1、Designation: D6081 98 (Reapproved 2014)Standard Practice forAquatic Toxicity Testing of Lubricants: Sample Preparationand Results Interpretation1This standard is issued under the fixed designation D6081; the number immediately following the designation indicates the year oforiginal adoption or, in t
2、he 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.1. Scope1.1 This practice covers procedures to be used in thepreparation of lubricants or their c
3、omponents 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 suitable for use withadditives, i
4、f 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 material will be tested, and bek
5、nown 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 invertebrates, or algae.This practice d
6、oes 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).21.5 This practice is intended
7、 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 The values stated in SI units are to be regarded asstandard. No other units of measurement are included i
8、n thisstandard.1.8 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.
9、2. Referenced Documents2.1 ASTM Standards:3D1129 Terminology Relating to WaterD1193 Specification for Reagent WaterE729 Guide for Conducting Acute Toxicity Tests on TestMaterials with Fishes, Macroinvertebrates, and Amphib-iansE943 Terminology Relating to Biological Effects and Envi-ronmental Fate3.
10、 Terminology3.1 Definitions:3.1.1 acute toxicity test, na comparative toxicity test inwhich 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 chro
11、nic toxicity test, na comparative toxicity test inwhich a representative subpopulation of 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 produ
12、ceadverse behavioral, biochemical, or physiological effects innon-human organisms or populations.3.1.4 effect load XX (ELXX), na statistically or graphicallyestimated loading rate of test material that is expected to causeone or more specified effects in XX % of a representativesubpopulation of orga
13、nisms under specified conditions.3.1.4.1 DiscussionThis terminology should be used in-stead of the standard ECXX when the test material is notcompletely soluble at the test treat rates.1This practice is under the jurisdiction of ASTM Committee D02 on PetroleumProducts, Liquid Fuels, and Lubricants a
14、nd is the direct responsibility of Subcom-mittee D02.12 on Environmental Standards for Lubricants.Current edition approved Oct. 1, 2014. Published November 2014. Originallyapproved in 1998. Last previous edition approved in 2009 as D608198(2009).DOI: 10.1520/D6081-98R14.2The boldface numbers in pare
15、ntheses 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 serviceastm.org. For Annual Book of ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM we
16、bsite.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States13.1.5 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 a
17、sgrowth or reproduction) that has an analog as opposed to adigital measure.3.1.5.1 DiscussionAn example of a digital measure wouldbe 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 loa
18、d XX (LLXX), na statistically or graphi-cally estimated loading rate of test material that is expected tobe lethal to XX % of a representative 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 n
19、ot com-pletely soluble at the test treat rates.3.1.7 loading rate, nthe ratio of test material to aqueousmedium used in the preparation 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
20、 mechanical dispersion, na low energy aqueous me-dium produced by continuous stirring of the test solution andcontaining both dissolved and undissolved components of thetest material.3.1.9 toxicity, nthe propensity of a test material to produceadverse behavioral, biochemical, or physiological effect
21、s in aliving organism.3.1.10 water accommodated fraction (WAF), nthe pre-dominantly aqueous portion of a mixture of water and amaterial poorly soluble in water, which separates in a specifiedperiod of time after the mixture has undergone a specifieddegree of mixing and which includes water, dissolve
22、dcomponents, and dispersed droplets of the poorly solublematerial.3.1.10.1 DiscussionThe composition of the WAF dependson the ratio 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 or cen-t
23、rifugate of the water accommodated fraction, which includesall parts of the WAF, except the dispersed droplets of the poorlysoluble 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 i
24、norganiccomponents depending on the test requirements.3.2.2 exposure matrix, nthe WAF, WSF, or mechanicaldispersion to which test 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
25、 defined by Type II ofSpecification D1193.3.3 Definitions of additional terms used in this practice canbe found in Terminologies D1129 and E943.4. Summary of Practice4.1 This practice discusses various techniques for the prepa-ration of test material for toxicity testing of poorly water-soluble lubr
26、icants or their components under acute or chronicexposure conditions with fish, large invertebrates, or algae.5. Significance 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 lubri
27、cant compo-nents are difficult to evaluate in toxicity tests because they aremixtures of chemical compounds with varying and usuallypoor solubility in water. Lubricants or lubricant componentmixtures should not be added directly to aquatic systems fortoxicity testing because the details of the addit
28、ion procedurewill have a large effect on the results of the toxicity test. Useof the techniques described in this practice will producewell-characterized test systems that will lead to tests withmeaningful and reproducible results.5.2 The toxicity of mixtures of poorly soluble componentscannot be ex
29、pressed in the usual terms of lethal concentration(or the similar terms of effect concentration or inhibitionconcentration) because 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 result
30、sexpressed in terms of loading rate, which is a practical andmeaningful concept for expressing the toxicity of this type ofmaterial.5.3 One of the recommended methods of material prepara-tion for lubricants or their components is the mechanicaldispersion technique. This particular technique generate
31、sturbulence, and thus, it should not be used for poorly swim-ming organisms.6. Preparation of Exposure Matrix6.1 Experimental design considerations for fish and inver-tebrates will determine the test species, number of loading ratestested, and number of organisms per treatment. Experimentaldesign co
32、nsiderations for algae will determine the test species,number of loading rates tested, and appropriate initial biomassper treatment.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
33、least fiveloading rates of test material.6.3 Test material should be added directly to the dilutionwater to prepare the exposure 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 ecot
34、oxicity relationships.6.4 Preparation of Water-Accommodated Fraction (WAF):6.4.1 Components dissolved in the water phase or entrainedas 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 WA
35、F are notD6081 98 (2014)2appropriate due to differential solubility of constituents at lowexposure loads (6, 7). A schematic of the WAF setup 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 siz
36、e to maintain the vortex intensity andoptimize surface area contact between the test material and thewater. Height to diameter ratio for the WAF preparation atinitiation should be between 1:1 and 2:1, and solutions shouldnot exceed 20 Lper individual preparation. If larger volumes oftest solution ar
37、e required for experimental design, separateWAF preparations of the same test load may be combined.Aspirator bottles or other vessels with outlet ports at the bottomare preferable for WAF collection, if the test material is lessdense than water. If a glass siphon is used to remove the WAF,it should
38、be in place prior to the introduction of the testmaterial. WAF solutions from test materials that are denserthan water should 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 stirpla
39、te that stirs the test solution from the bottom of the vessel.In all cases, care should be taken to ensure that non-dispersedtest 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 des
40、cribed in 6.4.2. Test material should be slowlyadded to the top of each vessel after dilution water addition.The vessel should 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 nee
41、d to be housed in water baths or incubators tomaintain the desired temperature. If photodecomposition of thetest material is likely, the stirring phase should be conducted inthe dark. Preparation vessels should be inspected occasionallyto ensure an appropriate vortex is maintained.FIG. 1 Water Accom
42、modated Fraction (WAF) SchematicD6081 98 (2014)36.4.4 The amount of test material added to each vesselshould be determined by the desired nominal exposure load.Nominal exposure loads may be based on direct weight ofmaterial added per volume solution (wt/vol) or calculated onthe basis of test materia
43、l 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 vessel. The vortex in all WAFpreparation vessels for any one experiment should be the same.Exercise care to ensure
44、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 depend on theresults of an optional equilibrium/stability study (see 6.7). Inthe absence of this study, a standard
45、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 settlingperiod, 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 bedisca
46、rded. The remaining solution should be used undiluted inthe exposure vessels.6.5 Preparation of Water-Soluble Fraction (WSF):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 o
47、rby centrifugation to remove undissolved material. Preliminarytesting may be necessary to ensure that the filter disc will notremove 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 not
48、appropriate due to differential solubility of constituents at lowexposure loads. A schematic of the WSF setup is provided inFig. 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 settlingperio
49、d, the aqueous solution should be decanted from belowthe surface of each preparation vessel. The first 5 to 10 mL ofsolution 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
