	Office of Chemical Safety and Pollution Prevention

(7101)	EPA 712-C-16-007

October 2016

	Ecological Effects

Test Guidelines





OCSPP 850.1075:

	Freshwater and Saltwater Fish 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 850.1075: Freshwater and saltwater fish
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, EPA 540/9-82-024,
1982) (see paragraph (j)(4) of this guideline); Standard Evaluation
Procedure: Acute Toxicity Test for Freshwater Fish (see paragraph (j)(5)
of this guideline); Standard Evaluation Procedure: Acute Toxicity Test
for Estuarine and Marine Organisms (see paragraph (j)(6) of this
guideline); EPA Pesticide Reregistration Rejection Rate Analysis:
Ecological Effects, EPA 738-R-94-035, 1994 (see paragraph (j)(7) of this
guideline); OECD 203 Fish Acute Toxicity Test, 1992 (see paragraph
(j)(3) of this guideline); and ASTM E729-96 (07), Standard Guide for
Conducting Acute Toxicity Tests on Test Materials with Fishes,
Macroinvertebrates, and Amphibians, 2007 (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 and saltwater fish are exposed to a test substance in
static, static-renewal, or flow-through systems. For freshwater testing,
data on cold and warm water species are generally required (40 CFR 158).
Rainbow trout, Oncorhynchus mykiss, and bluegill sunfish, Lepomis
macrochirus, are the preferred species to meet this requirement for cold
and warm water tests, respectively. These species are preferred because
they are sensitive indicator species, and there is a large environmental
contaminant-response database available on them. For saltwater tests,
Menidia sp. (silversides) are the preferred species. Other species as
identified in paragraph (e)(3)(i) of this guideline may be used. The
Environmental Protection Agency will use data from this test in
assessing the hazards and risks a test substance may present in the
aquatic environment. 

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

Death is defined as the lack of visible movement (i.e., respiratory
movements) or the lack of reaction when the caudal peduncle is touched.

(d) General considerations.

(1) Summary of test. Individuals of a particular species of fish 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 organism
survival and other toxic effects. The test is designed to determine the
relationship between aqueous concentrations of the test substance and
mortality of fish 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 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 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-h
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 2 of this guideline. Test
validity elements are listed in Table 3.

(5) Limit test. In some situations, it is only necessary to ascertain
that the 96-h LC50 is above a certain limit (i.e., 96-h LC50 greater
than (>) limit concentration). In a limit test, at least 7 fish 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 2 of this
guideline), and have both a dilution water control and a vehicle
(solvent) control, if a vehicle is used. Limit tests, like definitive
tests, should include analytical confirmation of the dissolved
concentration of the test substance. Clinical signs of toxicity such as
abnormal appearance and behavior, if any, should be reported. For
pesticides, at test termination, if any fish 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, then 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 organisms.

(i) Species. Data on both a warm and a cold freshwater species are
generally required for 40 CFR Part 158. If a marine or estuarine system
may be affected, data on a saltwater species are also generally
required. Freshwater and saltwater species that are recommended include:


(A) Freshwater species. Atlantic salmon, Salmo salar; bluegill sunfish,
Lepomis macrochirus; brook trout, Salvelinus fontinalis; channel
catfish, Ictalurus punctatus; coho salmon, Oncorhynchus kisutch; common
carp, Cyprinus carpio; fathead minnow, Pimephales promelas; guppy,
Poecilia reticulata; rainbow trout, Oncorhynchus mykiss; medaka, Oryzias
latipes; and zebrafish, Danio rerio. 

(B) Saltwater species. Atlantic silverside, Menidia menidia; inland
silverside, Menidia beryllina; tidewater silverside, Menidia peninsulae;
and sheepshead minnow Cyprinodon variegatus;

The preferred warm water species is the bluegill sunfish. The rainbow
trout is the preferred cold water species. When data on a marine or
estuarine species are desired, a Menidia spp. (silverside) is preferred.

Juvenile fish less than (<) 3.0 grams, and old enough to be actively
feeding, should be tested. It is recommended that the wet weight and
length of at least 7 fish from the batch of fish used in a particular
test be measured and that the mean values and ranges be reported. The
longest fish should not be more than twice the length of the shortest
fish. Since these fish should not be fed during the test and 24 to 48
hours prior to test initiation, they should be of sufficient size to
survive and not exhibit signs of stress during this time period without
food.

Fish may be cultured in the laboratory, purchased from culture
facilities, or collected from the wild in a relatively unpolluted area.
Wild caught fish should be quarantined 7 days in addition to the defined
minimum holding and acclimation periods (see paragraph (e)(3)(ii)).
Whenever salmon or trout are used, they should be obtained from a
hatchery that has been certified disease-free. Fish captured by
electroshocking, chemical treatment, or gill nets should not be used.
Fish used in a particular test should be obtained from the same source
and population, be of similar age, and be of normal size and appearance.
Records should be kept regarding the source of the initial stock and/or
culturing techniques. 

(ii) Holding and acclimation. Fish brought into the laboratory should be
held for a minimum of 12 days prior to use. The recommended holding
period, however, is 14 days. A minimum of 7 days of this period are used
for acclimation to environmental conditions (e.g., temperature, light
intensity, temperature, dilution water) similar to those used in the
test. To maintain organisms in good condition and avoid unnecessary
stress, they should not be crowded or subjected to rapid changes in
temperature or water quality. Acclimation water should be from the same
dilution water source as used in the test; if not, acclimation to the
dilution water should be done gradually over a 48-hour settling-in
period. Within a 24-hour period, changes in water temperature during
holding or acclimation should not exceed 3 degrees Celsius (°C), and
for saltwater species, changes in salinity change should not exceed 2
parts per thousand (ppt). 

Following a 48-hour settling-in period, mortalities should be recorded,
and the following guidelines 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. Fish should not be used for a test: 

(A) If more than 5% of the culture or acclimating group dies or shows
signs of stress (e.g., disease, physical damage, or abnormalities)
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;

(C) If disease treatments were administered within 48 hours of test
initiation. Fish should not receive treatment for a disease during a
test. 	

(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. Any disturbance which might change the behavior of the test
fish should be avoided. Detailed instructions for the care and handling
of fish such as those described under paragraph (j)(8) of this guideline
can be followed during the culturing, holding, acclimating, and testing
periods.

(v) Diet and feeding.  During holding and acclimation, different food
sources may be used depending on species and size (e.g., flake fish
food, pelleted food, live Artemia brine shrimp). 

Test fish should not be fed during testing. During the acclimation
period, fish should be fed daily until 24 hours prior to test
initiation. If fish are larger than 0.5 grams fish should be fed until
48 hours prior to test initiation.

	(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
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 fish per treatment level is 7. The use of
10 fish per treatment level and 2 replicates per treatment level is
preferred to obtain a more statistically accurate representation of the
concentration-response curve. Each test vessel should contain an equal
volume of test solution and an equal number of fish. Replicate test
vessels should be physically separated, since the test vessel is the
experimental unit. 

(i) Loading. The number of fish placed in each 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. The test should be started by
introducing juvenile fish, which have been acclimated to the test
conditions, 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 fish that are well ventilated and free of fumes and disturbances
which may affect the test organisms. There should also be flow-through
or recirculation tanks for culturing and acclimating fish. Equipment for
culturing and/or handling food sources for fish.

(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
loosely covered to reduce the loss of test solution or dilution water
due to evaporation, to minimize the entry of dust or other particulates
into solutions, and to prevent loss of test fish. A flow-through system,
if used, should contain an appropriate test substance delivery system.

Many different sizes of test vessels have been used successfully. The
size, shape, and depth of the test vessel is appropriate if the
specified flow rate and loading requirements can be achieved. 

(vi) Dilution water. Clean surface water, ground water, reconstituted
water, or natural or artificial seawater (for saltwater species) are
acceptable as dilution water if the test species will survive in it for
the duration of the culturing, holding, acclimation, and testing periods
without showing signs of stress. 

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.
For saltwater species, a salinity should be selected from a range of 15
and 25 ppt. For artificial seawater or natural seawater that is diluted
with freshwater, salinity should be maintainable within a weekly range
of 2 ppt.  

Dechlorinated tap water is not recommended (either as the freshwater
source, preparation of artificial seawater, or dilution of natural
seawater) because some forms of chlorination are difficult to remove
adequately. If dechlorinated tap water is used, recommended maximum
chlorine levels as well as other ways to demonstrate suitability as a
dilution water source are in OCSPP 850.1000. 

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

For freshwater testing, hardness, alkalinity, and conductivity should be
measured in the dilution water at the beginning of the test. For
saltwater testing, salinity 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, pH, and salinity) in test solutions during the test
are described in detail in OCSPP 850.1000. 

(i) Temperature. Recommended test temperatures by species are shown in
Table 1. During a given test, the selected temperature should be
constant within plus or minus (±) 2 °C. 

Table 1.—Recommended Test Temperatures by Species in the Freshwater
and Saltwater Fish Acute Toxicity Test

Species	Temperature (°C)

Atlantic salmon (Salmo salar)	12

Atlantic silverside (Menidia menidia)	22

Bluegill sunfish (Lepomis macrochirus)	22

Brook trout (Salvelinus fontinalis)	12

Channel catfish (Ictalurus punctatus)	22

Coho salmon (Oncorhynchus kisutch)	12

Common carp (Cyprinus carpio)	22

Fathead minnow (Pimephales promelas)	22

Guppy (Poecilia reticulata)	22

Inland silverside (Menidia beryllina)	22

Medaka (Oryzias latipes)	22

Rainbow trout (Oncorhynchus mykiss)	12

Sheepshead minnow (Cyprinodon variegatus)	22

Tidewater silverside (Menidia peninsulae)	22

Zebrafish (Danio rerio)	22



(ii) pH and salinity. The pH should be between 6.0 and 8.5 for
freshwater species and between 7.5 and 8.5 for saltwater species and
should vary less than 1 pH unit during the test within a test vessel and
between test concentrations (including control(s)). During a given test,
the salinity (selected from a range of 15 to 25 ppt) should be constant
within ± 2 ppt. 

(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 to1080 lux (approximately 50-100 foot-candles
(ft-c)). A 15- to 30-minute transition period between light and dark is
recommended.

(iv) Dissolved oxygen. The dissolved oxygen concentration should be
between 60 and 100% saturation during the test. If aeration is needed to
achieve an appropriate dissolved oxygen level, it should be done before
addition of the test substance. 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 the test solutions during the
test is not recommended. Gentle aeration of test vessels during the
exposure period is permitted only in cases where the dissolved oxygen
levels are in danger of dropping below 60% saturation. In such cases,
assurances should be made that the use of aeration does not stress the
test organisms; test substance concentrations should be measured during
the test to ensure that they are not affected by the use of aeration;
and all treatment and control vessels should be given the same aeration
treatment.	

(v) Flow in a flow-through system. During a test, the flow rates should
not vary more than 10% between any one replicate and another. The
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 fish 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 fish 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 fish exposed at test initiation in each
treatment and replicate and the cumulative number of dead fish should be
summarized in tabular form by time of observation, treatment, and
replicate.

(ii) Appearance and behavior. The number of fish 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 fish 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 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 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 2 lists the important
conditions that should prevail during the multiple-concentration
definitive test. The same conditions are recommended for a limit test,
except for differences in the number of test concentrations. Meeting
these test conditions will help ensure the satisfactory performance of
the test.

Table 2.—Summary of test conditions for freshwater and saltwater fish
acute toxicity test

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

Test duration	96 hours

Temperature	12 °C for Atlantic salmon, Brook trout, Coho salmon,
Rainbow trout

22 °C for Atlantic silverside, Bluegill sunfish, Channel catfish,
Common carp, Sheepshead minnow, Inland silverside, Tidewater silverside,
Fathead minnow, Guppy, Medaka, Zebrafish

Constant during test within ±2 °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 for freshwater testing; between 7.5 and 8.5 for
saltwater testing (constant during test within ±1 pH unit)

Water hardness (as CaCO3) 

≤2 mg/L

Age/size of test organisms	Juvenile fish <3.0 grams

Number of test organisms per concentration	7 minimum, 10 preferred

Number of replicate test vessels per concentration	1 minimum, 2
preferred

Loading	Static or static-renewal tests: ≤0.8 g wet weight per liter;

≤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 3
occurred. These parameters are not the only elements considered when
evaluating the acceptability of a test, and it is possible that a test
could be found unacceptable or invalid based on other considerations.
However, except for the conditions listed in Table 3 and in OCSPP
850.1000, it is unlikely that a test will be rejected when there are
only slight variations from guideline environmental conditions and study
design unless the control organisms are significantly affected and/or
significant biases are introduced in defining the magnitude of effect on
measurement endpoints as compared to guideline conditions. Before
departing significantly from this guideline (such as deviating from the
organism size), the investigator should contact the Agency to discuss
the reason for the departure and the effect the change(s) may have on
test acceptability. In the test report, all departures from the
guideline should be identified, reasons for the changes given, and any
resulting effects on test endpoints noted and discussed.

Table 2.—Test validity elements for the Freshwater and Saltwater Fish
Acute Toxicity Test

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. Fish 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 fish 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 (mean and range).

	(v) Weight of test organisms at test initiation (mean and range).

(vi) Length of test organisms at test initiation (mean and range). 

(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 (saltwater); pH; hardness and
alkalinity (freshwater); dissolved oxygen; total organic carbon or
chemical oxygen demand; particulate matter; conductivity; metals,
pesticides, and residual chlorine concentrations (mean, standard
deviation, range). Describe the frequency and sample date(s) for
documenting dilution water quality and consistency.

(iv) Use of aeration, if any, and location within exposure system of
aeration (e.g., test solution or dilution water prior to test substance
addition).

(v) Number of test organisms added to each test vessel at test
initiation.

(vi) Number of test vessels (replicates) per treatment level and
control(s).

(vii) Methods used for treatment randomization and assignment of test
organisms to test vessels.

(viii) Date of introduction of test organism 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 (if applicable), and light intensity. 

(xii) Methods and frequency of measuring the dissolved test substance to
verify 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 (if applicable), 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 fish 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
fish 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 fish, 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 the 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,
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) Organization of Economic Cooperation and Development, 1992.
Guidelines for Testing of Chemicals, Guideline 203, Fish Acute Toxicity
Test, Adopted July 17, 1992. Updated version October 12, 2009.

(4) 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.

(5) U.S. Environmental Protection Agency, 1985. Hazard Evaluation
Division Standard Evaluation Procedure: Acute Toxicity Test for
Freshwater Fish, EPA-540/9-85-006, Office of Pesticide Programs, Office
of Prevention, Pesticides and Toxic Substances, U.S. Environmental
Protection Agency, Washington DC. Revised June 1985.

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Evaluation Procedure: Acute Toxicity Test for Estuarine and Marine
Organisms (Estuarine Fish 96-Hour Acute Toxicity Test), EPA
540/9-85-009, Office of Pesticide Programs, Office of Prevention,
Pesticides, and Toxic Substances, U.S. Environmental Protection Agency,
Washington DC. Revised June 1985.

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

(8) U.S. Environmental Protection Agency, 2002. Methods for measuring
the acute toxicity of effluents and receiving waters to freshwater and
marine organisms, Fifth edition, October 2002. EPA-821-R-02-012.

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