1、Designation:D586405 Designation: D5864 11Standard 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 c
2、ase 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 test method covers the determination of the degree of aerobic aquatic biodegradation
3、 of fully formulated lubricants ortheir components on exposure to an inoculum under laboratory conditions.1.2 This test method is intended to specifically address the difficulties associated with testing water insoluble materials andcomplex mixtures such as are found in many lubricants.1.3 This test
4、 method is designed to be applicable to all lubricants 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 the safety concerns, if any, associated with its use. It is the responsibilityof
5、the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatorylimitations prior to use. Specific hazards are discussed in Section 10.2. Referenced Documents2.1 ASTM Standards:2D1193 Specification for Reagent WaterD1293 Test Methods for pH
6、 of WaterD4447 Guide for Disposal of Laboratory Chemicals and SamplesD5291 Test Methods for Instrumental Determination of Carbon, Hydrogen, and Nitrogen in Petroleum Products and LubricantsE943 Terminology Relating to Biological Effects and Environmental Fate2.2 ISO Standard:34259:1992(E) Petroleum
7、ProductsDetermination and Application 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 that are not described herein appear in th
8、e Compilation of ASTMStandard Definitions, 19902ASTM Online Dictionary of Engineering Science and Technology5or Terminology E943.3.1.2 aerobic, adj(1 ) taking place in the presence of oxygen, (2) living or active in the presence of oxygen.3.1.3 biodegradation, nthe process of chemical breakdown or t
9、ransformation of a substance caused by organisms or theirenzymes.3.1.4 biomass, nany material, excluding fossil fuels, which is or was a living organism or component of a living organism.biological material including any material other than fossil fuels which is or was a living organism or component
10、 or product ofa living organism.1This test method is under the jurisdiction of ASTM Committee D02 on Petroleum Products and Lubricants and is the direct responsibility of Subcommittee D02.12 onEnvironmental Standards for Lubricants.Current edition approved Nov.March 1, 2005.2011. Published December
11、2005.March 2011. Originally approved in 1995. Last previous edition approved in 20002005 asD586400.D586405. DOI: 10.1520/D5864-05.10.1520/D5864-11.2For referenced ASTM standards, visit the ASTM website, www.astm.org, or contact ASTM Customer Service at serviceastm.org. For Annual Book of ASTM Standa
12、rdsvolume information, refer to the standards Document Summary page on the ASTM website.3Available from American National Standards Institute (ANSI), 25 W. 43rd St., 4th Floor, New York, NY 10036.4From Standard Methods for the Examination of Water and Wastewater, latest edition. Available from the A
13、merican Public Health Association, 1015 18th St., N.W.,Washington, DC 20036.5Adapted from McGraw-Hill Dictionary of Scientific and Technical Terms, 4th ed., 1989.5ASTM Online Dictionary of Engineering Science and Technology (Stock#DEFONLINE) is available on the ASTM website, www.astm.org, or contact
14、 ASTM CustomerService at serviceastm.org.1This document is not an ASTM standard and is intended only to provide the user of an ASTM standard an indication of what changes have been made to the previous version. Becauseit may not be technically possible to adequately depict all changes accurately, AS
15、TM recommends that users consult prior editions as appropriate. In all cases only the current versionof the standard as published by ASTM is to be considered the official document.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.3.1.4.
16、1 DiscussionIn biology and environmental science, biomass is typically expressed as density of biological material perunit sample volume, area, or mass (g biomass/g(or/mLor/cm2) sample); when used for products derived from organismsbiomass is typically expressed in terms of mass (kg, MT, etc.) or vo
17、lume (L, m3, bbl, etc.).3.1.4.2 DiscussionProducts of living organisms include those materials produced directly by living organisms as metabolites(for example, ethanol, various carbohydrates and fatty acids), materials manufactured by processing living organisms (for example:pellets manufactured by
18、 shredding and pelletizing plant material) and materials produced by processing living organisms, theircomponents or metabolites (for example, transesterified oil; also called biodiesel).3.1.5 blank, na flask containing the test medium and the inoculum with no additional carbon source added.3.1.6 in
19、oculum, nspores, bacteria, single celled organisms, or other live materials, that are introduced into a test medium.3.1.7 lag phase, nthe period of physiological activity and diminished cell division following the addition of microorganismsto a new culture medium.63.1.8 log phase, nthe period of gro
20、wth of microorganisms during which cells divide at a constant rate.63.1.9 mixed liquor, nthe contents of an aeration tank including the activated sludge mixed with primary effluent or the rawwastewater and return sludge.3.1.10 pre-adaptation, nthe incubation of an inoculum in the presence of the tes
21、t substance which is done prior to theinitiation of the test and under conditions similar to the test conditions.3.1.10.1 DiscussionThe aim of pre-adaptation is to improve the precision of the test method by decreasing variability in therate of biodegradation produced by the inoculum. Pre-adaptation
22、 may mimic the natural processes which cause changes in themicrobial population of the inoculum leading to more rapid biodegradation of the test substance.3.1.11 supernatant, nthe liquid above settled solids.3.1.12 theoretical CO2, nthe amount of CO2which could hypothetically be produced from the co
23、mplete biological oxidationof all of the carbon in a substance.3.1.13 ultimate biodegradation, ndegradation achieved when the test substance is totally utilized by microorganisms resultingin the production of CO2, (and possibly methane in the case of anaerobic biodegradation), water, inorganic compo
24、unds, and newmicrobial cellular constituents (biomass or secretions, or both).3.1.14 ultimate biodegradation test, na test that estimates the extent to which the carbon in a product has been converted toCO2or methane, either directly, by measuring the production of CO2or methane, or indirectly, by m
25、easuring the consumption ofO2.3.1.14.1 DiscussionThe measurement of new biomass is not attempted.4. Summary of Test Method4.1 Biodegradation of a lubricant or the component(s) of a lubricant is measured by collecting and measuring the CO2producedwhen the lubricant or component is exposed to microorg
26、anisms under controlled aerobic aquatic conditions. This value is thencompared to the theoretical amount of CO2which could be generated if all of the carbon in the test material were converted toCO2.CO2is a product of aerobic microbial metabolism of carbon-containing substances and so is a direct me
27、asure of the testsubstances ultimate biodegradation. CO2production is quantified by trapping it in a Ba(OH)2solution and titrating the solutionto calculate the amount of CO2absorbed.4.2 The carbon content of the test substance is determined by Test Method D5291 or an equivalent method and the theore
28、ticalCO2is calculated from that measurement. It is necessary to directly measure the carbon content of the test substance instead ofcalculating this number, because of the complexity of the mixture of compounds present in lubricants.4.3 Biodegradability is expressed as a percentage of theoretical CO
29、2production.5. Significance and Use5.1 Results from the test method suggest, within the confines of a controlled laboratory setting, the degree of aerobic aquaticbiodegradation of a lubricant or components of a lubricant by measuring the evolved carbon dioxide upon exposure of the testmaterial to an
30、 inoculum. The plateau level of CO2evolution in this test method will suggest the degree of biodegradability of thelubricant. Test substances that achieve a high degree of biodegradation in this test may be assumed to easily biodegrade in manyaerobic aquatic environments.5.2 Because of the stringenc
31、y of this test, a low yield of CO2does not necessarily mean that the test substance is notbiodegradable under environmental conditions, but indicates that further testing is necessary to establish biodegradability.5.3 Information on toxicity to the inoculum of the test substance may be useful in the
32、 interpretation of low biodegradationresults.5.4 Activated sewage-sludge from a sewage-treatment plant that principally treats domestic waste is considered an acceptableactive aerobic inoculum available over a wide geographical area in which to test a broad range of lubricants. An inoculum derived6R
33、eagent Chemicals, American Chemical Society Specifications, American Chemical Society, Washington, DC. For Suggestions on the testing of reagents not listed bythe American Chemical Society, see Annual Standards for Laboratory Chemicals, BDH Ltd., Poole, Dorset, U.K., and the United States Pharmacope
34、ia and NationalFormulary, U.S. Pharmacopeial Convention, Inc. (USPC), Rockville, MD.6Adapted from McGraw-Hill Dictionary of Scientific and Technical Terms, 4th ed., 1989.D5864 112from soil or natural surface waters, or both, or any combination of the three sources, is also appropriate for this test
35、method.NOTE 1Allowance for various and multiple inoculum sources provides access to a greater diversity of biochemical competency and potentiallyrepresents more accurately the capacity for biodegradation.5.5 A reference or control substance known to biodegrade is necessary in order to verify the act
36、ivity of the inoculum. The testmust be regarded as invalid and should be repeated using a fresh inoculum if the reference does not demonstrate a biodegradationof 60 % of the theoretical CO2evolution within 28 days.5.6 A total CO2evolution in the blank at the end of the test exceeding 75 mg CO2per 3
37、L of medium shall be considered asinvalidating the test.5.7 The water solubility or dispersibility of the lubricant or component may influence the results obtained and hence theprocedure may be limited to comparing lubricants or components with similar solubilities.5.8 The ratio of carbon incorporat
38、ed into cellular material to carbon released as CO2will vary depending on the organicsubstrate, on the particular microorganisms carrying out the conversion, and on the environmental conditions under which theconversion takes place. In principle, this variability complicates the interpretation of th
39、e results from this test method.6. Apparatus6.1 Carbon Dioxide Scrubbing Apparatus(see Fig. 1):6.1.1 The following are required to produce a stream of CO2-free air of sufficient volume to test up to three materials and theaccompanying reference and blank controls in triplicate:A = NaOHB = EmptyC=Bla
40、nkS = StandardD = Ba(OH)21 = Test substance 12 = Test substance 23 = Test substance 3FIG. 1 Aerobic Aquatic Biodegradation Testing SchematicD5864 1136.1.1.1 Five 1-L plastic bottles, containing 700 mL of 10-M sodium hydroxide (NaOH),6.1.1.2 Two empty 1-L Erlenmeyer flasks, to prevent liquid carryove
41、r, and6.1.1.3 One 1-L Erlenmeyer flask, containing 700 mL of 0.0125 M barium hydroxide Ba(OH)2 solution.6.1.2 Connect the bottles in series, as shown in Fig. 1, using vinyl, or other suitable non gas-permeable tubing, to a pressurizedair system, and purge air through the scrubbing solution at a cons
42、tant rate.6.1.3 For each additional test substance to be tested, add one additional 1-L plastic bottle filled with 700 mL of 10 M sodiumhydroxide.6.1.4 The CO2scrubbing apparatus upstream of the Erlenmeyer flask containing the Ba(OH)2solution may be replaced by analternative system which effectively
43、 and consistently produces CO2free air (that is, containing less than 1 ppm CO2).6.2 Incubation/Biodegradation ApparatusEach test material, reference, or control requires the following:6.2.1 Three 4-L Erlenmeyer flasks,6.2.2 Stoppers, which are non-permeable to CO2.6.2.3 Flexible Plastic Tubing, whi
44、ch is non-permeable to CO2.6.2.4 Agitators or Stirrers, for each 4-L Erlenmeyer flask.6.3 Analytical Balance, to weigh out test material or reference material before or as adding to the test flask,6.4 Trapping Apparatus for Measuring Production of CO2For each incubation apparatus, the following are
45、required:6.4.1 Several 200-mL Bottles, fitted with gas bubblers and containing 100 mL 0.0125 M Ba(OH)2carbon dioxide scrubbingsolution.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 Reagen
46、tsReagent grade chemicals shall be used in all tests. Unless otherwise indicated, it is intended that allreagents conform to the specifications of the Committee on Analytical Reagents of the American Chemical Society where suchspecifications are available.7Other grades may be used, provided it is fi
47、rst ascertained that the reagent is of sufficiently high purityto permit its use without lessening the accuracy of the determination.7.2 Purity of WaterUnless otherwise indicated, references to water shall be understood to mean reagent water as defined byType II of Specification D1193.7.3 Prepare th
48、e following stock solutions:7.3.1 Ammonium Sulfate Solution (40 g/L)Dissolve 40 g of ammonium sulfate (NH4)2SO4) in water and dilute to 1 L.7.3.2 Calcium Chloride Solution (27.5 g/L)Dissolve 27.5 g of calcium chloride (CaCl2) in water and dilute to 1 L.7.3.3 Ferric Chloride Solution (0.25 g/L)Dissol
49、ve 0.25 g of ferric chloride hexahydrate (FeCl36H2O) in water and dilute to1L.7.3.4 Magnesium Sulfate Solution (22.5 g/L)Dissolve 22.5 g of magnesium sulfate heptahydrate (MgSO47H2O) in water anddilute to 1 L.7.3.5 Phosphate BufferDissolve 8.5 g potassium dihydrogen phosphate (KH2PO4), 21.7 g potassium monohydrogenphosphate (K2HPO4), 33.4 g sodium monohydrogen phosphate dihydrate (Na2HPO42H2O), and 1.7 g ammonium chloride(NH4Cl) in water and dilute to 1 L.7.4 The test medium will contain the following reagents diluted to 1 L with water.7.4.1 A
