	Office of Chemical Safety and Pollution Prevention

(7101)	EPA 712-C-16-009

October 2016

	Ecological Effects

Test Guidelines





OCSPP 850.1045:

	Penaeid Acute Toxicity Test





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 850.1045: Penaeid acute toxicity test

(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)(7) of this guideline); Standard Evaluation Procedure: Acute Toxicity
Test for Estuarine and Marine Organisms (Shrimp 96-Hour Toxicity Test)
(see paragraph (j)(8) 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 penaeid shrimp are exposed to a test substance in static,
static-renewal, or flow-through test systems. The Environmental
Protection Agency will use data from this test in assessing the hazard
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:

Adult refers to a post-larval life stage of the penaeid shrimp where
total length is 90 millimeters (mm) or more.

Death is defined as the lack of visible movement or the lack of reaction
of a test organism to gentle prodding.

Larvae refers to a life stage of the penaeid shrimp where total length
is less than (<) 0.4 mm.

Juvenile refers to a post-larval life stage of the penaeid shrimp where
total length is from 0.4 mm up to approximately 25 mm.

Subadult refers to a post-larval life stage of the penaeid shrimp where
total length is from 25 mm up to approximately 90 mm.

(d) General considerations.

(1) Summary of the test. Post-larval juvenile penaeid shrimp
(Farfantepenaeus aztecus, F. duorarum, or Litopenaeus setiferus) are
exposed to the test substance and to appropriate controls (i.e.,
dilution water control and a vehicle (vehicle) control if a vehicle is
used) for 96 hours, during which observations are made on organism
survival and other toxic effects. The test is designed to determine the
relationship between aqueous concentrations of the test substance and
mortality of penaeid shrimp over the full concentration-response curve.
The results of the test are expressed as the 96-hour median lethal
concentration (96-h LC50) and the slope of the concentration-response
relationship. Although the 96-h LC50 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 mortality; the
96-h LC50, its standard error and 95 percent (%) confidence interval;
and the slope of the concentration-response curve, its standard error
and 95% confidence interval. Where sufficient data are available, these
values are also calculated for the 24-, 48-, and 72-hour
concentration-response curves. A minimum of 5 concentrations of the test
substance, plus appropriate controls, should be tested. The selected
test concentrations should bracket the 96-h LC50. 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 96-h LC50 is above a certain limit concentration (i.e., 96-h
LC50 greater than (>) limit concentration). In a limit test, at least 20
shrimp, divided equally into a minimum of 2 replicates, 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, 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. For pesticides, if any shrimp dies
in the limit test 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. This test should be conducted using one of three species of
penaeids: Farfantepenaeus aztecus (brown shrimp, formerly Penaeus
aztecus); Farfantepenaeus duorarum (pink shrimp, formerly Penaeus
duorarum); or Litopenaeus setiferus (white shrimp, formerly Penaeus
setiferus) (see paragraphs (j)(3), (j)(4), (j)(5), and (j)(6) of this
guideline for additional information on penaeids). If another penaeid
species is being considered for use in this test, it is suggested that
the Agency be contacted prior to test initiation. Post-larval juvenile
shrimp should be used to start the test.

Shrimp may be reared from eggs in the laboratory or obtained directly as
juveniles or adults. Taxonomic verification should be obtained from the
commercial supplier or conducted by experienced laboratory personnel or
an outside expert. Records should be kept regarding their source and/or
culturing techniques. If collected from the wild, the specific location
should be identified. Shrimp used in a particular test should originate
from the same source or culture population, be of similar age, and be of
normal size and appearance.

(ii) Holding and acclimation. If shrimp are collected from the wild,
they should be held for at least 10 days for observation and
acclimation. Upon arrival at the test facility, the shrimp should be
transferred to water closely matching the temperature and salinity of
the transporting medium. If the culture or holding water for the
penaeids is not from the same source as the dilution water to be used in
the test, acclimation to the dilution water should be done gradually
over a 48-hour settling-in period. During culturing and acclimation to
the dilution water, stock animals should be maintained in environmental
conditions (e.g., temperature, light intensity) similar to those to be
used in the test. The shrimp should be maintained in dilution water at
the test temperature for at least 7 days prior to the start of the test.
Within a 24-hour period, changes in water temperature should not exceed
1 degree Celsius (°C), and salinity changes should not exceed 2 parts
per thousand (ppt).

Shrimp should be held in glass tanks of 30 liter (L) capacity or larger.
No more than 22 to 24 shrimp should be placed in a 30 L tank unless the
flow-through apparatus can maintain dissolved oxygen concentrations
above 60% of saturation. With species of the genera Farfantepenaeus and
Litopenaeus, a minimum flow rate of 7.5 liters per gram body weight per
day (L/g-bw/day) should be provided. Larger flows, up to 22 L/g-bw/day,
may be desirable to ensure dissolved oxygen concentrations above 60% of
saturation and the removal of metabolic products. 

Following the 48-hour settling-in period, mortalities should be
recorded, and the following recommendations should be applied:

(A) Mortalities of greater than 10% of the population in the 7 days of
acclimation: rejection of entire batch;

(B) Mortalities of between 5 and 10% of the population during the 7 days
of acclimation: acclimation continued for additional 7 days;

(C) Mortalities of less than 5% of the population during the 7 days of
acclimation: acceptance of batch.

(iii) Health status and condition. Shrimp should not be used for a test:

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

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

(iv) Care and handling. Organisms should be handled as little as
possible, but when necessary, it should be done as carefully and quickly
as possible. During culturing, holding, and acclimation, shrimp should
be observed carefully for signs of stress, physical damage, and
mortality. Dead and abnormal individuals should be discarded. Organisms
that touch dry surfaces or are dropped or injured in handling should
also be discarded. Detailed instructions for the care and handling of
penaeid shrimp such as those described in paragraph (j)(1) of this
guideline can be followed during the culturing, holding, and testing
periods.

(v) Diet and feeding. Penaeid shrimp should not be fed during testing.
Young shrimp that have been actively feeding for less than 20 days
should be fed up to the beginning of the test. If older and larger
shrimp over 0.5 grams (g) each are used, they should not be fed within
24 hours prior to test initiation. If the test substance tends to bind
to organic material, shrimp should not be fed within 48 hours prior to
test initiation. Every 2 or 3 days during the holding and acclimation
periods, shrimp may be fed pieces of fish approximately 1 square
centimeter (cm2) ad libitum. Other suitable sources of food (e.g.,
commercial feed, brine shrimp, Artemia salina) may also be used. Uneaten
food should be removed daily.

	(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 may be conducted using one of three
basic exposure techniques: static, static-renewal, or flow-through.
Guidance on the selection of the appropriate exposure 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., LC50), 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 more than 10% of the organisms in any
control shows signs of disease or stress (e.g., discoloration, unusual
behavior, immobilization) and/or death. 

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

(i) Loading. The number of shrimp 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. In static or
static-renewal tests, loading should not exceed 0.8 grams wet weight of
organism per liter (g/L). In flow-through tests, loading requirements
will vary depending upon the flow rate of dilution water, but should not
exceed 0.5 g/L of test solution passing through the test vessel in 24
hours or 5 g/L at any time. 

(ii) Introduction of test organisms. Prior to the start of the test, a 2
to 3 centimeter (cm) layer of sand should be placed on the bottom of
each test vessel. Penaeid shrimp, which have been acclimated to the test
conditions, should be introduced 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, and
testing penaeids that are well ventilated and free of fumes and
disturbances which may affect the test organisms. There should also be
flow-through tanks for holding and acclimating penaeid shrimp. Equipment
for culturing and/or handling food sources for penaeid shrimp.

(ii) Environmental control equipment. Mechanisms for controlling and
maintaining the water temperature, lighting, and salinity during the
culturing, holding, acclimation, and test periods. Apparatus for
aerating dilution water and removing gas bubbles as necessary. For
flow-through tests, apparatus for aerating the dilution water in the
head box before mixing with the test substance or delivery to test
vessels. Apparatus for providing a 30-minute transition period. The
lighting transition period is recommended for penaeid shrimp, which can
become easily agitated by sudden light changes and attempt to jump out
of the tanks.

(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, minimize the entry of dust and other particles into
the solutions, and prevent escape of the shrimp. A flow-through system,
if used, should contain an appropriate test substance delivery system. 

Test vessel size and test solution volume recommendations include (see
paragraph (j)(8) of this guideline): 

(A) For static tests, larger organisms (0.5 g each or more) should be
exposed in at least 19.6-L test vessels with 15 L of solution. Smaller
organisms may be exposed in at least 3.9-L test vessels with 2 to 3 L of
test solution; 

(B) For flow-through tests, test vessels may be slightly larger for each
size category mentioned above. 

Acid-washed sand, free of excess organic matter, should be placed in the
bottom of the test vessels to a depth of 2 to 3 cm.

(vi) Dilution water. Clean natural or artificial seawater is acceptable
as dilution water if shrimp will survive in it for the duration of the
culturing, holding, acclimation, and testing periods without showing
signs of stress, such as unusual behavior or discoloration. Natural
seawater should be filtered through a filter with a pore size of <20
micrometers (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
ground 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 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 23 °C. During a
given test, the temperature should be constant within plus or minus (±)
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
within ±2 ppt during the test.	

(iii) Lighting and photoperiod. While a photoperiod can be selected from
regimes of 12 hours light:12 hours dark to 16 hours light:8 hours dark,
a 12:12 light:dark cycle is preferred to allow for equalizing the time
between exposure in the water column and the substrate (shrimp may
burrow in substrate during light period). For any given test, the light
regime should be constant.  Penaeid shrimp are sensitive to the
transition between light and dark. Therefore, there a 30-minute
transition period between light and dark is recommended. Light intensity
should range from 540 to 1080 lux (approximately 50-100 foot-candles
(ft-c)).

(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. For flow-through exposures, 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 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
minimum number of test vessel volume replacements should be five per
24-hour period. 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 he appearance of surface slicks, precipitates,
or substance 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 number of dead shrimp in each test vessel should be
counted and recorded at 24, 48, 72, and 96 hours. An observation period
at <12 hours is desirable. Dead shrimp should be removed from the test
vessels at the time of observation. 

(B) Appearance and behavior. In addition to mortality, any abnormal
behavior or appearance, and the number of individuals exhibiting these
characteristics, should be counted and recorded at the same time as
observations of mortality.

(f) Treatment of results.

(1) Summary statistics.

(i) Mortality. The number of shrimp exposed at test initiation in each
treatment and replicate and the cumulative number of dead shrimp should
be summarized in tabular form by time of observation, treatment, and
replicate.

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

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

(3) Evaluation of limit test results. For pesticides, at test
termination, if any shrimp dies in the limit concentration, a
multiple-concentration 96-hour test should be conducted.

	(4) Evaluation of multiple-concentration definitive test.

(i) Concentration-response curve, slope, and LC50. Statistical
procedures should be employed to calculate the 96-h LC50 (standard error
and 95% confidence interval) based upon mortality. If a
concentration-response curve model (e.g., probit) was fit to the data to
determine the LC50, the model parameters (e.g., slope) and their
uncertainty estimates (e.g., standard error) should be recorded. The
24-, 48-, and 72-h LC50 values should also be calculated if the
magnitude of the mortality allows.

(ii) No observed effect concentration (NOEC). While calculation of the
NOEC and the lowest observed effect concentration (LOEC) is usually not
part of the experimental design for the regression-based definitive
test, reporting these values when possible is useful when testing
industrial and pesticide chemicals for understanding the toxic response.

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

(g) Tabular summary of test conditions. Table 1 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. Meeting
these test conditions will help ensure the satisfactory performance of
the test.

Table 1.―Summary of Test Conditions for the Penaeid Acute Toxicity
Test

Test type	Static, static-renewal or flow-through

Test species	Farfantepenaeus aztecus (brown shrimp); Farfantepenaeus
duorarum (pink shrimp); or Litopenaeus setiferus (white shrimp)

Test duration	96 hours

Temperature	23 °C (constant during test within ±1 °C) 

Light quality	Ambient laboratory illumination

Light intensity	540-1080 lux (approximately 50-100 ft-c)

Photoperiod	Prefer 12 hours light:12 hours dark with a recommended
30-minute transition period

Salinity	20 ppt (constant during test within ±2 ppt)

pH	Between 7.5 and 8.5 (constant during test within ±1 pH unit)

TOC	Less than or equal to (≤) 2 mg/L

Age of test organisms	Post-larval juveniles

Number of test organisms per concentration	20 (minimum)

Number of replicate test vessels per concentration	2 (minimum) 

Loading	Static or static-renewal tests: ≤0.8 g/L; 

Flow-through test: ≤0.5 g/L per 24 hours and <5 g/L at any time

Feeding regime	No feeding during test

Test vessel aeration	Not recommended; 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)

Measure of effect or measurement endpoint	96-h LC50 based on mortality



(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 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 2.―Test Validity Elements for the Penaeid Acute Toxicity Test

1. All test vessels were not identical.

2. Treatments were not randomly 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.,
discoloration, unusual behavior, immobilization), and/or death.

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, Kow. 

(v) If a vehicle (solvent) is used to prepare stock or test substance,
provide the name and source of the vehicle (solvent), 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 penaeids 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). 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) Age of test organisms at test initiation and method of
verification.

(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, static-renewal,
flow-through, open or closed system. If static-renewal, the frequency of
test solution renewal, and if flow-through, 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 vessel 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 number of dead test organisms
and measuring any other toxic symptoms.

(xiv) For the 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
the number and percentage of dead shrimp 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 the number and percentage of dead
shrimp 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 the number and percentage of
dead shrimp 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) Graphs of the concentration-response data for percent mortality.

(viii) For limit test, conclusion about the 96-h LC50 being above the
limit concentration.

(ix) For definitive test, where sufficient data exist to fit a model
(e.g., probit), 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.

(x) For definitive test, the 96-h LC50 value, its standard error and 95%
confidence interval. 

(xi) For definitive test, results for the 24-, 48-h, and 72-h LC50
values if the magnitude of the mortality allows.

		(xii) If definitive tests, the 96-hour NOEC for mortality, if
determined.

(xiii) Description of statistical method(s) used for point estimates,
including the software package for determining LC50 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 method.

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

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8In 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) Nizinski, M.S., 2003. Annotated checklist of decapod crustaceans of
Atlantic coastal and continental shelf waters of the United States.
Proc. Biological Society of Washington 116(1): 96-157. 

(4) Perez Farfante, I. and B.F. Kensley, 1997. Penaeid and sergestoid
shrimps and prawns of the world. Keys and diagnoses for the families and
genera. Memoires du Museum National d’Histoire Naturelle 175:1-233.

(5) Rose, C.D., 1975. Extensive culture of Penaeid shrimp in Louisiana
salt marsh impoundments. Trans. Amer. Fish. Soc. 105:296-307.

(6) U.S. Environmental Protection Agency, 1978. Bioassay Procedures for
the Ocean Disposal Permit Program. EPA Report No. 600-9-78-010, Gulf
Breeze, FL.

(7) U.S. Environmental Protection Agency, 1982. Pesticide Assessment
Guidelines, Subdivision E, Hazard Evaluation, Wildlife and Aquatic
Organisms, EPA 540/09-82-024, U.S. Environmental Protection Agency,
Washington, DC.

(8) U.S. Environmental Protection Agency, 1985. Hazard Evaluation
Division Standard Evaluation Procedure: Acute Toxicity Test for
Estuarine and Marine Organisms (Shrimp 96-hour Acute Toxicity Test),
EPA-540/9-85-010. Office of Pesticide Programs, June 1985.

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