1、Designation: E 729 96 (Reapproved 2007)Standard Guide forConducting Acute Toxicity Tests on Test Materials withFishes, Macroinvertebrates, and Amphibians1This standard is issued under the fixed designation E 729; the number immediately following the designation indicates the year oforiginal adoption
2、 or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon (e) 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. Sco
3、pe1.1 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 2to
4、 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. Althou
5、gh 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 pro
6、vide 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 on
7、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 s
8、hould 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 andco
9、ncentrations 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 rene
10、wal 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 t
11、o 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 bet
12、aken 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 p
13、ractical 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 fo
14、rth 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 e
15、ffective 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 (tim
16、e-independent, threshold, incipient) value, ifone exists. In some situations, it might only be necessary todetermine whether a specific concentration is acutely toxic tothe test species or whether the LC50 or EC50 is above or belowa specific concentration.1This guide is under the jurisdiction of AST
17、M Committee E47 on BiologicalEffects and Environmental Fate and is the direct responsibility of SubcommitteeE47.01 on Aquatic Assessment and Toxicology.Current edition approved Oct. 1, 2007. Published October 2007. Originallyapproved in 1980. Last previous edition approved in 2002 as E 729 96(2002).
18、2The boldface numbers in parentheses refer to the list of references at the end ofthis standard.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.1.7 This guide is arranged as follows:SectionReferenced Documents 2Terminology 3Summary o
19、f 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 8.3Characterization 8.4Test Material 9General 9.1Stock Solution 9.2T
20、est 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 11.3Loading 11.4Beginning the Test 11.5Feeding 11.6Duration of
21、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 informationonly.1.9 This standard does not purport to ad
22、dress 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. Specific hazardstatements are given in Section 7.2. Re
23、ferenced Documents2.1 ASTM Standards:3E 724 Guide for Conducting Static Acute Toxicity TestsStarting with Embryos of Four Species of SaltwaterBivalve MolluscsE 943 Terminology Relating to Biological Effects and En-vironmental FateE 1023 Guide for Assessing the Hazard of a Material toAquatic Organism
24、s and Their UsesE 1191 Guide for Conducting Life-Cycle Toxicity Testswith Saltwater MysidsE 1192 Guide for Conducting Acute Toxicity Tests onAqueous Ambient Samples and Effluents with Fishes,Macroinvertebrates, and AmphibiansE 1203 Practice for Using Brine Shrimp Nauplii as Food forTest Animals in A
25、quatic ToxicologyE 1563 Guide for Conducting Static Acute Toxicity Testswith Echinoid Embryos,E 1604 Guide for Behavioral Testing inAquatic ToxicologyIEEE/ASTM SI 10 Standard for Use of the InternationalSystem of Units (SI) (the Modernized Metric System)3. Terminology3.1 Acute toxicity tests are gen
26、erally 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 detected for many species, the mostcommon acute toxicity test is t
27、he acute lethality test. Experi-mentally, effect on 50 % of a group of test organisms is themost reproducible and easily determined measure of toxicity,and 96 h is often a convenient, useful exposure duration.Therefore, the measure of acute toxicity most often used withfishes, macroinvertebrates, an
28、d amphibians is the 96-h LC50.However, because immobilization is a severe effect and is noteasy to distinguish from death for some species, the measure ofacute toxicity most often used with daphnids and midge larvaeis the 48-h EC50 based on death plus immobilization. Theterms LC50 and EC50 are consi
29、stent with the widely usedtoxicological terms LD50 (median lethal dose) and ED50(median effective dose), respectively. The terms LC50 andEC50 should be used whenever results are calculated based onthe concentration of test material in dilution water, whereas theterms LD50 and ED50 should be used whe
30、never 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 with poorly water-soluble materials, theterms LL50 (median lethal l
31、evel) 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 percentage of the individual organisms that wereaffected, the term
32、 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 least four techniques:3.2.1 In the static technique, test solutions a
33、nd 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 throughan apparatus designed to maintain water quality, and possibly3For referenced ASTM standards, vi
34、sit 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.E 729 96 (2007)2remove degraded, but not undegraded, test material by suchmeans as aeration, fi
35、ltration, and sterilization and then returnedto the test chamber.3.2.3 The renewal technique is like the static techniqueexcept that test organisms are periodically exposed to fresh testsolution of the same composition, usually once every 24 h,either by transferring the organisms from one test chamb
36、er 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-through 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 beginningo
37、f the test, and these solutions flow through the respectivechambers. In the second and more common procedure, freshtest solutions are prepared continuously or every few minutesjust before they enter the respective test chambers. In bothprocedures a metering system controls the flow of test solution,
38、and in the latter procedure the test solutions are prepared by themetering system. Both of the procedures may be used toconduct continuous-flow tests. Many tests conducted using thesecond procedure, however, are intermittent-flow tests becausethe metering system cycles and delivers test solution eve
39、ry 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, 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
40、 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” refers to chlorpyrifos, demeton, diazinon, disulfo-ton, fenitrothion, malathion, methyl parathion, and parathion;the term “organochlorine pesticide
41、s” refers to aldrin, chlor-dane, DDD, DDE, DDT, dieldrin, endosulfan, endrin, hep-tachlor, heptachlor epoxide, lindane, methoxychlor, mirex, andtoxaphene; and 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
42、 chlorine” refers tochlorine that would be detected if, when samples are preparedfor gas chromatographic analysis for polychlorinated biphenyls(PCBs) and the organochlorine pesticides listed above, achloride detector is used instead of an electron capture detectorto measure compounds that elute from
43、 just before lindane tojust after mirex on the gas chromatograph being used. Organicchlorine does not refer only to chlorine associated withorganochlorine pesticides and PCBs; it refers to all chlorinethat elutes within the specified period.3.5 reconstituted watera dilution water that is prepared by
44、adding sea salt or appropriate amounts of selected chemicals towater, which is usually prepared using deionization, distilla-tion, or reverse osmosis, so that the concentrations and ratios ofthe major ions in the dilution water are similar to those incomparable natural surface waters.3.6 The words “
45、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 the testought to be designed to satisfy the specified condition, unlessthe purpose of the test requires a different design. “Must” isonly used
46、 in connection with factors that directly relate to theacceptability of the test (see 13.1). “Should” is used to statethat the specified condition is recommended and ought to bemet if possible. Although violation of one “should” is rarely aserious matter, violation of several will often render the r
47、esultsquestionable. Terms such as “is desirable,” “is often desirable,”and “might be desirable” are used in connection with lessimportant factors. “May” is used to mean “is (are) allowedto,”“ can” is used to mean “is (are) able to,” and “might” isused to mean “could possibly.” Thus the classic disti
48、nctionbetween “may” and “can” is preserved, and “might” is neverused as a synonym for either “may” or “can.”3.7 IC50a statistically or graphically estimated concen-tration of test material that is expected to cause a 50 %inhibition of one or more specified biological processes (suchas growth or repr
49、oduction), for which the data are not dichoto-mous, under specified conditions.3.8 For definitions of other terms used in this guide, refer toTerminology E 943 and Guide E 1023. 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, theorganisms are maintained in dilution water to which no testmaterial has been added in order to provide (1) a measure ofthe acceptability of
