Document ID: EPA-HQ-OPPT-2009-0154-0039
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-16-010

October 2016

	Ecological Effects

Test Guidelines

OCSPP 850.1025:

	Oyster Acute Toxicity Test (Shell Deposition)



 

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 850.1025: Oyster acute toxicity test (shell
deposition)

(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)(3) of this guideline); Standard Evaluation Procedure: Acute Toxicity
Test for Estuarine and Marine Organisms (Mollusc 96-Hour Flow-Through
Shell Deposition Study) (see paragraph (j)(4) of this guideline); EPA
Pesticide Reregistration Rejection Rate Analysis: Ecological Effects
(see paragraph (j)(5) of this guideline); and ASTM E729-96 Standard
Guide for Conducting Acute Toxicity Tests on Test Materials with Fishes,
Macroinvertebrates, and Amphibians (see paragraph (j)(1) 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 Eastern oysters, Crassostrea virginica (Gmelin), are exposed to a
test substance in a flow-through system. The Environmental Protection
Agency will use data from this test to assess the hazards and risks a
test substance may present in the aquatic environment.

(c) Definitions. The definitions in OCSPP 850.1000 apply to this test
guideline. In addition, the following more specific definitions apply to
this guideline:

Shell deposition is the measured length of shell growth that occurs
between the time the shell is ground at test initiation and test
termination 96 hours later. The measurement is made from the peripheral
ground edge to the tip of the longest finger of newly deposited shell.
In this guideline, a reduction in shell deposition is used as the
measure of toxicity.

Inhibitory concentration, median (IC50) is the experimentally derived
concentration of test substance in dilution water that would be expected
to cause a 50 percent (%) decrease or inhibition effect on growth of a
group of test organisms under specified exposure conditions. In this
guideline, the effect measured is inhibition of shell deposition.

Umbo is the narrow end (apex) of the oyster shell.

Valve height is the greatest linear dimension of the oyster as measured
from the umbo to the ventral edge of the valves (the farthest distance
from the umbo).

(d) General considerations.

(1) Summary of the test.  Oysters (Crassostrea virginica), which
previously had a portion of the shell periphery ground away, 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 96
hours, during which observations of any toxic effects are made on the
organisms. At test termination, the increment of new shell growth is
measured on all live oysters. The results of the test are expressed as
the shell deposition 96-hour median inhibition concentration (96-h
IC50), i.e., the concentration resulting in a 50% decrease or inhibition
effect on growth, and the slope of the concentration-response
relationship. Although the 96-h IC50 for shell deposition is the primary
toxicity endpoint, information on other signs of toxicity such as
abnormal appearance and behavior and concentration-response curves is
useful in understanding the toxic response.

(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 primary goal of the definitive test is to
determine the 96-hour concentration-response curve for shell deposition;
the 96-h IC50, 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. Concentrations
should be selected so that: very little shell growth relative to
control(s) occurs in oysters at the highest test concentration; shell
growth is slightly less than control(s) at the lowest test
concentration; and there are at least two additional test concentrations
that bracket the 96-h IC50. Clinical signs of toxicity such as abnormal
appearance and behavior, if any, should be reported. Analytical
confirmation of dissolved test concentrations should be performed as
described in OCSPP 850.1000. Summaries of the test conditions are
presented in Table 1 of this guideline. Test validity elements are
listed in Table 2.

(5) Limit test. In some situations, it is only necessary to ascertain
that the shell deposition 96-h IC50 is above a certain limit (i.e., 96-h
IC50 greater than (>) limit concentration). In a limit test, at least 32
oysters, divided into 4 replicates of 8 organisms each, are exposed to a
single “limit concentration,” with the same number of organisms in
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 1 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 the test substance. Clinical signs of toxicity such as
abnormal appearance and behavior, if any, should be reported. If the
effect level for the inhibition of shell growth at the limit
concentration compared to the control(s) is 50% or greater, then a
multiple-concentration 96-hour test should be conducted. For pesticides,
if there is a statistically significant reduction (inhibition) in shell
growth at the limit concentration as compared to the control(s) (i.e.,
no observed effect concentration (NOEC) less than (<) limit
concentration), a multiple-concentration 96-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 96 hours.

	(3) Test organism.

(i) Species. The test species is the Eastern oyster, Crassostrea
virginica. Oysters used in the same test should be 30 to 50 millimeters
(mm) in valve height and should be as similar in age and size as
possible to reduce variability. The standard deviation of the valve
height should be <20% of the mean. Small individuals are preferred
because they are less affected by temperature and thus continue to grow
in spite of temperature fluctuations as opposed to older individuals
that tend to become less active at temperature extremes. Oysters should
be in a pre-spawn condition of gonadal development prior to and during
the test. This may be determined by direct or histological observation
of the gonadal tissue for the presence of gametes or may be inferred
based upon size and condition. If evidence of spawning is observed
during the test, the test should be repeated. 

Oysters 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.
Oysters used in the test should be from the same source and from the
same holding and acclimation tanks.

(ii) Holding and acclimation. Oysters should be attended to immediately
upon arrival at the test facility. Oyster shells should be brushed clean
of fouling organisms. Oysters should be held and acclimated in dilution
water from the same origin as that used for testing. If the organisms
arrive in water, the transfer of the oysters to the holding water should
be gradual to reduce stress caused by differences in water quality
characteristics and temperature. Oysters should be held for a period of
time long enough to demonstrate that they are not diseased or stressed
and are capable of obtaining minimum control shell growth after 96
hours. Holding times of 10 to 12 days before testing have been used.
During holding and acclimation, the oysters should not be crowded, and
the dissolved oxygen concentration should be above 60% saturation.
Holding tanks should be kept clean and free of debris. All oysters
should be maintained in dilution water at the test temperature for at
least 48 hours before they are used. 

Mortalities should be recorded, and the following recommendations should
be applied:

(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/holding 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. Oysters should not receive treatment
for disease during a test.  Oysters should not be used in a test:

(A) If more than 5% of the culture or stock dies or shows signs of
stress (e.g., exhibits abnormal behavior; excessive mucus) during the 48
hours preceding the test;

(B) If the shell is cracked, chipped, bored, or gaping;

			(C) If the adductor muscle is damaged during grinding;

(D) If so much of the shell rim has been removed after grinding that an
opening into the shell cavity is visible;

(E) If infested with mudworms (Polydora sp.) or boring sponges (Cliona
celata);

(F) If they have been used in a previous test, either in a treatment or
in a control group.

(iv) Care and handling. Oysters should be handled as little as possible,
but when necessary, it should be done as carefully and as quickly as
possible. To prepare each oyster for testing, within 24 hours of test
initiation (preferably within 1-2 hours of test initiation),
approximately 3 to 5 mm of the shell periphery should be ground away at
the rounded (ventral) end using a small electric disc grinder or other
appropriate device, taking care to remove the shell rim uniformly to
produce a smooth, rounded, blunt profile. The oyster's valves should be
held together tightly during grinding to avoid vibrating the shell and
injuring the adductor muscle.

(v) Diet and feeding. Oysters should be provided enough food to support
survival and growth during holding, acclimation, and testing. Holding
and acclimating oysters 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 oyster 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 mollusks 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)(2) 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
flow-through exposure technique. Additional guidance on flow-through
exposures is provided in OCSPP 850.1000.

(iii) Treatment concentrations. At least 5 test solution concentrations
should be 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., IC50), 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:

(i) More than 10% of the organisms in any control showed signs of
disease, stress (e.g., abnormal behavior, excessive mucus), and/or
death. 

(ii) An overall mean of at least 2 mm of new shell growth (i.e., overall
mean of all replicates) was not observed in each control group (dilution
water control and vehicle (solvent) control, if a vehicle was used).

(6) Number of test organisms and replicates. For definitive tests, the
minimum number of test organisms per test concentration is 20 divided
into a minimum of 2 replicates, each with 10 oysters. For limit tests,
the minimum number of test organisms is 32 divided into 4 replicates,
each with 8 oysters. Each test vessel should contain an equal volume of
test solution and an equal number of oysters. Replicate test vessels
should be physically separated, since the test vessel is the
experimental unit.

(i) Loading. The loading rate should not crowd oysters and should permit
adequate circulation of water around each shell while avoiding physical
agitation of the oysters by water currents. Flow rates should be
sufficient to promote adequate oyster shell growth and maintain
environmental conditions. A flow rate of 1 L/h/individual has been
demonstrated to allow for adequate shell growth and is recommended as
the minimum flow rate if using unfiltered natural seawater that is not
supplemented with additional algae. 

(ii) Introduction of test organisms. The test should be started by
introducing oysters that have been acclimated to test conditions and
have had approximately 3 to 5 mm of the shell periphery ground away at
the rounded (ventral) end into the test vessels after the test system
has equilibrated. Within a test vessel, the oysters should be spread out
equidistant from one another so that the entire test vessel is used. The
oysters should all be placed with the left (cupped) valve down and the
open, unhinged ends all oriented in the same direction facing the
incoming flow of test solution. 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, and
testing oysters that are well ventilated and free of fumes and
disturbances which may affect the test organisms. There should be
flow-through tanks for holding and acclimating oysters and a system for
culturing algae, if prepared on-site. 

(ii) Environmental control equipment. Mechanisms for controlling and
maintaining the water temperature, lighting, salinity, and flow during
the culturing, holding, acclimation and test periods. Apparatus for
aerating dilution water and removing gas bubbles as necessary. Apparatus
for aerating the dilution water in the head box before mixing with the
test substance or delivery to test vessels. 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 substance delivery systems and 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. The flow-through system should contain an
appropriate test substance delivery system.

(vi) Dilution water. A dilution water is acceptable if oysters 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. 

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 oysters. 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.

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. 

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 recommended water temperature is 20 °C. During a
given test, the temperature should be constant within plus or minus (±)
2 °C. However, if unfiltered natural seawater that has not been
previously held is used, temporary fluctuations (less than 8 hours) of
±5 °C may occur and be tolerated by oysters (i.e., not affect control
performance) due to their adaptations to fluctuating tidal habitats.

(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)). For unfiltered natural seawater
that is not diluted with freshwater to reduce salinity, a salinity of
>12 ppt, with a range of <5 ppt, is recommended. For artificial seawater
or natural seawater that is diluted with freshwater, 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
addition of the test substance. The dilution water may be aerated
vigorously prior to delivery to the test vessels (e.g., in the diluter
head box) such that the dissolved oxygen concentration is at or near 90
to 100% saturation. If the water is heated, precautions should be taken
to ensure that supersaturation of dissolved gases is avoided. Aeration
of the test solutions during the test is not recommended. Gentle
aeration of test vessels during the exposure period is permitted only 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.

(v) Flow in a flow-through system. During a test, the flow rates should
not vary more than 10% between any one replicate and another. The flow
rate should be sufficient to promote adequate oyster shell growth and
maintain environmental conditions. A flow rate of 1 L/h/individual has
been shown to provide adequate environmental conditions. If unfiltered
natural seawater that is not supplemented with additional algae is used,
then the recommended flow rate is 1 L/hour/individual. It is recommended
that diluter systems be monitored for proper adjustment and operation at
least twice daily throughout the test period to better ensure that the
target test concentrations are achieved and maintained. The flow rate to
each test vessel should be measured at the beginning and end of the
test.

	(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) Mortality. The oysters should be inspected at least after 24, 48,
72, and 96 hours. An observation period at <12 hours is desirable.
Oysters should be considered dead if touching of the gaping shell
produces no reaction. Dead oysters should be removed from the test
vessels at the time of observation, and mortalities should be recorded. 

(B) Appearance and behavior. Visible abnormalities such as loss of
feeding activity (failure to deposit feces), excessive mucus production
(stringy material floating suspended from oysters), spawning, or closure
or gaping of shell should be recorded.

(C) Shell growth. Shell growth is the primary criterion used in this
test to evaluate the toxicity of the test substance. Shell growth
increments in all oysters should be measured after 96 hours of exposure.
The length of the longest “finger” of new shell growth, from the
ground edge to the tip, should be recorded to the nearest 0.1 mm.

(f) Treatment of results.

(1) Summary statistics.

(i) Mortality and shell growth. The number of oysters exposed at test
initiation in each treatment and replicate and the cumulative number of
dead oysters should be summarized in tabular form by time of
observation, treatment, and replicate. Shell growth (mean, standard
deviation) after 96 hours of exposure should be summarized in tabular
form by treatment and replicate. 

(ii) Appearance and behavior. The number of oysters exhibiting abnormal
appearance or behavioral symptoms should be summarized in tabular form
by time of observation, treatment, and replicate.

	(2) Percent mortality and shell growth inhibition. 

(i) The percent mortality at each treatment level and in the controls at
24, 48, 72, and at test termination (96 hours) should be calculated.

(ii) The mean percent inhibition of shell growth at each treatment level
compared to the control(s) at test termination (96 hours) should be
calculated.

(3) Evaluation of limit test results. If the effect level for the
inhibition of shell growth at the limit concentration compared to the
control(s) is 50% or greater, then a multiple-concentration 96-hour test
should be conducted. For pesticides, if there is a statistically
significant reduction (inhibition) in shell growth at the limit
concentration compared to the control(s) (i.e., NOEC < limit
concentration), then a multiple-concentration 96-hour test should be
conducted.

	(4) Evaluation of multiple-concentration definitive test.

(i) Concentration-response curve, slope, and IC50. Statistical
procedures should be employed to calculate the 96-h IC50 (standard error
and 95% confidence interval) based on reduction in shell deposition. If
a concentration-response curve model was fit to the data to determine
the IC50, the model parameters (e.g., slope) and their uncertainty
estimates (e.g., standard error, 95% confidence interval) 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
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 for shell deposition is
designed for hypothesis testing.

(iii) Statistical methods. Statistical procedures for modeling
continuous data should be used. Additional discussion about endpoints
and statistical procedures can be found in OCSPP 850.1000. 

―Summary of Test Conditions for Oyster Acute Toxicity Test (Shell
Deposition)

Test type	Flow-through

Test species	Crassostrea virginica

Test duration	96 hours

Temperature	20 °C (constant during test within ±2 °C) 

However, if unfiltered natural seawater that has not been previously
held is used, temporary fluctuations (less than 8 hours) of ±5 °C may
occur and be tolerated by oysters (i.e., not affect control performance)
due to their adaptations to fluctuating tidal habitats.

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);

Unfiltered natural seawater that 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

Size of test organisms	30-50 mm in valve height; similar in age and
size; standard deviation of valve height <20% of the mean

Number of organisms per concentration	Multiple-concentration definitive
test : 20 (minimum) 

Limit test: 32 (minimum)

Number of replicate test vessels per concentration
Multiple-concentration definitive test: 2 (minimum) 

≤0.1 mL/L for recommended solvents (see OCSPP 850.1000)

Measure of effect or measurement endpoint	96-h IC50 based on reduction
in shell growth

(h) Test validity elements. This test would be considered to be
unacceptable or invalid if one or more of the conditions in Table 2
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 2 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 size), 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 2. ―Test Validity Elements for the Oyster Acute Toxicity Test
(Shell Deposition)

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. More than 10% of the organisms in either the dilution water or
vehicle (solvent) controls showed signs of disease, stress (e.g., shell
gaping, excessive mucus), and/or death. 

5. An overall mean of at least 2 mm of new shell growth (i.e., overall
mean of all replicates) was not observed in each control group (vehicle
(solvent) control and dilution water control).

6. 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.)

7. Evidence of spawning was observed.	

(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 which 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 oysters used in the test: source, culture
practices, and holding and acclimation procedures and conditions,
including acclimation period, water used, feeding history, and health
status (mortality before test initiation and any preventative or disease
treatments). 

(iv) Duration of quarantine for test organisms collected from a natural
population.

	(v) For oysters receiving disease treatment, hours prior to testing
with no sign of disease.

		(vi) Age of test organisms and method of confirmation of prespawn
condition.

		(vii) Size of test organisms at test initiation (i.e., mean valve
height).

(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: flow-through, open or closed
system. A description of the flow-through system, including flow rate
and test vessel turnover rate. For closed systems, a description of the
closed system design. For all systems, a description of the calibration
and validation methods.

(iii) Description of the dilution water and 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 of aeration within exposure
system (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) Detailed information on feeding (e.g., type of feed, source, amount
given, and frequency). 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.

(xii) Methods and frequency of environmental monitoring performed during
the definitive or limit test for test solution temperature, dissolved
oxygen, pH, salinity, and light intensity.

(xiii) Methods and frequency of measuring the dissolved test substance
to confirm exposure concentrations.

(xiv) Methods and frequency of measuring shell growth and observing
mortality or any other toxic symptoms.

(xv) 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, a tabulation of
shell growth (mm) of each oyster and the number and percentage of dead
oysters in each test vessel, for all treatment levels and control(s), at
each observation period. A description and count of any other appearance
or behavioral effects, if recorded, at each treatment level and in the
control(s). 

(iv) For limit test, a tabulation of shell growth (mm) of each oyster
and the number and percentage of dead oysters 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). 

(v) For definitive test, a tabulation of shell growth (mm) of each
oyster and the number and percentage of dead oysters in each test
vessel, for all treatment levels and control(s), at each observation
period (provide the raw data) and descriptive statistics (mean, standard
deviation, minimum, maximum).

(vi) For limit and definitive tests, a description and tabulation of
abnormal appearance and behavioral signs of toxicity by test vessel,
treatment, and observation time (provide raw data).

(vii) The percent inhibition of shell growth for organisms in each
replicate test vessel for test concentrations compared to the control(s)
at each observation period.

(viii) Graphs of the concentration-response data for percent inhibition
of shell growth.

(ix) For limit test, conclusion about the 96-h IC50 for shell growth
being above the limit concentration.

(x) For definitive test, where sufficient data exist to fit a model, a
tabulation of the 96-hour slope of the concentration-response curve, its
standard error and 95% confidence interval, and any goodness-of-fit
results. 

(xi) For definitive test, the 96-h IC50 value for shell growth, and its
standard error and 95% confidence interval.

		(xii) For definitive test, the 96-hour NOEC for shell growth, if
determined.

(xiii) Description of statistical method(s) used for point estimates,
including the software package for determining IC50 values and fitting
the concentration-response model, and the basis for the choice of
method. Provide results of any goodness-of-fit tests.

(xiv) Description of statistical method(s) used for NOEC and LOEC
determination, including the software package, and the basis for the
choice of the method.

(j) References. The following references should be consulted for
additional background material on this test guideline.

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0(1) American Society for Testing and Materials. ASTM E729-96, Standard
Guide for Conducting Acute Toxicity Tests on Test Materials with Fishes,
Macroinvertebrates, and Amphibians. In Annual Book of ASTM Standards,
Vol. 11.06, ASTM, West Conshohocken, PA. Current edition approved
October 1, 2014. 

(2) 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.

(3) U.S. Environmental Protection Agency, 1982. Pesticide Assessment
Guidelines Subdivision E, Hazard Evaluation: Wildlife and Aquatic
Organisms, Office of Pesticide and Toxics Substances, Washington, DC,
EPA 540/9-82-024.

(4) U.S. Environmental Protection Agency, 1985. Hazard Evaluation
Division Standard Evaluation Procedure, Acute Toxicity Test for
Estuarine and Marine Organisms (Mollusc 96-Hour Flow-Through Shell
Deposition Study). Office of Pesticides Programs, Washington, DC,
EPA-540/9-85-011.

(5) U.S. Environmental Protection Agency, 1994. Pesticides
Reregistration Rejection Rate Analysis: Ecological Effects, Prevention,
Pesticides, and Toxic Substances, Washington, DC, EPA 738-R-94-035

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