1、Designation: D 5864 05An American National StandardStandard Test Method forDetermining Aerobic Aquatic Biodegradation of Lubricantsor Their Components1This standard is issued under the fixed designation D 5864; the number immediately following the designation indicates the year oforiginal adoption o
2、r, 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 the determination of the degreeof aerobic aquatic bio
3、degradation of fully formulated lubri-cants or their components on exposure to an inoculum underlaboratory conditions.1.2 This test method is intended to specifically address thedifficulties associated with testing water insoluble materialsand complex mixtures such as are found in many lubricants.1.
4、3 This test method is designed to be applicable to alllubricants that are not volatile and are not inhibitory at the testconcentration to the organisms present in the inoculum.1.4 This standard does not purport to address all of thesafety concerns, if any, associated with its use. It is theresponsib
5、ility 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. Specific hazards arediscussed in Section 10.2. Referenced Documents2.1 ASTM Standards:2D 1193 Specification for Reagent WaterD 1293 Test M
6、ethods for pH of WaterD 4447 Guide for Disposal of Laboratory Chemicals andSamplesD 5291 Test Methods for Instrumental Determination ofCarbon, Hydrogen, and Nitrogen in Petroleum Productsand LubricantsE 943 Terminology Relating to Biological Effects and En-vironmental Fate2.2 ISO Standard:34259:1992
7、(E) Petroleum ProductsDetermination and Ap-plication of Precision Data in Relation to Methods of Test2.3 APHA Standard:42540B Total Solids Dried at 103105C9215 Heterotrophic Plate Count3. Terminology3.1 Definitions:3.1.1 Definitions of terms applicable to this test method thatare not described herei
8、n appear in the Compilation of ASTMStandard Definitions, 19902or Terminology E 943.3.1.2 aerobic, adj(1) taking place in the presence ofoxygen, (2) living or active in the presence of oxygen.3.1.3 biodegradation, nthe process of chemical break-down or transformation of a substance caused by organism
9、s ortheir enzymes.3.1.4 biomass, nany material, excluding fossil fuels,which is or was a living organism or component of a livingorganism.3.1.5 blank, na flask containing the test medium and theinoculum with no additional carbon source added.3.1.6 inoculum, nspores, bacteria, single celled organ-ism
10、s, or other live materials, that are introduced into a testmedium.3.1.7 lag phase, nthe period of physiological activity anddiminished cell division following the addition of microorgan-isms to a new culture medium.53.1.8 log phase, nthe period of growth of microorganismsduring which cells divide at
11、 a constant rate.53.1.9 mixed liquor, nthe contents of an aeration tankincluding the activated sludge mixed with primary effluent orthe raw wastewater and return sludge.3.1.10 pre-adaptation, nthe incubation of an inoculum inthe presence of the test substance which is done prior to theinitiation of
12、the test and under conditions similar to the testconditions.3.1.10.1 DiscussionThe aim of pre-adaptation is to im-prove the precision of the test method by decreasing variabilityin the rate of biodegradation produced by the inoculum.1This test method is under the jurisdiction of ASTM Committee D02 o
13、nPetroleum Products and Lubricants and is the direct responsibility of SubcommitteeD02.12 on Environmental Standards for Lubricants.Current edition approved Nov. 1, 2005. Published December 2005. Originallyapproved in 1995. Last previous edition approved in 2000 as D 586400.2For referenced ASTM stan
14、dards, 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.3Available from American National Standards Institute (ANSI), 25 W. 43rd St.,4th Floor
15、, New York, NY 10036.4From Standard Methods for the Examination of Water and Wastewater, latestedition. Available from the American Public Health Association, 1015 18th St.,N.W., Washington, DC 20036.5Adapted from McGraw-Hill Dictionary of Scientific and Technical Terms, 4thed., 1989.1Copyright ASTM
16、 International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.Pre-adaptation may mimic the natural processes which causechanges in the microbial population of the inoculum leading tomore rapid biodegradation of the test substance.3.1.11 supernatant, nthe liquid
17、above settled solids.3.1.12 theoretical CO2, nthe amount of CO2which couldhypothetically be produced from the complete biologicaloxidation of all of the carbon in a substance.3.1.13 ultimate biodegradation, ndegradation achievedwhen the test substance is totally utilized by microorganismsresulting i
18、n the production of CO2, (and possibly methane inthe case of anaerobic biodegradation), water, inorganic com-pounds, and new microbial cellular constituents (biomass orsecretions, or both).3.1.14 ultimate biodegradation test, na test that estimatesthe extent to which the carbon in a product has been
19、 convertedto CO2or methane, either directly, by measuring the produc-tion of CO2or methane, or indirectly, by measuring theconsumption of O2.3.1.14.1 DiscussionThe measurement of new biomass isnot attempted.4. Summary of Test Method4.1 Biodegradation of a lubricant or the component(s) of alubricant
20、is measured by collecting and measuring the CO2produced when the lubricant or component is exposed tomicroorganisms under controlled aerobic aquatic conditions.This value is then compared to the theoretical amount of CO2which could be generated if all of the carbon in the test materialwere converted
21、 to CO2.CO2is a product of aerobic microbialmetabolism of carbon-containing substances and so is a directmeasure of the test substances ultimate biodegradation. CO2production is quantified by trapping it in a Ba(OH)2solutionand titrating the solution to calculate the amount of CO2absorbed.4.2 The ca
22、rbon content of the test substance is determinedby Test Method D 5291 or an equivalent method and thetheoretical CO2is calculated from that measurement. It isnecessary to directly measure the carbon content of the testsubstance instead of calculating this number, because of thecomplexity of the mixt
23、ure of compounds present in lubricants.4.3 Biodegradability is expressed as a percentage of theo-retical CO2production.5. Significance and Use5.1 Results from the test method suggest, within the con-fines of a controlled laboratory setting, the degree of aerobicaquatic biodegradation of a lubricant
24、or components of alubricant by measuring the evolved carbon dioxide uponexposure of the test material to an inoculum. The plateau levelof CO2evolution in this test method will suggest the degree ofbiodegradability of the lubricant. Test substances that achievea high degree of biodegradation in this
25、test may be assumed toeasily biodegrade in many aerobic aquatic environments.5.2 Because of the stringency of this test, a low yield of CO2does not necessarily mean that the test substance is notbiodegradable under environmental conditions, but indicatesthat further testing is necessary to establish
26、 biodegradability.5.3 Information on toxicity to the inoculum of the testsubstance may be useful in the interpretation of low biodegra-dation results.5.4 Activated sewage-sludge from a sewage-treatment plantthat principally treats domestic waste is considered an accept-able active aerobic inoculum a
27、vailable over a wide geographi-cal area in which to test a broad range of lubricants. Aninoculum derived from soil or natural surface waters, or both,or any combination of the three sources, is also appropriate forthis test method.NOTE 1Allowance for various and multiple inoculum sources pro-vides a
28、ccess to a greater diversity of biochemical competency andpotentially represents more accurately the capacity for biodegradation.5.5 Areference or control substance known to biodegrade isnecessary in order to verify the activity of the inoculum. Thetest must be regarded as invalid and should be repe
29、ated usinga fresh inoculum if the reference does not demonstrate abiodegradation of 60 % of the theoretical CO2evolutionwithin 28 days.5.6 A total CO2evolution in the blank at the end of the testexceeding 75 mg CO2per 3 L of medium shall be consideredas invalidating the test.5.7 The water solubility
30、 or dispersibility of the lubricant orcomponent may influence the results obtained and hence theprocedure may be limited to comparing lubricants or compo-nents with similar solubilities.5.8 The ratio of carbon incorporated into cellular material tocarbon released as CO2will vary depending on the org
31、anicsubstrate, on the particular microorganisms carrying out theconversion, and on the environmental conditions under whichthe conversion takes place. In principle, this variability com-plicates the interpretation of the results from this test method.6. Apparatus6.1 Carbon Dioxide Scrubbing Apparatu
32、s(see Fig. 1):6.1.1 The following are required to produce a stream ofCO2-free air of sufficient volume to test up to three materialsand the accompanying reference and blank controls in tripli-cate:6.1.1.1 Five 1-L plastic bottles, containing 700 mL of 10-Msodium hydroxide (NaOH),6.1.1.2 Two empty 1-
33、L Erlenmeyer flasks, to prevent liquidcarryover, and6.1.1.3 One 1-L Erlenmeyer flask, containing 700 mL of0.0125 M barium hydroxide Ba(OH)2 solution.6.1.2 Connect the bottles in series, as shown in Fig. 1, usingvinyl, or other suitable non gas-permeable tubing, to a pres-surized air system, and purg
34、e air through the scrubbingsolution at a constant rate.6.1.3 For each additional test substance to be tested, add oneadditional 1-L plastic bottle filled with 700 mL of 10 M sodiumhydroxide.6.1.4 The CO2scrubbing apparatus upstream of the Erlen-meyer flask containing the Ba(OH)2solution may be repla
35、cedby an alternative system which effectively and consistentlyproduces CO2free air (that is, containing less than 1 ppmCO2).D58640526.2 Incubation/Biodegradation ApparatusEach test mate-rial, reference, or control requires the following:6.2.1 Three 4-L Erlenmeyer flasks,6.2.2 Stoppers, which are non
36、-permeable to CO2.6.2.3 Flexible Plastic Tubing, which is non-permeable toCO2.6.2.4 Agitators or Stirrers, for each 4-L Erlenmeyer flask.6.3 Analytical Balance, to weigh out test material or refer-ence material before or as adding to the test flask,6.4 Trapping Apparatus for Measuring Production ofC
37、O2For each incubation apparatus, the following are re-quired:6.4.1 Several 200-mL Bottles, fitted with gas bubblers andcontaining 100 mL 0.0125 M Ba(OH)2carbon dioxide scrub-bing solution.6.5 Titration Apparatus for Measuring Production of CO2:6.5.1 100-mL burette.6.6 Glass Wool, for filtering the i
38、noculum.7. Reagents and Materials7.1 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 ava
39、ilable.6Other grades may be used,provided it is first ascertained that the reagent is of sufficientlyhigh purity to permit its use without lessening the accuracy ofthe determination.6Reagent Chemicals, American Chemical Society Specifications, AmericanChemical Society, Washington, DC. For suggestion
40、s 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 Pharmacopeiaand National Formulary, U.S. Pharmacopeial Convention, Inc. (USPC), Rockville,MD.A = NaOHB = EmptyC=BlankS = Standar
41、dD = Ba(OH)21 = Test substance 12 = Test substance 23 = Test substance 3FIG. 1 Aerobic Aquatic Biodegradation Testing SchematicD58640537.2 Purity of WaterUnless otherwise indicated, referencesto water shall be understood to mean reagent water as definedby Type II of Specification D 1193.7.3 Prepare
42、the following stock solutions:7.3.1 Ammonium Sulfate Solution (40 g/L)Dissolve 40 gof ammonium sulfate (NH4)2SO4) in water and dilute to 1 L.7.3.2 Calcium Chloride Solution (27.5 g/L)Dissolve 27.5g of calcium chloride (CaCl2) in water and dilute to 1 L.7.3.3 Ferric Chloride Solution (0.25 g/L)Dissol
43、ve 0.25 gof ferric chloride hexahydrate (FeCl36H2O) in water and diluteto1L.7.3.4 Magnesium Sulfate Solution (22.5 g/L)Dissolve22.5 g of magnesium sulfate heptahydrate (MgSO47H2O) inwater and dilute to 1 L.7.3.5 Phosphate BufferDissolve 8.5 g potassium dihydro-gen phosphate (KH2PO4), 21.7 g potassiu
44、m monohydrogenphosphate (K2HPO4), 33.4 g sodium monohydrogen phosphatedihydrate (Na2HPO42H2O), and 1.7 g ammonium chloride(NH4Cl) in water and dilute to 1 L.7.4 The test medium will contain the following reagentsdiluted to 1 L with water.7.4.1 Ammonium Sulfate Solution, 1 mL,7.4.2 Calcium Chloride S
45、olution, 1 mL,7.4.3 Ferric Chloride Solution, 4 mL,7.4.4 Magnesium Sulfate Solution, 1 mL, and7.4.5 Phosphate Buffer Solution,10mL.7.5 Barium Hydroxide Solution, 0.0125 M, is prepareddissolving 4.0 g Ba(OH)28H2O per litre of distilled water.Filter free of solid material, confirm molarity by titratio
46、n withstandard acid, and store under nitrogen sealed as a clearsolution to prevent absorption of CO2from the air. It isrecommended that 5 L be prepared at a time when running aseries of tests.7.6 Difco Vitamin-free Casamino Acids.7.7 Yeast Extract.7.8 Phenolphthalein.7.9 Standardized Hydrochloric Ac
47、id (0.04800.0520 M).8. Inoculum Test Organisms8.1 Sources of the InoculumThe following provides sev-eral options for where and how to obtain an appropriateinoculum:8.1.1 Inoculum from Activated SludgeActivated sludgefreshly sampled (that is, less than 24 h old) from a well-operated domestic sewage t
48、reatment plant (that is, one with norecent upsets and operating within its design parameters) maybe used. This sewage treatment plant should receive minimalor no effluent from industry.8.1.1.1 Using CO2-free air, aerate sludge in the laboratoryfor 4 h. Remove and homogenize 500 mL of the mixed liquo
49、rfor 2 min at medium speed in a blender or equivalent highspeed mixer.8.1.1.2 If using sludge supernatant as the inoculum, allowthe homogenized sludge to settle for 30 min. If the supernatantstill contains high levels of suspended solids at the end of 30min, allow to settle for a further 30 to 40 min or adaptlaboratory conditions to obtain better settling. Once sufficientsettling is achieved, decant sufficient volume of the supernatantto provide a 1% (by volume) inoculum. Avoid carry-over ofsludge solids which might cause inconsistencies in the mea-
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