1、Designation: E 1720 01Standard Test Method forDetermining Ready, Ultimate, Biodegradability of OrganicChemicals in a Sealed Vessel CO2Production Test1This standard is issued under the fixed designation E 1720; the number immediately following the designation indicates the year oforiginal adoption or
2、, 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 procedures for determining theready, ultimate, aerobic
3、 biodegradability of organic chemicalsby monitoring CO2production in sealed vessels containing thetest compound and a dilute sewage inoculum. Because of thestringency of the test conditions, it can be assumed that achemical that is 60 % or better biodegraded in this test methodwill biodegrade in mos
4、t aerobic environmental compartments.1.2 This test method is derived from the sealed vesselprocedures of Birch (1),2Struijs (2), Boatman (3), and Peterson(4), which were developed as simpler, more economicalalternatives to the CO2production techniques reported byGledhill (5) and Sturm (6), the Sturm
5、 report being the basis ofthe Modified Sturm Test of the Organization for EconomicCooperation and Development (OECD) (7).1.3 The procedures are applicable to pure materials, includ-ing sparingly solubles, which can be dissolved or dispersedhomogeneously in aqueous stock solutions of at least 25 ppmo
6、f carbon, or which can be introduced reproducibly to testbottles as pure test material in 1 to 2-mg portions. The testchemical should be nontoxic to sewage microorganisms at 10ppm of carbon. The test may be applied to volatile materialswith Henrys Law Constants of up to approximately 102atm/m3/mole.
7、 The testing of mixtures, extracts, or fully formu-lated products can lead to serious problems in data interpreta-tion.1.4 The procedures involve incubation of the test chemicalwith a dilute inoculum of microbes from domestic wastewatersecondary sewage treatment effluent in small, sealed vesselsfor
8、up to 28 days. Biodegradability is determined by monitor-ing CO2production as dissolved inorganic carbon (DIC) in theliquid phase, and as gaseous CO2in the head space. Alterna-tively, analysis can be performed on just the liquid phase afterthe addition of alkali, or on just the headspace followingac
9、idification. The determinations are made using commercialcarbon analyzers based on the IR detection of CO2. Thedetermination of CO2production provides unequivocal proofof biodegradation, barring the unlikely event of abiotic pro-duction of CO2from the test material.1.5 For water-soluble materials th
10、at do not adsorb to glassor biological solids, biodegradation may be confirmed furtherby monitoring the disappearance of dissolved organic carbon(DOC) in the liquid phase.1.6 The simplicity of the sealed vessel method permitsample replicate sampling for rate determination or statisticalevaluation, o
11、r both.1.7 For a chemical that fails the test as written, the strin-gency of the test may be reduced by substituting an acclimatedinoculum in order to provide a measure of inherent biodegrad-ability.1.8 Materials that are toxic to the microbial inoculum at 10ppm of carbon may not be amenable to test
12、ing by this testmethod, or they may require special method modification suchas reducing the test concentration if instrumental sensitivitypermits. For some cationics, complexing the test material witha nondegradable anionic may reduce toxicity.1.9 The values stated in SI units are to be regarded as
13、thestandard.1.10 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 of regulatory limitations prior to use. F
14、or specificprecautionary statements, see Section 6.2. Summary of Test Method2.1 Biodegradation testing of organic chemicals is per-formed by monitoring CO2production in small sealed vesselsinoculated with microbes from secondary sewage treatmenteffluent obtained from a local domestic sewage treatmen
15、t plant.The types of test chemicals for which the test is recommended,and those for which special considerations may be required, aresummarized in 1.3.2.2 Alternatively, smaller vessels (40-mL VOA vials or20-mL serum vials) containing 25 or 13 mL of medium,respectively may be used if headspace CO2is
16、 to be measuredusing a carbon analyzer equipped with an autosampler.2.3 Vessels (160-mL gas-tight bottles) are charged with thetest chemical and sewage inoculum in a dilute mineral salts1This test method is under the jurisdiction of ASTM Committee E47 onBiological Effects and Environmental Fateand i
17、s the direct responsibility ofSubcommittee E47.06on Environmental Fate of Chemical Substances.Current edition approved Oct. 10, 2001. Published November 2001. Originallypublished as E 1720-95. Last previous edition E 1720-95.2The boldface numbers in parentheses refer to the list of references at the
18、 end ofthis standard.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.solution to a volume of 100 mL. The vessels are sealed withbutyl rubber or neoprene septa and incubated on a gyrotoryshaker at 20C for up to 28 days.2.4 Test vessel
19、s are sacrificed periodically for analysis ofDIC in the liquid phase and analysis of gaseous CO2in theheadspace, using commercial carbon analyzers.2.5 The amount of CO2resulting from biodegradation of thetest chemical is determined by comparing the total CO2contentof the test vessels with that of bl
20、anks containing no testchemical. The extent of biodegradation is determined bycomparing the actual CO2produced with the theoreticalamount that would be produced by complete conversion of thetest chemical carbon to CO2.2.6 The duration of the sealed vessel test is typically fourweeks, with periodic s
21、acrifice of the vessels for analysis.Preadapted inoculum may be used in a subsequent test for testchemicals that fail to degrade within that time, but a positiveresult would classify the chemical only as “inherently” biode-gradable rather than “readily” biodegradable.3. Significance and Use3.1 As a
22、ready biodegradability test, when using non-adapted inoculum, the sealed vessel method provides only alimited opportunity for biodegradation and acclimatization tooccur. It may therefore be assumed that a chemical yielding apositive result in this stringent test will biodegrade rapidly andultimately
23、 in the environment. Generally, no further biodegrad-ability testing would be required for a chemical that passes thistest unequivocally.3.2 The sealed vessel test is applicable to the testing ofvolatile test chemicals because the biodegradative formation ofCO2occurs in a closed system.3.3 The seale
24、d vessel test is also appropriate for testingsparingly soluble chemicals and for chemicals that bind toinoculum, since biodegradability is based on the analysis of asoluble formation product rather than on the disappearance ofthe sparingly soluble substrate.3.4 Ample replicate sampling for rate dete
25、rmination orstatistical evaluation, or both, is feasible because of the speed,economy, and space efficiency of the sealed vessel test.3.5 The sealed vessel test is ideal for the comparative testingof groups of chemicals and for generating structure-activitydata bases also because of its speed, econo
26、my, and spaceefficiency.4. Apparatus4.1 The apparatus, reagent concentrations, and proceduresdescribed in the following sections are appropriate for testingboth soluble and sparingly soluble materials, and for volatilematerials with Henrys Law Constants of up to approximately102atm/m3/mole. Stock so
27、lution concentrations and volumescan be varied in practice in any convenient manner that resultsin the final concentrations indicated in 10.6 and permits theaccurate and reproducible introduction of test chemical to thereaction vessels. Some materials, such as insoluble or viscousliquids, are more e
28、ffectively added directly to the test bottles bythe alternative techniques described in 5.5.4.2 Gas-Tight Glass Vessels, 160-mL capacity,3with alumi-num crimp caps and neoprene or butyl rubber septa. Approxi-mately 30 vessels per test group, plus an additional 30 forblanks, will provide triplicate s
29、ampling at time 0 and sevensemiweekly time points, plus six bottles for Day 28 to permitend point statistics. The actual number of bottles will dependon the objectives of the particular experiment since there canbe great flexibility both in the sample timing and samplereplication needs.4.2.1 Bottles
30、 may be reused after thorough cleaning, forexample, in a 60C ultrasonic bath, rinsing with copiousamounts of water (final distilled) and drying.4.3 Large, Heavy-Duty Gyrotory Shaker,4equipped with auniversal platform.4.4 Carbon Analyzer(s):4.4.1 Capable of measuring DIC and DOC in aqueousmedia over
31、the range from 0 to 20 ppm;5and4.4.2 Capable of measuring CO2in gas over the range from0 to 1 g carbon.64.4.2.1 The same analyzer, for example, the Ionics 1555b,can be used for both analyses, with some loss of speed andconvenience.4.4.2.2 Alternatively, analysis can be performed on just theliquid ph
32、ase after the addition of 1 mL 10N NaOH, or on justthe headspace following acidification with 1 mL 10N HCl (4).4.5 Gas-Tight Cemented Needle Syringe, 1000 L with a22 beveled bent point, for piercing the butyl rubber orneoprene septa and injecting into the gas phase analyzer.74.5.1 Spring-Loaded Hami
33、lton Syringe, with a “square” endfor injecting liquid samples into Ionics-type analyzers, if used.4.6 Filter ApparatusTwo- or three-litre filter flask, 20-cmBuchner funnel, 18.5-cm coarse filter paper,8and a vacuumsource, for filtering sewage effluent inoculum.4.7 Compressed CO2-Free Air or Nitrogen
34、, for sparging theinoculum free of CO2. The delivery line should be equippedwith a large gas diffusing stone,9for maximum spargingefficiency.4.8 pH Meter.4.9 Volumetric Flasks, three 100-mL and one 1-L capacityfor preparation of mineral salts stock solutions.4.10 Glass Bottles or Flasks, 6-L capacit
35、y, for preparationof mineral salts solution. Sufficient media is provided by 6 L ofmineral salts for approximately 99 test vessels (that is, approxi-mately three and one-third test groups) for this test method aswritten.4.11 Volumetric Flasks, 2-L capacity, one flask per testmaterial, for preparatio
36、n of test material stock solutions.4.11.1 More concentrated stock solutions may be used forsoluble test chemicals that do not precipitate in the presence ofthe mineral salts medium; that is, smaller volumetric flasks willbe appropriate. In this case, volumes and concentrations of the3Pierce Chemical
37、 Co. 125-mL Hypo-Vials, or equivalent.4New Brunswick Scientific Model G10, or equivalent.5OI Corp. Model 700 TOC analyzer, or equivalent.6Ionics Model 1555b TOC analyzer with Horiba Model PIR2000 NDIR CO2detector (approximately $17 000 complete), or equivalent.7Hamilton No. 1001 with No. 81317 tip,
38、or equivalent.8Whatman No. 41, or equivalent.9FisherBrand Catalog No. 11-139A, or equivalent.E 17202mineral salts must also be adjusted accordingly, or an appro-priate volume of pure water must be added to each test vesselto bring the total to 100 mL.4.12 Magnetic Stirrer(s), for media and sample pr
39、eparation.4.13 Automated Pipetting Devices, to deliver variable vol-umes up to 100 mL, with an accuracy of 6 1%.104.14 Large Laboratory Oven, for drying glassware.4.15 Ultrasonic Processor (optional), for dispersing spar-ingly soluble test chemicals.5. Reagents5.1 InoculumNon-chlorinated secondary e
40、ffluent from anactivated sludge plant treating predominantly domestic sewageis obtained fresh on the day of initiation of the experiment,approximately 200 to 250 mL per test group of 30 vessels. Theundiluted inoculum should contain approximately 106organ-isms per millilitre.5.2 Alternatively, 40-mL
41、VOA vials or 20-mL serum vialsmay be substituted.5.3 Deionized or Distilled Water, free from calcium andtoxic substances, particularly metals such as copper. It may bedesirable to air-saturate the water by aerating strongly forapproximately 20 min with clean, filtered, compressed air.5.4 Mineral Sal
42、ts Stock SolutionsThe following stocksolutions should be stored in the dark and discarded at the firstsign of sediment, turbidity, or biological growth:5.4.1 Calcium Chloride Dihydrate, 3.64 g CaCl22H2O/100mL water.5.4.2 Magnesium Sulfate Heptahydrate, 2.25 gMgSO47H2O/100 mL water.5.4.3 Ferric Chlor
43、ide Hexahydrate + EDTA Disodium Salt,0.020 g FeCl36H2O/100 mL water and 0.040 g EDTANa2/100mL water.5.4.4 Potassium Phosphate, Monobasic + Potassium Phos-phate, Dibasic, + Sodium Phosphate,DibasicHeptahydrate, + Ammonium Chloride:8.50 g KH2PO4/L water21.75 g K2HPO4/L water50.30 g Na2HPO47H2O/L water
44、0.50 g NH4Cl/L water5.5 Test Chemical Stock Solutions or Stable DispersionsTest chemical stock solutions for a wide range of materials,including sparingly soluble molecules, are normally preparedto contain 25 mg carbon from the test chemical per litre ofdeionized or distilled water. The dispersion o
45、f sparinglysoluble test chemicals in the stock solutions may be improvedby the use of ultrasonic processing. Two litres of test chemicalstock solution is more than sufficient to dose 30 vessels. ThepH of the test chemical stock solution may be adjusted withHCl or NaOH to pH 7.2 6 0.2, provided that
46、no precipitationor reaction of the test material occurs.5.5.1 Alternatively, some materials, such as insoluble liq-uids, are better added directly to the test bottles by means of agood-quality microlitre syringe. Very viscous materials may bespread thinly on a tared coverslip that is then added to t
47、he testbottle.5.5.2 Materials known to be toxic to bacteria at 10 ppmcarbon (final) may be tested at lower concentrations, to aminimum of 2 to 5 ppm (final), depending on individualinstrument sensitivities, by adjusting the stock solution con-centrations appropriately.5.5.3 For analyses of headspace
48、 CO2in 40mL VOA vials, asuitable headspace auto-sampler (Tekmar M-7000) coupledwith a gas chromatograph may be used. For analyses ofheadspace CO2in 20-mL serum vials, a carbon analyzerequipped with an autosampler (ThermoGlas model 1200 car-bon analyzer) may be used.5.6 Reference Compound Stock Solut
49、ionA reference com-pound such as sodium benzoate, glucose, or sodium acetatemay be prepared as for the test chemicals. A control of similarsolubility, for example, sodium stearate, should be used forsparingly soluble or insoluble materials.5.7 Calibration Gas for Headspace Analysis, certified stan-dard, approximately 0.25 % (v/v) carbon dioxide, balancenitrogen.5.8 Calibration Solution for Liquid Phase DIC Analysis,standard solutions of sodium hydrogen carbonate in the rangefrom 0 to 20 ppm as TIC.5.9 Calibration Solution for Liquid Phase DOC Analysis,standard solutions
