1、Designation: E 2170 01Standard Test Method forDetermining Anaerobic Biodegradation Potential of OrganicChemicals Under Methanogenic Conditions1This standard is issued under the fixed designation E 2170; the number immediately following the designation indicates the year oforiginal adoption or, in th
2、e 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 screening procedures for thedetermining the biodegradation po
3、tential of organic chemicalsunder methanogenic conditions using an inoculum fromanaerobic sewage treatment. The conditions described in thistest method are not necessarily optimal for biodegradation,since it involves a dilute inoculum and a relatively highconcentration of test chemical.1.2 The test
4、method is applicable to most organic chemicals,assuming that they can be accurately dosed into the testsystems. The test is not applicable to chemicals that areinhibitory at the concentration used in the test or those whosevolatility generates significant head pressure in the test vessels.1.3 This s
5、tandard 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 of regulatory limitations prior to use. Specific hazardstatement
6、s are given in Section 8.2. Referenced Documents2.1 ASTM Standards:2D 1193 Specification for Reagent WaterD 5904 Test Method for Total Carbon, Inorganic Carbon,and Organic Carbon in Water by Ultraviolet, PersulfateOxidation, and Membrane Conductivity DetectionD 5907 Test Method for Filterable and No
7、n-filterable Mat-ter in Water3. Terminology3.1 Definitions:3.1.1 biodegradation potentialthe ability of an organicchemical to undergo partial or complete biodegradation underthe conditions of the test. In the context of this test, biodegra-dation is evidenced by the production of methane and carbond
8、ioxide, which is measured as an increased pressure in theheadspace and increased dissolved inorganic carbon in themedium.3.1.2 digester sludgesludge which is obtained from ananaerobic reactor used to reduce the solids recovered andproduced during the treatment of wastewater. Such reactors aregeneral
9、ly operated at 35C with a typical retention time of 25to 30 days.4. Summary of Test Method4.1 The test chemical and an anaerobic digester sludgeinoculum are suspended in a defined anaerobic medium andincubated in a sealed vessel at 35C. The increase in headspacepressure resulting from the production
10、 of carbon dioxide andmethane is measured at various time intervals using a pressuremeasuring device. At the termination of the test, the level ofdissolved carbon dioxide is assayed by measuring dissolvedinorganic carbon in the medium. The amount of total gasproduced from the test chemical is determ
11、ined by comparingtotal gas production in the experimental treatments and theblanks.5. Significance and Use5.1 Biodegradation is an important process for the removalof many chemical substances in anaerobic environments. Inmethanogenic environments, this process involves dissimila-tion to carbon dioxi
12、de and methane.5.2 This test method has been developed to screen organicchemicals for anaerobic biodegradation potential under metha-nogenic conditions, where there is no ingress of oxygen. Ahigh-biodegradability result in this test method is good evi-dence that the test substance will be biodegrada
13、ble in wastetreatment plant anaerobic digesters and in many natural envi-ronments. Conversely, a low-biodegradation result may havecauses other than poor biodegradability of the test substance.Inhibition of the microbial inoculum by the test substance mayhave occurred at the concentrations tested, o
14、r conditions in thetest may have been inappropriate for the development of anacclimated microbial population. Toxicity should be suspectedwhen the gas production in the blanks exceeds that in the test1This test method is under the jurisdiction of ASTM Committee E47 onBiological Effects and Environme
15、ntal Fate and is the direct responsibility ofSubcommittee E47.04 on Environmental Fate of Chemical Substances.Current edition approved Oct. 10, 2001. Published December 2001.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For
16、Annual Book of ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM website.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.vessels. In such cases, further work is needed to assess theanaerobic bi
17、odegradation potential of the chemical. An esti-mate of the expected environmental concentration will help toput any observed toxic effects into perspective.6. Apparatus6.1 Incubator, water or sand bath, thermostatically con-trolled at 35 6 2C.6.2 Pressure-Resistant Glass Test Vessels, each fitted w
18、ith agas-tight septum, capable of withstanding about 2 bar. From apractical point of view, the use of 160 mL serum bottles, whichare often described in catalogs as 125 mL serum bottles, sealedwith butyl rubber serum stoppers with crimped aluminum capsis recommended.6.3 Pressure Measuring DeviceAn ex
19、ample of such adevice would be a pressure transducer attached to a syringeneedle, which may include a three-way gas-tight valve forreleasing excess pressure. The device should be calibratedunder conditions relevant to the test setup.6.4 Carbon Analyzer, suitable for the direct determinationof organi
20、c and inorganic carbon in the test vessels.6.5 System for preparing and manipulating media, inoculaand samples under anaerobic conditions. The system could bean anaerobic chamber or gas sparging system.7. Reagents and Materials7.1 Purity of ReagentsReagent grade chemicals should beused in all tests.
21、 Unless otherwise indicated, it is intended thatall reagents should conform to the specifications of theCommittee on Analytical Reagents of the American ChemicalSociety, where such specifications are available. Other gradesmay be used, provided it is first ascertained that the reagent isof sufficien
22、tly high purity to permit its use without lesseningthe accuracy of the determination.7.2 Purity of the WaterUnless otherwise indicated, refer-ence to water shall be understood to mean reagent water asdefined by Type IV of Specification D 1193.7.3 Test MediumThe test medium consists of the follow-ing
23、 constituents in water.Anyhdrous potassium dihydrogen phosphate (KH2PO4) 0.27 g/LDisodium hydrogen phosphate dodecahydrate (NaHPO4-12H2O) 1.12 g/LAmmonium chloride (NH4Cl) 0.53 g/LCalcium chloride dihydrate (CaCl2-2H2O) 0.075 g/LMagnesium chloride hexahydrate (MgCl2-6H2O) 0.10 g/LIron (II) chloride
24、tetrahydrate (FeCl2-4H2O) 0.02 g/LResazurin (oxygen indicator) 0.001 g/LSodium sulfide nonahydrate (Na2S-9H2O) 0.1 g/L7.4 With the exception of sodium sulfide, concentrated (1003) stock solutions can be prepared for each constituent.8. Hazards8.1 This method includes the use of hazardous chemicals.A
25、void contact with the chemicals and follow manufacturersinstructions and Material Safety Data Sheets.8.2 This test involves the use of digester sludge from awastewater treatment plant.Avoid contact with sludge by usinggloves and other appropriate protective equipment. Use goodpersonal hygiene to min
26、imize exposure to potentially harmfulmicrobiological agents.8.3 Since pressure can build-up in the test vessels, selectinoculum levels, test substance concentrations and head spacevolumes that will avoid headspace pressures that could exceedsafe pressure specifications established for the test vesse
27、ls.9. Preparation of the Test Medium9.1 All constituents with the exception of the sodium sulfideare added to the water. The constituents can be added neat oras stock solutions. The use of stock solutions generallyimproves accuracy and convenience. The solution is auto-claved or alternatively heated
28、 to a boil while being mixed witha magnetic stirrer and being sparged with oxygen-free nitro-gen. After being autoclave or reaching a boil, the medium iscooled in an anaerobic chamber or with continued nitrogensparging. Once the medium has cooled to approximately 35C,the sodium sulfide is added to t
29、he medium at which point, theresazurin in the medium should be colorless.10. Inoculum10.1 Collect anaerobic sludge from a well functioninganaerobic digester at a sewage treatment plant receivingpredominantly domestic wastewater. Sludge must be sampledand transported in a manner that preserves its an
30、aerobicintegrity. One approach is to collect sludge in a wide neckedpolyethylene bottle filled to within 1 cm of the top and sealedtightly. Since pressure can build up in the bottle, the use ofglass should be strictly avoided, and excess pressure must beperiodically released during transport and sto
31、rage.10.2 If the sludge contains large particulates, it may beappropriate to sieve it through a 2-mm mesh screen or a singlelayer of cheese cloth prior to use. To reduce background gasproduction, it also may be appropriate to allow the sludge todigest in the laboratory without addition of nutrients
32、orsubstrates for up to 10 days. Work by ECETOC (1)3suggeststhat 5 days gives an optimum decrease in background gasproduction without an unacceptable increase in lag or incuba-tion period. Some laboratories monitor gas production and usethe inoculum once gas production has reached a plateau. Allproce
33、dures must be conducted under conditions that preservethe anaerobic integrity of the sludge (that is, in an anaerobicchamber).10.3 Immediately prior to use, it may be appropriate to washthe sludge inoculum with anaerobic test medium to reducelevels of inorganic and organic carbon. This is accomplish
34、edby centrifuging sludge samples at a low speed (that is, 3000 g)and decanting the supernatant. The supernatant is discarded,and the pellet resuspended in an equal volume of the testmedium. This process can be repeated as needed to reducebackground DIC below 10 mg DIC/L (2), which is usuallyaccompli
35、shed with two washings. It is critical that all washingprocedures be performed in a manner that preserves theanaerobic integrity of the sludge.10.4 Prior to use, the total solids level of the sludge isdetermined so upon dilution in the test medium, the final TSconcentration is 1-3 g/L in the inocula
36、ted medium.3The boldface numbers in parentheses refer to a list of refernces at the end of thistest method.E217001211. Preparation and Addition of Test Substances11.1 In the case of water soluble or dispersible test chemi-cals, prepare an aqueous stock solution, suspension or emul-sion, and dose ali
37、quots to each test vessel. Ideally, the stockshould be prepared in anaerobic medium. Since addition of atest solution can alter the ratio of liquid to headspace and thetotal solids content of the test vessels, the amount of addedliquid should be controlled or minimized. The volume of liquidand the l
38、evel of sludge solids must be identical in the blankvessels and those receiving test material. Test substances thatare insoluble can be directly added to the test vessels. Testsubstance may be weighed directly into tared empty testvessels prior to purging oxygen from the vessels. Sinceweighing such
39、and transferring such low levels can be inaccu-rate, the test substance can be ground into a powder and mixedwith a known excess of fine sand, which will increase the massbeing added.Alternatively, the test material can be dissolved ina volatile solvent, and aliquots transferred to the empty testves
40、sels with subsequent evaporation of the solvent. Dosing ofdeoxygenated test substance stock solutions to the test vesselsshould be done in an anaerobic chamber or in a manner thatmaintains the anaerobic integrity of all solutions.12. Procedure12.1 Preparation of Test VesselsAt least three test vesse
41、lsshould be prepared for each test chemical. In addition, at leastthree blanks should be prepared for each test. Multiple testchemicals can be included in a single test. At least one vesselshould be prepared for each reference compound. An optionalinhibition control can be included that is dosed wit
42、h equalconcentrations of both the test and reference chemical. Follow-ing the additions of test substances and solvent blanks asneeded, inoculated anaerobic test medium is added to eachvessel. Constant mixing of inoculated medium using a mag-netic bar is recommended during this process to ensure uni
43、formsolids addition to each vessel. The recommended final addedconcentration of the sludge inoculum is between 1 and 3 g/L.The typical concentration of test material is 100 mg C/L.Lower concentrations can be tested if an adequate signal can beachieved above the background in the blanks. All manipula
44、-tions should be conducted in an anaerobic chamber or in amanner that preserves the anaerobic integrity of the testsystems.12.2 Every test vessel should contain the same final volumeof liquid and have the same headspace volume. The ratio ofliquid volume to headspace should be based upon the predicte
45、dpressure increase resulting from the complete mineralization ofthe chemical and the measurement range of the transducer. Atno time should ratios be utilized that could result in thedevelopment of pressures that exceed the pressure tolerance ofthe test vessels. Furthermore, high headspace pressures
46、couldcontribute to leakage. A typical configuration is 100 mL of testmedia in a 160 mL serum bottle.12.3 Once inoculated and dosed, each vessel should bestoppered and crimp sealed. The vessels are allowed toequilibrate for1hat356 2.At this point, the starting pressureis recorded or equilibrated to a
47、tmospheric pressure by insertinga needle through the stopper to release any excess pressurewithin the bottles.12.4 IncubationIncubate the sealed bottles at 35 6 2inthe dark. After 24 to 48 h, examine the bottles and eliminateany vessels that exhibit pink coloration. Vessels can beincubated staticall
48、y or with intermittent or continual gentlemixing.Acommon practice is to gently swirl or mix the bottles2 or 3 times per week. Mixing resuspends the inoculum andensures gaseous equilibrium. It is recommended that vessels bethoroughly mixed prior to each measurement.12.5 Pressure MeasurementsEach vess
49、el can be outfittedwith an individual pressure measurement device that is peri-odically monitored.Alternatively, a single measurement devicecan be used by inserting the syringe needle through the septumand taking a reading. It is critical that the test vessel bemaintained at the incubation temperature during the measure-ment process. After recording the pressure, the pressure can bere-equilibrated to atmospheric pressure by releasing any head-space pressure through the 3-way valve before removing thesyringe needle from the septum. It is recommended thatmeasurements be take
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