Document MGmnGQ1mwbxxaqgDzG4MRZZwV

1260 Study number AR 236. 2711 414 | AR 05.820 AMI: StL 22 FINAL REPORT LED Biodegradation study of( C81) by microorganisms ASEM 8 `September 18, 2003 Chemicals Evaluanad tReiseaorcnh Institute, Japan Company Sanitized 000001 RAK 22D WE" Sponsor Tide Study number 14114 STATEMENT Kurume Laboratory Chemicals Evaluation and Research Institute, Japan DAIKIN INDUSTRIES, LTD Biodegradation study of ( C81) by microorganisms 14114 the 1, the undersigned, Final Report (Study heteby declare that No 14114, issued this report provides a correct English translation of on September 18, 2003, amended on October 10, 2003) Date Study Director December 2, 2003 ZY Miatam_zbn Yasuko Matsunobu 000003, on GLP STATEMENT Kurume Laboratory Chemicals Evaluation and Rescarch Institue, Japan Sponsor DAIKIN INDUSTRIES, LTD Tide Biodegradation study of C81) by microorganisms Study number 14114 This study was performed in compliance with "OECD PrinciplesofGood Laboratory. Practice" (Novemmben 26, 1997) "Thisfinalreportreflectsthe 1awdataaccuratelyan ithasbeenconfirmed thatthetestdata are validity Date Study Director Septembes 18, 2003 Signed i oiginal Yasuko Matsunobu 000003 006002-- 14114 GLP STATEMENT Kurume Laboratory Chemicals Evaluation and Research Institute, Japan Sponsor Title Study number 'DAIKIN INDUSTRIES, LID. Biodegiadation study of ( CBI) by microorganisms 1414 Amendment tothe Final Report was performed in compliance with "OECD Principles of Good Laboratory Practice" (November 26, 1997) This GLPstatementwas issued as supplement to the statement issued on September 18, 2003 becausofe the amendofmtheeFnintal Report Date Sudy Director October 10,2003 Signed in original `Yasuko Matsunobu 000004 1114 QUALITY ASSURANCE STATEMENT Kurume Laboratory `Chemicals Evaluation and Research Institute, Japan Sponsor DAIKIN INDUSTRIES, LTD Tide Biodegradstautdyioofn( C81)bymicroorganisms Study number 14114 facility The inspections management and otfhteiSstsutduydDyiwreecrteorcabryriQeudaoluittyaAnsdstuhrearnceesuUlnsitweorfeKrueprourmteedL1a0botrhaettoerys,t `Chemicals Evaluation and Reseach Institute, apan as follows. . Daofrteporetto |Date ofmpoiorfetst. typo Tuly 17,2003 [Cyis,2005 August 1,200 July 18,2003 September 10,2003 | September 10,2003 | uly 18,2008 hy 15,2003 `August26,2003 `August26, 2003 "Test Conduct | August15,2003 `August 18, 2003 `August 21,2003 August 26,200 agen 20,200 | August26,2003 `August 26,2003 "August 26,2003 | August 26,2003 August 26, 2003 `August 26,2003 `August 26, 2003 `August 26,2003 details in1tthhaesrebpeoernt aasxseuirnedcotmhaptlithaencfeinwalitrheptohret sdteusdcyripbleasnaaccnudraSttealnydtahrd test method used, that Operating Procedures and thatth final report reflects the raw data accurately Date September 18,2003 Quality Assurance Ui, Head Signed in original Kyoshiro Hori 000005 1114 QUALITY ASSURANCE STATEMENT Kurume Laboratory Chemicals Evaluation and Research Insitute, Japan Sponsor DAIKIN INDUSTRIES, LTD. Tide Biodegradation study of C81) by microorganisms Study number 14 Study inspectionofthe corrected part in the Final Report was cared out and it was confirmed that the correction has no problem. The result was reported to the test facility management and the Study Director as follows Dey etinspust:on DStautdeyofDrierpecotroirto |Dfaalcieliotyfrmaenapgeo0mretentstt "This statement was issued as a supplement to the quality asswance statement issued on September 18,2003 Date Quality Assurance Unit, Head October 10,2003 Signed in original Kyoshiro Hori 000006 CONTENTS Title Sponsor Test facility Objective Test method Applied GLP Dates Stoorftaestgteem, awdata,etc Personnel Approval of inal report SUMMARY 1 Testitem 2. Item supplied by sponsor 3 Activated sludge 4 Performance of biodegradatiteosnt 5 Valofitesdtcoindittioyns 6. Factors possibly affecting accuracy 7. Results Remarks 14114 page 1 1 1 1 1 1 2 2 3 3 4 5 6 7 9 2 2 2 2 000007 14114 Contentsoftables and figures Contents of tables. Table! Calculation table for percentage biodegradation by BOD Table2 Calculation table for amount of dissolved organic carbon by TOC Table:3 Calculation tableforpercentage biodegradatiboynweightmeasurement Tables Calculation tableforconcentration ofperfluorooctanoic acid by LC-MS. Tables Calculation able for recovery ateby GC-MS (2-perluorooctyethanol) Tables Calculationtableforconcentiation of 2-(perfluorooctyllbeytGhCa-nMoSl Contents of figures Fig 1 Chart of BOD Fig 2 IR specoftrresiudume Fig 3 Mass fragmentogram of LC-MS analysisforcalibration curve and calibration curve (perfluotooctanoic acid) Fig 4 Mcaalsisbrfaitiaognmceunrtvoegra2mo-(fpeGrCfl-uMorSooacntayllyesitshafnoor)lcalibrationcurveand Fig 5 MassfragmentoofgGCr-aMmSanalysis orrecoandvbleanrkteyst (analysisof 2-pexfluorooctyllethanc)l Fig 6 Mass fragmentogram of LC-MS analysisfo test solution (perfluorooctancic acid) Fig 7 M(2a{spserffilaugomreonotcotgyrlaetmhoafnoGlC-MS analysisfo test solution Fig 8 Massspectrumofperfluorooctancic acid Fig 9 Mass spectrumof2-(perfluorooctyDethanol Fig 101 IRspectorfutemst tem meabefs oreeu xperr imenetal d start Fig 102 IR spectrumoftest item measured ates experimental completion Reference IRspectrum suppliedbysponsor 000008 14114 StudyNo 14114 Title Biodegradation study of ( C81) by microorganisms Sponsor 'DAIKIN INDUSTRIES, LTD 1-1 Nishi-hitotsuya,Setisu,Osaka 566-8585, Japan Test facility Kurume Laboratory Chemicals Evaluationand Research Institute, Japan 19-14 Chuomachi, Kurume, Fukuoka 830-0023, Japan Objective This study was pefomed to evaluate the biodegradability of ( CBI ) by microorganisms Test method "This study was performed according to "Ready Biodegradablity: Modified MITI Test (D (Guideline 301C, Revised July 17, 1992)" in the OECD Guideline for Testing of Chemicals Applied GLP "This study was performed in compliance with "OECD Principles of Good Laboratory Practice" (November 26, 1997). -1- 000009 14114 Dates Study initiation date Experimentalstartingdate Experimentalcompletiondate Studycompletiondate July 17,2003 July 18,2003 August 15,2003 September 18,2003 Stoorftaetigteem, rawdata,etc. W)TestAitbeomut 5 gofthe item supplied bythesponsorissealedin a store vesseland storedinarchivesinthislaboratoryfortenyearsafterthepublicationofthe final dsepiorts. cTwuirtsehsasptonemsoodefr.nthte1iittemissnuoptpsliteadbbfyloterhtehessptoonrsaogreapfertirotdh,eitstiossrtaogreepdewrhiioldeiist iskeptstableand iidisposedwith approfosvpoanslor (2)Rawdataand materialest,c thefRinaalwredpaotrat,atnhdesnteucedsyspalraynm,adtoerciuamlesantesasbtooruetdtihneasrtcuhidvyepsriensetahitsedlbaybortahtoersypooarstohre,: sameterm asthe est fom Treatmentofrawdataandmateriaeltc,.aftrthe storageperiod i discussedwithsponsor. -2. 000010 Personnel StudyDirector Study personnel (Operation of biodegradation est) Staff for cultivation of activated sludge Approval of final report Study Director Date Signature 14114 Yasuko Matsunobu Kazuhiro Oyama Takakazu Kayashima Hiroto Nishijima `September 18, 2003 Signed in original `Yasuko Matsunobu -3. 000011 1114 SUMMARY Title ' Biodegradation study of ( CBI) by micioorganisms Conditionsofcultivation (1) Concentrationoftes item (2) Concentrationofactivated sludge (asthe concentation of suspended solid) ((@3)) CVuolltuivmaetioofntteesmtpseorlauttuiroen: (5) Cultivation duration 100mg 30mgL 300 mL 281C 28 days Measurement and analysis for percentage biodegradation (1) Measurement of biochemical oxygen demand (BOD) by means of a closed system oxygen consumption measuring apparatus (2) Deteaminatiofntest itembymeansof weightmeasurement Other measurement and analysis (1) Determinatioofn dissolved organic carbon by meansoftotal organic carbon (10C) (2) Maenaalsyusrisement of IR spectrum by means of a fourier transform infrared spectiophatometer (3) Determination of perfluorooctanoic acid by means of liquid chiomatogiaphy-mass spectrometry (4) Determination of 2perfluorooctyl)ethanol by means of gas chiomatogiaphy-mass spectrometry Results (1) Percentagebiodegradationby BOD~~ 2%, 4%, 6% aveiage 4% (2) Percentage biodegradation by weight measurement 0% 0% 0% aveiage 0% Conclusion The test item was not biodegraded by microorganisms under the present test conditions -4- 000012 14114 1 Testitem Inthis eport,( C81)hasthe following chemical name, etc 11 Chemical name'! car ) 12 Chemical structure, etc." Structural formula Molecular weight Weight-average (cI) "1 Information supplied by the sponsor -s- 000013 14114 2 Itemsuppliedby sponsor 21 Supplierand lot number"! (1) Supplier (2) Lot number DAIKIN INDUSTRIES, LTD. cel ) 22 Purity" (0) Testitem Cm @) Impuity cor ) Thetestitemwastreateads 100 %inpurity 23 Confirmationoftest item this laTbwooraitnofrryawreerde(IcRo)nsfpiezcmterdaotfo tbeheitdeensttiictaelm(psreoevFiidge1d0bayntdheResfpeornesncoer)andmeasuredat 24 PhysiAcpopcehaesmainccael property" Ca *1 Informationsuppliedbythe sponsor 25 Storage and stability (1) Storage condition Dark storage place at room temperature @) stability The test item was stable under the storage conditions, asshownby the finding. that TR spectia of the test item before the experimental start and after the `experimental completionwereidentical (see Fig 10). -6- 000014 14114 3 Activated sludge 31 Sludge sampling ites and date: (1) Sampling sites Onsite sludge sampling was canied out atth following10locations in Japan. Fushikogawa ity sewage plant (Sappoto-shi, Hokkaido) Fukashibaindustial sewage plant (Kashima-gun, Ibaraki) Nakahama city sewage plant (Osaka-shi, Osaka) Ochiai city sewage plant (Shinjuku-ku, Tokyo) KSihtiankaanmoiRRiivevrer(N(isihgianto-msahkii,-Nsihiig,aMtai)yagi) `LYaoksehiBniovwRaiv(eOrtsTuo-skhuis,hSihmiag-as)hi, Tokushima) Hiroshima Bay (Hiroshirma-shi, Hiroshima) Dookai Bay (Kitakyushu-shi, Fukuoka) Date June,2003 32 Sludge sampling 2) City sewage, Return sludges fom sewage plants were collected (2) Rivers, lake and sea colleScuterdf.acewaterandsurface oilwhichwasincontactwiththeatmospherewere 33 Preparation of activated sludge `Activated sludge was prepared as follows to maintain it uniformity. monthTshevifaisltmraitxee(d5 wLi)othftthheemsiuxpeedrnfatlauntteof(t5hLe)oacfttihvaetesdupselrundagtea?ntcoulaftivsaltueddgaebocoultlefcotre3d `nateeacwhlolcatiyon. mixture was adjusted to T70h4e1.m0ixedfiltrate (10 L)wasaerated"afterthe pHvalueofthe *2 The activated sludge cultivated the mixed filrate (10 L)ofthe supernatant of sludge collected at the ten locations #3 Prefilered open ai was used 7. 000015 18114 34 Cultivation Roughly 30 minutes after ceasing aeration of the sludge mixture, supernatant conesponding to about 13ofthe whole volume was removed Dechloinated wate: wasaddedtotheremainingportion sothatthe foalvolume reached 101. Thismixtwe m`wiaxstuareerasteodt,haandthethcenona cpreendettaeortmfiitnohened asymnotuhnetticofsesywnatgheetwicasse0.w1agwei"%wiansthaedvdoeldutmoetohfe dechlorinated wate added. This procedue was repeated once every day. Cultivation was canied out at 2522 C +4 Synthetic sewage Glucose, peptone and potassium dihydrogenphosphate were dissolved in `purifiedwater o obisin 50 g/Lofthesolutifoonr cach component. The pHofthe Solution was adjusted to 7 021 0 with sodium hydroxide. 35 Control and use During cultivation, the appearanceofthe supernatant, sedimentation of the sludge, formationofflock, pH, dissolved oxygen concentation in the solution and temperature were checked to maintain a normal sate of siudge. It was confimed that these were within the scope of the control standard stipulated in the "Testing Methods for New Chemical Substances", and these resuls were stored as raw data. Micuoflora in the activated sludge was microscopically observed and sludge with no abnormal symptoms was used for the test 36. Inspectionofactivity and date of initiation of useofactivated sludge (1) Inspection of activity Activity of the sludge was assessed using a reference item (2) Dateofinitiation of use Tuy 15,2003 "3. 000016 1414 4 Performanceofbiodegradation test 41 Preparations for test (1) Measurementofconcentation ofsuspended solid `The concentationofsuspended solidwasmeasuretdo determine theamount of activated studge 1 add. Method Date Result In accordance with Japanese Industial Standards (1S) K 0102-1998 section 14.1 July 15,2003 Concentration of suspended solid in the activated sludge was 3200 mg/L. (2) Preparation of basal culture medium Each 3mLofsolutions (8),(b), ()and ()shownbelowwere made upto IL with purified water Thefollowingstocksolutionswere preparedbyuscofanalyticalgraderegents: (@)Potassiumdibydrogenphosphate, KF,PO, 850g Dipotassium hydiogenphosphate, K;HPO, 2175 Disodium hydrogenphosphate dodecabydiate, NoHPO:- 12H, 460g Ammonium chloride, NiCl 170g Dissolved in water and filled upto 1 L (t) Magnesium sulphate beptahydrate, MgSO. THO 2250 Disinswatoeralndfvilledeuptdo1 L. () Calcium chloride anhydrous, CaCly 2750, Dissoilnvwaetedrandfilled upto 1 L. (@Dlrions(IsI)ocihlnlovwraietdeedthaenxadbfyidlreadteu,pFteoCI1 3L-6H:0 025 g (3) Reference item Aniline (reagent grade, Showa Chemicals Inc Lot No. HO-2729D) was used as a reference tem to conf thatth sludge was sufficiently active. "Thistestiseffective whenthepercentagebiodegradationcalculatedfromthe BOD ofanilineafter 7and 14daysexceeds40 %and65%,respectively,according to regulationoftheOECD Guideline for Testing of Chemicals. "9. 000017 144 42 Preparation of test solutions Test solutions for analysis of the test item and perfluorooctanoic acid and test solutions for analysis of 2-(perfluorooctyethanol were prepared, because th test tem and converted products (perflustooctanoic acid and 2-(perfluorooctyhethanol), which wasexpected 0 formintetsolutions,couldnotbeanalyzed by thesamepreteatment. Eleven test vessels were prepared The following test solutions were prepared and cultured under the conditions described in section 43 (1) Addiofttesit toemnor aniline (a) Testsolution (water + test tem) (n=2I:nVoesnseetleNstov6esasenld,a3t0emstgsoolfuttihoen fiotremasnuaplpylsiiesdofb2y-tphesefslpuoonrsooorcwtyasetahcacnuorla)tely weighedandaddedto300mLofpurifiedwater,5 thattheconcentrationofthe test em reached 100 mg/L (b) Test solution (sludge + test tem) 2((ap6er:fluVoersosoelctyNetohla,nol2), 3 and three of test solutions for analysis of ! Ineachtestvessel, 30mg oftheitemsuppliedbythe sponsorwasaccuately `weighedandadded tothebasalculture medium(thevolumewas fessthan300mL. by the volume (2.81 mL) of activated sludge inoculated), so that the concentation ofthetet item reached 100 mg/L () Test solution (sludge + aniline) (1=1: Vessel No 4) anilinIenwoanseatdedstevdeisnsteolt,h2e9b5esuaLlcu[l3t0urmegme=d2i9u.m5 (utLhe xv1o0l2um2egwlaesm'l(esdsetnhsiatny)3]0o0f mL by thevolu(m28e1mL)ofactivatedsludge inoculsaottehadt t)h,e concentration reached 100 mg/L (6) Test solution (contol blank) (=2:InVeossneeltNeos.t5vaesnsdela,tnesotthsoilnugtwioansfoardadneadlytsiostohfe2b-a(spaelcflcuuolrtouorcetmyeDedtibuamnol()the volume was less than 300 mL by the volume (281 mL) of activated sludge inoculated). (2) Tnoculation of activated sludge The activated sludge cultivatedunderthe conditions described in section 3 was added to each test vessel, (5), () and (), so tht the concentiationofthe suspended solid reached 30 mg/L "10- 000018 1414 43 Instruments and conditions ofcultivation (1) (Ian)stTreustmesnotlsutfioorncfuolrivaanatliyosnis of tet tem and perfluorooetanoic acid Closed system oxygen consumption measuring appesatus `Temperature controlled bath and measuing unit Oba Electic Co, Lid Data sampler : Asahi Techocion Co, Ld Vessel 300 mL in volume (improved type) `Absorbentforcarbon dioxide Soda ime No.1 (fr absorption of carbon dioxide, `Wako Pure Chemical Industies, Lid) (6) Test solution for analysisof2-(perfluorooctyethanol Closed system oxygen consumption measuring apparatus `Temperature controlled bath and measuring unit (Only the temperature controlled bath vas used) Ohkura Electric Co, Ltd Vessel 300 mL in volume (sealed improved type) (2) Conditions of cultivation Cultivation temperature 251C Cultivation duration 28 days Stining method Eachtestsolutionwassiredby amagneticstirrer ) Room Appaosoam NtouSs11 44 Observation and measurement of test conditions (1) Observationoftest solution Duing the cultivation period, the appearanceofthe test solution was observed periodically and conditions of the instruments were checked properly. 2) MeasurDuermienngtotfhebicouclhtievmaitciaoln opxeyrigoedn, dtehemacnhdan(gBeODin)BOD of the test solutions was measured by autorecoiding using a data sampler. Cultivation temperature was measured and recorded once a day. BOD of the test solutions for analysis of 2-(perfluorooctyDethanol was not measured. a. 000013 wns 45 cAonnvaer"ltAeyodRsfpttirheoessdttuescrtmosiun(oaptneisolnuoofrtohoecacnuoliicvataicoind,ndids2s-ol{vpeedslorugoarnoiocsctayrebtohn,antah)e,eswtheicmh wainsd expected productioninessolutions,weredtemined 45.1 Preteatmoeftnestt solutions for analysis (1)Pr"eAfrtthertorexeamnialaaytsiitosonofmfehseetctuenlmivtnadiopn,ehueoersotsoolcuttiaonnac(cvidai + tes tehmet,est s{loelmutaionnds (pselrudlgueo+rotosctaencmi)caacnidtdhweetersetpsroelturtieoante(dcofnotrrotlobtaalosrkgfoa)niacnalcyasribsoon(hTeOtCes)t a(nLaCly-sMiSs)onandliysssoslvoefd poercglaunoircocoacribaonnoiacncdildiuaisdfcolhlroowms.atogWieaipghhyt-mmeaassusrsempeencttooftmheey test temwasperformed. IR spectrumoftheresiduewasmeasured. Taking out 10mL + (Cemnetaisfuuignagipoinpe) (1000 x, 10min) FSiitlerteod nby(uTshee otfesmtemsborutaonnesfiwlelree" Takingow2SmL (caaster pipe) | wveishselssucwtaisontranTsfheerrerdeonstiihdnitutheeer ~F(imletiasngculp,toSml, |andwawsithpuhifieedwatder) volumetie Bask) TSOamCpaienfaolry_s|is ||LC[MSaSmpalneafloyrsis pDeefeleumoirnoacttaonncoife sed [Residu|e [ Filue +DCroyoilnigng(v(daecsuiuccmaotvoern,,o4o0mt,em1p37ehruast)e, about 30 min) weighStammpelaesuorrement *S MILLIPORE type HA (poresize 0.45 ym) Sampleolfoor messzement SIR spectrum 2- 000020 1114 ) Pretreatment for analysis of 2-(perfluorooctylethanl After the termination of the cultivation, the test solution (wate: + test item), the test solutions (sludge + test item) and the test solution (control blank) for analysis of 2-(perfluorooctyljethanol were cooled at S C for 66 hrs. and pretreated for gas chromatography - mass spectrometry (GC-MS) analysis as follows. --Ammonium sulfate 200 g (even balance) Chloroform * Shaking (10 9m0inm)L ( gr a d ua t e d cy l in d e r ) *2 + Filtration and dehydration (No 2 filter paper, sodium sulfate 30 g) * Filling up to 200 mL (chloroform, volumetric flask) Sample for GC-MS analysis 1. 000021 14114 452 Quantitativeand qualitative analysis (1) Determinationofdissolved organic carbon by meansof TOC analysis "The dissolved organic carbon (DOC) in the samples for TOC analysis was analyzed under th following conditions "The concentrationofDOC was calculated by subtracting concentration of the inorganic. carbon (IC) from concentration of the total carbon (TC). The concentrationofTC and IC in the test solutions was proportionally calculated from the peak area of the tet solution by comparison with that of 80.0 mgC/L ofa standard solution for TC analysis and 80.0 mgC/Lof a standard solution for IC analysis, respectively (see Tabie-2). "The standard solution for TC analysis was prepared by dissolving potassium hydrogenphthalate in purified water. The standard solution for IC analysis was prepared by dissolving sodium hydrogencarbonate and sodium carbonate. in purifi"eTdhewactoenrcentrationofdissolved organic carbon corresponding to the minimum determinationlimitwas regardeasd 1.0 mgC/L. Analytical conditions Instrument Temperature of fumace Flow rate Injection volume Sensitivity "Total organic carbon analyzer Shimadzu Corporation type TOC-5000 680C 150 mL/min 3p Range 5 (2) Determination oftest tbeymemansofweight measurement `Weight of the samples for weight measurement was measured under the following conditions (sce Table-3). Analytical conditions Instrument Electronic analytical balance Mettler Toledo type AT201 Minimum measuring weight 0.1 mg. (3)MeasurementofIRspectrumbymeansoffourier ransform infraredspectrophotometer IR spectrum of the samples for measurement of IR spectrum was measured under the following conditions (see Fig 2). Analytical conditions Tnstrument Measuring method Fourier transform infrared spectrophotometer `Shimadzu Corporation. type FTIR-5200PC KBr tablet Sas 000022 1114 (4) DeterTmihneatsiaomnplofespefrofrluLoCr-ooMeStaannoaclaycsiisdbwyemre eanaolayfzLendC-fsMorSpesluorooctanoic acid under the following conditions. The concentuation of pesfluoiooctancic acid in the sample for LC-MS andlyss was proportionally calculated by comparing the pethaekmaarsesaofangtmheemnatsosgaGrmaogmfent0.o5g00rmagm/oLf.tshteasndaamrpdlseolfuotriLoCn -(eMeSaTneablley-s4iasnwidthFitgha6t)on The lowestdetectablepeakareawasregardedas 2000considering thenoise level, which conespondteod 0 0098 mg/Lofperfluorocctanci acid @ AnalyItniscatlucmoendnittions 1c Ms ConditonsofLC Column `FElluoewntrae Semple size Liquid chromatograph-mass spectrometer JASCOCorporation type PU-980 Mictomass type Quatzoll L.column ODS 15cmx46 mm1D. M1e0tmhLainmoli"n/ purified water (80/20 V/V) 1040 ConIodniiztatioiofonMnmesSthod Detection mode Monitoring method Monitoringm/z CSoonuesvotletmapgeerature Electospray (ESD) Negative ion Selected ion monitoring (STM) 413 (sec Fig 8) 215v0C #6 Containi0n.g1 % formic acid -15- 000023 ns (b) Preparation of standard solution The standard solution to determine the concentiation of perfluorooctanoic: acid in the sample for LC-MS analysis was prepared as follows. 100 mg of the standard substance" supplied by the sponsor was accurately weighed and dissolved in methanol 10 obtain 2000 mg/L solution of pecfluorooctanic acid 0.500mg/Lstandard solution was then prepared from this solution by dilution with methanol / purified water (1/1 V/V). +7 Name, lot mumber and purity Name Perfluorooctanoic acid (C-1700) Lot number CI7001401 Purity Perfluacoidrwaostroeatcedats1a00n%ionpiuricty () Calibr0a1t2io5n,cu0r2v5e0 and 0.500 mg/L standard solutions were prepared by the `same methodas descriibn(ebd) Thesesolutionswereanalyzedaccording to the analytical conditions described in (8). A calibration curve vis drawn batsheerdeospnectthieverecloantcieonntbieattiwoense(tseheeFpiega3k).areaonthemass fagmentograms and -16- 000024 dean (5) DeterTmihneatsiaomnploefs2f{oprerGfCl-uaMrSoaoncatlyyestihsawneorlebyanmaelaynzsedoffoGr C2--(MpSefhuorooctyDethanol uthnedesramthpelefoflolrowGiCng-cMoSndiatniiolnyssis wTahse cpornocpoernttiaotniaolnloyf2ca-l(cpuelraftleudarboyocctoymlpeatrhianngolthien pethaekmaazsesa orangtmheenmtaosgsrfarmaogmfen0t.5o0g0 ammg/oLftshteasndaamrpdlseoflourtiGonC(-sMeeSaTnaabllyes-i6sawnidthFtihga7t)on level,Twhheiclhowceosrtredseptoencdtaebdle0.p0e0a2k1amiega/wLaosf 2r-e(gpaercdfelduoarsoo3c0t0ylceotnhsaindoelring the noise (@) AnalyItniscatlrucomnednittions GSahsimcahdrzoumaCtoorgporraapthi-omnasstsyppeecQtPo-m5e0t0e0r `CoConlduimtnoifoGnCs HP-FFAP (Agilen) Column temperature 2550mCx(04.m3i2nmy--m17ID0,mCa(dRaetoef3f0us`Ceidmsiilni)ca Carries gas Herium PInrjeescstuiroen otfemcpaemreartugraes 100kPa 200C Sample size 24 Sampling method ~~ Splitless Sampling time: 20min Conti Ionization method Detection mode MMoonniittoorriinngg mm/ezthod Interface temperature Electron ionization (ED) PSoeslietcitveedoionn monitoring (SIM) 29530(seCe Fig) 1. 000025 4114 (0) Preparationofstandard solution The standard solution to determine the concentationof2-(perfluorooctyl)ethano1l0i0nmtgheosfatmhpelsetafonrdaGrCd-suMbSstaananlcyes"isswupapsliperdebpyartehdeassfpoolnlsoowrswas taken out accurately and dissolved in chloroform to obtain 2000 mg/L solution of 2-(perfiuoroocyllethanol. 150 mg/L standard solution was then prepared rom this solution by dilution with chloroform +8 Name, lot mumber and pusity Name 2(pesfluorooctyllethancl (A-1280) Lot number AI8201X01 Puity 2(pexfluorooctywlaesttrheaatnedoals 1%0 inpu0 sity. () Calibr0a3ti7o5n,c0u7rv5e.0and 1 50mg/Lstandardsolutionswerepreparedbythesame. meastdesh cribo edind(b).Thesesolutionswereanalyzed according tothe analytical conditions descriibne(2d). Acalibration curve was drawn based on therelation betweenthepeakarea onthemassfiagmentogiamsandthe respective concentiations (see Fig 4) -18- 000026 14114 453 Recteostavndbelarnktyest Each two test solutions (water +2-(perfluoroocty ethanol) and two test solutions (sludge + 2-perfiuorooctyl)ethanol) for recovery test were prepared according 10 the `methods describedinsection 42. 0 300mgof2-(exfluorooctylw)aestahddaendotlo eachtestvessel,sothattheconcentiation ofitreached 1 mg/L The testsolutions, werepreneatedin accordancewiththe methoddescribedinsection 45.12),then analyzed accotroadnailytnicgal conditions describedinsection 4.5 2(). Atestsolutionforblank tetwaspreparedaccording10themethoddescribedin section 42. Thetestsolutionforblanktestwasanalyzedinthesame wayasthe recoverytest. Asfortheblanktest,nopeakswereappeated aroundthepeakofthe 2-(perfluoronotchetmaysls efitaghmeantnogoialm Twoindividual recoveryratesandtheiraveragesontheanalyticalprocedureac shownbelow. Theaveragerecoveryrateswereusedascorrectionfactor,forthe. determination of2-perfluorooctylethanolinthetet solutions see Table-5andFig 5) Recoverynateofth testsolutions (wate +2-(pexluorooctyDethanol) 996%, 982% avenge 989% Recoratveofethretyest solutions (sludge + 2-(perfluorooctyDethanol) 805%, 815% avenge 810% 19- 000027 18114 46 Calculation ofpercentage biodegradation The percentage biodegradation was calculated by the following equations and expressed in whole umbers (1) Percentage biodegradation by BOD Percenage biodegdaton (0) = gE x 100 BoD Biochemical oxygen demand inth test solution (sludge + est tem) (experimental) (mg) B + Bioocxygehndeemanmdinitheccontraolbllank (experimental) (mg) TOD": Thietoermewtiacsaclomopxlyegteenldyeomxaidnidzreedq(utihreeodrwethiecanlt(hmegt)est "9. Thepuritywasregardeads 100% andTODwascalcfuromlcoamptosietidon formula ( cal ) which was calculated by use of n=8, monomer valuesof(C80 regarded as mol% in section 1 2 @) Percentage biodegradation by weight measurement Percentagebioy degradatiNon(4) = Swg-nss x 100 ss + Residualamountofthetestiteminthetest solution sw + R(seisniddgueal+aemsotuntetmo)ft(heexpteertimietnetmailn)t(hmeg)estsolution (wate + test tem) (experimental) (mg) 47 Treatmentofnumerical values `Valueswereroundedoffinaccordawnicteh 11ZS 8401:1999rule B 20. 000028 om 5 Validity of test conditions 72.9% Paetrceen7taagned b1i4oddaeygsa,draeispoencstiovfelaynilitwnaseccaolcunlatced lbytuhthaedtBhieODedstvaclouensdiwieornes w6e8re%vaalnidd (see Table-l and Fig1) 6 Factors posibly affecting accuracy Noadverseeffects onthe lability ofthis estwere noted. 7 Results Tre|wT] 71 Appearancesoftestsolutions `Appearancesoftest media in cultivation vessels were as follows Atthe stat Wate +testitem | The test temwas not dissolved. of cultivation Slud+ge estitem |Thetest itemwasnotdissolved. Atthe termination Wa+ttesetitrem |Insolublecompoundvas observed. | - | --_---- Trsoluble compound except for he Slu+dtesgtiteem | G1row2thwa3sofob8steher2vesdludge was not observed. La. 000029 1114 ToT] 72 A`nAnarleaystuilctrlsesouflyttsesotfstotlheutteisontssolution after 28dayswerea follows testitem Sludgep+eterstitonea" l rape| rig | [wor TorJoeTusTurJem oo] 03 | can deebionofBoC| % 3 - Rmesipduselamgounst |L1gT| 285 | 00R | 96E | m9 L | 300 due %|os wo | 9 | - } En pCeornfclveonrtoaocttoanno_ioef | mel| 50020 id CCS) i nr Thea 1a = stem[1 2[3 amount CSoonmooeenprtaattoin.o_noo,ff| mgt. soo? | so017| 0017 7 thanol (GC-MS) +10. T(helvaulu+edteosftgceoemnt)rol blank was subtracted fom the values of the test solutions #11 Mi=n(iMimnuimmduemtdsemtienrmaitnoantliiomnimtotfnpefslaomrpoloecftoarnLcCi-aMcSiadliysiesst)soon =0(0i0n9a8lvmolLumex)(5(mvoLlAu0me0omfLte)sts2olu5t3o0n0)) /= a0t02o0ofmpgo/rtLionusd fr analysis) #12 M=i(nMiimnuimmudmetedretmeirnmaitniaotniolnimliitmoift2in(spaemfpiloerfoooxcGtCye-tMbSaannoalliynsiess)t solution (finalvolume) (volume oftest solution) (averagerecovery ate) 100)/ Te(statsiooloutfipoonrt(ivonatese+defsoraenaml)ysis) Test so0l0ut2i1onmsgl/uLdgxe 200mL/300mL) + test tem): / (98 97100) 1=0014 mg/L 0021mg/L x 200mL 300 mL)!(81 4100) 120017mglL 2. 000030 in 73 Percentage biodegradation Percentage biodegiadations after 28 days were as follows Method Percentage biodegradation (%) Table [wo [2[eTeT} v] [vsos0[mmJmomTmoe|n5[] 74 Results ofmeasurementofIR spectrum after NosignificantdifferencebetweenIRspectrumof th weight measuremewnats observed (see Figs 2, 10). tet temandtha of th residue 75 Discussion item Determination of the test tem by chromatography was not completely dissolved in water and organic was difficult, because the solvents (tetahycrofuran test and Acshioaltofeolrm,)thepTehrecreefnotraeg,tehreestiedsuestoemftwhaestdeteittemewerrbmey9im5en%aniesnodtfhewteeisgthsotlmuteiaosnu(ewmaetnetr +test item)and 100, 99and96 %inthetestsolutions percentage detections ofDOCwere 0 %inthetestsolution (sludge (water + +test item). est tem)and The 0, 3 and item 3%in thetestsolutions (sludge +test wasnot disisntoh ltestvsoleutidon item). Theseresulsindicate that the test Nosignificantdifferencebetween IRspecra ofthetest tem before andafter the cultivation was observed 2(perfluorooctyl)ethanol), which Converted products (perfluorooctanoic acid was expected o form in test solutions, was. nd not detected cultivation These results indicate that the test item was not converted during the Iti consideredthatthe test itemwasnot biodegraded by microorganisms, because twheeigahvteorfagtehepetersctentteagmebbeifoodreeagrnaddaafttieornthbeycBulOtDivwaatisonw4a%saonbdsenrovseidgaifican! changeinthe 76 Conclusion The test item was not biodegraded by microorganisms under the present test conditions 2. 000031 14114 8 Remarks 81 Instruments used for est Fourier transform infrared spectrophotometer Shimadzu Corporation type FTIR-8200PC Closed system oxygen consumption measuring apparatus see page 11 Total organic carbon analyzer: see page 14 Liquid chromatograph. mass spectrometer see page 15 Gass chromatograph- mass spectrometer see page 17 Electronic analytical balance: ~~ Mettler Toledo type AT201 Sartorius AG type CP324S Sartorius AG type BP210S Refrigerated centrifuge Shimadzu Corporation _ type CST-060LF Mechanical shaker : TIETECH Co. Lid. type SR-2w Vaccume oven : SIBATA SCIENTIFIC TECHNOLOGY LTD. type VS-300 82 Reagents used for analysis Methanol (HPLC grade) `Wako Pure Chemical Industries, Lid. Purified water Takasugi Seiyaku Co, Lid. Chloroform (reagent grade) Kishida Chemical Co., Lid Ammonium sulfate (reagent grade): Kanto Chemical Co, Inc. Sodium sulfate (reagent grade): Kanto Chemical Co, Inc. Formic acid (reagent grade): Wako Pure Chemical Industries, Lid. `Sodium hydrogencarbonate (reagent grade): `Wako Pure Chemical Industries, Ld. Sodium carbonate (reagent grade): Wako Pure Chemical Industries, Lid Potassium hydrogenphthalate (reagent grade) `Wako Pure Chemical Industries, Ltd. Potassium bromide (for measurementofinfrared absorption) NACALAI TESQUE, INC. Perluorooctanoic acid Standard substance supplied by the sponsor 2(perfluorooetyethanal Standard substance supplied by the sponsor 24. 000032 BT waloo]wa]nl (mg) (%) (mg) | (3%) wos] (mg) mw](%) mail(mg) mw](%) |% | TTeo[eeaallo a]eaa l=olo] eool] 1 oa]a] = 88[[oooollT3o]oos]]Ta]oowsl]a1 oaaa] 1a] | Vessel no. @ : Sludge + Aniline Vessel no. : Control blank [B] Vessel no. I @ @: Sludge + Test substance Vessel no. B : Water + Test substance TOD of test substance : (CBI) (mg) 1 TOD = 30.0 x (CBI) = (CBI) (mg) TOD of aniline 90.3 (mg) 2003.08.15 NKamie fpmer 000033 Table-2 Calculation table for amount of issolved organic carbon by TOC Stody No 14116 Water blank (6) Water + est em 0) Side + stem 2) Sludge+ test item (31Studge + esteem 51 Control bark nd nd 0 o na 0 o 11s 03 3 2 12 03 3 nd Amooftesttem added 30 (mg) Vohumoftest soon: 300 (ml) : Theorameoutntiofccaablon (CB (mg) (30) x ( oa ) A: Measured value(mgCL) B: AmoutofDOC Bw (A(Water +testte) A(Waterblak) x 3001000(ng) B= (A(Sudge + test te) - A(Contol bank) x 300/ 1000 (mC) : Persenage detection (4%) CB (Theoarmoeunttoifccaanl)x100 (6) D: Avesags percentage detection (4) pig) Name F Qrawce 000034 Table3 Calculation table for percentage biodegradation by weight measurement Sty No. 1anss 16 Water + ester 0) Stdge+tes em 2) Side +test em 3)Side +test tem 15) Convolac us 5 ss 59 300 10 5s 1s ES o 28 29 9% 0 35 Amoustoftest te added:30(ag) Voloftuessmouieon 300 (nL) A+ Weightofresidue (og) 2:Residualamountfest em (mg) B=A(Wa+ tsteter) Bi =AGludge- st em) A(Coutolbark) : Percentageresidue04) CB(Amountottest emadded) 100 D:Pecenage biodegradation 04) D=@w-Bs) Bux 100 B Avengepercetagsbiodegradation 04) roms 0 Nee Get 000035 Table4 Calculation table for concentration of perfluorooctanoic acid by LC-MS Study No. 14114 `Sundardsouion0 S00mgL [61 Water+est em [1]Siudge + teste 21Sludge+ stem 13]She + tstitem (5)Control lak 102018 nd 0(s0020) * nd (0020) na o0(s0020) 2 nd. o(s0020) ad. Volume of estsoon 300(nL) As Peakarea(-) B: Finalvolume: 5(mL) C: Ratioofpordon sedforanalysis :2 57300 D: Concentration ofpefuoarcidootesetwaatenr(mog/iL) Dx (A/AlSianx(d8/a3r00d) ) Minimum determiiitn:0a02t0(imgo/nL) E: Averageconcoefpnerotroorcuanolteaicd(omny) F: Concoef stnandatrdsrolutaiont:0.i500(ogLn) Seeig 6 Spenber 18,2000 Name K. Byam 000036 Table-5 Calculation table for recovery rate by GC-MS ( 2-(perfluorooctyl)ethano)l SVoeoornp:et0me.st wsomm am r Ve-- ot 7186 atmm r iyed mes va oo mwee ws wkei me Artaette ns Amountof 2perfiuorooctyDethaadndoeld:0.300 (mg) C1 rR--o--tttistsii :Recooro CnAAramir (813031 ReE vereB t++ aAsntre 0 EY 000037 Table-6 Calculation table for concentrationof2-(perfluorooctyl)ethanol by GC-MS. omtuotinont 0m. pio om Baees aBeEs I neem w we ee poeom rt sesn 2001 B+: FiS ntalevsotl:e2s u08t 0m 3(m0e L) riee tot + CominT asIsetY SLRSAT) Minimumdeterminationlimit: 0.014 (mg/L)(Water +testitem) 1 Am 0017(mg/L)(Sludge +testitem) wares G: Conce of sn tandt ardsr olua tiont:1i 50(omg/nL) `September 18,2003 Namek. Qyamg" 000038 TPeinetlFo chaeriseor mpFeet wer =] 7 Cultivating conditions: rJaaiFfTmeceestfrtierarrseesrnueecbdestasinucbeauSa UPR B21|2a50%05d(3imFg.n/Yh7.) 10m we 15.2000) Note: Regular condition Vesnosee| Semple description re Lathasy [[80[St[suiduiegee ++ Toesset sswbusrbmnseetlan22c..e17]|| 20..15||5 40||7 ssiis]] [ [8 5[[Ssiiudseee +Teansitiisneubst[aano5c:e60]| r|i:s||4 hon||7 Tr6i.s5|] [8[wCaocnetrrol+Tbeisetnsu(51bst|a2me00 .s0l|| ozi s ||a 0:0||4 0:s1s]] To 1 :z ol i.: i i : espe - @ Pel il = 20- ; rare) T corerventd=s ine o-aT fihoy F 2003.08 15 Nae baer 000039 sap _ = PRE A Ga Fig2a-1 IR spectrum of residue. Study No. : 14114 Sample : [6] Water + test item Method : KBr tablet Date August 26, 2003 Name TkGames oi 2 oe sta 000040 Poa ar aFBoYs SaLdn pot x 2 ped a . eo REa Fl li EEA Fig.2-2 IR spectrum of residue Sty bo: 14114 Sample : [1] Sludge + test item Method KBr tablet DNaamtee AEugGusyt a26n, 2003 000041 ha n Hd 2 Fig2-3 IR spectrum of residue. Study No. : 14114 Sample [2] Sludge + test item Method KBr tablet Date August 26, 2003 Name #Symes ;o PES 000042 Fig 2-4 IR spectrum of residue. SStaudmyplNoe.: : 1[34]114Sludge + test iten Method : KBr tablet Date Name August 26, 2003 Operas 000043 Trp | wl | | I ests pandWN] wo - | w me we we em Fig2-5 IR spectrum of residue. Study No. : 14114 Sample: [5] Control blank Method : KBr tablet Date August 26, 2003 Name LE Gyms 000044 Ts"olovent, oe 8o; EY articeShardss solution 0.128mgl |i1 100- ae Peak position 7 |: |% Sn0nd.aarsd 1} s2olu!tion 080mg 4. A ass 6. !iI I i a18Tindard addoufio=n 0.5 I :| Ss5s5s5 7 5 14085s 1S5R-oAf |uGgr-an2e0:S0e0 80085 3 aTiOm; y SIRof 1 Change 52 0e0Ja8c he 8 SIR of | GraT0nEA0gSe05. 0N0Te8IaC 5 RiTgoTyime SIRof | Changes)oS5ic. Area Iq 3 Z TE Tine - RZ 2003 3 15k Gans Fig3-1 M a(psosrffuroroaorgocomanneccenatnocgiraacmiofd LC-MS acid) analysis for calibratciurovne 000045 120000 | Lam | | SudyNo 14114 o 03 06 Concentration (mg/L) y=2014140 r-100 Coscennaton Pekan (mal) ) [o[ zs o Ts ms s]| os 1 io oosas | Fig.3-2 Calibration curve of LC-MS analysis forperfluorooctanoic acid August 22,2003 New fo fpoen 000046 StyNo 14114 ! { DSiupaet"e:Satveentrascoss inSsooo ----------------------------------50 2500 20000 1500} Peak ston i ag 3000 3 5 SDimaplfe :Sa41n14acd0ssotutien 0375p. ny 2. 5000 x 15000 I sa: 7 ain : RETT oes ens 3 DSaiampflele:Sa14o1d1a4rd0s6a8tion 0 750mg infetoSood00 15000 I som. 7 500 PaUk eRT Pamkwa 3 i DeSiamplfel"e{:Staen1d14aanssoelution 1 mg. inentySooo B00 1500 1 so 3 7 ss00 PaTk eRmI Pawkeem 7 Due: Aga, 2003 Name: K Dyan Figd-1 MassSagmentogiamof GC-MSanalysisforcalibration curve (2-(perflorocetyethanol 000047 - {i 3 | : ss oe (mg) ) omTeme| om TT im so Tasos Fig 4-2 Calibration curveof GC-MS analysis for 2-(pefluorooctylethanol August 11,2003 Namek. Cromer 000048 suo te -- i ryoYno!: 5a ao.3 'wo 3 fDuot us Ywoor intensity i | 7 7 me Hrs bolus so. | 1 7 7 7 page pi iy rtrd 2a30000' so 10000- I1 7 T 7 no ropalt RT rate i| 95.00 ro* k kr pare no TE hn Peak RT Peakarm - Date: August7,2003 Name: }D . amas FigS-1 Mas SagmentogamofGCMS analyes orrcoverysnd bktos (analysis of 2-(perfluorooctylethano)l. 000049 Sutyo 14114 SDoanmieeSune im it aT"ATEto, Kpmere " I 250007: stpeibereaetent 5 200 som] | x20 1 7 7 a , nis fort "Sooo --" 1500 I sao T peas 1114072 inscstySous 25000 1500 7 7 peakpositon 2a 95.00 PaTxoeRTs Pankiei ma m PaTxoeRIs Pankeem nia sm Bi ---------- 3 : 7 =a Dus: Avgust7,2000 Name: Gaumors Fig$-2 MassfagmeotogramofGC-MSanalysis fo recoveryand blank test (analysis of2perfiuoroocrylethanol ). 000050 1144Stan0dar1d 5 solution 0.500mglL 195 TT "SI"R of {1 8C.hAanunge-l2050.3 Ti 80r0eea5 140280878 f[ ia01.14e d016Wa 2. t--er 4 st6est8!i -- SiRai l11SCAo huW agn2hm o 0y05:3 |i 1 800i65 i* 0 7 j| ,k Shfuodoge + otem | ae Peak position J 7 [3 - a 7000 Sirol Co\ugr-: Tic 8.0065. 2d `Poak position. i 199 | L i o TANGT abe R aks foToime. 160 3653 808 F fms Fig6-1 MassfragmentoofgLCr-aMmS anafolrty estssoliutison ( perfluorooctancic aci)d. 000051 RSlTudgeFlestiem TTTSRof1qsCh.aanuigaa050. ] 800S6i5e . { oe]Pas postion ad P aioaldI 0t Sh 2! d--g --4evtal osat t SIRi of --1 ho an--0TEAugmse0sn 0"5- a| 800S6i3c * | 1% 2101;40020 BlCombarBank 2! I i i z`Peak position. | z 3 ; 56 5%Time _SRof ChTSaAGAnm i5a. 80065 ||| % at Pask position. | 158 _OBTTTTRN TARsk Bho 1 3) ptt Figh-2 Ma(psesrffvrcraoogsamnecinsotidfo)L.gC-aMmS anafortyestssolutsion ioTime klpmaens 000052 DuSaigle:Spangae one inon13d 0mg. ins Joos] 2008 oe) 15000: ood so, 5 7 Da uasie ona en tySooo 2500 Satyo tants . 7 2s%e|FakeRI Pak | i wn 5500 | 15000: Ey `Poa position so, i 7 pSathie tHaris n styYoo 2500 x 7 PA 5500 15000: \a a|t Peakposition | : 7 7 - Du Aug e18,:003 NamJoou : Fig7-1 MassfragomfGeC-MnSantalysois gortrestsaolutmion (2perfucrooetylethancl) 000053 pBeEymT,on r sos n uo om i . !- =| 13000: `Peakposition | EH] ` oEeEYo ns pos 7 -| I" i pifrn i" | pepmuEtdeossFem 6 200 7 - x | - 5| 15000: `Pek position Date: August13,2003 Name: Gomes FT-2 SemmGaCSpwaaysrneoifn (24perfluorooctyl)ethano)l. 000054 Stay No. _ 14114 Instrument_ MS: Micromass Quatro, HPLC : Jasco PU-980, 45-050 Sample __ Perfuorssotansic acid HPLC Conditions Column _L-co0lDuSmn Size _15om x _48 mm 1D. Elent_M_ eth/a purfn iedwoatelr""( _8/2 V/V) 1 containing 01% formic acid Flow rate _10 mL/min Sample size _20_ul (Sobvert. _bthanel MS Conditions Tonizaton mode _ESI_ Detection mods_Negativ_e Function _MS_ Source conditions ( Cupilary _30_K, Cone Mass range m/s) __100 = 1000 _20 V, Temperature _150_G) Ore die 10.2003 Opurster {Gel Kurume Laboratory, Chemicals Evakoton and Research sti, Japen Fig8-1 Massspectrumof perfluorooctanoicacid (analytical condition). 000055 Sata std 20.0mg/L. to- ) | | % p0l8,4 149 265 485 se gr Te17-Jun-2003 199e6. 85 3 7 5 siSp T.0myTossot) om 6875p82H6861200 Men) wT ee To 11A-Jrun-Ti2o0se0s3 (M-co-aR5Y (pMy+H00) FT Fig8-2 amen " ar BH oy T EE mz SsoYny pkpee Massspectrumofperfluoracoiod(cmatssaspnecotriumc). 000056 So $d20.0mg/l. wo, - ve -- Sines,te TTT 77 474un-2003 199e6 #] omy" 288 a0 |\ 435 5% er a:i 43507 as ] --- | TeanTeish. 35008 : fr 141144001 a|m T 2'ScaTsnEaEsss- oid mamas * % Tap5es. BE gear por oP TsW.h = Figte3 2 7 5 ToTime SZLa kr Mas pecoum of pflroosasie aid mss chromsogam) 000057 Study No. __14114 Instrument Sample _Shimadzy GP-5000 2-{perfivorooctyDethanol GC Conditions Column __HP-FFAP (_Fused sila Size __25 mx _032 mmID, Fimthickness 052 pm Columntemp. _50 C (_3_min) = _170 C, Temp.rate _30_'C/min Carri gas _He Columnhead pressure __100 kPa Injection temp. __200 C Sample size _1_uL (Solvent Methano)l Inlet mode _Spitess Sampling time __20 min MS Conditions fonization Mods _El Detection Mode _Positive Mass range (m/z) __50 = 600. Interface temp. __230 C Date _June 11,2003 Operator __. Qroma/. Kurume Laboratory, Chemicals Evaluation and Research Institute, Japan Fig-1 Massspectrumof 2-perfluorooctylethanol( analytical conditio)n 000058 Suto 4114 DSuiam ls e vaa aisnnon n10d 0mg. nasStyooo ssaio00 3500000 Toa on chromogen 3 *2500000 | 150000 | some - 7 7 TESm : ain7 MSsperm ijPi etiytymRliig es m as2sRY o : el pmo : | " KA X A Novo | 1 E ] |r m x 16810 ssa9 us09 13 ws 408 a SiR Te He B00 Te a0 60 5 0 5mwe Date: June 11,2008 Name a Fig9-2 Massspectumof 2perluorcosty(mlaesstspheactnrcuml) 000059 DSamuplie::a1nadna1ards00etlon 10 0m. nnsity f 250000. 1500000 || I 5000. 7 SttyNo 14114 .Mas chomsiog a 3 H | 4ni5"c 00 7 a)2To0n0 7 mi Date:June 11,2003 Names KGans Fig-3 Mass spectrum of2-(perfluorooctylethanol mass chromatogram) 000060 P TEReTE RAT Fad EFEInTreR Het Pr re Fig.10-1 IR spectrum of test item measured before exper imental start. Study No.: Sample Method Date Name 14114 Test item KBr tablet July 10, 2003 kGromer ae asl i 000061 Fig.10-2 exIRperspiemcetnrtuaml ofcomtpelsettioint,em measured after Study No.: 14114 SNeatmhopdle:: KTBersttaibtleent fate" August 21, 2003 \ame ke OGyyoo" 000062 TS Aha = Ei Ha et Reference TR spectrum supplied by sponsor 000063 Amendment to Final Report Kurume Laboratory CRehseemairccahlsInEsvtailtuuea,tiJoanpaann.d 1 Tite 2 Study number Biodegradsatutdiyoofn C81) bymicroorganisms 1114 3. Content 453 Recoverytestandblanktest(page 19) 4 Reason `Writing error 5. Content of correction " Recovery 1ateofthe test solutions (water + test item) Recovery9t9at6%of,the t9es8t 2so%lutionasve(rfaugdege 9+ t8e9st%item) 805%, 815% avenge 810%" is corrected to Recovery rate of the test solutions (water + 2-(perfluorooctyl)ethanol) 996%, 982% average 989% Recovery tate of the test solutions (sludge + 2-(perfluorooctyDethanol) 805%, 815% average 810%" 6. Approval Date Study Disector October 10,2003 Signed in original `Yasuko Matsunobu 000064 fECEIVED 054" 27 Ais wd FINAL REPORT aw A 6 Biodegradationstudyof ( CBI ) by microorganisms 32 RE z 8 September 18, 2003 Chemicals EvalKuurautmiaeondnLaRbeosreaatrocrhyInstitute, Japan Company Sanitized MRI4TT33 000065 1411s STATEMEN Sponsor Title Stuy number Kurume Laboratory Chemicals Evaluation and Research Institute, Japan DAIKIN INDUSTRIES, LTD `Biodegradationstudyof ( CB ) by microorganisms 111s 1, theundersigned,hereby declare that this report provides a comect English translation of the Final Report (Study No.14115, issued on September 18, 2003, amended on October 10, 2003). Date sb December 2, 2c03 . Yasuko Matsunobu 000066 111s GLP STATEMENT Kurume Laboratory `Chemicals Evaluation end Research Institute, Japan Sponsor DAIKIN INDUSTRIES, LTD. Title Biodegradationstudyof ( CBI)bymicroorganisms Sty number 14115 Thisstudy wasperformedincompliancewith "OECD Principles of GoodLaboratory Practice" (November 26, 1997) Thisfinalreportreflectsthe rawdataaccuratelyand ithasbeenconfirmedthatthetestdata awe validity Date StdyDitector September 18,2003 Signed in original Yasuko Matsunobu 000067 141s GLP STATEMENT Kurume Laboratory Chemicals Evaluation and Research Institute, Jspen Sponsor DAIKIN INDUSTRIES, LTD Title Biodegradasttuidyonf CB) bymicroorganisms Study number ns Amendment to the Final Report was performed in compliance with "OECD Principles of Good Laboratory Practice" (November 26, 1997) "ThisGLPstatementwasissuedassupplementtothestatementissuedonSeptember 18, 2003 because of the amendment of the Final Report. Date Study Director October 10,2003 Signed in original Yasuko Matsunobu 000068 111s QUALITY ASSURANCE STATEMENT KChuermuimcealLsabEovraaltuoartyion and Reseach Institute, Japan Sponsor Title DAIKIN INDUSTRIES, LTD. Biodegradasttuidyonf ( CB )bymicroorganisms Study number 14115 EEE ll "Theinspectionsofthsstudywerecaredoutandtheresultswere reported10the test ChfaecmiliictyalmsaEnvaagleumaetnitonaannddtRheeseSatrucdhyIDnistrictcuttoer, bJyapQaunaalsiftoylAlsoswusrance UnitofKurume Laboratory, } Study plan July 17,2003 August 11,2003 July 18,2003 August 11,2003 July 18,2003 August 11,2003 September 10,2003| September 10,2003 encores [ug 8,20 Aug772.2005 EEE |so|se yr|sieva] . Ithasbeenassuredthatthe fina reportdescribes accuratelythetestmethodused,that details in the report are in compliance with the study plan and Standard Operating Procedures and thatth final report reflects the raw data accurately Date September 18,2003 Quality Assurance Uni, Head Signed in original Kyoshiro Hori 000069 14115 QUALITY ASSURANCE STATEMENT Kurume Laboratory Chemicals Evaluation and Research Institute, Japan `Sponsor Title DAIKIN INDUSTRIES, LTD. Biodegradationstudyof ( C81 ) by microorganisms Study number 14115 `Studyinspectionof thecorrectedpartsinthe FinalReportwas cartied outanditwas confirmedthatthecorrectionhasnoproblem. Therews asru epol rtedt tothetestfacility `mananadtgheSetudmyDierecntortas follows. Dateof inspection ET ove 102 `StudyDirector TT facility management SoThis statement was issued as a supplement to the quality assurance statement issued on ue onsen 08 Quality Assurance Unit, Head Signed in original Kyoshiro Hori 000070 CONTENTS Tide Sponsor Test facility Objective Test method Applied GLP Dates. Storageoftest tem,rawdaa,etc. Personnel `Approval of final report SUMMARY 1 Testitem 2 Item supplied by sponsor 3 Activated shudge. 4 Performance of biodegradation test 5 Validityoftest conditions 6. Factors possiblyaffectingaccuracy 7 Results 8 Remaiks 1411s page 1 1 1 1 1 1 2 2 3 3 4 5 6 7 9 2a 2 2 2% 000071 1411s Contents of tables and figures Contents of tables Table-1 Calculationtableforpercentage biodegradbaytBiOoDn Table2 Calculationtableforamoofudissnolvted organiccarbonby TOC Table Calculationtableforpercentage biodegrabdyawteiigohnt measurement Table4 Calculationtableforconcentrationofpesfluorooctanoic acby LiC-dMS Tables Calculation table for recovery rate by GC-MS. (2-(perfluorooctyethanol) Tables Calculation table for conceofn2(pterfrluoarootctyietohannolby GC-MS. Contentsoffigures Fig. 1 Chart of BOD Fig2 IR spectrum of residue Fig3 `caMlaistsraftriaognmceunrtvoeg(rpaemsfoufLorCo-ocMtSanaoniaclyasciisdf)orcalibrationcurveand Fig 4 Massfiagmentogam ofGC-MSanalysisforcalibrationcurveand calibration curve (2-(perfluorooetyDethanol ) Fig 5 Massfragmentogram ofGC-MSanalysisforrecoveryandblanktest (analysis of 2-perfiuorooctyDethanol Fig 6 Mass fragmentogram of LC-MS analysis for test solution (perfluorooctanoic acid) Fig 7 MassfragmentogramofGC-MS analysis fortestsolution (2-(perfluorooctylethanol ) Fig 8 Mass speofcperftluorroocutanmcic acid Fig 9 Mass spectrum of2-(pecfluoroocty ethanol Fig 10-1 IRspectumoftest itemmeasuredbeforeexperimentalstart Fig. 102 IRspectrumoftest temmeasuredaferexperimentalcompletion Reference IR spectrum supplied bysponsor 000072 14115 Study No. 14115 Tide Biodegradation study of ( C81) by microorganisms Sponsor DAIKIN INDUSTRIES, LTD. 1-1 Nishi-hitotsuya, Settsu, Osaka 66-8585, Japan Test facility Kurume Laboratory Chemicals Evaluation and Research Insitute, Japan 19-14 Chuomachi, Kurume, Fukuoka 830-0023, Japan Objective This sudy wasperformed to evaluate the biodegradabilityof (CBI ) by mictoorganisms Test method () (TGhuiisdesltiundey 3w0a1sC,perRfeovrimseedd according July 17, to "Ready 1992)" in Biodegradabilty: Modified the OECD Guideline for MITT Test Testing of Chemicals Applied GLP This study was performed in compliance with "OECD Principles of Good Laboratory Practice" (November 26, 1997) 1. 000073 14115 Dates `Studyinitiation date `Experimental starting date Experimentalcompletiondate. Studycompletion date Joly 17,2003. Tuly 18,2003 August 15,2003 `September 18,2003 Stoorfteastigteem,rawdata,etc (1)Testitem About 5goftheitemsuppliedbythesponsor issealedin astorevesselandstored in archives in this laboratory for ten years after the publication of the final report Treatmentoftheitemsuppliedbythesponsorafterthestorageperiodisdiscussed `with sponsor. If itis notstableforthestorageperiod, itisstored while itiskept stableanditisdisposedwithapprovalofsponsor. (2)Rawdataandmaterialtsc,. Rawdata,thestudyplan,documentsaboutthestudypresentedbythesponsor, thefinalreportandnecessarymaterialsarestoredinarchivesinthis laboratoryforthe same term asthetest tem. Treatmentofrawdataandmaterialest,c.aferthestorage. periodisdiscwuitshssponesodr. 2. 000074 Personnel Study Director i Study personnel (Opeorf baiodtegriadaotin on test) `Stafffor cultivation of activated sludge `Approval of inal report Study Director Date Signature 14115 Yasuko Matsunobu Kazuhiro Oyama Takakazu Kayashima Hiroto Nishijima September 18, 2003 Signed in original `Yasuko Matsunobu -3- 000075 111s SUMMARY Title Biodegradation study of( C81) by microorganisms Conditionsofcultivation (1) Concentrationoftest item (2) C(oansctehnectornacteinotnroaftaicotniovaftseudspselunddgeedsolid) (3) Volumeoftest solution ((54)) CCuullttiivvaattiioonn dtuermapteiroanture 100mg 30mgL 300mL 241C days Measuremeanntd analysis fopercentagebiodegradation (1) Measurement of biochemical oxygen demand (BOD) by meansof a closed system oxygen consumption measuring apparatus 2) Determination of test tbeymem ans of weight measurement. Othes measurementandanalysis (1) Determinationofdissolvedorganiccarbon bymeansof total organiccarbon (TOC) analysis (2) MeasuremenotfIRspecbtyrmeuanmsof a four transform infrared spectrophotometer 3) Determination of perfluorooctancic acid by means of liguid chiomatogaphy-mass spectiomeny (4) Determination of 2-perfluorooctylethanol by means of gas chiomatography-mass spectrometry Results (1)Percentagebiodegradationby BOD ~~ 0%, 0%, 0% average 0% (2) Percentage biodegradation by weight measwement 0% 0% 0% vege 0% Conclusion The test item was not biodegiaded by micioorganisms under the present test conditions 4 000076 1411s | Testitem Inthis report, C81 )hasthefollowingchemicalname, etc 11 Chemical name! car ) 12 Chemicalstuctue, etc." Structural formula Molecular weight Weightaverage (col) "1. Informationsuppliedbythe sponsor .5- 000077 Lams 2 lemsuppblysipoensdor 21 Supapndllot iumebesr" (@1))LSuoptplniuemrber DAIKININDUS--TRIES, LID. 22 Pain" (0) Testitem Cw @)Impurity cal ) The test femwastreatedas 100%inpurty 23 Confirmofattesitiotenm Twoinfrared (IR)spect ofthetest itemprovidedbythesponsorandmeasuredat tislaboratory were confirmed tobe identical seFig10andReference) 24 Physi`cAopcpheeamriacncaelproperty" Coe "1 Information suppliedbythesponsor i 25 Stoanrdsatabgiliety (1)StoraDgaecroknsdtiotriaogneplace a room temperature @subilTithyetest itemvasstableunderthestorageconditions,asshownbythe finding thatIR spectraofth tet itembefor theexperimentalstart andafertheexperimental completion were identical (se Fig 10). "6- 600078 ns 3 Activated sludge 31 Shudge samplsitiesnangd date (1) Sampl`Oinnsgistietesshudge sampling was carried out atthe following 10 locations in Japan. Fushikogawacity sewage plant (Sapporo-shi, Hokkaido) Fukashiba industial sewage plant (Keshima-gun, Ibaraki) NOcahkiashiacritay csietwysaegweapgleanptl(aSnhi(nOjsuakkua--ksuh,i,ToOksyaok)a) Kitakami River (Tshinomaki-shi, Miyagi) Shinano River (Niigate-shi, Niigete) `YoshinoRiver(Tokusbima-shi, Tokushima) Lake Biwa (Otsu-shi, Shiga) Hiroshima Bay (Hiroshima-shi, Hiroshima) Dookai Bay (Kitakyushu-shi, Fukuoka) @Date June, 2003 32 Shdge sampling (1) City sReewtaugren sludges fiom sewage plants were collected. (@) RiverSsu,rlfaakceeawnadtseeraandsufacesoilwhichwasincontactwiththeatmospherewere collected 33 Preparation ofactivated shudge Activatedsludgewaspreparedas follows to maintain itsuniformity. mont`hTshwefaislmtirax(tee5dLwi)tohftthheemsiuxpeedmafitlatnrattoe(f5thLe)acotfitvhateesdsuludpgee"cuorlftainvsaltaueddtgaebcoaoultlnfeocrtte3d newlyateach location. Themixedfiltrate (10L)wasaerated >after thepH valueofthe `mixtuse wasadjustot7e02d10 +2 The activated sludge cultivatheed mixed filtrate (10 L) of the supernatant of sludge collecteda the ten locations #3 Prefilteed open ar was used -7- 000079 1411s 34 Cultivation corRroeugshploytno3da0imbionnugutt1e/s3oaffthlewcheoaslienvgoaleurmaetwioansorfetmhoevseld.udgDeecmhilxotruinraet,esdwupaetmeartwaanst. aadedraetdedt,aotnhdethteemnaiapnriengdpeotretrimoinnseodtahmaottuhnteotfostyanlvtoheltuimcesreaechwedaw1a0gsLa.eddTe"hdistomtihxetmuirxetwuarse. Sdoecthhlaotritnhateecdownacteenrtaatdidoedn.ofTthhies spyrnotcheedtuirce sweawsargeepewaatesd 0o.n1ceWeiv%eriyndatyh.evCoulltuimvaetoiofn was carried out at 2542 C *4 Synthetic sewage puGliwuactoesfre,toipoebptetaoinnde50an/dLpooftatshsesiuomludtihiyfodornroegaecnphhcoosmpphaotneenwtere ThdeispsoHlovefdthien solution was adjusted 07 01 0 with sodium hydroxide. 35 Control and use formatDiuorninogf cfullotcikv,atpiHo,n,dtihsesoalpvpeedasoaxnycgeenofctohnecesnutpreartniaotnanitn,tsheedismoelnuttiaotnioannodftthemepesrlautdugree, wweirtheicnhethcekesdctopoe moafintthaeincoantnroolrmsatlasntdaatredosftispluuladtgeed. iInttwhaes"cTesotinng fMettihhaotrdtshemfsoerewNederew aCchteimviatceadlsSluubdsgteanwcaess"m,icarnodsctohpeiscealrleysuolbtsserwveerde asntdoresdludagserwaiwthdantao.abnMoircmroafllosryampitnotmhse was used fo the test 36 Inspeocftacitiovinty anddateofinitiation ofuseofactivatedsludge (1) Inspectionofactivity Activity ofthe sludge was assessed using a reference item. (2)Dateof initiaotfiusoen: July15,2003 3 000080 14115 4 Performanceofbiodegradatiteosnt 41 Preparations for test (1)Measofcuoncer ntate ion omfsusepenn dedst olid The concentration of suspended solid was measured to determine the amount of activated sludge to add Method Date Result In accordance with Japanese Industial Standards (11S) K 0102-1998 section 14.1 July 15,2003 Concentration of suspended solid in the activated sludge was 3200 mg/L. (@) PrepEaraactihono3fmbLasoaflscoulluttuireonmse(da)i,u(mb), c)and (d)shownbelowweremadeupto IL with purified water The following stock solutions were prepared by useofanalytical grade regents: (2) Potassium dihydrogenphosphate, KH:PO, 850g Dipotassium hydrogenphosphate, KaHPO, 21758 Disodium hydrogenphosphatedodecahydiate, NasHPO,12H:0 4460 Ammonium chloride, NHiCl 170g Disinswatoeralndfvilleed updto 1 L (b)Magnesiumsulphateheptahydrate,MgSOu-THO 22.50% Disinswatoeralnd fviledeuptdo1 L (c) Calcium chloride anhydrous, CaCly 27.508 Dissoilnvwaetedrandfilledupto 1 L (@ lron(II chloride hexahydrate, FeCI3-6H:0 025g Dissoilnwvaetedr and filled upto 1 L. (3) ReferAennicleiniete(mreagent grade, Showa Chemicals Inc Lot No. HO-2729D) was used as a reference item to confirmthtthe sludge was sufficiently active. This test is effective when the percentage biodegradation calculated from the BODofanilineafer 7and 14daysexceeds40 %and 65%,respectively,according to regulation of the OECD Guideline for Testing of Chemicals -9- 000081 111s 42 Preparation of est solutions solutiToensstfosrolauntailoynssisfoorf a2v-a(lpyesrifsluoofrotohectyteestthaintoelm waenrde pperrefplauroerdo,ocbteacnaouisceatchied taesntditteesmt eaxnpdecctoendvetrotfeodrpmriondutcetsss(opleuftliuoonrso,cocotualndonioctabceidanaanldy2z-e(dpbeyrftlhueosroaomcetypDreetthraenaot)m,enwth.ich was Eleventestvesselswereprepared Thefollowingtestsolutionswerepreparedand cultured under the conditions described in section 43 (1) Addofitesttteim oroaninline (@) Test solution (water +test tem) (=2: Vessel No.6 and a test solution for analysis of 2-(perfluorooctyl)ethanol) weigheIndoannedteasdtdevedstseol,33000mmgLooffthpeuriitfeimedswuaptpelri,esdbotyhtahtetshepocnosncoernwtarsataicocnuorafttehley test item reached 100 mg/L. (b) Test solution (sludge + test tem) (@=6: Vessel Nol, 2, 2(perfluorooctyDethanol) 3 and thee of test solutions for analysis of weigheIndaeancdhatdedstevdetsostelh,e3b0asmaglocfulttuhreeimteemdisuumpp(ltiheedbvyotluhmeeswpaonssolrewssathsaacnc3u0r0atmelLy by the volume (2.81 mL) of activated shudge inoculated), so that the concentiation ofthe test item reached 100 mg/L ()Testsolution (sludge + aniline) (v=1: Vessel No 4) anilinIewnaosnaedtedsetdvienstsoetl,he2b9a.5sailcLu(lt3u0rmemged=i2u9m5(thke Lvoxlu1m0e2w2agsllees' t(hdeannsi3t0y0)m] Lof by the volume (2.81 reached 100 mg/L mL) of activated sludge inoculated), sothatthe concentration (@ Test solution (contol blank) (=2: Vessel No.5 and atest solution for analysisof2-(perfluotooetylethanol) volumIenownaestelsestsvetshsaenl,3n0o0thmiLngwbyasthaeddveodlutmoeth(2e8b1asamlLc)ulotfuracetmiveadtiedumslu(dthgee inoculated) (2) Inoculationofactivated sludge addedTthoecaaccthivtaetsetdvselssuedlg,e(c)u,lt(iv)ataenddun(4d)e,rstohethcaotntdhieticoonnscednetsrcartiiboendoifn tsheectsiuosnp3enadesd solid reached 30 mg/L. -10- 000082 14115 43 Instruments and conditionsofcultivation. (1) (Ian)stTreusmtesnotlsutfioorncfuolrivanaatliyosniso test item and perfluorooctanoic acid Closed system oxygen consumption measuring appasatus Temperature contolled bath and meesuring unit: Ohikura Electic Co, Lid Datasampler: Asahi TechaCeoi,oLund. VAebsssoerlbent3f0o0cmarLboinnvdoiolxuimdee (improved type) `Soda lime No 1 (for absorptionofcarbon dioxide, `Wako Pure Chemical Industies, Lid) () Test solution for analysis of 2-(perfluorooctyllethancl Closed system oxygenconsumptionmeasuring spperatus `Temperature controlled bath and measusing unit (Only the temperature controled bath was used) : `Ohara Electric Co, Lid. | Vessel 300 mL in volume (sealed improved type) @ Conditions of cultivation Cultivation temperature Cultivation duration Stiningmethod 251C 28days Bachtest solution wassiredby amagnetic stirrer 3) Room Apparatus room No S11 44 Observation and measurementof est conditions (1) Observation of test solution During the cultivation period, the appearanceofthe est solution was observed periodically and conditions of the instruments were checked properly. (2) Measurement of biochemical oxygen demand (BOD) During the cultivation period, the change in BOD of the test solutions was. measured by autorecording using a data sampler. Cultivation temperature was measured and recorded once a day BOD of the test solutions for analysis of 2(perfluoroocryethanol was not measured. "1. 000083 1s 45 Analysisoftest solution conver`Atfetder ptrhoedutcetrsmin(apteirofnivoofrotohceiacnucltiicvaatciiond, adnids2s-o(lvpeedxfolrugoarnoioccctayrebtonh,atnh)e,teswthiitcehm waansd `expected production in test solutions, were determined. 451 Pretofrtee stsoalutt ionsmfoe rann alyt sis (1)Pretreatmentforanalysisoftest itemand perfluorooctanaociicd `Aftertheterminationofthecultivation,the testsolution (water +testitem),thetest solutions (sludge + test item) and the test solution (control blank) for analysis of the test item and perfluorooctanoic acid were pretreatedfortotal organic carbon (TOC) analysis on dissolved organic carbon and liquid chromatography - mass spectrometry (LC-MS) paanaaflyosrimsedo.f peIrRfslpueorcotorcutmaonfoitchaecriedsiasdufeowllaoswsm.easWueriegdht measuremoeftnhte test item was Taking owt 10 mL (measuring pipe) + Centrifugation (1000x g, 10 min) Supernatant ing ou aml i (methanol, volumetric flask) * Filuation (The test solutions were filtered by use of membrane filter | wveistshelssuwctaisonwanTshteerdesoindueteinfitehre `andwashedwithpurifiedwater) TSaOmCpalneafloyrsi_s|| LCS-aMmSplaenatloysis Determination of au Drying (vaocveun,u40mC, 137hs) + `Caobooluitn3g0 (mdie8nsi)ccator, room temperature, `weighStammepalseufroerment +5 MILLIPORE typeHA (pore size 0.45 um ) `SampOlfe IfRorspmeecatsruurme.ment ne 000084 Ee rae rar oe mn (2) Pretreatment for analysisof2-(perfluorooctyl)ethanol Aftertheterminatioofn the cultivatiothne,testsolution (water +test itethme)te,st 2-(perfluorweoreoccootleydalt)5eCtfhora6n6ohrls.and pretrefaotrgeads chromatography - mass spectrometry (GC-MS) analysis as follows. "--Ammonium sulfate 200 g (even balance) "Chloroform 90 mL (graduated cylinder) + Shaking (10 min) *2 *Filtration and dehydration (N2ofilterpaper, sodium sulfate 30g) +Filling up to 200 mL (chloroform, volumetric flask) `Sample for GC-MS analysis i. 000085 1415s 452 Quantandiqutaliatattiveianavlyseis (1) Determinationofdissolved organic carbonby meansofTOCanalysis "The dissolved organic carbon (DOC) in the samples for TOC analysis was analyzed under the following conditions. "The concentrationofDOC was calculated by subtracting concentrationofthe inorganic. carbon (IC) from concentration of the total carbon (TC). The concentration of TC and IC in th test solutions was proportionally calculated from the peak area of the fest solution by comparison with that of 80.0 mgCLofa standard solution for TC analysis and 80.0 mgC/L of a standard solution for IC analysis, respectively (see Table-2). "The standard solution for TC analysis was prepared by dissolving potassium hydrogenphthalate in purified water. The standard solution for IC analysis wes prepared by dissolving sodium hydrogencarbonate and sodium carbonate in purified water. "The concentration ofdissolved organic carbon corresponding t the minimum determination mitwasregardeads 1.0 mgC/L. Analytical conditions Instrument Temperatureoffumace Flow rate Injection volume Sensitivity "Total organic carbon analyzer Shimadzu Corporation. typeTOC-5000 680C 150 mLimin Bul Range 5 (2) Determinationoftest itembymeoafwneigsht measurement Weight of the samples for weight measurement was measured under the following conditions (see Table-3). Analytical conditions Instrument Electronic analytical balance MeterToledo type AT201 Minimum measuring weight 0.1 mg. (3) MeasuIrRemsepnetcotfrIumRsopfecttherusmabmyplmeesanfsoromffeoausruireertmreanntsfoofrIiRnfsrpareecdtsrpuemctwraosphmoteoamseutreerd underthe following conditions (sec Fig 2). Analytical conditions Instrument Measuring method Fourier transform infrared spectrophotometer Shimadzu Corporation type FTIR-3200PC KBr tablet Sa- 000086 ns (4) Determination of perfluorooctanoie acid by means of LC-MS The samples for LC-MS analysis were analyzed for perfluorooctanoic acid under the following conditions. ~ The concentation of pesfluorooctanoic acid in the sample for LC-MS analysis was proportionally calculated by comparing the peak areaon themassfragmentogramofthesampleforLC-MSanalysiswiththatonthe `mass fragmentogram of 0.500 mg/L standard solution (see Table-4 and Fig 6) "The lowest detectable peak area was regarded as 2000 considering the noise level, which corresponded to 0.0097 mg/L of perfluorooctancic acid. (a) Analytical conditions Instument Lc Ms Liquid chromatogsaph-mass spectrometer JASCO Corporation type PU-980 Micromass type Quattoll CoCnodliutminoofLnsC Eluent Flow rate Sample size Lecolumn ODS 15cm x 4 mm ID. Methanol / purified water" (80/20 V/V) 10 mimin 10uL `CoInondiziattoiiofonMnmesSthod Detection mode. Monitoring method Monitoring m/z Source temperature Cone voltage Electrospray (ESD Negative ion Selieonmcontitoeridng (SIM) 413 (sec Fig 8) 150C 20v #6 Containing 0.1 % formic acid S15. 0600087 1115 (b) Preparation of standard solution The standard solution to detesmine the concentation of perfluotoocianoic acid in the sample for LC-MS analysis was prepared as follows. 100 mgofthestaidardsubstance"?suppliedby thesponsorwas accurately weighed and dissolved in methanol to obtain 2000 mg/L. solution of perfluorooctancic acid. 0500 mg/L standard solution was then prepared fiom this solutionbydilution with methanol / uified water (1/1 V/V) *7 Name, lot numanbdpeuitry Name Pesfluorooctanoic acid (C-1700) Lotnumber C17001401 Puity Perfluacoidrwaostroeatced tas1a00n% ionpiusicty () Calibration curve 0.125, 0250and0.500mg/L standardsolutionswerepreparedbythesame. `method as described in (b) These solutions were analyzed according to the analytical conditions described in (8) ~ A calibration curve was drawn based on therelation between thepeakareaonthemass fSagmentograms andthe respective concentations (se Fig 3). -16- 000088 14115 (5) Deteaminationof2-(perflucrooctyDethanol by meansof GC-MS `The samples for GC-MS analysis were analyzed for 2-(pefluorooctylethanol `under the following conditions. The concentration of 2-(perfluorooctyl)ethanol in the sample for GC-MS aialysis was proportionally calculated by comparing the t`hpeeamk aarsesafoangtmheeamtaosgsrfarmagomfe0n5t0o0grmagm/oLftshteansdaamrpdsloelfuotiroGnC(-soMe STaenballey-s6iasnwditFhitgh7a).ton The lowest detectable peak area was regarded as 300 considering the noise level, which corresponded to 0.021 mg/Lof2-(perfluorooctylhethanal. (8) AnalyItnicsatlrcuomnedinttions Gas chiomatograph-mass spectrometer Shimadzu Corporation type QP-5000 Condens erGe Column HP-FFAP (Agilent) 25 mx 032mim 1D,madeoffusedsilica Columntemperature 50C(4min)C (R--ate130C7in0) Carer gas Heiium Pressure of carer gas 10.0kPa Injection temperature. 200C Sample size 2uL Sampling method ~~ Splitless Sampling time 20min . Roe {onizaton ) Detection mode, Monitoring method Monitoring m/z Interface temperature Positive ion Selected ion monitoring (SIM) 95 (sec Fig) 230C a7. 000089 14115 (b) Preparationofstandard solution The standardsolutionto determine the concentrationof2-(perfluotooctyl)ethanol in the samplefor GC-MS analysis was prepared as follows 100 mgofthestandard substance TM suppliedbythesponsorwastaken out accurately and dissolved in chlotoform to obtain 2000 mg/L solution of 2-(perfluorooctyl)ethanol. 1.50 mg/L standard solution was then prepared fiom this solution by dilution with chloroform *8 Name, lot number and purity Name 2-(perfluorooctyljethanol (A-1280) Lotnumber A18201X01 Purity 2-{perfluorooctwyalsettrehaatendoals 100 %inpurity () Calibration curve 0375, 0.750 and1.50mg/Lstandardsolutionswereprepared bythesame `methodasdescriibne(bd). Thesesolutionswereanalyzedaccordingtothe analytical conditions described in (2). A calibration curve was drawn based on the relationbetweenthepeakareaonthemassfiagmentogramsandthe respective concentrations (see Fig 4). 18 000090 453 Recteo st avnde blar nkty est re Eachtwotest solutions (water + 2-(perfluoroocatnyd ltw)oettesht saonluotilon)s (sludge + 2-(perfluorooctyl)ethanol)forrecoverytestwere preparedaccordingtothe `eachtestvessel,sothattheconcentration of itreached 1 mg/L. Thetestsolutiwoenres om ls ems220 20) pretreatedinaccordancewiththemethoddesctibedinsection `according toanalyticalconditions described insection 45.2(5). odds 45.1(2),thenanalyzed section 42. Thetestsolutionforblanktestwasanalyzedinthesame wayasthe recoverytest Asfor the blanktest,nopeakswereappearedaroundthepeakofthe 2-(perflucroooncthteymalss)feratgmhenatongroaml. `Twoindividualrecoveryrates andtheiraveragesontheanalyticalprocedureare shownbelow. Theaveragerecoveryrateswereusedascorrection factor,forthe deterofm2-(iperfnluoarootctyil)eothannolinthetestsolutions (see Table-5 and Fig 5). Recoveryrateofthetestsolutions(water + 2-(perfluorooctyl)ethanol) 996%, 982% average 989% Recoveryrate of thetestsolutions (sludge +2-(perfluorooctyl)ethanol) 80.5%, 815% average 810% 000091 1411s 46 Calculation of percentage biodegradation expreTssheedipnerwcheonlteagneumbbeirosd.egradation was calculated by the following equations and (1) Percentage biodegradation by BOD Peccentage biodegradation (4) = ps x 100 BOD B TOD" Biooc xygh endee manm dini thetc esta solul tion (sludge + test item) (experimental) (mg) : B (expei rimeo ntaolc x)y(gmegh )ndee manm dini thecc ontra olbll ank Thieteomrewtaiscacloomxplyegteenlydeoxmiadnidzerd (etcoqreuwthcaieln)rt(hmeegt)edst *9 Thepuritywasregardedas 100 %andTODwascalculatedfromcomposition formula ( cal ) which was calculated by use of n=8, `monomer values of (CB) regarded as mol% in section 1 2. (2) Percentage biodegradation by weight measurement `Percentagebiodegradati"on (%) = --S--wg-uS=s-- x 100 Ss (Rselsuiddguea+lamteosut ntteomf)t(heextpeesrtiimtenetmailn)t(hmegt)estsolution Sw. Residualamountofthe testiteminthetestsolution (water + test item) (experimental) (mg) 47 Treatmentofmumerical values Values were rou offn inad ccoe rdad nce with JZI84S 01:1999 rule B. 20. 000092 14115 Percentagebiodegradationsof anilinecalculated by theBODvalueswere65 %and69% after 7 and 14 days, respectively. It was concluded that this test conditions were valid (see 6 Factorspossiblyaffectingaccuracy _--Noadverseeffectsonthereliabilityof thistestwerenoted. 71 Appeaorftaesn tsocluteiosns [Te | wm m e Ta] `Appearancesoftestmediaincultivation vesselswereasfollows. ew [rem Wa+t tese titr em |Thetest itemwasnotdissolved - Atthestart of cultivation mmm. [amrematease | Atthetermination " Insoluble compoundexceptforthe i | ludge "M |Growth of the sludge was not observed. 000093 ans 72 An"Anaalryetsuillctseoysfttsetsotfsotilhuetticeosntssoutonafter 28dayswereas follows = ln Water + To . T= alse Le] = deckimotBOC | % Te Residualamount. [a [wr[mn| m2 | 0 | Suna sa | 100 97 - 5CC6ondco(eLntpCrMataSi)onno_off| gt. soos Water + lu+egstietem | Theoreeal| | wien [ 1T 2T 3 | emo CSoomenncateionnt,ration._ooff| mgt| 0014 | 0017| S0017| S0017 7 ebanol (GC-MS) +10. T(hecvall+uueedostf gtcoenemt)rol blank was subracted fiom the values of the test solutions #11 Mi=n(iMimnuimmduemtedremtienramtiinoantiolinlmiitmoiftpinersfaimoprloeofcotraLnCic-McSidanianlytseits)sxouon = 0(0G0n9a7vmogl/uLmex)(S(mvo.lu/3me0o0ftmeLs)t s/ o2.t5o30)0)/=G0at0o1o9fmpgo/tLion used for analysis) #12 Mi=n(iMminuimmduemtedremtienramtiinoantiloinmliitmoift2inpsaemlploefroroGCo-cMStaynaleiynstiseb)stasonluotioln ((afiinoaolfvpoolrutmioen) s(evodlfuomraeonafltyessits)olution)/ (average recovery te)/100} / Test so0l0u2i1omng(/vLatex (2e0s0tmtLem)300mL)/ (989/100) 1=0014mglL Test so0l0ut2i1onm(gsllLudgxe +20e0smt Lte/m3)00mL) (81 0100) /1=0017 mgL 2. 600094 111s 73 Percentagebiodegradation Percentage biodegradations after 28 days were as follows Method "Percentage biodegradation (%) Table [[mroome[o0e|[0|o 0[To 0o[T5] 74 Results ofmeasurofeIRmspeectnrutm No significantdifferencebetweenIRspectrum ofthetestitemandthatof theresidue after weight measuremewnast observed(seeFigs 2, 10). 75 Discussion Determinationofthe testitemby chromatographywasdifficult,becausethetest item was not completely dissolved in water and organic solvents (tetrahydrofuran and chroloform). Therefore,thetest iterwasdetermbiymneeandsofweight measurement As a result, thepercentageresiduesofthetestitemwere94 %inthetestsolution(water + testitem) and 100, 103 and97 %inthetestsolutions (sludge +testitem). The percentagedetections of DOCwere 0 %inthetestsolution(water+testitem) and 0 %in alltestsol(uslutdgei+toestnitesm) These resultsindicatethat thetestitem was not disisnto he tlestvsoleutid on. No significantdifferencebetween [R spectraofthe testitem before and after the cultivation was observed. Converted products (perfluorooctanoic acid and 2-{perfluorooctyl)ethanol), wh wasi expc ecteh dtoformintestsolutions,wasnotdetected. `TheseItreissultcsonisnididcearteedthtahtatthethteestteisttemtweamswnaostconnovterbtieoddedgurraidnge tdhuerciunlgtivmaitciroonorganisms, because the average percentage biodegradation by BOD was 0 % and 10 significant change in theweightofthe testitem before and afterthe cultivationwasobserved. 76 Conclusion Thetest item wasnotbiodegiadedbymicroorganisms underthepresenttest conditions. 23. 600095 iis Remarks 81 InstrFuomeunrtiseutsreadnsfofortmesitnfrared sectrophotometer Shimadzu Corporation type FTIR-8200PC Closed system oxygen consumption smeeeapsaugrieng11apparatus TLoitqaulidorcgharnoimcactaorgbroanpha-namlyazsesr:spectrosmeeeteprage 14 seepage 15 `Gass chromatograph- mass spectromesteeerp:age 17 Electronic analytical balance MSeatrttloeriruTsoAlGe.do tytpyepeCPA3T2240S1 Sarorius AG type BR210S MReefcrhiagneircaatledscheankterri:fuge Vaccume oven TShIiEmTaEdzCuHCCoor,poLrtadt.iontypetySpRe-C2SwT-060LF SIBATA SCIENTIFIC TtEypCeHVNS-O3L0O0GY LTD. 82 Reage`nMtesthuasneodlf(oHrPanLaClygsriasde) Wako Pure Chemical Industries, Lid PCuhrliofrioedfowramte(rreagent grade): TKaikshaisduagiChSeimyiackaul CCoo., LLdd. ASomdmiounmisuulmfistuelf(irteeag(ernetaggernatdeg)r:ade): KantoKCahnetmoicCahlemCioc,alIcC.o. Inc. FSoordmiiucmahcyiddr(orgeeangceantrbgornadaet)e:(reagentWgraakdoe)Pure Chemical Industries, Lid. Sodium carbonate (reagent grade): Wako Wako Pure Pure Chemical Chemical Industries, Industries, LLiidd Potassium hycdrogenphthalat (reagent grade): Potassium bromide (for measurement`WoafiknofPruarreedChabesmoircpatlioInn)d:ustries, Ltd. NACALAI TESQUE, INC. P2-e(rpfelrufolruooorcotoacntoyilceatchiadnol SSttaannddaarrdd ssuubbssttaannccee ssuupppplliieedd bbyy tthhee ssppoonnssoorr 24. 000096 TEsose(mga]) l(Ts5%)s]woo(mrg)o](T3s%)ool mo(nag)ll (9%0)|] v0(mg0)a] 10] ]| 9%) |[% TT a[eae l eo]wa walloolsl suolo oe l a vo]] ooll7|] B 8[ooe l Toa eorl]Tolol soslloe ]7ooa]] o1] || Vessel no. @ : Sludge + Aniline Vessel no. B : Control blank [B] Vessel no. (I @ @: Sludge + Test substance Vessel no. @& : Water + Test substance 2003.08.15 Name kramer 000097 Table2 Calculation table for amount of dissolved organic carbon by TOC Study No.14115 Waterblank (6)Water+ test em. 11)Studge +test tem 2) Studge +test tem 3) Shudge +test tem 5)Controlblank ae ad 0 nd 101 o 102 114 0 0 Amountoftes te added:30(ag) Volumeoftes soliton :300 (ml) Theosnoruteof ctarboin(CcB)a(mlg) (30x) cal ) A:Measvauter(@egCldL) B: Aouof DtOC Bw = (A(Water + teste) - A(Water bank) x300. 1000(gC) Bs= (A(Sludge + test em) -A(Controlbark)x 300/ 1000 (gC) C: Percentagedetection (%) C=B/(Theamoountrofecardbon)cx1a00(l3%) D: Averagepercentagedetection (%) September3,2003 __ Nemo b Brome 000098 Table:3 Calculation tableforpercentagebiodegradationby weight measurement studyNo, 14115 16) Water+ esttem 11)Sindge +testem 12)Side +test em 0) Sudge +testem 15)Control lak #2 #2 " 7 s01 100 ss s08 103 28 2 5 36 0 o o Amosof esttemadded: 30 (mg) Volum of estsousio:n 300 (aL) A: Weightofresidue (wg) 1B: Residual amountof testfem (mg) BoeAWa+teset erm) Ba =A(Sludge + et em)-A(Contolbask) C: Percemagereside(%) CB (Amountoftest emadded)x 100 D: Percentagebiodegradation(5) D=@w-BBewx)10/0 B+ Averagepercentagebiodegradation (%) Septem3,b2e00r3 _ Namek. Ooms" 000099 Table-4 Calculation tableforconcentrationofperfluorooctanoic ac by LiC-dMS Study No 11s Standardsolaion 00mg [6] Water +testitem (1)Sludge +testitem [2] Shudge +testitem [3]Sludge +testitem 15)Control blank 103049 nd 0(S0019) - nd 0(S0019) nd 0(S0019) - andd 0(s50019) Volumeof test soksi30o0(nnL:) A: Peakarea(-) B:Final vohu5mael:) C:Rathofpartonuedforalysis 2.5300 1D: Concentrationofperlcxacsidfontecstwaatenr(egLc) D=Fx(AJACSastt) (B/300)/C Minimumdeterminationlimit: 0.019(mg/L) E: Averageconcoefpn erfltuororoctaanoitcaciido (mg/nL) F: Concoe fstanndat rdsr oluta ion t :0.5i00o (mgn /L) seeig 6 September 18,2003 New fb Gueer? 000100 Tables Calculationtableforrecovery ratebyGC-MS (2-(perfiuorooctyDethanol Standard soln 1 50m 269 Water +2Gerisoroocyetase | 2m 0m Water+ 2peruoroocyethaol 2 25861 os Studge+2 perhoroocyDethnol-1 2096 02 Sige+2 petoroceyechnol 2 2149 ous Controlbank ad Suayvo 11s ws oo m2 ws mo ws Aout of 2perfsooocatddeedh0a30n0o(agl) Volumeoftetsolut3i00o(nmL:) A: Pak(a-) B: Final vol:2u00m(meL) C:Ratio ofportion sedforsaa: 1 DsRecovery amount (0g) DG x(A/A(Sunx(d8a/3s00t)/)C E: Recoveryrte (4) E=D/0m3p)0x1000 Fi Averagerecovery ate(4) seefig.5 `September 11,2003 Name k. Suu 000101 Table-6 Calculationtableforconcentrationof2-(perfluorooctylbeytGhCa-nMoSl. SDitYs 0m. IieMs pitatin "w ~ m S nw OsNeeS n . wnn am atFtem tre 208 a 3 emt C:Ratioofportion usedforanalysis:1 D:: Recovery rate:98.9(%)(Wa+tteste iterm) + CA s --A T----A SS) 81.0 (%)(Sh+atedstigteem) 73 Amo SEor T Miniumdetermination limit: 0.014(mg/L)(Water +testitem) pba 6: Concenoftstranadartdsoiluotionn: 1.50 (mg/L) ar 000102 Fig Test Noo Ch1e4r1e15of 8(0TDest substance CApupSlaetrniatvteauntsiirnagticoonnditions: . AReecsftteirveasntucebedseassnuicbnesgteance( aniline ) TDeumrpaetriaotnure Note: poguiar condition ew Yoo ow 11003000 (((sseee/pm) 2S5oa0a%vsT(dCu1 18~aug 15,2000) Vesisoe1| sample description 500 (me) Lathdsy 28hday [ [Sludge + Test substance] 2.8 | 4.4 | s8| 7.4| Sludge + Test subst2.a1 | nc3e.6]| [__@ [Sludge + Aniline [[control blank (B] [20[ as| 60[72| [8[Water +Testsubstance] 0.0| 0.0 | 0.0| 0.0| Too 3 wo - 3i . 2 ow i iw ; eee 2 ; 20 : cn ei 328 7 CultiTvatihon Time ( 0~285thdny) PA 2003.08.15 Name _ Qymmars 000103 Figa-1 IR spectrum of residue. SSatmupdlye:No. 1[46]115Water + tost item DNaettehod : AKBurgusttabf26e,t 2003 Name CE pens 000104 Fig2-2 IR spectrum of residue. SSatmupdlye:No.: 1[14]115Sludge + test iten DMaettehod AKuBgrusttabl26e,t 2003 Nene Flan 600105 Fig2-3 IR spectrum of residue. Study No. : 14115 Sample [2] Sludge + test item DaMettehod KABurgusttab2e6,t 2003 Name: kfm" 0600106 Fig2-4 IR spectrum of residue. SStaumdpyle No : 14115 [3] Sludgo + test iten Method KBr tablet DNamaete: AkugGusote2r6, 2003 000107 : i Te rapppppee i wo | | | | i t | | an i | ono somo won 00 won "- Fig2 5 IR spectrum of residue. SStaumdyplNeo:. 1[45]115Control blank DMeathtode:| AKuBgrusttabl26e,t 2003 Nee: 4 gon" 000108 `1S1o1l4vdeor21 400 I% ' 8 Tn Poiin nd Sal3on OZ al 3] 7 rT0ane.da s suion24 0.25 iI| K || | | Ter&darrd ien SaTalo7n 08 p 4023889 4. 6 ase 50821 7) 143.85s D5 yrs fo SI1R 5o-1fACuhagn-ne2l0<0= 3 8.00e5 a Rot C0A ra we m 8. Sot] Cro00os0ay 000g0s = Sot GoiaoeTi2mae soit a [Ji 7 7 7 TE#rReiTr oHTime 2003 puffGusera Fig3-1 (petuaroocanci acid). `Massfragofm LCe -MSn analt ysiso forcg alibr ratia oncum rve 000109 120000 : i peed. #22 36 Epp Zs | -, Concentration (mg/L) Faptu. (mg) [o|u oms| [oz sess| oso iooss | Fig.3-2 wn ve ger CalibcurrvaeotfLiC-oMSn. ana forl perfy luos rooci tans oic acid. 000110 sntyve 1011s DSpuia": {tSaonnhanoss nastySoapT------------------------------------2255000 25000}i . T 1500] Pakpsn so0- 3 i DSieea nSianndtven075mg. ry 7 5500 50%) 1500: soo A 7 7 DSupae: {anSnuashtne0i10mtg. itySono du 7 a 5500 1500 6) $7 2 bon ens Pk RE pa 1 6357 6136 PaTkooRTn Paikma 5000A : Il ] i : Dea; nvasss 7 an Sole Sani easy tea 130301. Soong 7 ss00 00 3 150m so0 oiT : 7 FTk eRnT pamkei Due: Moga1,2000 Nese: K Dyn Figd-1 MassSagmentogramofGC-MSanalysisforcalibrationcurve (2(perfuorooctylethanol) 600111 30000 : uso Wn ieConcentration(mg/L) (mg) ) [o TTaenn e | CsTose]| Fig.4-2 Calibration cuor fGv C-MeS.`analysisfor 2-(perflucrooctyl)ethanol. August 11,2003 Name k. Cramer 000112 2----A_--HE -- ae0]r;} hp sore aie 3 mt e es s a fofrshesmdd hoSooos" po rporr prenk i. . Ths Pak RT Mew Peak: - 150 roe Pek RI Peskares 7 6 pate srBeam -mg. . ro } wo 1 7 - mn * Pek RT. Peskares - Duis: August7,2003 Name: A, Dypmen Fig5-1 Massfragmentogram of GC-MSanalysis for recoveryandblank test (analysisof2-(perfluorooctyl)ethano)l. 000113 say, 1s DSoela"s:Sereieim 1i" atariKee sy Srlbrton Stag : i Ca 200002 A 15000 | I sam 1 7 7 pene yom emt He"ar 1500 se 9.1136 Emin ren so 2 Pak RT pakem Toes aie a on Pak aRsTs Punskeim sao! 3 i pSuotee:{anEiaoer mySooo 2500. 7 =a 5500 1500 Poutpostion ne -------------------- i 7 = Duo: Augus7,2008 NamGream:a Fig5-2 Massfragmentogram ofGC-MS analysisforrecoveryandblanktest (analysisof2peruorooctyetbasol ) 000114 S1t4a11n5d4a0r0d4solu0 tion500mgIlTT S=IRof 11 5C.haanungel2005.3 = 800T0i5c 488 Area 103449 |= i || lo | ol 2 al a 850 70h0y 0 141154005 Waters testitem ---- S--IfRo5fA1 uChgaannmamESs. |I 800T6i5c | Pakapdu.tin ge i [1 + Z mw |L a- o Team 8 700T0ime ug: 8.0005. | | Put tn 1 o --ob 200 4b eb "78h Time 1000 1905 2003 P45 ke gen" Fig6-1 MassSagmentogramofLC-MS analysisfortetsolution (perfluorooctanoieacid) 000115 T6t350l0u0d%ge + test item tT SiRa"f15-CAruga-2ng0e0o3 E . x i..0. 2. | a2 [hess Pak position. 4 6 8. 10.00 SIRof 1 Chane ES i M|di ow]o 1 of `Peak position. h| l | ow]ee | of 21 nd 4 80 - ug: 8.0085. Time 8bo 1000 ef ParkGon DStaovle"e nSainstainsulon 1 $001. esi 2-- 500]i x 1500 I so 3 i DSeoael :Hn1se co0srem imiToo 2100 Suv nis 3 7 Lr l 1500 Peak pestin Too ne 3 i 7 DSpui"a :S1ed1gaeeos amt stSyooo 2500 15000 Peak postion i nd oo l 7 i 7 5500 |! re onerm [nSeen P 5500 wn 5500 | Due: Aug 10.2009 NaksGuea : Fig7-1 MassGragomfGeC-MnS atnalyosisfgortrestsaolutmion (2-pectucrooctyethasol). 00011.7 Std 101s wo | DSlue"e S:1h1essa0esaten 2 neShnog------------------------------------= 25000] i 20000] ) | 1500 Peakpostin | x ae 5 i DSpul"e :{S1endsgaen0ryien 3 insStoo 25000 7 an =0 = | 1500 Pa postin 2d 3 i DuSiaapgllee :C1o1n5vo4l0b0a8k sity 2500 7 win sso0 - | 15000 Pus postion 1000 = 5 i 7 =o Date: August 18,2003 Name Gps Fig7-2 MassfagmofeGCn-MStaloysisgforrtestasolumtion (2perflaorooctybethasol). 0001318 erg 138 En Teor Study No. tetra Instant MS: Micromass GuatI, PLD : asso PU-080 45-950 Sample __Prfuorsoctangic acid HPLC Conditions Colum _Loco0l0uSmn Sie 15 em x _48 om ID. Eluent__ Methanol" /ourified water Flow rate _10_nl/min ( _8/2 V/V) #1 containing 0.1% formic acid Sample size 204 (Sobvent_ than MS Conditions lorzaton mode _ESI_ Detection mode_Negative Function _uts Source conditions ( Capilry _30_W, Gone Mass range (m/z) __100 =1000 _20_V. Temparstre _i50_")0 Dete_hne11,2000 Oper _L.Graven Karu Liberatory Charicals Evaatio ad Rovere bins, Jamin Fig8-1 Massspectrumofperfluoarcido(aonalcytticaalcnondoitiionc). 600119 1s4t1d21400.000m1giL - 100. 38 2h E72 Kin rent 172-SJcuna-n20s0.3 19T0i6c * 089,44 149 265 3% 485 500 g72 E 14o1f144r 0T0i1m70(33!.C-- m(856:673-- -612P:Y6.)8x1.200) a oo Bh ToRe e iTduna Tia 413 2Sean39E0S6.5 (Mm) (M-COs-H)" 369 (M+HC00) 450 (M-R) 1a] ar 106 280300 4% 8 TOE S80 100m0z Ssenly ips Figh-2 Massspectrumofpefiuoraociod(cmatssaspnecotrfumc). 000120 s14@11R44I00m1 gL as we 089141949 226%5 304 485 50 672 1:4114001 as wz=369 NE 1 ys 17-#Jsuena-n2e0s0i.3c 199e6 Tanass.o 360e8 `141743067 wz=a13 ow a Eanes. 14114007 i mizmdsy 141143007 1 38 CO] wees FSanes. TSao2nr5ee5ss8 ob - Fig8=3 3 0 55% 5 70T0ime Fnldy1) prs Massspectrum ofperfluccridomoascscthraomnatoogriamo). 000121 30d 1 2 Epa Jens Study No. _1e11s Instrument ___ Shimadzu0P-5000 Sample ____2-{perfuoraootyfthancl GO Conditions Column __H-FFAP(Fusedalles) Size _25 mx_032mm iD, Fim ticks _052 um CoCalruemrtgoasme. H_o50_%C (_3_min=) _110_"C,Tomo. rate _30_'G/rmin Column head pressure __100 kPa Injection temp. _ 200 C Sample size _1 ul (Solvent _Methano)l nit mode _Spitess Sansiing time _20 in MS Conditions lonzaton Mode _EL_ Detection Mads _Positve Mass range (m/s) _5=6000 Interface temp. __230 C Date _ ne 11,2000 Operator __& Dymma Kurume Laboratory, Chemicals Evaustion and Rosearsh Insite, Japan Fig9-1 Massspectumof2perfeuthanaol ranaloyticaalconeditiyon). 000122 DSunelesoetisadaoSnhien10mg. ] S"asdoem ss0m0. x 20000. [rs i S000 7 FJmoyRaT t esscs2 Smoyvo atte Totin chrorsiogan 3 3 61 26 Ee vr rc 7 7 7 "io 35pr aPAHnAoY nAo2a NA " w of [7 40 Be 1 FssHmsBsuse asReusT48Rue RTwe Date: June 11,2000 Name: .Gown) Fig9-2 Massspectrumof2perfucroty(meatsshspaepctorulm) 600123 satrdo 1110 SeDa lele nei1d10aon 100mg. ty Mas avonsopin x 250000 x 150000 \ s0000 7 --7 ? seed 4050 he tens .neSo 7 7 omSoo 7 wn Dulwe112:00 Name: K Dyn" Fig9-3 Massspectrumof 2pefioroostmaysecthrbomaatnogoralm) 00124 Fig.10 -1 eIxRpesrpiemcetnrtuaml ofstatrets.t item measured before SStaumdyplNoe. :: 14115 Test item fNaettshod | Kbr tablet July 10, 2003 lame. pur 000125 Fig.10-2 IR spectrum of test exper imental complet iont.em measured after Study No. : 14115 jMeatmhloed : TKBorsttaibtleemt Date: August 27, 2003 Neme # pun 600126 | nn Relenmce IR pecttum supplied by sponsor 500127 Amendment to Final Report KuumeLaboratory ChemicalsEvaluation and ResearchInstitue, Japan 1 Tite Biodegradationstudyof C81)bymicroorganisms 2. Studynumber 1115 3 Content 453 Recoverytestandblanktest(page 19) 4 Reason Wiiting exror 5 Conotfcoerenctton *Recovery9a9t6e%o,fthe t9es8ts2o%lutioanvse(rwaagtee +9e8t9i%tem) Recovery tat ofthetestsolutions(sludge +tetitem) 805% 815% aveuge 810%" iscomectetdo *Recovery ate ofthetestsolutions (water +2-(perflvorooctybethanal) `Recovery 996%, 982% avenge rate ofthetestsolutions (sludge 989% + 2-(perfluorooctylethasol) 805%, 815% average 310%" 6 Approval Date Study Director October 10,2003 Siginnoriegindal Yasuko Matsunobu 000128