1、Designation: D 2667 95 (Reapproved 2001)Standard Test Method forBiodegradability of Alkylbenzene Sulfonates1This standard is issued under the fixed designation D 2667; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the year of las
2、t revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon (e) indicates an editorial change since the last revision or reapproval.This standard has been approved for use by agencies of the Department of Defense.1. Scope1.1 This test method2covers the determinati
3、on of the degreeof biodegradability of alkylbenzene sulfonates. It serves as anindex of the suitability of the sulfonate for general use as asurfactant.1.2 In general, this test method distinguishes between sul-fonates in which the alkyl side chains are linear and those inwhich they are branched, si
4、nce the former are more readilybiodegradable. If the alkylbenzene sulfonate in fully formu-lated products is to be examined, it must be extracted using themethod noted in Annex A1. (See Appendix X1 for data.)1.3 This standard does not purport to address all of thesafety concerns, if any, associated
5、with its use. It is theresponsibility of the user of this standard to establish appro-priate safety and health practices and determine the applica-bility of regulatory limitations prior to use. Material SafetyData Sheets are available for reagents and materials. Reviewthem for hazards prior to usage
6、.2. Referenced Documents2.1 ASTM Standards:3D 1293 Test Methods for pH of WaterD 2330 Test Method for Methylene Blue Active SubstancesE 1625 Test Method for Determining Biodegradability ofOrganic Chemicals in Semi-Continuous Activated Sludge(SCAS)3. Summary of Test Method3.1 The sample is first subj
7、ected to a presumptive test basedon shake culture. When necessary, the sample may be sub-jected to a confirming test based on semicontinuous treatmentwith activated sludge.3.2 In the presumptive test, microorganisms are inoculatedinto a flask that contains a chemically defined microbial growthmedium
8、 (basal medium) and the surfactant to be tested.Aeration is accomplished by continuous shaking of the flask.Following two adaptive transfers, biodegradation is determinedby measuring the reduction in surfactant content during the testperiod.3.3 In the confirming test, activated sludge obtained from
9、asewage treatment plant is used. The sludge, the surfactant to betested, and a synthetic sewage used as an energy source for thesludge microorganisms are all placed in a specially designedaeration chamber. The mixture is aerated for 23 h, allowed tosettle, and the supernatant material removed. The s
10、ludgeremaining in the aeration chamber is then brought back tovolume with fresh surfactant and synthetic sewage and thecycle repeated. Biodegradation is determined by the reductionin surfactant content during each cycle.4. Significance and Use4.1 This test method is designed to determine whether the
11、sulfonate tested will be removed sufficiently by usual methodsof sewage treatment for the effluent to be safely discharged tothe environment without further treatment.4.2 If the surfactant reduction in the presumptive test equalsor exceeds 90 %, the material is considered to be adequatelybiodegradab
12、le without further testing.4.3 If the surfactant reduction in the presumptive test isbetween 80 and 90 %, the material should be subjected to theconfirming test.4.4 If the surfactant reduction in the presumptive test isbelow 80 %, the material is considered inadequately biode-gradable.4.5 If it is n
13、ecessary to run the confirming test, the surfactantreduction in this test must be at least 90 % for the material tobe considered adequately biodegradable.4.6 An example of data from both the presumptive andconfirming test can be found in Appendix X4.1This test method is under the jurisdiction of AST
14、M Committee E47 onBiological Effects and Environmental Fate and is the direct responsibility ofSubcommittee E47.04 on Environmental Fate and Transport of Biologicals andChemicals.Current edition approved May 14, 2002. Published December 1995. Originallypublished as D 2667 67 T. Last previous edition
15、 D 2667 89.2This test method is based on “AProcedure and Standards for the Determinationof the Biodegradability of Alkyl Benzene Sulfonate and Linear Alkylate Sulfonate”by the Committee on Biodegradation Test Methods of the Soap and the DetergentAssociation, Journal of the Americal Oil Chemists Soci
16、ety, Vol 42, 1965, p. 986.3For referenced ASTM standards, visit 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.1Copyright ASTM International, 100
17、Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.PRESUMPTIVE TEST (SHAKE CULTURE)5. Apparatus5.1 Shaking MachineA reciprocating shaker operating atabout 128 strokes of 51 to 101.6 mm (2 to 4 in.)/min or agyrator shaker operating at 225 to 250 r/min with an amplitudeof
18、25 to 51 mm (1 to 2 in.) should be used. (Other shakers maybe used if equivalent aeration can be demonstrated.)6. Reagents and Materials6.1 Purity of WaterEither distilled or deionized water maybe used in this test. It must be free of bacteriostatic materials.Water derived from steam condensate in m
19、any cases willcontain amines which are inhibitory to microbial growth.6.2 Basal Medium:6.2.1 The composition of the basal medium shall be asfollows:NH4Cl 3.0 gK2HPO41.0 gMgSO47H2O0.25KCl 0.25 gFeSO47H2O 0.002 gYeast extract 0.30 gWater 1.0 L6.2.2 The basal medium may be prepared by sequentiallydisso
20、lving the NH4Cl, K2HPO4, KCl, and FeSO4in approxi-mately 800 mL water and adjusting the pH to 7.2 6 0.2 witha dilute solution of hydrochloric acid or sodium hydroxide. Theyeast extract and MgSO4dissolved in 200 mL water are thenadded with stirring to the former solution. Alternatively, themedium may
21、 be prepared using suitable stock solutions of thesalts, but the pH must be adjusted before the MgSO4is added.In either case, the yeast extract must be added in dry formimmediately before use. It is important to use the basal mediumimmediately after preparation to avoid bacterial growth. Thebasal me
22、dium shall be dispensed into one of the followingErlenmeyer flasks: 500 mL in a 1-L flask, 1000 mL in a 2-Lflask, and 1500 mL in a 4-L flask.NOTE 1The 1-L and 2-L flasks are best suited for a gyratory shakerand the 4-L flask for a reciprocating shaker.NOTE 2The pH of the medium should be checked bef
23、ore use andadjusted to pH 6.8 to 7.2 if necessary.6.2.3 The flasks shall be stoppered with cotton plugs or theequivalent to reduce contamination and evaporation.6.3 Microbial Culture:6.3.1 SourceThe microbial inoculum may be obtainedfrom any of the following sources:6.3.1.1 Natural sources (soil, ri
24、ver/lake water, sewage, acti-vated sludge, secondary effluent, and so forth).6.3.1.2 Laboratory cultures (activated sludge, river die-away, and so forth).6.3.2 Maintenance of CultureIf desired, the culture maybe maintained as a shake flask culture by weekly transfers inthe basal medium plus 10 mg/L
25、linear alkyl sulfonate (LAS).4For each weekly transfer use 1 mL of 7-day culture for each100 mL of fresh medium.7. Standardization7.1 As a control on the culture and test conditions used, thetotal run is invalid if the result with a suitable reference sampleof linear alkyl sulfonate4is less than 90
26、% removal asmeasured by methylene blue active substance (MBAS) loss.8. Procedure8.1 Addition of Surfactant to Basal Medium:8.1.1 Add 10 mg/L of surfactant5(active basis) to the flaskscontaining basal medium. If surfactant stock solutions are used,stability during storage must be confirmed.8.1.2 Use
27、one flask for each surfactant being tested, plus onecontrol flask for LAS,4additional controls if desired (see Note3), and one blank flask containing all basal medium compo-nents but with no surfactant.NOTE 3A reference LAS sample that meets the standards of biode-gradability of both the presumptive
28、 and confirming tests is availablethrough the Environmental Protection Agency (EPA). This sample is acomposite of several commercially available products, believed to betypical (from a biodegradability standpoint) of LAS surfactants incommercial use. It is suggested that a control test should be con
29、ductedusing this material, whenever surfactant biodegradability determinationsare undertaken. Biodegradation values for the EPA LAS standard arenoted in the accompanying data from the EPA. A more complete andrecent analysis may be found in Appendix X2.NOTE 4Failure to repeatedly attain prescribed bi
30、odegradation valuesfor the surfactant control (LAS) indicates that conditions are unfavorablefor normal microbial activity or that an analytical problem exists. Suchproblems should be investigated by an experienced microbiologist or ananalytical chemist.8.2 InoculationUsing the culture described in
31、6.3, inocu-late the flasks. Use the same culture for all flasks includingcontrol and blank. Use 1 mL of inoculum for each 100 mL ofbasal medium in the flask.8.3 IncubationPlace flasks containing basal medium, sur-factant, and inoculum on a shaking machine that will produceacceptable aeration and mix
32、ing for biodegradation. Maintainthe temperature of the flask contents at 25 6 3C, and measure,and, if necessary, adjust the pH of the media at the start of eachincubation period to pH 6 to 8.8.4 Adaptation (acclimation)Prior to beginning the bio-degradation test, make two 72-h acclimation transfers
33、from theflasks from 8.3 according to the following illustrative drawingdescribing the sequence comprising 8.2, 8.3, and 8.4.8.5 Analysis (see Test Method D 2330):NOTE 5It is important to follow Test Method D 2330 exactly since itis known to eliminate the effects of interfering ions that might be pre
34、sent.8.5.1 To follow the course of biodegradation, removesamples from the shake flasks for analysis. Samples must betaken during the 8-day test at zero time (immediately afterinoculation and mixing of the flask contents) and on theseventh and eighth days. Samples at zero time of the twoadaptive tran
35、sfers are desirable to ensure proper initial concen-tration. Unless analyses are run immediately, the addition of 14LAS may be obtained through the EPA, Quality Assurance Branch of theEnvironmental Monitoring and Support Laboratory, Cincinnati, OH 45268.5Supporting data suggest that concentrations i
36、n excess of 10 mg/L may beinhibitory to the microorganisms in the shake culture. Data are available fromASTM Headquarters.D 2667 95 (2001)2mL of formaldehyde/100 mL of sample should be used forpreservation for any sample (0 time or 7 or 8 days). Whenpreservative is used, add to all samples including
37、 blank, andstore the samples at 4C.8.5.2 Since the analytical result from the blank sample isused to correct the results from the other flasks, use the samesample size (or dilution factor) for the blank as is used for theother samples.9. Calculation9.1 Calculate the net surfactant concentration by s
38、ubtractingthe analyzed blank values from the analyzed values for theother flasks.9.2 Calculate the percentage removal from the reduction insurfactant concentration as follows:Percentage removal Day x!5S02 B0! 2 Sx2 Bx!/S02 B0!#3 100 (1)where:S0and Sx= analyses of test surfactant cultures, andB0and B
39、x= analyses of blank cultures, on Days 0 and x,expressed as concentrations of MBAS, mg/L.9.3 The result of the test shall be calculated as the averageof the seventh and eighth day percentage removals.10. Precision and Bias10.1 SummaryStatistical analyses were employed to de-termine the reproducibili
40、ty of the methods and the bestestimate of the true percentage removal. Using these statisticsfor each surfactant, confidence limits around the true percent-age removal and lower tolerance limits for individual resultswere calculated.10.2 Statistical Approach Used:10.2.1 Three cooperative experiments
41、 were conducted dur-ing a 15-month period. Each experiment was designed toprovide for replicate units within each run and replicate runsfor each laboratory. Additionally, in the first experiment,replicate analyses for each unit were obtained, Thus, fourlevels or sources of variability were investiga
42、ted: laboratory-to-laboratory, run-to-run within laboratories, unit-to-unitwithin runs, and analysis-to-analysis within units.10.2.2 Since all the participating laboratories did not havethe facilities to conduct the entire testing scheme, the statisticalanalysis was performed recognizing the varying
43、 number ofdegrees of freedom in the experimental design. Test results ateach level of variability were averaged to yield the average forthe next higher level; for example, the grand mean is theaverage of laboratory means rather than the average of indi-vidual runs or unit means. It is believed that
44、any slight loss inprecision of the confidence limits is of less importance thanunduly biasing the results when a few laboratories submit alarger proportion of the determinations.10.2.3 It was observed from the first set of data thatvariaility increased as the percentage removal decreased, andthat th
45、e distribution of results was skewed toward the lowerpercentage removal values. As a variance stabilizing step, thesquare root transformation attributed to Yates and discussed byBartlett6was applied to the data prior to analysis. Thetransformation used was:X 5 100 2 Y 1 Z! (2)where:Y = observed perc
46、entage removal value, andZ = small value.As all calculations were done by computer, a range of Zvalues from 0 to 2.0 was explored. It was found that Z = 0.1successfully stabilized the variance. In the transformed statethe population was found to approach normality.10.2.4 After transformation, means
47、were determined and ananalysis of variance performed to estimate the components ofvariance for the sources listed above. Using these statistics,confidence limits around the true percentage removal andlower tolerance limits for individual results were calculated.10.3 Results:10.3.1 Components of Vari
48、anceDuring the early work,analyses of the components of variance indicated no need forduplicate analyses and only single analyses were run for theremainder of the study. Considering the other sources ofvariability, laboratory-to-laboratory variations were signifi-cantly greater than variation betwee
49、n runs in the same labora-tory. Table 1 summarizes the relative importance of the sourcesof variability. These data are pooled variances from five LASmaterials.10.3.2 Confidence and Tolerance LimitsTable 2 presentsthe means and limits obtained. The lower tolerance limit is thatvalue above which 95.0 % of the results of single determina-tions are expected to fall (with 95 % confidence).CONFIRMING TEST(SEMICONTINUOUS ACTIVATED SLUDGE)11. Apparatus11.1 Aeration Chambers (see Fig. 1):11.1.1 ConstructionUse methyl methacrylate tubing 83mm (314 in.) in insid
