1、Designation: D 6081 98 (Reapproved 2004)An American National StandardStandard Practice forAquatic Toxicity Testing of Lubricants: Sample Preparationand Results Interpretation1This standard is issued under the fixed designation D 6081; the number immediately following the designation indicates the ye
2、ar 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 practice covers procedures to be used in theprep
3、aration of lubricants or their components for toxicitytesting in aquatic systems and in the interpretation of the resultsof such tests.1.2 This practice is suitable for use on fully-formulatedlubricants or their components that are not completely solubleat the intended test treat rates. It is also s
4、uitable for use withadditives, if the additive is tested after being blended into acarrier fluid at the approximate concentration as in the intendedfully formulated lubricant. The carrier fluid shall meet theabove solubility criterion, be known to be minimally toxic inthe toxicity test in which the
5、material will be tested, and beknown to have a chemical composition similar to the rest of theintended fully formulated lubricant.1.3 Samples prepared in accordance with this practice maybe used in acute or chronic aquatic toxicity tests conducted infresh water or salt water with fish, large inverte
6、brates, or algae.This practice does not address preparation of samples for planttoxicity testing other than algae.1.4 Standard acute and chronic aquatic toxicity proceduresare more appropriate for lubricants with compositions that arecompletely soluble at the intended test treat rates (1, 2, 3, 4, 5
7、).21.5 This practice is intended for use with lubricants orlubricant components of any volatility.1.6 This practice does not address any questions regardingthe effects of any lubricant or lubricant component on humanhealth.1.7 This standard does not purport to address all of thesafety concerns, if a
8、ny, 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 1129 Terminology Relating to WaterD 1193 Specif
9、ication for Reagent WaterE 729 Guide for Conducting Acute Toxicity Tests on TestMaterials with Fishes, Macroinvertebrates, and Amphib-iansE 943 Terminology Relating to Biological Effects and En-vironmental Fate3. Terminology3.1 Definitions:3.1.1 acute toxicity test, na comparative toxicity test inwh
10、ich a representative subpopulation of organisms is exposedto different treat rates of a test material and is observed for ashort period, usually not constituting a substantial portion oftheir life span.3.1.2 chronic toxicity test, na comparative toxicity test inwhich a representative subpopulation o
11、f organisms is exposedto different treat rates of a test material and is observed for aperiod of time that constitutes a major portion of their life span.3.1.3 ecotoxicity, npropensity of a test material to pro-duce adverse behavioral, biochemical, or physiological effectsin non-human organisms or p
12、opulations.3.1.4 effect load XX (ELXX), na statistically or graphi-cally estimated loading rate of test material that is expected tocause one or more specified effects in XX % of a representativesubpopulation of organisms under specified conditions.3.1.4.1 DiscussionThis terminology should be used i
13、n-stead of the standard ECXX when the test material is notcompletely soluble at the test treat rates.3.1.5 inhibition load XX (ILXX), na statistically or graphi-cally estimated loading rate of test material that is expected tocause a XX % inhibition of a biological process (such asgrowth or reproduc
14、tion) that has an analog as opposed to adigital measure.1This practice is under the jurisdiction of ASTM Committee D02 on PetroleumProducts and Lubricants and is the direct responsibility of Subcommittee D02.12 onEnvironmental Standards for Lubricants.Current edition approved June 1, 2004. Published
15、 June 2004. Originallyapproved in 1998. Last previous edition approved in 1998 as D 6081 - 98.2The boldface numbers in parentheses refer to the list of references at the end ofthis standard.3For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at servi
16、ceastm.org. For Annual Book of ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM website.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.3.1.5.1 DiscussionAn example of a digital measure wouldb
17、e alive/dead. This terminology (ILXX) should be used insteadof the standard ICXX when the test material is not completelysoluble at the test treat rates.3.1.6 lethal load XX (LLXX), na statistically or graphi-cally estimated loading rate of test material that is expected tobe lethal to XX % of a rep
18、resentative subpopulation of organ-isms under specified conditions.3.1.6.1 DiscussionThis terminology should be used in-stead of the standard LCXX when the material is not com-pletely soluble at the test treat rates.3.1.7 loading rate, nthe ratio of test material to aqueousmedium used in the prepara
19、tion of WAF, WSF, or mechanicaldispersion and in the interpretation of the results of a toxicitystudy with a poorly water-soluble lubricant or lubricant com-ponent.3.1.8 mechanical dispersion, na low energy aqueousmedium produced by continuous stirring of the test solutionand containing both dissolv
20、ed and undissolved components ofthe test material.3.1.9 toxicity, nthe propensity of a test material to pro-duce adverse behavioral, biochemical, or physiological effectsin a living organism.3.1.10 water accommodated fraction (WAF), nthe pre-dominantly aqueous portion of a mixture of water and amate
21、rial poorly soluble in water, which separates in a specifiedperiod of time after the mixture has undergone a specifieddegree of mixing and which includes water, dissolved compo-nents, and dispersed droplets of the poorly soluble material.3.1.10.1 DiscussionThe composition of the WAF dependson the ra
22、tio of poorly soluble material to water in the originalmixture as well as on the details of the mixing procedure.3.1.11 water soluble fraction (WSF), nthe filtrate orcentrifugate of the water accommodated fraction, which in-cludes all parts of the WAF, except the dispersed droplets of thepoorly solu
23、ble material.3.2 Definitions of Terms Specific to This Standard:3.2.1 dilution water, nthe water used in the aquatic testunder consideration, which may contain organic or inorganiccomponents depending on the test requirements.3.2.2 exposure matrix, nthe WAF, WSF, or mechanicaldispersion to which tes
24、t organisms are exposed for toxicitytesting.3.2.3 test systems, nthe test organisms plus the exposurematrix and the test chambers themselves.3.2.4 water, na reagent that is defined by Type II ofSpecification D 1193.3.3 Definitions of additional terms used in this practice canbe found in Terminologie
25、s D 1129 and E 943.4. Summary of Practice4.1 This practice discusses various techniques for the prepa-ration of test material for toxicity testing of poorly water-soluble lubricants or their components under acute or chronicexposure conditions with fish, large invertebrates, or algae.5. Significance
26、 and Use5.1 This practice gives techniques to use in the preparationof lubricants or lubricant components for acute or chronicaquatic toxicity tests. Most lubricants and lubricant compo-nents are difficult to evaluate in toxicity tests because they aremixtures of chemical compounds with varying and
27、usuallypoor solubility in water. Lubricants or lubricant componentmixtures should not be added directly to aquatic systems fortoxicity testing because the details of the addition procedurewill have a large effect on the results of the toxicity test. Useof the techniques described in this practice wi
28、ll producewell-characterized test systems that will lead to tests withmeaningful and reproducible results.5.2 The toxicity of mixtures of poorly soluble componentscannot be expressed in the usual terms of lethal concentration(or the similar terms of effect concentration or inhibitionconcentration) b
29、ecause the mixtures may not be completelysoluble at treat levels that lead to toxic effects. The test materialpreparation techniques given in this practice lead to test resultsexpressed in terms of loading rate, which is a practical andmeaningful concept for expressing the toxicity of this type ofma
30、terial.5.3 One of the recommended methods of material prepara-tion for lubricants or their components is the mechanicaldispersion technique. This particular technique generates tur-bulence, and thus, it should not be used for poorly swimmingorganisms.6. Preparation of Exposure Matrix6.1 Experimental
31、 design considerations for fish and inver-tebrates will determine the test species, number of loading ratestested, and number of organisms per treatment. Experimentaldesign considerations for algae will determine the test species,number of loading rates tested, and appropriate initial biomassper tre
32、atment.6.2 An acute or chronic test intended to allow calculation ofan LLXX, ILXX, or ELXX will typically consist of a dilutionwater or test medium control and a series of at least fiveloading rates of test material.6.3 Test material should be added directly to the dilutionwater to prepare the expos
33、ure matrix. Use of a solvent orchemical dispersant is not recommended because these mayalter the nature of the exposure to the test material and lead tonon-representative ecotoxicity relationships.6.4 Preparation of Water-Accommodated Fraction (WAF):6.4.1 Components dissolved in the water phase or e
34、ntrainedas stable droplets in the water phase are evaluated in a WAFstudy (6). Individual WAFs must be generated for each testexposure loading. Serial dilutions of a single WAF are notappropriate due to differential solubility of constituents at lowexposure loads (6, 7). A schematic of the WAF setup
35、 isprovided in Fig. 1.6.4.2 Vessels for WAF preparation should be of suitableshape and size for the experimental design. The vessels shouldbe of sufficient size to maintain the vortex intensity andoptimize surface area contact between the test material and thewater. Height to diameter ratio for the
36、WAF preparation atinitiation should be between 1:1 and 2:1, and solutions shouldnot exceed 20 Lper individual preparation. If larger volumes oftest solution are required for experimental design, separateWAF preparations of the same test load may be combined.Aspirator bottles or other vessels with ou
37、tlet ports at the bottomD 6081 98 (2004)2are preferable for WAF collection, if the test material is lessdense than water. If a glass siphon is used to remove the WAF,it should be in place prior to the introduction of the testmaterial. WAF solutions from test materials that are denserthan water shoul
38、d be decanted from the top of the vessel. Forthese dense fluids, a propeller-driven system that stirs the testsolution from the top of the vessel may be preferable to a stirplate that stirs the test solution from the bottom of the vessel.In all cases, care should be taken to ensure that non-disperse
39、dtest material is not collected with the WAF.6.4.3 Vessels for WAF preparation should be filled with theappropriate volume of dilution water, and be prepared forstirring as described in 6.4.2. Test material should be slowlyadded to the top of each vessel after dilution water addition.The vessel shou
40、ld be capped with foil or a non-reactivecovering and stirred at the test temperature. For test tempera-tures different than room temperature, the WAF preparationvessels may need to be housed in water baths or incubators tomaintain the desired temperature. If photodecomposition of thetest material is
41、 likely, the stirring phase should be conducted inthe dark. Preparation vessels should be inspected occasionallyto ensure an appropriate vortex is maintained.6.4.4 The amount of test material added to each vesselshould be determined by the desired nominal exposure load.Nominal exposure loads may be
42、based on direct weight ofmaterial added per volume solution (wt/vol) or calculated onthe basis of test material density and added on a vol/vol basis.6.4.5 The stir plates should be run at a sufficient speed toensure a vortex depth of 10 to 35 % of the test solution heightin the WAF preparation vesse
43、l. The vortex in all WAFpreparation vessels for any one experiment should be the same.Exercise care to ensure that test material is not pulled down tothe bottom of the vessel, and that the rate of stirring is not sovigorous as to promote emulsification.6.4.6 The duration of WAF preparation may depen
44、d on theresults of an optional equilibrium/stability study (see 6.7). InFIG. 1 Water Accommodated Fraction (WAF) SchematicD 6081 98 (2004)3the absence of this study, a standard 20 to 24-h mixing and 1to 4-h settling period should be used (6, 8).6.4.7 Upon completion of the WAF mixing and settlingper
45、iod, the aqueous solution should be drained or decantedfrom below the surface of each preparation vessel. The first 5to 10 mL of solution decanted from the vessel should bediscarded. The remaining solution should be used undiluted inthe exposure vessels.6.5 Preparation of Water-Soluble Fraction (WSF
46、):6.5.1 Only components dissolved in the water phase areevaluated in a WSF study (6). WSFs are prepared by filtrationof WAFs through a 0.45-m nominal cellulose-acetate filter orby centrifugation to remove undissolved material. Preliminarytesting may be necessary to ensure that the filter disc will n
47、otremove test material constituents that are dissolved in the waterphase. Individual WSFs must be generated for each testexposure load. Serial dilutions of a single WSF are notappropriate due to differential solubility of constituents at lowexposure loads. A schematic of the WSF setup is provided in
48、Fig. 2.6.5.2 The procedures described in 6.4.3-6.4.7 for the WAFpreparation are the initial steps of the WSF procedure.6.5.3 Upon completion of the WAF mixing and settlingperiod, the aqueous solution should be decanted from belowthe surface of each preparation vessel. The first 5 to 10 mL ofsolution
49、 decanted from the vessel should be discarded, with theremaining solution used to prepare the WSF. Filtration througha 0.45-m filter or low speed centrifugation (to removedroplets but not to break an emulsion) is performed on thesolution to remove undissolved material. The filtrate or cen-trifugation supernatant is the WSF exposure matrix and shouldbe used undiluted in the exposure vessels.6.6 Preparation of a Mechanical Dispersion:6.6.1 In the mechanical dispersion technique, a motor-driven propeller assembly is used to continually move testmaterial and dilution
