Document ID: EPA-HQ-OPPT-2009-0154-0036
Agency: epa
Document Type: Supporting & Related Material
Title: 
Posted Date: 2016-12-28T05:00Z

Office of Chemical Safety and Pollution Prevention

(7101)	EPA 712-C-16-006

October 2016

	Ecological Effects

Test Guidelines

OCSPP 850.1055:

	Bivalve Acute Toxicity Test (Embryo-Larval)



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  HYPERLINK
"http://www.epa.gov/ocspp" http://www.epa.gov/ocspp  and select “Test
Methods & Guidelines” on the navigation menu. You may also access the
guidelines in  HYPERLINK "http://www.regulations.gov"
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-OPPT-2009-0576.

  SEQ CHAPTER \h \r 1 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.

―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)(1) of this guideline); and EPA
Pesticide Reregistration 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)(1) of this guideline). Completely developed, empty shells (i.e.,
with no 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)(1) 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 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. 

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)(1) 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)(1) 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.

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)(1) 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 (µm). 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 need to be made prior to the
addition of test organisms. Hydrochloric acid (HCl) 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 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 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 µm 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.	

(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.

(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; N0), 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, i, 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, N48,
from the number of embryos in the test vessel at test initiation, N0
(assumed to be normal), and multiplying by 100 (Equation 1).

 	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.

	(4) Evaluation of multiple-concentration definitive test.

(i) Concentration-response curve, slope, and EC50. 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 seawater that is diluted with freshwater:
20 ppt (range of ±2 ppt during test);

≤2 mg/L

Age of test organisms	Embryos at 2- to 8-cell stage at test initiation

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 EC50 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.)

(i) Reporting.

(1) Background information. Paragraph (k)(1) 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.

		(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 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.

(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.

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 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
Reregistration Rejection Rate Analysis: Ecological Effects, EPA
738-R-94-035, Office of Prevention, Pesticides and Toxic Substances,
December, 1994. 

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