Document VeNVD5k3B4VJZedLjmVzq3Kg

BIODEGRADATION (301E) TES -SUBSTA JCE Identity:A mixturecontainingperfluorooctanesulfonatweh,ich may alsobe referredtoas PFOS, FC-95, or as a component of FC-206. (1Octanesulfoniaccid)(CAS # 2795-39-3). Remarks: The 3M productionlotnumber was 2330, 10/79.The test sample isFC-206 (FM-3824). Currentinformatioinndicatesitisa mixtureof 0.67% PFOS, 17.5% diethyleneglycolbutylether,78.91% water,1.33% Sultonefoamer,1% sodium octylsulfate0,.04% sodium laurylsulfate0,.5% polyoxyethylenemonooctylphenylether,and 0.05% benzotriazole. The followingsummary applies toa mixture with incompletely characterized concentrations ofimpurities.Data may not accurately retlecttoxicityof the fluorochemical component of the testsample. METHOD Method: ModifiedOECD Screening Test,OECD 301E withDOC Analysis Test type: Ready Biodegradability GLP: No Year Completed: 1980 Analyticalmonitoring: Dissolvedorganiccarbon (DOC) Statisticamlethods: Resultswere determinedby calculatioonfthe % DOC removal and graphicinterpretation. Test organism source: A 50:50 mix ofsoilextractand secondary effluent. The secondary effluenwtas from an activatedsludgeaerationbasinattheSt. Paul,MN Metro Wastewater Treatment Plant,whilethesoilwas from the city ofWhite Bear Lake,Ramsey County, MN. Test condition: Dilutionwater: Deionizedwater. Mineral NutrientMedium: Nutrientmedium per OECD 301 E method (1979).InitipaHl 7.0. Test and referencesolutionpreparation:The testmateriawlas preparedby dissolving426 mg FC-206 inone liteorfmineralnutrient medium. Thissolutiognivesa finatlestconcentratioonf40 mg DOC/L. The referencesubstance,hydroquinone,was prepared by dissolvin3g0.6mg inone liteorfmineralnutrienmtedium. This solutiongivesa finaltestconcentrationof20 mg DOC/L. Test vessels:500 mL Erienmeyer flaskscontaining250 mL test solutionand capped withfoam plugs. Incubation conditions: Lighting:Constantdark conditions. Temperature: 23.5 - 240C AgitationC:ontinuously Number ofconcentration1s:plushydroquinon(ereference substance)and blank,allinduplicate. lnoculum conditionon testinitiationsN:ot given.(No measurements are requiredper method referenced.) Element Basis: Decrease indissolvedorganiccarbon compared to the blanks. Test substance flaskconditions:Not given. RESJLTS Nominal concentrations: Blankcontrolh,ydroquinoneat-20 mg DOC/L and at-20 mg DOC/L plusHgC12 (inhibitetde,stmaterialat- 40 mg DOC/L and at-40 mg DOC/L plusHgC12,allinduplicate. Element values: 28-day degradation= 91.4% duplicate1 and 92.3% duplicate2. Mean value= 92% Remarks: Testingwas conducted on the mixtureas describedinthe Test Substance Remarks field.The valuesreportedapplytothatmixtureand not the fluorochemicalproportionalone. CONCLUSIONS- The testsubstance % degradationbased on the mean DOC removal value was 92% after28 days. Submifter: 3M Company, EnvironmentalLaboratory,P.O. Box 33331, St. Paul,Minnesota,55133 DA k QU KLITY ReliabilitKyl:imischranking2. Thisstudymeets the criterifaorquality testing.However, thesample puritwyas notpropedy characterizedand the studylacksanalyticaclonfirmationoftheamount offluorochemicalproportion insolution. REFERENCES The studieswere conducted by the 3M Company, EnvironmentalLaboratory, Lab Request number 5541 S, 1980. OTHER Last changed: 6/27/00 ii14 1.11 A TECHNICAL REPORT TO: TECHNICAL COMMUNICATIONS CENTER - 201-2CN SUMMARY .ti)i.ior-tIafirietporitsprinteodn bothsidesofpapesre,ndtwocopiestoTCC.) 6/30/8=C 1,"nvironm(--ntal Laboratory (EE 0"u1c t Commercial tio)pr%-Tl-tra@ Chemicals Division & PC) Biodegradation of "LIGHT WATER" Products John A. Pignato Au tharis) Commercial Chemicals in OECD Division Test-6/80 - 236-2A Oopt- .1-bw@ 0535 Proloc gum 9970012600 Report NumBWr- 040 (01) Eric A. Reiner Not ---- -..w,ance Environmental Lab (EE&PC) - 21-BW None - See 3M Environmental Lab Request I;o. 5541S SECURITY 10, Open KEYWORDS: (Select terms from 3M Thes.aurus. Suggost other applicawe terms.) 1,*nvironmental Eaboratory (EE & PC) CURRENT 0 Closed OBJECTIVE: 3R4CHEMICAL si@. REGISTRY P' To use the internationally recognized Test with DOC analysis to show that products are highly biodegradable. Empi (631 ----Ru---b-s-rrsT' 47816 No. iil"-@- lncj,,dl@nCgO@1.1-@hst@ 31 Now Chemicals Reported Yes No OECD "LIGHT Screening WATER" I-F Iroc RaElPeOrRt3TM'erAsBtSoTCRoAnCVT&:ny (R2&0D0.-250 words)This abstractinfornutio.insdistributebdy the TechnicalCommunications Center to 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. I-n 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. InformationL' lnittals: 114T!@ODUCTIO(4 BIODEGRADATION OF "LIGliT WATER" ItJ OECD TEST PRODUCTS JM Lnviconitientai Laboratory chose to attetapt to destionstrate t biudeyradaoility of "LIGHT WATER" products using the OECD screeninhge iiietho(bJecause the test has wide international acceptance and the Lnvironittental Lab has had experience using the OECD test 'as a participant in an international ring test. These resul t coiiijilL,jiitehneL existing SOD data that show these products,to have a tiigh degree of biodegradability. s will 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 tesling, the OECD test is an appropriate method for measuring % total degradation. The TOC analysis method even measures the perfluorinated portion of these products. mc,rilODS AND MATERIALS methods and Conditions - Wade A. Scheil performed this testing f-07-Lo-w-l@tnhge September 19, 1979 version of the Modified OECD Screening Test with DOC Analysisltl). A copy of this method is attached (Appendix 1). l@eductions in the scale of the test were necessary to adapt it to availaule shaking equipment. Five hundred-ml Erlenmeyer flasks replaced 2-liter flasks, 250 iniof inoculated nutrient solution replaced 90U mi, and the sainple size was 25, not 30 ml. As in the prescribed procedure, the analyst used only the last 10 mi 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). Chei(ticals- All chemicals were reagent grade unless otherwise noted. The 3M "LIGilT WATER" products used were samples of commercial material of Antwerp iiianufacture. Their label identified them as FC-203 NFP, FC-3017 Lot 2303, and FC-206 (FM 3824) Lot 2330 10/79. '2ne hydroquinone used as the calibration compound was Aldrich reagent yrade 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 signficant effect on results. The ii,tpuritieshave 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 inoculuni, was St. Paul city water passed thcougli a carbon bed, inacroporous anionic resin bed, and 2 mixed bed deionizers, and then filtered tnrouyh a 0.2-uia filter. This water was also used to make up losses to evaporation. Stocks of water collected at the start of the uxpuriioent ensured consistent water quality. In prepariny nutrient solutions, 26.4 g of anhydrous Na21lPO4 replaced ttie prescribed 27.5 9 CaCI2. 33.4 9 of N[121IP04--21120,36.3 ( j of CaC I2-21120 replaced 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 The 100 my of Fe-chelate used in the trace element sanodlut7iUoniticgoEnDsTiA)s.ted of equimolar amounts of PeC13 and EDTA (30 mg FeC13 'r(@A.@_q2acetnra tions - DOC measurements of sample stock dilutions served ai--Ebt'iaesis- for adjusting initial test concentrations to approximately 40 Lug 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 ineasureiiients. The low toxicity of "LIGEIT WATER" products to iuiniliickreoloyr.ganisinsmade significant inhibition at this concentration 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%. Tcwoorrebcltainokn,sc.ontrols, instead of 1, were utilized in making blank DOC Inoculum - The inoculum was 50-50 mixture prepared according to steps !.-6.2@.1and 1.6.2.2 of the OECD test using fresh soil from E. A. l@einer'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. !@terile Controls - Replicate vessels containing 400 ing/l flgCI2 served @T@s'Ee-El'Te--Con@trOlf-osr each test material and the calibration coitipound. flandling of these sterile controls was identical to the handling of the viable cultures. They had the same innoculum and sainpling frequency, but the analyst only made DOC U- and 2d-day sainples. measureinents on the @6@al@ Preservation - Tne sample preservation method used was that drescrioiid-oy the IZOC manufacturer(4). The method involves adding i drop of concentrated HCI to the 10 ml filtered samp saitipleto <LjLl2, and storing the samples under refrilgeesr,atbiroinngiinng vitahles 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 plitlialate,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 elimination of the llgCl2preservation technique described in the OECD method. All TOC analyses were made within a iaontliuf the E.)reservationof the saiaples. Instruitiontsand egpipment - The organic carbon analyzer used was a 5-o-li'FiTian-DC-52A. per liter. Its sensitivity limit is between 1 and 2 mg of carbon pTltuigess.toppers for the reaction flasks were clean, porous, plastic foam Gelman 0.2 uinmeinbrane filters, (Part No. 64814) boiled 3 atniailiyiseisAsn.d stored in deionized water, served to filter samples for DOC Glassware cleaning involved soaking in chromic acid cleaning solution (CtiroiiiertiefRo)llowed by 6 deionized water rinses. LTESULTS AND DISCUSSION Tai)ie I summarizes the results of this study. Appendices 1 and 2 contain the actual data sheets and plots of the degradation as a cfoujnicitpiloenteolfy.tiate. All 3 "LIGFIT 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 L-'C-206 23 FC-3017 37 ilydro- 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 "LIGIITWATER" products, it is very unlikely that physical means such as adsorption, volatilization, ur i)ruci,,jitatiocnaused the loss of soluble TOC (DOC). This is suostantiated by the relatively high oxygen demand observed in BOD tests of the 3M Environmental Laboratory (PC-203 B005/COD - 0.5, FC-206 BOL)5/COD = 0.5). The sterile control data in Table 2 provide further substantial evidence that the soluble TOC loss from the test samples containing "LIGLIIZWATER" 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. 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 inajor factor, at least for nonmetabolized "LIGHT WATER" coinponents. This control doesn't prove that physical processes did nut reiiiove"LI(;klTWATER" inetabolites in the test runs, since no taqtabolisinoccurred in these sterile controls, but the catabolic* [(-)rjaatioonf less water soluble materials is unlikely because QaLabolic products are usually inore polar and sinaller. TABLE 2 DOC Loss from Sterile Controls Product DOC Remaining at-Da)Z 28 FC-203 I"C-20() t.'C-3017 ilydro.quinone 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 sliglitly brownish, as was the solution in the hydroquinone sterile control after 21 days. aL -Least This suggests that hydroquinone is not stable, OECD experIinmetnhte. presence of llcjci;lt,hroughout the course This apparent tendency of hydroquinone of the 28-day to spontaneously form insoluble humus-like material makes it an inappropriate control compound for the OECD test. Its partial precipitation with 119CI2 also casts some doubts about the ai,pipertolpiroida.teness of using the I]gCI2 preservation procedure in the OECD COL4CLUSION The present study conclusively demonstrates 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. ItL'L-'Li@L';iN C E S Organization for Economic Co-operation and Development Chemicals Testing Prograin Expert Group on Degradation/Accumulation, Dec., 1979, Test Guideline for the Modified OECD Screening Test with DOC Analysis (Level I) (ii.G. tiosler)Revision of Sept. 19, 1979. Dohrinan DC-52A Operating Manual, 4th Ed., 1978, p. 3-1. Appendices; 1 - OECD Test Guidelines (Sept. 19, 1979) 2 - Data Sheets 3 - Graphs Cmaotlaebcoulliessi.a is biologically facilitated breakdown to less complex Ai.)I-,ENL)I1X Tokyo, December 197 C 118/79/lnt. OECD Expert Chemicals Testing Programme Group C, Degradation/Accumulation *4bdified OECD Screening with DOC Analysis Test Test Guideline C 118/79/In-.. Test Guideline for the Modifi4d OECD Screening Test witli DOC Analysis Date of last revision: Sept. 19,1979 Lev6l I test for ready biodegradability 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 i@snot unequivocally requircd but constitutes useful information for the conduction of the test. 3. QunlifN,inm Stntements The nethod is suited for the measurement of the aerobic ultitna*.obiodegradibility of water soluble, non-volatile organic compounds. It is unsuited for the biodegradability evaluation of mix%-ures. Sept. Ia. 1979 Modified OECD Screening Test with DOC Analysis Preamble It has to be realized that the following procedu.-e for the modified OECD Screening Test has to be regarded in some points as provisional since 3everal important features such as the calibration compound and the new test duration of 28 days are completely untried yet. H@wever, since the test constitutes a modification the of&'icial, widely practi3ed and well accepted OECD Screening Test for the biodegradability evaluation of surfactants there should exist a good prospect for success. September 19., 1979 OECD Chemic als Testing P-@ogramme Test Guideline for the Modified OECD Screening Test with DOC Analysis 0. TI.,,t4eest procedure constitutes a modifica tion of the OECD Screening Test (OECD Environment Directorate, Proposed Method for the Determination of the Biodegradability of Surractant3 Used in Synthetic Detersents, Paris 1976, and council directive of Nov. 22, 1973, on the approximation of the laws of the member states relating to methods of testing the b4-odegrada- b.'&'&itoyf a.-&ionicsurfactaots (73/405/EEC), Official wournal of the Eu.-opean Communities No. 4 347/53 of Dec. 17,,1973) for the application of the dissolved organic carbon(DOC)analyzi3. 1. Methcd 1.1 introduction: tion of test Purpose. scope, and exnlanation rel evance, of 1.*LMit3. and aoolica- The purpose of the method is the mea3urement of the ultimate biodegradability of water 3olubles non - volatile organic compounds in an aerobic, aqueou3 medium at a starting test concentration corresponding to 5 - 40 mg DOC/I ( In order to avoid inhibitory effec*.sit is in the investigator*o3wn interest 2 2 to choose as low a starting concentration analytical capability permits as his 1.2 Definitions and units 1.2.1 Definition of biodegradability D= t L where Ct - Cblti x 100 co - c bl oi D t = degradation at time t in.pqrcent DOC-removal c 0 = starting DOC concentration culture medium (mg DOC/1) of the c t = DOC concentration of the culture at time t (mg DOC/1) medium c bl= starting DOC concentration 0 blank (mg DOC/1) odO the c bl = DOC concentration t of the blank at time t (mg DOC/1) 1.2.2 Units The degradation is stated as the percentage DOC-removal within 28 days with respect to the test material DOC-removal 3 3 1.3 Relerence compounds .6-3.1 Calibration compound The calibration compound used in this test is hydroquinone at a concentration corre3pordingto 20 mg DOC/l. Hydroquinone has to exhibit a DOC-removal of@@60 % within 28 days, otherwise the test is regarded as invalid. 1.4 The orinciple of the method A predetermined amount of the compound is di3solved in an inorganic medium (mineral nutrien4* solution, fortified with a trace element and essential vitamin solution), providing a concentration corresponding to 5 - 40 mg DOC/l. The solution is inoculated with a small number of microorganisms from a mixed population and aerated at 293 - 298 (20 - 250C) in the dark or at least in diffuse light only. The de,-,radationis'followed by DOC analysis over a 28 day pe.-iod. The procedure is checked by means of a standard (hydroquinone). A control with inoculation but without either tezt material or standard is run parallel for the deter,nination of DOC blanks. 1.5 Qualitv criteria 1.5.1 Reproducibility The reproducibility of the method i3 appropriate for a screen,4..Xtlegst which has solely an acceptance but no rejective function. 4 1.5.2 Sensitivity The sensitivity of the method i3 largely determined 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 evaluat-oion of water soluble, non - volatile organic compounds. 1.5.4 Possibilit-.yof standardization The test versiom with specific analyses.-for anionic and nonionic surfactants 4-s stardard;-zed as "OECD Screening Testn. 1.5.5 Possibility of automation Parts of*the test,, e.g.., the analys4hs.can be automated, although hardly the total procedure. The procedure is, though, well suited for be4&ng operated w4&th whole series of te3t materials. 1.5.6 Costs ( in 1978 Swiss Franc3 1.5.6.1 Equipment Glassware 3haki.ng machine Carbon analyzer Miscellaneous provision for (PH air 20002- 6000 - 20000,- meter, balance, dond'itioning) 42000,- 5000,- 5 5 1.5.6.2 :.5.6.3 Person hours 10 Approximate total cost per test750s(i-nvestments assumed to be amortized) 'OOOS- 1.6 Descript;-on of the method 1.6.1 ReaZents and materials 1.6.1.1 Deionized water Deion4-zed or distilled water free of toxic substances (copper in particular), for general use a3 a solvent. Wate--which has been dei-onized by distillation or ion exchange is suitable. A high purity of this test water is necessa.-y in view of the DOC analyses in the concent@-ation range of 0 - 40 mg/l. I&nhecontaminations result from inherent impurities but al30 from the ion exchange resins and microbial devel opn-,ent(sbacteria, 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 necO33&ry, suitable water may be gained by UV irradiation or other means. 1.6.1.2 Nutrient solution Mix 1 ml each of the followirg solutions (a) to (f) and make up to a volume of 1 1 with water 1.6.1.1 6 6 (a) Kh2 FO 4 x 2 F.FO 4 Na 2 liFO4 NH4cl A.R. A.R. 2 H20 A.R. 8.5 9 21-759 A..R. 33.4 g 20-0 9 in 1000 ml of water 1.6.1.1 the PH value should be 7.2 (b) 22.59 of M9504- 7 H20A.R.dissolvien1d00m0l of water (3-3-1) (c)27.5 s of CaCl2 A.R. dissolved in 1000 ml of water (d)0.25 g of FeCl3 * 6H20 A.R. dissolved in I-OCOml of water (3-3-1) 'Lh4-solution is prepared freshly immed4-ately before .,se. (a) T.-ace element solution .MnSO4 0 4 H20 H 3BO 3 ZnS04 - 7 H20 (NH4)6MO7024 39.9 mg (30.23 mg MnSo. . H20) 57,2 mg 42.8 mg 34.7 mg (36-85 ms (NH4)6mo7024*4H 20) Fe - chelate (FeCl 3# EDTA) water 1.6.1.1 100 mg 1000 ml S%"erilizationof the trace element stock solution a'.-393 (.7%()1200C)t 2 atm., 20 m4in. 7 7 Vitan,in solution Biotin Nicotinic acid Thiamine p-Aminobenzoic acid Pantothenic acid Py4-idoxamine Cyanocobalamine Pol.-6c acid water 1.6.1.1 0. 2 mg 2.0 mg 1.0 mg 1.0 mg 1.0 mg 5.0 mg 2.0 mg 5.0 mg 100 ml The Solution is filtered sterile (0.2jum). Instead Of solution 1.6.1.2 (f) 15 mg of yeast extraxt ,,iaybe used per 100 ml of water 1.6.1.1. i.6-1.3 Biodegradability standard Hydroquinone 99.5 % DAB Erg. B. 6 1.6.1.4 Mercuric chloride solution 1 per cent or Hgcl 2 in water 1.6.1.5 Shaking machine accomodating 2 ltr. Erlenmeyer rlasks either with automatic temperature control or used in a conr."ant temperature room at 293 208(K) (20 - 250C) 1*6*1.6 Narrow neck 2 ltr, Erlenmeyer flasks. (Creased flasks are reco=ended) The flask3 muat be carefully cleaned withs e.g., alcoholic hydrochloric fluted 8 ..-euses rinsed and dried in order to avoid -nation with residues from previous tests. lasks also have to be cleaned berore their -.rstuse since they may be contaminated, 1.6.1.7 Membrane filtration apparatus Membrane filters 0. 2,um 1.6.1.9 Carbon analyzer 1.6.2 Inoculation ZiEher ol the following three alternatives may-be used as inoculum or a composi%'Pe sample thereof. 1*6%291 Y.no..,ulufrrnom secondarv e-Flfluent The inoc"ium is gained preferentially from a secDndar,,,effluent of good quality collected &Ir"om a treatment plant dealing with a predominantly docestic sewage. The effluent must be kept under ae-robic conditions in the period between sampling a.iduse. To prepare the inoculum the sample 4&a 6'ilteredthrough a coarse filter, the rir3t 200 ml )eing discarded. The filtrate is kept aerobic until 4sed. The inoculum must be used on the day of collec'.-ion. 1.6.2.2 inoculum fro-m 3oil 100 g of soil (fertile, not sterile) are susperded in 1000 ml of chlorine-free drinking water (soils with an extremely large content of clay, 3&nd or organic carbon are ur.3uited)..After stirring the suspen3icn is allowed to settle for 30 minutes. 9 9 The supernatant is filtered through a coarse filter paper, the first 200 ml being discarded. The A'ilttra#-i.se aerated immediately and until use. The inoculum must be used on the day of collection. 1.6-2.3 1.6-2.4 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-muzt be used on the day of collection. Composite inoculum Equal volumes of the 3 inoculum samples are united, mixed well, and the final inoculum drawn b'!romthis mixture. The suitability of the inoculum is checked by means of the standard hydroquinone. i.6.3 Conduction of the test i.6-3.1 Proceduz-e The test materials are evaluated simultaneously 4n duplicates together with the standard (1.6.1-3) and a control test with inoculation but without either test or standard material for the determination of DOC blanks. The standard material has to attain 1 60 % DOC re- moval within 28 days at a starting concentration corresponding to 20 mg DOC/1 - if < 60 % DOC removal are achieved the whole series ha3-to be discarded +) - +) T is imit '3 -b 3e on present experience wit-h- the 19 day version of the test A revision of this limit or even of the stand*ard might have to be considered after the accumulation of experien- ce 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/l. 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 openirz of the vessel is covered with, e.g., alumi.num foil in such a wa:y'thatthe exchange of iir between the flask and the surrounding atM03phere is not unduly impeded (Cotton wool is unsuited because of the DOC analysis). The ve33el3 are t.",.einnserted in the shaking machine. The temperature of 293 -298K ( 20 -250C) Must be mainta4ned 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 beperformed in rather regular time intervals and -21. day) 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- 4ugation preceeding the carbon determination. The latter requires differing volumes for the different inat Evaporation losses 'ruments. of the culture medium are to be made up by adding water (1.6.1.1.) in the required amount3. A'he culture medium a sar,.ple.Material is to be mixed well before wl'thdrawing adhering to the wall of the vezzel has to be dissolved or suspended before samp;ing. The' membrane filt6-at4-onor centrifugation has to be.@Xone immediately. The filtered or centrifuged samples,have to be analyzed on the same day, otherwise they must be pre3erved with 0.05 ml of the HgCl 2 solution (1.6.1.4) for each 10ml of nutrient medium or by storing them at 2 - 40C. The biodegradability test i3 valid provided the atandard exhibits a degradation rate within the specified range. The test can be*finished before the 28. day it complete mineralization is accomplished. All 3teps require great care and cleanline3a of the vessel3, pipettes etc. but not 3terili-ty. 12 12 1.6-3.2 Calcula-ion of results The degradation at the time t is calculated from the determinations of the DOC concentrations at the beginning (Co and the time t (Ct) according to c t - Cblt D@= ix 100 co - c bl 0 where D t = degradation in per cent at time t c 0 = measured starting DOC concentration of the inoculated culture medium (mg DOC/1) ct = DOC concentration of the culture medium at time t (mg DOC/1) Cb!O = starting DOC blank of the mineral nutrient solution with inoculation but without test material (mg DOC/1) c blt = 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 031 ;. The means of the D t values are calculated and reported to the nearest full per cent. Results - 13 13 ending in 0,5 are rounded up to*the neares,,%w,h#ole number. The course of the degradation test is followed graphically in a diagram as shown in the at-otachedexample. 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 '-@l6-0 1*DOC-removal. 1.6.4 JA%LnicaL.Lyticmaelans 1.6.4.1 Membrane filter O@2,um, 25 mm 0 (1.6.1.6). Pre- paration'of the filters: membrane filters are impregnated with surfactants for hydrophilizat4&on. Thus each filter contair.3up to several mg of soluble carbon which would interfere in the biodegradab4-litydeterminations. Therefore the filters are purified from surfactants and other soluble organic:**interferencesby boiling them 3 times I 1,.r each in deioni'zed water. These filters may be stored in water (1.6.1.1) for at least one week. Other membrane filters are suitable if it is assured that they neither release carbon nor adsorb the co.npour.idn the filtration step. Ir the samples are centrifuged, this has to be done at 40,000 m 3ac-2 ( 4000 g) for 15 minute3, preferably in a refrigerated centrifuge, in any case <400C. (Remark: the differertiation 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 or the bacterial plasma 13 redissolved. At hi.&her test concentrations ( @10 mg C/1) and the same small inoculation the centrifugation error seems to be comparatively small Form Sheet for the Modified OECD Scrpening Test I;Ap. no.: r ' of 3tart or Lq::.L: Tezt / 3tandard material: Theoretical test conc.: Inaculum; Carbon analyzer: A.: Controls: itock 30lUtlon of thl* test material 1000 mg/l. dilution ... /1000 'nl of nutrient solution) TOC DOC U: C-irbon detprmination5: Culture medium Xineral nutrient -.olution Mith test material and with inoculum Flask no. AnaLyses a a 2 a+ I 2 1 2 ct Theor. -DOC - concentrazions ast4tr x 4mays conc. ag/i I mg/i 0 (Cf)).1 @7 -14 1-21 1 27. 1 28@ .'41neral nutrienz *inlut&on without te3t materialUut -,vlth lnoculum 2 131ank b2 b + bp 2 2 ct ) c 2 c + C, M3 @2 cbl) C: Evaluation of raw data,. DOC - concentrations blanks Flask no. U3 . zoo am rjmova@@at-z*r a "ys 7 14 21 27 28 2 1&2 A3 2 . loo mean JV%- -ft" -T"C D for day x 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 mean3 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 mea3urement3 (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. AIJIIIENL)IX2 3M EnvironmentalLaboratoryData Sheet -results of the Modified OECD Screening Test- Lab Request No.: 5541 s Data of Start of Test: 4 -%%-,go Analyst: Wade A. Scharl- ast MaterialA:w-rWr-PP--@@ MFP fhooreticat OC of Test Material:-(-No-rC-kLC-tAt-A---rSrt-amn%dard Material: hydroquinone,98.5%. Lot Measured TOCY of Test Material:--147..000 Inoculum: soil mg TOC kg and_non-chlorinated !Cac@ndar-OYff luent frcma municloal was-townter-treatmentplant Carbon Analyzer: Bohrmann DC-52A Total Organic CarD*n Analyzer 100147, Aldrich A. Date mination -of OCI a Test Material: A stock solutionof the test materialwas preparedby diluting If70 with dolonizedwater. :"Mg too final volume Of500 ml - conc.test material TOC In stock solution stock solution mg/i mg/i Toc test material mg/kg This result was used to calculate the necessary amount of test matorlql to prepare a culture medium of 40 ms/I DOCI. ?,34C) 332, 142,.Iooo 0. Carbon Determinations:2 Culture medium Flask no. Msionleurtailonnwuittrhient I test material and with lnoculum 2 Mineral nutrient solutionwithout test material but with Inoc7iu-m Blank 1 Blank 2 C. Evaluation of Raw Data: Conc. of test material In flask. mg/i ?,Iaz CalculatedDOC' DOC DOC - conc. of test material Anal- 0 (CO) 7 In flask.EL/I X303 --- al *Z.$t 37,0 40-0 a ti, al + a2 ...an c n zsz @to. 0 I + b7 ...b c t2 n q/.S* 37.q b bl& b -1 Ci + C I Cbll" n (Cblo /-$- F d-+ 1 Cbl2@ 2 0 c -0.1 fter x days lncub tion, /I 14 21 27 28 co-ZT- 7' C -'1 5-1- 4-1 Z'E s,.r s-..0 z.- (o.o 14- Z.E 0.11 -0.1 4D. i e3.I 0.9' o., 1-3 0.6 -o. 1-2. o-6 Flask 2 Degrade ton OC resoval after 7 iz % 21 1 27 I& %43..Tq Zo. 1 6.. 91.0 dals Incubation a 13.0 Cl 3-'D 93 See attached formula used section 1.6.3.2for the to calculatedegradation. Results of blank no.1 and no. 2 were ftera"41for"" dayx. rw *"ra" values were used In the calculationk Cbla" Cbll Cbl2 (from above) 2 1. The test material Is c=Wlotely soluble In water. Therefore, the TOC a DOC. 2. DOC analyses performed more than twice on the same sample were run for quality assurance purposes* 3M Environmental Laboratory Data Sheet -results of the Modified OECD Screening Test- Lola Request No.: 5541 S Date of Start of Test:_.+ - It-ad Analyst: wade A. Schell 5st no tor;at :-I)TOJLHI--f-c.-?,rj-fFi,@ a; i'hooratcla 70C of Test Material: tol-ri-StandardMotorIaI: hydroqu In", ALC&4J-A-rei>) 98.SX. Lot Measured TOC of Test Material:- 113,900 mg TOC/kg Inoculum: soil and-non-chlorinated secondary effluent fr(ma munlci"l wastewater treatment Plant Carbon Analyzer: Oohrmann DC-5ZA Total 100147, A IdrIch A. Determination of- OC I of Test Material; A stock solution of the test material was prepared by diluting /,ocio with dolonized water. to a f lnal volume of 5'00 mi conc.test material In stock solution mo/I TOC stock solution ing/i TOC test material mg/kg This result was used to calculate the necessary amount of test materill to prepare a culture mdlum of 40 mg/i DOC z@ /ZO 199 1,3j,@oD 0. Cnrbon Determinat-lonso2 Culture medium mineral nutrient solution with test material and with Inoculum Flask no. I 2 Mineral nutrient solution without test material but with lnoculum Blank I Blank 2 C. Evaluation of Raw Data: Conc. of test material In flask. mg/l Calculated DOC I of test material In flask mg/l DOC Analyses at DOC - conc. 0 (co) 7 I '3/00 fter x days lncub tion, mg/i 14 21 27 2E 'S *74.r4 F ctl=al + 82 ...an n J/o, o bi + b7 --.-b c t2. n cl + C2 Cbll' n q2.o 3/,0 2.3 !b ql,3 30-7 --b;14 b til- Ci I $.1 c 9 id d 4@-d 2 Cb]20 n- c AVP- V* I L/ a 3. q 4t, 2-C gli 3-M ?. 3.4 3.6 7 - u Flask no. Degrade ton - % DOC 7 1 14 21 23.3 q4.9 9411 I after dais Incubation 27 1 9311 L7 '914.1 1:@3 '741 13 92 $*a attached section 1.6-3.2 for the formula used to calculate degradation. ltesultsof blank no.1 and no. 2 were averaged for each day x. The average values were used In the calculations. Cbll+ Cbl2 (from above) Cbl&ve 2- 1. The test material Is completely soluble In water. Therefore, the TOC - DOC. 2. DOC analyses performed more then twice on the so= sample were run for quality assurance purposes. 3M Environmental Laboratory Data Sheet -results of the Modified OECD Screening Test- LiibRequest No.:-5541 s Date of Start of Test: +-11-17io Nnalyst:_.!@adeA. Schell i*est Material :-A-Wrwcplp.r-c - @tnt-7 L. or - 7-'5o Theoretical OC of Test Material. NOT CALetALAIC-0@ Standard Material: hydroquinone, 98.5%. Lot tieasured TOC7 of Test Material: lq2 -000 ag TOC/kg lnoculum: so!! and non-chlorinatod socondetz-@ffluent frcma munlctipalwastowe-ter treatment plant Carbon Analyzer: Dohrmann OC-52A Total Orqanl; (;SrbonAnalyzer 100147. Aldrlrh A. Determination of TOC I of Test Material: A stock solution of the test material was prepared by diluting with delonized water. to a fIna I vo Iume of !!,;Oo mi conc.tast material In stock solution mo/i TOC stock solution mg/i TOC test material mg/kg This result was used to calculate the necessary amount of test matorill to prepare a culture madlum of 40 mgll DOC )4190 -1471 1921 ooo U. Carbon Determinations: 2 Culture medium Flask no. Mineral nutrient I solution with test material and with Inaculum 2 Conc. of test material In flask, mg/l--- Calculated DOCI of test material In flask mg/l DOC Analyses DOC - conc. after x 0 (CO)i 7 14 @(0.0 -I.L- 13/-.a a WO.M- .2.T' days lncub tion. mgll 21 27 1 21 . g--- 2 -,Z.z I + &ft2... ctl n 203, ell)0. bj_ V.7,3 b 2-4 -Y 300 2.2 Mine ral nutrient solution without test material but with lnoculum Blank Blank 2 C. Evaluation of Raw Data: b c ti- b@@b n ci + C2 Cbllm a a +d I2 Cbl2" n- C6) L /I-?-3,(p f 0.1 Of Cz- 0.3- dl4!P 1,5- -017. -0. I 0-4/ 0.1 089 0.5' o, 0 0.14 V4 0.'(o jq. -o'l o,y ri/l-.Z 0-6 Flask no. 2 F,mean Degrade Ion - % 7 14 i;.0 52. 92,1 .3r7 114 romval after 21 27 -- S, 95*,.a IS-2 94 Incubation 2. 15' See attached section 1.6.3.2 for the formula used to calculate degradation. Results of blank no.1 &W no. 2 were averaged for each day x. TM &,forage values were used In the calculations. Cbli + Cblz (from above) Cblave 1. The test material Is comlotely soluble In water. Therefore, the TOCn 0%. 2. DOC analyses perfo. more than twfco on the same s&vplo were run for quality assurance purposes. 3M Environmental Laboratory Data Sheet -results of the Modified OECD Screening Test- Lab Request No.- 5541 S Date of Start of Test: 4--1 Analyst: Wade A. Schel I est Material:-- st ard Material:. hydre3ulnone,-98.5%, Lot II)RIIL Aldrich .hooratical OC of Test Material: hoasured TOC7 of To t Material: 6.S.4+4% C RALC C KE-OTJASSUMF-D mg TOC kg *10 L-$L SOLU6LE OkCvANIC C lnoculum: sot? an'dsnon-chlorinatad secondary effluent frCMa municipal wastewater treatmen-t-plant Carbon Analyzer:-Etrm-ann OC-52A Total Organic- Cart@Ao-nnal-yzer A. Daterminatlon of TOC of -Test material: A stock solution of the test material was prepared by diluting mg to a f Ina) Volume of with dolonized water. ml conc.test material In stock solution mg/i TOC stock solution mg/i Toc test material mg/kg This result was used to calculate the necessary amount of test matorli! to prepare a culture medium of 40 mg/i DOC U. Carbon Daterminationso Culture medium Flask no. mineral nutrient 1 solution with test material and with lnoculum 2 Mineral nutrient solution without material tbeusttwith lnoculum Blank I Blank C. Evaluation of Raw Data: Conc. of test material In flask, mg/l '7. &ia-riOAI 4@Aw %eolAri'I1-13It11710o eomftuA@,D.- @66 S7&AA)DARD t4k-i RIA L AISOVE I Calculated DOC DOC DOC - conc. of test material Anal- 0 (Co) 7 In flask. mg/l yses - fter x days Incubation, mg/i 14 21 27 2 ZO. 0 at '7.1 z ]f@-a 1-7 IK.4- Ir-- ly /.3 1,7- 2@. - cti, at a2 ...an n 2.o /.4 2, ? g 3o.r. ZL). 0 b" /6.2 0. .2.-CA bb: -a. ci /.4 b ::0:@bb bi + bz ... b c t2 m n /7.0 /,2- /.e o.s q C3 0.9 I Ci + C 2 Cbllm n rb l@ro d-I dli c b)2 61&ve C I+ ci.%2. -0.) Flask no. Degrade [on % COC removal after 7 14 21 1 27 d @s Incubation 1s 7 102 gq.9 qq.9 q 2- 1? q3 Soo attached section 6.3.2 for the formula used to calculate degradation. awr"ed Results of blank no.1 and no. 2 were for each day x. no average values were used In the calculations Cbll c bIZ (frm above) Cb I.,,. 1. The test material Is completely soluble In water. Therefore, the TOC - DOC. 2. DOC analyses performed more then twice on the same sample were run for quality assurance purpos*$. 00Y. 90%- so%. 70%- 60%:H 50%, koy 4c OK 3M Environrental Laboratory 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:At4TWEPP FC -203 14;*P AF -T NUT,SER OF DAYS INCUBATED 10 OY, - --------------------80%,. 60X 50%. 40% W 30%- 20% 10% .-T --law ........... 3,4Environ.-nentaLlaboratory OECD Modified Screening Test with DOC Analysis Analyst: Wade A. Schell Daie of Start of Test: 4-11-1980 Lab Request No.: 5541S Test Material:-AN -rweg? FC- 2-0(; F,M 38 1-07'- 2-330 loo@29 21 NUT,.BER OF DAYS INCUBATED IOVA 90% 80%,. 71VL w 60% 1?> V4 50%. 40,A W 30% .. 0 3.4Environ.-entalLaboral@ory OECD Modified Screening Test with DOC Analysis Analyst: Wade A. Schell Date of Start of Test-.4-11-1980 Lab Request No.: 5541 s Te st Mate ria I AN*rWEiz? F C. 3017 t-oT-23o5 iE OF . ..... 14 2t NUP.SER OF DAYS lt,'CUBATED T-- t 2 IOVA 9TALL 70:/.: Lw 6 (YL 50Z z 3WAO 2 0 3.4Environ,-entalLaboratory OECD Modified Screening Test with DOC Analysis Analyst: UladeA. Schell Date of Start of Test: 4-11-1980 Lab Request No.: 5541 S Test Material:__HYDRC)4,tAI?JO14E @CALii;.'ZAToot4 7-- 4 14 21 NUP.;'oc,ORF DAYS 1,%ICUBATED -2" F LC-'-@A3@3R0EM@RE-;P"W@OIUEBSOTOK - 42.5W Ref: ENVI R. ASSESP. X-Oir., PRODUCT. ING #-g6 zcen PRODUCT REQUEST i DIV[-qlr)N - ?- ECOLOGICAL ASSESSMENT FOR LABORATORY WORK C/le LAB REQUEST 3M%ctiowtoorapoi S Name: 'Fri Request Date: @.tvroiectNo.: Code No.: tn A @ECIUESTER 6c ru Phone No. Prob. No. Sample Date: 1))w, 1% t IC ItJ Date Needed: Descript(i-o)n,:C4c"ot, /t, -;tr-e,. Purpose of Tests: 7-a le@ven t,.r l@ll- 5-0 7!@, 2,4;6 11 . Date ReceivedSam I Analyst: Date Completed:-"- Hours Spent: IS-A y B_P LABORATORY 3-iso PleaseCall Requester IfDelays Are Encountered. PHYSICAL & CHEMICAL PROPERTIES c Heatof Combustion Cal/G (BTU/Lb., v Density Ash Other WATER POLLUTION PH COD BOD5 BOD, Ultiffhate TOC GC Oxygen Uptake DehydrogenaseActi-itv(TTC) Other ; fii,QofA11 I L) SIOASSAY SpecifyOrganism and Method: A, '.I -7 1307o A- IfKA. @-cC-,,-jorz. 09-3 ify do,* LEACHATE p COD BOD5 FROM LEACHING BOD, Ultiniate TOC GC TEST Other_ OTHER Bioaccumulation Persistence Degradation Products 1-3 17,3 !2!Z!7j2 1707, 4,'011, 3 White - R enrad - nob,-