1、Designation: E 1625 94 (Reapproved 2001)Standard Test Method forDetermining Biodegradability of Organic Chemicals in Semi-Continuous Activated Sludge (SCAS)1This standard is issued under the fixed designation E 1625; the number immediately following the designation indicates the year oforiginal adop
2、tion or, 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 the determina-tion of the biodeg
3、radability or removability, or both, ofnonvolatile organic chemicals (Henrys Constant 103atm/m3/day); however, ithas been used for chemicals that are not completely volatilizedbetween additions to the SCAS system (5, 7). For chemicals ofmoderate volatility, volatilization losses during a cycle may b
4、eevaluated by scrubbing of aeration off-gases through a solventtrain (usually three consecutive traps containing acetone,methylene chloride, or hexane) or polymeric trap (for example,Tenax7or SEP-PAK8). Specific chemical analysis of eachsolvent trap or polymeric trap is then carried out.8.2.7 Highly
5、 polar non-extractable test chemicals that areassociated with the mixed liquor solids require specializedtesting and analytical procedures that cannot be fully docu-mented in this test method, for example, use of14C-labeledmaterials and special apparatus. However, the basic SCASoperating system can
6、be employed if appropriate mass balancescan be obtained.9. Procedure9.1 The activated sludge mixed liquor suspended solids(MLSS) are generally obtained from the treatment plant severalweeks prior to the start of the SCAS test. Source of MLSS iseither the aeration basin or return stream from the seco
7、ndaryclarifier.9.2 Filter MLSS through a 20-mesh stainless steel screen toremove extraneous particulate matter and concentrated bysettling and decanting of supernate to a suspended solidscontent of 4000 to 6000 mg/L. This concentration is typicallyfound in the activated sludge return line from the s
8、econdaryclarifier.9.3 Mixed liquor suspended solids (MLSS) from 9.1 (4000to 6000 mg/L) are then charged to a 10-L reservoir where theyare aerated prior to test initiation. Maintain the reservoir in thesame manner as the smaller SCAS test chambers using 24 and72-h weekend time cycles. At the end of t
9、he time-cycle,aeration is halted, solids are allowed to settle, and supernatedrained to one third of the original mixed liquor suspendedsolids volume. The reservoir is then charged with natural orsynthetic sewage to the original volume and aeration, at a rateto maintain vigorous agitation of MLSS, i
10、s resumed. Deter-mine stabilization of the activated sludge by monitoring DOCremoval until steady-state removal is achieved.9.4 When natural sewage (obtained either before or afterprimary clarification) is used to maintain the reservoir andSCAS units, it is obtained generally on a weekly basis from
11、theDWTP. Filter the sewage through glass wool to remove largeparticulates and stored in a refrigerator at 5C until use.9.5 The SCAS units are charged with MLSS from thereservoir generally one week prior to the start of a test.9.5.1 The MLSS of the reservoir is determined on a weeklybasis using a 5-m
12、L aliquot of the mixed liquor. Collect solidson a weighed glass-fiber filter contained in a Gooch crucibleand dry in an oven at 105C.9.5.2 Add sufficient MLSS to each SCAS unit so that theMLSS level is maintained in the 2000 to 3000-mg/Lrange. Usesynthetic or natural sewage for dilution, if necessar
13、y.9.5.3 Check the MLSS of the individual SCAS units usinga single 10 to 20-mLmixed liquor aliquot prior to initial dosingand on a once per week basis thereafter. The nutrient level ofthe natural sewage should also be checked on a weekly basis.If the MLSS level should fall below 2000 mg/L, the additi
14、on of7Tenaxt, available from Alltech Association Inc., 2051 Waukegan Road,Deerfield, IL 60015-1899, has been found suitable for this purpose.8SEP-PAKt, available fromWater Chemical Products Department, Milford, MA01757, has been found suitable for this purpose.E 1625 94 (2001)4non-acclimated MLSS fr
15、om the reservoir may be advisable ifthe study is to continue for any extended period of time. IfMLSS concentrations builds to 4000 mg/L, remove activatedsludge to lower the concentration to the 2000 to 3000-mg/Lrange.9.6 Connect the SCAS units to a compressed air manifoldand aerate each unit at a fl
16、ow rate sufficient to maintaindissolved oxygen (DO) levels of at least 2 mg/L (about 0.3mL/min/mL liquid). Reduced flow may be necessary forvolatile chemicals. Removal by way of volatility or adsorption,or both, can also be assessed in a separate SCAS unitmetabolically poisoned, for example, with 1
17、% mercuric chlo-ride. Additional mixing for the 1500-mL SCAS units can beprovided by magnetic stirring as shown in Fig. 2. Maintaintemperature at 22 6 3C.9.7 The basic time cycle used in this test is 24 h (optional72-h cycle on weekends is permitted).9.7.1 At the beginning of each cycle, stop aerati
18、on orstirring, or both, (if used) and allow the sludge solids to settleuntil they are less than one third of the liquid volume, forexample, 500-mL for 1500-mL units. Settling time is usually30 min to 1 h.9.7.2 Remove aqueous supernate amounting to two thirds ofthe liquid volume, for example, 1000 mL
19、 for 1500- mL units,from the unit by means of the stopcock or by application ofvacuum. Then resume aeration and stirring. Then fill units withfresh sewage (natural or synthetic) feed. Retain a sample ofsewage feed for DOC and pH analysis. Retain aqueoussupernate samples and analyze for dissolved DOC
20、 and pH. Ifdesired, also determine the concentration of test chemical inthe supernatant or on the sludge by means of specific analyticalprocedures previously developed.9.7.3 Then dose the test chemical to the unit as an aqueousor nonaqueous (for water-insoluble chemicals) stock solution.9.7.3.1 For
21、water-soluble test chemicals, the standard doselevel is a concentration equivalent to 10 mg carbon/L (C/L)based on the volume of wastewater replaced. The normaldosing volume is 5 mL equating to stock solution concentra-tions of 2 mg C/mL (1500-mL units). An incremental buildupto 10 mg C, for example
22、, Day 0 = 2 mg; Day 1 = 4 mg; Day2 = 6 mg; Day 3 = 8 mg; Day 4 = 10 mg, may be employed ifinhibitory effects are anticipated. This approach, however, maycause difficulties in determination of adsorption. At the end ofthe 24 or 72-h cycle, repeat the maintenance operations. It isalso acceptable to te
23、st at higher test chemical concentrations ifthey are not inhibitory to the activated sludge.9.7.3.2 For water-insoluble hydrophobic chemicals, lowertest chemical levels are normally employed, for example, 1 to3 mg/cycle and specific analytical procedures to measure theconcentration of chemical bound
24、 to sludge are required.Hydrophobic chemicals are generally sorbed to the mixedliquor solids and, if not biodegradable may not be removed toany significant degree when supernate is drained. Conse-quently, with repetitive additions of test chemical, there will bebuildup in the concentration of the ch
25、emical on the solids untila steady state is achieved, that is, amount degraded or removedduring a cycle equals the input level. If degradation or removalis complete during a cycle, no buildup occurs. If initial dataindicate higher test chemical levels can be tolerated, testing athigher levels may be
26、 subsequently carried out. Water-insolublechemicals are generally added in 200 L of acetone stocksolution, for example, 1 mg/200 L. If acetone solubility isinadequate, other solvents such as methylene chloride may beemployed.9.8 The duration of the SCAS test is dependent on a numberof factors, for e
27、xample, the time required for acclimation orachieving steady-state conditions, number of concentrations,consistency of removal data, inhibitory or toxic effects, deple-tion of sludge solids due to sampling, and use of mixed liquoras inoculum for other tests.9.9 In order to prevent accumulation of so
28、lids on the wallsof the units, periodic cleaning with a scraper or brush should bedone just after feeding. Use separate brushes for each unit toprevent cross contamination. Do not scrape the walls duringthelast8hofthecycle.10. Calculation10.1 Dissolved Organic Carbon (DOC):10.1.1 Calculate the perce
29、nt DOC loss or removal for thetest chemical during a cycle from the following equation:% DOC removal 5 100 2EffDOCTC2 EffDOCCNT!concentrationTC3 100 (1)where:EffDOCTC= dissolved organic carbon (mg/L) in ef-fluent from test chemical SCAS unit,EffDOCCNT= dissolved organic carbon (mg/L) in ef-fluent fr
30、om control SCAS unit, andConcentrationTC= mg test chemical organic carbon addedto SCAS unit per litre of wastewaterinfluent.10.1.2 ConcentrationTCcan be calculated from the follow-ing equation:ConcentrationTC5 (2)concentration stock concentration,mg/mL 3 volume, mL 3 %C/100influent wastewater volume
31、, mL/1000where:%C = percent carbon content oftest chemical calculatedfrom DOC analysis ofstock dilution (see Note2), andinfluent waste-water volume= volume of natural sewageadded to SCAS unit atbeginning of cycle, forexample, 180 or 1000mL.NOTE 2It is highly advisable to determine the DOC of the tes
32、tchemical solution.10.2 Specific Chemical Analysis:10.2.1 Calculate the percent loss or removal for the testchemical during a cycle from the following equation:E 1625 94 (2001)5% removal 5Co2 CnCo3 100 (3)where:Co= concentration of test chemical after addition, mg/L,andCn= concentration of test chem
33、ical at end of cycle, mg/L.NOTE 3If control unit has a background concentration of test chemi-cal, this value should be subtracted from Cn.11. Interpretation11.1 Organic chemicals are considered ultimately biode-gradable if DOC removal equals or exceeds 70 % and inher-ently biodegradable if DOC remo
34、val equals or exceeds 20 %(6). As previously indicated, because of the tendency of somechemicals to partition to activated sludge, one must be carefulto distinguish biodegradation from removal by means ofadsorption, volatility, or chemical transformation (hydrolysis,oxidation, etc.). However, the 70
35、 % value has been challengedand a more thorough discussion is presented by Painter (7).Depending on molecular structure, DOC removal values of upto 95 % have been suggested.11.2 Information on the toxicity of the test chemical orpotential toxic transformation products to activated sludgemacroorganis
36、ms may be useful to the interpretation of lowbiodegradation results and in selection of appropriate testchemical concentrations. Indications of toxicity can be gainedfrom observations of significant elevation of effluent DOC overthe control SCAS unit or from standard tests such as theOrganization fo
37、r Economic Cooperation and Development(OECD) 209 respiration inhibition test.11.3 Information on the physical-chemical properties of thetest chemical may be useful for interpretation of results and inthe selection of appropriate test chemical concentrations.Theseproperties include molecular weight,
38、vapor pressure, octanol-water partition coefficient, adsorption isotherm, surface ten-sion, water solubility, and Henrys constant. Knowledge ofhydrolytic or oxidative properties of the test chemical is alsoimportant for differentiation of biodegradation from chemicaltransformation processes.11.4 Use
39、 of synthetic versus natural sewage is an importantconsideration. Synthetic sewage was thought to lead to morereproducible results; however, the microbial population thatdevelops differs from that which is present in domesticactivated sludge plants. Generally, the most rapidly growingmicroorganisms
40、evolve and the more slowly growing popula-tions that are present in domestic activated sludge plantsdecline. Natural domestic sewage varies from source to sourceand in nutrient content. However, it provides the naturalnutrients that support the natural microbial population and asemi-continuous suppl
41、y of fresh microorganisms to the testsystem. DOC values in many plants, however, can be as low as20 ppm and may be insufficient to maintain the 2000 to 3000mg/L biomass required for the test. A blend made by supple-mentation of natural sewage with synthetic sewage to achievea DOC level in the 150 to
42、 200 mg/L and a 100:12:2 ratio ofC:N:P may be desirable.11.5 Reference chemicals may also be useful in monitoringthe performance of the activated sludge and in comparingresults between laboratories. While specific reference chemi-cals cannot be recommended, data are available for severalchemicals. T
43、able 1 summarizes data from intralaboratory testsso that calibration may be determined from time to time. Otherreference chemicals may also be appropriate, especially if thereare historical data.12. Quality Assurance12.1 To ensure the integrity of data developed using this testmethod and to comply w
44、ith current regulatory requirements, aquality assurance program meeting EPA, FDA, or OECDguidelines should be followed. This may require replicates(three or more) to be run for Good Laboratory Practice (GLP)compliance and assessment of variability.13. Report13.1 Aprotocol giving a general overview o
45、f the study goalsand procedures must be prepared before the study is initiated.If a substantive modification of this test method is deemednecessary for the test chemical, deviation from this test methodshould be documented in the protocol.13.2 Document final results of this study in a final report.R
46、eport the following information in the final report:13.2.1 Names of study, investigator(s), and laboratory,13.2.2 A brief description of the test material including itslog number, chemical name(s), composition, and other appro-priate parameters,13.2.3 Summary of test method including deviations from
47、the written method,13.2.4 Summary of specific analytical methods, if em-ployed,13.2.5 Tabular and graphical presentation of DOC removaldata (if determined) as a function of time after test initiation.Data are expressed as % DOC removal (weekly mean for 24-hcycles and individual values for 72-h weeke
48、nd cycles),13.2.6 Tabular and graphical presentation of specific chemi-cal analysis data (if determined) as a function of time (cyclenumber) after test initiation. Data are expressed as concentra-tion of test chemical at beginning and end of the cycle andpercent removal or primary biodegradation dur
49、ing the cycle,and13.2.7 A listing of relevant references including all note-book pages containing raw data from this study.TABLE 1 Results from SCAS Tests on Various Chemicals Usedin the OECD/EEC Ring Test (5)ATest Chemical% Biodegradation orBioelimination4-Acetylaminobenzene sulfonate 85Tetrapropylenebenzene sulfonate 514-Nitrophenol 95Diethylene glycol 99Aniline 96Cyclopentane tetracarboxylate 81ADuration of test was 40 days, except 120 days for cyclopentane tetracarboxy-late.E 1625 94 (2001)6REFERENCES(1) Soap and DetergentAssociation Subcommittee on Biodegradation TestMe
