EPA-540/9-85-006
June 1985
HAZARD EVALUATION DIVISION
STANDARD EVALUATION PROCEDURE
ACUTE TOXICITY TEST FOR FRESHWATER FISH
Prepared by
Elizabeth Zucker, M.S.
Standard Evaluation Procedures Project Manager
Stephen L. Johnson
Hazard Evaluation Division
Office of Pesticide Programs
United States Environmental Protection Agency
Office of Pesticide Programs
Washington, D.C. 20460
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30273-101
REPORT DOCUMENTATION
PAGE
1. REPORT HO.
1 Recipient's Accession No.
PB8 k 1 2 9 ? ? 7 /AS
4. TltM tftd Subtitk
HAZARD EVALUATION DIVISION, STANDARD EVALUATION PROCEDURE
Acute Toxicity Test for Freshwater Fish
5. Report Date
June 1985
7. Authors)
Elizabeth Zucker
*- P»rtannific Oresnlletion Rept. No;
epa-51+0/9-85-006
9. Performing Organization Nam* and Addrats
U.S. Environmental Protection Agency/0PP/HED/(TS-769C)
401 M Street SW
Washington, D.C. 20460
10. Proloct/Task/Work Unit No.
11. Contract« of Rapoft & Period Covared
14.
IS. Supptamantary Not as
Supplement to Pesticide Assessment Guidelines Subdivision E - Hazard Evaluation:
Wildlife and Aquatic Organisms (EPA-540/9-82-024 and NTIS Order Number PB83-153908),
October, 1982.
IS. Abstract (Limit 200 words)
The Standard Evaluation Procedure (SEP) for the Acute Toxicity Test for Freshwater
Fish is a guidance document primarily intended for Agency reviewers and the regulated in-
dustry who evaluate ecological effects data specified in 40 CFR Part 158.145. The SEP is
also intended to provide information to the general public indicating how the Agency evalu-
ates these types of studies. As such, it is designed to supplement Subdivision E of the
Pesticide Assessment Guidelines: Hazard Evaluation - Wildlife and Aquatic Organisms.
This SEP provides an Introduction, Materials and Methods, Reporting Requirements, Reviewer
Evaluation, and Appendix of appropriate methods to guide the review and scientific evalu-
ation of pesticide effects on fish.
—The SEP for the Acute Toxicity Test for Freshwater Fish is only one of a number of
SEP's published by the National Technical Information Service as a supplement to Subdivi-
sion E of the Pesticide Assessment Guidelines.
17. Document Analysis a. Descriptors
b. Identifiers/Open-Ended Terms
c. COSATt Field/Group
1>. Availability Statement
Unclassified and freely available,
19. Security Clan (This Report)
Unclassi fied.
20. Security Class (This Page)
Unclassified.
21. No. of Pages
18
22. Price
(Sn ANSt—239.18)
See Instructions on Reverse
OPTIONAL FORM 272 (4-77)
(Formerly NTIS—35)
Department of Commerce
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STANDARD EVALUATION PROCEDURE
PREAMBLE
This standard Evaluation Procedure (SEP) is one of a set of
guidance documents which explain the procedures used to evaluate
environmental and human health effects data submitted to the
Office of Pesticide Programs. The SEPs are designed to ensure
comprehensive and consistent treatment of major scientific topics
in these reviews and to provide interpretive policy guidance
where appropriate. The Standard Evaluation Procedures will be
used in conjunction with the appropriate Pesticide Assessment
Guidelines and other Agency Guidelines. While the documents were
developed to explain specifically the principles of scientific
evaluation within the Office of Pesticide Programs, they may also
be used by other offices in the Agency in the evaluation of
studies and scientific data. The Standard Evaluation Procedures
will also serve as valuable internal reference documents and will
inform the public and regulated community of important consider-
ations in the evaluation of test data for determining chemical
hazards. I believe the SEPs will improve both the quality of
science within EPA and, in conjunction with the Pesticide Assess-
ment Guidelines, will lead to more effective use of both public
and private resources.
John w. Melone, Director
Hazard Evaluation Division
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TABLE OF CONTENTS
Page
I. INTRODUCTION
A. When Required 1
B. Purpose 1
C. Test Material 1
1. Technical Grade I
2. End-Use Product 2
II. MATERIALS AND METHODS: TESTING STANDARDS/DATA
ACCEPTABILITY
A. Recommended Protocols 2
B. Test Organisms 3
1. Acceptable Species 3
2. Size/Age/Physica.1 Condition 3
3. Source/Acclimation 4
C. Test System 4
1. Source of Dilution Water 4
2. Temperature 5
3. Test Vessels 5
4. Photoperiod 5
5. Load ing 5
6. Solvents 6
D. Test Design 6
1. Test Levels 6
2. Number of Test Animals 6
3. Controls 6
4. Beginning the Test 7
5. Measuring Temperature/DO/pH 7
6. Chemical Analysis 7
III. REPORTING REQUIREMENTS
A. Test Material 8
B. Dilution Water/Test Vessels 8
C. Test Organisms 8
D. Range Finding Tests 8
E. Definitive Tests 9
F. Calculated LC50 9
G. Temperature/DO/pH 9
H. Chemical Analysis 9
I. Testing Protocols 9
IV. REVIEWER'S EVALUATION
A. Review of Test Conditions 9
B. Verification of Statistical Analyses 9
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TABLE OF CONTENTS (Continued)
Page
C. Conclusions 10
1. Categorization of Results 10
2. Rationale 11
3. Repairability 11
D. Descriptive Classification 11
E. References 12
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ACUTE TOXICITY TEST FOR FRESHWATER FISH
I. INTRODUCTION
A. When Required
Acute toxicity testing on freshwater fish is required to
support registration of manufacturing-use pesticide products and
end-use pesticide products intended for outdoor application.
B. Purpose
Acute toxicity studies on freshwater fish determine the
lethal concentration (LC50) of a chemical which will kill fifty
percent of the test population in 48 to 96 hours. These acute
tests have attained broad acceptance among environmental toxicolo-
gists as relatively rapid, uncomplicated, inexpensive, and
statistically reliable methods for assessing immediate, short-
term, adverse effects of chemicals on fish.
The Ecological Effects Branch regularly requires that results
of two freshwater fish acute toxicity tests be submitted to
support the registration of a pesticide. One study should be
performed on a coldwater fish species and one study on a warmwater
fish species. The data from these tests are used:
0 To establish acute toxicity levels of the active ingredient
to nontarget freshwater fish?
0 To assess potential impact to fish by comparing toxicity
information with measured or estimated pesticide residues
in the freshwater environment;
0 To provide support for precautionary label statements
that will minimize adverse effects to fish when the pesti-
cide is used according to directions; and
0 To indicate the need for further laboratory testing and/or
field studies.
C. Test Material
1. Technical Grade
Tests must be conducted with the technical grade of the active
ingredient. If more than one active ingredient constitutes a
technical product, then the technical grade of each active ingredient
must be tested separately.
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2. End-Use Product
In addition to technical product testing, the applicant may
be required to test the end-use product as well if:
0 The end-use product will be introduced directly into an
aquatic environment when used as directed;
° The freshwater fish LC50 of the technical grade of the
active ingredient is equal to or less than the expected
environmental concentration in the freshwater environment
when the end-use product is used as directed;
0 An ingredient of the formulated end-use product is expected
to enhance the toxicity of the end-use product beyond
that expected from the active ingredient(s) alone; or
0 The technical product is insoluble in water but the formu-
lated product is soluble in water. In this situation/
the test design should include a control where organisms
are exposed to just the inert ingredients and carriers.
II. MATERIALS AND METHODS: TESTING STANDARDS/DATA ACCEPTABILITY
A. Recommended Protocols
Because the acute test is an established technique for assessing
toxicity of a chemical to freshwater fish species, much of the
methodology for performing these studies, as well as the procedures
for statistical analyses of results, have been carefully outlined
and documented in the published literature. Notably, the information
to be discussed in this Standard Evaluation Procedure (SEP) is
presented in greater detail in the following references:
Committee on Methods for Toxicity Tests with Aquatic
Organisms. 1975. Methods for Acute Toxicity Tests with
Fish, Macroinvertebrates and Amphibians. U.S. Environmental
Protection Agency, Ecol. Res. Series, EPA 660/375-009. 61 pp.
American Society for Testing Materials. 1980. Standard
Practice for Conducting Acute Toxicity Tests with Fishes,
Macroinvertebrates and Amphibians. E 729-80. Published by
ASTM Committee on Standards, 1916 Race Street, Philadelphia,
PA, 19103.
Peltier, William. 1978. Methods for Measuring the Acute
Toxicity of Effluents to Aquatic Organisms. U.S. Environmental
Protection Agency, Ecol. Res. Series, EPA 600/4-78-012. 52 pp.
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U.S. Environmental Protection Agency. Pesticide Assessment
Guidelines Subdivision E. Hazard Evaluation; Wildlife
and Aquatic Organisms. EPA-540/9-82-024.
These referenced protocols are presented as flexible guidance to
help researchers design scientific protocol and to help the
reviewer validate studies. It is important to recognize that
freshwater fish tests are validated as to whether they provide
scientifically sound information on the acute toxicity of the
test material to fish and whether the results of the study will
fulfill guideline reguirements. This is more important than
whether a study completely conforms to referenced protocols. It
is sometimes necessary to alter the procedures presented in
published protocols to meet the characteristics of the chemical
or test organisms used.
The static test is the standard technique for obtaining LC50
values for fish, however flow-through testing may be needed when
toxicants are highly volatile or otherwise unstable in the aqueous
environment, or when a chemical has a high biochemical oxygen
demand. The information as it is presented below will focus on
static testing protocols. Specific references to acceptable
flow-through methods are indicated when necessary.
B. Test Organisms
1. Acceptable Species
The preferred test species for the coldwater fish toxicity
test is the rainbow trout (Salmo gairdneri). The bluegill sunfish
(Lepomis macrochirus) is the recommended species for warmwater
fish toxicity testing. Other acceptable species include:
Coldwater species
Brook trout (Salvelinus fontinalis)
Coho salmon (Oncorhynchus kisutch)
Warmwater species
Channel catfish (Ictalurus punctatus)
Fathead minnow (Pimephales promelus)
2. Size/Age/Physical Condition
Individual fish should weigh between 0.5 and 5 grams and
should be of the same year class. The standard length of the
longest fish should not be more than twice the length of the
shortest fish. Very young (not actively feeding), sexually
mature, spawning, or recently spent fish should not be utilized.
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3. Source/Acclimation
All organisms must be from the same source. This may include
laboratory or commercial stocks. Animals captured in the wild
are acceptable provided they meet the requirements pertaining to
physical condition and age/size criteria mentioned above.
Organisms captured via chemical treatment must not be used.
When test animals are brought into the laboratory, they should
be quarantined for at least seven days. It is recommended that
fish be acclimated to study conditions for at least two weeks
prior to testing.
Test organisms must be observed prior to testing for signs of
disease, stress, physical damage, and mortality. Injured, dead
and abnormal individuals must be discarded. If the organisms
show signs of disease or stress, remedial action must be taken
during the acclimation period and not during the test phase.
Organisms that have been treated for disease are acceptable for
testing provided they do not exhibit signs of disease during the
48-hours pretest. Organisms must not be used if they appear to
be diseased or stressed or if more than 3% die during the 48
hours immediately prior to testing.
If possible, feeding of the fish should be limited to the time
just prior to testing. This reduces the amount of organic
material generated in the test vessels. Organic matter may
alter the test results by adsorbing the chemical or increasing
the oxygen demand of the test solution.
C. Test System
1. Source of Dilution Water
Whenever possible, soft, reconstituted water should be used for
freshwater studies. Reconstituted water should be aged one or two
weeks and intensely aerated prior to use. Detailed descriptions
of acceptable procedures for preparing diluent are found in the
protocols by the American Society for Testing Materials (1980) or
the Committee on Methods for Toxicity Tests with Aquatic Organisms
(1975).
Use of a natural dilution water with a hardness of 40 to 48 mg/L
as CaC03 can be used in lieu of reconstituted water. If possible,
natural dilution water should be obtained from an uncontaminated
well, spring, or surface water source. Dechlorinated water should
not be used because removal of chlorine is rarely complete and
residual chlorine can be quite toxic to aquatic organisms.
The dilution water must be able to support the test animals
without observable signs of stress such as discoloration or
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unusual behavior.
2. Temperature
The recommended temperature for testing coldwater fish species
is 12°C. Warmwater fish tests should be performed at 17°C or 22°C.
Testing facilities should have a constant temperature area or a
recirculating water bath for the test vessels.
3. Test Vessels
Test containers should be constructed from welded stainless
steel or glass. Vessels with dimensions of about 300 x 600 x 300 mm
depth are acceptable for both static and flow-through tests.
Frequently, static tests are performed in 18.9 L (5 gallon)
wide-mouth glass jars containing 15 L solution.
If test vessels are constructed from materials other than glass
or stainless steel, solutions must be analyzed to determine exact
toxicant concentrations. Past studies have shown that some test
vessel materials (e.g., polyethylene) can adsorb residues of the
pesticide being tested.
The metering system chosen for flow-through studies must
reproducibly supply appropriate toxicant concentrations at a
consistent flow rate. Metering systems should be calibrated
before and after each study and checked twice daily during the
test period. Flow rates should be five to ten volume additions per
24-hours. Systems should be constructed so that the organisms
are not stressed by turbulence.
4. Photoperiod
A 16-hour light and an 8-hour dark photoperiod with a 15- to
30-minute transition period between light and dark is recommended.
5. Loading
The size of the test container should be such that the loading
factor (test organism mass per volume of test solution) is no
greater than 0.8 g/L in static tests performed at or below 17°C.
At higher temperatures, a biological loading of 0.5 g/L of
test solution is acceptable. For flow-through tests, the
loading should be no greater than 1 g/L of solution passing
through the chamber in 24 hours, and must not exceed 10 g/L
of test solution at any time at or below 17°C or 5 g/L at higher
temperatures.
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6. Solvents
Whenever possible, the toxicant should be introduced into the
test solution without the use of solvents other than water. If
alternative solvents are necessary, they should be used sparingly,
not to exceed 0.5 ml/L in any static test solution and 0.1 ml/L
under flow-through conditions. The following solvents are preferred:
dimethyl formamide
triethylene glycol
methanol
acetone
ethanol
D. Test Design
1. Test Levels
Initially, range finding tests may be necessary to define
concentrations of the toxicant needed for definitive studies. If
results from a range finding study indicate a low toxicity for
the chemical, a definitive test need not be performed. However,
it must be determined that the chemical will have an LC50 greater
than 100 mg/L, by exposing at least 30 individuals to a concentration
of 100 mg/L or greater.
Definitive acute toxicity tests normally are designed to include
one or more control groups and a geometric series of at least five
toxicant concentrations to be tested. Each designated treatment
group should be exposed to a concentration of toxicant that is at
least 60% of the next highest concentration.
2. Number of Test Animals
In definitive tests, at least ten test organisms should be
exposed to each treatment level. Treatment groups can be divided
into two or more containers. All organisms must be randomly
assigned to test vessels.
3. Controls
Each test requires a concurrent control using the same dilution
water and the same number of organisms per test level. If a solvent
other than water is used, a solvent control should be established.
The highest concentration of the solvent that was added to any of
the study chambers should be tested as the control.
A study is not acceptable if more than 10% of the control
organisms die during a static test or 5% during a flow-through
study.
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4. Beginning the Test
Static acute tests are initiated either by adding the test
material to the test chambers after the fish are added or by
adding the fish to the test chambers within 30 minutes after the
test material is added to the dilution water.
5. Measuring Temperature/DO/pH
Temperature should be measured continuously (hourly) in at least
one test vessel during the entire study period. If temperature
is controlled by a water bath, measurements can be recorded every
six hours. Temperature should not vary more than one degree Centi-
grade (C) during the entire study period.
The dissolved oxygen (DO) concentration must be measured at the
beginning of the test and every 48 hours thereafter to the end
of the test. Measurements should be taken from the control
and the high, medium, and low concentrations as long as animals
are present at those levels. The DO level during the first 48
hours should be between 60% and 100% of saturation and between
40% and 100% saturation after 48 hours. In the flow-through
test, the DO concentrations in each chamber should be between 60%
and 100% saturation at all times during the study.
The pH should be measured at the beginning and end of the test
in the control and the high, medium, and low toxicant concentrations.
6. Chemical Analysis
It is preferred that test solutions be chemically analyzed to
determine exact concentrations of pesticides. It is particularly
important that residues are measured if:
° The test solutions were aerated (aeration may cause
volatilization of the pesticide);
° The test material was volatile, insoluble, or precipitated
out of solution;
° The test containers were not made of stainless steel or
glass;
° The test chemical is known to adsorb to the test container's
structural material; or
° A flow-through system is used (measurement verifies accuracy
of metering system).
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III. REPORTING REQUIREMENTS
The test report submitted to the Agency must fully describe
the materials and methodology used to perform the study. The
reviewer must be able to establish from the report that the study
was performed under conditions that render the results acceptable
for use in a risk assessment and/or for fulfilling the guidelines
requirement. The following information is particularly important
for a complete evaluation.
A. Test Material
If the study is to be performed with the technical grade
product, the test material should be clearly identified as to
source, batch, and exact purity. Simply identifying the material
as technical may not be acceptable because the percent active
ingredient of some newer products may increase with time as the
manufacturing process is improved to produce greater yield.
For studies involving the end-use product, the exact percent of
the active ingredient and the type of formulation (e.g., granular,
wettable powder) of the test material should be described. It
should be clearly stated in the test report whether results are
expressed in terms of active ingredient or as total formulated
product.
B. Dilution Water/Test Vessels
Test reports submitted to the Agency should include a complete
description of dilution waters used in the toxicity studies.
Descriptions should include identification of the source, the
chemical characteristics of the water, and information on any
pretreatments.
Test vessels should be described as to construction material
and size, as well as depth and volume of solution.
C. Test Organisms
Test reports should provide complete descriptions of source,
holding, and acclimation conditions including information on
feeding schedules and disease treatment procedures.
The age, average weight, and length of test fish should
be reported. Species should be identified by scientific name.
D. Range Finding Tests
Test reports should provide information describing range finding
study procedures and results. The information should include sample
sizes, concentrations tested, and mortality data.
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E. Definitive Tests
Procedures used to prepare toxicant stock solutions and test
aliquots should be thoroughly described.
The criteria for determining effects must be defined. The raw
data or percentage of deaths at each level as well as the number
of fish tested per level must be reported for each 24-hour period
of the study. Toxic symptoms (physical and behavioral} should
be described throughout the test period.
F. Calculated LC5Q
The statistically calculated LC50 with 95% confidence limits
and method of calculation must be presented. The slope of the
dose-response line should be calculated and reported.
In lieu of a calculated 1X50* the study may show that the LC50
is greater than 100 pprti.
G. Temperature/DQ/pH
Dissolved oxygen and pH measurements should be reported along
with the range and average temperature.
H. Chemical Analyses
If chemical analyses are conducted, the test report should
provide information on the methods (or references to established
methods) utilized and results of analyses. Residues found at the
beginning and end of the study should be reported.
I. Testing Protocols
The test report should include references to any protocols
followed during the test.
IV. REVIEWER'S EVALUATION
A. Review of Test Conditions
The reviewer should note any important information missing
from the submitted report. Also noted are conditions of the study
that are inconsistent with recommended methodologies as discussed
in this SEP or in designated references.
B. Verification of Statistical Analyses
An integral part of the data evaluation process is the veri-
fication of statistical analyses. The reviewer should ensure that
the LC50 has been properly derived by recalcuating data through
currently available statistical programs.
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An acceptable acute toxicity test should provide additional
important information other than the LC50 (EC50). Results from
a valid study should provide a zero mortality level and a slope
of the dose-mortality response line. These data can give further
insight into the toxicological characteristics of the chemical
such as whether the response is gradual over a wide concentration
range or relatively rapid over a narrow range.
If the recalculated results differ substantially from the
submitted results, the reviewer should note this and attempt
to explain the discrepancies.
A test can be considered unacceptable if more than 10% of
the control organisms die during the study period. An inadequate
number of test organisms per test level can also produce unreliable
results.
C. Conclusions
1. Categorization of Results
The significance of inconsistencies in the test procedures
must be determined by the reviewer so that the results of the
study can be categorized as to their usefulness in a risk assess-
ment. Categories are described as:
0 Core: All essential information was reported and the
study was performed according to recommended protocols.
Minor inconsistencies with standard methodologies may be
apparent; however, the deviations do not detract from the
study's soundness or intent. Studies within this category
fulfill the basic requirements of Part 158 of the regu-
lations and are acceptable for use in a risk assessment.
0 Supplemental: Studies in this category are scientifically
sound; however, they were performed under conditions that
deviated substantially from recommended guideline protocols.
Results do not meet regulatory requirements; however, the
information may be useful in a risk assessment.
Some of the conditions that may place a study in a supple-
mental category include:
- Unacceptable test species;
Inappropriate test material;
Dosage levels tested were less than 100 ppm but not
high enough to produce an effect on the organisms
or a precise LC50 (EC50); or
Deviations from recommended test solution characteristics
(variations in DO, temperature, hardness, and pH can
affect toxicological response).
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° Invalid: These studies provide no useful information.
They are not scientifically sound, or they were performed
under conditions that deviated so significantly from
recommended protocols that the results will not be
useful in a risk assessment.
Examples of studies placed in this category commonly
include those where the test system was aerated, test
vessels were constructed from materials other than
glass, or there were problems of solubility or volatility
of the test material. Unless acceptable chemical analyses
of actual toxicant concentrations were performed in
studies such as these, the reviewer cannot be sure that
organisms were actually exposed to nominally designated
residues. Also, a study where the test material was
not properly identified can be invalidated.
2. Rationale
To support a supplemental or invalid category, the reviewer
must list and explain all test conditions that deviated from
standard protocols.
If any or all of the deviations can be re-examined and found
acceptable (i.e., the study category can be upgraded), the reviewer
also discusses this. Usually to upgrade a study additional
information must be acguired.
D. Descriptive Classification
Valid aquatic LC50 (EC50) toxicity values can be categorically
compared to LC50 (EC50) values determined for other chemicals
and/or species by the following descriptive classification:
3. Repairability
LC50 (EC 50)
(PPm)
Description
Category
< 0.1
very highly toxic
highly toxic
moderately toxic
slightly toxic
practically non-toxic
0.1 - 1
> 1 < 10
> 10 <_ 100
> 100
These descriptive categories are for inter-chemical comparisons
only and do not reflect actual environmental hazard to freshwater
organisms.
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E. References
The reviewer should reference any information used in the
validation procedure. This should include protocol documents,
statistical methods, or information taken from files of other
division branches.
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