1、Designation: E729 96 (Reapproved 2014)Standard Guide forConducting Acute Toxicity Tests on Test Materials withFishes, Macroinvertebrates, and Amphibians1This standard is issued under the fixed designation E729; the number immediately following the designation indicates the year oforiginal adoption o
2、r, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon () indicates an editorial change since the last revision or reapproval.This standard has been approved for use by agencies of the U.S. Department of Defense.1. S
3、cope1.1 This guide (1)2describes procedures for obtaininglaboratory data concerning the adverse effects (for example,lethality and immobility) of a test material added to dilutionwater, but not to food, on certain species of freshwater andsaltwater fishes, macroinvertebrates, and amphibians during 2
4、to 8-day exposures, depending on the species. These proce-dures will probably be useful for conducting acute toxicity testswith many other aquatic species, although modifications mightbe necessary.1.2 Other modifications of these procedures might be justi-fied by special needs or circumstances. Alth
5、ough using appro-priate procedures is more important than following prescribedprocedures, results of tests conducted using unusual proceduresare not likely to be comparable to results of many other tests.Comparison of results obtained using modified and unmodifiedversions of these procedures might p
6、rovide useful informationconcerning new concepts and procedures for conducting acutetests.1.3 This guide describes tests using three basic exposuretechniques: static, renewal, and flow-through. Selection of thetechnique to use in a specific situation will depend on the needsof the investigator and o
7、n available resources. Tests using thestatic technique provide the most easily obtained measure ofacute toxicity, but conditions often change substantially duringstatic tests; therefore, static tests should not last longer than 96h, and test organisms should not be fed during such tests. Statictests
8、 should probably not be conducted on materials that havea high oxygen demand, are highly volatile, are rapidly trans-formed biologically or chemically in aqueous solution, or areremoved from test solutions in substantial quantities by the testchambers or organisms during the test. Because the pH and
9、concentrations of dissolved oxygen and test material aremaintained at desired levels and degradation and metabolicproducts are removed, tests using renewal and flow-throughmethods are preferable and may last longer than 96 h; testorganisms may be fed during renewal and flow-through tests.Although re
10、newal tests might be more cost-effective, flow-through tests are generally preferable.1.4 Acute tests may be performed to meet regulatory datarequirements or to obtain time-independent estimates of toxic-ity.1.4.1 If the objective is to obtain data to meet regulatoryrequirements, it may be necessary
11、 to limit the number ofobservation times based on stipulations of the regulatoryagency and cost considerations.1.4.2 If the objective of an acute toxicity test is to determinea time-independent (that is, incipient, threshold, or asymptotic)toxicity level, an appropriate number of observations must b
12、etaken over an exposure duration of sufficient length to establishthe shape of the toxicity curve or allow the direct or math-ematically estimated determination of a time-independent tox-icity value (1), or both.1.5 In the development of these procedures, an attempt wasmade to balance scientific and
13、 practical considerations and toensure that the results will be sufficiently accurate and precisefor the applications for which they are commonly used. Amajor consideration was that the common uses of the results ofacute toxicity tests do not require or justify stricter require-ments than those set
14、forth herein. Although the tests may beimproved by using more organisms, longer acclimation times,and so forth, the requirements presented herein should usuallybe sufficient.1.6 Results of acute toxicity tests should usually be reportedin terms of an LC50 (median lethal concentration) or EC50(median
15、 effective concentration) at the end of the test, but it isdesirable to provide information concerning the dependence ofadverse effects on both time and concentration. Thus, whenfeasible, flow-through and renewal tests should be conductedso that LC50s or EC50s can be reported from6htoanasymptotic (t
16、ime-independent, threshold, incipient) value, ifone exists. In some situations, it might only be necessary to1This guide is under the jurisdiction ofASTM Committee E50 on EnvironmentalAssessment, Risk Management and CorrectiveActionand is the direct responsibilityof Subcommittee E50.47 on Biological
17、 Effects and Environmental Fate.Current edition approved Oct. 1, 2014. Published December 2014. Originallyapproved in 1980. Last previous edition approved in 2007 as E729 96(2007). DOI:10.1520/E0729-96R14.2The boldface numbers in parentheses refer to the list of references at the end ofthis standard
18、.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States1determine whether a specific concentration is acutely toxic tothe test species or whether the LC50 or EC50 is above or belowa specific concentration.1.7 This guide is arranged as follow
19、s:SectionReferenced Documents 2Terminology 3Summary of Guide 4Significance and Use 5Apparatus 6Facilities 6.1Special Requirements 6.2Construction Materials 6.3Metering System 6.4Test Chambers 6.5Cleaning 6.6Acceptability 6.7Hazards 7Dilution Water 8Requirements 8.1Source 8.2Treatment 8.3Characteriza
20、tion 8.4Test Material 9General 9.1Stock Solution 9.2Test Concentration(s) 9.3Test Organisms 10Species 10.1Age 10.2Source 10.3Care and Handling 10.4Feeding 10.5Disease Treatment 10.6Holding 10.7Acclimation 10.8Quality 10.9Procedure 11Experimental Design 11.1Dissolved Oxygen 11.2Temperature 11.3Loadin
21、g 11.4Beginning the Test 11.5Feeding 11.6Duration of Test 11.7Biological Data 11.8Other Measurements 11.9Analytical Methodology 12Acceptability of Test 13Calculation of Results 14Report 151.8 The values stated in SI units are to be regarded as thestandard. The values given in parentheses are for inf
22、ormationonly.1.9 This standard does not purport to address all of thesafety concerns, if any, associated with its use. It is theresponsibility of the user of this standard to establish appro-priate safety and health practices and determine the applica-bility of regulatory limitations prior to use. S
23、pecific hazardstatements are given in Section 7.2. Referenced Documents2.1 ASTM Standards:3E724 Guide for Conducting Static Acute Toxicity TestsStarting with Embryos of Four Species of SaltwaterBivalve MolluscsE943 Terminology Relating to Biological Effects and Envi-ronmental FateE1023 Guide for Ass
24、essing the Hazard of a Material toAquatic Organisms and Their UsesE1191 Guide for Conducting Life-Cycle Toxicity Tests withSaltwater MysidsE1192 Guide for Conducting Acute Toxicity Tests on Aque-ous Ambient Samples and Effluents with Fishes,Macroinvertebrates, and AmphibiansE1203 Practice for Using
25、Brine Shrimp Nauplii as Food forTest Animals in Aquatic Toxicology (Withdrawn 2013)4E1563 Guide for Conducting Static Acute Toxicity Testswith Echinoid EmbryosE1604 Guide for Behavioral Testing in Aquatic ToxicologyIEEE/ASTM SI 10 Standard for Use of the InternationalSystem of Units (SI) (the Modern
26、ized Metric System)3. Terminology3.1 Acute toxicity tests are generally used to determine theconcentration of test material that produces a specific adverseeffect on a specified percentage of test organisms during a shortexposure. Because death is an obviously important adverseeffect and is easily d
27、etected for many species, the mostcommon acute toxicity test is the acute lethality test.Experimentally, effect on 50 % of a group of test organisms isthe most reproducible and easily determined measure oftoxicity, and 96 h is often a convenient, useful exposureduration. Therefore, the measure of ac
28、ute toxicity most oftenused with fishes, macroinvertebrates, and amphibians is the96-h LC50. However, because immobilization is a severe effectand is not easy to distinguish from death for some species, themeasure of acute toxicity most often used with daphnids andmidge larvae is the 48-h EC50 based
29、 on death plus immobili-zation. The terms LC50 and EC50 are consistent with thewidely used toxicological terms LD50 (median lethal dose) andED50 (median effective dose), respectively. The terms LC50and EC50 should be used whenever results are calculated basedon the concentration of test material in
30、dilution water, whereasthe terms LD50 and ED50 should be used whenever results arecalculated based on the quantity of test material that enters oris applied directly to test organisms. For toxic agents or testsfor which neither concentration nor dose is appropriate, such astests on temperature or wi
31、th poorly water-soluble materials, theterms LL50 (median lethal level) and EL50 (median effectivelevel) should be used, if the effect is dichotomous. For tests inwhich the effect is expressed as a percent inhibition comparedto the control, for example, a percent inhibition in growth, andnot as the p
32、ercentage of the individual organisms that wereaffected, the term IC50 should be used to denote the concen-tration that causes a 50 % inhibition compared to the control.3.2 Acute toxicity tests in which test organisms are exposedto test solutions containing a test material can be conducted byat leas
33、t four techniques:3For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM website.4The last approved version of this historic
34、al standard is referenced onwww.astm.org.E729 96 (2014)23.2.1 In the static technique, test solutions and organismsare placed in chambers and kept there for the duration of thetest.3.2.2 The recirculation technique is like the static techniqueexcept that each test solution is continuously circulated
35、 throughan apparatus designed to maintain water quality, and possiblyremove degraded, but not undegraded, test material by suchmeans as aeration, filtration, and sterilization and then returnedto the test chamber.3.2.3 The renewal technique is like the static techniqueexcept that test organisms are
36、periodically exposed to fresh testsolution of the same composition, usually once every 24 h,either by transferring the organisms from one test chamber toanother or by replacing nearly all the test solution.3.2.4 In the flow-through technique, test solution flowsthrough the test chamber on a once-thr
37、ough basis throughoutthe test.3.2.4.1 Two procedures may be used. In the first a largevolume of each test solution is prepared before the beginningof the test, and these solutions flow through the respectivechambers. In the second and more common procedure, freshtest solutions are prepared continuou
38、sly or every few minutesjust before they enter the respective test chambers. In bothprocedures a metering system controls the flow of test solution,and in the latter procedure the test solutions are prepared by themetering system. Both of the procedures may be used toconduct continuous-flow tests. M
39、any tests conducted using thesecond procedure, however, are intermittent-flow tests becausethe metering system cycles and delivers test solution every fewminutes.3.2.5 With any of these techniques a pump or stirrer can beused to create a current in the test chamber to accommodateparticular species,
40、but the current will often increase bothaeration and volatilization.3.3 In flow-through tests a “volume addition” is the intro-duction into the test chamber of a volume of test solution equalto the volume of solution in the chamber.3.4 For the purposes of 8.4.1, the term“ organophosphoruspesticides”
41、 refers to chlorpyrifos, demeton, diazinon,disulfoton, fenitrothion, malathion, methyl parathion, and para-thion; the term “organochlorine pesticides” refers to aldrin,chlordane, DDD, DDE, DDT, dieldrin, endosulfan, endrin,heptachlor, heptachlor epoxide, lindane, methoxychlor, mirex,and toxaphene; a
42、nd the term “chlorinated phenoxy herbicides”refers to the free acids, salts, and esters of 2,4-D, dicamba,silvex, and 2,4,5-T. The term “organic chlorine” refers tochlorine that would be detected if, when samples are preparedfor gas chromatographic analysis for polychlorinated biphenyls(PCBs) and th
43、e organochlorine pesticides listed above, achloride detector is used instead of an electron capture detectorto measure compounds that elute from just before lindane tojust after mirex on the gas chromatograph being used. Organicchlorine does not refer only to chlorine associated withorganochlorine p
44、esticides and PCBs; it refers to all chlorinethat elutes within the specified period.3.5 reconstituted watera dilution water that is prepared byadding sea salt or appropriate amounts of selected chemicals towater, which is usually prepared using deionization,distillation, or reverse osmosis, so that
45、 the concentrations andratios of the major ions in the dilution water are similar to thosein comparable natural surface waters.3.6 The words “must,” “should,”“ may,” “can,” and “might”have very specific meanings in this guide. “Must” is used toexpress an absolute requirement, that is, to state that
46、the testought to be designed to satisfy the specified condition, unlessthe purpose of the test requires a different design. “Must” isonly used in connection with factors that directly relate to theacceptability of the test (see 13.1). “Should” is used to statethat the specified condition is recommen
47、ded and ought to bemet if possible. Although violation of one “should” is rarely aserious matter, violation of several will often render the resultsquestionable. Terms such as “is desirable,” “is often desirable,”and “might be desirable” are used in connection with lessimportant factors. “May” is us
48、ed to mean “is (are) allowedto,”“ can” is used to mean “is (are) able to,” and “might” isused to mean “could possibly.” Thus the classic distinctionbetween “may” and “can” is preserved, and “might” is neverused as a synonym for either “may” or “can.”3.7 IC50a statistically or graphically estimated c
49、oncentra-tion of test material that is expected to cause a 50 % inhibitionof one or more specified biological processes (such as growthor reproduction), for which the data are not dichotomous, underspecified conditions.3.8 For definitions of other terms used in this guide, refer toTerminology E943 and Guide E1023. For an explanation ofunits and symbols, refer to Standard IEEE/ASTM SI 10.4. Summary of Guide4.1 In each of two or more treatments, test organisms of onespecies are maintained for 2 to 8 days in one or more testchambers. In each of the one or more control treatments, the
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