1、Designation:D613900 (Reapproved 2005) Designation: D6139 11Standard 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
2、 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 () indicates an editorial change since the last revision or reapproval.1. Scope1.1 This test method covers the determina
3、tion of the degree of aerobic aquatic biodegradation of fully formulated lubricants ortheir components on exposure to an inoculum under controlled laboratory conditions. This test method is an ultimatebiodegradation test that measures carbon dioxide (CO2) evolution.1.2 This test method is intended t
4、o specifically address the difficulties associated with testing water insoluble materials andcomplex mixtures such as are found in many lubricants.1.3 This test method is designed to be applicable to all non-volatile lubricants or lubricant components that are not toxic andnot inhibitory at the test
5、 concentration to the organisms present in the inoculum.1.4 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.1.5 This standard does not purport to address all the safety concerns, if any, associated with its use. It is the resp
6、onsibility ofthe user of this standard to establish appropriate safety and health practices and to determine the applicability of regulatorylimitations prior to use. Specific hazards are discussed in Section 10.2. Referenced Documents2.1 ASTM Standards:D1129Terminology Relating to Water ASTM Standar
7、ds:2D1193 Specification for Reagent WaterD1293 Test Methods for pH of WaterD4447 Guide for Disposal of Laboratory Chemicals and SamplesD5291 Test Methods for Instrumental Determination of Carbon, Hydrogen, and Nitrogen in Petroleum Products and LubricantsD5864D5864 Test Method for Determining Aerobi
8、c Aquatic Biodegradation of Lubricants or Their ComponentsE943 Terminology Relating to Biological Effects and Environmental Fate2.2 ISO Standard:34259:1992(E) Petroleum ProductsDetermination and application of precision data in relation to methods of test2.3 APHA Standards:42540B Total Solids Dried
9、at 103105C9215 Heterotrophic Plate Count3. Terminology3.1Definitions:3.1.1Definitions of terms applicable to this test method which are not described herein, appear in the Compilation of ASTMStandard Definitions (1990) or Terminology3.1 Definitions:3.2 Definitions of terms applicable to this test me
10、thod that are not described herein appear in the ASTM Online Dictionary of1This 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 N
11、ov.Jan. 1, 2005.2011. Published November 2005.March 2011. Originally approved in 1997. Last previous edition approved in 20002005 asD613900.D613900(2005). DOI: 10.1520/D6139-00R05.10.1520/D6139-11.2For referenced ASTM standards, visit the ASTM website, www.astm.org, or contact ASTM Customer Service
12、at serviceastm.org. For Annual Book of ASTM Standardsvolume information, refer to the standards Document Summary page on the ASTM website.3Available from American National Standards Institute, 11 West 42nd St., 13th Floor, New York, NY 10036.4Methods from Standard Methods for the Examination of Wate
13、r and Wastewater, latest edition. Available from the American Public Health Assoc.Association (APHA),1015 18th St., N.W.,800 I Street, NW, Washington, D.C. 20036. DC 20001.1This document is not an ASTM standard and is intended only to provide the user of an ASTM standard an indication of what change
14、s have been made to the previous version. Becauseit may not be technically possible to adequately depict all changes accurately, ASTM 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 officia
15、l document.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.Engineering Science and Technology5or Terminology E943.3.1.2activated sludge, n3.3 activated sludge, nthe precipitated solid matter, consisting mainly of bacteria and other aq
16、uatic microorganisms, that isproduced at a domestic wastewater treatment plant; activated sludge is used primarily in secondary sewage treatment to microbiallyoxidize dissolved organic matter in the effluent.3.1.3aerobic, adj.3.4 aerobic, adj(1 ) taking place in the presence of oxygen; (2) living or
17、 active in the presence of oxygen.3.1.4biodegradation, n3.5 biodegradation, nthe process of chemical breakdown or transformation of a test material caused by organisms or theirenzymes.3.1.4.1Discussion3.5.1 DiscussionBiodegradation is only one mechanism by which substances are removed from the envir
18、onment.3.1.5biomass, nany material, excluding fossil fuels, which is or was a living organism or component of a living organism.3.1.6blank, nin biodegradability testing3.6 biomass, nbiological material including any material other than fossil fuels which is or was a living organism orcomponent or pr
19、oduct of a living organism.3.6.1 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 ter
20、ms of mass (kg, MT, etc.) or volume (L, m3, bbl, etc.).3.6.2 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 ex
21、ample:pellets manufactured by shredding and pelletizing plant material) and materials produced by processing living organisms, theircomponents or metabolites (for example, transesterified oil; also called biodiesel).3.7 blank, nin biodegradability testing, a test system containing all system compone
22、nts with the exception of the test material.3.1.7inoculum, n3.8 inoculum, nspores, bacteria, single celled organisms, or other live materials, that are introduced into a test medium.3.1.8lag phase, n3.9 lag phase, nthe period of diminished physiological activity and cell division following the addit
23、ion of microorganismsto a new culture medium.3.1.9log phase, n3.10 log phase, nthe period of growth of microorganisms during which cells divide at a positive constant rate.3.1.10mixed liquor, n3.11 mixed liquor, nin sewage treatment, the contents of an aeration tank including the activated sludge mi
24、xed with primaryeffluent or the raw wastewater and return sludge.3.1.11pre-adaptation, n3.12 pre-adaptation, nthe incubation of an inoculum in the presence of the test material which is done prior to the initiationof the test and under conditions similar to the test conditions.3.1.11.1Discussion3.12
25、.1 DiscussionThe aim of pre-adaptation is to improve the precision of the test method by decreasing variability in the rateof biodegradation produced by the inoculum. Pre-adaptation may mimic the natural processes which cause changes in the microbialpopulation of the inoculum leading to a more rapid
26、 rate of biodegradation of the test material but is not expected to change theoverall extent of biodegradation of the test material.3.1.12pre-condition, n3.13 pre-condition, nthe pre-incubation of an inoculum under the conditions of the test in the absence of the test material.3.1.13supernatant, n3.
27、14 supernatant, nthe liquid above settled solids.3.1.143.15 suspended solids (of activated sludge or other inoculum samples), nsolids present in activated sludge or inoculumsamples that are not removed by settling under specified conditions.3.1.153.16 theoretical carbon dioxide (ThCO2), nthe amount
28、of CO2which could theoretically be produced from thecomplete biological oxidation of all of the carbon in a test material.3.1.16ultimate biodegradation, n3.17 ultimate biodegradation, ndegradation achieved when the test material is totally utilized by microorganisms resulting inthe production of CO2
29、(and possibly methane in the case of anaerobic biodegradation), water, inorganic compounds, and new5Gledhill, W. E., “Screening Test for Assessment of Ultimate Biodegradability: Linear Alkyl Benzene Sulfonate,” Applied Microbiology Vol 30, 1975, pp. 992929. Alsosee description of Gledhill shake flas
30、k unit in EPA Chemical Fate Testing Guidelines for Aerobic Aquatic Biodegradation, EPA Publication 560/6-82-003, No. CG-2000(August 1982); Federal Register, September 27, 1985, p. 39277, Section 796.3100; 40 CFR 796.3100, 1994.5ASTM Online Dictionary of Engineering Science and Technology (Stock#DEFO
31、NLINE) is available on the ASTM website, www.astm.org, or contact ASTM CustomerService at serviceastm.org.D6139 112microbial cellular constituents (biomass and secretions).3.1.17ultimate biodegradation test, n3.18 ultimate biodegradation test, na test which estimates the extent to which the carbon i
32、n a product has been converted toCO2or methane, either directly by measuring the production of CO2or methane, or indirectly by measuring the consumption ofO2.3.1.17.1Discussion3.18.1 DiscussionThe measurement of new biomass is not attempted.4. Summary of Test Method4.1 Biodegradation of a lubricant
33、or the component(s) of a lubricant is estimated by collecting and measuring the CO2producedwhen the lubricant or component is exposed to microorganisms under controlled aerobic aquatic conditions. This value is thencompared to the theoretical amount of CO2which could be generated if all of the carbo
34、n in the test material were converted toCO2. Carbon dioxide is a product of aerobic microbial metabolism of carbon-containing materials and so is a direct measure ofthe test materials ultimate biodegradation. The evolved CO2is trapped in a Ba(OH)2or other alkaline solution and the amountof CO2absorb
35、ed is determined by titrating the remaining hydroxide in solution.4.2 The carbon content of the test material is determined by Test Methods D5291 or another appropriate method and thetheoretical CO2is calculated from that measurement. It is necessary to directly measure the carbon content of the tes
36、t materialinstead of calculating this number, because of the complexity of the mixture of compounds present in lubricants.4.3 Biodegradability is expressed as a percentage of theoretical CO2production.5. Significance and Use5.1 Results from this CO2evolution test method suggest, within the confines
37、of a controlled laboratory setting, the degree ofultimate aerobic aquatic biodegradability of a lubricant or components of a lubricant. Test materials which achieve a high degreeof biodegradation in this test method may be assumed to easily biodegrade in many aerobic aquatic environments. (See also
38、TestMethod D5864.)5.2 Because of the stringency of this test method, a low yield of CO2does not necessarily mean that the test material is notbiodegradable under environmental conditions, but indicates that further testing needs to be carried out in order to establishbiodegradability.5.3 Information
39、 on the toxicity of the test material to the inoculum may be useful in the interpretation of low biodegradationresults.5.4 Activated sewage-sludge from a sewage treatment plant that principally treats domestic waste may be used as an aerobicinoculum. An inoculum derived from soil or natural surface
40、waters, or any combination of the three sources, may also be used inthis test 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
41、 control material known to biodegrade under the conditions of this test method is necessary in order to verifythe activity of the inoculum. The test method must be regarded as invalid and should be repeated using a fresh inoculum if thereference does not demonstrate biodegradation to the extent of 6
42、0 % of the theoretical CO2within 28 days.5.6 The water solubility or dispersibility of the lubricant or components may influence the results obtained and hence theprocedure may be limited to comparing lubricants or components with similar solubilities.5.7 The ratio of carbon incorporated into cellul
43、ar material to carbon metabolized to 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 the results f
44、rom this test method.5.8 The behavior of complex mixtures may not always be consistent with the individual properties of the components. Thebiodegradability of the components may be suggestive of whether a mixture containing these components (that is, a fullyformulated lubricant) is biodegradable bu
45、t such information should be used judiciously.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 for aeration and for sparging aqueous solutions andmixtures (for example, test medium, sewage inoculum):6.1.1.1 Erlenmeye
46、r flask, one 1-L with side arm containing 500 mL of 10 M sodium hydroxide (NaOH), and fitted with a rubberstopper and an inlet tube that extends below the level of the NaOH solution or an equivalent apparatus or system.6.1.1.2 Erlenmeyer flask, one 1-L with side arm containing 500 mL of distilled wa
47、ter and fitted with a stopper and inlet tube,or an equivalent apparatus or system.6.1.1.3 It is optional to add an empty 1-L Erlenmeyer flask in series with the flasks to prevent liquid carryover.6.1.1.4 It is optional to add a 1-L Erlenmeyer flask containing 500 mL of 0.1M barium hydroxide Ba(OH)2
48、solution to monitorfor possible breakthrough CO2.D6139 1136.1.2 Connect the flasks 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.6.1.3 The CO2scrubbing apparatus upstream of the Erlenmeyer
49、 flask containing the Ba(OH)2may be substituted with analternative system which effectively and consistently produces CO2-free air (that is, containing 1 ppm CO2).6.2 Incubation/Biodegradation Apparatus Gledhill-type Shake Flask Units6(see Fig. 2)Each test material, reference, orblank control requires the following:6.2.1 Erlenmeyer Flasks, 2-L2-L Erlenmeyer flasks are used to hold the 1 L of total final aqueous volume but larger volumeErlenmeyer flasks (as large as 3 to 4 L) may be used if 2 to 3-L final aqueous volumes are required. The amount
