Document 1yYwXg4GD0E01Eao5B7yKqEnK

BIODEGRADATION (301E) TEST SUBSTANCE Identity:A mixturecontainingperfluorooctanesulfonatweh,ich may alsobe referredto as PFOS, FC-95, oras a component of FC-3017. (1Octanesulfoniaccid)(CAS # 2795-39-3). Remarks: The 3M productionlotnumber was 2330. The testsample is FC-3017 produced inAntwerp, Belgium. CurrentinformationindicatesFC3017 isa mixtureof0.67% PFOS, 20% diethyleneglycolbutylether,55.41% water,21% ethyleneglycol,1.33% Hydroxy foamer, 1% sodium octylsulfate, 0.5% polyoxyethylenemonooctylphenylether,0.04% sodium laurylsulfate, and 0.05% tolyltraizole. The followingsummary applies to a mixture with incompletely charactedzed concentrations of impudties. Data may not accurately reflectthe biodegradation potentialof the fluorochemical component of the testsample. METHOD Method: ModifiedOECD ScreeningTest,OECD 301E withDOC Analysis, 1979 version Test type: Aerobic Ready Biodegradation GLP: No Year Completed: 1980 Analyticalmonitoring: Dissolvedorganiccarbon(DOC) Statisticamlethods: Resultswere determinedby calculabonofthe% DOC removed as compared tothe blankcontrols. Test organism source: A 50:50 mix ofsoilextractand secondary effluent. The secondary effluenwtas from an activatedsludge aerationbasin atthe Metro Wastewater Treatment Plant,St.Paul,MN, whilethe soilwas garden soilfrom the town ofWhite Bear Lake, Ramsey County,MN. Test condition: Dilutionwater: Deionizedwater Mineral NutrientMedium: Nutrientniedium per OECD 301E method (1979).InitipaHl 7.0. Test and referencesolutionpreparation:The testmateriawlas prepared by dissolvin2g08 mg inon liteorfmineralnutrienmtedium. This solutiongivesa finaltestconcentrabon of40 nigDOC/L. The referencesubstance,hydroquinone,was prepared by dissolving30.6 mg inone liteorfmineralnutrienmtedium. This solutongivesa final testconcentrationof20 mg DOC/L Test vessels: 500 mL Erienmeyer flaskscontaining250 mL test solutionand capped withfoam plugs. Incubation conditions: Lighting: Constant dark conditions. Temperature: 23.5- 24*C Agitation: Continuously Number ofconcentrations1:plushydroquinon(ereference substand)and blank,allinduplicate. lnoculum conditionon testinitiationN:ot given(No measurements are requiredperthe referencedmethod). Totalsuspended solidsand pH on day of testing:Not given. Element Basis: Decrease indissolvedorganiccarbon compared to the blanks. Test substance flaskconditions: Not given. RESULTS Nominal concentrations: Blank control,hydroquinone at-20 mg DOC/L and at-20 mg DOC/L plusHgC12 (inhibitedt)e,stmaterialat- 40 mg DOC/L and at-40 mg DOC/L plusHgC12, allinduplicate. Element values: 28-Day Degradation= 96.2% DOC removal induplicate1 and 94.6% DOC removal induplicate2. Mean value = 95%. Remarks: Testingwas conducted on the mixtureas describedinthe Test Substance Remarks field.The valuesreportedapplytothatmixtureand not the fluorochemicalproportionalone. CONCLUSIONS Percent biodegradationof FC-3017 was based on the mean DOC removal valuewas 95 after28 days. Submifter: 3M Company, EnvironmentalLaboratory,P.O. Box 33331, St. Paul,Minnesota,55133 DATA QUALITY ReliabilitKyJ:imischranking= 2. Thisstudy meets the criterifaorquality testing.However, the sample puritywas not propedy characterizedand the study lacksanalyticaclonfirmatioonfthe amount offluorochemicalproportion insolution. REFERENCES The studieswere conducted by the 3M Company, EnvironmentalLaboratory, St.Paul,MN, Lab Request number 5541 S, 1980. OTHER Last changed: 6/26/00 frn6747-1I-A TECHNICAL REPORT SUMMARY Date 6/30/80 TECHNICALCOMMUNICATIONSCENTER - 201-2CN nportenItf-reporitsprinotnebdo6 s;desofpawpnedrt," copietsoTCC.) vision . Environmental Laboratory (EE & olect Commercial lp-o-rt-r-tio Chemicals Division Biodegradation of "LIGHT WATER" PC) Products in OECD Test-6/80 Dept.Number 0535 Project N umber 9970012600 W;Fo-rt-wu-m-wr 040 John A. Pignato Commercial Chemicals Division - 236-2A (01) ,jthor(s) Employ4m Number(*) Eric A. Reiner Environmental Lab (EE&PC) - 21-BW (63 47816 @tabook Reforenc* No6 of PagaitIncludingCovershost None - See 3M Environmental Lab Request No. 5541S 31 ECURITY 21 Open 0 Closed ICAL RY Now Chemicals Reported 0 Yes CH No EYWORDS: ;electterrm from 3M liesauna.Sugpst other VlicaWteerms.) vironmental .boratory IE & PC) CURRENT OBJECTIVE: To use the internationally recognized OECD Screening Test with DOC analysis to show that "LIGHT WATER" products are highly biodegradable. IFFF REPORT ABSTRACT: (20D-250 words) This abstract information isdistributed by the Technical Communications Center to alert 3M'ers to Company R&D. This study used the "Modified OECD Screening Test With DOC Analysis" and supplemental parallel sterile controls to conclusively demonstrate the extensive biodegradability of "LIGHT WATER" Brand AFFF products: FC-203, FC-206, and FC-3017. In 14 days, the dissolved organic carbon (DOC) levels of FC-206 degraded by 90% and FC-3017 was 94% degraded. FC-203 showed 93% DOC-degradation in 21 days. The parallel sterile controls proved that this DOC loss was not due to chemical or physical-processes such as adsorption, volatilization, or precipitation of the parent material. iformationL- 'so nitials: FC - Z-4 Form 6747-1 I.A TECHNICAL REPORT SUMMARY D.- 6/30/8C TO: TECHNICAL COMMUNICATIONS CENTER - 201-2CN )nportant - Ifreport isprinted on bodi sidesof paper, send t" copies to TCC.) Oivision Environmental Laboratory (EE & PC) Dijitu.@mbw@ 0535 ProJ*ct Commercial Report Title Chemicals Division ProlectNumber 9970012600 Repcwt Number - Biodegradation of "LIGHT WATER" Products in OECD Test-6/80 040 To John A. Author(*) Eric A. Notebook Reference Pignato Reiner Conunercial Chemicals Division 236-2A (01) Employee Numberts) Environmental Lab (EE&PC) - 21-BW (63'1 47816 No6 of Pages IncludingC37orsh"@t None See 3M Environmental Lab Request No. 5541S 31 SECURITY 10- FKIOpen KEYWORDS: (@Seletcetrmsfrom 3M Thesaurus. Suggest other applicablteerms.) Environmental Laboratory (EE & PC) 0 Closed CURRENT OBJECTIVE: WA CHEMICAL REGISTRY I To use the internationally recognized Test with DOC analysis to show that products are highly biodegradable. @zc-D-o3 Now ChemicalsReported 0 Yes IN No OECD Screening "LIGHT WATER" -@FF TOC REPORT ABSTRACT(:200-2w5o0rdsT)hiasbstraicntformatidoi.snt.riisbbuyttehdeTechniCcoamlmunicatsCieonntteor ale3rMt'ortsoCompanRy&D. This study used the "Modified OECD Screening Test with DOC Analysis" and supplemental parallel sterile controls to conclusively demonstrate the extensive biodegradability of "LIGHT WATEP," Brand AFFF products: FC-3017. In 14 days, the dissolved FC-203, organic FC-206, and carbon (DOC) levels of FC-206 degraded by 90% and FC-3017 was 94% degraded. FC-203 showed 93% DOC degradation in 21 days. The parallel sterile controls proved that this DOC loss was not due to chemical or physical ptocesses--such as adsorption, material. volatilization, or precipitation of the parent @j3t, r 2,@3 TJFIO s)"Ilcse -ru InformatLion Initial$: -2- BIODEGRADATION OF "LIG11T WATER" IN OECD TEST PRODUCTS INTRODUCTION The 3M. Environmental Laboratory chose to attempt to demonstrate the biodegradability Of "LIGHT WATER" products using the OECD screening method because the test has wide international acceptance, and the Environmental Lab has had experience using the OECD test as a participant in an international ring test. These results will complement the existing BOD data that show these products to have a high degree of biodegradability. The OECD test is run at 20-250C and it.measures only dissolved organic carbon. For these water-miscible "LIGHT WATER" products, which are not expected to volatilize, precipitate, or be adsorbed during testing, the OECD test is an appropriate method for measuring % total degradation. The TOC analysis method even measures the perfluorinated portion of these products. METIIODS A14D MATERIALS Methods and Conditions - Wade A. Scheil performed this testing following the September 19, 1979 version of the Modified OECD Screening Test with DOC Analysis(l). A copy of this method is attached (Appendix 1). Reductions in the scale of the test were necessary to adapt it to available shaking equipment. Five hundred-ml Erlenmeyer flasks replaced 2-liter flasks, 250 ialof inoculated nutrient solution replaced 900 ml, and the sample size was 25, not 30 ml. As in the prescribed procedure, the analyst used only the last 10 ml of sample filtrate for DOC analysis. A calibrated chart recorder monitored the temperature which stayed within the 20-250C range during the 28-day test, except for a 3-4 hr. period when it reached 25.50C. The temperature was most frequently near the high end of the range (23.5-240C). Chemicals - All chemicals were reagent--grade unless otherwise noted. The 3M "LIGHT WATER" products used were samples of commercial material of Antwerp manufacture. Their label identified them as FC-203 NFP, FC-30.17 Lot 2303, and FC-206 (FM 3824) Lot 2330 10/79. The hydroquinone used as the calibration compound was Aldrich reagent grade hydroquinone 98.5%, Lot 100147. Four chemical supply houses were contacted in an attempt to obtain the 99.5% pure hydroquinone prescribed by the Modified OECD Screening Test, but this purity is apparenty not readiy available in the USA. We expect that this reduced purity will have no signficajit effect on results. The impurities have no apparent inhibitory effect on microorganisms and their presence could make only a trivial change in TOC measurements. -3- Water used in the preparation of all solutions, except the inoculum, was St. Paul city water passed through a carbon bed, macroporous anionic resin bed, and 2 mixed bed deionizers, and then filtered through a 0.2-um filter. This water was also used to make up losses to evaporation. Stocks of water collected at the start of the experiinent ensured consistent water quality. In preparing nutrient solutions, 26.4 g of anhydrous Na2liPO4 replaced the prescribed 33.4 9 Of NI121IP04'*211203,6.3 g of CaC12-21120 replaced 27.5 g CaC12. These replacements yielded solutions and concentrations identical to those required by the OECD Method. The optional yeast extract solution was used in place of the vitamin solution (1.6.1.2(f)). The 100 mg of Fe-chelate used in the trace element solution consisted of equimolar amounts of FeC13 and EDTA (30 mg FeCl3 and 70 mg EDTA). Test Concentrations - DOC measurements of sample stock dilutions served as the basis for adjusting initial test concentrations to approximately 40 mg of carbon per liter. This is the.highest concentration in the range recommended by the OECD test. Use of this high initial concentration increased the sensitivity of % DOC removal measureinents. The low toxicity of "LIGHT WATER" products to microorganisms made significant inhibition at this concentration unlikely. Adjustment of hydroquinone, the calibration compound, to 20 mg of carbon per liter was done by weight based on its known carbon concentration of 65.4%. Two blankcontrols, instead of 1, were utilized in making blank DOC corrections. Inoculum - The inoculum was 50-50 riiixtureprepared according to steps 1.6.2.1 and 1.6.2.2 of the OECD test using fresh soil from,E. A. Reiner's garden and the supernatant of a sample from an activated sludge aeration basin at the St. Paul Metro Plant. The filter paper used was WhatinanR 54, and the water for soil extraction was chlorine-free well water. Sterile Controls - Replicate vessels containing 400 mg/l ligCl2 served as sterile controls for each test material and the calibration coinpound. flandlingof these sterile controls was identical to the handling of the viable cultures. They had the same innoculum and sampling frequency, but the analyst only made DOC measureiiientson the U- and 28-day sainples. Sample Preservation - The sample preservation method used was that prescrioed by the TOC manufacturer(4). The method involves adding I drop of concentrated HC1 to the 10 ml filtered samples, bringing the sample to <pll2, and storing the samples under refrigeration in vials with aluminum foil-lined caps. The method is an effective preservation method. The manufacturer indicates that the method can stabilize calibration solutions made from potassium hydrogen phthalate, a readily biodegradable material, for several months. -4- Since the acidification in this preservation method was also a necessary step in the TOC analysis protocol, its use was the most practical and allowed elim"ination of the llgCl2 preservation technique described in the OECD method. All TOC analyses were made within a month of the preservation of the samples. Instruments and equipment - The organic carbon analyzer used was a Dohrman DC-52A. per liter. Its sensitivity limit is between 1 and 2 mg of carbon The stoppers for the reaction flasks were clean, porous, plastic foam plugs. Gelman 0.2 um membrane filters, (Part tio. 64814) boiled 3 times and stored in deionized water, served to filter samples for DOC analysis. Glassware cleaning involved soaking in chromic acid cleaning solution (ChroinergeR) followed by 6 deionized water rinses. RESULTS AND DISCUSSION Table 1 summarizes contain the actual function of time. coinpletely. the results of this study. Appendices 1 and 2 data sheets and plots of the degradation as a All 3 "LIGHT WATER" AFFF products degraded nearly Percent TABLE I Degradation of flydroquinone and "LIGHT WATER" FC-203, FC-206, and FC-3017 With Time in Modified OECD Screening Test AFFF Products Product 7 FC-203 13 FC-206 23 FC-3017 37 Hydro- 89 quinone % Degradation at Day: 14 21 27 28 85 93 91- 93 90 94 93 92 94 96 96 95 92 97 94 93 In the case of the highly water soluble "LIGIIT WATER" products, it is very unlikely that physical means such as adsorption, volatilization, or precipitation caused the loss of soluble TOC (DOC). This is substantiated by the relatively high oxygen demand observed in BOD tests of the 3M Environmental Laboratory (FC-203 BOD5/COD = 0.5, FC-206 BOD5/COD = 0.5). The sterile control data in Table 2 provide further substantial evidence that the soluble TOC loss from the test samples containing "LIGHT WATER" products is not due to adsorption, volatilization, or precipitation. These samples were handled identically to the test samples except that they contained HgCl2 to prevent microbial growth. -5- At the end of the 28-day test period, they still contained nearly all the initial DOC. Thus loss of soluble TOC by these physical modes was not a major factor, at least for nonmetabolized "LIGHT WATER" components. This control doesn't prove that physical processes did not reinove "LIGHT WATER" metabolites in the test runs, since no metabolism occurred in these sterile controls, but the catabolic* formation of less water soluble materials is unlikely because catabolic products are usually more polar and smaller. TABLE 2 DOC Loss from Sterile Controls Product DOC Remaining at Day 28 FC-203 FC-206 FC-3017 liydroquinone 91.3 94.2 88.1 77.5 An off-white precipitate formed in the hydroquinone sterile control on day 0 prior to filtering for DOC analysis. The precipitate was slightly brownish, as was the solution in the hydroquinone sterile control after 21 days. This suggests that hydroquinone is not stable, at least in the presence of llgC.L2,throughout the course of the 28-day OECD experiment. This apparent tendency of hydroquinone to spontaneously form insoluble humus-like material makes it an inappropriate control compound for the OECD test. Its partial precipitation with IigCl2 also casts some doubts about the appropriateness of using the HgCl2 preservation procedure in the OECD method. CONCLUSION The present study conclusively demonstra-tes that "LIGHT WATER" products, FC-203, FC-206, and FC-3017, are 'nearly completely degraded in 21 days under the conditions of the modified OECD Screening Test. REFERENCES (1) Organization for Economic Co-operation and Development Chemicals Testing Program Expert Group on Degradation/Accumulation, Dec., 1979, Test Guideline for the Modified OECD Screening Test with DOC Analysis (Level I) (H. G. Nosler) Revision of Sept. 19, 1979. (2)Dohrinan DC-52A Operating Manual, 4th Ed., 1978, p. 3-1. Appendices: 1 - OECD Test Guidelines (Sept. 19, 1979) 2 - Data Sheets 3 - Graphs Catabolism is biologically facilitated breakdown to less complex molecules. APPENDIX 1 Tokyo, December Ist, 1975P C 118/79/Int. OECD Chemicals Testing Programme Expert Group C, Degradation/Accumulation Mbdified OECD Screening Test with DOC Analysis Test Guideline C 118/79/In@-.. Test Guideline for the Modifi@d OECD Screening Test witli DOC Analysis Date of last revision: Sept. 19,1979 Lev6l I test for ready biodegradability 1. Prercqxiisites It has to be known whether the test material is soluble in the concentration range employed in the test (corresponding to 5 - 40 mg DOC/1). 2. Guidance Information Knowledge of the bacterial toxicity or inhibitory properties of the test material is not unequivocally required but constitutes useful information for the conduction of the test. 3. Qualifyina Statements The method is suited for the measurement of the aerobic ultima*.c biodegradability of water soluble, non-volatile organic compounds. It is unsuited for the biodegradability evaluation of ,nix@@.ures. Sept. 1.0. 197,Q Modified OECD Screening Test with DOC Analysis Preamble It has to be realized that the following procedure for the modified OECD Screening Test has to be regarded in some points as provisional since several important features such as the calibration compound and the new test duration of 28 days are completely untried yet. However, since the test constitutes a modification the of4o6icial,widely practised and well accepted OECD Screening Test for the biodegradability evaluation of surfactants there should exist a good prospect for success. -September 19., 1979 OECD Chemicals Testing P.-ogramme Test Guideline for the Modified OECD Screening Test with DOC Analysis 0. The test procedure constitutes a modification of the OECD Screening Test (OECD Enviro=ent Directorate, Proposed Method for the Determination of the B4-odegradability of Surfactants Used in Synthetic Detergents, Paris 1976, and council directive of Nov. 22, 1973, on the approximat4-onof the laws of the member states relating to methods of testing the b4-odegradab4&lityof anionic surfactau@s (73/405/EEC), Official Journal of the European Communities No. 4 347/53 0*." Dec. 17, 1973) for the application of the dissolved organic carbonCDOC)analysis. 1. Methcd 1.1 Introduction: Purpose_,___scopree,levance, and acllication of test and exglanation of-limits. The purpose of the method is the measurement of the ultimate biodegradability of water soluble, non volatile organic compounds in an aerobic, aqueous medi= at a starting test concentration corresponding to 5 - 4o mg Doc/i ( In order to avoid inhibitory effects it is in the investigator'sown interest 2 2 to choose as low a starting concentration analytical capability permits 1.2 Definitions and units 1.2.1 Definition of biodegradability as his D= t L where Ct - Cblt! x loo co - c bl 01 D = degradation t at time t in.pqrcent DOC-removal c = starting DOC concentration 0 culture medium (mg DOC/1) of the c = DOC concentration t of the cul'%-Puremedium at time t (mg DOC/1) c = starting DOC concentration bl 0 blank (mg DOC/1) o,."the c bl = DOC concentration of the blank a-. t time t (mg DOC/1) 1.2.2 Units The degradation is stated as the percenta&e DOC-removal within 28 days with respect to the test material DOC-removal 3 3 1.3 Reference compounds 1.3.1 Calibration compound The calibration compound used in this test is hydroquinone at a concentration corresponding to 20 mg DOC/l. Hydroquinone has to exhibit a DOC-removal of,@@t6o% within 28 days, otherwise the test is regarded as invalid. 1.4 The principle of the method A predetermined amount of the compound is disso'A.ved in an inorganic medium (mineral nutrient solution, fortified with a trace element and essential vitamin solution'),providing a concentration corresponding to 5 - 40 mg DOC/li-The solution is inoculated with a small number of microorganisms from a mixed population and aerated at 293 - 2108 (20 - 250 C) in the dark or at least in diffuse light only. The degradation is followed by DOC analysis over a 28 day period. The procedure is checked by means of a standard (hydroquinone). A control with inoculation but without either test material or standard is run parallel for the determination of DOC blanks. 1.5 Quality criteria 1.5.1 Reproducibility The reproducibility of the method is appropriate for a screening test which has solely an acceptance but no rejective function. 4 UY .7 4 1.5.2 Sensitivity The sensitivity of the method is largely deter- mined by the sensitivity limit of the organic carbon analysis which is 0.5 mg C/1 at the present state of the art. 1.5.3 Specificity,applicability Applicable for the biodegradability evalua-wion of water soluble,,non - volatile organic compounds. 1.5.4 Possibility of standardization The test versiom w-ithspecific analyses ?A*or anionic and nonionic surfactants is standard4-zed as "OECD Screening Test". 1.5.5 Possibility of automation Parts of the test, e.g., the analys4@s,can be automated, although hardly the total procedure. The procedure is, though, w-ellsuited for be4hng operated with whole series of test materials. 1.5.6 Costs ( in 1978 Swiss Francs 1.5.6.1 Equipment Glassware 20000- shaking machine 6000 - 200000- Carbon analyzer 420OOs- Miscellaneous (PH meter, balance, provision for air donditioning) 5000.,- 5 5 1.5-6.2 1.5-6.3 Person hours 10 Approx4-mate total cost per tm-,750s(investments assumed to be amortized) 46.0009- 1.6 Descripti-on of the method 1*6.1 Reagents and materials 1.6.1.1 Deionized water Deion4-zed or distilled water fr.ee of tcxic substances (copper in particular), for general use as a solvent. Water which has been de4-onized by distillation or ion exchange is suitable. A high purity of this test water is necessary in view of the DOC analyses in the concentration range of 0 - 4o ng/j. Amhe contaminations result from inherent impurities but also from the ion exchange resins and microbial devel opments (bacteria., algae under the influence of light etc). Only one water charge must be used for one test series which is to be'controlled beforehand by DOC analysis. If necessary, suitable water may be gained by UV irradiation or other means. 1.6.1.2 NutrienL'Osolution Mix 1 ml each of the following solutions (a) to (f) and make up to a volume of 1 1 with water 1.6.1.1 6 6 (a) KH 2PO 4 K 2 HFO 4 Na 2liPO4 NH4cl A.R. A.R. 2 H20 A..ct. A.R. 8.r.0, 21-75g 33.4 g 20-0 9 in 1000 ml of water 1.6.1.1 the pH value should be 7.2 (b) 22.5 9 of M9504 - 7 H20 A.R. dissolved in 1000 ml of water (3-3-1) (c)27.5 g of CaCl 2 A R. dissolved in 1000 ml of wate.-(3-3-1) (d)0.25 g of FeCl 3 " 6H 2 0 A.R. dissolved ir. I-OCO ml of water (3.3-1) 'Lh4-ssolution is prepared freshly i=ed4-ately before -,se. (e)T.-ace element solution MnSO4 H BO 33 ZnS04 04H 0 2 s 7 H2 0 (NH4)6MO7 0 24 39.9 mg (30.23-mg 57,2 mg MNSO 4 0 H 2 0) 42.8 mg 34.7 mg (36.85 mg (NH4)6MO7 0 240 4H 2 0) Fe - chelate (FeCl EDTA) 32 water 1.6.1.1 100 mg 1000 ml ,"-@f-,erilisatioofn the trace element stock solution a-. 393 (.71.():200C) , 2 atm., 20 -n4in. 7 --7 Vitamin solution Biotin 0.2 mg Nicotinic acid 2.0 mg Thiamine 1.0 mg p-Aminobenzoic acid 1.0 mg Pantothenic acid 1.0 mg Pyridoxamine Cyanocobalamine 5.0 ms 2.0 mg Fol4Lc acid 5.0 mg water 1.6.1.1 100 ml The solution is filtered sterile (0.2,um). Instead of solution 1.6.1.2 (f) 15 mg of yeast extraxt may be used per 100 ml of water 1.6.1.1. 1.6.1.3 Biodegradability standard Hydroquinone 99.5 % DAB Erg. B. 6 1.6.1.4 Mercuric chloride solution I per cent of Hgcl 2 in water 1.6.1.5 Shaking machine accomodating 2 ltr. Erlenmeyer flasks either with automatic temperature control or used in a conr.ant temperature room at 293 - 2008(K) (20 - 250 C) 1.6.1.6 Narrow neck - 2 ltr. Erlenmeyer flasks. (Creased flasks are reco=ended) The flasks must be carefully cleaned withs e.g., alcoholic hydrochloric fluted .:e use.,rinsed and dried in order to avoid I -i.nationwith residues from previous tests.. ;..t-lasks also have to be cleaned before their . .rst use since they may be contaminated. 1.6.1.7 Membrane filtration apparatus 1.60.1.8 Membrane filters 0 2 um 1.6.1.9 Carbon analyzer 1.0'.2 Inoculation Either of the following three alternatives nay.-be used as inoculu m or a composite sample thereof. 1. 6. 2. 1, '&no-.,ulumfrom secondary @effluent The inoc;,@lumis gained preferentially from a secondar.,,e,ffluent of good quality collected &"rom a treatment plant dealing with a predominantly docestic sewage. The effluen t must be kept under aerobic conditions in the per' iod between sampling aad use. To prepare the inoculum- the sample is &'iltered through a coarse filter, the first 200 ml .@eingdiscarded. The filtrate is kept aerobic until ased. The inoculum must be used on the day of collec'.-ion. 1.6.2.2 inoculum from soil 100 g of soil (fertile, not sterile) are suspended in 1000 ml of chlorine-free drinking water (soils with an extremely large content of clay, sand or organic carbon are unsuited). After stirring the suspension is allowed to settle for 30 minutes* - 9- The supernatant is filtered tl-.rougha coarse filter paper, the first 200 ml being discarded. The filt.-ate is aerated immediately and until use. The inocul= must be used on the day of collection. 1.6.2.3 Inoculum from a surface water An inoculum is drawn from a suitable surface water. The sample is filtered through a coarse paper, the first 2oo ml being discarded. The filtrate is kept aerobic until used. The inoculum must be used on the day of collection. 1.6.2.4 Composite inoculum Equal volumes of the 3 inoculum samples are unit@.ed, mixed well, and the f4.nal inoculum drawn @lromthis mixture. The suitability of the inoculum is checked by means of the standard hydroquinone. 1.6.3 Conduct4.onof the test 1.6-3.1 Procedure The test materials are evaluated simultaneously 4-n duplicates together with the standard (1.6.1-3) and a control test with inoculatiorlbutwithout either test or standard material for the-determina%-,ioofn DOC blanks. The standard material has to attain '--6:0. % DOC removal within 28 days at a starting concentration corresponding to 20 mg DOC/l. if <60 % DOC removal are achieved the whole series has to be discarded +) +) This limit is based on present experience with the 19 day version of the test. A revision of this limit or even of the standard might have to be considered after the accumulation of experience with the new 28 day version of the test. 10 -10 A stock solution Of the test material in water (1.6.1.1) is prepared. So much of this stock solution is added to the nutrient solution (1.6.1.2) that a carbon concentration of 5 - 40 mg DOC/1 is attained. The starting concentration of the standard hydroquinone is, though, 20 mg DOC/I. Two reaction vessels (1.6.1-5) are each filled with 900 ml of the nutrient solution and inoculated with 0,5 ml/l of the inoculum (1.6.2). The opening of the vessel is covered with, e.g., aluminum foil in such a way 'that the exchange of gir between the flask and the surrounding atmosphere is not unduly impeded (Cotton wool is unsuited because of the DOC analysis). The vessels are then inserted in the shaking machine. The temperature of 293 -298K ( 20 -250C) must be maintained unchanged during the test, and the vessels should be shielded from light. The air should-be free..of pollutants and toxic materials (chlorinated solvents etc). in-the course of the biodegradation test the DOC concentrations are determined in duplicate (1.6.4.2) at the beginning (day 0). and on the 27. and 28. day. Three additional analyses have to be.performed in rather regular time intervals -21. day) 14., and The analyses are registered in the attached form sheet and evaluated. Only the necessary volumes of culture medium may be drawn for each determination; however, they have to be large enough for the membrane filtration or centri.Llugationpreceeding the carbon determination. The latter requires differing volumes for the different instruments. Evaporation losses of the culture medium are to be made up by adding water (1.6.1.1) in the required amounts. The culture medium,is to.b@-.*mixedwell before wi*thdrawing a sample. Material adhering to the wall of the vessel has to be dissolved or suspended before sampxing. The'membrane filtrat4-onor centrifugation has to be done immediately. The filtered or centrifuged samples..have to be analyzed on the same day, otherwise they must be preserved with 0,05 ml of the HgCl.@ solution (1.6.1.4) for each 10ml of nutrient medium or by storing them at'2 - 40c.-The biodegradability test is valid provided the standard exhibits a degradation rate within the specified range. The test can be*finished before the 28. day if complete miner'ali-zationis accomplished. All steps require great care and cleanliness of the vessels, pipettes etc. but not sterili-ty. 12 12 1.6.3.2 Calculation of results The degradation at the time t is calculated from the determinations of the DOC concentrations at the beginning (C0) and the time t (C ) according to t c t c bl t x 100 c 0 c bl 0 where D, = degradation in per cent at time t c 0 = measured starting DOC concentration of the inoculated culture medium (mg DOC/1) c t = DOC concentration of the culture medium at time t (mg DOC/1) Cb!O = starting DOC blank of -.hemineral nutrient solution with inoculation but without test material (mg DOC/1) Cblt = DOC blank of the mineral nutrient solution with inoculation but without test material at the time t (mg DOC/1) The degradation rates are calculated to the nearest 021 Z. The means of the Dt values are calculated and reported to the nearest full per cent. Results 13 - 13 ending in 0,5 are rounded up to*the nearest whole number. The course of the degradation test is followed graphically in a diagram as shown in the attached example. The results are reported on the attached data sheet. The results of the degradation test are valid if the condition is met that in the same test series the standard yields *a:60 ; DOC-removal. 1.6.4 Analytical means 1.6.4.1 Membrane filter 0;2/um, 25 mm 0 (1.6.1.6). Pre- paration'of the filters; membrane Loilters are impregnated with surfactants &Oor hydrophilizat4.cn. Thus each filter contains up to several mg of soluble carbon which would interfere in the biodegradabi-lity determinations. Therefore the filters are purified from surfactants and other soluble organic '*interferences by boiling them 3 times 1 1,,r each in deionized water. These filters way be stored in water (1.6*,1.1)for at least one week. Other membrane filters are su4.tab-leif it is assured that they neither release carbon-nor adsorb the compound in the filtration step. If the samples are centrifuged, this has to be done at 40 000 m sec- 2 ( 4000 g) for 15 minutes, preferably in a ref.-igerated centrifuge, in any case <40 0 C. (Remark: the differentiation TOC:DOC by centrifugation at very low concentrations does not seem to work well since either not all bacteria are removed or carbon as part of the bacterial plasma is redissolved. At higher test concentrations ( >/10 mg C/1) and the same small inoculationthe cen4wrifugat4-eornror seems to be comparatively small ). Form Sheet for the Xodified OECD Screening Test Exp. no.: :1 Of start of test; ) Te,.@t / standard material: rheoretical test conc.: Inoculum; :arbon analyzer: mg DOC/I A: Controls: tock solution of the test material 1000 mg/l, dilution .../1000 ml of nutrient--solution) TOC* I)OC mg/i B: Carbon determinations: Culture medium Flask no. Analyses Mineral nutrient solution with test material and with inocuium .*Aineral nutrient solution without t03t material but with lnoculuzo I 2 Blank - a I a2 a +a mI 2 ct ) b b2 b b, m 2 - --= ( ct ) c c2 c c2 m 3 -2 ( cbi) Theor. DOC - concentrations alter x Gays conc. mg/i mg/i 0 (CO). 7 -14 1-21 27, .8 C: Evaluation of raw data: DOC - concentrations minus blanks Flas no. m m3 1 D 100 DOC 7 rpmoval 14 alter 21 x days 27 1 28 m 2-- M3 2 D 2 2 100 2 mean D Di + D2 for day x t thp t p@tt 14 1.6.4.2 The DOC measurement The sample withdrawn frcm the culture medium (about 30 ml) is centrifuged or membrane filtered immediately in the filtration apparatus (1.6.1.6) using the membrane filters prepared acc. to 1.6.4.1. The first 20 ml of the filtrate are discarded*. The DOC concentration is determined twice in the remaining filtrate (about 10 ml) by means of the TOC/DOC instrument (1.6.1.8). If the filtrate cannot be analyzed on the same day it has to be conserved acc. to 1.6-3.1. The DOC measurements (mgC/1) obtained'are registered on the.*attached data sheet and the DOC concentrations of the culture medium and of the blanks calculated for each sampling time. APPENDIX 2 3h EnvironmentalLaboratoryData stmet -resultsof the ModifiedOECD ScreeningTest- Lab Request ma.: 5541 s Dateof Startof Test:.4 - tt-no Analyst:Wede A. Scholl est MeterIa 1:.ANrwf-Kp FC-2.o-s, MFP tandardMaterial:-hydroquinons,98.5%.Lot 100147,Aldrich ihooretlcalqOC of TestMaterial:(tJO-rC-ALCtAl-ATEt)) MeasuredTOCI of Test haterIaI: I Z .000 mg TOC/kg Inoculum: sofI and p"-chlorinatedifecondaroyffluentfrcma mjnlcID81wastewater-t-reatmepaltant CarbonAnalyzer:DohrmannUC,,5ZATotal organicCarbonAnalyzer A. Determinationof TOCI of-TestMaterial: A stock solutionof the testmaterialwas preparedby diluting1170 mo.to a finalvolumeof 5700 ml with dolonizedwater. cone.testmaterial -yoc- TOC In stocksolution stocksolution testmaterial mg/i mg/i mg/kg This resultwas used to calculatethe necessary amountof testmotorlqlto preparea culture medlm of 40 mg/i DOC'* Z'340 332, 11+2,,ooo a. Carbon Daterminations.2 Culture modlum Flask no. Mineralnutrient solutionwith est material atnd with lnoculum 2 Mineralnutrient Blank solutionwithout I testmaterial but with inac7lu-m Blank 2 C. Evaluationof Raw Data* Conc. of test CalculatedDOC] DOC DOC - corvc.afterx d materialIn of testmaterialAnal- 0 (CO) 7 14 flask,mg/i ln_flaskm,g/1- yses - I al 4Z.V 37,-D 6.1 40-0 --a!2 -rft a Incubationm.g/l 21 27 28 G.Z 4.1 z.,l a, + a2 .#.an CtIm n o bi + b2 ... bn ct2 n cbllm-Cl +n C 2 d 1+ d2 Cbl?m n lil-S' 37-14 -1 A/-/0. .a]-re 46.5' !blli -32.1 -5..r ql.o (32..o C) 1 (Cblo@ 0 /.S- di- G d "Z -0./ 0./ -@bl-or 0 0.2. 0 -r 0-/ 0-6 0.4 d4 I 5-'c@ 2.8 l@,.o 3 2.9 SI-0 -Z-9 -V-6 --7.Lffa- Z-5 1.5- 0.9 o., o./ -0.1 /.0 6.9 -6.1 0.6 -o./ 1+ o 1-2- 6 Flask no. 2 mean Degrade ton 7 2-o.1 OC removalafter D1 21 27 1 COC1I5.q 1.0 d 9 Incubation al8 13.0 q 3.ID 93 See attached action1.6.3.2for the formula used to calculatedegradation. Resultsof blankno.1 and no. 2 were averagedfor each day x. The average valueswere used In thecalculations. cbli+ cbl2 (fromabove) Cblav." 2 1. The testmaterialIs completelysolubleIn water. Therefore,the TOC - DOC. 2. DOC analysesperformedmore than twice on the same samplewere run for qualityassurancepurposes. 3M Envirormental LaboratoryData Shoot -results of the Modified OECD Screening Test- Lob Roque$ t No.: 5541 s Date of Start of-Test: Od slyst: Wade A. Scherl st Matorlal:^PITVXKII,_FC,-2,OC,. standard Material:-hydrogulnone, 98.5%. Lot I eoratical qOC of Test moterial:?'POT AL.Ct4l.Alr&b)- -- Measured TOCI of Test material: 24-100 _mg TOC/kg lnoculum: soll and non-chlorinatedsecondary effluent frcma municipal wastewater treatment plant Carbon Analyzer: Dohrmann OC-52A T;tal-orqanic carbon Analyzer 100147, Aldrich A. Determination of TOC I of-Test Material: A stock solutionof the test material was prepared by diluting IJOCOO mg to a final volume of -5'00ml with dolonized water. cone.test material TOC In stock solution stock solution mg/l mg/l TOC test material mg/kg This result was used to calculate the necessary amount of test materill to prepare a culture madilm of 40 mg/i ooc o z@ /ZO 19q 2 0. Carbon Determinations* Culture madlum Flask no. Mineral nutrient solution with test material and with lnoculum 2 I Minera nutrient solution without test material but with Inocuiu-m Blank 1 Blank C. Evaluation of Raw Data: Conc.of test Calculated DOC I material In of test material flask, mg/i In flask. mg/i &I + a2 ...an n 4o'D b + bz b c t2 n -7 Ci + C c2 blia n DOC Analyses ai a :ia--- ac bl- b b b C-Ci DOC - conc. after x days Incubation. 0 (CO) 7 14 21 27 qZ.3 3/10 r.13 11-ro mgll 28 .0 s- q.?o,3/,0 7 112, 3 30--y All, 3 !@.!r 3, '2 -- -9,0 3. q -3.X 32E,@; 30,2 3-@ 1. 0-t 0-2. (Cblo) -6. 2. O'V 3+.4 3.(o 3.1 /.s- o, 1-2- 40,S- 0-/ d +d c I2 bl2' n d /,,2. n - t of 0.8 -0./ (Cbi-07 /.4 0 p3//.,.3 CbIAVP-!z I.q I -O,t r6.L0-!)4 1.2-2 0.(. 1 1 Flask no. 1 2 mean Dograda 7 2-3.3 Ion l@ 94.9 DOC removal after dis Incubation 21 1 27 1 8 93,1 c@ 3 93 9 See attached section 1.6 3.2 for the formula used to calculat; degradation. Results averaged values of blank for each were used no.1 and no. 2 were day x. The average In the calculations. c Cbll + Cbl2 (from above) blave a 2 1. The test material Is completely soluble In water. Therefore, the TOC - DOC. 2. DOC analyses performed more than twice on the same sample were run for quality assurance purposes. 3H EnvironmentalLaboratory Data Shoat -resultsof the ModifiedOECD Scre*nlngTest- Lab Request No.: 5541 S Dateof Startof Test:- +-Il-fO nalyst: weds A. Schell lostmat7erial:ANTWEItPr.-e--IolL'-7(,rr-7--43 tandardMaterial: hydroquinone,98.5%, Lot 100147,Aldrich Theoretics OC of Test Material':CNOTCALCtAt-Al,ED) Measured TOIC7 of Test Material: 192-WO mg TOC kg lnoculum: soil and non-chlorinatedsecondaryeffluentfr(ma munictpal_wastewotartreatmentplant Carbon Analyzer: Dohrmann OC-52A Total_Orqanic Carbon nalyzer A. Determinationof TOCI of Test Material.L A stock solutionof the test materialwas preparedby diluting 1)2-qS'MQ to a finalvolumeof 5'00 mi. with dolonizedwater* conc.testmaterial TOC TOC In stock solution stock solution test material mg/l mg/l mglkg This resultwas used to calculatethe necessary amountof testmatorlitto preparea culture madlum of 40 mg/i DOC 2.)440 479 112J000 S. Carbon Do-torm-inatlon2s- Culture medium Flask no. Mineral nutrient 1 solutionwith testmaterial and with lnoculum 2 Conc. of test CalculatedDOCI DOC -DOC- conc. after x materialIn flask.mg/i of testmaterial Anal- 0 (CO) 7 In flask.'mg/l yses - 14 I al 1/1-IV 3/.z .2-v 209 410.0 a2- 270-l' a ae a, + &2 ...a. CtI. n 2 2-4 b 4o(.7,3 /'6y. 20,3 b.3 days Incubation.mgll 21 27 28 I 3.9 /.17 2,0 Z -2 1.0 2.5- 3.o 2,r /#7 2. 2 .2. Mineral nutrient solutionwithout test material but with lnoc=UM Blank Blank 2 C. Evaluationof Raw Data: bi + b2 ...b ct2m n I Ci + C2 Cbl n d I + d2 Cbl2" n C3 0-1 0- :k r 0. t4 I 0.!r o.9 ldj- 1. dg 1 -0.1 0 (CbIO7 ).#4 LO 0 L Cii 4ve- )lq C>.q 1- 1. 0 c>.(4 019 1 0. 1 0.5' 0-Y 0. A 0,(* 6 :. ;L Ol/ e>. 0- a Flask no. 2 Fmoan Degrade Ion DOC ram*val after 7 iz 21 27 x 9!;.o 52-,2- 92%1 3'7--- 114 dais Incubation 8 Iq 15- See attached action 1.6.3.2for the formula used Sto calculatedegradation. Resultsof blank no.1 and no. 2 were averagedfor each day x. The average valueswere used In the calculations. Cblsve Cbll + Cbl2 (fromabove) 2- 1. The test material Is completely soluble In water. Thereforetthe TOC - DOC. 2. DOC analysesperformedmore than twice on the same sample were run for qualityassurancepurposes. 3H Environmental Laboratory Data Sheet -results of the Modified OECD Screening Test- Lab Request No.: 5541 s Date of Start of- Test: 4. - 11-10 Analyst-._W&Ad.e SchalI st Material: toward Material:,h-ydroquinane,98.5%. Lot 100147,Aldrich looratical 7DC of Test Material: IR@C1.44%r-- LL FBL*J ^S5tAMF-D *14>L-@rS-OLu6LE ORC"IC- r.A%Rr6,ow_@ MeasuredTOC of Test Material: mg TOC kp lnoculum: soil and 9"-chlortnated second of lunicloal wast ter treatmentplant Carbon Analyzer: Dohrmann DC-52A Total Orqa'n07l1c, Carbon Analyzer-- A. Determination of TOC I of Test Material: A stock solutionof the test materialwas preparedby diluting with dolonizedwater. mg to a final volume of mi conc.test material TOC In stock solution stock solution mg/l mg/l TOC test material mg/kg This result was used to calculate the necessary amount of tost materi?] to prepare a culture medium of 40 mg/l DOC - -I I 2 8. Carbon Determinations, CV.4tt4A-roAl COMPOUt,)D- Mk-reRIAL. Pic $66 A36VE .%ar-t-,di.1i1f3A7olod SarAAJDARD - Culture modlum Flask no. Mineral nutrient solutionwith test material and with Inoculum 2 Mineral nutrient solutionwithout test material but with inoc7lu-m Blank I Blank 2 C. Evaluation of.Raw Data: Conc. of test CalculatedDOC] material In of test material flask, mg/l In flask. mg/l -70.6 zo, 0 al + 02 ...an Ctin n 3o. G zo. 0 DOC DOC - cone. fter x Anal- 0.(C.) 7 14 yses - I )'7.1 Z-3 1 2.0 1-7 1.7- a a-j-_ IG.?- - as I a /?,:5 2.0 . - -17.6 b -161.2 y- .7.4 days Incubation,mg/l 21 27 28 z. /-7 1.40 ? 1.4 0.!;- 0. R )-Z I C. b1 + b2 bn t2- n b /7.0 2_ /.S q 6-8) I Ci + C 21 Cbll" n d I + d2 Cbl2' n Ci c C- blo /@.5 - d d, I /,,,4ap 0./ (CbIO7 1.4 0 0.4 40 Ll /.2 0.(/ at 1 0. 4v 0,/ 0.1 C C-6toLve + Ciil.Z Flask no. 2 Dograda [on 71 DOC removal after dals Incubation 21 1 27 8 q /,7 gq.9 qq. 9 q 7- TI'7 9,tl q See attached action 1 6.3.2 for the formula used Sto calcuI;to degrad&tlori. Results of blank no.) and no. 2 were averaged for each day x. The average values were used In the calculations Cblave Cbll + Cb)2 (from above) 2 1. The test material is completely soluble In water. Therefore, the TOC - DOC. 2. DOC analyses performed more than twice on the same sample were run for quality Assurance purposes. L < gLi <cc L 0 3M Environr.-entaLlaboratory OECD Modified Screening Test with DOC Analysis Analyst: Wade A. Schell Date of Start of Test: 4-11-1980 Lab Request No.: 5541 S Test material:At4TWERP FC -2-03 T- iA IF ff 7 14 21 NUI,.BEROF DAYS INCUBATED $I:;%,: X ot OW/I- 9TI, 7VA ui 6 0:/. z- 4 D:/, ui 3VI-. - ------ -7- --------------- 3M Environn-entalLaboratory OECD Modified Screening Test with DOC Analysis Analyst: Wade A. Scheil Date of Start of Test: 4-11-1980 Lab Request No.: 5541S Test Material: AN-rwEp,@ FC--:LCG F:t-i--382+ L-OT - 2-3-3,-) 10129 -4- 7 21 27 NUI,.BEROF DAYS INCUBATED IDo:/-, 80%. 7 0:/. 6 0,:@ 5 (P/. 40:/. cc 30Y. 20Y, 0 3M Environrental Laboratory OECD Modified Screening Test with Analyst: Wade A. Schell Date of Start of Test: 4-11-1980 Lab Request No.: 5541 S DOC Analysis Test Material:Ati-rweiz-p Fc,-'4017 L-OT -230,S- or; 14 21 NUP,SER OF DAYS li%,CUBATED 27-1 10 0:/-. L 9alL.8VIL 7D:/.!- 5TI..z 3VIt@ 2O:A,-- 0 3M EnvironrentalLaboratory OECD Modified Screening Test with DOC Analysis Analyst: Wade A. Schell Date of Start of Test: 4-11-1980 Lab Request No.: 5541 S Test Material: HYDRopal?JogE ALii3RATiDI4Cot-i?@6ti9b) 21 NUIIOSER OF DAYS 1.%ICUBATED