1、Designation: E 2591 07Standard Guide forConducting Whole Sediment Toxicity Tests withAmphibians1This standard is issued under the fixed designation E 2591; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the year of last revision.
2、A number in parentheses indicates the year of last reapproval. Asuperscript epsilon (e) indicates an editorial change since the last revision or reapproval.1. Scope1.1 This standard covers procedures for obtaining labora-tory data concerning the toxicity of test material (for example,sediment or hyd
3、ric soil (that is, a soil that is saturated, flooded,or ponded long enough during the growing season to developanaerobic (oxygen-lacking) conditions that favor the growthand regeneration of hydrophytic vegetation) to amphibians.This test procedure uses larvae of the northern leopard frog(Rana pipien
4、s). Other anuran species (for example, the greenfrog (Rana clamitans), the wood frog (Rana sylvatica), theAmerican toad (Bufo americanus) may be used if sufficientdata on handling, feeding, and sensitivity are available. Testmaterial may be sediments or hydric soil collected from thefield or spiked
5、with compounds in the laboratory.1.2 The test procedure describes a 10-d whole sedimenttoxicity test with an assessment of mortality and selectedsublethal endpoints (that is, body width, body length). Thetoxicity tests are conducted in 300 to 500-mL chamberscontaining 100 mLof sediment and 175 mLof
6、overlying water.Overlying water is renewed daily and larval amphibians are fedduring the toxicity test once they reach Gosner stage 25(operculum closure over gills). The test procedure is designedto assess freshwater sediments, however, R. pipiens can toler-ate mildly saline water (not exceeding abo
7、ut 2500 mg Cl-/L,equivalent to a salinity of about 4.1 when Na+is the cation) in10-d tests, although such tests should always include a con-current freshwater control. Alternative test durations and sub-lethal endpoints may be considered based on site-specificneeds. Statistical evaluations are condu
8、cted to determinewhether test materials are significantly more toxic than thelaboratory control sediment or a field-collected referencesample(s).1.3 Where appropriate, this standard has been designed tobe consistent with previously developed methods for assessingsediment toxicity to invertebrates (f
9、or example, Hyalella az-teca and Chironomus dilutus toxicity tests) described in theUnited States Environmental ProtectionAgency (USEPA, (1)2freshwater sediment testing guidance, Test Methods E 1367and E 1706, and Guides E 1391, E 1525, E 1611, and E 1688.Tests extending to 10 d or beyond, and inclu
10、ding sublethalmeasurements such as growth, are considered more effective inidentifying chronic toxicity and thus delineating areas ofmoderate contamination (1-3).1.4 Many historical amphibian studies, both water andsediment exposure, have used tests of shorter duration (5 daysor less) (for example,
11、4-7) and, although both survival andsublethal endpoints were often assessed, there is substantiveevidence that tests of longer duration are likely to be moresensitive to some contaminants (8, 9). Research performed todevelop and validate this test protocol included long-term(through metamorphosis) i
12、nvestigations and other researchershave also conducted long-duration tests with anurans (7-11).Inthe development of these procedures, an attempt was made tobalance the needs of a practical assessment with the importanceof assessing longer-term effects so that the results will demon-strate the needed
13、 accuracy and precision. The most recentsediment toxicity testing protocols for invertebrates haveencompassed longer duration studies which allow the measure-ment of reproductive endpoints (1, 12). Such tests, because ofincreased sensitivity of the sublethal endpoints, may also behelpful in evaluati
14、ng toxicity. Full life-cycle studies withanurans (including reproduction) are usually not feasible fromeither a technical or monetary standpoint. However, if site-specific information indicates that the contaminants present arelikely to affect other endpoints (including teratogenicity), thenthe dura
15、tion of the toxicity test may be increased throughmetamorphosis or additional sublethal endpoints may be mea-sured (for example, impaired behavior, deformities, time-to-metamorphosis). The possible inclusion of these endpoints andextension of test length should be considered during develop-ment of t
16、he project or study plan (see 8.1.1).1.5 The methodology presented in this standard was devel-oped under a Department of Defense (DoD) research programand presented in a guidance manual for risk assessment staff1This guide is under the jurisdiction of ASTM Committee E47 on BiologicalEffects and Envi
17、ronmental Fate and is the direct responsibility of SubcommitteeE47.03 on Sediment Assessment and Toxicology.Current edition approved Nov. 15, 2007. Published December 2007.2The boldface numbers in parentheses refer to the list of references at the end ofthis standard.1Copyright ASTM International, 1
18、00 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.and state/federal regulators involved in the review and ap-proval of risk assessment work plans and reports (13).Todevelop this method, a number of tests with spiked sedimenttests were conducted (13, 14). Since develo
19、pment of themethodology it has been used operationally to evaluate field-collected sediments from several state and federal environmen-tal sites (15, 16). For most of these studies the preferred testorganisms, Rana pipiens, was used. At a lead-contaminatedstate-led site, operated by the Massachusett
20、s Highway Depart-ment, Xenopus laevis (African clawed frog) was used in thesediment test system because of availability problems withRana pipiens (17), The test method was also used to evaluatesediment toxicity at a cadmium-contaminated USEPA Region4-led site in Tennessee (18). The methodology was u
21、sed to helpcharacterize potential effects of contaminants on amphibiansand to help develop preliminary remedial goals, if warranted.All tests evaluated survival and growth effects after 10 d ofexposure in accordance with the methods presented in thisstandard.1.6 The use of larval amphibians to asses
22、s environmentaltoxicity is not novel. Researchers have used tadpoles toexamine toxicity of metals and organic compounds. Most ofthese studies have been through water exposure, usually in amanner similar to fish or invertebrate exposure as described inGuide E 729 (19-29). Fewer studies have focused o
23、n exposureof anuran larvae to sediments, and the methods employed varywidely, from in situ enclosures (30) to laboratory tests usingvariable exposure conditions and organism ages (4, 8, 31-33).No studies were identified that used the same test conditions asdescribed in this standard. However, severa
24、l laboratory-basedevaluations of sediment effects on amphibians are described inthe following subsections.1.6.1 Sediment toxicity tests conducted in the laboratorywith amphibians were performed over a range of test durationsfrom4d(4, 31, Guide E 1439-98 Appendix X2) to 12 d (33)and through metamorph
25、osis (8, 32). Sediment toxicity testswith anurans native to North America were started with larvaltadpoles between Gosner stages 23 and 25 (8, 32, 33). Testtemperatures were between 21 and 23C and feeding beganafter tadpoles reached Gosner stage 25. Food sources wereTetraminy (8), boiled romaine let
26、tuce (32), or boiled romainelettuce and dissipated rabbit food pellets (33). Tests wereconducted in static renewal mode with water replacementsconducted at varying rates (daily (31, 33), weekly (8), every 3to5d(32). Test design (number of replicates, test vessel size,number of organisms per replicat
27、e) varied depending on theobjective of the study with several tests conducted in aquaria(32), large bins (8), or swimming pools (33). Endpointsevaluated at test termination included survival (4, 8, 31-33),growth (8, 31-33), bioaccumulation of metals (8), developmen-tal rates (8, 32), deformities (31
28、, 32), swimming speed (33) andforaging activity levels (32).1.6.2 To assess the effect of direct contact with the sedi-ments containing PCBs, Savage et al. (32) exposed larvaltadpoles (Gosner stage 23 to 25; wood frogs (R. sylvatica) tofield-collected sediments under conditions that allowed bothdire
29、ct contact with the sediment and separation from thesediment with a 500 m mesh barrier. The study found thatlethal and sublethal effects on tadpoles observed throughmetamorphosis were more pronounced when direct contactwith the sediment was allowed. The test conditions describedin this standard allo
30、w tadpoles to maintain direct contact withthe sediment.1.6.3 Sediment toxicity testing with Xenopus laevis hasfocused on evaluating the developmental effects of sedimentextracts, as opposed to whole sediments, on frog embryos.Methods have been developed which expose blastula stageembryos to sediment
31、 by enclosing the embryos in a Teflonmesh insert that rests over the top of the sediment in thesedimentwater interface region (31, Guide E 1439-98 Appen-dix X2). These studies are conducted evaluate survival,growth, and physical malformations of the embryos after a 4-dexposure period. The test condi
32、tions described in this standardallow more direct contact with the sediment, using older testorganisms, and a longer exposure duration.1.7 Sediment toxicity tests are an effective means forevaluating the impact of sediment contamination on amphib-ians in a multiple lines of evidence paradigm. The ev
33、aluation ismost powerful when toxicity testing sampling stations areco-located with sediment analytical chemistry samples andecological surveys, allowing for a detailed evaluation of theco-occurring data in the ecological risk assessment. The spatialand temporal co-location of toxicity testing and a
34、nalyticalsamples is particularly important for establishing contaminant-specific effects and assessing contaminant bioavailability.1.8 In order for a sediment toxicity test to be sensitive itmust be of sufficient duration to measure potential toxicity andit must be conducted during the appropriate d
35、evelopmentalstage of the test organisms life cycle. Using recently hatchedtadpoles and conducting the sediment exposure test for 10 d toallow the evaluation of growth endpoints meets both of thesesensitivity requirements.1.9 This standard does not purport to address all of thesafety concerns, if any
36、, 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.2. Referenced Documents2.1 ASTM Standards:3D 4447 Guide for Disposal of Laboratory Chemicals a
37、ndSamplesE 177 Practice for Use of the Terms Precision and Bias inASTM Test MethodsE 691 Practice for Conducting an Interlaboratory Study toDetermine the Precision of a Test MethodE 729 Guide for Conducting Acute Toxicity Tests on TestMaterials with Fishes, Macroinvertebrates, and Amphib-iansE 943 T
38、erminology Relating to Biological Effects and En-vironmental Fate3For 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 webs
39、ite.E2591072E 1367 Test Method for Measuring the Toxicity ofSediment-Associated Contaminants with Estuarine andMarine InvertebratesE 1391 Guide for Collection, Storage, Characterization, andManipulation of Sediments for Toxicological Testing andfor Selection of Samplers Used to Collect Benthic Inver
40、-tebratesE 1439 Guide for Conducting the Frog Embryo Teratogen-esis Assay-Xenopus (FETAX)E 1525 Guide for Designing Biological Tests with Sedi-mentsE 1611 Guide for Conducting Sediment Toxicity Tests withPolychaetous AnnelidsE 1688 Guide for Determination of the Bioaccumulation ofSediment-Associated
41、 Contaminants by Benthic Inverte-bratesE 1706 Test Method for Measuring the Toxicity ofSediment-Associated Contaminants with Freshwater Inver-tebrates3. Terminology3.1 The words “must”, “should”, “may”, “can” and “might”have very specific meanings in this guide. “Must” is used toexpress an absolute
42、requirement, that is, to state that the designof a test ought to be in a manner that satisfies the specifiedconditions, unless project goals dictate needed alterations inorder to address the study hypotheses. “Should” is used to statethat the specified condition is recommended and ought to bemet if
43、possible.Although the violation of one “should” is rarelya serious matter, violation of several could render the resultsquestionable. Terms such as “is desirable”, “is often desirable”and “might be desirable” are used in association with lessimportant factors, the alteration of which will probably n
44、othave substantive effects on test outcome. “May” means “is(are) allowed to,” “can” means “is (are) able to” and “might”means “could possibly.” In this manner, the classic distinctionbetween “may” and “can” is preserved and “might” is neverused as a synonym for either “may” or “can.”3.2 DefinitionsF
45、or definitions of general terms related totoxicity testing and used in this guide, refer to Guide E 943.3.3 Definitions of Terms Specific to This Standard:3.3.1 IC25 (25 % inhibition concentration),nconcentration at which there is a 25 % reduction in organ-ism performance, relative to the control. P
46、erformance may besurvival or a sublethal measurement such as growth.3.3.2 overlying water, nwater that is placed over thesediment for the duration of the study. Overlying water may besurface water collected from the project site or from a cleanlake or reservoir, or may be reconstituted water prepare
47、d in thelaboratory (for example, moderately hard water; (34).3.3.3 reference-toxicant test, na test conducted with areagent-grade reference chemical to assess the sensitivity of thetest organisms. Deviations outside an established normal rangemay indicate a change in the sensitivity of the test orga
48、nismpopulation. Reference-toxicity tests are most often performedin the absence of sediment.3.3.4 test sediment or test material, nsediment that maycontain contaminants, which is being evaluated using this testprocedure.4. Summary of Guide4.1 Each test consists of eight replicates of the test materi
49、al(for example, field-collected sediment or spiked sediment) andoverlying water with five test organisms (recently-hatchedtadpoles) per replicate. A laboratory control sediment (some-times called a negative control) is used to provide (1) ameasure of the acceptability of the test by indicating the qualityof tadpoles, test conditions and handling procedures, and (2) abasis for interpreting data from other treatments. The testduration is ten days with an assessment of mortality andselected sublethal endpoints (that is, body width, body length)at the end of the test. Assessm
