1、Designation: D6139 18Standard Test Method forDetermining the Aerobic Aquatic Biodegradation ofLubricants or Their Components Using the Gledhill ShakeFlask1This standard is issued under the fixed designation D6139; the number immediately following the designation indicates the year oforiginal adoptio
2、n or, in the 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. Scope*1.1 This test method covers the determination of the degreeof aerobic aquatic
3、biodegradation of fully formulated lubri-cants or their components on exposure to an inoculum undercontrolled laboratory conditions. This test method is an ulti-mate biodegradation test that measures carbon dioxide (CO2)evolution.1.2 This test method is intended to specifically address thedifficulti
4、es associated with testing water insoluble materialsand complex mixtures such as are found in many lubricants.1.3 This test method is designed to be applicable to allnon-volatile lubricants or lubricant components that are nottoxic and not inhibitory at the test concentration to theorganisms present
5、 in the inoculum.1.4 The 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
6、to establish appro-priate safety, health, and environmental practices and deter-mine the applicability of regulatory limitations prior to use.Specific hazards are discussed in Section 10.1.6 This international standard was developed in accor-dance with internationally recognized principles on standa
7、rd-ization established in the Decision on Principles for theDevelopment of International Standards, Guides and Recom-mendations issued by the World Trade Organization TechnicalBarriers to Trade (TBT) Committee.2. Referenced Documents2.1 ASTM Standards:2D1193 Specification for Reagent WaterD1293 Test
8、 Methods for pH of WaterD4447 Guide for Disposal of Laboratory Chemicals andSamplesD5291 Test Methods for Instrumental Determination ofCarbon, Hydrogen, and Nitrogen in Petroleum Productsand LubricantsD5864 Test Method for Determining Aerobic Aquatic Bio-degradation of Lubricants or Their Components
9、E943 Terminology Relating to Biological Effects and Envi-ronmental Fate2.2 ISO Standard:34259:1992(E) Petroleum ProductsDetermination and ap-plication of precision data in relation to methods of test2.3 APHA Standards:42540B Total Solids Dried at 103105C9215 Heterotrophic Plate Count3. Terminology3.
10、1 Definitions:3.2 Definitions of terms applicable to this test method thatare not described herein appear in theASTM Online Dictionaryof Engineering Science and Technology5or TerminologyE943.1This test method is under the jurisdiction of ASTM Committee D02 onPetroleum Products, Liquid Fuels, and Lub
11、ricants and is the direct responsibility ofSubcommittee D02.12 on Environmental Standards for Lubricants.Current edition approved June 1, 2018. Published June 2018. Originallyapproved in 1997. Last previous edition approved in 2017 as D6139 17. DOI:10.1520/D6139-18.2For referenced ASTM standards, vi
12、sit 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, New Yor
13、k, NY 10036, http:/www.ansi.org.4Methods from Standard Methods for the Examination of Water and Wastewater,latest edition. Available from the American Public Health Association (APHA), 800I Street, NW, Washington, DC 20001.5ASTM Online Dictionary of Engineering Science and Technology(Stock#DEFONLINE
14、) is available on the ASTM website, www.astm.org, or contactASTM Customer Service at serviceastm.org.*A Summary of Changes section appears at the end of this standardCopyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United StatesThis international st
15、andard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for theDevelopment of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.13.3
16、 activated sludge, nthe precipitated solid matter, con-sisting mainly of bacteria and other aquatic microorganisms,that is produced at a domestic wastewater treatment plant;activated sludge is used primarily in secondary sewage treat-ment to microbially oxidize dissolved organic matter in theeffluen
17、t.3.4 aerobic, adj(1) taking place in the presence of oxy-gen; (2) living or active in the presence of oxygen.3.5 biodegradation, nthe process of chemical breakdownor transformation of a material caused by organisms or theirenzymes.3.5.1 DiscussionBiodegradation is only one mechanismby which materia
18、ls are transformed in the environment.3.6 biomass, nbiological material including any materialother than fossil fuels which is or was a living organism orcomponent or product of a living organism.3.6.1 DiscussionIn biology and environmental science,biomass is typically expressed as density of biolog
19、ical 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.).3.6.2 DiscussionProducts of living organisms includethose materials pro
20、duced 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 material) and materials produced byprocessing living organisms, the
21、ir components or metabolites(for example, transesterified oil; also called biodiesel).3.7 blank, nin biodegradability testing, a test systemcontaining all system components with the exception of the testmaterial.3.8 inoculum, nspores, bacteria, single celled organisms,or other live materials, that a
22、re introduced into a test medium.3.9 lag phase, nthe period of diminished physiologicalactivity and cell division following the addition of microorgan-isms to a new culture medium.3.10 log phase, nthe period of growth of microorganismsduring which cells divide at a positive constant rate.3.11 mixed
23、liquor, nin sewage treatment, the contents ofan aeration tank including the activated sludge mixed withprimary effluent or the raw wastewater and return sludge.3.12 pre-adaptation, nthe pre-incubation of an inoculumin the presence of the test material under conditions similar tothe test conditions.3
24、.12.1 DiscussionThe aim of pre-adaptation is to improvethe precision of the test method by decreasing variability in therate of biodegradation produced by the inoculum. Pre-adaptation may mimic the natural processes which causechanges in the microbial population of the inoculum leading toa more rapi
25、d rate of biodegradation of the test material but notto a change in the final degree of biodegradation.3.13 pre-condition, nthe pre-incubation of an inoculumunder the conditions of the test in the absence of the testmaterial.3.14 supernatant, nthe liquid above settled solids.3.15 suspended solids (o
26、f activated sludge or other inocu-lum samples), nsolids present in activated sludge or inocu-lum samples that are not removed by settling under specifiedconditions.3.16 theoretical CO2,nthe amount of CO2which could intheory be produced from the complete oxidation of all of thecarbon in a material.3.
27、17 ultimate biodegradation, ndegradation achievedwhen a material is totally utilized by microorganisms resultingin the production of CO2(and possibly methane in the case ofanaerobic biodegradation), water, inorganic compounds, andnew microbial cellular constituents (biomass and secretions).4. Summar
28、y of Test Method4.1 Biodegradation of a lubricant or the component(s) of alubricant is estimated 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 o
29、f CO2which could be generated if all of the carbon in the test materialwere converted to CO2. Carbon dioxide is a product of aerobicmicrobial metabolism of carbon-containing materials and so isa direct measure of the test materials ultimate biodegradation.The evolved CO2is trapped in a Ba(OH)2or oth
30、er alkalinesolution and the amount of CO2absorbed is determined bytitrating the remaining hydroxide in solution.4.2 The carbon content of the test material is determined byTest Methods D5291 or another appropriate method and thetheoretical CO2is calculated from that measurement. It isnecessary to di
31、rectly measure the carbon content of the testmaterial instead of calculating this number, because of thecomplexity of the mixture of compounds present in lubricants.4.3 Biodegradability is expressed as a percentage of theo-retical CO2production.5. Significance and Use5.1 Results from this CO2evoluti
32、on test method suggest,within the confines of a controlled laboratory setting, thedegree of ultimate aerobic aquatic biodegradability of a lubri-cant or components of a lubricant. Test materials which achievea high degree of biodegradation in this test method may beassumed to easily biodegrade in ma
33、ny aerobic aquatic envi-ronments. (See also Test Method D5864.)5.2 Because of the stringency of this test method, a lowyield of CO2does not necessarily mean that the test material isnot biodegradable under environmental conditions, but indi-cates that further testing needs to be carried out in order
34、 toestablish biodegradability.5.3 Information on the toxicity of the test material to theinoculum 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 may be used as anaerobic inoculum. An i
35、noculum derived from soil or naturalD6139 182surface waters, or any combination of the three sources, mayalso be used in this test method.NOTE 1Allowance for various and multiple inoculum sources pro-vides access to a greater diversity of biochemical competency andpotentially represents more accurat
36、ely the capacity for biodegradation.5.5 A reference or control material known to biodegradeunder the conditions of this test method is necessary in order toverify the activity of the inoculum. The test method must beregarded as invalid and should be repeated using a freshinoculum if the reference do
37、es not demonstrate biodegradationto the extent of 60 % of the theoretical CO2within 28 days.5.6 The water solubility or dispersibility of the lubricant orcomponents may influence the results obtained and hence theprocedure may be limited to comparing lubricants or compo-nents with similar solubiliti
38、es.5.7 The ratio of carbon incorporated into cellular material tocarbon metabolized to CO2will vary depending on the organicsubstrate, on the particular microorganisms carrying out theconversion, and on the environmental conditions under whichthe conversion takes place. In principle, this variabilit
39、y com-plicates the interpretation of the results from this test method.5.8 The behavior of complex mixtures may not always beconsistent with the individual properties of the components.The biodegradability of the components may be suggestive ofwhether a mixture containing these components (that is,
40、a fullyformulated lubricant) is biodegradable but such informationshould be used judiciously.6. Apparatus6.1 Carbon Dioxide Scrubbing Apparatus (see Fig. 1):6.1.1 The following are required to produce a stream ofCO2-free air for aeration and for sparging aqueous solutionsand mixtures (for example, t
41、est medium, sewage inoculum):6.1.1.1 Erlenmeyer flask, one 1 L with side arm containing500 mL of 10 M sodium hydroxide (NaOH), and fitted with arubber stopper and an inlet tube that extends below the level ofthe NaOH solution or an equivalent apparatus or system.6.1.1.2 Erlenmeyer flask, one 1 L wit
42、h side arm containing500 mL of distilled water and fitted with a stopper and inlettube, or an equivalent apparatus or system.6.1.1.3 It is optional to add an empty 1 L Erlenmeyer flaskin series with the flasks to prevent liquid carryover.6.1.1.4 It is optional to add a 1 L Erlenmeyer flask contain-i
43、ng 500 mL of 0.1 M barium hydroxide Ba(OH)2 solution tomonitor for possible breakthrough CO2.6.1.2 Connect the flasks in series as shown in Fig. 1, usingvinyl or other suitable non-gas-permeable tubing, to a pressur-ized air system and purge air through the scrubbing solution.6.1.3 The CO2scrubbing
44、apparatus upstream of the Erlen-meyer flask containing the Ba(OH)2may be substituted with analternative system which effectively and consistently producesCO2-free air (that is, containing 1 ppm CO2).6.2 Incubation/Biodegradation ApparatusGledhill-typeShake Flask Units6(see Fig. 2)Each test material,
45、 reference,or blank control requires the following:6.2.1 Erlenmeyer Flasks, 2 L2 L Erlenmeyer flasks areused to hold the 1 L of total final aqueous volume but largervolume Erlenmeyer flasks (as large as 3 L to 4 L) may be usedif 2 L to 3 L final aqueous volumes are required. The amountsdescribed her
46、e are for 1 L final aqueous volumes carried out in2 L Erlenmeyer flasks; scale procedure accordingly if largerfinal aqueous volumes and larger Erlenmeyer flasks are neces-sary.6.2.2 StoppersEach stopper is fitted with a conical alka-line trap, an outlet and an inlet vent tube (see Fig. 2). Ensuretha
47、t the stopper fits tightly in the Erlenmeyer flask to preventany leaks.6.2.3 Conical Alkaline Trap Tube or UnitGlass, 40 mLconical tube (borosilicate glass, No. 8120 centrifuge tube orequivalent) welded to a glass support rod, or an equivalentapparatus, will be used to hold the Ba(OH)2solution fortr
48、apping the evolved CO2from aerobic biodegradation. Theopening in the alkaline trap tube is large enough to permit CO2diffusion into the barium hydroxide solution. The support rodof the conical trap shall fit tightly in the stopper.6.2.4 Inlet and Outlet Vent TubesThe inlet vent tubeattached to the s
49、topper extends down into the flask so that itwill be immersed below the surface of the aqueous mediumand will be used for sparging. The outlet vent tube will besituated significantly above the level of the aqueous mediumand will be used for venting. The two vent tubes shall fit tightlyin the stopper.6.2.5 Flexible tubing which is non-permeable to CO2will beused to connect the tops of inlet and outlet vent tubes to forma closed system.6Gledhill, W. E., “Screening Test for Assessment of Ultimate Biodegradability:LinearAlkyl Benzene Sulfonate,” Applied
