1、Designation: D5864 17Standard Test Method forDetermining Aerobic Aquatic Biodegradation of Lubricantsor Their Components1This standard is issued under the fixed designation D5864; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the
2、 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. Scope*1.1 This test method covers the determination of the degreeof aerobic aquatic biodegradation of fully formulated
3、 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.1.3 This test method is designed
4、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 theresponsibility of the user of this stand
5、ard 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:2D1193 Specification for Reagent WaterD1293 Test Methods for pH of WaterD4447 Guide
6、 for Disposal of Laboratory Chemicals andSamplesD5291 Test Methods for Instrumental Determination ofCarbon, Hydrogen, and Nitrogen in Petroleum Productsand LubricantsE943 Terminology Relating to Biological Effects and Envi-ronmental Fate2.2 ISO Standard:34259:1992(E) Petroleum ProductsDetermination
7、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 herein appear in the ASTM Online Dictiona
8、ryof Engineering Science and Technology5or Terminology E943.3.1.2 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 material caused by organisms ortheir enzymes.3.1.3
9、.1 DiscussionBiodegradation is only one mechanismby which materials are transformed in the environment.3.1.4 biomass, nbiological material including any mate-rial other than fossil fuels which is or was a living organism orcomponent or product of a living organism.3.1.4.1 DiscussionIn biology and en
10、vironmental science,biomass is typically expressed as density of biological materialper unit sample volume, area, or mass (g biomass/g(or/mLor / cm2) sample); when used for products derived fromorganisms biomass is typically expressed in terms of mass (kg,MT, etc.) or volume (L, m3, bbl, etc.).3.1.4
11、.2 DiscussionProducts of living organisms includethose materials produced directly by living organisms asmetabolites (for example, ethanol, various carbohydrates andfatty acids), materials manufactured by processing livingorganisms (for example: pellets manufactured by shreddingand pelletizing plant
12、 material) and materials produced by1This test method is under the jurisdiction of ASTM Committee D02 onPetroleum Products, Liquid Fuels, and Lubricants and is the direct responsibility ofSubcommittee D02.12 on Environmental Standards for Lubricants.Current edition approved Jan. 1, 2017. Published F
13、ebruary 2017. Originallyapproved in 1995. Last previous edition approved in 2011 as D5864 11. DOI:10.1520/D5864-17.2For 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 t
14、he standards Document Summary page onthe ASTM website.3Available from American National Standards Institute (ANSI), 25 W. 43rd St.,4th Floor, New York, NY 10036, http:/www.ansi.org.4From Standard Methods for the Examination of Water and Wastewater, latestedition. Available from the American Public H
15、ealth Association, 1015 18th St.,N.W., Washington, DC 20036.5ASTM Online Dictionary of Engineering Science and Technology(Stock#DEFONLINE) is available on theASTM website, www.astm.org, or contactASTM Customer Service at serviceastm.org.*A Summary of Changes section appears at the end of this standa
16、rdCopyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United StatesThis international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for theDevelopment of Int
17、ernational Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.1processing living organisms, their components or metabolites(for example, transesterified oil; also called biodiesel).3.1.5 blank, na flask containing the test medium
18、and theinoculum with no additional carbon source added.3.1.6 inoculum, nspores, bacteria, single celledorganisms, or other live materials, that are introduced into atest medium.3.1.7 lag phase, nthe period of physiological activity anddiminished cell division following the addition of microorgan-ism
19、s to a new culture medium.63.1.8 log phase, nthe period of growth of microorganismsduring which cells divide at a constant rate.63.1.9 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-adaptatio
20、n, nthe pre-incubation of an inocu-lum in the presence of the test material under conditions similarto the test conditions.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.Pre-
21、adaptation may mimic the natural processes which causechanges in the microbial population of the inoculum leading tomore rapid biodegradation of the test material but not to achange in the final degree of biodegradation.3.1.11 supernatant, nthe liquid above settled solids.3.1.12 theoretical CO2,nthe
22、 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 in the production of CO2, (and possibly methane in
23、the case of anaerobic biodegradation), water, inorganiccompounds, and new microbial cellular constituents (biomassor secretions, or both).3.1.14 ultimate biodegradation test, na test that estimatesthe extent to which the carbon in a product has been convertedto CO2or methane, either directly, by mea
24、suring 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 is measured by collecting and measuring the CO2prod
25、uced 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 to CO2.CO2is a product of aerobic microbialmetabol
26、ism 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 carbon content of the test substance is determinedby
27、Test Method D5291 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 mixture of compounds present in lubricants.4.3 Biodegrad
28、ability 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 or components of alubricant by measuring the evolved
29、 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 test may be assumed toeasily biodegrade in many aero
30、bic 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 biodegradability.5.3 Information on toxicity to the
31、 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 available over a wide geographi-cal area in which to
32、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 access to a greater diversity of biochemical competen
33、cy 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 repeated usinga fresh inoculum if the reference does not
34、 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 or dispersibility of the lubricant orcomponent may
35、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 organicsubstrate, on the particular microorganisms carr
36、ying out the6Adapted from McGraw-Hill Dictionary of Scientific and Technical Terms, 4thed., 1989.D5864 172conversion, 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. Appa
37、ratus6.1 Carbon Dioxide Scrubbing Apparatus(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 Mso
38、dium hydroxide (NaOH),6.1.1.2 Two empty 1-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 tubi
39、ng, to a pres-surized air system, and purge air through the scrubbingsolution at a constant rate.6.1.3 For each additional test substance to be tested, add oneadditional 1 Lplastic bottle filled with 700 mLof 10 M sodiumhydroxide.6.1.4 The CO2scrubbing apparatus upstream of the Erlen-meyer flask con
40、taining the Ba(OH)2solution may be replacedby an alternative system which effectively and consistentlyproduces CO2free air (that is, containing less than 1 ppmCO2).6.2 Incubation/Biodegradation ApparatusEach testmaterial, reference, or control requires the following:6.2.1 Three 4 L Erlenmeyer flasks
41、,6.2.2 Stoppers, which are non-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.A = NaOHB = EmptyC=BlankS = StandardD = Ba(OH)21 = Test substance 12 = Test substance 23 = Test substance 3FIG. 1 Aerobic Aquatic Bio
42、degradation Testing SchematicD5864 1736.3 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 ofCO2For each incubation apparatus, the following are re-quired:6.4.1 Several 200 mL Bottles, fitted w
43、ith 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 inoculum.7. Reagents and Materials7.1 Purity of ReagentsReagent grade chemicals shall beused in all
44、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.7Other grades may be used,provided it is first ascertained that the reagent is of sufficient
45、lyhigh purity to permit its use without lessening 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 D1193.7.3 Prepare the following stock solutions:7.3.1 Ammonium Sulfate
46、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)Dissolve27.5 g of calcium chloride (CaCl2) in water and dilute to 1 L.7.3.3 Ferric Chloride Solution (0.25 g L)Dissolve 0.25 gof ferric chloride hexahydrate (FeCl36H2O) i
47、n water and diluteto 1 L.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 potassium monohydrogenphosphate (K2HPO4), 33.4 g sodium mon
48、ohydrogen 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 Solution, 1 mL,7.4.3 Ferric Chloride Solution, 4 mL,
49、7.4.4 Magnesium Sulfate Solution, 1 mL, and7.4.5 Phosphate Buffer Solution, 10 mL.7.5 Barium Hydroxide Solution, 0.0125 M, is prepared dis-solving 4.0 g Ba(OH)28H2O per litre of distilled water. Filterfree of solid material, confirm molarity by titration 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 Hydrochl
