PB86-129335
Hazard Evaluation Division, Standard
Evaluation Procedure: Acute Toxicity
Test for Estuarine and Marine Organisms
(Mollusc 48-Hour Embryo Larvae Study)
(U.S.) Environmental Protection Agency
Washington, DC
Jun 85
J.S. DEPARTMENT OF COMMERCE
ional Technical Information Service
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Pabb-129335
EPA-540/9-85-012
June 1985
HAZARD EVALUATION DIVISION
STANDARD EVALUATION PROCEDURE
ACUTE TOXICITY TEST FOR ESTUARINE AND MARINE ORGANISMS
(Mollusc 48-Hour Embryo Larvae Study)
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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50273-101
REPORT DOCUMENTATION
PAGE
1. REPORT NO.
3. Recipient** Accession No.
PBSfc 129335/AS
4. Title and Subtitle
HAZARD EVALUATION DIVISION, STANDARD EVALUATION PROCEDURE
Acute Toxicity Test for Estuarine and Marine Organisms (Mollusc
48-Hour Embryo Larvae Study)
5. Report Oat*
June 1985
EPA-5W9-85-012"
7. Authord)
Daniel Reider
8. Performing Organization Rapt. No.
9. Performing Organization Nam* and Addrata
U.S. Environmental Protection Agency/OPP/HED/(TS-769C)
401 M Street SW
Washington, D.C. 20460
10. Pro|«et/T»«k/Work Unit No.
H. Contracted or Grant(O) No.
(O
(G)
12. Sponsoring Organization Nam* and Address
Same as #9.
13. Type of Report & Period Covered
14.
IS. Supplementary Notes
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.
16. Abstract (Limit: 200 words)
The Standard Evaluation Procedure (SEP) for the Mollusc 48-Hour Embryo-Larvae Toxicity
Test is a guidance document primarily intended for Agency reviewers and the regulated in-i
dustry who evaluate ecological effects data specified ;in 4*0 CFR Part: 1158.145. ilhe^SEPits
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 estuarine/marine fish.
The SEP for the Estuarine Fish 9&--ffiour Acute Toxicity Test is only one of a number
of SEP's published by the National Technical Information Service as a supplement to Sub-
division E of the Pestici'de Assessment Guidelines.
17. Document Analysis a. Descriptors
Reproduced from
best available copy.
b. Identlfiers/OpeivEnded Terms
c. COSAT1 Field/Group
18. Availability Statement
Unclassified and freely available.
19. Security Cless (This Report)
Unclassified.
20. Security Class (This Page)
Unclassi fied.
21. No. of Pages
17
22. Prlcfl
(See ANSI-239.18)
See Instructions on Reverse
OPTIONAL FORy 272 (4-77)
(Formerly NTIS-3S)
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.
[. Melone, Director
Hazard Evaluation Division
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TABLE OF CONTENTS
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 3
1. Source of Dilution Water 3
2. Temperature 4
D. Testing System 4
1. Test Vessels 4
2. Photoperiod 4
3. Load i ng 4
4. Solvents 5
5. Aeration 5
E. Test Design 5
1. Test Levels 5
2. Number of Test Animals 5
3. Controls 6
4. Beg inning the Test 6
5. Measurement of Diluent Characteristics . 6
6. Chemical Analysis 6
III. REPORTING REQUIREMENTS
A. Test Material 7
B. Dilution Water 7
C. Holding of Test Organisms 7
D. Observable Effects Criteria 8
E. Calculated EC50 ~. 8
F. Temperature/DO/pH 8
G. Chemical Analysis 8
H. Testing Protocols 8
IV. REVIEWER'S EVALUATION
A. Review of Test Conditions 9
B. Verification of Statistical Analysis 9
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TABLE OF CONTENTS (Continued)
Page
C. Conclusions 9
1. Categorization of Results 9
2. Rationale 10
3. Repairability 10
D. Descriptive Classification 10
E. References 11
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ACUTE TOXICITY TEST FOR ESTUARINE AND MARINE ORGANISMS;
MOLLUSC 48-HOUR EMBRYO LARVAE STUDY
I. INTRODUCTION
A. When Required
Acute toxicity studies with mollusc are required to support
registration of an end-use product intended for direct application
to the estuarine or marine environment. This study is 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
0 To establish acute toxicity levels of the active
ingredient to nontarget marine and estuarine organisms;
0 To compare toxicity information with measured or
estimated pesticide residues in the estuarine or marine
environment to assess potential impact to invertebrates;
0 To provide support for precautionary label statements
to minimize adverse effects to estuarine or marine non-
target 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 mollusc ECso of the technical grade of the active
ingredient is equal to or less than the expected environ-
mental 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
0 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/RECOMMENDATIONS
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 mollusc acute toxicity tests. These
protocols include:
American Public Health Association, American Water Works
Association and Water Pollution Control Federation. 1981.
Standard Methods for the Examination of Water and Wastewater.
Fifteenth edition. Publication office: American Public
Health Association, 1015 18th Street-NW, Washington, DC
20036.
American Society Testing Materials. 1980. Standard Prac-
tice for Conducting Static Acute Toxicity Tests with Larvae
of Four Species of Bivalve Molluscs. E 724-80, Published by
the ASTM Committee on Standards, 1916 Race Street, Philadelphia,
PA, 19103.
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.
Woelke, C.E. 1967. "Measurement of Water Quality with the
Pacific Oyster Embryo Bioassay." Water Quality Criteria, ASTM
STP 416, p. 112.
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 reeognize that
mollusc tests are validated based on whether they provide scienti-
fically sound information on the acute toxicity of the test
material to mollusc that is useful in risk assessments 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 that represent exposed species.
Numerous species are specifically referenced in "Methods of
Acute Toxicity Tests with Fish, Macroinvertebrates, and
Amphibians" (Committee on Methods for Toxicity Tests with Aquatic
Organisms, 1975, and ASTM E 729-80). The following species are
preferred because there is a substantial data base available on
them:
Crassostrea giga Pacific oyster
Crassostrea virginica Eastern oyster
Mytilus edulis Mussel
Mercenaria mercenaria Quahog
Some other species may be acceptable if they have been shown to be
sensitive in acute toxicity tests.
2. Size/Age/Physical Condition
Embryos should be tested within one hour of spawning and after
fertilization. The eggs are fertilized as they are discharged from
the female; they are then added to treated test solution.
The adults must be from clean water that is not subject to point
or non-point pollution so they will not have developed genetic
resistance to toxicants that would make their offspring less
sensitive to toxicants.
3. Source/Acclimation
Mollusc larvae are obtained from adult brood stock in the labora-
tory. This brood stock is obtained either from the wild or from
commercial sources and caused to spawn. The referenced protocols
provide procedures for causing molluscs to spawn.
All organisms must be from the same source. This may include
laboratory or commercial stocks. Organisms captured in the wild
are acceptable provided they meet the requirements pertaining to
physical condition and age/size criteria mentioned above.
C. Test Solution
1. Source of Dilution Water
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An adequate supply of dilution water that meets the minimum
requirements must be available. The water may be natural or
reconstituted but must be able to support the test animals without
stress. Specifically, control larvae spawned by adults conditioned
in the dilution water must not incur more than 10% abnormal
development or 30% mortality in 48 hours.
Natural or reconstituted seawater of 30 to 34% salinity and
pH of 8 to 8.3 should be used when testing marine (stenohaline)
mollusc, and 10 to 17% salinity and pH 7.7 to 8.0 with estuarine
(euryhaline) mollusc species. Natural seawater is considered to
be of constant quality if the weekly range of salinity is less
than 6% and if the monthly pH range is less than 0.8 of a pH
unit. See the protocols 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
Most protocols recommend 20°C as the temperature. However
it is possible that a higher temperature (e.g., 25°C) may be accept-
able when the adult molluscs were collected from warmer waters as
in southern areas. Temperature should not vary more than 2°C
during the test. Laboratories using molluscs collected in the
south where ambient temperatures may already be 27-28°C may have
difficulty raising the temperature the 5-10°C necessary to induce
spawning without exceeding the lethal limit (33-34°C) for adult
molluscs.
D. Testing System
1. Test Vessels
Glass 1 Liter beakers are preferred. It is strongly recommended
that each treatment be tested in duplicate or even triplicate.
There should be 20,000 to 30,000 fertilized eggs per liter of
test solution.
2. Photoperiod
A 16-hour light and an 8-hour dark photoperiod should be
provided, with a 15 to 30 minute transition period between light
and dark.
3. Loading
There should be 20,000 to 30,000 fertilized eggs per liter of
test solution.
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4. Solvents
If solvents other than water are necessary, they should be
used sparingly, not to exceed 0.5 ml/L in any test solution.
The following solvents are preferred:
dimethyl formamide
triethylene glycol
methanol
acetone
ethanol
5. Aeration
Aeration is not recommended because it could cause.volatili-
zation of test material. However if aeration is necessary, the
residues in the test solution must be measured at the beginning
and near the end of the study.
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 there is a
problem with solubility or volatibility, if the test containers
are aerated, or if it is a flow-through test, the residue levels
must be measured at the beginning and at the end of the test
period.
If it can be shown that a chemical will have an £€50 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
Numbers of mollusc larvae per level are determined as a
certain density per volume of test solution. If oyster larvae
are used, normally, 20,000 to 30,000 are tested per liter.
Replicate test chambers may be used, but the number of organisms
would still be measured as number of larvae per volume of test
solution.
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3. Controls
Each test requires a concurrent control using the same dilution
water and same concentrations of larvae at each test level. The
number of control replicates should be four or the equivalent of
10% of the total number of treatment replicates, whichever is
greater. If any solvent other than water is used a solvent
control should be conducted. This solvent control should contain
the highest concentration of the solvent that was added to any of
the test solutions. A test is not acceptable if there is more
than 30% mortality in the control. If mean mortality exceeds 2%
in controls, each treatment response should be corrected for the
mean level of control using the following equation:
Net Percent ( percent of surviving larvae for )
Mortality = ( each treatment replicate ) x iQO
For Each ( mean percent of surviving control )
Level ( larvae per control replicate )
4. Beginning the Test
Fill each test container with the same volume of test solution
and toxicant. The larvae are then mixed with the test solution at
approximately 20,000 to 30,000 per liter.
5. Measurement of Diluent Characteristics
Temperature should be recorded hourly throughout the acclimation
and test periods 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. The range and average temperature must
be recorded.
The dissolved oxygen (DO) concentration must be measured at the
beginning of the test and at the end of the test in the control
and the high, medium, and low concentration. The DO level must
be between 60% and 100% of saturation.
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 necessary to chemically analyze test solutions to
determine exact concentrations of pesticides. It is particularly
important that residues are measured if:
0 The test solutions were aerated;
0 The test material was volatile, insoluble, or precipitated
out of solution;
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0 The test containers were not made of glass or stainless
steel;
0 The test chemical is known to adsorb to the test container's
structural material; or
0 A flow-through system is used (measurement verifies accuracy
of metering system).
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.
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D. Observable Effects Criteria
According to Woelke, fertilized eggs develop into free-swimming,
fully shelled veliger larvae in the 48 hours covered by this
protocol. He assumes that failure to .do so will break the life
cycle of, in this case, the Pacific oyster.
The measured response in this test should be the percentage
of larvae dying or failing to develop complete shells. Larvae
possessing misshapen shells or otherwise malformed shells are
considered normal (survivable) as long as the shell is complete.
Incidences of misshapen shells or otherwise malformed shells
should be reported.
To determine percent response per level, samples containing
about 150 to 250 larvae taken from each culture are examined. The
number of fully developed larvae compared to not fully developed
larvae are counted and used in the following equation to obtain
percent response per level.
Percent ( # fully dev. in control - )
reduction = ( ft fully dev. in test culture ) x IQO
per test ( # fully dev. in control )
level ( culture )
E. Calculated EC50
The statistically calculated ECsg with 95% confidence limits
and the method of calculation must be presented. In lieu of a
calculated EC$Q the study may show that the ECso is greater than
100 ppm. The mean percent abnormal larvae from each test level
should be plotted on probability paper, with the concentrations
converted to logs. The EC^Q may then be calculated by linear
regression.
F. Temperature/DO/pH
Dissolved oxygen, pH measurements and the range and average
temperature during the study should be reported.
G. Chemical Analysis
The test report should provide information on the methods
(references) utilized and the results of analyses. Residues
found at the beginning and end of the study should be reported.
H. Testing Protocols
The test report should include reference to the testing
protocol(s) used during the study.
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IV. REVIEWER'S EVALUATION
A. Review of Test Conditions
The reviewer should identify each aspect of the reported procedure
that is inconsistent with recommended protocol. The significance
of these deviations must be determined. The number of deviations
and their severity will determine the validity of the study and
the interpretation of the results.
B. Verification of Statistical Analysis
The reviewer should ensure that the ECso has been properly derived
by recalculating these values. An acceptable acute toxicity test
should provide not only an EC$Q but also a NOEL (no observed
effect level) and a slope of the dose/effect response. The
NOEL is the highest test level at which no effects were observed.
If the recalculated results differ substantially from the submitted
results, the reviewer should note this and attempt to explain the
differences .
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:
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 sound-
ness 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 supplemental category i.nclude:
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 EC^Q; and
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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 made invalid.
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 given 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 ECso with 95% confidence limits.
This should allow classifying the test material based on the
following scheme:
Category
(ppm) Description
< 0.1 very highly toxic
0.1-1 highly toxic
1-10 moderately toxic
10 - 100 slightly toxic
> 100 practically non-toxic
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These descriptive categories are for inter-chemical comparison
only and do not reflect actual environmental hazard 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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