EPA-540/9-85-009
June 1985
HAZARD EVALUATION DIVISION
STANDARD EVALUATION PROCEDURE
ACUTE TOXICITY TEST FOR ESTUARINE AND MARINE ORGANISMS
(Estuarine Fish 96-Hour Acute Toxicity Test)
Prepared by
Daniel Rieder, 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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30272-10)
REPORT DOCUMENTATION l- "eport no. 2.
PAGE
3. Recipient** Accession No. "~
PB8 fe 129301 /AS
4. Title and Subtitle
HAZARD EVALUATION DIVISION, STANDARD EVALUATION PROCEDURE
Acute Toxicity Test for Estuarine and Marine Organisms
(Estuarine Fish 96-Hour Acute Toxicity Test)
5. Report Oat*
June 1985
&
7, AuttioKs)
Daniel Reider
8. Performing Organization Rapt. No.
epa-$W9~8^ooq
9, Performing Organization Name and Address
U.S. Environmental Protection Agency/0PP/HED/(TS-769C)
401 M Street SW
Washington, D.C. 20460
10. Projact/Tatk/Work Unit No.1
11. Contract(C) Or Grant(G> No.
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 1
2. End-Use Product 1
II. MATERIALS AND METHODS: TESTING STANDARDS/
RECOMMENDATIONS
A. Acceptable Protocols 2
B. Test Organisms 3
1. Acceptable Species 3
2. Size/Age/Physical Condition 3
3. Source/Acclimation 3
C. Test Solution 4
1. Source of Dilution Water 4
2. Temperature 4
D. Testing System 5
1. Test Vessels 5
2. Photoperiod 5
3. Loading 5
4. Solvents 5
5. Flow-Through Tests 5
E. Test Design 6
1. Test Levels 6
2. Number of Test Animals 6
3. Controls 6
4. Beginning the Test 7
5. Measurement of Diluent Characteristics.. 7
6. Chemical Analysis 7
III. REPORTING REQUIREMENTS
A. Test Material 8
B. Dilution Water 8
C. Holding of Test Organisms 8
D. Mortality/Observable Effects 8
E. Calculated LC50 8
F. Temperature/DO/pH 9
G. Chemical Analysis 9
H. Testing Protocols 9
IV. REVIEWER'S EVALUATION
A. Review of Test Conditions 9
)< i
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TABLE OF CONTENTS (Continued)
Page
B. Verification of Statistical Analyses 9
C. Conclusions 10
1. Categorization of Results 10
2. Rationale 11
3. Repairability 11
D. Descriptive Classification 11
E. References 11
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ACUTE TOXICITY TEST FOR ESTUARINE AND MARINE ORGANISMS:
ESTUARINE FISH 96-HOUR ACUTE TOXICITY
I. INTRODUCTION
A. When Required
Acute toxicity studies with estuarine fish are requirec] to
support registration of an end-use product intended for direct
application to the estuarine or marine environment. These studies
are also necessary when it is expected that the pesticide would
enter this environment in significant concentrations because of
its expected use or mobility.
B. Purpose
° To establish acute toxicity levels of the active ingredient
to nontarget marine and estuarine organisms;
° To compare toxicity information with measured or estimated
pesticide residues in the estuarine or marine environment
to assess potential impact to fish;
° To provide support for precautionary label statements to
minimize adverse effects to estuarine or marine nontarget
organisms; and
0 To indicate the need for further 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.
2. End-Use Product
The applicant may be required to test the end-use product as
well if:
0 The end-use product will be introduced directly into the
marine or estuarine environment when used as directed;
° The estuarine fish LC50 of the technical grade of the
active ingredient is equal to or less than the expected
environmental concentration in the marine or estuarine
environment when the end-use product is used as directed;
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0 An ingredient of the end-use product is expected to
enhance the toxicity of the end-use product beyond that
expected from the active ingredient(s); or
° The technical product is insoluble in water but the
formulated product is soluble in water. In that case
the carriers or inerts of the formulated product must be
tested in a solvent control.
II. MATERIALS AND METHODS: TESTING STANDARDS/RECOMENDATIONS
A. Acceptable Protocols
EEB does not endorse any one protocol. It is sometimes
necessary and desirable to alter the procedures presented in
published protocols to meet the needs of the chemical or test
organisms used. However, EEB does recommend some protocols as
guidance for developing estuarine fish acute toxicity tests.
These protocols include:
Bahner, L.H., C.D. Craft, and D.R. Nimmo. 1975. A
Saltwater Flow-through Bioassay Method with Controlled
Temperature and Salinity. Prog. Fish Cult. 37(3): 126-129.
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.
Peltier, William. 1978. Methods for Measuring the Acute
Toxicity of Effluents to Aquatic Organisms. U.S. Environment-
al Protection Agency, Ecol. Res. Series, EPA 600/4-78-012.
5 2 pp.
Anonymous. 1978. Bioassay Procedures for the Ocean Disposal
Permit Program. U.S. Environmental Protection Agency, Office
of Research and Development. EPA-600/9-78-010. 121 pp.
American Society for Testing and 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.
These referenced protocols are presented as flexible guidance
to help researchers design scientific protocols and to help the
reviewer validate studies. It is important to recognize that
fish tests are to be validated based on whether they provide
scientifically sound information on the acute toxicity of the
test material to estuarine fish that is useful in risk assess-
ments and whether they fulfill guideline requirements. This is
more important than whether they follow a referenced protocol
step-by-step.
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B. Test Organisms
1. Acceptable Species
The selected species should have a demonstrated sensitivity
to known toxicants. If possible they should be species that
occur in the area of exposure or are closely related to exposed
spec ies.
Several species are specifically referenced in "Standard
Practice for Conducting Acute Toxicity Tests with Fishes,
Macroinvertebrates, and Amphibians" (ASTM, 1980). The preferred
species are the silverside (Menidia sp.) and the sheepshead
minnow (Cyprinodon variegatus). Other species may be acceptable
if they are shown to be sensitive in acute toxicity tests.
2. Size/Age/Physical Condition
Use of fish weighing between 0.5 and 5.0 grams is usually
desirable. Very young fish (not actively feeding) should not
be used, nor should sexually mature, spawning, or recently spent
fish. All fish should be from the same year class, and the
standard length (tip of snout to end of caudal peduncle) of the
longest fish should be no more than twice that of the shortest
f ish.
Test fish 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
period. Organisms treated for disease may be used, but care
should be taken to avoid using diseased animals. Organisms
must not be used if they appear to be diseased, stressed, or if
more than 3% die during the 48 hours immediately prior to
testing.
3. Source/Acclimation
All organisms must be from the same source. This may include
laboratory or commercial stocks. Fish captured in the wild are
acceptable provided they meet the requirements pertaining to
physical condition and age/size criteria mentioned above. Fish
captured via gill netting, electro-shocking or chemical treatment
must not be used.
After the fish have been received from the supplier or collected
they must be held for at least seven days for observation and acclim
ation. To avoid unnecessary stress, rapid changes in water
temperature and quality must be avoided. Feeding, handling and
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other maintenance must be such that the fish are not stressed,
diseased/ or damaged. All organisms must be maintained under
actual test conditions (temperature and water quality) for at
least 48 hours before the test actually begins. If used, young
fish that have been actively feeding for less than 20 days must
be fed up to the beginning of the test. If they are larger
(over 0.5 grams each) they should not be fed for 48 to 96 hours
before the beginning of the test.
B'eeding of the organisms should be limited to the time just
prior to testing. This reduces the amount of organic material
generated in the test containers. Organic matter may alter the
test results by either adsorbing the chemical or increasing the
oxygen demand of the test solution. Experience will dictate the
exact "time pretest without feed" necessary and appropriate; it
may vary between species. If the test material tends to bind to
organic material, fish species that can survive at least 48
hours without feed before testing should be used. Pretest feed-
ing information must be provided in the report.
C. Test Solution
1. Source of Dilution Water
An adequate supply of dilution water that meets the minimum
requirements mentioned below must be available. The water may
be natural or reconstituted but must be able to support the test
animals without stress (i.e., <5% mortality in 48-hour period
pretest). Natural water must be of constant quality as described
below.
Natural or reconstituted seawater of 30 to 34% salinity and
pH of 8 to 8.3 should be used when testing marine (stenohaline)
fish, and 10 to 17% salinity and pH 7.7 to 8.0 with estuarine
(euryhaline) fish species. Natural seawater is considered to be
of constant quality if the weekly range of salinity is less than
6%, and if the monthly range is less than 0.8 of a pH unit.
See the guidance by the American Society for Testing Materials
(1980), or the Committee on Methods for Toxicity Tests with Aquatic
Organisms (1975), for guidance on specific amounts of minerals in
reconstituted seawater. Commercial sources of seawater mixture
are acceptable provided they do not adversely affect the test
organism or alter the toxicity of the test material.
2. Temperature
The recommended temperature for the silverside and the
sheepshead minnow is 22°C. The temperature for other species
should be determined on a case-by-case basis. The actual measured
temperature should not deviate more than 1°C during the test.
Greater deviations could affect test results.
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D. Testing System
1. Test Vessels
Test containers should be of welded stainless steel or glass.
If other materials such as polyethylene are used, the toxicant
concentrations must be measured. For static tests fish (0.5 g
each or more) are to be exposed in 19.6 liter containers with
15 liters of solution. Flow-through containers may be slightly
larger.
2. Photoperiod
A 16-hour light and 8-hour dark photoperiod should be provided,
with a 15- to 30-minute transition period between light and dark.
3. Loading
The loading factor or ratio of mass of test organisms to
volume of test solution must not be high enough to affect test
results. 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 of test solution in static
tests at or below 17°C or 0.5 g/L of test solution at higher
temperatures. For flow-through tests, the loading should be no
greater than 1 g/L of test 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 of test solution at higher
temperatures.
4. Solvents
If solvents other than water are necessary, they should be
used sparingly, not to exceed 0.5 ml/L in any static test solution,
and 0.1 ml/L in flow-through systems. If solvents are necessary,
the following are acceptable:
dimethyl formamide
triethylene glycol
methanol
acetone
ethanol
5. Flow-Through Tests
Flow-through tests are not usually reguired. They may be
necessary if the test chemical is relatively insoluble, has a
short half-life, or is volatile. The metering system chosen for
a flow-through study must reproducibly supply appropriate toxicant
concentrations at a consistent flow rate. Metering systems
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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. Flow-through systems should
be constructed so that the organisms are not stressed by turbulence.
Concentrations must be measured in a flow-through system to
verify the metering system.
E. Test Design
1. Test Levels (Nominal/Measured)
Initially, range finding tests may be necessary to define
concentrations of the toxicant needed for definitive studies.
Test reports should provide information describing range finding
study procedures and results. The information should include
sample sizes, dosage levels, and mortality data.
If it can be shown (by testing at least 30 individuals) that a
chemical will have an LC50 greater than 100 ppm, a definitive
study need not be performed.
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. If a formulated
product is tested, it should be clearly stated in the test report
whether results are expressed in terms of active ingredient alone
or as total formulated product.
2. Number of Test Animals
The test vessels must contain no less than ten organisms per
level in a static test and no less than 20 organisms per level
in a flow-through design. These 10 or 20 organisms may be in
one container or may be divided between two or more replicate
containers. In every case, they must be randomly assigned to
the test containers.
3. Controls
Each test reguires a concurrent control using the same dilu-
tion water and same number of organisms at each test level. If
any solvent other than water is used a solvent control should
also be conducted. This solvent control should contain the high-
est concentration of the solvent that was added to any of the
test chambers. A test is not acceptable if more than 10% of
the control organisms die during a test.
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4. Beginning the Test
Static acute tests are begun either by adding the test material
to the test chambers after the organisms have been added or by
adding the organisms to the test chambers within 30 minutes after
the test material has been added to the dilation water.
5. Measurement of Diluent Characteristics
Temperature should be measured hourly throughout the acclima-
tion and test period in at least one test chamber if the test
containers are not in a temperature controlled water bath
because air temperature may change more frequently and to a
greater extent than water, thus affecting the test container
temperature. If the temperature is controlled by a water bath,
the temperature of the bath may be recorded every six hours.
Temperature should not vary more than one degree 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 in the control and the high, medium, and
low concentration as long as fish are present at those concentra-
tions. The DO level during the first 48 hours must be between
60% and 100% saturation and between 40% and 100% saturation
after 48 hours. In the flow-through test, the DO concentrations
in each chamber must 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 may be preferred to chemically analyze test solutions to
determine exact concentrations of pesticides. It is particularly
important that residues are measured if:
° The test solutions were aerated;
° The test material was volatile, insoluble, or precipitated
out of solution;
0 The test containers were not made of glass or stainless
steel;
° The test chemical is known to adsorb to the test containers
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 methods used to perform the study. The
reviewer must be able to determine 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 a
guideline 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 sufficient because the percent active
ingredient of some newer products may increase with time as the
manufacturing process is improved to produce greater purity.
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 clearly state in the test report whether the results
are expressed in terms of active ingredient or as formulation.
B. Dilution Water
Test reports submitted to the Agency should include a complete
description of dilution waters used in the test. Descriptions
should include identification of the source, the chemical
characteristics of the water, and information on any pretreatment.
C. Holding of Test Organisms
Test reports should include complete information on holding
and acclimation conditions including feeding schedules and
treatment for diseases.
D. Mortality/Observable Effects
The criteria for determining effects must be defined. The
raw data or percentage of deaths at each level as well as the
number of organisms tested per level must be reported for each
24-hour period. Toxic symptoms (physical and behavioral)
should be described.
E. Calculated LC5Q
The statistically calculated LC50 with 95% confidence limits
and the method of calculation must be presented. In lieu of a
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calculated LC50, the study may show that the LC5Q is greater
than 100 ppm. The slope of the dose-response line should be
calculated and reported. Some reports have a graphic (log
probit/dose concentration) presentation which is helpful.
F. Temperature/DO/pH
Dissolved oxygen, pH measurements and the range and average
temperature during the study should be reported.
G. Chemical Analysis
If chemical analyses were conducted, the test report should
provide information on the methods (references) utilized and the
results of analyses. Residues found at the beginning and end of
the study should be reported.
H. Testing Protocols
The test report should include reference to the testing
protocol(s) used during the study.
III. REVIEWER'S EVALUATION
A. Review of Test Conditions
The reviewer should identify each aspect of the reported
procedure that is inconsistent with recommended protocol. The
significance of these deviations must be determined. The
number of deviations and their severity will determine the
validity of the study and the interpretation of the results.
B. Verification of Statistical Analyses
The reviewer should ensure that the LC50 has been properly
derived by recalculating these values. An acceptable acute
toxicity test should provide not only an LC50 but also a NOEL
(no observed effect level) and a slope of the dose/mortality
response. The NOEL is the highest concentration at which no
mortality or other toxic signs occurred. These data can give
further insight into the toxicological characteristics of the
chemical such as whether the response is gradual, over a wide
range, or rapid.
If the recalculated results differ substantially from the
submitted results the reviewer should note this and attempt to
explain the differences.
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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 test can
be categorized as to whether they fulfill Part 158 regulations
and are useful in performing a risk assessment. Categories are
described as:
° 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 current guidelines 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 protocols. Results
do not meet guideline 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?
Concentrations tested were less than 100 ppm but
not high enough to produce an effect on the organ-
isms or a precise LC5Q? and
Deviations from recommended test solution character-
istics (variations in DO, temperature, hardness,
and pH can affect toxicological response).
0 Invalid: These studies provide no useful information.
They may be scientifically unsound, 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 test organisms were actually
exposed to nominally designated concentrations.
A study where the test material was not properly identified
can also be invalidated.
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2. Rationale
Identify what makes the study supplemental or invalid. While
all deviations from recommended protocol should be noted, the
reviewer is expected to exercise judgment in the area of study
categorization.
3. Repairability
Indicate whether the study may be upgraded or qiven a higher
validation category if certain conditions are met. Usually this
would involve the registrant submitting more data on the study.
D. Descriptive Classification
The reviewer should indicate what the results were and how much
information can be drawn from them. At a minimum, an acute toxicity
test will provide an LC50 with 95% confidence limits. This should
allow classifying the test material based on the following scheme:
LC50
(ppm)
< 0.1
0.1 - 1
1-10
10 - 100
> 100
Ca tegory
Description
very highly toxic
highly toxic
moderately toxic
slightly toxic
practically non-toxic
These descriptive categories are for inter-chemical comparison
only and do not reflect actual environmental risk to the test
organism. The results may provide other useful information such as
slope or a no observed effect level (NOEL). These additional data
are useful in a risk assessment.
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
branches.
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