United States Prevention, Pesticides EPA 712-C-96-118
Environmental Protection and Toxic Substances April 1996
Agency (7101)
&EPA Ecological Effects Test
Guidelines
OPPTS 850.1075
Fish Acute Toxicity Test,
Freshwater and Marine
'Public Draft"
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INTRODUCTION
This guideline is one of a series of test guidelines that have been
developed by the Office of Prevention, Pesticides and Toxic Substances,
United States Environmental Protection Agency for use in the testing of
pesticides and toxic substances, and the development of test data that must
be submitted to the Agency for review under Federal regulations.
The Office of Prevention, Pesticides and Toxic Substances (OPPTS)
has developed this guideline through a process of harmonization that
blended the testing guidance and requirements that existed in the Office
of Pollution Prevention and Toxics (OPPT) and appeared in Title 40,
Chapter I, Subchapter R of the Code of Federal Regulations (CFR), the
Office of Pesticide Programs (OPP) which appeared in publications of the
National Technical Information Service (NTIS) and the guidelines pub-
lished by the Organization for Economic Cooperation and Development
(OECD).
The purpose of harmonizing these guidelines into a single set of
OPPTS guidelines is to minimize variations among the testing procedures
that must be performed to meet the data requirements of the U. S. Environ-
mental Protection Agency under the Toxic Substances Control Act (15
U.S.C. 2601) and the Federal Insecticide, Fungicide and Rodenticide Act
(7U.S.C. I36,etseq.).
Public Draft Access Information: This draft guideline is part of a
series of related harmonized guidelines that need to be considered as a
unit. For copies: These guidelines are available electronically from the
EPA Public Access Gopher (gopher.epa.gov) under the heading "Environ-
mental Test Methods and Guidelines" or in paper by contacting the OPP
Public Docket at (703) 305-5805 or by e-mail:
guidelines@epamail.epa.gov.
To Submit Comments: Interested persons are invited to submit com-
ments. By mail: Public Docket and Freedom of Information Section, Office
of Pesticide Programs, Field Operations Division (7506C), Environmental
Protection Agency, 401 M St. SW., Washington, DC 20460. In person:
bring to: Rm. 1132, Crystal Mall #2, 1921 Jefferson Davis Highway, Ar-
lington, VA. Comments may also be submitted electronically by sending
electronic mail (e-mail) to: guidelines@epamail.epa.gov.
Final Guideline Release: This guideline is available from the U.S.
Government Printing Office, Washington, DC 20402 on The Federal Bul-
letin Board. By modem dial 202-512-1387, telnet and ftp:
fedbbs.access.gpo.gov (IP 162.140.64.19), or call 202-512-0135 for disks
or paper copies. This guideline is also available electronically in ASCII
and PDF (portable document format) from the EPA Public Access Gopher
(gopher.epa.gov) under the heading "Environmental Test Methods and
Guidelines."
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OPPTS 850.1075 Fish acute toxicity test, freshwater and marine.
(a) Scope—(1) Applicability. This guideline is intended to meet test-
ing requirements of both 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).
(2) Background. The source material used in developing this har-
monized OPPTS test guideline are 40 CFR 797.1400 Fish Acute Toxicity
Test; OPP 72-1 Acute Toxicity Test for Freshwater Fish and 72-3 Acute
Toxicity Test for Estuarine and Marine Organisms (Pesticide Assessment
Guidelines, Subdivision E—Hazard Evaluation; Wildlife and Aquatic Or-
ganisms) EPA report 540/09-82-024, 1982; and OECD 203 Fish Acute
Toxicity Test.
(b) Purpose. The purpose of the acute toxicity test with fish species
is to help in the assessment of possible risk to similar species in natural
environments, as an aid in determination of possible water quality criteria
for regulatory purposes, and for use in correlation with acute testing of
other species for comparative purposes. Data on a cold and warm fresh-
water species are generally required. The rainbow trout, Oncorhynchus
mykiss, and bluegill sunfish, Lepomis macrochirus, are preferred species
to meet this requirement since they are sensitive indicator species and a
large data base which characterizes the response to environmental contami-
nants is available. Other species as identified in paragraph (e)(4)(i)(A) of
this guideline may be used. However, under certain circumstances, when
potential environmental exposures may lead to significant risks, data on
the preferred species may be required for risk assessment purposes so that
the Agency can conduct comparative analyses with alternative chemical
substances. Historically, it appears that many chemical classes are subject
to comparative analyses. Development of a good data base could ulti-
mately result in the use of other species in comparative analyses. In any
case, the results of such a study should not be construed to represent be-
havior of the test material in the natural environment where other factors
may come into play, but rather as a indicator of effects which might occur
under comparable conditions as those utilized in the study.
(c) Principle of the test—(1) Definitive test. The goal of the defini-
tive test is to determine concentration-response curves for fish mortality,
the LC50's, and the 95 percent confidence intervals for each species tested
at 24, 48, 72, and 96 h in a static, static-renewal, or flow-through system.
(2) Range-finding or limit testing. Definitive testing may be waived
if limit testing with at least 30 organisms shows LC50 levels to be greater
than 1,000 mg/L based on 100 percent active ingredients (AI), or the limits
of water solubility or dispersibility. For pesticides, a lower level of 100
mg AI/L may be tested when estimated environmental concentrations are
not expected to exceed 100 mg/L (ppm) as might occur with pesticide
use. Prior to selection of definitive test concentrations it may be advisable
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to conduct a range-finding test. Results of any range-finding and limit tests
should be reported with results of the definitive test.
(3) Information on the test substance. The material to be tested
should be technical grade unless the test is designed to test a specific for-
mulation, mixture, or effluent. The degree of purity must be recorded for
technical ingredients and mixtures. The percentage of each impurity should
be reported and percentages should total 100 percent. A complete descrip-
tion of physicochemical characteristics (i.e. solubility, vapor pressure, hy-
drolysis in pH 5, 7, and 9) should be included with description of the
AI used in specific chemical testing. A reliable analytical method for quan-
tification of test substance concentrations must be available.
(d) Validity of the test. (1) Maximum-allowable control or solvent
control mortality is 10 percent (or 1 mortality if 7 to 10 control fish are
used) for a 96-h period of testing. If the test is continued past 96 h, the
maximum-allowable additional mortality is 10 percent.
(2) Constant conditions must be maintained throughout the test pe-
riod. Flow-through procedures are preferred over static-renewal or
semistatic procedures and static-renewal procedures are preferred over a
static test procedure.
(3) In static tests, the dissolved oxygen (DO) in each replicate should
at all times be greater than 60 percent saturation. In flow-through tests,
the DO should be maintained above 75 percent saturation.
(4) Measured concentrations are required if the test chemical is unsta-
ble or a flow-through diluter system is employed. Exception may be made
in cases where hydrolysis studies indicate chemical to be stable (<5 per-
cent degradation) in 96 h at a pH comparable to test dilution water. In
any case there must be evidence that test concentrations remained at least
80 percent of the nominal concentrations throughout the test or that mean
measured concentrations are an accurate representation of exposure levels
maintained throughout the test period.
(e) Description of the method—(1) Apparatus. Normal laboratory
equipment and especially the following is necessary:
(i) Equipment for determination of water hardness, etc.
(ii) Adequate apparatus for temperature control.
(iii) Tanks constructed of chemically inert material and of suitable
capacity to allow recommended loading levels.
(2) Water, (i) Clean surface or ground water, seawater (for estuarine
or marine species), and reconstituted water are acceptable as dilution
water. Dechlorinated water should not be used because some forms of
chlorination are difficult to remove adequately. If dechlorinated tap water
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is used, then daily chlorine analysis should be performed. Reconstituted
or natural water is preferred.
(ii) Chemical analysis of water used in testing should include the fol-
lowing elements and limitations on maximum concentrations based on at
least biannual testing:
Suhstanrp Maximum con-
Substance centration
Particulate matter 20.0 mg/L
Chemical oxygen demand (COD) 5.0 mg/L
Total organic carbon (TOC) 2.0 mg/L
Boron and fluoride <100.0 mg/L
Residual chlorine 0.003 mg/L
Un-ionized ammonia 0.020 mg/L
Aluminum, arsenic, chromium, cobalt, copper, iron, lead, nickel, and 0.001 mg/L
zinc.
Cadmium, mercury, and silver <0.100 u,g/L
Total organophosphorus pesticides 0.050 u,g/L
Total organochlorine pesticides + PCBs or organic chlorine 0.050 u,g/L
Specific conductivity <1.0 u,ohms
(iii) Salinity should be 20 + 5 ppt for estuarine species.
(iv) Hardness should range between 40 and 180 mg/L as CaCOs for
freshwater species.
(v) Water hardness or salinity, as appropriate, should be measured
at the beginning of each test.
(vi) In marine flow-through tests, salinity should be recorded at the
beginning of the test, on day 4, and if extended, on days 7 and 14.
(3) Solutions of test water, (i) Distilled water should be used in mak-
ing stock solutions of the test substance. If the stock volume is more than
10 percent of the test solution volume, dilution water should be used. If
a carrier, i.e. a solvent and/or dispersant, is absolutely necessary to dissolve
the test substance, the amount used should not exceed the minimum vol-
ume necessary to dissolve or suspend the test substance in the dilution
water. If the test substance is a mixture, formulation, or commercial prod-
uct, none of the ingredients is considered a carrier unless an extra amount
is used to prepare the stock solution.
(ii) Solvent concentration may not exceed 0.5 mL/L in static-renewal
or static testing, and 0.1 mL/L in flow-through testing.
(iii) Preferred solvents are dimethyl formamide, triethylene glycol,
methanol, acetone, or ethanol. Solvent use should be avoided if possible.
(iv) Solvent concentrations selected should be kept constant in the
solvent control and all test solutions. The concentration of solvent in high-
est treatment level should be used in the solvent control.
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(v) The use of a solubility (saturation) column is permitted in the
preparation of stock solutions. This may help to ensure the aqueous solu-
bility limit is attained for poorly soluble test materials.
(vi) The pH should not be adjusted after the addition of the test chem-
ical or stock solution into dilution water.
(vii) The pH should be measured in each replicate at the beginning
of the test and every 24 h thereafter.
(viii) The pH must be monitored in low, medium, and high test con-
centrations and must remain > 6.0 and < 8.0 for freshwater testing and > 7.5
and < 8.5 for marine testing.
(ix) The pH may be adjusted in stock solutions to match the pH of
dilution water if pH change does not affect stability of compound in water.
HC1 and NaOH may be used for this adjustment if warranted.
(4) Selection of test species—(i) Test species. One or more of the
following species may be used:
(A) Freshwater species—Atlantic salmon, Salmo salar; bluegill sun-
fish, Lepomis macrochirus; brook trout, Salvelinus fontinalis; channel cat-
fish, Ictalurus punctatus; coho salmon, Oncorhynchus kisutch; common
carp, Cyprinus carpio; fathead minnnow, Pimephales promelas; guppy,
Poecilia reticulata; rainbow trout, Oncorhynchus mykiss; red killifish,
Oryzias latipes; threespine stickleback, Gasterosteus aculeatus; and zebra-
fish, Brachydanio rerio.
(B) Saltwater species—Atlantic silverside, Menidia menidia; sheeps-
head minnow, Cyprinodon variegatus; and tidewater silverside, Menidia
penisulae.
(C) Data on both a warm and a cold freshwater species are generally
required. 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 is desired, the Atlantic silversides is preferred.
(ii) Acclimation. (A) A minimum 12-day acclimation period is re-
quired with 14 days recommended. A minimum of 7 days of the acclima-
tion period must be performed in test dilution water.
(B) Holding water should come from the same source as the test dilu-
tion water, if not, acclimation to the dilution water should be done gradu-
ally over a 48-h period.
(C) No disease treatments may be administered within 48 h of test
initiation or during testing.
(D) No feeding is permitted within 48 h of test initiation.
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(E) Pretest mortality must be < 5 percent during acclimation. If pretest
mortality is > 10 percent, then the entire batch must be rejected and a
new batch begun in acclimation.
(F) Any changes in water temperature should not exceed 3 °C per
day. Fish should be held for a minimum of 7 days at the test temperature
prior to testing.
(G) During the final 48 h of acclimation fish should be maintained
in facilities with background colors and light intensities similar to those
of testing area.
(iii) Age and size of test fish. (A) Juvenile fish must be tested. Juve-
nile fish <3.0 g should be used and the longest should not be more than
twice the length of the shortest. The fish should be of normal size and
appearance for their age. All fish must be of the same age.
(B) Wild caught fish may be used to satisfy testing guidelines if size,
age, and source requirements are satisfied. Wild caught fish should be
quarantined 7 days before acclimation procedures begin.
(C) Fish must originate from the same source and population. Records
should be kept regarding the source of the initial stock and/or culturing
techniques.
(D) Fish should not be used for a test if they appear stressed, or
if more than 5 percent die during the 48 h immediately prior to the test,
or if they were used in previous tests for treatments or controls.
(iv) Temperature. The recommended test temperatures are:
Species Temperature, °C
Atlantic salmon 12 + 2.0
Atlantic silverside 22 + 2.0
Bluegill sunfish 22 + 2.0
Brook trout 12 + 2.0
Channel catfish 22 + 2.0
Coho salmon 12 + 2.0
Common carp 22 + 2.0
Fathead minnnow 23 + 2.0
Guppy 23 + 2.0
Rainbow trout 12 + 2.0
Red killifish 23 + 2.0
Sheepshead minnow 22 + 2.0
Threespine stickleback 10 + 2.0
Tidewater silverside 22 + 2.0
Zebra-fish 23 + 2.0
(v) Feeding. Feeding of test fish daily until 48 h prior to test initiation
is suggested.
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(f) Performance of the test—(1) Test design—(i) Test duration.
Acute testing must be performed for a minimum of 96 h.
(ii) Controls. Every test should include controls consisting of the
same dilution water, conditions, procedures, and test population, except
that no test substance is added. Solvent (carrier) controls are also required
if a solvent was used.
(iii) Introduction of fish. Fish should be added to test chambers with-
in 30 min of addition of the test material to dilution water. Fish may be
added prior to addition of test material. Fish should be introduced ran-
domly to individual replicates.
(iv) Number of test organisms. A minimum of seven fish per rep-
licate is required. The use of 10 fish per replicate is preferred to obtain
a more statistically accurate representation of the dose-response curve, to
allow for mortality which may occur, yet be unrelated to chemical effect,
and to avoid unnecessary repetitions of the test due to excessive control
mortality.
(v) Replicates. (A) Two replicates per test concentration are preferred
to avoid test repetition due to system failures, and to provide a stronger
statistical baseline.
(B) Each test chamber should contain an equal volume of test solution
and equal numbers of test fish. Replicate test chambers should be phys-
ically separated.
(vi) Loading. (A) The number of fish placed in each replicate should
not be so great as to affect the test results.
(B) In static or static-renewal tests, loading should not exceed
0.8 g (fresh weight) of fish per liter of test solution in a replicate at any
one time.
(C) In flow-through tests, loading should not exceed 0.5 g fresh
weight of fish (FWF) per liter of test solution passing through a replicate
within 24 h.
(vii) Test chambers and support equipment. (A) Construction ma-
terials and equipment that contact the stock solution, test solution, or dilu-
tion water should not contain substances that can be leached or dissolved
into aqueous solutions in quantities that can affect the test results. Mate-
rials and equipment that contact stock or test solutions should be chosen
to minimize sorption of test chemicals. Glass, no. 316 stainless steel, nylon
screen, and perfluorocarbon plastic (e.g. Teflon) are acceptable materials
and should be used whenever possible. Concrete, fiberglass, or plastic (e.g.
PVC) may be used for holding tanks, acclimation tanks, and water supply
systems, but they should be thoroughly conditioned before use. Rubber,
copper, brass, galvanized metal, epoxy glues, lead, and flexible tubing
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should not come in contact with the dilution water, stock solution, or test
solution.
(B) Test chambers should be loosely covered to reduce evaporation
and to minimize the entry of dust or other particulates into solutions and
to prevent loss of test fish.
(C) Size. Many different sizes of test chambers have been used suc-
cessfully. The size, shape, and depth of the test chamber is acceptable
if the specified flow rate and loading requirements can be achieved. Test
vessels must be of adequate size to maintain a load rate of
FWF > 0.8 g FWF/L for static or static-renewal tests, or
FWF > 0.5 g FWF/L for flow-through tests.
(D) Test substance delivery system. (7) In flow-through tests, propor-
tional diluters, metering pump systems, or other suitable systems should
be used to deliver the test chemical to the test chambers. The choice of
a specific delivery system depends on the specific properties and require-
ments of the test substance.
(2) The system should be calibrated before and after each test. Cali-
bration includes determining the flow rate and test concentration in each
replicate. The apparatus used should accurately and precisely deliver the
appropriate amount of stock solution and dilution water to each replicate.
(3) A closed flow-through system may be used to test volatile com-
pounds when more than 20 percent of the test substance would be lost
through volatility or the test substance would cause oxygen levels may
fall below 60 percent of the saturation level. A design description of this
type of system should be included in the study report.
(E) Aeration. Gentle aeration of test vessels used in static systems
during the exposure period is permitted only in cases where oxygen levels
are in danger of dropping below 60 percent saturation due to chemical
characteristics of the test material. Test concentrations must be measured
during the test if aeration is used. No aeration of actual test vessels may
be utilized in flow-through tests.
(viii) Light. (A) The photoperiod with 15 to 30 min transition periods
is suggested. Photoperiods may range from 12D/12N to 16D/8N, where
D = day, and N = night.
(B) Light intensity should range from 30 to 100 1m at the water sur-
face; the intensity selected should be duplicated as closely as possible in
all replicates.
(ix) Temperature. (A) Temperatures must be recorded in all rep-
licates at the beginning of the test and every 24 h thereafter. The tempera-
ture should be recorded at least hourly in one replicate throughout the
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test. Temperature should vary no more then 1.0 °C in any given 24-h
period.
(B) The test system should be equipped with an automatic alarm sys-
tem to alert staff of temperature changes in excess of 2.0 °C.
(C) If the water is heated, precautions should be taken to ensure that
supersaturation of dissolved gases is avoided.
(x) Dissolved oxygen. DO concentrations should be measured in each
replicate at the beginning of the test and every 24 h thereafter.
(xi) Feeding. Fish may not be fed during the treatment period.
(xii) Disturbances. Any disturbance which might change the behavior
of the test fish should be avoided.
(2) Test concentrations, (i) A minimum of five test concentrations
must be employed.
(ii) Five or more concentrations in a geometric series should be tested.
Test concentrations must be at least 50 percent greater than the next lowest
test concentration (not to exceed 120 percent). Range-finding studies prior
to testing may allow more accurate selection of test concentrations.
(iii) No more than 25 percent variation is allowed between test con-
centrations within the same treatment during the test.
(iv) Concentration selection. (A) Test concentrations should be se-
lected to produce a no-observable-effect concentration (NOEC) and, pref-
erably, at least two partial mortalities, i.e. one greater than and the other
less than 50 percent, after 96 h. The highest test concentration should not
exceed the chemical's aqueous solubility limit if the chemical is not a
surfactant or the chemical's self-dispersibility limit if the chemical is a
surfactant or a charged polymer.
(B) Exceptions may be required in testing certain pesticide AIs as
products. Product formulations may increase the solubility of the AI be-
yond its aqueous solubility limit.
(v) Concentration analysis. (A) Concentration analysis must be per-
formed at initiation and every 48 h of the study thereafter.
(B) In static tests, the test substance concentration should be measured
in each replicate minimally at the beginning (0-hour, before test organisms
are added), at 48 h, and at the end of the test.
(C) In static-renewal tests, the test substance should be measured in
each replicate at the beginning and end of test and just before and after
each renewal.
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(D) In flow-through tests, the test substance should be measured as
follows:
(7) In each replicate at 0, 48, and 96 h, and every 96 h thereafter,
as long as the test is continued.
(2) In at least one appropriate chamber whenever a malfunction is
detected in any part of the test substance delivery system.
(3) Collection of samples for measurement, (i) Water samples must
be removed from a central point within the test vessel, not from inflow
or outflow points.
(ii) These samples should not contain any surface particulates or ma-
terial dislodged from the bottom or sides. Samples should be analyzed
immediately, or handled and stored in a manner which minimizes loss of
test substance through microbial degradation, photo degradation, chemical
reaction, volatilization, or sorption.
(iii) The test solution volume should not be reduced during the test
by more than 10 percent as a result of sampling.
(iv) Samples from each test concentration replicate should not be
pooled for analyses.
(v) Diluter systems must be monitored for proper adjustment, and
operation every 24 h, and should be monitored during the first hour of
operation.
(vi) Surface films and precipitates must be reported should they occur.
(vii) The flow rate to each replicate should be measured at the begin-
ning and end of each test.
(viii) During a test, the flow rates should not vary more than a factor
of 10 from any one replicate to another.
(ix) Minimum number of test vessel replacements should be 6 to 10
per 24-h period for flow-through testing.
(4) Observations, (i) Mortality observations should be recorded at
6, 24, 48, 72, and 96 h.
(ii) If the test is continued past 96 h, additional observations should
be made every 24 h until termination.
(iii) In addition to mortality, any abnormal behavior should be re-
corded, such as, but not limited to, erratic swimming, loss of reflex, in-
creased excitability, lethargy, and changes in appearance or physiology
such as discoloration, excessive mucous production, hyperventilation,
opaque eyes, curved spine, or hemorrhaging.
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(g) Data and reporting—(1) Treatment of results. The cumulative
percentage mortality for each exposure period is plotted against concentra-
tion on logarithmic paper. Normal statistical procedures are then employed
to calculate the LC50 for the appropriate exposure period. Confidence lim-
its (CI) with p = 0.95 for the calculated LC50 values are to be included.
(2) Test report, (i) The test report must include the following:
(ii) Test facilities, test dates, and personnel must be reported.
(iii) Identification of the test substance and purity.
(iv) Water quality characteristics as reported in the laboratory records
for the study. These must include 24-h records of DO, pH, and tempera-
ture.
(v) Methods of stock solution preparation and the concentrations used
in definitive testing.
(vi) All test concentrations measured during the test and at termi-
nation.
(vii) The number of test organisms in each replicate and/or test con-
centration.
(viii) The LC50 concentration-response curves, LC50 values, and as-
sociated 95 percent CI should be determined for 24, 48, 72 and 96 h,
whenever sufficient data exist.
(ix) A graph of the concentration-mortality curve at test termination.
Any control mortality observed during the acclimation or study period.
(x) An NOEL for the 96-h test should also be reported.
(xi) If no LC50 value is determined, but it can be demonstrated that
the concentrations tested were the highest possible due to the test chemi-
cal's aqueous solubility limit, self-dispersibility limit, or other physico-
chemical limitations, then the data will be considered for acceptance. Ex-
planation should include details of the solvents which were tried prior to
initiation of the final study.
(xii) Any abnormal behavior displayed by the test fish.
(xiii) Any protocol deviations or occurrences which may have influ-
enced the final results of the test.
(xiv) A quality control methods and quality assurance statement
should accompany all final study reports.
(xv) Raw data must be available to support study author's conclusions
and should be presented with the study report.
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(xvi) Methods of statistical analysis should be reported.
(xvii) Methods used in analysis of test concentrations of the test
chemical should be described. The accuracy of the method (i.e. detection
limit and quantification limit) should be given.
(h) References. The following references should be consulted for ad-
ditional background material on this test guideline.
(1) Standard Guide for Conducting Acute Toxicity Tests with Fishes,
Macroinvertebrates, and Amphibians, E 729-88a. American Society Test-
ing Materials, Philadelphia, PA. Approved Nov. 21, 1988.
(2) Organization of Economic Cooperation and Development, Guide-
lines for Testing of Chemicals, Guideline 203 "Fish Acute Toxicity Test."
Adopted July 17, 1992.
(3) Test Guideline EG-9, Fish Acute Toxicity Test, Office of Pollu-
tion Prevention and Toxics, Office of Prevention, Pesticides and Toxic
Substances, U.S. Environmental Protection Agency, Washington DC.
(4) Standard Evaluation Procedure Acute Toxicity Test for Freshwater
Fish, EPA-540/9-85-006, Office of Pesticide Programs, Office of Preven-
tion Pesticides and Toxic Substances, U.S. Environmental Protection
Agency, Washington DC. Revised June 1985.
(5) Acute Toxicity Test for Estuarine and Marine Organisms (Estua-
rine Fish 96-Hour Acute Toxicity Test), EPA 540/9-85-009, Office of
Pesticide Programs, Office of Prevention, Pesticides, and Toxic Sub-
stances, U.S. Environmental Protection Agency, Washington DC. Revised
June 1985.
(6) Federal Insecticide, Fungicide, Rodenticide Act, Subdivision E,
Hazard Evaluation, Wildlife and Aquatic Organisms, U.S. Environmental
Protection Agency. October 1982.
(7) Finney, D.J., Probit Analysis. 3rd Edition. Cambridge University
Press: London and New York (1971).
(8) Stephen, C.E., "Methods for Calculating an LC50" Aquatic Toxi-
cology and Hazard Evaluation, ASTM STP 634, American Society of Test-
ing and Materials, Philadelphia, PA (1977).
(9) Canada, Environment Canada. Biological test method: acute
lethality test using threespine stickleback (Gasterosteus aculeatus). Envi-
ronmental Protection, Conservation and Protection, Environment Canada,
Report EPS l/RM/10 (1990).
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