1、Designation: E 1279 89 (Reapproved 2001)Standard Test Method forBiodegradation By a Shake-Flask Die-Away Method1This standard is issued under the fixed designation E 1279; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the year of
2、 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 describes procedures for assessing thebiodegradation of chemicals in natural surface water samples.
3、1.2 This test method provides an opportunity to evaluaterates of biodegradation in the presence and absence of naturalsediment materials. It also may provide limited information onthe abiotic degradation rate, and sorption to sediment andvessel walls.1.3 This test method allows for the development o
4、f afirst-order rate constant, based on the disappearance of the testcompound with time, and a second-order rate constant, nor-malized for changes in microbial biomass.1.4 This test method requires a chemical specific analyticalmethod and the concentrations of test substance employed aredependent on
5、the sensitivity of the analytical method.1.5 This test method is designed to be applicable to com-pounds that are not inhibitory to bacteria at the concentrationsused in the test method, which do not rapidly volatilize fromwater, that are soluble at the initial test concentration and thatdo not degr
6、ade rapidly by abiotic processes, such as hydrolysis.1.6 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 to establish appro-priate safety and health practices and determine the applica-bility o
7、f regulatory limitations prior to use.2. Referenced Documents2.1 ASTM Standards:2D 1193 Specification for Reagent WaterD 4129 Test Method for Total and Organic Carbon in Waterby Oxidation and Coulometric DetectionE 895 Practice for Determination of Hydrolysis Rate Con-stants of Organic Chemicals in
8、Aqueous SolutionsE 896 Test Method for Conducting Aqueous Direct Pho-tolysis TestsE 1194 Test Method for Vapor PressureE 1195 Test Method for Determining a Sorption Constant3. Summary of Test Method3.1 The shake-flask die-away biodegradation method issimilar to river water die-away tests described b
9、y manyauthors, including Degens et al (1),3Eichelberger and Licht-enberg (2), Saeger and Tucker (3), Paris et al (4), and Cripe etal (5). It differs from most die-away methods by providing foran evaluation of the effects of natural sediments on thetransformation of the test compound and by the use o
10、f shakingto ensure a dissolved oxygen supply. Each test compound(substrate) is dissolved in water collected from a field site, withand without added natural sediment and with and withoutsterilization. Initial substrate concentrations typically are rela-tively low (g/L), analytical capabilities permi
11、tting. Loss oftest compound with time is followed by an appropriate,chemical-specific analytical technique. Changes in microbialbiomass also may be followed by the use of an appropriatetechnique such as bacterial plate counts. Data obtained duringuse of the test method are used to provide the follow
12、inginformation: (a) the abiotic degradation rate in the presenceand absence of sediment and (b) the combined biotic andabiotic degradation rate in the presence and absence ofsediment.4. Significance and Use4.1 Most of the simpler methods used to screen chemicalsfor biodegradation potential employ me
13、asurements that are notspecific to the test substance, such as loss of dissolved organiccarbon, evolution of respiratory carbon dioxide, or uptake ofdissolved oxygen. Such methods generally are used to evaluatethe transformation of the test substance to carbon dioxide,water, oxides or mineral salts
14、of other elements, or productsassociated with the normal metabolic processes of microorgan-isms (ultimate biodegradability), or both. These methods re-quire the use of relatively high initial concentrations of the testsubstance, generally 10 mg/L or higher, unless the tests areconducted using14C-rad
15、iolabeled test compounds. Biodegra-dation tests measuring14C-CO2evolution, for example, can be1This specification is under the jurisdiction of Committee E47 on BiologicalEffects and Environmental Fate and is the direct responsibility of SubcommitteeE47.04 on Environmental Fate of Chemical Substances
16、.Current edition approved Jan. 27, 1989. Published March 1989.2For 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
17、.3The boldface numbers in parentheses refer to the list of references at the end ofthis test method.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.conducted using initial concentration of test compound at partsper billion. These tes
18、ts, however, require specialized equip-ment and the custom preparation of appropriately labeledcompound is often very expensive.4.2 Die-away biodegradation methods are simple simulationmethods that employ water collected from natural watersources and follow the disappearance of an added amount ofthe
19、 test substance resulting from the activity of microorganismsin the water sample. The chemical-specific analytical tech-niques used to follow the disappearance of the test substance,typically are employed using relatively low initial concentra-tions of the test substance. Most environmental pollutan
20、ts arepresent in the environment at relatively low concentrations(less than 1 mg/L) and it has been observed that biodegradationrates obtained using high test compound concentrations may bequite different from those observed at lower concentrations (6).4.3 The transformation of the test substance to
21、 an extentsufficient to remove some characteristic property of the mol-ecule, resulting in the loss of detection by the chemicalspecificanalytical technique, is referred to as primary biodegradation.For many purposes, evidence of primary biodegradation issufficient, especially when it is known or ca
22、n be shown thattoxicity, or some other undesirable feature, associated with thetest compound is removed or significantly reduced as a resultof the primary biodegradation. A determination of ultimatebiodegradation, on the other hand, is usually required onlywhen treatability or organic loading are is
23、sues of concern.Furthermore, many of the simpler methods, such as thosemeasuring CO2evolution (see 4.1), may not detect primarybiodegradation.4.4 The use of low substrate concentration enhances theprobability of observing first-order, or pseudo first-order,kinetics. Thus, a rate constant for the pri
24、mary biodegradationreaction and a half-life can be derived from the test compoundunder defined incubation conditions. Rate constants are re-quired in many environmental fate mathematical models.5. Apparatus5.1 Carefully Cleaned Glass or Plastic Carboys, requiredfor the collection and transport of fi
25、eld water samples.5.2 Field Sediment Samples, obtained using scoop, beaker,or box sampler, as appropriate.5.3 A Rotary Shaker, capable of holding 2-L Erlenmeyerflasks and shaking at 140 to 150 r/min is required for theincubation of test flasks. Temperature control (62C) may beincorporated in an incu
26、bator/shaker unit or may be obtained byplacing the shaker in a temperature controlled space. The flasksshould be constructed of material that minimizes sorption oftest or reference compound to the walls of the flasks. Ingeneral, glass is the best choice.5.4 A Gas Chromatograph, or other suitable ins
27、trumentequipped with a detector sensitive to the test compound(s) andreference compound is required for the chemical-specificanalysis of the test and reference compounds.6. Reagents and Materials6.1 Reference compounds are desirable to evaluate thebiodegradation potential of the microbial population
28、.Asuitablereference compound will be biodegradable under the testconditions but not so readily biodegradable that it is completelydegraded within a small fraction of the normal test period.7. Sampling7.1 Take samples from each flask according to a scheduleappropriate to the rate of biodegradation of
29、 the test andreference substances. Sampling should be sufficiently frequentto establish plots of degradation versus time and to permit thedetermination of rate constants. Take a minimum of sixsamples from time zero until completion of the test. A nominaltest time of 28 days allows a reasonable perio
30、d for observationswith slowly degraded substances. The test period may beextended beyond 28 days if necessary to calculate a half-life.Tests may be terminated prior to 28 days when more than 50 %of the starting material has disappeared from solution, due tobiodegradation.7.2 Remove duplicate samples
31、 of a sufficient size from eachflask at appropriate intervals from day 1 (t = 24 h) untilcompletion of the test. Centrifuge each sample to removesuspended particulates. Analyze the supernatant (or a suitableextract of the supernatant) to determine the concentration oftest or reference compound. A re
32、cord is maintained of com-pound concentration versus time for each flask. If adsorption tosediment solids is a significant factor, extract the sediment plugand analyze the extract to more fully account for untrans-formed test compound.7.3 If microbial adaptation (a lag phase with little or no lossof
33、 test compound followed by relatively rapid loss) is sus-pected, add additional test compound to that flask and thecorresponding control flask, at or near the normal end of thetest period.Adaptation is indicated if the microorganisms in thetest flask degrade the added compound without a lag period a
34、ndthe control flask, to which test compound has been added,exhibits a lag prior to degradation. Do not use the lag period inthe calculation of the biodegradation rate. If there is a lagperiod due to adaptation, use the end of the lag period as timezero when calculating the first-order constant (see
35、section8.2.1). For an example, see Cripe et al (5).7.4 If desired, samples also may be taken for biomassdeterminations. Sampling times should coincide with the timesof sampling for chemical concentrations.8. Procedure8.1 Field Sampling:8.1.1 Collect water and sediment from a selected field site(for
36、example, river, lake, or estuary), the day before testinitiation. Measure the salinity (when appropriate), watertemperature, and pH at the time of sampling. Collect water,from approximately 60 mm below the air/water surface, inclean glass or plastic carboys. Remove floating or suspendedparticulates,
37、 preferably by filtering the water through a 3-mmembrane filter. Collect the upper 5 to 10 mm of underlyingsediment by skimming with a beaker, scoop, or box sampler.Screen the sediment through a sieve with 2 mm-openings toremove larger particles and biota. Omit sand by resuspendingdetritus and fine
38、particles and decanting. This is necessarybecause sand cannot be quantitatively transferred from a slurrywith a pipet. Add field water to or decant it from the sievedsediment until there is approximately a 1:1 ratio betweenE 1279 89 (2001)2sediment and water volumes. Return the water sample and thes
39、ediment slurry to the laboratory in closed containers.8.1.2 If there is no sediment layer at the field site (forexample, the stream or lake bed is all rock), omit the sedimentcollection and use procedures.8.2 Handing of Field Samples:8.2.1 Stir the sediment slurry and the site water continu-ously at
40、 room temperature until use in the test method.8.2.2 Measure the concentration of sediment in the slurry byfiltering 5-mL samples of well-mixed slurry through predried(105, 1 h) 0.45-m membrane filters. The slurry must bestirred vigorously during sampling to ensure homogeneity.Rinse the slurry sampl
41、ing pipet, sediment, and filter with 2 to3 mL of deionized water. The filter and sediment are then driedat 105 for 1 h. Determine the weight of the sediment after thedried filter and sediment have cooled to room temperature in adessicator. Use the weight of sediment per mL of slurry tocalculate the
42、volume of slurry to be used in test flasks.8.3 Preparation of Flasks:8.3.1 Initial test compound concentration in the methodtypically is 200 g/L. This concentration is generally highenough for analytical sensitivity and low enough to be envi-ronmentally realistic. Choose other concentrations as appr
43、opri-ate.8.3.2 Control Water FlasksAdd 1 L of site water to eachof two 2-L Erlenmeyer flasks.8.3.3 Control Sediment FlasksAdd 900 to 950 mL of sitewater to each of two 2-LErlenmeyer flasks. Sufficient sedimentslurry is added to each flask to achieve a final (following asecond addition of site water)
44、 suspended sediment concentra-tion of 500 mg/L (on a dry weight of sediment basis). Addadditional site water to achieve a final volume of site water plussediment equal to 1 L.8.3.4 Amended Site WaterAdd sufficient test compound(or reference compound) to 9 to 10 L of site water to producethe desired
45、initial concentration. Generally, analytical sensitiv-ity permitting, the desired initial concentration is 200 g/L and2.0 mg of test compound are added to 10 L of site water.Addition of test compound may be accomplished through theaddition of a solution of the test compound in a volatile solvent(for
46、 example, acetone) to a clean, empty vessel, removal of thevolatile solvent by flushing with a clean air or nitrogen stream,and addition of 10 L of site water. Analyze the final solution todetermine the concentration of test compound. To compensatefor the volume of sediment slurry and formalin added
47、 later(8.3.6-8.3.8) an excess of test compound may be added to yielda concentration greater than 200 g/L. The amount of amendedsite water added to the active water, active sediment, sterilewater, and sterile sediment flasks is then adjusted to yield afinal concentration of 200 g/L test substance. Un
48、amended sitewater is used, as necessary, to produce a final volume of 1 L ineach flask.8.3.5 Active Water FlasksAdd 1 L of amended water toeach of two 2-L Erlenmeyer flasks.8.3.6 Active Sediment FlasksAdd 900 to 950 mL ofamended water to each of two 2-L Erlenmeyer flasks. Addsufficient sediment slur
49、ry to each flask to achieve a final(following a second addition of amended site water) suspendedsediment concentration of 500 mg/L. Add additional amendedsite water to achieve a final volume of water plus sedimentequal to 1 L.8.3.7 Sterile Water FlasksAdd 900 to 950 mL of amendedwater to each of two 2-L Erlenmeyer flasks. Add 20 mL of37 % formaldehyde solution (formalin) to each flask to act asa sterilant. Add additional amended site water to each flask toachieve a final volume of 1 L. If an interaction betweenformalin and the test or reference compound is likely orsusp
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