1、Designation: D6139 17Standard 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 the safetyconcerns, if any, associated with its use. It is the responsibilityof the user of this standard to
6、establish appropriate safety andhealth practices and to determine the applicability of regula-tory limitations prior to use. Specific hazards are discussed inSection 10.2. Referenced Documents2.1 ASTM Standards:2D1193 Specification for Reagent WaterD1293 Test Methods for pH of WaterD4447 Guide for D
7、isposal 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 ComponentsE943 Terminology Relating to Biological E
8、ffects 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. Terminology3.1 Definitions:3.2 Definitions of terms ap
9、plicable to this test method thatare not described herein appear in theASTM Online Dictionaryof Engineering Science and Technology5or TerminologyE943.3.3 activated sludge, nthe precipitated solid matter, con-sisting mainly of bacteria and other aquatic microorganisms,that is produced at a domestic w
10、astewater treatment plant;activated sludge is used primarily in secondary sewage treat-ment to microbially oxidize dissolved organic matter in theeffluent.3.4 aerobic, adj(1 ) taking place in the presence ofoxygen; (2) living or active in the presence of oxygen.3.5 biodegradation, nthe process of ch
11、emical breakdownor transformation of a material caused by organisms or theirenzymes.1This 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.Cu
12、rrent edition approved Jan. 1, 2017. Published February 2017. Originallyapproved in 1997. Last previous edition approved in 2011 as D6139 11. DOI:10.1520/D6139-17.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book
13、 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 York, NY 10036, http:/www.ansi.org.4Methods from Standard Methods for the Examination of Water and Wastewa
14、ter,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) is available on the ASTM website, www.astm.org, or contactASTM Customer Service at serviceastm.org.*A
15、 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 standard was developed in accordance with internationally recognized principles on standardization establ
16、ished 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.13.5.1 DiscussionBiodegradation is only one mechanismby which materials are transformed in the environment.
17、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 biological materialper unit sample volume, a
18、rea, 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 produced directly by living organisms asm
19、etabolites (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, their components or metabolites(for examp
20、le, 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 are introduced into a test medium.3.9 l
21、ag 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 liquor, nin sewage treatment, the cont
22、ents 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.3.12.1 DiscussionThe aim of pre-adaptat
23、ion 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 rapid rate of biodegradation of the test m
24、aterial 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 (of activated sludge or other inocu-lum
25、samples), nsolids present in activated sludge or inocu-lum samples that are not removed by settling under specifiedconditions.3.16 theoretical carbon dioxide (ThCO2), nthe amount ofCO2which could theoretically be produced from the completebiological oxidation of all of the carbon in a test material.
26、3.17 ultimate biodegradation, ndegradation achievedwhen the test material is totally utilized by microorganismsresulting in the production of CO2(and possibly methane in thecase of anaerobic biodegradation), water, inorganiccompounds, and new microbial cellular constituents (biomassand secretions).3
27、.18 ultimate biodegradation test, na test which estimatesthe extent to which the carbon in a product has been convertedto CO2or methane, either directly by measuring the productionof CO2or methane, or indirectly by measuring the consump-tion of O2.3.18.1 DiscussionThe measurement of new biomass is n
28、otattempted.4. Summary 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
29、 theoretical amount of 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 trapp
30、ed in a Ba(OH)2or other 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.
31、 It isnecessary to directly 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
32、 from this CO2evolution 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 ea
33、sily biodegrade in many 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
34、 carried out in order 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 ana
35、erobic inoculum. An inoculum derived from soil or naturalsurface waters, or any combination of the three sources, mayalso be used in this test method.D6139 172NOTE 1Allowance for various and multiple inoculum sources pro-vides access to a greater diversity of biochemical competency andpotentially re
36、presents more accurately 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 freshinoculu
37、m if the reference does 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 wi
38、th similar solubilities.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 princ
39、iple, this variability 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
40、components (that is, 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 mix
41、tures (for example, test 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 Erlenmey
42、er flask, one 1 L with 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 Erlen
43、meyer flask contain-ing 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.
44、1.3 The CO2scrubbing 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.
45、2)Each test material, 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
46、 amountsdescribed here 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 (s
47、ee Fig. 2). Ensurethat 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
48、Ba(OH)2solution fortrapping 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
49、tubeattached to the stopper 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 Benz
