluation Record on the Effects of [Chemical] on Amphibian [Species]
Parameter
Details
Remarks
Were concentrations in water or diet
verified by chemical analysis?
Measured test concentrations should be reported in Table
A.II.2 above.
Yes No
Indicate media:
Were test concentrations verified by
chemical analysis in tissue?
Measured test concentrations can be verified in test
organism tissue (e.g., blood, liver, muscle) alone if a
dose-response relationship is observed.
Measured test concentrations should be reported in Table
A.II.2 above.
Yes No
Indicate tissue type:
If test concentrations were verified in test organism
tissue, was a dose-response relationship observed?
Were stability and homogeneity of test
material in water/diet determined?
Yes No
Was test material regurgitated/avoided?
Yes No
£:
O
*3
Test Chemical Solubility in Water:
• List units and conditions (e.g.. 0.01% at 20°C)
s:
£
Cl
tq
"3
s:
£
Solvent/Vehicle Type:
• When used, a carrier solvent should be kept to a
minimum concentration [2]
o Should be restricted to situations where no other
acceptable method of media preparation is available
[1]
• Should not affect either survival or growth of test
organisms [2]
• Should be reagent grade or better [2]
• Should not exceed 0.5 ml/L (static) or 0.1 ml/L (flow
through), unless it was shown that higher
concentrations do not affect toxicity [USEPA
Guidelines Addendum - 7]
• Should not exceed 0.1 mL/L [OCSPP - 1]
o Solvent concentration as low as 0.02 mL/L
recommended [1]
• Examples of preferred solvents include
dimethylformamide, triethylene glycol, methanol,
acetone, and ethanol 111
Negative Control:
Yes No
Reference Toxicant Testing:
Yes No
If Yes, identify substance:
Other Control: If any (e.g. solvent control)
U.S. EPA OWAMPHIBIANDER
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Data Evaluation Record on the Effects of [Chemical] on Amphibian [Species]
Parameter
Details
Remarks
For Controlled Experiments Only
Biomass Loading Rate:
• Loading should be limited so as not to affect test
results [2]
• Loading will vary depending on temperature, type of
test (static vs. flow-through), species, food/feeding
regime, chamber size, test solution volume, etc.
• This maximum number would have to be determined
for the species, temperature, flow rate or test solution
volume, chamber size, food, feeding regime, etc.
• For all species, loading should be sufficiently low to
ensure:
o Dissolved oxygen is at least 60% of saturation (40%
for warm-water species) [2,9]
o Unionized ammonia does not exceed 35 (.ig/L
o LTptake by test organisms does not lower test
material concentration by >20% [2]
o Growth of organisms is not reduced by crowding
• Generally, at the end of the test, the loading (grams of
organisms; wet weight; blotted dry) in each test
chamber should not exceed the following:
o Static tests: >0.8 g/L (lower temperatures); >0.5 g/L
(higher temperatures) [2]
o Flow through tests: >1 g/L/day or >10 g/L at any
time (lower temperatures); >0.5 g/L/day or >5 g/L at
any time (higher temperatures) [2]
o Lower temperatures are defined as the lower of
17°C or the optimal test temperature for that species
121
U.S. EPA OWAMPHIBIANDER
Part B: Detailed Review
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Data Evaluation Record on the Effects of [Chemical] on Amphibian [Species]
Parameter
Details
Remarks
For Controlled Experiments Only
Feeding:
• Unacceptable for acute tests [4]
o Exceptions:
¦ Data indicate that the food did not affect the
toxicity of the test material [4]
¦ Test organisms will be severely stressed if they
are unfed for 96 hours [4]
¦ Test material is very soluble and does not sorb or
complex readily (e.g., ammonia) 141
Yes No
Describe diet as provided:
Lighting:
• No specific requirements for lighting
o Embryos should be incubated under dim
incandescent lighting (<20 fc) or total darkness
during early life-stage toxicity testing
o Embryos must not be subjected to prolonged
exposure to direct sunlight, fluorescent lighting, or
high intensity incandescent lighting
• Generally, ambient laboratory levels (540-1080 lux or
50-100 foot candles) or natural lighting should be
acceptable, as well as a diurnal cycle consisting of 50%
daylight or other natural seasonal diurnal cycle;
• Artificial light cycles should have a 15-30 minute
transition period to avoid stress due to rapid increases
in light intensity [2]
• Depends on the type of test (acute or chronic) and
endpoint (e.g., reproduction) of interest.
• LAGDA: recommends fluorescent bulbs (wide
spectrum), 600-2000 lux (lumens/nr) at the water
surface and photoperiod of 12 h light: 12 h dark [61
Study Design/Methods Classification: (Place Xbv One Based on Ch>erall Study Design/Methods Classification)
Provide details of Major or Minor Deficiencies/Concerns with Study Design in Associated Sections of Part A: Overview
This classification should be taken into consideration for the overall study classification for aquatic life criteria development in Part A.
Study Design Acceptable for Quantitative Use
Study Design Acceptable for Qualitative Use
Study Design Not Acceptable for Use
Additional Notes: Provide additional considerations for the classification of study use based on the study design.
Clarifying Questions for Study Authors and the Other Pertinent Information/Notes from Discussion:
Provide clarifying questions for study authors.
U.S. EPA OWAMPHIBIANDER
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Data Evaluation Record on the Effects of [Chemical] on Amphibian [Species]
OBSERVATIONS: Provide information under Details and any relevant information in Remarks. This information should be
consistent with the Results Section in Part A.
Parameter
Details
Remarks
Parameters measured including sublethal
effects/toxicity symptoms:
Common Apical Parameters Include:
Acute
• EC50 based on percentage of organisms exhibiting
loss of equilibrium plus the percentage of organisms
immobilized plus percentage of organisms killed
0 If not available, the 96-hr LC50 should be used [4]
FETAX
• Mortality, malformation, and growth inhibition [5]
Chronic
• Partial Life-cycle test:
0 Survival and growth of adults and young,
maturation of males and females, eggs spawned
per female, embryo viability, and hatchability [4]
• LAGDA: Mortality (and abnormal appearances), time
to NF stage 62, growth (weight and length) [6]
• AMA: mortality, hind limb length, snout to vent
length, developmental stage, wet weight, thyroid
histology 131
List parameters:
Egg Collection Interval:
Egg Storage Conditions:
Temperature:
Relative Humidity:
Was control survival acceptable?
Acute
• >90% control survival at test termination [4]
Chronic
• >80% control survival at test termination [41
Yes No
Control survival (%):
Were individuals excluded from the
analysis?
Yes No
If yes, describe justification provided:
Were exposure conditions or water quality
in test chambers acceptable?
• If appropriate, describe any water quality issues
(e.g., dissolved oxygen level below 60% of
saturation)
Yes No
U.S. EPA OWAMPHIBIANDER
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Data Evaluation Record on the Effects of [Chemical] on Amphibian [Species]
Parameter
Details
Remarks
Availability of concentration-response
data:
• Were treatment level concentration-response
data included in study publication (can be from
tables, graphs, or supplemental materials)?
specify endpoints in remarks
Yes
No
• Were replicate level concentration-response
data included in study publication (can be from
tables, graphs, or supplemental materials)?
specify endpoints in remarks
Yes
No
• If treatment and/or replicate level
concentration-response data were included, how
was data presented? (check all that apply)
Tables
Graphs
Supplemental Files
• Were concentration-response data estimated
from graphs study publication or supplemental
materials?
Yes No
If yes, indicate software used:
Yes
No
• Should additional concentration-response data
be requested from study authors?
If concentration-response data are available, complete
Verification of Statistical Results (Part C) for sensitive
species.
Requested by:
Request date:
Date additional data received:
U.S. EPA OWAMPHIBIANDER
Part B: Detailed Review
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Data Evaluation Record on the Effects of [Chemical] on Amphibian [Species]
Part C: Statistical Verification of Results
I. Statistical Verification Information: Report the statistical methods (e.g., R, EPA TRAP, BMDS, other) used to verify the
reported study or test results for the five (5) most sensitive genera and sensitive apical endpoints (including for tests where such
estimates were not provided). If values for the LC50, LT50 and NOEC are greater than the highest test concentration, use the "> "
symbol.
Primary Reviewer: Date: EPA Contractor {PlaceXby One)
Secondary Reviewer: Date: EPA Contractor {PlaceXby One)
{At least one reviewer should be from EPA for sensitive taxa)
Endpoint(s) Verified:
Additional Calculated Endpoint(s):
Statistical Method (e.g., TRAP, BMDS, R, other):
II. Toxicity Values: Include confidence intervals if applicable
NOEC:
LOEC:
MATC:
ECs:
EC10:
EC20:
ECso or LCso
Dose-Response Curve Classification: (PlaceXby One)
This classification should be taken into consideration for the overall study classification for aquatic life criteria development in Part A
Dose-Response Curve Acceptable for Quantitative Use
Dose-Response Curve Acceptable for Qualitative Use
Dose-Response Curve Not Acceptable for Use
Summary of Statistical Verification: Provide summary of methods used in statistical verification.
Additional Notes:
Attachments:
1. Provide attachments to ensure all data used in Part C is captured, whether from study results reported in the publication
and/or from additional data requested from study authors
• Data from study results of the publication should be reported in Results section of Part A
• Additional data provided upon request from study authors should be reported in Table C.II.l below and original
correspondence with study authors should be included as attachments
2. Model assessment output (including all model figures, tables, and fit metrics)
3. Statistical code used for curve fitting
U.S. EPA OWAMPHIBIANDER
Part C: Statistical Verification of Results
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Data Evaluation Record on the Effects of [Chemical] on Amphibian [Species]
III. Attachments: Include all attachments listed above after the table below.
Additional Data Used in Response-Curve: Provide all data used to fit dose-response cur\'e not captured in Results section of PER above in Part A, rows as needed. First
row in italicized text is an example.
Table C.II.1 Additional Data Used in Dose-Response Curve.
Curve ID
Species
Endpoint
Treatment
Replicate
[Standard
Deviation
or Standard
Error!
# of
Survivors
Na
ka
na
Response
Response
Unit
Cone
Cone units
Alchronicl
Ceriodaphnia dubia
#of
voimg/female
0
6
10
10
1
18
count
0.03
nig/L
a N = number of individuals per treatment; k = number of replicates per treatment level; n = number of individuals per replicate
U.S. EPA OWAMPHIBIANDER
Part C: Statistical Verification of Results
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Data Evaluation Record on the Effects of [Chemical] on Amphibian [Species]
Part D: References to Test Guidance
43. U.S. EPA. 2016. OCSPP 850.1000: Background and special consideration-tests with aquatic and
sediment-dwelling fauna and aquatic microcosms. Ecological effects test guidelines. Office of Chemical
Safety and Pollution Prevention. EPA 712-C-16-014. October 2016.
44. ASTM Standard E 729, 1980. 2002. Standard guide for conducting acute toxicity tests on test materials
with fishes, macroinvertebrates, and amphibians. ASTM International, West Conshohocken, PA.
45. OECD 407. 2008. T est No. 407: Repeated Dose 28-day Oral Toxicity Study in Rodents, OECD
Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris,
https://doi.org/10.1787/9789264070684-en.
46. Stephan, C.E., D.I. Mount, D.J. Hansen, J.H. Gentile, G.A. Chapman and W.A. Brungs. 1985.
Guidelines for Deriving Numerical National Water Quality Criteria for the Protection of Aquatic
Organisms and their Uses. PB85-227049. National Technical Information Service, Springfield, VA.
47. National Toxicology Program (NTP) Interagency Center for the Evaluation of Alternative Toxicological
Methods (NICEATM). 2000. Frog embryo teratogenesis assay -Xenopus (FETAX). Background
Review Document. National Institute of Environmental Health Sciences (NIEHS). Research Triangle
Park, NC, 273 pp.
48. OECD 241.2015. The larval amphibian growth and development assay (LAGDA). OECD Guidelines
for the Testing of Chemicals, Section 2, OECD Publishing, Paris,
https://doi.org/10.1787/9789264242340-en.
49. Stephan, C.E. 1995. Review of results of toxicity tests with aquatic organisms. Draft. U.S. EPA, MED.
Duluth, MN. 13 pp.
50. Boudreau, T.M., Sibley, P.K., Mabury, S.A., Muir, D.G.C., and Solomon, K.R. 2003. Laboratory
Evaluation of the Toxicity of Perfluorooctane Sulfonate (PFOS) on Selenastrum capricornutum,
Chlorella vulgaris, Lemna gibba, Daphnia magna, and Daphnia pulicaria. Archives of Environmental
Contamination and Toxicology. 44: 307-313.
51. American Public Health Association (APHA). 2012. Standard methods for the examination of water and
wastewater. Part 8000 - Toxicity. APHA. Washington, DC.
U.S. EPA OWAMPHIBIANDER
Part D: References to Test Guidance
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Data Evaluation Record on the Chronic Effects of [Chemical] on Avian [Species]
Attachment J Avian Data Evaluation Record (DER) Template
(September 2024)
-Page 159 of 178-
-------
Data Evaluation Record on the Chronic Effects of [Chemical] on Avian [Species]
Part A: Overview
I. Test Information
Chemical name:
CAS name:
Purity:
Solubility in Water (units):
CAS Number:
Storage conditions:
Controlled Experiment
(,manipulated)
Primary Reviewer:
Secondary Reviewer:
Field Study/Observation
(inot manipulated)
Date:
Date:
(At least one reviewer should be from EPA for sensitive taxa)
{Place X by One)
EPA
EPA
Contractor
Contractor
(Place X by One)
(Place X by One)
Citation: Indicate: author (s), year, study title, journal, volume, and pages.
(e.g., Heinz, G. H. 1979. Methylmercury: reproductive and behavioral effects on three generations of mallard ducks. J. Wildl. Manage. 43(2): 394 - 401.)
Companion Papers: Identify any companion papers associated with this paper using the citation format above.
Were other DERs completed for Companion Papers?
Yes
(Ifyes, list file names of
No DERs below)
Study Classification for Aquatic Life Criteria Development: Place X by One Based on Highest Use
Acceptable for Quantitative Use
Acceptable for Qualitative Use
Not Acceptable for Use/Unused
General Notes: Provide any necessary details regarding the study's use classification for all pertinent endpoints,
including non-apical endpoints within the study (e.g., note all study classifications for each endpoint if the use varies)
Major Deficiencies (note any stated exclusions): Check all that apply. Checking any of these items make the study "Not
Acceptable for Use"
,A ^ ii , . , , . No Controls (for controlled experiments
Mixture (tor controlled experiments only) only)
Excessive Control Mortality (dependent on test type and species)
, . . . . Bioaccumulation: steady state not
Diet not adequately characterized , ,
reached
Dermal or Injection Exposure Pathway
Review paper or previously published without modification
Other: (if an, list here)
U.S. EPA OWAVIANDER-
Part A. Overview
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Data Evaluation Record on the Chronic Effects of [Chemical] on Avian [Species]
POTENTIAL CHEMICAL MIXTURES: Describe any potential chemicals mixtures as characterized by study authors
(including any confirmation of chemical mixtures).
General Notes:
Minor Deficiencies: List and describe any minor deficiencies or other concerns with test. These items may make the study
"Acceptablefor Qualitative Use" (exceptions may apply as noted)
DESCRIPTION OF UNMEASURED TEST CONCENTRATIONS: Describe concerns with unmeasured test
concentrations and the influence of the study classification.
DESCRIPTION OF CONCERNS WITH DILUTION WATER: Describe concerns with characterization of and/or
deficiencies with dilution water (e.g., uncharacterized stream or lake water, potential presence of unknown containments,
high organic content, extreme hardness, pH, etc).
For Field Studies/Observations: A field study/observation may be considered "Acceptable for Quantitative Use" if it
consisted of a range of exposure concentrations and the observed effects are justifiably contributed to a single chemical
exposure
Mixture (observed effects not justifiably contributed to single chemical exposure)
Uncharacterized Reference Sites/Conditions
POTENTIAL CHEMICAL MIXTURES PRESENT AT SITE: Describe any potential chemicals mixtures present at
the site as characterized by study authors (including any confirmation of chemicals present at study site).
EXPOSURE VARIABILITY ACROSS STUDY SITE(S): Describe any exposure variability across study site(s) as
characterized by study authors (i.e., description of study design with reference and contaminated sites).
General Notes:
Reviewer's Comments: Provide additional comments that do not appear under other sections of the template.
U.S. EPA OWAVIANDER-
Part A. Overview
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Data Evaluation Record on the Chronic Effects of [Chemical] on Avian [Species]
ABSTRACT: Copy and paste abstract from publication.
SUMMARY: Fill out for the most sensitive endpoint (apical and or non-apical) and modify as needed. If study is classified as ''Not Acceptable for
Use " DO NOT complete summary tables.
Species (lifestage)
Duration
Exposure
Media3
Measured-M;
Unmeasured-U;
Form
Measuredb
Chemical
Form
Exposure
WW/
I)W /
IWW
Moisture
Content
(%)
Test
Endpoint
and Effectd
Reported
Effect
Concentration
frig/g or ppm)
Verified Effect
Concentration6
(units)
Classification
Quantitative /
Qualitative
a Diet, tissue type, etc.
b In addition, note if maternal transfer (MT)
0 WW=wet weight, DW=dry weight, FWW=fresh wet weight.
d Where Test Endpoint = ECx, NOEC, LOEC, MATC, etc., and Effect = growth, survival, reproduction, etc.
e Verification following completion of Part C of the DER
U.S. EPA OW AVIAN DER -
Part A. Overview
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Data Evaluation Record on the Chronic Effects of [Chemical] on Avian [Species]
II. Results Provide results as reported in the publication (including supplemental materials). Include screen shots of tables and/or
figures reporting results from the article following tabulated data table in each associated results section for all studies. Complete
tabulated data tables for all studies for studies marked "Acceptable for Quantitative Use" and "Acceptable for Qualitative Use".
Test Condition Parameters: If only general summary data of test condition parameters is provided by study authors (i.e., no
specific details of test condition parameters on a treatment level is provided), summarize any information regarding test condition
parameters under General Notes below.
General Notes:
Table A.II.1. Measured Test Condition Parameters.
Dissolved oxygen, temperature, pH and [other parameters (hardness, salinity, DOC)] in test solutions during the /117-day
exposure of [test organism] to [concentration of treatment(s)] of [test substance] under [static renewal/flow-through]
conditions.
Parameter
Treatment
Mean
Range
[1]
Photoperiod
[2]
J
J
[11
Temperature (C)
[2]
J
J
[11
Humidity
[21
(%)
J
J
Other (e.g.,
ventilation,
lighting)
[11
[21
J
I
U.S. EPA OWAVIANDER -
Part A. Overview
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Data Evaluation Record on the Chronic Effects of [Chemical] on Avian [Species]
Chemical Concentrations: Summarize the concentration verification data from test solutions/media. Expand table to include
each measured concentration data for each media type (i.e., muscle, liver, blood, etc.).
General Notes: Provide any necessary detail regarding the measured concentrations, including any identified cause for
substantial differences between nominal and measured concentrations, if samples were collected on separate days (and if so provide
details), and any potential cross contamination.
Table A.II.2. Measured (and Nominal) Chemical Concentrations in Test Solutions/Media.
[Analytical Method] verification of test and control concentrations during an [X]-day exposure of [test organism] to [test
substance] under [static renewal/flow-through] conditions.
[Mean]
Number of
[Standard
Nominal
Measured
Samples
Deviation or
Concentration
Concentration
Number of
Non-
Below Non-
Standard
Treatment
(units)
(units)
Samples
Detect3
Detect
Error]
Range
Control
[1]
[2]
[3]
[4]
[5]
[6]
J
aNon-Detect: 0 = measured and detected; l=measured and not detected; if not measured or reported enter as such
U.S. EPA OWAVIANDER -
Part A. Overview
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Data Evaluation Record on the Chronic Effects of [Chemical] on Avian [Species]
Mortality: Briefly summarize mortality results (if any).
General Notes: Comment on concentrations response relations and slope of response ifprovided. Compare mortality with control
treatment and/or the reference chemical.
Table A.II.3. Mean Percent [Mortality or Survival].
Mean percent mortality [or number of immobilized] or survival of [test organism] exposed to [test substance] for [test
duration] under [static/renewal/flow-through] conditions. Superscript(s) used to identify the values reported to be
significantly different from control as p value of [0.05/ or any other provided by authors].
Treatment
[Mean % Mortality]
Sample Size
[Standard Deviation
or Standard Error]
Control
[1]
[2]
[3]
[4]
[5]
[6]
[LCx]
NOEC
LOEC
a Use superscript(s) to identify the values reported to be significantly different from control.
U.S. EPA OWAVIANDER -
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Data Evaluation Record on the Chronic Effects of [Chemical] on Avian [Species]
Growth: Briefly summarize growth results (if any).
General Notes: Comment on concentrations response relations and slope of response ifprovided. Compare growth endpoints with
control treatment and/or the reference chemical.
Table A.II.4. Mean [Growth].
Mean growth [length and/or weight] of [test organism] exposed to [test substance] for [test duration] under
[static/renewal/flow-through] conditions. Superscript(s) used to identify the values reported to be significantly different from
control as p value of [0.05/ or any other provided by authors].
[Standard
[Standard
Mean
[Standard
Mean Growth
Deviation
Mean Growth
Deviation
Percent
Deviation
[Adult/Offspring]
or
[Adult/Offspringl
or
Change in
or
[Weight]
Sample
Standard
[Length]
Sample
Standard
[Length/
Sample
Standard
Treatment
(units)
Size
Error!
(units)
Size
Error!
Biomassl
Size
Error!
Control
[11
[21
[31
[41
[51
[61
./
[ECxl
NOEC
LOEC
a Use superscript(s) to identify the values reported to be significantly different from control.
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Data Evaluation Record on the Chronic Effects of [Chemical] on Avian [Species]
Reproductive: Briefly summarize reproduction endpoint results (if any). For multi-eenerational studies, copy and vaste Table
A.II.5 below for each generation with reproductive effects data.
General Notes: Comment on concentrations response relations and slope of response ifprovided. Compare reproduction
endpoints with control treatment and/or the reference chemical.
Table A.II.5. Mean [Reproductive] Effect.
Mean [reproductive] effects for [generation] of [test organism] exposed to [test substance] for [test duration] under
[static/renewal/flow-through] conditions. Superscript(s) used to identify the values reported to be significantly different from
control as p value of [0.05/ or any other provided by authors].
[Standard
[Standard
[Standard
[Standard
[Mean
Deviation
Deviation
Deviation
Deviation
Number
or
[Mean
or
[Mean
or
[Mean
or
Treatment
of
Sample
Standard
Clutch
Sample
Standard
Number of
Sample
Standard
Number of
Sample
Standard
(units)
Clutchesl
Size
Error!
Sizel
Size
Error!
Hatchlingsl
Size
Error!
Fledglingsl
Size
Error!
Control
rn
T21
T31
T41
rsi
rei
J
|ECxl
NOEC
LOEC
a Use superscript(s) to identify the values reported to be significantly different from control.
b PerEPA's Ecological Effects Test Guidelines - OCSPP 850.2300: Avian Reproduction Test, the following general
requirements apply to controls.
(ii) For a satisfactory test, the following values for response variables in controls should be met or at least
approached at test termination. There is likely to be a problem with test procedures or conditions that should be
investigated and corrected when these values are not met.
(A) Eggs laid - Normal values for both northern bobwhite and mallards are 29 to 61 eggs per hen for a 10
week egg laying period.
(B) Eggs cracked - Normal values for northern bobwhite are 0 to 7.0% of eggs laid. Normal values for
mallards are 0 to 4.0% of eggs laid.
(C) Fertility (viable embryos) - Normal fertility values for northern bobwhite and mallards are 80 to 100%
of eggs set.
(D) Live 18-d or 21-d northern bobwhite and mallard embryos, respectively (as a percentage of viable
embryos) - Normal values for northern bobwhite are 97 to 100%. Normal values for mallards are 94 to
100%.
(E) Hatchability (percentage of 18-d or 21-d northern bobwhite and mallard embryos, respectively
that hatch) - Normal values for northern bobwhite are 85 to 100%. Normal values for mallards are 52 to
100%.
(F) Percentage of eggs set that hatch - Normal values for northern bobwhite are 71 to 95%. Normal values
for mallards are 44 to 92%.
(G) 14-day-old survivors of eggs hatched - Normal values for northern bobwhite are 77 to 100%. Normal
values for mallards are 94 to 100%.
(H) Eggshell thickness - Normal average values for northern bobwhite are 0.20 to 0.24 mm. Normal values
for mallards are 0.316 to 0.372 mm.
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Part A. Overview
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Data Evaluation Record on the Chronic Effects of [Chemical] on Avian [Species]
Sublethal Toxicity Endpoints: Include other sublethal effect(s), including behavioral abnormalities or other signs of toxicity,
if any. Copy Table A.II.6 as needed to provide details for each sublethal effect obser\>ed.
General Notes: Briefly summarize obser\>ed sublethal effects otherwise not captured in the results table(s) below.
Table A.II.6. Mean [Sublethal] Effect.
Mean /Sublethal effect, (e.g., behavioral abnormalities, etc.)] in [test organism] during [test duration (acute/chronic)]
exposure to [test substance] under [static/renewal/flow-through] conditions. Superscript(s) used to identify the values
reported to be significantly different from control as p value of [0.05/ or any other provided by authors].
Treatment
[Mean Sublethal Response]
(units)
Sample Size
[Standard Deviation or
Standard Error]
Control
[1]
[2]
[3]
[4]
[5]
[6]
[ECx]
NOEC
LOEC
a Use superscript(s) to identify the values reported to be significantly different from control
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Part A. Overview
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Data Evaluation Record on the Chronic Effects of [Chemical] on Avian [Species]
Reported Statistics: Copy and paste statistical section from publication.
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Data Evaluation Record on the Chronic Effects of [Chemical] on Avian [Species]
Part B: Detailed Review
I. Materials and Methods
Protocol/Guidance Followed: Indicate if provided by authors.
•
Deviations from Protocol: If authors report any deviations from the protocol noted above indicate here.
Study Design and Methods: Copy and paste methods section from publication.
TEST ORGANISM: Provide information under Details and any relevant or related information or clarifications in Remarks.
Parameter
Details
Remarks
Species:
Useful sites include:
• httDs://www.itis.eov/
• httt>s://www.fws.eov/endaneered/
• httt>s://www.fisheries.noaa.eov/fmd-st>ecies
Common Name:
Scientific Name:
Order Name:
Family Name:
North American species?
Surrogate for North American
Taxon?
Is this species Threatened or
Endangered?
(Place A' if applicable)
Strain/Source:
• May be laboratory-reared or purchased from a
breeder [1-3]
• All birds should be from the same source and
breeding population [1-3]
• Test birds should be phenotypically indistinguishable
(except for size) from wild stock [1-3]
Age at Study Initiation:
• Acute test: Young adults of both sexes, not mated, at
least 16 weeks old [1]
• Dietary test: Not too old to be able to avoid eating
(e.g., mallard - 5 days old, bobwhite quail - 10-14
days old [2])
• Reproduction test: approaching first breeding season,
at least 16 weeks old, all within 1 month age [31
Was body weight or length recorded at
test initiation and/or at regular intervals?
• For field observations, was body weight measured in
a consistent manner (e.g., during blood sample
collection)
Yes No
Was body weight or length recorded at
regular intervals?
Yes No
If yes, describe regular inten'als:
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Data Evaluation Record on the Chronic Effects of [Chemical] on Avian [Species]
STUDY PARAMETERS: Provide information under Details and any relevant information of deficiencies in Remarks.
Complete for both Controlled Experiments and Field Studies/Obsen'ations
For Both Controlled Experiments and Field Obsen'ations
Parameter
Details
Remarks
Number of Replicates per Treatment
Group:
• Acute and Dietary tests: 1-2 per treatment level
[1-2]
• Reproductive test: 16 per treatment level [3]
Control(s):
Treatment(s):
Number of Birds per Replicate/Test
Condition:
• Acute and Dietary tests: at least 10 per treatment
level (equal numbers from each sex) [1,2]
• Reproductive test: one pair (1 M, 1 F) [31
Body Condition:
• Birds should be healthy without excess mortality
[1-3]
• Deformed, abnormal, sick, of injured birds should
not be used [1-3]
• Birds used in a previous test, or offspring of birds
used in a test treatment group, should not be used
[31
Good:
Poor:
Number of individuals excluded from
analysis:
Exposure Pathway:
• Should be dietary exposure [31
Exposure Duration:
Exposure Time:
Breeding
Non-breeding
Year round
Observation Intervals:
• No specific guidance on number of observation
intervals for changes in survival, deformities,
behavior, etc. of study organisms during a test.
Should be an appropriate number of observations over
the study to ensure test conditions are being properly
maintained [41
Test Concentrations (remember units):
Recommended test concentrations include at least two
concentrations other than the control; three or more
will provide a better statistical analysis.
Nominal:
Measured:
Media measured in:
What analytic methods were used to
measure test concentrations?
What was the recovery of the test material?
What was the reporting limit of the
analytical method used to measure the test
concentrations?
Were standards used as part of the
analytical method?
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Data Evaluation Record on the Chronic Effects of [Chemical] on Avian [Species]
EGG COLLECTION AND INCUBATION (if applicable):
For Both Controlled Experiments and Field
Obsen'ations
Parameter
Details
Remarks
Collection Interval:
• Recommend daily [31
Egg Storage Conditions:
• Temperature recommend 13-16°C (55-61°F) [3]
• Relative humidity recommend 55-80% [31
Temperature:
Relative humidity:
Were eggs candled for cracks prior to
setting for incubation?
Yes No
Were eggs set weekly?
Yes No
When was candling done to check for
fertility?
When were eggs transferred to the
hatcher?
Hatching Conditions:
• Temperature recommend 37.5-39°C (100-102°F)
[2.3]
• Relative humidity recommend -70% 12,31
Temperature:
Relative humidity:
What day was hatched eggs removed and
counted?
(e.g., removed on day 27)
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Data Evaluation Record on the Chronic Effects of [Chemical] on Avian [Species]
CONTROLLED EXPERIMENT STUDY PARAMETERS: Provide information under Details and any relevant
information of deficiencies in Remarks. Complete for Controlled Experiments only.
Parameter
Details
Remarks
Acclimation Period:
• Recommend at least 2 weeks [1,3]
• Dietary test: 3 days - mallard, 7 days - bobwhite
quail [2]
• Acute test: Should not be if mortality during
acclimation >5% (lab, breeder) or 10% (wild) [1]
• Dietary test: Should not be used if >5% mortality
during acclimation
• Reproduction test: Should not be used if >3% dead or
debilitated during acclimation [31
Acclimation followed published guidance?
Describe, if any
Yes No
Food Type:
• Recommend commercial feed or nutritional
equivalent [1-31
Test Chemical Solubility in Water:
• List units and conditions (e.g.. 0.01% at 20°C)
£
o
*3
£
£
Solvent/vehicle Type:
• Recommended solvents include (acetone, methylene
chloride, table grade com oil. propylene glycol, gum
arabic)[3]
• Should not comprise more than 2% of diet by weight
[3]
• Should be completely evaporated before feeding [3]
• Equivalent amount should be added to control diets
[31
Negative Control:
Yes No
"3
s:
£
Reference Toxicant Testing:
Yes No
If Yes, identify substance:
Other Control: If any (e.g. solvent control)
Describe preparation of test diet:
Were concentrations in diet verified by
chemical analysis?
Yes No
Indicate whether stability and
homogeneity of test material in diet
determined:
Yes No
Indicate if the test material was
regurgitated/avoided:
Yes No
Pen Size:
• Acute test: At least 75 in.2 / bird (quail) or 150 in.2 /
bird (mallard) surface area [1]
o Should be at least 9.5 in. height (quail) or 12.5 in.
height (mallard) [1]
• Dietary test: At least 50 in.2 / bird (quail) or 100 in.2 /
bird (mallard) surface area, and pens should be
arranged to prevent cross contamination [2]
• Reproductive test: should be of sufficient size to
prevent stress, and pens should be arranged to prevent
cross-contamination [3]
• Outdoor pens should only be used during breeding
season.[31
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Data Evaluation Record on the Chronic Effects of [Chemical] on Avian [Species]
Parameter
Details
Remarks
For Controlled Experiments Only
Number of Birds per Pen:
• Acute and Dietary tests: at least 10 per pen (equal
numbers from each sex) [1,2]
• Reproductive test: one pair (1 M, 1 F) [31
Male:
Female:
Number of Pens per Group/Treatment:
• Acute and Dietary tests: 1 or 2 per treatment 1,2]
• Reproductive test: 16 per treatment level [31
Control:
Treatment:
Test Conditions:
• Recommended temperature:
n Adults:
¦ 15-27°C(59-81°F) [1]
¦ 15-30°C(59-86°F) [3]
o Flatchlings:
¦ 22-38°C (72-100T) [2]
¦ 22-35°C (72-95°F) [1.3]
• Relative humidity recommend 45-70% [1-31
Temperature:
Relative humidity:
Photoperiod:
Feeding:
• Should be administered ad libitum throughout the
study [1-3]
Lighting:
• All pens should receive equal illumination [1-3]
• Acute and Dietary tests: indoor lighting
recommended [1,2]
o Incandescent of fluorescent acceptable [1,2]
o 14 Light: 10 Dark photoperiod recommended for
most species [1,2]
¦ Should be adjusted as appropriate for test
species[1]
o Light intensity not specified [1,2]
• Reproductive test.
o Should be full spectrum simulating daylight (avoid
shorter wavelength "cool-white" fluorescent) [3]
o Photoperiod should be acceptable for the test and
species[3]
o Recommended illumination (10-65 lux) [3]
o Outdoor lighting acceptable but not recommended
[31
Study Design/Methods Classification: (Place Xbv One Based on Ch>erall Study Design/Methods Classification)
Provide details of Major or Minor Deficiencies/Concerns with Study Design in Associated Sections of Part A: Overview
This classification should be taken into consideration for the overall study classification for aquatic life criteria development in Part A.
Study Design Acceptable for Quantitative Use
Study Design Acceptable for Qualitative Use
Study Design Not Acceptable for Use
Additional Notes: Provide additional considerations for the classification of study use based on the study design.
Clarifying Questions for Study Authors and the Other Pertinent Information/Notes from Discussion: Provide clarifying
questions for study authors.
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Data Evaluation Record on the Chronic Effects of [Chemical] on Avian [Species]
OBSERVATIONS: Provide information under Details and any relevant information in Remarks. This information should be
consistent with the Results Section in Part A.
Parameter
Details
Remarks
Parental:
(e.g., mortality, body weight, mean feed consumption )
n-3i
List parameters:
Reproductive Success:
(e.g., eggs laid/pen, nestlings produced, juvenile body
weight) PI
List parameters:
Was control survival acceptable?
• Unacceptable if >10% control birds dead or moribund
n-3i
Yes No
Control survival (%):
Were individuals excluded from the
analysis?
Yes No
Lfves, describe justification provided:
Was test condition parameters
acceptable?
(see notes under Reproductive Effects of Results
Section for test validity considerations) fl-3/
Yes No
Availability of concentration-response
data:
• Were treatment level concentration-response
data included in study publication (can be from
tables, graphs, or supplemental materials)?
specify endpoints in remarks
• Were replicate level concentration-response
data included in study publication (can be from
tables, graphs, or supplemental materials)?
specify endpoints in remarks
Yes No
Yes No
• If treatment and/or replicate level
concentration-response data were included, how
was data presented? (check all that apply)
Tables
Graphs
Supplemental Files
• Were concentration-response data estimated
from graphs study publication or supplemental
materials?
Yes No
Lfves, indicate software used:
Yes No
• Should additional concentration-response data
be requested from study authors?
Requested by:
Request date:
Date additional data received:
If concentration-response data are available, complete
Verification of Statistical Results (Part C) for sensitive
species.
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Part B. Detailed Review
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Data Evaluation Record on the Chronic Effects of [Chemical] on Avian [Species]
Part C: Statistical Verification of Results
I. Statistical Verification Information: Report the statistical methods (e.g., EPA TRAP, BMDS, R, other) used to verify the
reported study or test results for the five (5) most sensitive genera and sensitive apical endpoints (including for tests where such
estimates were not provided). If values for the LC50, LT50 and NOEC are greater than the highest test concentration, use the "> "
symbol.
Primary Reviewer: Date: EPA Contractor {PlaceXby One)
Secondary Reviewer: Date: EPA Contractor {PlaceXby One)
{At least one reviewer should be from EPA for sensitive taxa)
Endpoint(s) Verified:
Additional Calculated Endpoint(s):
Statistical Method (e.g., TRAP, BMDS, R, other):
II. Toxicity Values: Include confidence intervals if applicable
NOEC:
LOEC:
MATC:
ECs:
EC10:
EC20:
ECso or LCso
Dose-Response Curve Classification: (PlaceXby One)
This classification should be taken into consideration for the overall study classification for aquatic life criteria development in Part A
Dose-Response Curve Acceptable for Quantitative Use
Dose-Response Curve Acceptable for Qualitative Use
Dose-Response Curve Not Acceptable for Use
Summary of Statistical Verification: Provide summary of methods used in statistical verification.
Additional Notes:
Attachments:
1. Provide attachments to ensure all data used in Part C is captured, whether from study results reported in the publication
and/or from additional data requested from study authors
• Data from study results of the publication should be reported in Results section of Part A
• Additional data provided upon request from study authors should be reported in Table C.II.l below and original
correspondence with study authors should be included as attachments
2. Model assessment output (including all model figures, tables, and fit metrics)
3. Statistical code used for curve fitting
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Part D. References to Test Guidance
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Data Evaluation Record on the Chronic Effects of [Chemical] on Avian [Species]
III. Attachments: Include all attachments listed above after the table below.
Additional Data Used in Response-Curve: Provide all data used to fit dose-response cur\'e not captured in Results section of PER above in Part A, rows as needed. First
row in italicized text is an example.
Table C.II.1 Additional Data Used in Dose-Response Curve.
Curve ID
Species
Endpoint
Treatment
Replicate
[Standard
Deviation
or Standard
Error!
# of
Survivors
Na
ka
na
Response
Response
Unit
Cone
Cone units
Alchronicl
Ceriodaphnia dubia
#of
voting/female
0
6
10
10
1
18
count
0.03
mg/L
a N = number of individuals per treatment; k = number of replicates per treatment level; n = number of individuals per replicate
U.S. EPA OWAVIANDER-
Part D. References to Test Guidance
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Data Evaluation Record on the Chronic Effects of [Chemical] on Avian [Species]
Part I): References to Test Guidance
52. U.S. EPA. 2012a. OCSPP 850.2300: Avian acute oral toxicity test. Ecological effects test guidelines.
Office of Chemical Safety and Pollution Prevention. EPA 712-C-025. January 2012
53. U.S. EPA. 2012b. OCSPP 850.2300: Avian dietary toxicity test. Ecological effects test guidelines.
Office of Chemical Safety and Pollution Prevention. EPA 712-C-024. January 2012
54. U.S. EPA. 2012c. OCSPP 850.2300: Avian reproduction test. Ecological effects test guidelines. Office
of Chemical Safety and Pollution Prevention. EPA 712-C-023. January 2012
55. American Public Health Association (APHA). 2012. Standard methods for the examination of water and
wastewater. Part 8000 - Toxicity. APHA. Washington, DC.
U.S. EPA OWAVIANDER-
Part D. References to Test Guidance
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