1、Designation: D 6139 00 (Reapproved 2005)An American National StandardStandard Test Method forDetermining the Aerobic Aquatic Biodegradation ofLubricants or Their Components Using the Gledhill ShakeFlask1This standard is issued under the fixed designation D 6139; the number immediately following the
2、designation indicates the year oforiginal adoption or, 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 t
3、he determination of the degreeof aerobic aquatic 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
4、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 to be applicable to allnon-volatile lubricants or lubricant components that are nottoxic and not inhibitory
5、at the test concentration to theorganisms present 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 t
6、he responsibilityof the user of this standard to 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:2D 1129 Terminology Relating to WaterD 1
7、193 Specification for Reagent WaterD 1293 Test Methods 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 LubricantsD 5864 Test Method for Determining Aerobic Aquatic
8、Bio-degradation of Lubricants or Their ComponentsE 943 Terminology Relating to Biological Effects and En-vironmental 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 103
9、105C9215 Heterotrophic Plate Count3. Terminology3.1 Definitions:3.1.1 Definitions of terms applicable to this test methodwhich are not described herein, appear in the Compilation ofASTM Standard Definitions (1990) or Terminology E 943.3.1.2 activated sludge, nthe precipitated solid matter,consisting
10、 mainly of bacteria and other aquatic microorgan-isms, that is produced at a domestic wastewater treatmentplant; activated sludge is used primarily in secondary sewagetreatment to microbially oxidize dissolved organic matter in theeffluent.3.1.3 aerobic, adj.(1) taking place in the presence ofoxygen
11、; (2) living or active in the presence of oxygen.3.1.4 biodegradation, nthe process of chemical break-down or transformation of a test material caused by organismsor their enzymes.3.1.4.1 DiscussionBiodegradation is only one mechanismby which substances are removed from the environment.3.1.5 biomass
12、, nany material, excluding fossil fuels,which is or was a living organism or component of a livingorganism.1This test method is under the jurisdiction of ASTM Committee D02 onPetroleum Products and Lubricants and is the direct responsibility of SubcommitteeD02.12 on Environmental Standards for Lubri
13、cants.Current edition approved Nov. 1, 2005. Published November 2005. Originallyapproved in 1997. Last previous edition approved in 2000 as D 6139 00.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStand
14、ards volume information, refer to the standards Document Summary page onthe ASTM website.3Available from American National Standards Institute, 11 West 42nd St., 13thFloor, New York, NY 10036.4Methods from Standard Methods for the Examination of Water and Wastewa-ter, latest edition. Available from
15、the American Public Health Assoc. (APHA), 101518th St., N.W., Washington, D.C. 20036.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.3.1.6 blank, nin biodegradability testing, a test systemcontaining all system components with the ex
16、ception of the testmaterial.3.1.7 inoculum, nspores, bacteria, single celled organ-isms, or other live materials, that are introduced into a testmedium.3.1.8 lag phase, nthe period of diminished physiologicalactivity and cell division following the addition of microorgan-isms to a new culture medium
17、.3.1.9 log phase, nthe period of growth of microorganismsduring which cells divide at a positive constant rate.3.1.10 mixed liquor, nin sewage treatment, the contents ofan aeration tank including the activated sludge mixed withprimary effluent or the raw wastewater and return sludge.3.1.11 pre-adapt
18、ation, nthe incubation of an inoculum inthe presence of the test material which is done prior to theinitiation of the test and under conditions similar to the testconditions.3.1.11.1 DiscussionThe aim of pre-adaptation is to im-prove the precision of the test method by decreasing variabilityin the r
19、ate 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 material but isnot expected to change the overall extent of biodegradation ofthe test
20、 material.3.1.12 pre-condition, nthe pre-incubation of an inoculumunder the conditions of the test in the absence of the testmaterial.3.1.13 supernatant, nthe liquid above settled solids.3.1.14 suspended solids (of activated sludge or other inocu-lum samples), nsolids present in activated sludge or
21、inocu-lum samples that are not removed by settling under specifiedconditions.3.1.15 theoretical carbon dioxide (ThCO2), nthe amountof CO2which could theoretically be produced from thecomplete biological oxidation of all of the carbon in a testmaterial.3.1.16 ultimate biodegradation, ndegradation ach
22、ievedwhen the test material is totally utilized by microorganismsresulting in the production of CO2(and possibly methane in thecase of anaerobic biodegradation), water, inorganic com-pounds, and new microbial cellular constituents (biomass andsecretions).3.1.17 ultimate biodegradation test, na test
23、which esti-mates the extent to which the carbon in a product has beenconverted to CO2or methane, either directly by measuring theproduction of CO2or methane, or indirectly by measuring theconsumption of O2.3.1.17.1 DiscussionThe measurement of new biomass isnot attempted.4. Summary of Test Method4.1
24、 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 of CO2which could be
25、 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 other alkalinesolution
26、 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 D 5291 or another appropriate method and thetheoretical CO2is calculated from that measurement. It isnecessary to directly measure the
27、 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 CO2evolution test method sug
28、gest,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 many aerobic aquatic
29、 envi-ronments.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 toestablish biodegradability.5.3 Information on
30、 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 inoculum derived from soil or naturalsurface wate
31、rs, 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 accurately the capacity for biodegradation.5.5 A reference or co
32、ntrol 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 does not demonstrate biodegradationto the extent of 60 % of
33、 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 solubilities.5.7 The ratio of carbon incorporated into cellular mat
34、erial 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 variability com-plicates the interpretation of the results from thi
35、s 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 ofD 6139 00 (2005)2whether a mixture containing these components (that is, a fullyformulated lubricant) is biodegra
36、dable 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, test medium, sewage inoculum):6.1.1.1 Erl
37、enmeyer 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 with side arm containing500 mL of distilled
38、 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-ing 500 mL of 0.1M barium hydroxide Ba(OH
39、)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 apparatus upstream of the Erlen-meyer fla
40、sk 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 Apparatus Gledhill-typeShake Flask Units5(see Fig. 2)Each test material, reference,or blank control requires the
41、 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 to 4 L) may be used if2 to 3-L final aqueous volumes are required. The amountsdescribed here are for 1-L final aqueous volumes carried
42、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). Ensurethat the stopper fits tightly in the Erlenmeyer
43、 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 fortrapping the evolved CO2from aerobic biodegrad
44、ation. 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 stopper extends down into the flask so that i
45、twill 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-permeabl
46、e to CO2will beused to connect the tops of inlet and outlet vent tubes to forma closed system.6.2.6 AgitatorsIncubator-shaker table unit or equivalent,or stirrers may be used to agitate the aqueous mixture in theErlenmeyer flasks.6.3 Analytical Balance, to weigh out test material or refer-ence mater
47、ial to be added to the test flask (capable of weighingto appropriate precision and accuracy, for example, 60.0001 g)6.4 Titration Apparatus for Measuring the Production ofCO2:6.4.1 Appropriate graduated burette filled with standardHCl solution.6.4.2 Alternatively, an automatic titration apparatus in
48、which the burette dispenser is filled with standard HCl solu-tion. Automatic titrations are carried out to a potentiometricend point of pH 8.3 (that is, phenolphthalein end pointequivalent)6.5 Glass Wool, for filtering the inoculum.7. Reagents and Materials7.1 Purity of ReagentsReagent grade chemica
49、ls 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 where5Gledhill, W. E., “Screening Test for Assessment of Ultimate Biodegradability:LinearAlkyl Benzene Sulfonate,” Applied Microbiology Vol 30, 1975, pp. 992929.Also see description of Gledhill shake flask unit in EPA Chemical Fate TestingGuidelines forAerobicAquatic Biodegradation, EPAPublication 560/6-82-003, No.CG-2000 (August 1982); Federal Register, Septe
