1、Designation: D 6731 01 (Reapproved 2005)An American National StandardStandard Test Method forDetermining the Aerobic, Aquatic Biodegradability ofLubricants or Lubricant Components in a ClosedRespirometer1This standard is issued under the fixed designation D 6731; the number immediately following the
2、 designation 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 test method covers
3、a procedure for determining thedegree of biodegradability of lubricants or their components inan aerobic aqueous medium on exposure to an inoculum undercontrolled laboratory conditions. This test method is an ulti-mate biodegradation test that measures oxygen demand in aclosed respirometer.1.2 This
4、test method is suitable for evaluating the biodeg-radation of volatile as well as nonvolatile lubricants or lubri-cant components.1.3 This test method is applicable to lubricants and lubricantcomponents which are not toxic and not inhibitory to the testmicroorganisms at the test concentration.1.4 Th
5、e values stated in SI units are to be regarded asstandard. No other units of measurement are included in thisstandard.1.5 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-pria
6、te safety and health practices and determine the applica-bility of regulatory limitations prior to use. Specific hazards aregiven in Section 10.2. Referenced Documents2.1 ASTM Standards:2D 1129 Terminology Relating to WaterD 1193 Specification for Reagent WaterD 1293 Test Methods for pH of WaterD 41
7、75 Terminology Relating to Petroleum, PetroleumProducts, and LubricantsD 4447 Guide for Disposal of Laboratory Chemicals andSamplesD 6384 Terminology Relating to Biodegradability and Ec-otoxicity of LubricantsE 943 Terminology Relating to Biological Effects and En-vironmental Fate2.2 ISO Standards:3
8、ISO 4259:1992(E) Petroleum ProductsDetermination andApplication of Precision Data in Relation to Methods ofTestISO 6107-2:1997 Water QualityVocabularyPart 2ISO 8192:1986 Water QualityTest for Inhibition of Oxy-gen Consumption by Activated SludgeISO 9408:1999 Water QualityEvaluation of UltimateAerobi
9、c Biodegradability of Organic Compounds inAqueous Medium by Determination of Oxygen Demandin a Closed Respirometer2.3 OECD Standards:4OECD 301F:1992 Ready Biodegradability-ManometricRespirometry2.4 APHA Standards:52540B Total Solids Dried at 103-105C9215 Heterotrophic Plate Count3. Terminology3.1 De
10、finitions:3.1.1 Definitions of terms applicable to this test methodappear in the Compilation of ASTM Standard Definitions andthe following terminology standards: D 1129, D 4175, D 6384,E 943, and ISO 6107-2:1997.3.1.2 activated sludge, nthe precipitated solid matter,consisting mainly of bacteria and
11、 other aquatic microorgan-isms, that is produced at a domestic wastewater treatment plantand is used primarily in secondary sewage treatment tomicrobially oxidize dissolved organic matter in the effluent.3.1.3 aerobic, adj(a) taking place in the presence ofoxygen; (b) living or active in the presenc
12、e of oxygen.1This test method is under the jurisdiction of ASTM Committee D02 onPetroleum Products and Lubricants and is the direct responsibility of SubcommitteeD02.12 on Environmental Standards for Lubricants.Current edition approved Nov. 1, 2005. Published November 2005. Originallyapproved in 200
13、1. Last previous edition approved in 2001 as D 6731 01.2For 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 website.3Avail
14、able from American National Standards Institute, 25 W. 43rd St., 4thFloor, New York, NY 10036.4Available from the Head of Publications Service, Organization for EconomicCooperation and Development (OECD), 2, rueAndre-Pascal, 75775 Paris Cedex 16,France.5From Standard Methods for the Examination of W
15、ater and Wastewater, latestedition. Available from the American Public Health Assoc., 1015 18th St., NW,Washington, DC 20036.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.3.1.4 biochemical oxygen demand (BOD), nthe massconcentratio
16、n of dissolved oxygen consumed under specifiedconditions by the biological oxidation of organic or inorganicmatter, or both.3.1.4.1 DiscussionBOD determination is performed usingempirical tests employing standardized laboratory procedures.These tests measure oxygen utilization during a specifiedincu
17、bation period for the biochemical degradation of organicmaterial (carbonaceous demand) in water.3.1.5 biodegradation, nthe process of chemical break-down or transformation of a test material caused by microor-ganisms or their enzymes.3.1.5.1 DiscussionBiodegradation is only one mechanismby which mat
18、erials are removed, transformed, or both, in theenvironment.3.1.6 lag phase, nthe period of diminished physiologicalactivity and cell division following the addition of microorgan-isms to a new culture medium.3.1.7 log phase, nthe period of growth of microorganismsduring which cells divide at a posi
19、tive constant rate.3.1.8 pre-adaptation, nthe incubation of an inoculum inthe presence of the test material which is done prior to theinitiation of the test and under conditions similar to the testconditions.3.1.8.1 DiscussionThe aim of pre-adaptation is to im-prove the precision of the test method
20、by decreasing variabilityin the rate of biodegradation produced by the inoculum.Pre-adaptation may mimic the natural processes which causechanges in the microbial population of the inoculum leading tomore rapid biodegradation of the test material but is notexpected to change the overall extent of bi
21、odegradation of thetest material.3.1.9 pre-condition, nthe pre-incubation of an inoculumunder the conditions of the test in the absence of the testmaterial.3.1.10 sludge, na water-formed sedimentary deposit.3.1.11 suspended solids (of an activated sludge or otherinoculum samples), nsolids present in
22、 activated sludge orother inoculum samples that are not removed by settling underspecified conditions.4. Summary of Test Method4.1 Biodegradation of a lubricant or the component(s) of alubricant is determined by measuring the oxygen consumedwhen the lubricant or component is exposed to microorganism
23、sunder controlled aerobic aquatic conditions. This value is thencompared to the theoretical amount of oxygen (ThO2) which isrequired to oxidize all of the elements (that is, carbon,hydrogen, nitrogen, and so forth) in the test material. This testmethod mixes the test material (lubricant or component
24、) withaerobic microorganisms in a closed respirometer containing adefined aquatic medium and measures the biodegradation ofthe test material by following the decrease in oxygen in therespirometer.4.2 The test material is the sole source of carbon and energyin the medium.Areference material known to
25、biodegrade, suchas low erucic acid rapeseed oil (LEAR or canola oil) is runalongside the test material to confirm that the inoculum isviable and capable of biodegrading suitable materials under thetest conditions. The test material or reference material concen-tration is normally 50 to 100 mg/L, pro
26、viding a theoreticaloxygen demand of at least 50 mg O2/L but no more than 200mg O2/L. The ThO2of the test and reference materials will bedetermined from measured elemental compositional analysisand will be calculated as in 13.1.4.3 The inoculated medium is stirred in a closed flask andthe consumptio
27、n of oxygen is determined either by measuringthe amount of oxygen required to maintain a constant gasvolume in the respirometer flask, or by measuring the changein volume or pressure (or a combination of the two) in theapparatus.4.4 Evolved CO2(carbon dioxide) is absorbed in an alkalinetrap solution
28、 (for example, 10 M NaOH or KOH) or otherCO2-absorbing system suspended within the test vessel, typi-cally in the headspace of the test vessel.4.5 Biodegradation is followed over a specified period bydetermining the consumption of oxygen. The amount ofoxygen utilized in oxidation of the test and ref
29、erence materialis corrected for oxygen uptake by the inoculum in the blankcontrols and is expressed as a percentage of the theoreticaloxygen demand (ThO2) calculated from the empirical formulaof the material. Evaluation of the biodegradability of the testmaterial is made on the basis of these data.
30、Normally the testduration is 28 days; however, the test may be terminated ifoxygen consumption has plateaued. The test may be extendedas long as the systems integrity is maintained and theinoculum in the blank systems is viable. The duration of thetest will be dependent on the length of time require
31、d for the rateof test material biodegradation to achieve a plateau. A graphi-cal illustration of the test results for a biodegradable material ispresented in Fig. 1.5. Significance and Use5.1 Results from this test method suggest the degree ofaerobic, aquatic biodegradation of a lubricant or lubrica
32、ntcomponent. The rate and extent of oxygen consumption ismeasured upon exposure of the test material to an inoculumwithin the confines of a controlled laboratory setting. Testmaterials which achieve a high degree of biodegradation in thistest may be assumed to easily biodegrade in many aerobicaquati
33、c environments.5.2 Because of the stringency of this test method, lowresults do not necessarily mean that the test material is notbiodegradable under environmental conditions, but indicatethat further testing is necessary to establish biodegradability.5.3 If the pH value at the end of the test is ou
34、tside the rangefrom 6 to 8 and if the percentage degradation of the testmaterial is less than 50 %, it is advisable to repeat the test witha lower concentration of the test material or a higher concen-tration of the buffer solution, or both.5.4 A reference or control material known to biodegradeunde
35、r the conditions of this test method is necessary in order toverify the activity of the inoculum. The test must be regardedas invalid and shall be repeated using a fresh inoculum if thereference material does not demonstrate biodegradation to theextent of 60 % of the ThO2within 28 days.D 6731 01 (20
36、05)25.5 Information on the toxicity of the test material to theinoculum may be useful in the interpretation of low biodegra-dation results. Toxicity of the test material to the inoculum maybe evaluated by testing the test material in combination withthe reference material in inhibition control syste
37、ms. If aninhibition control is included, the test material is assumed to beinhibiting if the degradation percentage of the referencematerial is lower than 40 % (ISO 8192:1986). In this case, it isadvisable to repeat the test with lower concentrations of the testmaterial.5.6 Total oxygen utilization
38、in the blank at the end of thetest exceeding 60 mg O2/L invalidates the test.5.7 The water solubility or dispersibility of the lubricant orcomponent may influence the results obtained and hencecomparison of test results may be limited to lubricants orcomponents with similar solubilities.5.8 The beha
39、viors of complex mixtures are not alwaysconsistent with the individual properties of the components.Test results for individual lubricant components may be sug-gestive of whether a mixture containing these components (thatis, fully formulated lubricants) is biodegradable, but suchinformation should
40、be used judiciously.6. Apparatus6.1 Closed Respirometer:6.1.1 The principle of a closed respirometer is given in Fig.2. When testing volatile compounds, the apparatus used shallbe appropriate or adapted to this particular purpose in accor-dance with the manufacturers instructions. Exercise care that
41、the closed respirometer apparatus is well sealed to prevent anyloss (for example, leakage) of volatile compounds from thesystem or of oxygen into the system.6.1.2 The test mixture is stirred by a magnetic stirrer in thetest flask, which is filled with sufficient volume to minimizeheadspace and preve
42、nt delay of O2and CO2diffusion throughthe air-water phases. This volume is dependent on the selectedflask size, and is normally specified by the manufacturer of therespirometer. If biodegradation takes place, the microorgan-isms consume oxygen and produce carbon dioxide. Oxygenfrom the headspace is
43、then dissolved in the liquid to reestablishchemical equilibrium. The carbon dioxide produced by themicroorganisms diffuses into the headspace where it is trappedin an absorbent solution or material and the total pressure in theflask then decreases.6.1.3 This pressure drop is detected by a manometer,
44、 whichproduces a signal that results in the electrolytic generation ofoxygen. When the original pressure is re-established, the signalis stopped and the quantity of electricity used is measured. Theamount of electricity used is proportional to the amount ofconsumed oxygen. This is indicated on a plo
45、tter or a printer, orthe data are collected using an appropriate software program.6.2 Water-Bath or Constant Temperature Room, to complywith 11.2.6.3 Centrifuge.6.4 pH-meter.6.5 Analytical Balance, capable of weighing to appropriateprecision and accuracy (for example, 60.0001 g).7. Reagents and Mate
46、rials7.1 Purity of ReagentsReagent grade chemicals shall beused in all tests. Unless otherwise indicated, it is intended thatall reagents conform to the specifications of the Committee onAnalytical Reagents of the American Chemical Society wheresuch specifications are available.6Other grades may be
47、used,provided it is first ascertained that the reagent is of sufficientlyhigh purity to permit its use without decreasing the accuracy ofthe determination.7.2 Purity of WaterUnless otherwise indicated, referencesto water shall be understood to mean reagent water as definedby Type II of Specification
48、 D 1193.6Reagent Chemicals, American Chemical Society Specifications, AmericanChemical Society, Washington, DC. For suggestions on the testing of reagents notlisted by the American Chemical Society, see Analar Standards for LaboratoryChemicals, BDH Ltd., Poole, Dorset, U.K., and the United States Ph
49、armacopeiaand National Formulary, U.S. Pharmacopeial Convention, Inc. (USPC), Rockville,MD.FIG. 1 Respirometric TestBiodegradation CurveD 6731 01 (2005)37.3 Prepare the following stock solutions:7.3.1 Calcium Chloride SolutionDissolve 27.5 g of anhy-drous calcium chloride (CaCl2) or 36.4 g of calcium chloridedihydrate (CaCl22H2O) in water and dilute to 1 L.7.3.2 Ferric Chloride SolutionDissolve 0.25 g of iron(III) chloride hexahydrate (FeCl36H2O) in water and dilute to1 L. Prepare this solution just before use or add a drop ofconcentrated hydrochloric ac
