1、Designation: E 1676 04Standard Guide forConducting Laboratory Soil Toxicity or BioaccumulationTests with the Lumbricid Earthworm Eisenia Fetida and theEnchytraeid Potworm Enchytraeus albidus1This standard is issued under the fixed designation E 1676; the number immediately following the designation
2、indicates the year oforiginal adoption 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.1. Scope1.1 This guide covers procedures for obta
3、ining laboratorydata to evaluate the adverse effects of contaminants (forexample, chemicals or biomolecules) associated with soil toearthworms (Family Lumbricidae) and potworms (FamilyEnchytraeidae) from soil toxicity or bioaccumulation tests. Themethods are designed to assess lethal or sublethal to
4、xic effectson earthworms or bioaccumulation of contaminants in short-term tests (7 to 28 days) or on potworms in short to long-termtests (14 to 42 days) in terrestrial systems. Soils to be testedmay be (1) reference soils or potentially toxic site soils; (2)artificial, reference, or site soils spike
5、d with compounds; (3)site soils diluted with reference soils; or (4) site or referencesoils diluted with artificial soil. Test procedures are describedfor the species Eisenia fetida (see Annex A1) and for thespecies Enchytraeus albidus (see Annex A4). Methods de-scribed in this guide may also be use
6、ful for conducting soiltoxicity tests with other lumbricid and enchytraeid terrestrialspecies, although modifications may be necessary.1.2 Modification of these procedures might be justified byspecial needs. The results of tests conducted using atypicalprocedures may not be comparable to results usi
7、ng this guide.Comparison of results obtained using modified and unmodifiedversions of these procedures might provide useful informationconcerning new concepts and procedures for conducting soiltoxicity and bioaccumulation tests with terrestrial worms.1.3 The results from field-collected soils used i
8、n toxicitytests to determine a spatial or temporal distribution of soiltoxicity may be reported in terms of the biological effects onsurvival or sublethal endpoints (see Section 14). These proce-dures can be used with appropriate modifications to conductsoil toxicity tests when factors such as tempe
9、rature, pH, andsoil characteristics (for example, particle size, organic mattercontent, and clay content) are of interest or when there is aneed to test such materials as sewage sludge and oils. Thesemethods might also be useful for conducting bioaccumulationtests.1.4 The results of toxicity tests w
10、ith (1) materials (forexample, chemicals or waste mixtures) added experimentallyto artificial soil, reference soils, or site soils, (2) site soilsdiluted with reference soils, and (3) site or reference soilsdiluted with artificial soil, so as to create a series of concen-trations, may be reported in
11、 terms of an LC50 (median lethalconcentration) and sometimes an EC50 (median effect concen-tration). Test results may be reported in terms of NOEC (noobserved effect concentration), LOEC (lowest observed effectconcentration) or as an ECx (concentration where x % reduc-tion of a biological effect occ
12、urs. Bioaccumulation test resultsare reported as the magnitude of contaminant concentrationabove either the Day 0 tissue baseline analysis or the Day 28tissues from the negative control or reference soil (that is, 23,53,103) (see A3.9).1.5 This guide is arranged as follows:Scope 1Referenced Document
13、s 2Terminology 3Summary of Guide 4Significance and Use 5Interferences 6Apparatus 7Safety Precautions 8Soil 9Test Organism 10Procedure 11Analytical Methodology 12Acceptability of Test 13Calculation of Results 14Report 15AnnexesAnnex A1. Eisenia fetidaAnnex A2. Artificial Soil CompositionAnnex A3. Bio
14、accumulation Testing Utilizing Eisenia fetidaAnnex A4. Enchytraeid Reporduction Test (ERT)References1This guide is under the jurisdiction of ASTM Committee E47 on BiologicalEffects and Environmental Fate and is the direct responsibility of SubcommitteeE47.02 on Terrestrial Assessment and Toxicology.
15、An ASTM guide is defined as a series of options or instructions that do notrecommend a specific course of action.Current edition approved April 1, 2004. Published May 2004. Originallyapproved in 1995. Last previous edition approved in 1997 as E 1676 97.1Copyright ASTM International, 100 Barr Harbor
16、Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.1.6 The values stated in SI units are to be regarded as thestandard.1.7 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 es
17、tablish appro-priate safety and health practices and determine the applica-bility of regulatory limitations prior to use. While some safetyconsiderations are included in this guide, it is beyond the scopeof this standard to encompass all safety requirements necessaryto conduct soil toxicity tests. S
18、pecific precautionary statementsare given in Section 8.2. Referenced Documents2.1 ASTM Standards:2D 653 Terminology Relating to Soil, Rock, and ContainedFluidD 4447 Guide for the Disposal of Laboratory Chemicalsand SamplesE 380 Practice for Use of the International System of Units(SI) (the Modernize
19、d Metric System)E 943 Terminology Relating to Biological Effects and En-vironmental FateE 1023 Guide for Assessing the Hazard of a Material toAquatic Organisms and Their UsesE 1383 Guide for Conducting Sediment Toxicity Tests withFreshwater InvertebratesE 1688 Guide for Determination of the Bioaccum
20、ulation ofSediment-Associated Contaminants by Benthic Inverte-bratesE 1706 Test Methods for Measuring the Toxicity ofSediment-Associated Contaminants with Fresh Water In-vertebrates3. Terminology3.1 Definitions:3.1.1 The words “must,” “should,” “may,”“ can,” and“might” have very specific meanings in
21、 this guide. “Must” isused to express an absolute requirement, that is, to state that thetest must be designed to satisfy the specified condition, unlessthe purpose of the test requires a different design. “Must” isused only in connection with the factors that relate directly tothe acceptability of
22、the test (see Section 13). “Should” is usedto state that the specified condition is recommended and oughtto be met if possible. Although a violation of one “should” israrely a serious matter, the violation of several will often renderthe results questionable. Terms such as “is desirable,” “is oftend
23、esirable,” and “might be desirable” are used in connectionwith less important factors. “May” is used to mean “is (are)allowed to,” “can” is used to mean “is (are) able to,” and“might” is used to mean “could possibly.” Thus, the classicdistinction between “may” and “can” is preserved, and “might”is n
24、ever used as a synonym for either “may” or “can.”3.1.2 For definitions of terms used in this guide, refer toTerminology E 943 and Guide E 1023. For an explanation ofunits and symbols, refer to Practice E 380.3.2 Definitions of Terms Specific to This Standard:3.2.1 artificial soila synthetic soil, pr
25、epared with a spe-cific formulation, designed to simulate a natural soil (seeAnnex A2). Artificial soil may be used as a diluent medium toprepare concentrations of site or reference soil and may be usedas a negative control medium.3.2.2 batchthe total amount of test soil prepared for eachconcentrati
26、on in a test. A batch is any hydrated test soil readyfor separation into replicates.3.2.3 bioaccumulationthe net accumulation of a sub-stance by an organism as a result of uptake from all environ-mental sources. (See Guide E 1688.)3.2.4 bioaccumulation factor (BAF)the ratio of tissueresidue to sedim
27、ent contaminant concentration at steady-state.(See Guide E 1688.)3.2.5 bioaccumulation potentiala qualitative assessmentof whether a contaminant in a particular sediment is bioavail-able. (See Guide E 1688.)3.2.6 bioconcentrationthe net assimilation of a substanceby an organism as a result of uptake
28、 directly from aqueoussolution. (See Guide E 1688.)3.2.7 bioconcentration factor (BCF)the ratio of tissueresidue to water contaminant concentration as steady-state.(See Guide E 1688.)3.2.8 biota-sediment accumulation factor (BSAF)the ratioof lipid-normalized tissue residue to organic carbon-normaliz
29、ed sediment contaminant concentration at steady state,with units of g-carbon/g-lipid. (See Guide E 1688.)3.2.9 clitellumthe fleshy “ring” or “saddle” of glandulartissue found on certain mid-body segments of oligochaete(Lumbricidae and Enchytraeidae) worms. It is the most visiblefeature of an adult e
30、arthworm or potworm and secretes thecocoon into which eggs and sperm are deposited.3.2.10 concentrationthe ratio of the weight of test mate-rials to the weight of soil (artificial, reference, or site), usuallyexpressed on a dry weight basis as percent or milligram/kilogram.3.2.11 depurationloss of a
31、 substance from an organism asa result of any active (for example, metabolic breakdown) orpassive process.3.2.12 diluent soilthe artificial or reference soil used todilute site soils.3.2.13 enchytraeidpotworm members of the FamilyEnchytraeidae of the Class Oligochaeta of the Phylum Anne-lida.3.2.14
32、hydration waterwater used to hydrate test soils tocreate an environment with a moisture level suitable for thespecies being tested. The water used for hydration is often testwater (see 3.2.27); however, depending on the nature of the testbeing implemented, site surface water or groundwater may alsob
33、e utilized for hydration.3.2.15 lumbricidearthworm members of the Family Lu-bricidae of the Class Oligochaeta of the Phylum Annelida.3.2.16 negative control soilartificial or reference soil tobe used for evaluating the acceptability of a test.2For referenced ASTM standards, visit the ASTM website, w
34、ww.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.E16760423.2.17 reference soila field-collected soil that has physi-cochemical and biological properties as similar as
35、possible tothe site soil but does not contain the potentially toxic com-pounds of the site soil. It is used to describe matrix effects onthe test in question. It may be used as a diluent medium toprepare concentrations of site soil and may be used as anegative control medium.3.2.18 sampling stationa
36、 specific location, within a site orsampling unit, depending on the field study design, at whichsoil is collected for chemical, physical, and biological evalua-tion.3.2.19 sampling unitan area of land within a site distin-guished by habitat and topography.3.2.20 sitea delineated tract of land that i
37、s being consid-ered as a study area, usually from the standpoint of its beingpotentially affected by xenobiotics.3.2.21 site soila soil collected from the field to beevaluated for potential toxicity. A site soil may be a naturallyoccurring soil or one that has been influenced by xenobiotics.3.2.22 s
38、oilsediments or other unconsolidated accumula-tions of solid particles produced by the physical and chemicaldisintegration of rocks, and that may or may not containorganic material. (See Terminology D 653.)3.2.23 spikingthe experimental addition of a test materialto an artificial, site, or reference
39、 soil, such that the toxicity ofthe material added can be determined. After the test material isadded, which may involve a solvent carrier, the soil is mixedthoroughly to distribute the test material evenly throughout thesoil.3.2.24 test chamberan enclosed space or compartment inwhich environmental
40、parameters such as temperature andlighting are controlled (for example, incubator or modifiedroom). Test containers are placed in the test chamber forbiological evaluation.3.2.25 test containerthe experimental unit; the smallestphysical entity to which treatments can be assigned indepen-dently.3.2.2
41、6 test soila soil prepared to receive a test organism.Site or reference soil mixed with artificial soil or reference soilmixed with site soil in known concentrations for evaluation aretest soils. Artificial, site, or reference soils spiked with testmaterials such as chemicals, oils, or manufacturing
42、 productsare test soils. Once a site, reference, or artificial soil ishydrated, even though it is not mixed with artificial orreference soil or spiked with a material, it may be called a testsoil.3.2.27 test waterwater used to prepare stock solutions,rinse test organisms, rinse glassware, and appara
43、tus or for anyother purpose associated with the test procedures or culture ofthe test organism. Test water must be deionized or distilledwater or better, such as reagent-grade water produced by asystem of reverse osmosis, carbon, and ion-exchange car-tridges.4. Summary of Guide4.1 The toxicity of te
44、st soils or the bioavailability ofcontaminants are assessed during the continuous exposure ofterrestrial organisms. Soils tested may be the following: (1)soils collected from potentially contaminated sites, (2) soilscollected from reference sites, (3) artificial soil (see Annex A2)spiked with compou
45、nds, (4) site soil spiked with compounds,(5) reference soil spiked with compounds, (6) site soil dilutedwith artificial soil, (7) site soil diluted with reference soil, or (8)reference soil diluted with artificial soil. A negative control ofartificial or reference soil is used for the following: (1)
46、 to yielda measure of the acceptability of the test; (2) to provideevidence of the health and relative quality of the test organ-isms; (3) to determine the suitability of test conditions, food,and handling procedures; and (4) to provide a basis forinterpreting data obtained from the test soils. Spec
47、ified data areobtained to determine the toxic effects on survival or sublethalendpoints for 7 to 28-day exposures or containment bioaccu-mulation for 28-day exposures to terrestrial lumbricids and thetoxic effects on survival or sublethal endpoints for 4 to 42-dayexposures to enchytraeids.4.2 Summar
48、y of ChangesThis current version of thestandard is a revision of the E 1676-97 version. Changes madesince 1997 involve toxicity testing procedures for theEnchytraied potworm, Enchytraeus albidus. There has been anadditional annex added (Annex A4) and the main document hasbeen modified to include thi
49、s species.5. Significance and Use5.1 Soil toxicity tests provide information concerning thetoxicity and bioavailability of chemicals associated with soilsto terrestrial organisms. As important members of the soilfauna, lumbricid earthworms and enchytraeid potworms have anumber of characteristics that make them appropriate organ-isms for use in the assessment of potentially hazardous soils.Earthworms may ingest large quantities of soil, have a closerelationship with other soil biomasses (for example, inverte-brates, roots, humus, litter, and microorganisms),
