ASTM E729-96(2007)
(Guide)Standard Guide for Conducting Acute Toxicity Tests on Test Materials with Fishes, Macroinvertebrates, and Amphibians
Standard Guide for Conducting Acute Toxicity Tests on Test Materials with Fishes, Macroinvertebrates, and Amphibians
SIGNIFICANCE AND USE
An acute toxicity test is conducted to obtain information concerning the immediate effects on test organisms of a short-term exposure to a test material under specific experimental conditions. An acute toxicity test does not provide information about whether delayed effects will occur, although a post-exposure observation period, with appropriate feeding, if necessary, might provide such information.
Results of acute toxicity tests might be used to predict acute effects likely to occur on aquatic organisms in field situations as a result of exposure under comparable conditions, except that (1) motile organisms might avoid exposure when possible, and (2) toxicity to benthic organisms might be dependent on sorption or settling of the test material onto the substrate.
Results of acute tests might be used to compare the acute sensitivities of different species and the acute toxicities of different test materials, and to study the effects of various environmental factors on results of such tests.
Results of acute toxicity tests might be an important consideration when assessing the hazards of materials to aquatic organisms (see Guide E 1023) or when deriving water quality criteria for aquatic organisms (2).
Results of acute toxicity tests might be useful for studying the biological availability of, and structure-activity relationships between, test materials.
Results of acute toxicity tests will depend on the temperature, composition of the dilution water, condition of the test organisms, exposure technique, and other factors.
SCOPE
1.1 This guide () describes procedures for obtaining laboratory data concerning the adverse effects (for example, lethality and immobility) of a test material added to dilution water, but not to food, on certain species of freshwater and saltwater fishes, macroinvertebrates, and amphibians during 2 to 8-day exposures, depending on the species. These procedures will probably be useful for conducting acute toxicity tests with many other aquatic species, although modifications might be necessary.
1.2 Other modifications of these procedures might be justified by special needs or circumstances. Although using appropriate procedures is more important than following prescribed procedures, results of tests conducted using unusual procedures are not likely to be comparable to results of many other tests. Comparison of results obtained using modified and unmodified versions of these procedures might provide useful information concerning new concepts and procedures for conducting acute tests.
1.3 This guide describes tests using three basic exposure techniques: static, renewal, and flow-through. Selection of the technique to use in a specific situation will depend on the needs of the investigator and on available resources. Tests using the static technique provide the most easily obtained measure of acute toxicity, but conditions often change substantially during static tests; therefore, static tests should not last longer than 96 h, and test organisms should not be fed during such tests. Static tests should probably not be conducted on materials that have a high oxygen demand, are highly volatile, are rapidly transformed biologically or chemically in aqueous solution, or are removed from test solutions in substantial quantities by the test chambers or organisms during the test. Because the pH and concentrations of dissolved oxygen and test material are maintained at desired levels and degradation and metabolic products are removed, tests using renewal and flow-through methods are preferable and may last longer than 96 h; test organisms may be fed during renewal and flow-through tests. Although renewal tests might be more cost-effective, flow-through tests are generally preferable.
1.4 Acute tests may be performed to meet regulatory data requirements or to obtain time-independent estimates of toxicity.
1.4.1 If the objective is to obtain data to meet regulatory req...
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Designation: E729 − 96(Reapproved 2007)
Standard Guide for
Conducting Acute Toxicity Tests on Test Materials with
Fishes, Macroinvertebrates, and Amphibians
This standard is issued under the fixed designation E729; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision.Anumber in parentheses indicates the year of last reapproval.A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
This standard has been approved for use by agencies of the Department of Defense.
1. Scope concentrations of dissolved oxygen and test material are
2 maintained at desired levels and degradation and metabolic
1.1 This guide (1) describes procedures for obtaining
products are removed, tests using renewal and flow-through
laboratory data concerning the adverse effects (for example,
methods are preferable and may last longer than 96 h; test
lethality and immobility) of a test material added to dilution
organisms may be fed during renewal and flow-through tests.
water, but not to food, on certain species of freshwater and
Although renewal tests might be more cost-effective, flow-
saltwater fishes, macroinvertebrates, and amphibians during 2
through tests are generally preferable.
to 8-day exposures, depending on the species. These proce-
dureswillprobablybeusefulforconductingacutetoxicitytests
1.4 Acute tests may be performed to meet regulatory data
withmanyotheraquaticspecies,althoughmodificationsmight
requirements or to obtain time-independent estimates of toxic-
be necessary.
ity.
1.4.1 If the objective is to obtain data to meet regulatory
1.2 Other modifications of these procedures might be justi-
requirements, it may be necessary to limit the number of
fied by special needs or circumstances.Although using appro-
observation times based on stipulations of the regulatory
priate procedures is more important than following prescribed
agency and cost considerations.
procedures,resultsoftestsconductedusingunusualprocedures
1.4.2 Iftheobjectiveofanacutetoxicitytestistodetermine
are not likely to be comparable to results of many other tests.
Comparisonofresultsobtainedusingmodifiedandunmodified atime-independent(thatis,incipient,threshold,orasymptotic)
toxicity level, an appropriate number of observations must be
versions of these procedures might provide useful information
concerning new concepts and procedures for conducting acute takenoveranexposuredurationofsufficientlengthtoestablish
the shape of the toxicity curve or allow the direct or math-
tests.
ematically estimated determination of a time-independent tox-
1.3 This guide describes tests using three basic exposure
icity value (1), or both.
techniques: static, renewal, and flow-through. Selection of the
techniquetouseinaspecificsituationwilldependontheneeds
1.5 In the development of these procedures, an attempt was
of the investigator and on available resources. Tests using the
made to balance scientific and practical considerations and to
static technique provide the most easily obtained measure of
ensure that the results will be sufficiently accurate and precise
acute toxicity, but conditionsoftenchangesubstantially during
for the applications for which they are commonly used. A
static tests; therefore, static tests should not last longer than 96
majorconsiderationwasthatthecommonusesoftheresultsof
h,andtestorganismsshouldnotbefedduringsuchtests.Static
acute toxicity tests do not require or justify stricter require-
tests should probably not be conducted on materials that have
ments than those set forth herein. Although the tests may be
a high oxygen demand, are highly volatile, are rapidly trans-
improved by using more organisms, longer acclimation times,
formed biologically or chemically in aqueous solution, or are
and so forth, the requirements presented herein should usually
removedfromtestsolutionsinsubstantialquantitiesbythetest
be sufficient.
chambers or organisms during the test. Because the pH and
1.6 Resultsofacutetoxicitytestsshouldusuallybereported
in terms of an LC50 (median lethal concentration) or EC50
(median effective concentration) at the end of the test, but it is
ThisguideisunderthejurisdictionofASTMCommitteeE50onEnvironmental
desirabletoprovideinformationconcerningthedependenceof
Assessment, Risk Management and CorrectiveAction and is the direct responsibil-
adverse effects on both time and concentration. Thus, when
ity of Subcommittee E50.47 on Biological Effects and Environmental Fate.
Current edition approved Oct. 1, 2007. Published October 2007. Originally
feasible, flow-through and renewal tests should be conducted
approvedin1980.Lastpreviouseditionapprovedin2002asE729–96(2002).DOI:
so that LC50s or EC50s can be reported from6htoan
10.1520/E0729-96R07.
asymptotic (time-independent, threshold, incipient) value, if
Theboldfacenumbersinparenthesesrefertothelistofreferencesattheendof
this standard. one exists. In some situations, it might only be necessary to
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E729 − 96 (2007)
determine whether a specific concentration is acutely toxic to E724Guide for Conducting Static Acute Toxicity Tests
thetestspeciesorwhethertheLC50orEC50isaboveorbelow Starting with Embryos of Four Species of Saltwater
a specific concentration. Bivalve Molluscs
E943Terminology Relating to Biological Effects and Envi-
1.7 This guide is arranged as follows:
ronmental Fate
Section
E1023Guide for Assessing the Hazard of a Material to
Referenced Documents 2 Aquatic Organisms and Their Uses
Terminology 3
E1191Guide for Conducting Life-Cycle Toxicity Tests with
Summary of Guide 4
Saltwater Mysids
Significance and Use 5
Apparatus 6 E1192Guide for ConductingAcute Toxicity Tests onAque-
Facilities 6.1
ous Ambient Samples and Effluents with Fishes,
Special Requirements 6.2
Macroinvertebrates, and Amphibians
Construction Materials 6.3
Metering System 6.4 E1203Practice for Using Brine Shrimp Nauplii as Food for
Test Chambers 6.5
Test Animals in Aquatic Toxicology (Withdrawn 2013)
Cleaning 6.6
E1563Guide for Conducting Static Acute Toxicity Tests
Acceptability 6.7
Hazards 7 with Echinoid Embryos
Dilution Water 8
E1604Guide for Behavioral Testing in Aquatic Toxicology
Requirements 8.1
IEEE/ASTM SI10Standard for Use of the International
Source 8.2
Treatment 8.3
System of Units (SI) (the Modernized Metric System)
Characterization 8.4
Test Material 9
3. Terminology
General 9.1
3.1 Acute toxicity tests are generally used to determine the
Stock Solution 9.2
Test Concentration(s) 9.3
concentration of test material that produces a specific adverse
Test Organisms 10
effectonaspecifiedpercentageoftestorganismsduringashort
Species 10.1
exposure. Because death is an obviously important adverse
Age 10.2
Source 10.3
effect and is easily detected for many species, the most
Care and Handling 10.4
common acute toxicity test is the acute lethality test.
Feeding 10.5
Experimentally, effect on 50% of a group of test organisms is
Disease Treatment 10.6
Holding 10.7
the most reproducible and easily determined measure of
Acclimation 10.8
toxicity, and 96 h is often a convenient, useful exposure
Quality 10.9
duration. Therefore, the measure of acute toxicity most often
Procedure 11
Experimental Design 11.1
used with fishes, macroinvertebrates, and amphibians is the
Dissolved Oxygen 11.2
96-hLC50.However,becauseimmobilizationisasevereeffect
Temperature 11.3
and is not easy to distinguish from death for some species, the
Loading 11.4
Beginning the Test 11.5
measure of acute toxicity most often used with daphnids and
Feeding 11.6
midge larvae is the 48-h EC50 based on death plus immobili-
Duration of Test 11.7
zation. The terms LC50 and EC50 are consistent with the
Biological Data 11.8
Other Measurements 11.9
widelyusedtoxicologicaltermsLD50(medianlethaldose)and
Analytical Methodology 12
ED50 (median effective dose), respectively. The terms LC50
Acceptability of Test 13
Calculation of Results 14 andEC50shouldbeusedwheneverresultsarecalculatedbased
Report 15
on the concentration of test material in dilution water, whereas
1.8 The values stated in SI units are to be regarded as the
thetermsLD50andED50shouldbeusedwheneverresultsare
standard. The values given in parentheses are for information calculated based on the quantity of test material that enters or
only. is applied directly to test organisms. For toxic agents or tests
forwhichneitherconcentrationnordoseisappropriate,suchas
1.9 This standard does not purport to address all of the
testsontemperatureorwithpoorlywater-solublematerials,the
safety concerns, if any, associated with its use. It is the
terms LL50 (median lethal level) and EL50 (median effective
responsibility of the user of this standard to establish appro-
level) should be used, if the effect is dichotomous. For tests in
priate safety and health practices and determine the applica-
which the effect is expressed as a percent inhibition compared
bility of regulatory limitations prior to use. Specific hazard
to the control, for example, a percent inhibition in growth, and
statements are given in Section 7.
not as the percentage of the individual organisms that were
affected, the term IC50 should be used to denote the concen-
2. Referenced Documents
tration that causes a 50% inhibition compared to the control.
2.1 ASTM Standards:
3.2 Acutetoxicitytestsinwhichtestorganismsareexposed
to test solutions containing a test material can be conducted by
at least four techniques:
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
Standards volume information, refer to the standard’s Document Summary page on The last approved version of this historical standard is referenced on
the ASTM website. www.astm.org.
E729 − 96 (2007)
3.2.1 In the static technique, test solutions and organisms water, which is usually prepared using deionization,
are placed in chambers and kept there for the duration of the distillation, or reverse osmosis, so that the concentrations and
test. ratiosofthemajorionsinthedilutionwateraresimilartothose
in comparable natural surface waters.
3.2.2 The recirculation technique is like the static technique
exceptthateachtestsolutioniscontinuouslycirculatedthrough
3.6 Thewords“must,”“should,”“may,”“can,”and“might”
an apparatus designed to maintain water quality, and possibly
have very specific meanings in this guide. “Must” is used to
remove degraded, but not undegraded, test material by such
express an absolute requirement, that is, to state that the test
meansasaeration,filtration,andsterilizationandthenreturned
ought to be designed to satisfy the specified condition, unless
to the test chamber.
the purpose of the test requires a different design. “Must” is
3.2.3 The renewal technique is like the static technique
only used in connection with factors that directly relate to the
exceptthattestorganismsareperiodicallyexposedtofreshtest
acceptability of the test (see 13.1). “Should” is used to state
solution of the same composition, usually once every 24 h,
that the specified condition is recommended and ought to be
either by transferring the organisms from one test chamber to
met if possible.Although violation of one “should” is rarely a
another or by replacing nearly all the test solution.
serious matter, violation of several will often render the results
3.2.4 In the flow-through technique, test solution flows
questionable.Termssuchas“isdesirable,”“isoftendesirable,”
through the test chamber on a once-through basis throughout and “might be desirable” are used in connection with less
the test.
important factors. “May” is used to mean “is (are) allowed
to,”“ can” is used to mean “is (are) able to,” and “might” is
3.2.4.1 Two procedures may be used. In the first a large
volume of each test solution is prepared before the beginning used to mean “could possibly.” Thus the classic distinction
between “may” and “can” is preserved, and “might” is never
of the test, and these solutions flow through the respective
chambers. In the second and more common procedure, fresh used as a synonym for either “may” or “can.”
test solutions are prepared continuously or every few minutes
3.7 IC50—astatisticallyorgraphicallyestimatedconcentra-
just before they enter the respective test chambers. In both
tion of test material that is expected to cause a 50% inhibition
proceduresameteringsystemcontrolstheflowoftestsolution,
of one or more specified biological processes (such as growth
andinthelatterprocedurethetestsolutionsarepreparedbythe
orreproduction),forwhichthedataarenotdichotomous,under
metering system. Both of the procedures may be used to
specified conditions.
conduct continuous-flow tests. Many tests conducted using the
3.8 Fordefinitionsofothertermsusedinthisguide,referto
secondprocedure,however,areintermittent-flowtestsbecause
Terminology E943 and Guide E1023. For an explanation of
themeteringsystemcyclesanddeliverstestsolutioneveryfew
units and symbols, refer to Standard IEEE/ASTM SI10.
minutes.
3.2.5 With any of these techniques a pump or stirrer can be
4. Summary of Guide
used to create a current in the test chamber to accommodate
4.1 Ineachoftwoormoretreatments,testorganismsofone
particular species, but the current will often increase both
species are maintained for 2 to 8 days in one or more test
aeration and volatilization.
chambers. In each of the one or more control treatments, the
3.3 In flow-through tests a “volume addition” is the intro-
organisms are maintained in dilution water to which no test
ductionintothetestchamberofavolumeoftestsolutionequal
material has been added in order to provide (1) a measure of
to the volume of solution in the chamber.
the acceptability of the test by giving an indication of the
quality of the test organisms and the suitability of the dilution
3.4 For the purposes of 8.4.1, the term“ organophosphorus
water, test conditions, handling procedures, and so forth, and
pesticides” refers to chlorpyrifos, demeton, diazinon,
(2) the basis for interpreting data obtained from the other
disulfoton,fenitrothion,malathion,methylparathion,andpara-
treatments. In each of the one or more other treatments, the
thion; the term “organochlorine pesticides” refers to aldrin,
organisms are maintained in dilution water to which a selected
chlordane, DDD, DDE, DDT, dieldrin, endosulfan, endrin,
concentrationoftestmaterialhasbeenadded.Dataconcerning
heptachlor, heptachlor epoxide, lindane, methoxychlor, mirex,
effects on the organisms in each test chamber are usually
and toxaphene; and the term “chlorinated phenoxy herb
...
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