United States Prevention, Pesticides EPA712-C-96-160
Environmental Protection and Toxic Substances April 1996
Agency (7101)
&EPA Ecological Effects Test
Guidelines
OPPTS 850.1055
Bivalve Acute Toxicity
Test (Embryo-Larval)
'Public Draft"
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INTRODUCTION
This guideline is one of a series of test guidelines that have been
developed by the Office of Prevention, Pesticides and Toxic Substances,
United States Environmental Protection Agency for use in the testing of
pesticides and toxic substances, and the development of test data that must
be submitted to the Agency for review under Federal regulations.
The Office of Prevention, Pesticides and Toxic Substances (OPPTS)
has developed this guideline through a process of harmonization that
blended the testing guidance and requirements that existed in the Office
of Pollution Prevention and Toxics (OPPT) and appeared in Title 40,
Chapter I, Subchapter R of the Code of Federal Regulations (CFR), the
Office of Pesticide Programs (OPP) which appeared in publications of the
National Technical Information Service (NTIS) and the guidelines pub-
lished by the Organization for Economic Cooperation and Development
(OECD).
The purpose of harmonizing these guidelines into a single set of
OPPTS guidelines is to minimize variations among the testing procedures
that must be performed to meet the data requirements of the U. S. Environ-
mental Protection Agency under the Toxic Substances Control Act (15
U.S.C. 2601) and the Federal Insecticide, Fungicide and Rodenticide Act
(7U.S.C. I36,etseq.).
Public Draft Access Information: This draft guideline is part of a
series of related harmonized guidelines that need to be considered as a
unit. For copies: These guidelines are available electronically from the
EPA Public Access Gopher (gopher.epa.gov) under the heading "Environ-
mental Test Methods and Guidelines" or in paper by contacting the OPP
Public Docket at (703) 305-5805 or by e-mail:
guidelines@epamail.epa.gov.
To Submit Comments: Interested persons are invited to submit com-
ments. By mail: Public Docket and Freedom of Information Section, Office
of Pesticide Programs, Field Operations Division (7506C), Environmental
Protection Agency, 401 M St. SW., Washington, DC 20460. In person:
bring to: Rm. 1132, Crystal Mall #2, 1921 Jefferson Davis Highway, Ar-
lington, VA. Comments may also be submitted electronically by sending
electronic mail (e-mail) to: guidelines@epamail.epa.gov.
Final Guideline Release: This guideline is available from the U.S.
Government Printing Office, Washington, DC 20402 on The Federal Bul-
letin Board. By modem dial 202-512-1387, telnet and ftp:
fedbbs.access.gpo.gov (IP 162.140.64.19), or call 202-512-0135 for disks
or paper copies. This guideline is also available electronically in ASCII
and PDF (portable document format) from the EPA Public Access Gopher
(gopher.epa.gov) under the heading "Environmental Test Methods and
Guidelines."
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OPPTS 850.1055 Bivalve acute toxicity test (embryo-larval).
(a) Scope—(1) Applicability. This guideline is intended to meet test-
ing requirements of the Federal Insecticide, Fungicide, and Rodenticide
Act (FIFRA) (7 U.S.C. 136, et seq.).
(2) Background. The source material used in developing this har-
monized OPPTS test guideline is OPP 72-3 Acute Toxicity Test for Estua-
rine and Marine Organisms (Pesticide Assessment Guidelines, Subdivision
E—Hazard Evaluation; Wildlife and Aquatic Organisms) EPA report 5407
09-82-024, 1982.
(b) Purpose. This guideline prescribes tests to be used to develop
data on the acute toxicity of chemical substances and mixtures ("chemi-
cals") to Eastern oysters (Crassostrea virginica), Pacific oysters
(Crassostrea gigas), quahogs (Mercenaria mercenaria), or bay mussels
(Mytilus edulis). The Environmental Protection Agency will use data from
these tests in assessing the hazard of a chemical to the environment.
(c) Definitions. The definitions in section 3 of the Toxic Substances
Control Act (TSCA) and the definitions in 40 CFR Part 792—Good Lab-
oratory Practice Standards apply to this guideline. The following defini-
tions also apply to this test guideline.
Acute toxicity is the discernible adverse effects induced in an orga-
nism within a short period of time (days) of exposure to a chemical. The
effects (lethal or sublethal) occurring may usually be observed within the
period of exposure with aquatic organisms. In this test guideline, abnormal
development or death is used as the measure of toxicity.
48-h EC50 (Effective Median Concentration) is that experimentally
derived concentration of a chemical in water in which 50 percent of the
larvae exposed to test material are dead or abnormally developed compared
to larvae in the controls (not exposed to test material) after a 48-h expo-
sure.
Embryo is the stage between the fertilization of the egg and the
trochophore (2 to 8 cell stage).
Larva includes the trochophore and the straight hinge stage.
LOEC is the lowest observed effect concentration.
NOEC is the no observed effect concentration.
Veliger is the larval stage in which the ciliated velum (swimming
organ) is present.
(d) Test procedures—(1) Summary of the test, (i) The water solu-
bility and the vapor pressure of the test chemical should be known. Prior
to testing, the structural formula of the test chemical, its purity, stability
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in water and light, w-octanol/water partition coefficient, and pK values
should be known. The results of a biodegradability test and the method
of analysis for the quantification of the chemical in water is also desirable.
(ii) It may be possible to determine an EC50 for a chemical with
limited solubility under the test conditions. If the stability or homogeneity
of the test chemical cannot be maintained, care should be taken in the
interpretation of the results and a note made that these results may not
be reproducible.
(iii) This study consists of a static 48-h exposure that is used to evalu-
ate the proportion of living and normal D-shaped veligers exposed to the
test material compared to the proportion of the same in controls not ex-
posed to test material. The concentration-response curve and EC50 value
for the test chemical are developed from these data.
(2) Range-finding test. A range-finding test should be conducted to
establish test chemical concentrations for the definitive test. The test is
conducted in the same way as the definitive test except a widely spaced
chemical concentration series (i.e. log-interval) is used.
(3) Definitive test, (i) The test is started about 4 h after fertilization
while the embryos are in the 2- to 4-cell stage (determined microscopi-
cally). At this stage embryos (15-30 embryos/mL/replicate) are added to
the test solution. The endpoint for this test is the determination of a
48-h EC50. This will be based on the proportion of normal larvae (those
that are alive with completely developed shells containing meat) exposed
to test solution as compared to normal larvae in controls. An LOEC and
an NOEC are also to be calculated. Constant conditions should be main-
tained in the test facilities as much as possible throughout the test. The
preparation and storage of the test material, the holding of the oysters,
and all operations and tests should be carried out in an environmental free
from harmful concentrations of dust, vapors, and gases and in such a way
to avoid cross-contamination. Any disturbances that may change the be-
havior of the test organisms should be avoided.
(ii) The test chemical concentrations are to be documented in all tests.
At least five test concentrations are to be used with a dose separation
factor not to exceed 1.8 between concentrations.
(iii) Test organisms are to be impartially distributed among test cham-
bers in such a manner that the test results show no significant bias from
the distributions.
(iv) Test organisms are inspected at regular intervals. Dead bivalves
are removed when observed.
(v) The criteria for a valid definitive test are:
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(A) Mortality or aberrant development in the controls are not exceed
30 percent percent for oysters or 40 percent for clams at the end of each
test.
(B) The dissolved oxygen concentration should be at least 60 percent
of air saturation throughout all tests.
(C) Embryos were not more than 4-h old from fertilization at the
beginning of the test.
(D) The difference between the time-weighted-average (TWA) meas-
ured temperatures for any two test chambers from the beginning to the
end of the test should not be greater than 1 °C. No single measured tem-
perature in any test chamber should be more than 3 °C different from the
mean of the TWA measured temperatures for the individual test chambers.
The difference between the measured temperatures in any two test cham-
bers should not be more than 2 °C at any one time.
(e) Test conditions—(1) Test species—(i) Selection. (A) Eastern
oysters (C. virginicd) are the preferred test species, but Pacific oysters
(C. gigas), quahogs (M. mercenaria), or bay mussels (M. edulis) may also
be used.
(B) The test must begin with embryos within 4-h of fertilization when
embryos are in the 2- to 4-, and 8-cell stages.
(C) Embryos used to start a test should be obtained from females
and males that have been maintained for at least 2 weeks in the dilution
water in the laboratory before they are stimulated to spawn.
(D) The spawning of bivalve test organisms is induced by rapidly
elevating the temperature 5-10 °C above the conditioning temperature. An
added stimulus of heat-killed bivalve sperm may be used. To fertilize the
eggs, sufficient sperm suspension should be added to the egg suspension
to yield 105 to 107 sperm/mL in the final mixture. Additional guidance
may be found in paragraph (g)(l) of this guideline.
(ii) Acquisition. Bivalves may be cultured in the laboratory, pur-
chased from culture facilities or commercial harvesters, or collected from
a natural population in an unpolluted area free from epizootic disease.
(2) Test facilities—(i) Apparatus. (A) Test vessels, equipment and
facilities that contact stock solutions, test solutions, or any water into
which any brood stock or test organisms will be placed should not contain
substances that can be leached or dissolved by aqueous solutions in
amounts that adversely affect test organisms.
(B) Test chambers are defined as the smallest physical units between
which there are no water connections. Tests are usually conducted in glass
chambers that are 1- to 2-L in capacity.
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(ii) Dilution water. A constant supply of good quality unfiltered sea-
water should be available throughout the holding, acclimation, and testing
periods. The dilution water should be acceptable to adult bivalve molluscs
and their embryos and larvae. For oysters, at least 70 percent of the em-
bryos resulting from eggs and sperm of appropriately conditioned adults
result in normal larvae while being maintained in the dilution water for
48 h. For clams, this should be 60 percent of the embryos resulting in
normal larvae. Also, a dilution water is acceptable if adult oysters or clams
will survive and grow normally for 14 days without exhibiting signs of
stress, i.e., excessive mucus production (stringy material floating sus-
pended from oysters) lack of feeding, shell gaping, poor shell closing in
response to prodding, or excessive mortality. Natural seawater is rec-
ommended, although artificial seawater with food added may be used. The
dilution water is to have a salinity in excess of 12 ppt. A natural seawater
should have a weekly range in salinity of less than 10 ppt and a monthly
range in pH of less than 0.8 unit. Artificial seawater salinity should not
vary more than 2 ppt nor more than 0.5 pH unit. Oysters are to be tested
in dilution water from the same origin.
(3) Test parameters—(i) Carriers. Stock solutions of substances of
low aqueous solubility may be prepared by ultrasonic dispersion or, if nec-
essary, by use of organic solvents, emulsifiers or dispersant of low toxicity
to oysters. When such carriers are used the control oysters are to be ex-
posed to the same concentration of the carrier as that used in the highest
concentration of the test substance. The concentration of such carriers
should not exceed 0.1 mL/L.
(ii) Dissolved oxygen. The dissolved oxygen concentrations are to
be at least 60 percent of the saturation value and should be recorded daily.
(iii) Loading. The loading rate should not crowd oysters and should
permit adequate circulation of water while avoiding physical agitation of
oysters by water current.
(iv) Temperature. Tests with C. gigas should be conducted at 20 °C,
with C. virginica andM mercenaria at 25 °C, and withM edulis at 16 °C.
The temperature for C. gigas, C. virginica, and M. mercenaria should
never exceed 32 °C, nor 20 °C for M. edulis (even during spawning induc-
tion). Temperature should be recorded continuously.
(v) pH. The pH is to be measured at the beginning and end of the
test in each test chamber.
(f) Reporting. In addition to the reporting requirements prescribed
in 40 CFR Part 792—Good Laboratory Practice Standards, the report is
to contain the following:
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(1) The source of the dilution water, the mean, standard deviation
and range of the salinity, pH, temperature, and dissolved oxygen during
the test period.
(2) A description of the test procedures used (e.g., the flow-through
system, test chambers, chemical delivery system, aeration, etc.).
(3) Detailed information about the oysters used, including the age
and/or size (i.e., height), source, history, method of confirmation of
prespawn condition, acclimation procedures, and food used.
(4) The number of organisms tested, the loading rate, and the
flowrate.
(5) The methods of preparation of stock and test solutions, and the
test chemical concentrations used.
(6) The number of dead and live test organisms, the percentage of
organisms that died, and the number that showed any abnormal effects
in the control and in each test chamber at each observation period.
(7) The calculated 48-h EC50 and its 95 percent confidence limits
and the statistical methods used to calculate these values.
(8) The calculated LOEC and a NOEC must also be developed.
(9) Methods and data records of all chemical analyses of water quality
parameters and test substance concentrations, including method validations
and reagent blanks.
(10) Any incidents in the course of the test which might have influ-
enced the results.
(11) A statement that the test was carried out in agreement with the
prescriptions of the test guideline given above (otherwise a description
of any deviations occurring).
(g) References. The following references should be consulted for ad-
ditional background material on this test guideline.
(1) ASTM. Standard Guide for Conducting Static Acute Toxicity
Tests Starting with Embryos of Four Apecies of Saltwater Bivalve
Molluscs. E 724-89. American Society for Testing and Materials, Philadel-
phia, PA. 18 pp (1989).
(2) [Reserved]
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