United States
Environmental Protection
Iml M m Agency
Office of Chemical Safety EPA 712-C-16-009
and Pollution Prevention October 2016
(7101)
Ecological Effects
Test Guidelines
OCSPP 850.1045:
Penaeid Acute Toxicity
Test
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NOTICE
This guideline is one of a series of test guidelines established by the United States
Environmental Protection Agency's Office of Chemical Safety and Pollution Prevention (OCSPP) for
use in testing pesticides and chemical substances to develop data for submission to the Agency under
the Toxic Substances Control Act (TSCA) (15 U.S.C. 2601, et seq.), the Federal Insecticide,
Fungicide and Rodenticide Act (FIFRA) (7 U.S.C. 136, et seq.), and section 408 of the Federal Food,
Drug and Cosmetic Act (FFDCA) (21 U.S.C. 346a). Prior to April 22, 2010, OCSPP was known as
the Office of Prevention, Pesticides and Toxic Substances (OPPTS). To distinguish these guidelines
from guidelines issued by other organizations, the numbering convention adopted in 1994 specifically
included OPPTS as part of the guideline's number. Any test guidelines developed after April 22, 2010
will use the new acronym (OCSPP) in their title.
The OCSPP harmonized test guidelines serve as a compendium of accepted scientific
methodologies and protocols that are intended to provide data to inform regulatory decisions under
TSCA, FIFRA, and/or FFDCA. This document provides guidance for conducting the test, and is also
used by EPA, the public, and the companies that are subject to data submission requirements under
TSCA, FIFRA, and/or the FFDCA. As a guidance document, these guidelines are not binding on
either EPA or any outside parties, and the EPA may depart from the guidelines where circumstances
warrant and without prior notice. At places in this guidance, the Agency uses the word "should." In
this guidance, the use of "should" with regard to an action means that the action is recommended
rather than mandatory. The procedures contained in this guideline are strongly recommended for
generating the data that are the subject of the guideline, but EPA recognizes that departures may be
appropriate in specific situations. You may propose alternatives to the recommendations described in
these guidelines, and the Agency will assess them for appropriateness on a case-by-case basis.
For additional information about these test guidelines and to access these guidelines
electronically, please go to http://www.epa.gov/ocspp and select "Test Methods & Guidelines" on the
navigation menu. You may also access the guidelines in http://www.regulations.gov grouped by
Series under Docket ID #s: EPA-HQ-OPPT-2009-0150 through EPA-HQ-OPPT-2009-0159, and
EPA-HQ-OPPT-2009-0576.
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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.
(2) Background. The source materials used in developing this harmonized OCSPP test
guideline are 40 CFR 797.1970 Penaeid Shrimp Acute Toxicity Test; OPP 72-3 Acute
Toxicity Test for Estuarine and Marine Organisms Pesticide Assessment Guidelines,
Subdivision E — Hazard Evaluation: Wildlife and Aquatic Organisms (see paragraph
(j)(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)(l) 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.
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(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
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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
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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)(l) 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
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(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 (HC1)
and sodium hydroxide (NaOH) may be used for this adjustment if warranted.
See additional information about pH during testing in (e)(8)(ii).
(ii) Exposure technique. The test 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., LCso), 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
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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
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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 (|-im) prior to use in a test.
Artificial seawater can be prepared by adding commercially available
formulations or specific amounts of reagent-grade chemicals to reagent water
(deionized, distilled or reverse osmosis water), surface water, or 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.
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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.
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(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.
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(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 LCso. Statistical procedures
should be employed to calculate the 96-h LCso (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 LCso, the model
parameters (e.g., slope) and their uncertainty estimates (e.g., standard error)
should be recorded. The 24-, 48-, and 72-h LCso 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)
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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 LCso 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.
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5. A surfactant ordispersant 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)(l) of OCSPP 850.1000 describes the
minimum background information to be supplied in the report.
(2) Guideline deviations. Provide a statement of the guideline or protocol followed.
Include a description of any deviations from the test guideline or any occurrences that
may have influenced the results of the test, the reasons for these changes, and any
resulting effects on test endpoints noted and discussed.
(3) Test substance.
(i) Identification of the test substance: common name, IUPAC and CAS names,
CAS number, structural formula, source, lot or batch number, chemical state or
form of the test substance, purity {i.e., for pesticides, the identity and
concentration of active ingredient(s)), and radiolabeling, if any, including the
location of label(s) and radiopurity.
(ii) Storage conditions of the test chemical or test substance and stability of the
test chemical or test substance under storage conditions if stored prior to use.
(iii) Methods of preparation of the test substance and the treatment concentrations
used in the range-finding and definitive tests, or limit test. Identify whether the
nominal concentrations are corrected or uncorrected for purity of the test
substance.
(iv) Physicochemical properties of the test substance such as water solubility,
vapor pressure, UV absorption, pKa, 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.
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(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.
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(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
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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.
(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) 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'HistoireNaturelle 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.
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(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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