United States
Environmental Protection
Iml M m Agency
Office of Chemical Safety	EPA 712-C-16-006
and Pollution Prevention	October 2016
(7101)
Ecological Effects
Test Guidelines
OCSPP 850.1055:
Bivalve Acute Toxicity
Test (Embryo-Larval)

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NOTICE
This guideline is one of a series of test guidelines established by the United States
Environmental Protection Agency's Office of Chemical Safety and Pollution Prevention (OCSPP) for
use in testing pesticides and chemical substances to develop data for submission to the Agency under
the Toxic Substances Control Act (TSCA) (15 U.S.C. 2601, et seq.), the Federal Insecticide,
Fungicide and Rodenticide Act (FIFRA) (7 U.S.C. 136, et seq.), and section 408 of the Federal Food,
Drug and Cosmetic Act (FFDCA) (21 U.S.C. 346a). Prior to April 22, 2010, OCSPP was known as
the Office of Prevention, Pesticides and Toxic Substances (OPPTS). To distinguish these guidelines
from guidelines issued by other organizations, the numbering convention adopted in 1994 specifically
included OPPTS as part of the guideline's number. Any test guidelines developed after April 22, 2010
will use the new acronym (OCSPP) in their title.
The OCSPP harmonized test guidelines serve as a compendium of accepted scientific
methodologies and protocols that are intended to provide data to inform regulatory decisions under
TSCA, FIFRA, and/or FFDCA. This document provides guidance for conducting the test, and is also
used by EPA, the public, and the companies that are subject to data submission requirements under
TSCA, FIFRA, and/or the FFDCA. As a guidance document, these guidelines are not binding on
either EPA or any outside parties, and the EPA may depart from the guidelines where circumstances
warrant and without prior notice. At places in this guidance, the Agency uses the word "should." In
this guidance, the use of "should" with regard to an action means that the action is recommended
rather than mandatory. The procedures contained in this guideline are strongly recommended for
generating the data that are the subject of the guideline, but EPA recognizes that departures may be
appropriate in specific situations. You may propose alternatives to the recommendations described in
these guidelines, and the Agency will assess them for appropriateness on a case-by-case basis.
For additional information about these test guidelines and to access these guidelines
electronically, please go to http://www.epa.gov/ocspp and select "Test Methods & Guidelines" on the
navigation menu. You may also access the guidelines in http://www.regulations.gov grouped by
Series under Docket ID #s: EPA-HQ-OPPT-2009-0150 through EPA-HQ-OPPT-2009-0159, and
EPA-HQ-QPPT-2009-0576.
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OCSPP 850.1055: Bivalve acute toxicity test (embryo-larval)
(a)	Scope.
(1)	Applicability. This guideline is intended for use in meeting testing requirements of
the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) (7 U.S.C. 136, et seq.)
and the Toxic Substances Control Act (TSCA) (15 U.S.C. 2601, et seq.). It describes
procedures that, if followed, would result in data that would generally be of scientific
merit for the purposes described in paragraph (b) of this guideline.
(2)	Background. The source materials used in developing this harmonized OCSPP test
guideline are OPP 72-3 Acute Toxicity Test for Estuarine and Marine Organisms
(Pesticide Assessment Guidelines, Subdivision E—Hazard Evaluation: Wildlife and
Aquatic Organisms, see paragraph (j)(4) of this guideline); Acute Toxicity Test for
Estuarine and Marine Organisms Mollusc 48-Hour Embryo Larvae Study (Hazard
Evaluation Division Standard Evaluation Procedure, see paragraph (j)(5) of this
guideline); ASTM E724-98, Standard Guide for Conducting Static Acute Toxicity Tests
Starting with Embryos of Four Species of Saltwater Bivalve Molluscs (see paragraph
(j)(l) of this guideline); and EPA Pesticide Reregi strati on Rejection Rate Analysis:
Ecological Effects (see paragraph (j)(6) of this guideline).
(b)	Purpose. This guideline is intended for use in developing data on the acute toxicity of
chemical substances and mixtures ("test chemicals" or "test substances") subject to
environmental effects test regulations. This guideline describes an acute toxicity test in which
embryos of Eastern oysters (Crassostrea virginica), Pacific oysters (Crassostrea gigas), quahogs
(Mercenaria mercenaria), or blue mussels (Mytilus edulis) are exposed to a test substance in a
static system. The Environmental Protection Agency will use data from this test in assessing the
hazards and risks a test substance may present in the aquatic environment.
(c)	Definitions. The definitions in OCSPP 850.1000 apply to this guideline. In addition, the
following more specific definitions apply to this guideline:
Effect concentration, median (EC50) is the experimentally derived concentration of test
substance in dilution water that would be expected to cause a defined adverse effect in 50
percent (%) of a group of test organisms under specified exposure conditions. In this
guideline, the effect measured is the failure to develop into normal larvae as defined in
this guidance.
Embryo is the stage between the fertilization of the egg (2- to 8-cell stages) and the
trochophore.
Larva includes the trochophore through the straight hinge stage.
Normal larvae, as used in this guidance, include larvae with completely developed shells
containing meat and larvae with completely developed but misshapen or otherwise
malformed shells containing meat. The latter are included among normal larvae because a
malformed shell is considered unlikely to reduce survival in the natural environment (see
paragraph (j)(l) of this guideline). Completely developed, empty shells (i.e., with no
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meat) are not considered to be normal larvae because they are indicative of mortality. In
addition, larvae with incompletely developed shells containing meat are not considered to
be normal larvae because they are likely to have reduced survival in the natural
environment (see paragraph (j)(l) of this guideline).
(d) General considerations.
(1)	Summary of the test. Embryos of a particular species of bivalve mollusc are exposed
to the test substance and to appropriate controls (i.e., dilution water control and vehicle
(solvent) control, if a vehicle is used) for 48 hours, at which time observations are made
on the number of normal larvae (see paragraph (c) of this guideline for definition). The
test is designed to determine the relationship between aqueous concentrations of the test
substance and embryos that did not result in normal larvae over the full concentration-
response curve. The results of the test are expressed as the 48-hour median effect
concentration (48-h EC50) based upon the percentage of embryos that did not result in
normal larvae.
(2)	General test guidance. The general guidance in OCSPP 850.1000 applies to this
guideline except as specifically noted herein.
(3)	Range-finding test. A range-finding test is usually conducted to establish the
appropriate test solution concentrations to be used for the definitive test. In the range-
finding test, the test organisms are generally exposed to a series of widely-spaced
concentrations of the test substance (e.g., 1, 10, 100 milligrams per liter (mg/L)). The
details of the range-finding test do not have to be the same as those of definitive testing in
that the number of replicates, the number of test organisms, and duration of exposure may
be less than that used in definitive testing. In addition, the types of observations made on
test organisms may not be as detailed or as frequently observed as that of a definitive test.
(4)	Definitive test. The goal of the definitive test is to determine the 48-hour
concentration-response curve for the proportion of embryos that did not result in normal
larvae; the 48-h EC50, its standard error and 95% confidence interval; and the slope of the
concentration-response curve, its standard error and 95% confidence interval. A
minimum of 5 concentrations of the test substance, plus appropriate controls, should be
tested. The selected test concentrations should bracket the 48-h EC50. Analytical
confirmation of dissolved test concentrations should be performed as described in
OCSPP 850.1000. Summaries of the test conditions are presented in Table 2 of this
guideline. Test validity elements are listed in Table 3.
(5)	Limit test. In some situations, it is only necessary to ascertain that the 48-h EC50 is
above a certain limit (i.e., 48-h EC50 greater than (>) limit concentration). In a limit test,
at least 4 replicate test vessels, each containing 15-30 embryos per milliliter (mL), are
exposed to a single "limit concentration," with at least 4 replicates of the appropriate
controls. For most industrial chemicals, the lower of 100 mg/L or the limits of water
solubility or dispersion is considered appropriate as the limit concentration. For
pesticides, the lower of 100 milligrams active ingredient per liter (mg a.i./L), when
estimated environmental concentrations are not expected to exceed 100 mg/L, or the limit
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of water solubility may be used as the limit concentration. Except for the number of test
concentrations and the number of replicates, limit tests should follow the same test
procedures, have the same duration as the multiple-concentration definitive test (see
Table 2 of this guideline), and have both a dilution water control and a vehicle (solvent)
control, if a vehicle is used. Limit tests, like definitive tests, should include analytical
confirmation of the dissolved concentration of test substance. If the effect level for the
percentage of embryos that did not result in normal larvae at the limit concentration
compared to the control(s) is 50% or greater (i.e., 50% or more "affected" larvae), then a
multiple-concentration 48-hour test should be conducted. For pesticides, if there is a
statistically significant effect on the percentage of embryos that did not result in normal
larvae at the limit concentration compared to the control(s) (i.e., no observed effect
concentration (NOEC) less than (<) limit concentration), a multiple-concentration 48-
hour test should be conducted.
(e) Test standards.
(1)	Test substance. The substance to be tested should be technical or reagent grade
unless the test is designed to evaluate a specific formulation, mixture, or end-use product.
For pesticides, if more than one active ingredient constitutes a technical product, the
technical grade of each active ingredient should be tested separately, in addition to the
combination, if applicable. OCSPP 850.1000 lists the type of information that should be
known about the test substance before testing and discusses methods for preparation of
test solutions.
(2)	Test duration. The test duration is a minimum of 48 hours.
(3)	Test organism.
(i)	Species. Crassostrea virginica (Eastern oysters) is the preferred test species,
but Crassostrea gigas (Pacific oysters), Mercenaria mercenaria (quahogs, hard
clams) or Mytilus edulis (blue mussels, bay mussels) may also be used. The test
should start with embryos within 4 hours of fertilization when they are in the 2- to
8-cell stages.
Bivalves may be cultured in the laboratory, purchased from culture facilities or
commercial harvesters, or collected from a natural population in an unpolluted
area free from epizootic diseases. Records should be kept regarding their source
and/or culturing techniques. For any one test, all animals in the brood stock
should be from the same source and from the same holding and acclimation tanks.
(ii)	Holding and acclimation. 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. During
culturing, holding, and acclimation, adult bivalves should be maintained in
environmental conditions (e.g., temperature, light intensity) similar to those to be
used in the test and should not be crowded or subjected to rapid changes in
temperature (except to induce spawning) or water quality.
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Abrupt changes in water quality, rough handling, or extended periods of
dessication can induce spawning, so it is important to minimize these stimuli until
spawning is desired. If unplanned spawning occurs, discarding all individuals in
the trough is recommended.
The flow rate during holding, acclimation, and conditioning should be high
enough to prevent water quality degradation and provide adequate food.
Additional information on flow rates for each species can be found in paragraph
(j)(l) of this guideline. Holding, acclimating, and conditioning trays should be
drained and sprayed with clean water at least once a week to prevent
accumulation of organic matter and bacteria. The brood stock should be observed
daily for any signs of stress or mortality. Gaping molluscs that do not close when
touched or molluscs that never open or never produce feces or pseudofeces should
be discarded. Dead bivalves should be removed daily.
Mortalities should be recorded, and the following recommendations should be
applied to the brood stock:
(A)	Mortalities of greater than 10% of the population in the 7 days directly
preceding the test: rejection of entire batch;
(B)	Mortalities of between 5 and 10% of the population during the 7 days
directly preceding the test: acclimation continued for additional 7 days;
(C)	Mortalities of less than 5% of the population during the 7 days directly
preceding the test: acceptance of batch.
(iii)	Health status and condition. Adult brood stock should not be used for a test:
(A)	If they are injured during handling;
(B)	If they exhibit abnormal shell development;
(C)	If unplanned spawning occurs in the brood stock tank.
(iv)	Care and handling of brood stock. Organisms should be handled as little as
possible, but when necessary, it should be done as carefully and quickly as
possible.
The ripeness of the brood stock can be determined by sacrificing several animals
and examining the gonads, as described in paragraph (j)(l) of this guideline. If the
brood stock contains ripe gonads, the animals should be held in cool water (see
Table 1) to prevent deterioration of the quality of the gametes.
If the brood stock does not contain ripe gonads, the brood stock should be
conditioned prior to any attempt to induce spawning. To condition bivalves, the
temperature and water should be gradually changed to the conditioning
temperature (see Table 1) and the dilution water.
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The spawning of adult bivalves should be induced by rapidly elevating the
temperature 5 to 10 degrees Celsius (°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 per mL in the final mixture. Additional guidance may be found in
paragraph (j)(l) of this guideline.
Table 1.—Recommended Temperatures (°C) (from Paragraph (j)(1) of this Guideline)
Species
Holding
Conditioning
Induction
Never to be exceeded
Test
C. gigas
14-15
20
25-32
32
20
C. virginica
14-15
20-25
25-32
32
25
M. mercenaria
14-15
20-25
25-32
32
25
M. edulis
8
12-14
15-20
20
16
(v) Diet and feeding. Larvae should not be fed during testing. During holding,
acclimation, and conditioning, the brood stock should be provided with enough
food to support survival and growth and not cause stress. Holding, acclimating,
and conditioning bivalves in natural seawater that is not expected to contain
disease-causing organisms at adverse concentrations and contains as much natural
phytoplankton as possible is advantageous. Cultured algae may be added to the
water as necessary to support bivalve survival and growth. If natural seawater that
is not supplemented with an additional food source is used, it should not be
passed through an ultraviolet sterilizer or a filter of <20 micrometers (|im). If
unsterilized and unfiltered natural seawater is used without adding algae, at least 1
liter per hour per individual (L/h/individual) is usually the minimum flow rate for
molluscs of the size 40-50 mm (umbo to distal valve edge) to provide an adequate
food supply that supports the desired growth rate. If the presence of disease-
causing organisms is suspected in natural seawater, then filtration through a
smaller sized filter and addition of a supplemental algal source to the test system
may be necessary.
(4) Administration of test substance.
(i) Preparation of test solutions. Preparation of test solutions depends on the
solubility and stability of the test substance. Guidance for preparation of test
solutions, especially for difficult or low solubility test substances, is provided in
OCSPP 850.1000. Dilution water source and quality used in the test are described
in OCSPP 850.1000 and paragraph (e)(7)(vi) of this guideline.
The concentration of vehicle solvent should not exceed 0.1 milliliters per liter
(mL/L). A previous review recommends that solvent concentrations as low as 0.02
mL/L of dilution water be used (see paragraph (j)(3) of this guideline).
The pH of stock solutions may be adjusted to match the pH of dilution water or to
a neutral pH if pH change does not affect the stability of the test substance in
water. The pH of test solutions may be adjusted after the addition of the test
substance or stock solution into the dilution water. However, all pH adjustments
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need to be made prior to the addition of test organisms. Hydrochloric acid (HC1)
and sodium hydroxide (NaOH) may be used for this adjustment if warranted.
See additional information about pH during testing in (e)(8)(ii).
(ii)	Exposure technique. The test should be conducted using the static exposure
technique. Guidance on this technique is provided in OCSPP 850.1000.
(iii)	Treatment concentrations. At least 5 test solution concentrations should be
used for definitive testing, plus the appropriate control(s). A range-finding test
can be used to establish the appropriate test solution concentrations for the
definitive test (see paragraph (d)(3) of this guideline). For scientifically sound
estimates of a given point estimate (e.g., EC50), test substance concentrations
should immediately bracket the point estimate(s) of concern. OCSPP 850.1000
provides guidance on selection of test concentrations. For a limit test, there is a
single treatment concentration, plus the appropriate control(s). Guidance on the
limit concentration is provided in paragraph (d)(5) of this guideline.
(5)	Controls. Every test includes a dilution water control and a vehicle (solvent) control,
if a vehicle is used. Controls consist of the same dilution water, conditions, procedures,
and test population as the test solutions, except that no test substance is added.
A test is not acceptable if less than 70% of oyster embryos or less than 60% of hard clam
or mussel embryos in any control resulted in normal larvae (see paragraph (c) of this
guideline for definition) at the end of the test.
(6)	Number of test organisms and replicates. The number of embryos per test vessel
should be determined and expressed as a certain density per volume of test solution.
About 1 hour after adding the sperm suspension to the egg suspension, the concentration
of embryos in the resulting suspension should be determined by mixing the solution with
a perforated plunger, withdrawing a 1-mL sample, placing it in a Sedgwick-Rafter cell,
and counting the number of embryos that have developed to a 2-cell stage or beyond.
Based on the concentration of embryos in the suspension and the volume of test solution
in the test vessels, a volume of suspension that will result in a final density of 15-30
embryos/mL should be determined. The same volume of the well-mixed embryo
suspension should be added to each replicate test vessel using an automatic pipet. Each
test vessel should contain an equal volume of test solution and embryo suspension.
For definitive tests, the minimum of replicates per test concentration is two. Due to the
natural rate of incomplete development in bivalve embryos, additional replicates are
recommended for the control(s) to obtain a good estimate of the mean and variance and
provide a stronger statistical baseline. For limit tests, the minimum number of replicates
for the control(s) and limit concentration is four. Replicate test vessels should be
physically separated, since the test vessel is the experimental unit.
(i) Loading. The number of embryos placed in a test vessel should not be so large
as to cause the dissolved oxygen concentration to fall below the recommended
levels or affect the results of the test. Although a loading rate of up to 100
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embryos/mL can be used with Pacific oysters, the other 3 species develop
abnormally at loading densities of greater than 30 embryos/mL. Thus, 30
embryos/mL is the recommended loading limit for this guideline.
(ii) Introduction of test organisms. The test is started within 4 hours of
fertilization by introducing embryos in the 2- to 8-cell stage (determined
microscopically) into the test vessels after the test substance has been added. Test
vessels for treatment levels should be randomly or indiscriminately located within
the test area, and test organisms should be randomly or indiscriminately
distributed among test vessels. Further guidance is provided in OCSPP 850.1000.
(7) Facilities, apparatuses, and supplies. Normal laboratory equipment should be used,
especially the following:
(i)	Facilities. Facilities for culturing, holding, acclimating, conditioning, and
testing that are well ventilated and free of fumes and disturbances which may
affect the test organisms. There should be flow-through tanks for holding,
acclimating, and conditioning brood stock and a system for culturing algae.
(ii)	Environmental control equipment. Mechanisms for controlling and
maintaining the water temperature, lighting, and salinity during the holding,
acclimation, conditioning, and test periods. Apparatus for aerating dilution water
and removing gas bubbles as necessary. An apparatus providing a 30-minute
lighting transition period may be needed.
(iii)	Water Quality Testing Instruments. Equipment for determination of water
quality characteristics (pH, salinity, temperature, etc)
(iv)	Cleaning of test system. Test vessels should be cleaned before each test. See
OCSPP 850.1000 for further information.
(v)	Test containers and delivery system. Construction materials and equipment
that may contact the stock solution, test solution, or dilution water should not
contain substances that can be leached or dissolved into aqueous solutions in
quantities that can affect the test results. Construction materials and equipment
that contact stock or test solutions should be chosen to minimize sorption of test
substances. Refer to OCSPP 850.1000 for additional information on appropriate
construction materials. Test vessels, which should be constructed of chemically
inert material, should be of a capacity to maintain the loading rate and
environmental conditions. Test vessels should be loosely covered to reduce the
loss of test solution or dilution water due to evaporation and to minimize entry of
dust and other particles into the solutions.
Tests are usually conducted in glass test vessels that are 1 to 2 L in capacity.
(vi)	Dilution water. A dilution water is acceptable if bivalves will survive in it
for the duration of the culturing, holding, acclimation, and testing periods without
showing signs of stress. Clean natural unfiltered seawater may be used; such
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water should come from a thoroughly mixed common source to ensure each
oyster is provided equal amounts of food. Clean artificial seawater or filtered
natural seawater with food (algae) added may be used especially if the presence of
disease organisms is suspected in natural seawater. Natural seawater should be
filtered through a filter with a pore size of <20 |im prior to use in a test. Artificial
seawater can be prepared by adding commercially available formulations or
specific amounts of reagent-grade chemicals to reagent water (deionized, distilled,
or reverse osmosis water), surface water, or well water. Dechlorinated tap water is
not recommended for preparation of artificial seawater (or dilution of natural
seawater) because some forms of chlorination are difficult to remove adequately.
If dechlorinated tap water is used, recommended maximum chlorine levels as well
as other ways to demonstrate suitability as a dilution water source are in OCSPP
850.1000.
Dissolved oxygen in the dilution water (prior to use in a test) should be between
90 and 100% saturation. If necessary, the dilution water can be aerated before the
addition of the test substance.
Since embryos/larvae are not fed during the test, dilution water during testing
should be seawater that is filtered to remove potential food sources and is of the
same quality as that used for the holding, acclimating, and conditioning of adults.
The recommended salinity is 20 parts per thousand (ppt). However, if unfiltered
natural seawater is used, greater variability may be unavoidable but still allow for
adequate control performance due to the euryhaline life history of bivalves. For
unfiltered natural seawater that is not diluted with freshwater to reduce salinity,
salinity of >12 ppt is recommended with a weekly range in salinity of <5 ppt. For
artificial seawater or natural seawater that is diluted with freshwater, salinity
should be maintainable within a weekly range of 2 ppt.
Measurement of total organic carbon (TOC) or chemical oxygen demand (COD)
in the dilution water at the beginning of the test is recommended, but at a
minimum, TOC and COD should be analyzed periodically in the dilution water
source to document and characterize their magnitude and variability. For tests
with cationic substances, TOC or COD should be measured at the beginning of
the test.
Specifications for dilution water quality and constancy are described in OCSPP
850.1000.
(8) Environmental conditions. Environmental parameters during the test should be
maintained as specified below. The number and frequency of measurements
recommended for documenting and confirming the magnitude and variability of water
quality parameters (e.g., temperature, dissolved oxygen, pH, and salinity) in test solutions
during the test are described in detail in OCSPP 850.1000.
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(i)	Temperature. The water temperature for a given species should be as
described in Table 1. During a given test, the temperature should be constant
within ± 1 °C.
(ii)	pH and salinity. The pH should be between 7.5 and 8.5 and vary less than 1
pH unit during the test within a test vessel and between test concentrations
(including control(s)). Salinity should be 20 ppt and constant within ±2 ppt during
the test.
(iii)	Lighting and photoperiod. A photoperiod should be selected from regimes
of 12 hours light: 12 hours dark to 16 hours light: 8 hours dark. For any given test,
the light regime should be constant. Light intensity should range from 540 to 1080
lux (approximately 50-100 foot-candles (ft-c)). A 15- to 30-minute transition
period between light and dark is suggested.
(iv)	Dissolved oxygen. The dissolved oxygen concentration should be between 60
and 100% saturation during the test. If aeration is needed to achieve an
appropriate dissolved oxygen level, it should be done before the addition of the
test substance. Aeration of test solutions during the test is strongly discouraged
because bubbles can collect within the mantle cavity of a larva resulting in death.
Therefore, gentle aeration of test vessels during the exposure period may only be
utilized in cases where the dissolved oxygen levels are in danger of dropping
below 60% saturation. In such cases, assurances should be made that the use of
aeration does not stress the test organisms; test substance concentrations should
be measured during the test to ensure that they are not affected by the use of
aeration; and all treatment and control vessels should be given the same aeration
treatment.
(9) Observations.
(i)	Measurement of test substance. OCSPP 850.1000 describes the
recommended sampling methods, frequency of sampling, and sample processing
(especially of low solubility test substances) for analytical confirmation of
dissolved test concentrations and characterization of test substance stability
throughout the test. The analytical methods used to measure the amount of
dissolved test substance in a sample should be validated before beginning the test,
as described in OCSPP 850.1000, and the relevant method detection limit(s) and
limit(s) of quantification should be reported.
(ii)	Test solution appearance. Observations on test solution appearance and test
substance solubility should be made daily and at the beginning and end of the test.
The appearance of surface slicks, precipitates, or material adhering to the sides of
the test vessels or in any part of the mixing and delivery system should be
recorded at a minimum at the beginning and end of the test and during the test
when the test solution appearance changes.
(iii)	Measures of effect.
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(A)	Embryos at test initiation. At test initiation, the number of embryos
at the 2- to 8-cell stage and beyond should be determined in at least two
suspension samples where the volume of a sample is equivalent to that
added to each test vessel. For each suspension samples, at least 20 counts
with a Sedgwick-Rafter cell counter are recommended.
(B)	Normal larvae at test termination. Forty-eight hours after the start of
the test, the solution in each test vessel should be carefully mixed, and a
measured volume of sample should be immediately removed and
preserved with 5% buffered formalin. The embryos and larvae in the
samples should be placed in a Sedgwick-Rafter cell for counting. For each
replicate, at least 20 counts with the Sedgwick-Rafter cell counter are
recommended. All normal larvae (see paragraph (c) of this guideline for
definition) should be counted.
(f) Treatment of results.
(1) Summary statistics.
(i)	Embryos at test initiation. The mean (Sedgwick counts; No), standard
deviation, and coefficient of variation for the number of embryos at the 2-cell
stage or beyond at test initiation for a test vessel should be calculated.
(ii)	Normal larvae at test termination. For each test vessel, the mean (Sedgwick
counts), standard deviation, and coefficient of variation for the number of normal
larvae (see paragraph (c) of this guideline for definition) should be calculated and
summarized in tabular form.
(2) Percent affected. For each test vessel, z, the percentage of embryos that did not result
in normal larvae ("affected"), pi, is calculated by subtracting the number of normal larvae
at test termination, Nw, from the number of embryos in the test vessel at test initiation, No
(assumed to be normal), and multiplying by 100 (Equation 1).
Pt =
( M - M ^
1 y n	48
N„
(100)
Equation 1
For each test substance test vessel, the percentage of embryos affected should be adjusted
for the percentage affected in the control(s), using Abbotts' formula (see paragraph (j)(2)
of this guideline).
(3) Evaluation of limit test results. If the effect level for the percentage of embryos that
did not result in normal larvae at the limit concentration compared to the control(s) is
50% or greater (i.e., 50% or more "affected" larvae), then a multiple-concentration 48-
hour test should be conducted. For pesticides, if there is a statistically significant increase
in the percentage of embryos that did not result in normal larvae at the limit concentration
compared to the control(s) (i.e., NOEC < limit concentration), then a multiple-
concentration 48-hour test should be conducted.
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(4) Evaluation of multiple-concentration definitive test.
(i)	Concentration-response curve, slope, and ECso. Statistical procedures
should be employed to calculate the 48-h EC50 (standard error and 95%
confidence interval) based on the percentage of embryos that did not result in
normal larvae (see paragraph (c) of this guideline for definition). If a
concentration-response curve model (e.g., probit) was fit to the data to determine
the EC50, the model parameters (e.g., slope) and their uncertainty estimates (e.g.,
standard error) should be recorded.
(ii)	NOEC. While calculation of the NOEC and lowest observed effect
concentration (LOEC) is usually not part of the experimental design for the
regression-based definitive tests, reporting these values when possible is useful
when testing industrial and pesticide chemicals for understanding the toxic
response. The limit test is designed for hypothesis testing.
(iii)	Statistical methods. Statistical procedures for modeling quantal data should
be used. Additional discussion about endpoints and statistical procedures is found
in OCSPP 850.1000.
(g) Tabular summary of test conditions. Table 2 lists the important conditions that should
prevail during the multiple-concentration definitive test. The same conditions are recommended
for a limit test, except for differences in the number of test concentrations and replicates.
Meeting these test conditions will help ensure the satisfactory performance of the test.
Table 2.—Summary of Test Conditions for Bivalve Acute Toxicity Test (Embryo-Larval)
Test type
Static
Test species
Crassostrea virginica (Eastern oysters) is the preferred test
species; other test species that may be used include
Crassostrea gigas (Pacific oysters), Mercenaria mercenaria
(quahogs, hard clams), or Mytilus edulis (blue mussels, bay
mussels)
Test duration
48 hours
Temperature
Varies by species (see Table 1) (constant during test within ±1
°C of selected test temperature)
Light quality
Ambient laboratory illumination
Light intensity
540-1080 lux (approximately 50-100 ft-c)
Photoperiod
Selected from among 12 hours light: 12 hours dark to 16 hours
light:8 hours dark schemes
Salinity
Artificial or natural seawaterthat is diluted with freshwater: 20
ppt (range of ±2 ppt during test);
Natural seawaterthat is not diluted with freshwater to reduce
salinity: >12 ppt (range of <5 ppt during test)
PH
Between 7.5 and 8.5 (constant during test within ±1 pH unit)
TOC
<2 mg/L
Age of test organisms
Embryos at 2- to 8-cell stage at test initiation
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Number of replicate test vessels per
concentration
Multiple-concentration definitive test: 2 (minimum; more are
preferable for the control(s))
Limit test: 4 (minimum)
Number of organisms per test vessel
15-30 embryos/mL
Loading
<30 embryos/mL
Feeding regime
No feeding during test
Test vessel aeration
Strongly discouraged because bubbles can collect within the
mantle cavity of a larva resulting in death. Gentle aeration of
test vessels may only be used in cases where the dissolved
oxygen levels are in danger of dropping below 60% saturation.
In such cases, assurances should be made that the use of
aeration does not stress the test organisms; test substance
concentrations should be measured during the test; and all
treatment and control vessels should be given the same
aeration treatment.
Test concentrations
Definitive test: minimum of 5 test concentrations chosen in a
geometric series plus a dilution water control and a vehicle
(solvent) control, if a vehicle is used
Vehicle concentration, if used
<0.1 mL/L for recommended solvents (see OCSPP 850.1000)
Measures of effect or measurement
endpoints
48-h ECso based on the percentage of larvae that are dead or
failed to develop complete shells
(h) Test validity elements. This test would be considered to be unacceptable or invalid if one or
more of the conditions in Table 3 occurred. These parameters are not the only elements
considered when evaluating the acceptability of a test, and it is possible that a test could be found
unacceptable or invalid based on other considerations. However, except for the conditions listed
in Table 3 and in OCSPP 850.1000, it is unlikely that a test will be rejected when there are only
slight variations from guideline environmental conditions and test design unless the control
organisms are significantly affected and/or significant biases are introduced in defining the
magnitude of effect on measurement endpoints as compared to guideline conditions. Before
departing significantly from this guideline (such as deviating from the organism age), the
investigator should contact the Agency to discuss the reason for the departure and the effect the
change(s) may have on test acceptability. In the test report, all departures from the guideline
should be identified, reasons for the changes given, and any resulting effects on test endpoints
noted and discussed.
Table 3.—Test Validity Elements for the Bivalve Acute Toxicity Test (Embryo-Larval)
1.	All test vessels were not identical.
2.	Treatments were not randomly or indiscriminately assigned to individual test vessel locations, or
individual test organisms were not randomly or indiscriminately assigned to test vessels.
3.	A dilution water control (and vehicle (solvent) control, if a vehicle was used) was not included in the
test.
4.	Less than 70% of oyster embryos or 60% of hard clam or mussel embryos in either the dilution water
control or vehicle (solvent) control resulted in normal larvae (see paragraph (c) of this guideline for
definition) at test termination.
5.	A surfactant or dispersant was used in the preparation of a stock or test solution. (However, adjuvants
may be used when testing pesticide typical end-use products.)
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(i) Reporting.
(1)	Background information. Paragraph (k)(l) of OCSPP 850.1000 describes the
minimum background information to be supplied in the report.
(2)	Guideline deviations. Provide a statement of the guideline or protocol followed.
Include a description of any deviations from the test guideline or any occurrences that
may have influenced the results of the test, the reasons for these changes, and any
resulting effects on test endpoints noted and discussed.
(3)	Test substance.
(i)	Identification of the test substance: common name, IUPAC and CAS names,
CAS number, structural formula, source, lot or batch number, chemical state or
form of the test substance, purity (i.e. for pesticides, the identity and concentration
of active ingredient(s)), and radiolabeling, if any, including the location of label(s)
and radiopurity.
(ii)	Storage conditions of the test chemical or test substance and stability of the
test chemical or test substance under storage conditions if stored prior to use.
(iii)	Methods of preparation of the test substance and the treatment concentrations
used in the range-finding and definitive tests, or limit test. Identify whether the
nominal concentrations are corrected or uncorrected for purity of the test
substance.
(iv)	Physicochemical properties of the test substance such as water solubility,
vapor pressure, UV absorption, pKa, and Kow.
(v)	If a vehicle (solvent) is used to prepare stock or test substance provide: the
name and source of the vehicle, the nominal concentration(s) of the test substance
in the vehicle in stock solutions or mixtures, and the vehicle concentration(s) used
in the treatments and vehicle control. If different vehicle concentrations are used
at different treatment levels, the report should, at a minimum, identify the
maximum vehicle concentration used. It is helpful to support the vehicle choice
by including a description of any measures that were taken to identify an
appropriate vehicle for use in the test, such as the types and concentrations of
vehicles used and their corresponding effects on solubility during any preliminary
work.
(vi)	If a positive control is used, provide the name and source of positive control
and the nominal concentration(s) of the positive control material in stock
solutions or mixtures.
(4)	Test organism.
(i) Scientific name and common name.
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(ii)	Method for verifying the species.
(iii)	Information about the bivalves used as brood stock: source, age and/or size
(i.e., height), method confirmation of pre-spawn condition, culture practices, and
holding, acclimation, and conditioning procedures and conditions, including
acclimation period, water used, feeding history, and health status (mortality
before test initiation and any preventative or disease treatments). Feed should be
analyzed periodically to identify background contaminants such as heavy metals
(e.g., arsenic, cadmium, lead, mercury, and selenium) and persistent pesticides,
especially chlorinated insecticides.
(iv)	Description of the methods and handling used to obtain embryos. The
description should include the number of females and males from which gametes
were collected.
(vi) Age (cell stage) and time post-fertilization of embryos at test initiation.
(5) Test system and conditions. Provide a description of the test system and conditions
used in the definitive or limit test and any preliminary range-finding tests.
(i)	Description of the test vessels: size, type, material, and fill volume.
(ii)	Description of the exposure technique: static, open or closed system. For
closed systems, a description of the closed system design.
(iii)	Description of the dilution water an any water pretreatment: source/type;
temperature; salinity; pH; dissolved oxygen; total organic carbon or chemical
oxygen demand; particulate matter; conductivity; metals, pesticides, and residual
chlorine concentrations (mean, standard deviation, range). Describe the frequency
and sample date(s) for documenting dilution water quality and consistency.
(iv)	Use of aeration, if any, and location within exposure system of aeration (e.g.,
test solution or dilution water prior to test substance addition).
(v)	Number of test organisms added to each test vessel at test initiation.
(vi)	Number of test vessels (replicates) per treatment level and control(s).
(vii)	Methods used for treatment randomization and assignment of test organisms
to test vessels.
(viii)	Date of introduction of test organisms to test solutions and test duration.
(ix)	Loading rate.
(x)	Photoperiod and light source.
(xi)	Methods and frequency of environmental monitoring performed during the
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definitive or limit test for test solution temperature, dissolved oxygen, pH,
salinity, and light intensity.
(xii)	Methods and frequency of measuring the dissolved test substance to confirm
exposure concentrations.
(xiii)	Methods and frequency of counting the number of normal larvae and any
other symptoms.
(xiv)	For definitive and limit tests, description of all analytical procedures,
accuracy of the method, method detection limit, and limit of quantification.
(6) Results.
(i)	Nominal exposure concentrations and a tabulation of test substance analytical
results by treatment group and test vessel (provide raw data) and descriptive
statistics (mean, standard deviation, minimum, maximum, coefficient of
variation).
(ii)	Environmental monitoring data results (test solution temperature, dissolved
oxygen, pH, salinity, and light intensity) in tabular form (provide raw data for
measurements not made on a continuous basis) and descriptive statistics (mean,
standard deviation, minimum, maximum).
(iii)	For preliminary range-finding test, if conducted, and for limit and definitive
tests, a tabulation of the number of embryos at test initiation.
(iv)	For preliminary range-finding test, if conducted, a tabulation of the number of
normal larvae and percentage of embryos that did not result in normal larvae in
each test vessel, including all treatment levels and control(s), at each observation
period.
(v)	For limit test, a tabulation of the number of normal larvae and percentage of
embryos that did not result in normal larvae in each test vessel, for the limit
concentration and control(s), at each observation period (provide the raw data)
and descriptive statistics (mean, standard deviation, minimum, maximum).
(vi)	For definitive test, a tabulation of the number of normal larvae and percentage
of embryos that did not result in normal larvae, for all treatment levels and
control(s), at each observation period (provide the raw data) and descriptive
statistics (mean, standard deviation, minimum, maximum).
(vii)	Graphs of the concentration-response data for percentage of embryos that
did not result in normal larvae.
(viii)	For limit test, conclusion about the 48-h EC50 for embryos that did not result
in normal larvae being above the limit concentration.
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(ix)	For definitive test, where sufficient data exist to fit a model (e.g. probit), a
tabulation of the 48-hour slope of the concentration-response curve, its standard
error and 95% confidence interval, and any goodness-of-fit results.
(x)	For definitive test, the 48-h EC50 value based upon the percentage of embryos
that did not result in normal larvae, its standard error, and 95% confidence
interval.
(xi)	For definitive test, the 48-hour NOEC based upon the percentage of embryos
that did not result in normal larvae, if determined.
(xii)	Description of statistical method(s) used for point estimates, including the
software package for determining EC50 values and fitting the concentration-
response model, and the basis for the choice of method. Provide results of any
goodness-of-fit tests.
(xiii)	Description of statistical method(s) used for NOEC and LOEC
determination, including the software package, and the basis for the choice of
method.
(j) References. The following references should be consulted for additional background material
on this test guideline.
(1)	American Society for Testing and Materials. ASTM E724-98, Standard Guide for
Conducting Static Acute Toxicity Tests Starting with Embryos of Four Species of
Saltwater Bivalve Molluscs. In Annual Book of ASTM Standards, Vol. 11.06, ASTM,
West Conshohocken, PA. Current edition approved December 1, 2012.
(2)	Finney, D.J., 1971. Probit Analysis. Cambridge University Press.
(3)	Hutchinson, T.H., N. Shillabeer, M.J. Winter and D.B. Pickford, 2006. Acute and
chronic effects of carrier solvents in aquatic organisms: A critical review. Aquatic
Toxicology, 76, 69-92.
(4)	U.S. Environmental Protection Agency, 1982. Pesticide Assessment Guidelines,
Subdivision E, Hazard Evaluation, Wildlife and Aquatic Organisms, EPA 540/9-82-024,
U.S. Environmental Protection Agency, Washington, DC.
(5)	U.S. Environmental Protection Agency, 1985. Hazard Evaluation Division Standard
Evaluation Procedure: Acute Toxicity Test for Estuarine and Marine Organisms (Mollusc
48-hour Embryo Larvae Study), EPA-540/9-85-012, Office of Pesticide Programs, Office
of Prevention, Pesticides and Toxic Substances, U.S. Environmental Protection Agency,
Washington DC. June 1985.
(6)	U.S. Environmental Protection Agency, 1994. Pesticides Reregi strati on Rejection
Rate Analysis: Ecological Effects, EPA 738-R-94-035, Office of Prevention, Pesticides
and Toxic Substances, December, 1994.
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