	Office of Chemical Safety and Pollution Prevention (7101)	EPA
712-C-16-012

October 2016

	Ecological Effects

Test Guidelines





OCSPP 850.1020:

	Gammarid Amphipod 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 850.1020: Gammarid amphipod 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)(12) of this guideline); Acute Toxicity Test for Freshwater
Invertebrates Standard Evaluation Procedure (see paragraph (j)(13) of
this guideline); and ASTM E729-96 Standard Guide for Conducting Aquatic
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 freshwater, gammarid amphipods are exposed to a test substance in
static, static-renewal, or flow-through systems. 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
guideline. In addition, the following more specific definitions apply to
this guideline:

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

(d) General considerations.

(1) Summary of the test. Young gammarid amphipods 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 are made on survival and other toxic effects.
The test is designed to determine the relationship between aqueous
concentrations of the test substance and mortality of gammarids 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 information 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 in 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 of 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 soluble 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
amphipods, 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 amphipod
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 types 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 recommended test species include the freshwater
gammarid amphipods Gammarus fasciatus, Gammarus pseudolimnaeus, or
Gammarus lacustris. Immature gammarids, defined as newly-released young
less than (<) 24 hours post-release or early instar (first or second)
gammarids of similar age and size, should be used to start the test. For
G. fasciatus, the average time to the first molt is 6.3 days and the
average time to the second molt is 7.2 days at 18 degrees Celsius (°C)
(see paragraph (j)(4) of this guideline). Therefore, young released
within a 7-day period should be of the same instar and should be used to
begin a test with this species. The interval between molts will vary
with species and temperature; the literature should be consulted to
determine the appropriate timing. 

At test initiation, instar stage and size should be identified and
measured on a random sample of 30 individuals.

Immature gammarids to be used in acute toxicity tests may be cultured at
the test facility, purchased from commercial culture facilities, or
obtained from adults collected from a natural population in an
unpolluted area. If adults are collected from natural sources, this
parental stock should be held in the laboratory for at least 14 days
prior to obtaining newly released immatures for test initiation.
Immature gammarids of similar age can be collected from laboratory or
wild parental stocks using various techniques, some of which are
summarized below. 

(A) Isolate ovigerous females in beakers and remove newly released young
(see paragraph (j)(9) of this guideline);

(B) Isolate ovigerous females, or mixed-age culture or wild population
stocks in separation tanks, which have small holes or nylon mesh that
allow newly-released young to move into a separate compartment, and
remove all young from this compartment within an appropriate period (see
paragraphs (j)(5) and (j)(10) of this guideline); 

(C) Place adults in containers along with several pieces of artificial
plastic aquarium plants and pieces of cotton gauze as a substrate. In 7
days, shake the plants and gauze, filter the water through a nylon mesh
screen (275 microns), and rinse into petri dishes where the young can be
sorted and removed (see paragraph (j)(3) of this guideline).

Taxonomic verification should be obtained from the commercial supplier
or conducted by experienced laboratory personnel or an outside expert.
Records should be kept regarding the source of the initial stock and/or
culturing techniques. All organisms used for a particular test should
originate from the same source or culture population.

(ii) Holding and acclimation. If the culture or holding water for the
brood stock 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. The gammarid brood stock should be
maintained in dilution water at the test temperature for at least 7 days
prior to the start of the test and should be cultured in environmental
conditions (e.g., light intensity) similar to those to be used in the
test. Within a 24-hour period, changes in water temperature should not
exceed 1 °C per day. 

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

(A) Mortalities of greater than 10% of the population in the 7 days 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. Amphipods should not be used for a
test:

(A) If more than 5% of the culture or parental stock 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, gammarids
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 during handling should
be discarded. Detailed instructions for the care and handling of
gammarid amphipods such as those described in paragraphs (j)(2), (j)(3),
(j)(7), (j)(9), and (j)(10) of this guideline can be followed during the
culturing, holding, acclimation, and testing periods.

(v) Diet and feeding. The organisms should not be fed during testing.
Early instar gammarids should be taken out of the culture with the brood
stock, isolated, and then added to the test vessels just before the test
is begun. During culturing, holding, and acclimation of parental stock,
a sufficient quantity of food such as aged deciduous leaves, such as
maple, aspen, or birch, should be placed in the culture, holding, and
acclimation containers to cover the bottom with several layers. These
leaves should be aged for at least 30 days in a flow-through system
before putting them in aquaria. As these leaves are eaten, more aged
leaves should be added. Pelleted, flaked, and/or ground fish food, live
newly released cladocerans, or pieces of fresh or frozen fish may also
be added (see paragraphs (j)(8) and (j)(11) of this guideline).

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

(i) Loading. The number of gammarids 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 (e.g., should
not crowd gammarids as they can be cannibalistic). 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. The test should be started by
introducing newly released or early instar gammarids, from acclimated
parents, 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 gammarids that are well ventilated and free of fumes and
disturbances which may affect the test organisms. There should be
facilities for aging leaves under flow-through conditions. Equipment for
culturing and/or handling food sources for amphipods. 

(ii) Environmental control equipment. Mechanisms for controlling and
maintaining the water temperature and lighting 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. 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, hardness, 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
covered loosely to reduce the loss of test solution or dilution water
due to evaporation and to minimize the entry of dust or other
particulates into the solutions. Test vessels that contain at least one
liter of test solution have been previously recommended (see paragraph
(j)(14) of this guideline). A flow-through system, if used, should
contain an appropriate test substance delivery system.

For flow-through tests, gammarids can be retained in glass or stainless
steel containers (i.e., test compartments) with stainless steel or nylon
screen bottoms within the test vessels. The containers should be
suspended in the test vessel in such a manner as to ensure that the test
solution flows regularly into and out of the container and that the
gammarids are submerged in generally at least 5 centimeters (cm) of test
solution. A substrate, such as a bent piece of stainless steel screen,
should be placed on the bottom of each test vessel to provide cover for
the gammarids. 

(vi) Dilution water. Clean surface water, ground water, or reconstituted
water are acceptable as dilution water if gammarids will survive in it
for the duration of the culturing, holding, acclimation, and testing
periods without showing signs of stress. Dechlorinated tap water is not
recommended 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 can be found in OCSPP 850.1000. 

Dissolved oxygen in the dilution water (prior to use in a test) should
be between 90 and 100% saturation. If necessary, the dilution water can
be aerated before the addition of the test substance. 

Hardness, alkalinity, and conductivity should be measured in the
dilution water at the beginning of the test. 

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, and pH) in test solutions during the test are
described in detail in OCSPP 850.1000.

(i) Temperature. The recommended water temperature is 18 °C. During a
given test, the temperature should be constant within plus or minus (±)
1 °C.

(ii) pH. The pH should be between 6.0 and 8.5 and vary less than 1 pH
unit during the test within a test vessel and between test
concentrations (including control(s)).

(iii) Lighting and photoperiod. A photoperiod should be selected from
regimes of 12 hours light:12 hours dark to 16 hours light:8 hours dark.
For any given test, the light regime should be constant. Light intensity
should range from 540 to 1080 lux (approximately 50-100 foot-candles
(ft-c)). A 15- to 30-minute transition period between light and dark is
suggested.

(iv) Dissolved oxygen. The dissolved oxygen concentration should be
between 60 and 100% saturation during the test. If aeration is needed to
achieve an appropriate dissolved oxygen level, it should be done before
the addition of the test substance. 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 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 number of dead gammarids 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 gammarids should be removed from
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 gammarids exposed at test initiation in
each treatment and replicate and the cumulative number of dead gammarids
should be summarized in tabular form by time of observation, treatment,
and replicate.

(ii) Appearance and behavior. The number of gammarids 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 amphipod dies in the limit concentration, a
multiple-concentration acute 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.

―Summary of Test Conditions for Gammarid Acute Toxicity Test

Test type	Static, static-renewal, or flow-through

Test species	Recommended Gammarus fasciatus, Gammarus pseudolimnaeus, or
Gammarus lacustris

Test duration	96 hours

Temperature	18 °C (constant during test within ±1 °C)

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

pH	Between 6.0 and 8.5 (constant during test within ±1 pH unit)

Water hardness (as CaCO3) 	<250 mg/L (preferably <180 mg/L); for testing
with metals, 40-50 mg/L

Total organic carbon (TOC)	<2 mg/L

Age of test organisms	<24 hours post-release or early instar (first or
second)

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 Acute Toxicity Test, Gammarid
Amphipods

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.,
discoloration, unusual behavior, immobilization), and/or death.

5. Gammarids were fed during the test.

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.)

(i) Reporting.

(1) Background information. Paragraph (k)(1) of OCSPP 850.1000 describes
the minimum background information to be supplied in the report.

(2) Guideline deviations. Provide a statement of the guideline or
protocol followed. Include a description of any deviations from the test
guideline or any occurrences that may have influenced the results of the
test, the reasons for these changes, and any resulting effects on test
endpoints noted and discussed.

(3) Test substance. 

(i) Identification of the test substance: common name, IUPAC and CAS
names, CAS number, structural formula, source, lot or batch number,
chemical state or form of the test substance, purity (i.e., for
pesticides, the identity and concentration of active ingredient(s)), and
radiolabeling, if any, including the location of label(s) and
radiopurity.

(ii) Storage conditions of the test chemical or test substance and
stability of the test chemical or test substance under storage
conditions if stored prior to use.

(iii) Methods of preparation of the test substance and the treatment
concentrations used in the range-finding and definitive tests, or limit
test. Identify whether the nominal concentrations are corrected or
uncorrected for purity of the test substance.

(iv) Physicochemical properties of the test substance such as water
solubility, vapor pressure, UV absorption, pKa, and Kow.

(v) If a vehicle (solvent) is used to prepare stock or test substance,
provide the name and source of the vehicle, the nominal concentration(s)
of the test substance in the vehicle in stock solutions or mixtures, and
the vehicle concentration(s) used in the treatments and vehicle control.
If different vehicle concentrations are used at different treatment
levels, the report should, at a minimum, identify the maximum vehicle
concentration used. It is helpful to support the vehicle choice by
including a description of any measures that were taken to identify an
appropriate vehicle for use in the test, such as the types and
concentrations of vehicles used and their corresponding effects on
solubility during any preliminary work.

(vi) If a positive control is used, provide the name and source of
positive control and the nominal concentration(s) of the positive
control material in stock solutions or mixtures.

	(4) Test organism. 

		(i) Scientific name and common name.

		(ii) Method for verifying the species.

(iii) Information about the gammarids used in the test as well as the
brood stock: 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; pH; hardness and alkalinity; 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, 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 definitive and limit tests, description of all analytical
procedures used, 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, 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 gammarids 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
gammarids 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 gammarids 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-, and 72-h LC50 values
if the magnitude of the mortality allows.

		(xii) For definitive test, 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) Arthur, J.W., 1977. Review of freshwater bioassay procedures for
selected amphipods. In Aquatic Invertebrate Bioassays, ASTM STP 715,
A.L. Buikema and J.C. Cairns, Jr., eds., American Society for Testing
and Materials, Philadelphia, PA, pp. 98-108. 

(3) Borgmann, U., K. Ralph and W. Norwood, 1989. Toxicity test
procedures for Hyalella azteca, and chronic toxicity of cadmium and
pentachlorophenol to H. azteca, Gammarus fasciatus and Daphnia magna.
Archives of Environmental Contamination and Toxicology 18:756-764. 

(4) Clemens, H.P., 1950. Life cycle and ecology of Gammarus fasciatus
Say, The Ohio State University, F.T. Stone Lab, Contribution No. 12,
29-35.

(5) Emery, R., 1970. The comparative acute toxicity of cresol to two
benthic crustaceans. Water Research 4:485- 491.

(6) 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.

(7) Lawrence, S., 1982. Manual for the culture of selected freshwater
invertebrates, Canadian Special Publication of Fisheries and Aquatic
Sciences 54. 169 pp.

(8) Marines, B., C. Barton, P. Markle and J. Bottomley, 2004.
Investigation of the acute toxicity of diazinon using Gammarus
pseudolimnaeus. Poster presentation, Southern California SETAC Chapter
meeting.

(9) McCahon, C.P. and D. Pascoe, 1988. Use of Gammarus pulex (L.) in
safety evaluation tests: culture and selection of a sensitive life
stage. Ecotoxicology and Environmental Safety 15:245-252.

(10) McCahon, C.P. and D. Pascoe, 1988. Culture techniques for three
freshwater macroinvertebrate species and their use in toxicity tests.
Chemosphere 17(12):2471-2480. 

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, D.M., 1978. A comparison of the variability of Asellus communis
(Crustacea: Isopoda) and Gammarus pseudolimnaeus (Crustacea: Amphipoda)
and suitability for joint bioassays. Bulletin of Environmental
Contamination and Toxicology 20:461-469. 

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

(13) U.S. Environmental Protection Agency, 1985. Hazard Evaluation
Division Standard Evaluation Procedure: Acute Toxicity Test for
Freshwater Invertebrates, Office of Pesticides Programs, Washington DC,
EPA-540/9-85-005.

(14) U.S. Code of Federal Registration. Title 40- Protection of
Environment. Part 795.120. Gammarid Acute Toxicity Test.

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