Document aBw5Zav0bLMBbDyk9N1aMzezX

} `BIODEGRADATION STUDY REPORT i. . -- ETT Wednesday, Novemb0e6r, 2002 } Blodogradatin Srsan Styfr TelomerType Alcohols aco ConamaPptRrOojJeEcrCiT0N:uwEE0Bo1E.nR0s088 cacns - CoSstToUnDYDILRaEeC,TOPR A cuss } EJaGnmens naLunnL, eP1o0.n,y a } Soturi ataveeSSmot,o a as 4a J etEe tor: gat, 2000on sun . Clason. Loge, PhD rage 1130 cTeyPapcsnCAeG: Sdemign Screen Sot ToorEs" -Rovon Bi PaceAnalytical Services, Bio-AnalyticalServicesGroup CONTAIN NO OS CBI - Table of Contents . Tite Page Tableof Contents . -. -- 1 2 Listof Tables & Figures.............. I - Uistof Appendices... i 3 4 Executive SUMMary................. 1.0 Project Personne... . BE . 5 6 - 20 DataRequirementsandRevision Justfication 3.0Project Objective. A os BR 6 6 40Test Avice... - 5.0 ReferenceArticles -- er BE nr 8 9 6.0 ReCeipUGENEorf aSBlMiPIoEnS..........ccvrvrv 7.0Methods... FE ei m---- 9 9 - 7.1 Sample Preparaton............ BE BE 7.11 CollecoftSiIUoAGne ......... BE 910 7.1.2 CulurePreparation BE SE naz = 727I.n1s.t3rSuomlenitdaPlAhnaasleysEixtsra(cHtPiLoCnI(MSSP)E.). ersemesnseenm---------- EE -- 131 72.1 Instrument Parameters... FE 131s 77.22.32QQuuaalnittiattaitvieveAnAaRlyBsIisYSofSP.ar.e.nt.s.a.n.d ProGUCES .....w...m..cmcuws--iww--ies-- en 14-165 BN 7.3 D7a.t3.a1 MTorlaar nCalscuflaotiroannmsdaCatlciulaotonnssM....A.... BE 16 18 ~ 7.3.2 ConversionofngimLto micromolar and nanomolar 73.3Molar MassBalance Calculations. SE . 16 " 7.4 SoftwareVersions eons 80Results. JE SE JER 17 J 18 - 81 8.2 Sludge Qually Characterizaton.... Control/Sample Matrix Spike er Resuls .......... FR 18 18 8.3 Analytica Blanks Results. BE - 8.4 HPLCIMSAnGlySi....... FE 18 19 88..44.21 QQuuaalnittiattaitvieveH AnalyP sisaRnL deHPsfLC oOruMPSFlOIM AM.Ss..S RESU. ee 1924 2025 - 9.0 CONCLSIONS cr 100LiteratureCited J J SE 21 28 11.0 Sample and DataRetention ........ a 2 Page 20138 ace Project CAQBS: "Biodegradation Screen Stour Tedlomyes Alcohols" Revision 1 - WeBaanemsdoayy.eNnoSvaenmtbeere,0S,i20A0n2aicaSarvcesGroup 000004 - List of Tables & Figures Thee TTaabbllee 21.. PEaxrpeencttetdelpormoedrucaltcfolhuoolrienxapteedctfeatdtyfoancsd ProGUCIS 00-- ... 14 15 - Tablo Table 3. 4. HHPPLLCC//MMSSIDMaStaDaTtaablfeorfo-rOxPiardeanttioanndPrPordoudcutctlolnosnsobosbesrevrevded ...... 2 2 - Fioures - Figure Figure 1. 2. Chemical Chemical Representation of Telomer AIGONOI Structureofperluorooctanoate (PFOA). ......... BR 1 9 Figure Figure 3. 4. Potted Potted HPLCIMS HPLCIMS Integrated Integrated Peak Peak Areas Areas for for Cs, Cs, Cro Cys and and Ci Cys Telomer Telomer Alcohols... Alcohols. 20 20 - FFiigguurree 65.. PPllootttteedd PHePaLkCA/rMeSasInftoergTrraatnesdiPeentakPaAlryeflausorfionraPteerdflFuaotrtiynAaclieddsFaDtettyeAcctiedds Detected 222 . FFiigguurree 78.. PPlrootptoedsePdFBOiAodCeognrcaednattriaotniPonasthMweaaysuforreTdeilnoBmieordAelgcroahdoaltsion SampleI s (SPE eluate 2). 25 2 Page 30138 SPtaucdeyPOrocjtecot.CAlGeESs:io`nBiCo.dLegarnagdea,tiPoOn.ScreenStudyfo Telomer Alcohols" Revision 1 Pace Anata Senices. Bo-Analytical Senices Grove - `Wednesday, November 06, 2002 000005 - List of Appendices ~ Appendix A: Approval Signatures... . . 30 - Appendix B:TFaibnlael Q1.uanTteisttatCiuvletuRreessulFtisnaflorRePsFulOtAs (ng/mL) -- Table 2. Abiotic Cultures Final Resuls... . . 3 3 Table 3. Blank Cullures Final Resuls ....... J 31 Appendix C:"TIanbtleeg1r.ateTdelCohmreormaAltcooghroalms PPeeaakkAArreeaasDat.a for the Telomer AlcoholsJ 2 - Table 2. Percentage of Peak Areas For Telomer Alcohols Remaining at Time Poin...... 32 - Appendix D: Table 1. Integrated LC/MS (SIR) Peak Area Data for Perfluorinated Fatty ACids.........33 Appendix E: Table 1. Peak Area Data for Transient Formed Polfluornated Fatty Acids. 3 Appendix F: Table 1. Total lon Chromatogram (TIC) for SIR Data or Biodegradation Sampies........35 Appendix G: Table 1. TICs for SIR Data for Day0 and Day 16 Biodegradation Samples. 3 Appendix Hs Table 1. TICs for SIR Data for Day0 and Day 16Abiotic Control Samples... a Appendix i: Table 1. TICS for Extracted lon Data for Transient Polyflorinated Fatty Acids 3 Page dof 38 PSatcaeyPrreocjtecotr.CACOlBSe:so"nBiC.odLegarnagda,tiPohnO.Screen Study or Telomer Alcohol" Revision - WPeadcneaAsnaaabyc.aNloSveneimcabse.rBo0-6,A2n00a2lytica Sences Group 000006 = Executive Summary _ A(stcarmeeerniincgosntacy wdoesgruanddeasiwkheenn0eGxepaorsiedngmwuhneitcepralhweasftuecwratoecrheomaimcelenltosmeunee.rmeTdhieate toy nc he preporaionof cursfo a ss 53m pont comparative sty, an included - - ttaeusabttcnaulnatcuerrewosa,msbltaahdndkTecudulitouCrceisl,seanMsdoatcoonatWrpaopslrtocxuoliwtmusarttesee.lyTTrheeautMmmieccnrotonbcFieanoltcriiant.ioocnuTsl.eulmComufoelrrucrlueletsouwrneeasreweasts sludge. - caduabgartaecdavtoinsphraokdiuncgtsa1a2n5dCp.areSnotlad phneweaaxstreamceptiloonysoefd.curAnesHPfoLrGeIcMoSvearnyayoifcal med avandyoveoflcoplferodr aanrycspofrorvoitdelsoomenrgalvciodnaonsceanaetxphectoemd eprroduacltcso.holTshweeHrePLOMS - - boofifosodeervgirranedlaetde.xepdoT{chioeosdodbpaseterorunvoendnnelarotsmesedodfciattebelosorwmyeeirrcaelaccciobdhsso.elrsvUeondc.ecxupTrhereceoidxecidoydn,acotvmiaiotsnainpetnawyittfhoatrhmeweoadsppearance . ucsaprecotechsaiton bloontghie pmoeorrfooruatoeffbaottdygarciadd.atMoinnorresnuldtinpgri ahce,ob5seorvdedrouvmebne-rneudmbcearrebdon ) eacnidscoexnigdiantpone,raorminnaeerdpafthtwaayiosf,twyaacildsmeobtsearvbed ausnudalwleyroebsikearlvyehdeinpbrroadnucchts fchaaiyn feortyeaocsb. sAlonr iho 1fs6tacloutsp.roTdh,eseercormpaotunadscwaerrbeoynioc accidas nrasngiongbfarmk Cur0eCsr Pmearsfuobrlcaanctcandoastt (aPcFcOoAu),ntwaosrtaheporlayaclomapyou6n7d5%aufahnieatouealytainamleyrzeadlcaonhdo.lsb9nsoldyon resentin th est cultures Pagesorse acPerjcstGASGSe:nBnodoCraadptn,Sse Sucfor Tamer cna ain 1 - e Sevane oh3008 Seve Gp 000007 10 Proect Personnel - 1.1 Sponsor Company 12 Sponsor Representative am Or. JamesK. Lundberg - 13 Conlract Facilly Personnel: 13.1 Study Director Or. Cleston C. Lange - 132 Laboratory Management 133 Sample Preparation Mr. Bruce E. Warden Ms. AngelaL. Schuler Or. ClestoCn. Lange - 1133.54 HSPaLmCpIleMSCusAtnoadliyasnt: MDsr.. CAlnegsetloan LC.. SLcahnugleer - 136 Report Author Or. Cleston C. Lange 20 DR ataequirandeRevm isioenJunstifticatsion The sponsor representaiive desired an aerobic biodegradabilty screening study to be - conducted using the telomer alcohol mixture as test substrate. The study was initiated on Augus8t, 2000 as a non-GLP study. The report issued Friday, November 16, 2001 is revised to substitute "Dupont Zonyl BA type Telomer, or Zonyl type Telomer" with Telomer. The reason for the revision is to - more accurately reflect the impact of results to the general class of telomer alcohols. 30 Project Objective This msetrudyalwcaohsolc)onidsucbtioeddeginraodradbelre tounedleurcidaaetreobwihcetchoenrditthieonfsluuosriontgeloamermiicnrtoebrimaeldiraitceh 2 (telo inoculum of municipal wastewater treaiment sludge. This study was similar to earlier studies conducted for 3M by Pace as Pace projects CASS * and CADS7 and CA104 CA105 *, and CA132 %. The development of an analytical method and the analysis of parent analytes and possible products were critical parameters for the determination of biodegradation. Page 60f 38 `PSatcuedyPDriorjeectcotr:CACQl8eSs:to"nBCi.odeLgarnagdea.tiPoh.nD.Screen Stfour Tedlomyer Alcohols" ~Revisio1n - `PWaecdeneAnsadlayyi,caNloSverevmicbese0,r6B,i2o0-A0n2alycal Services Group. 000008 40 Tostanice Tunreacttoatntantlcemuesdeidto.r nTiwsossouutcveassofthteesZt omanteBriAa reetousmeedrfoarliosnestourdyo.chTahmeicraslt uoltdceswtamsatpreorviidaedblbeydthZeasnpyonBsAorNctoemlpoamneyr.s3cMa,n2, 9appraocxeibmiatleylynu2m5berofTNs-aAl-2i1n6e, oinnformlyati7o,n2f0o0r0.thTehset saproinls,ornodridannoetxppirroavtiidoen adnaeMSfoDrSt,he@mahteariia.of cTushtsodmya,tehreialpwrays . ore given aattes4t,C.contrMola,raintd rCeAf-erTeCnRceOZ(0T0C0R)wnausmbuesreatpPraicmearays fCoAr -mTeCtRh0o2d-0d0e9vealnodpmweanst. : sp066s of th sty, including th fst set of culbres prepared and orginal LCS `method development. Acocohmomlerucseida.l vMenedtore,rArwacshpCuhrachiacsaeldC2osmpZoannyy|,BprAoLvidfeudorhteelsoemceorndinstoeurrmeodfil(o19r89 . cAlhdarriacchtaCrastiaclsogwenruembebrlin42g,15p1o-i0n)t, 1a4l5so-2c4a5l,led FpeCrfFiuoCroHalCkyHletOhaHnol.wherTehen maetqeurailasl : pappiroaxinmatcelayt7antdo h8,eapnurdtym,oolrecpuelracreneticghotmpwoassir,apoofritede amsateMryialcav,er4e49n7f0prwio.videAdn PTancee,putrhcehamsateedrimaaltewraiasl gwiavsenrteacceanbeidtaytnPuamcbeeronCSAe-pTtNeCmOb0e-r26224,2a0n0d0.vasUpsoornedrecatop4t,at oles. A representa chemical compiaion of ie Zory BA-ype telomer alcohols . `shown in Figure 1. } BCPiaccheAPSnoeirec,aGAGGSSe:oenBs,dSaCog3Aa0n0diaPncDa.SeSeonniScP s Gaoao7rgTofoe 3oym8er Alcon Revlon 1 Ween. Novara 5, 302 : 000009 :| . TTT lo Co MMoolesccuuaarrwfeoimguna ==2C6H4,0F89,0 M+CH,CO0] =328 | p TTT T a , MMoocscumarrwfeoimgina =CHFs0 =seios || | (M+CH,CO0 = 423 : . T BE TTLTiIR or Go.C1 MoMloelcucluaarrWfeoimguhat == 4C64.F118O || ' [M+ CH,CO0] = 828 | 3 || SFA eePEEHE EEEREEL . TTTTTT TTT a On Moscuarfomua =CH,F0 || olecuiarwon =seanse | [M+ CH,C1 =06203 . Frere rere C, Moloorformia =C HELO || : TIT TTT TT aCO o (M+Cs HC0e 0 "=-s72s3ares || TIT T I TT 1 1 o o.O10 MoMlocluecaurarWfeiogmhuta ==7C6y4H.1,6F4y0 - * [M+ CH,CO01=823 - Figure 1. The telomer alcohols. Based on the HPLCMS average peak area response observed in the six abiotic control cultures, the composition of the test 3 material was 6.4% + 0.3% as Cs telomer alcohol, 39.3% + 1.5% as Cs. 27.7% 2.1% 85Cus, 17.6% 21.2%85 Cg, 7.2% +1.0%35 Cra, and 20% 0.2% 25Cus Pa8g01e38 J SPtaucceyPDriorjeeccttorC:AC0l8eSs:to"nBiCo.deLgarnagdea,tiPohn.DS.creen Study for Telomer Alcohols" Revision 1 WPeadcneeAsndaalyyt,icNalovSeenmibceers,06B,o2-0A0n2ayical Services Group. 000040 50 Reference Materials The sponsar provided reference material. The neat material was stored at -80C. NS: Expiration datenaspecified 5:1. Pefloroociancate Ammonium Sat (PFOR) Pury. 95.2% 3M: TCR99131-37; Pac#e CATORO2.001; Expires: NS Ferrer oo wg EEE ERN Figure 2. Perfluorooctanoate ammonium salt 60 ReceipUGenerationof Samples Samples were not received, but were generated as an inherent part of tis study. All of i ne experimental extraction (SPE) cules prepared for each culture set were exacted by sold methodology to generate three analytical samples per cule. phase The aenlaulaytteisca1l,2saomrpl3e.s,OafsthSePtEhrleuaetleuastecsolgleenceterdatfeodr feoracehacchuslaem,plew,ereelualtaebe1lewdsasheSP2E5 mL aqueous sample eluete and eluates 2 and 3 were 25 ml methanol eluates from the - SPE cartridge. Qualtaive and semiquantitative HPLMS analysis was conducled for eluate 2 only, and the resulting dala was evaluated o determine whether biodegradation ) occurred 70 Methods 74 Sample Preparation 7:41. Collection of Sludge. The sludge for tis study was obtained from the Twin Cites Municipal waste treatment facily in @ manner consistent with the sludge colleced . fporrepaortahteironsotfudtihees fslitstseedt oinf csuelcttuiroens w3a0s. collTehcetedsolnudJguelyu3s1,ed20f0o0r atnhde Page 9038 SPBiaavccaeasAPrrneojaceoicrtc:CaAsQS8oStn:icoBesin,odBeG.ogLarAnagneda.alPolncna.SlcSreeennceSstuGdryofuorpTeomer Akihols" Revision 1 Wednesday. Novembe06r, 3002 000011 . delivered with Pace chain of custody (COC) form 465254. The sludge for the second set of cultures was collected on September 18, 2000 and was accompanied with COC # 528791. Sludge used for the second set | of cultures in ths study was also used in preparation of cultures for other fluorochemical biodegradation studies (CATS * ang CA132 ), and was - shown to be active for biodegradation of other fluorochemical `compounds in those studies. To prepare cultures, sludge was obtained from the primary municipal waste treatment facilty in the Twin Cilies area. Amangements were : made for Pace personnel to relrieve fresh mixed liquor suspended solids (MLSS) from the aeration units at the Twin Ciles Metro Wastewater - Treatment Facily located in St. Paul, MN. Typically, Four ters of MLSS was collected by Pace laboratory personnel and delivered as four 1-iter . Nalgene polypropylene bottles containing MLSS, and were accompanied with a corresponding chain of custody with date collected. Upon receipt at Pace Science Solutions, the individual bottles were labeled #1 through | #4. The suspended solids in the bottles were allowed to sett a! least 24 hours at 4C + 3C. The settled solids "sludge" were then used to - prepare MLSS plus sludge for use in preparing test cultures. A sludge characterization analysis was not conducted as part of this screening . study. The setiied sludge in each bottle constituted approximately 20% of the | volume, or approximately 200 mL volume in a 1-ier bole, based on visual observations-and was consistent with observation of earlier sludge . collections. ) Page 100138 . P`SatcuedyPDriorjeeccttorCACBlSes:to"nBiCo.dLegarnagdea,tiPoh.nD.Screen Study for Telomer Alcohols" Revision 1 FaceArabic Sons,SoAniicl Soon Grup 000012 7.4.2. Culture Preparation. i The first setofclues were prepared August 9, 2000 and incubation of those cultures continued with intermittent culture harvests until the final " harvest on September 13, 2000, for a total incubation time of 35 days. `The second set of cultures were prepared on September 27, 2000 and incubation of those cultures continued with intermittent culture harvests until the final harvest on October 12, 2000, for a total of 16 days incubation. The culture preparation procedure described below Was : used, and was documented as Pace standard operating procedure (SOP) CAG-5P-03 Cultures were prepared using a mineral salts medium defined by EPA Guideline OPPTS 835.3200. The mineral salts medium pH was 7.4 and contained per liter, 0.334 g N&;HPO,-2H;0, 0.005 NH.CI, 02175 g KiHPO,, and 0.085 KH;PO,, 0.0275 g CaCl-anhydrous, 0.0225 = MgSO,-7H;0, and 0.00025 g FeCly-6H:O. : Two lters of a mineral salts medium containing 100 mL of setied sludge was prepared and contained 1 mL of methanol. To each test culture was `added 25 mLof this mineral medium containing sludge. A mineral salts medium, un-steriized and without sludge, was prepared. : "This mineral salts medium without sludge was sed to prepare the 25 mL. no-sludge (abiotic) control cultures. All cultures were prepared by dispensing 25 mL. of appropriate mineral salts medium solution into clear sterile 125 mL Naigene polycarbonate culture flasks containing labels with appropriate identification information Note: The mineral salts medium that contained sludge had to be swirled - regularly during dispensing in order to keep the mixiure homogenous and prevent the sludge from setting out of the solution. "The test substance, either Zonyl BA-N teiomer aiconol or culure set one (Stock ID CA0S8-5S-004 at 10,280 g/mL. in methanol) or Zonyl BAL Page 110138 . PStaucdeyPDriorjeeccttoCrACQ8lSe:st"oBCin.odeLgarnagdea.tiPoh.nD.Screen Study for Telomer Alcohols" ~Revision 1 PWeadcneeAsndalayyt.icaNlovSeermvbiecers0,6,Bio2-0A0n2alyical Services Group 000013 ) telomer alcohol for culture set two (Stock ID CAOSS-SW-001 at 7.288 g/mL in methanol), was added to the test cultures by transferring 5 ul and Bul. respectively. to the appropriate test cultures. The final test : concentration n cultures was 2.056 g/mL of Zonyl BAN telomer aicohol for sat one, and 2.332 pg/mL of Zonyl BA-L telomer alcohol for set two. Blank control cultures received 25 mL. of mineral medium solution, as did the test culture, but without addition of the test substrate. All of the day zero cultures were prepared and immediately frozen at 20C for storage until SPE preparation could be conducted. Al other cultures were placed in temperature controlled shaking incubators that were maintained at 25C + 3C. Cultures were removed from the - incubators at designated time points. Upon removal from the incubator, cultures were either immediately frozen, or immediately prepared for . `analysis by sold phase extraction. Al culture preparation information, including times, analyte additions, etc | were recorded in sample preparation worksheets and signed and dated by the preparation analyst. All original data sheets were maintained in - project specific binder labeled as Project CAOBS. . 71.3. Solid Phase Extraction of Cultures The solid phase extraction procedure described below was documented | as Pace standard operating procedure (SOP) CAG-SP-04 - All cultures and control cultures were prepared by solid-phase extraction methodology using SEP-VAC C18 6cc SPE cartridges from Waters Corporation (Part No. WAT036905). A sample abel was applied to each SPE cartridge prior to use, and each cartridge was packed with plug of quartz glass wool to deter plugging. Each SPE cartridge was washed | prior to use by drawing 5 mL of methanol and then 5 mL of aqueous 1% acetic acid solution through the cartridge. These wash solution eluates - were discarded to waste. All of the SPE eluates for this study were collected in ciear I-Chem vials with labels that identified them as eluate 1,20r3,as defined below. Page 120138 - `PSatucdeyDPirroejcetcotr:CACQlBeSs:to"nBCi.odeLgarnagdea,tiPohn.0Screen Study for Telomer Alcohols" Revision 1 aceArabclSoca,Srnicl Srvc Group 000014 Frozen cultures were thawed al ambient room temperature before extraction. Following thawing, and prior to solid phase extraction. 0.25 | mL of glacial acetic acid was added lo each of the cultures yielding a final concentration of 1% acetic acid. The content of each acidified cullure was wired lo mix, and then drawn by vacuum through the appropriately labeled SPE cartridge by carefully pouring the contents of ) the culture flask into the SPE cartridge. The aqueous eluate was collected in an 1-Chem vial labeled eluate 1, removed from the vacuum manifold, and capped. Then, 25 mL of methanol was added to the : culure flask, the flask sealed, and vigorously shaken. The cap was then removed from the flask, and the methanol content (25 mL) drawn through the SPE cartridge, collected in an 1-Chem vial labeled eluate 2. Eluate 2 was expected to contain a majority of the analyte that was in the original culture. As a precaution that some analyte may be retained in the SPE cartridge: . or in the culture flask, a second 25 mL methanol eluate was collected in a similar fashion 10 that collected for eluate 2, and was labeled eluate 3. 1 Aliquots of eluates 2 and 3 were transferred to autovials, capped, and then quantitatively analyzed by HPLC/MS. The remaining volume of each eluate was stored at 4C + 2C. The final SPE extractions of the day 7. 14 and 16 day cultures from set | two occurred on September 24, 2001 . 7.2 Instrumental Analysis (LC/MS) 7.2.1 Instrument Parameters. ' The HPLCIMS method used was a modified version of Pace method CAG- ORG:23*, as descrived below. Analysisofculure extracts from culture set one, prepared with Zonyl BA-N . telomer alcohol, was conducted using instrument "STING which included an HP1100 HPLC pump with Gilson 215 liquid handing system in line with a Micromass Quattro Il triple-quadrapole mass spectrometer detector. | Page 13038 . `PSatcudeyPDriorjeeccttorCACOlBeSs:to"nBiCo.dLegarnagdea,tiPoh.nD.Screen Study for Telomer Alcohols" Revision 1 ace Arabic Sons,SeArdcl Scns Group 000015 Ansys of culture extracts from cure st two, prepare wih Zon! BAL elmer aol, were anahzed both on instrument "STNG and Insttument "1OLCMSD3" which consisted of Waters 2690 HPLC system n : Ine wih a Micromass Quattro I tple-quadrapole mass spectrometer detector. Typical condilons for analysis of telomer aconols and : uarochemical acid products were as follows: rasasmSopenctcoamreatteans Tor LG sar.[ee SSnoorseuastBonamoaTemapAmeVpEaSae.T0a1t5e0 I5mm uoan 5us CSSoiFnevra0s5 10S5%00 H$oE0o 30 =0 FpChtiRrefso3.5n0: 1144 Flow11: 5%%mini, tasSrrooappro33%031 : CSloenepe?rr35 550 SSStaoavnem n5T:a3mmampndaoa!: oamnberacetate . SISSENSS.pCiCaotriiosnn75GE0onvrgygvoanra om 1010 40 fr un Expanimans Typical infection volumes or samples and calbraton standards were 50 JL. The 46 x 150 mm Betas C8 column used or the uaniaton of exact from cur se wo had serial number 1101567H and th 4x 35 . mlimteNrGw1ascoulsuemdnwiuhseadn haapdprosexriimaaltneuSmpbterrat1i5o1n0o4f. 21A(pWraesstseurMeS-r)egulated 722 Quallatve Parent Analyt and Products Analysis [we |a Bpewdmn mmi | I lr lahol state adda = . IeCoiootmeleormesr calocorhosl caceetmateesadddaucita|e 523 |1 . Ce lomeracahor ace ada | [Caer scores sides 75 | Table 1. Parent eames scans anaized. - Page 14 of 38 . PSPaacciee APOrornjaeacttouAaQS:esei"sBn,loidLeagnArgaen,dayPoOna.ScSreeenncSetsuGyrfooupTamer Aconls" Revision 1 Wednesday. November 06, 2002 000016 ) To conduct the analysis of parent compounds and expected biodegradation products, the HPLCIMS system was set up wih the same chromatographic configuration as that described in method CAG-ORG- i 23. The method utiized two columns in tandem with flow 10 a pressure reef valve that serves as a flow through spitter (0 the mass spectrometer Z-spray source. ) Speciic MS condiions for mass analysis of the telomer alcohols and acid products were developed during ths study. The mass spectrometer, Micromass iple-quadrapole mass spectrometer with Z-spray ion source, : was operated wih electrospray ionization n either selected ion-recording (SIR) mode or with mass-range (150-1000 m/z) scanning on MST . Discrete chromatographic peaks with singly charged negative ions, (M + Acetate)" for parent telomer alcohols and (M - H')" for expected acid biodegradation products, were observed and monitored: - | ExpectedProduct | Expesctrtuecdtaurneion | Expectedanions(mz) | Perfvorobubrate (C) 273.169 Peon CI THEFNC00 | 473.369 1 ) CFYCFHG00 wee 513.469 . ZH. 2H-perfuorodecancate (Co) wT 2H. ZHperiorododecancate (Ca) 577 Table 2. Expected products, and expected anions for each IEE Sewee 000047 ) Page 15 of 38 . SPtaucdeyPOrroejceicotrCACSlSe:st"oBCin.odLegarnagdea,tiPonn0S.creenStudyfor Telomer Alcohols" Revision 1 723 Quantitative Analysis. : Quantitative analysis was conducted only for perfluorooctanoate (PFOA) and only on extracts of cultre sel two. Quantitative analysis was . performed by the extemal standard method using SPE. extracted calibration standards (eluate 2 only) and quadratic calibration curves. i The HPLOIMS analysis of culture set one extracts was used primarily for method development purposes. : Typical injection volumes for samples and calibration standards were 50 JL. The 46 x 150 mm Betasil C8 column used for the quantiaton of - extracts from culture set two had serial number 1101567H and the 4 x 35 mm NG! column used had serial number 15104. The pressure: ) regulated spitter had no identiying number to distinguish it. The part number for ordering the pressure relief valve (spitter) was Altech catalog part number 39025. 73 Data Transformations and Calculations : 731 MolarCalculations: . Because al data was collected on an ngimL basis (part per billon. ppb), transformation from ng/mL to molar concentrations had to be ) conducted to obtain mass balance information when applicable. The mole conversion values for each analyte are as follows: - PFOA molecular weight, as ammonium salt is 431.10 Zonyi BAL telomer alcohol average molecular weight is 434.70. 732 Conversion of nglmL to micromolar (M) and nanomolar (1M). (Working Examples): - 2232 ngimL. telomer alcohol = (2:232 ngimL)"(1nmole / 434.7 ng) = 5.135 nmoleimL = 5.135 umolefiter = 5.135 uM--assuming 100% purity. 160 ngimi. PFOA (NH4+ salt) = (150 ngimL)* (1 amole 431.1 ng) ) = 0.3479 Amole/mL = 0.3479 umolelL = 0.3479 uM = 347.9 TM Page 16 0/38 . SPtaucdeyPOrorjeecctorCACOlBeSs:to"nBiCo.dLegarnagdea.tiPohnO.ScreenSiudyfor Telomer Alcanols" Revision 1 PWaecdeneAsndsaiyyi.caNloSverevimcoesa,06rB,o0-A0n2lyical Seni Group 000018 _ 7.33 Molar Mass Balance Calculations (Theoretical Yield): Gonvert all ngimi. values to their corresponding molar concentrations as 4M or nM (see section 7.32, above). Divide the sum of the analyte - concentrations by the Known concentration of staring compound and represent the final result as a percentage of the known starting - concentration _ (Working Example): 1, the starting concentration of BALL telomer alcohol was at 5.135 kM And, ate incubation, the following was determined If, PFOA was detected at 0.3479:M Then, the mass balance s as follows: - Mass balance = (03479 uM) 5.135 uM) X 100% Mass balance = 0.0678]X 100% = 6.78% Or, 6.78% of the telomer BAL alcohol was oxidized to form PFOA. - 74 Software Versions MicrosoftTM Excel 2000 was used for data processing and producing tables. MicrosoftTM Word 2000 was used for processing the analytical report text. `Adobe Acrobat 4.0 was used fo generation ofth final electonic report, Massiynx version 3.2 was used for data coliection and peak integration. ACD Chemsketch version 4.0 was used for chemical drawings. . Page 17.0f38 . PStaucdeyPOrroejcetcotr.CAlQ8eSs:anBioG.deLgarnagdea,tiPohno.Screen Sty forTelomer Alcohols Revision 1 WPeadcneoAsnsaalyyi.caNlovSeenmibceers,06B,o3-0A0n2ahical Sevices Goup 000013 80 Resuts 8.1 Sludge Characterization . A chemical analysis of the mixed liquor suspended soids used fo the samples prepared on August 9, 2000 was conducted, and characterization information - can be found in the final report for project CAOS8'. The sludge for samples prepared on September 27, 2000 was not characterized, but was obtained from the Tuin Cies Municipal wast eatment facilty in @ manner consistent wih the sludge collected and used or project CAOSE - 5.2 Quality ControliSample Matrix Spike Results. ; The determination of the anal recoveries from sample malrx spikes was not included 23 part of his screening study. Recoveries are any reflected n the aby to achieve molar mass balance based on expected parent and product yids from samples and by the use of a sludge-extacted curve for semi auantiative analysis of PFOA 5.3 Analytical Blanks Methanol sovent blanks were injected onto the HPLCMS column and quantiatvely anaized to determine the instrument background analte concantaton and cary ver uring the analysis : The no-sludge (abiol) controls containing mineral salts medium wih test analyte, and cure blank, containing mineral sats medium with test analyte 3 and no sludge, were prepared and analyzed. Abiotic controls and blank cultures were prepared and incubated in an identical manner to biodgradation test Gules. The results were used to determine whether the sample marx contained any of the anaes of interest and whether biodegradation coud be atiibuted to the bioactuiy of the sludge. Fluorochemical analytes were not - Getecte in blanks and biodegradation id not occur in abiotic cons: . Page 18 of 38. 3 PSPaaicceeaAPDrnoaejbecictcta.ClASGDeBenSv:seet`sB,Gi.oBdLoea-grAgrena,adyaPioncna.SlcSreeernveSsuGcrofourpTeomes Aono" Revision 1 000020 Wednesday Novemoe65r. 202 8.4 HPLCIMS Analysis Results - `Semiquantitative analysis was conducted only for the SPE eluate 2 extracts from the second set of cultures, prepared September 27, 2000, and were performed - on September of 2001 as sequence CAOBS_092401b.5pl (with full scan MS data), and CAB5_092701a.5pl (SIR data) on instrument 10LCMSO3 in the Bio- _ Analytical services group at Pace. The quallaive HPLCIMS analyses and HPLCIMS method development for this study were conducted in September and October of 2000 as analytical sequence runs S091500.spl, S091800.5pl, - $092000.5pl, S092100.5pl, S092600.5p, $1000200.5I, and S100400.5pl on Pace instrument `STING" 8.4.2 Qualitative HPLCIMS and HPLC/MS/MS Results "The first set of test cultures containing sludge and BA-N telomer alcohol substrate provided evidence that biodegradation occurred. The HPLCIMS - data showed near total [0ss of all of the telomer alcohol peaks in the HPLC/MS chromatogram, with formation of chromatographic peaks that - had mass spectra consistent with perfluorinated acids. However, at that time, the analytical test method was not complete. Concerns about the. 3 integorfitthey test material resulted in 2 second set of test cultures being prepared using commercially purchased Zonyl BAL telomer intermediate. - Although each sample, control, andblankfor the second set of cultures was prepared and incubated in duplicate, analysis was conducted for SPE - eluate 2 extracts from one sample, control and blank per time point. The data showed that the sample test cultures underwent rapid oss of the Cs. Cs. Cio,and Cyzelomer alcohols, and moderate loss of the Ctelomer alcohol over the 16 ays of incubation, as determined by decreasing peak area response for each telomer alcool ion at the sequential sampiing time. points (Figures 3 and 4). TheCytelomer alcohol was degraded very . Slowly and showed lite oss. . Page 19.038 . PStaucdeyPDriorjeeccttoCrACBlSes:to"nBiCo.deLgarnagdea,tiPho.nDS.creen Study for Telomer Alcohols" Revision 1 WPeadcneeAsndaalyy,icNalovSeermvbiceers0,6,Bio2-0A0n2alylical Services Group 000021 : per ------ wo ho boga ERES {| : ooo} C S SA Ae E R ACH wcoorrmiees 15000 Vin Si a A CT1 Apeak2 area ) BAIN A ee ) soo [| N NG a C a sai En Ea Ne s wo PA ) (FTiAg)u.rNee'.arHcPomLpCoItSe idneggrraadtaetdiopneoafk rrceworaaths bmsoesrteadb.undDaanttaptlloimeedrwcihohles ostmantouf hae ilta. sono NC[NPRESSIE Wl eReARESe ) B f1, aA HOONL GCEERIREREESRGES) COTApeakarea - PSanr] lI ioN iiG dnae<ogia ed cCoTi apoeae kana 3 Bo | hn RNS IS Md I SHES ae - o o : ww Incubation me (days) | . L Figura & FPLGIS ogra peak teas oe r 7los abundant tomar scons c(7eAg).racNaetaorncoomfptlheetC.d.eTgrAadwaatsioonbwsearsveodbsaenrvietdefodretghreadCaTtiAon.oMofhdeerCatTeA was osserved. Data plod wi he best manga of ne ita ' ) Pa20g ofe 38. SPaiceePDreojaeccotrC,AlQBeS:saBinCo.dLeagnrgaedPaonOScreenStuyfox Teamer Aonols" Revision 1 Sa Anyi Sontees, Bo Apical ences Gevp Wednesday. November 06, 2002 000022 ) Concomitant with the loss in telomer aicohols was the formation of compounds with HPLCIMS ions and retention times consistent with perflucrinated acids (PFAs), including perfluorooctancate (PFOA, Cy-PFA). : which was confirmed as an end product. The transformation of telomer alcohols to PFAS was rapid, with a large increase in al of the PFA peak area responses at day 1, and continuing increase through day-16 (Figure 5). No degradation to form PFAs was observed i the abiotic no-sludge controls. : Although PFOA was accurately quantified, many other perfiuorinated acids were qualtatively observed and their relative concentrations determined : based on the observed increasing peak area respons. The observed perfluorinated fatty acid end products were: perfluoropentanoic acd (Cs ' PFA), periuorohexanoic acid (C-PFA), perfluoroheptancic acid (Cr-PFA). perfluorooctanoic acid (C-PFA, PFOR), perfluorononancic acid (Cy-PFA), perfluorodecanoic acid (Cy-PFA), periuoroundecancic acid (Cy-PFA) and perfluorododecanoic acid (Cez-PFA). - Transiently formed intermediate compounds were also observed at early time points following the nial exposure of the sludge to the telomer alcohol substrate (Figure 6). The transient compounds were suspected to be: 2H, 2H-perfuorooctanoate; 2H, 2H-perfiuorodecanoate; 2H, 2H ) Perfluorododecanoate and the possible f-oxidation pathway intermediates: 2H-perfluoro-2-octenoate, 2H-perfluoro-2-decenoate, and 2H-perfiuoro-2- Godecenoate. Table 3 shows the MS ions and retention times observed for - the polyiuorinated and perfiuorinated acid products and the telomer alcohol parent substrates, . HPLG/MSIMS Data (Table 4) support the identification of the suspected metaboltes. - | Page 21 of 38 - PSatucdeyPDriorjeecctotrC. ACBeSs:toBnioC.deLgarnagdea,tiPoh.n0S.creen Study for Telomer Alcohols" Revision | FaceAndSeuca,Sohn Seis Gop 000023 250000 | : C,PFA STHiR] 3& S shi Ois s 0%.er------ Sd F 3 oLlliA n Bt LE ooo [E/E % RER R STa RSed OR } pe 2 + 5 5 -- Ww 7 ou wow Incubiamtaiaoe)n Foibgsuerreve5d nIemsetgauleldupseaekxaactrse.esEpvaennineusfmobetdo pcearrfbuonarcihaataidlfenagtthacciadsr(bPFoASa)cids : worethomost abundant carl acd peaks and wee: parforanexanoate (CePA). perorooctanoae (CrPFA) and pertuorodecante (Cs PFA). EH ---- ANGE on| oof ONES psGal ILININa SE scoS ne tEe)R 5wo| 6 SSE>EB<S KN ai NE c ar ra o r ae we | : i wo| f[m8Ng ENE: eaohni ) x0 W[eEEaS o:2 N C NN Ge s cs ntaTtim o0an Figur6. Taansiantpobluonated ay ai intermediates observed. The 24: 3 p{aepiturpoan nlalfecxptoysuarce0idseoameesrusapleccothlesd,oaxniddatthironweinrteoamedniasteos rthamttooterhednied cotesponding perfuorinated carboxy acids end paducts Pa22gofe 38. PSiavcaesPDraocjetcotr,CAlOBsS:to`BCni.odLaagnrgea.daPohnO.Screen Study for Teomer Akohols" Revision 1 SWaadcnamsdcaya.lNoevnevimebse6.6r,8530A0n2abica Sonices Coup 000024 Kaos Goad CRF Raion Tis| GsasonW eotw | Ce---- Crs CECH E00" Tw = [Crsmersr Crotelomer alcohol" [CCFR3O(CEFI2)OCRHc2GCHR;O0cHT.O.CHcosCOo0'JO[ |WsLs seJr| |NL5o23 [ CewmrserC o" o [orepoo Raon Ghk oo0 | e om |_|||| e sez N oG reeciot R0 c | |o T7e%| |WO e3m || ([ FerrerP s i [[i CeRo oeao0e0 m[|eNe wwLe ||WmvLeem | [PPotaerocoaranne [rm| oeeee e To ||wwmeaw| PPoees [J Gee Tm | we Perfiuorododeclayn as oate GTSS[paendhineg mTowecvaocsha TG RES|Ware T Ge BS| 613,568 Feeatbtieese3utPreasrcnoetnratinadmirnccuoSncictogamsritiaaonnndt iabhmacsrreedcassehnsblyisnHupPbroLtdautc.sagTahlg,yaMeanontfyaSoPnfEoesnsttigenmalZesadcaectorsenafsoed : Samaldecreased bolow deco sn stcurs - ace Project CASS: Blodigradaion Sree SPtaf2ou3gTofdoe 3m8ye.r cohol" -Fosin - S oSaressn.RaSveamE noan,0S6.r30a0n8 aca Srdcss Group 000025 Fradict yen Observed | ChomieaT Formals Retention| LGSIS | LISS daughior I Faree [Gero |To A Tan | Fiepouaro Toe SEsE=mRA TinaebHlPeL,CHIPVLSCIIAMSSaInMSaldaytoasffihoesr uSsPpEaclieadto2xiodattiaonrpormodtucetsd.ayPaorecnetgaanddadtaiuognhtsearmaprlieon(sCAfGoSmS- . d09e2g7r-aSdAa-t03i3onE2s)a.mpTiheespnalrye,ntwahniotnhsewemorsetobnseenrsveednaasltsranfsoirenttelyofnosrmoebdsearnvieodnsatindeaayrloyes.ampTlheepoint ) mMaSssSosdsatfarocomlilnecetpeadrfeontsanaihon,pra oductu wasocoonsssiofstCOewsinlth(tmhsre4c4)itnedd HchFem(imcaaslsf2o0)r,maw.eeCosbmsmeovned. : 8.4.2 Quantitative Analysis of PFOA ) The PLCS. multi-component caloraton standards used in tis study for FprOoSdAu,ct Nqu-aMnGiFiaOtSiAon. cMonStGaSi,neMdStThOe,taarngedt NanMaeeFsO:SEPFOaAlc,ohoPlF.OSuFlifvneatcea,ibPrFaOiSo,n astnaanldyairsdstow2erceultpurreepamreeddiubmy waidtdhitisloundgoef, aandknothwenn aexmtoaucnttonofbypersfolludorpinhaatseed - extraction in manner identical to the treatment of samples. The PFOA was the ony target analyte of that misture that was used for tis study. The exact concentrations of he standards were used in calibration curves for cuaniiatve analysis - . Page 24 of 38 PSahceyPDrorjaeccto.CAsQBSo:nBioC.deLgarnagdea.ti0on.Screen StforTuoomyer Alcona" Revision 1 Seaan Servoss Grup 000026 _ Al calibration standards and sample eluates were stored at 4C in refigeratIoDr 0213 unt analysis. Aliquots were transferred to autovials and cappedfo use in - HPLCIMS runs. The Instrument caibrations were performed for PFOA by use of a five point calibration wih quadratic fit of the data. The LLOQ was 11.0 ngimL _ PFOA, and the coefficient of determination (1) for PFOA was 0.965. Quantialve sequence runs contained calibration standards at the beginning and end of the wn - The biodegradation of the telomer alcohols resulted in an increase in the measured levels of PFOA dung the 16-day sudy (Figure 7) - LC ---- _-- 100 [e68 i % .op - 7 140.0 00 {ESE [ES ed = es 55 - oowo [[LDEiORT eb Sia e200o|[islkesli SHtAeA - wo {fa hd i . mo fordi Re Hk no AESA a 2 4 5 8 ow uw ow ow Incubation time (days) fFuiogruorteel7.omeTrheaicmoehaas.ureTdhepePrFflOuAorowoacstannootateme(aPsFuOrAab)lein icnulctuurlesescontthaatindiindg nsloudcgoentaanidn . sludge. Data plotted with the best manual fit of the data and cancenirations shown were not aust fo reerence materi purty of 85.2%. : Page 25of 38. : SPtaucdeyPDreojceocrt:CACQlBSe:si"oBGin.odLeognrgaed,atPionnScreen Study or Telomer Alcohals" Revision PWeodcnoeAsndaalyyi,caNloveenmcbeesr.06B,i2o-0A0n2ayical Services Group 000027 - | HHH C, Toomer Aah - [J-- v [~~ - " T 1 H PETT= _ " TIT Sndana a SNe {a-oxidation) p-Oxidation . wat IT "HH - | ! wa HL iI S LLnL m2 TT Pertuorononanaste Joann co, 2 wes HHL Peducrsociancate elon - Acette Figur . Proposed iodegradaan pathy for eames acon, shown for Cselomer acon 3 . n`eaxtaimpslne.o sThhoew-no,xidactlieaornlypagtihwcaythberarnacchuc(Htisb)oinbvolsvesebpryriHmPavrLyCeIfoMrSdm.atiTohnoeffaayfatatcyya-cCyolA-ChoiAethbiooensdteirs i readily ydrlyzed oye th corresponding attacidsobserved } Page 26 of 38 PSPaatccaee DAPrnroacjyeiicoct:aClAsDSBeStrv:iocBeinsGo..dBeLgoor-naAgdneaa,tyPiiorcna,SlcSraeneincSes GtrfoourpTueamyer Alcohols" Revision 1 Wednesday. November 66.2008 000028 - 9.0 Conclusions - Tahriesvsratnsutohramsedcetomopnesrutoartidattehda facuiodroecnhdempircoadluctos.meArltkhcouoghhlstheartaeblioomderegariadcaobhlaes aanrde _ supplied only peruorinated 35 acids oven numbered carbon chain consist of boih even number and compounds, odd number the end product carbon chains, win even numbered carbon chan lengh acids pradominalng as the major end products - fTohirsm otbhseerpvraitmiaorny csaurgbgaersytisctahatc, f(oellloowimneg ctaherbionnitaiatle)o,xidtawtoioonxiodfattihoen hmyedcrhoxaynli-scamrsboenxistto 3 a5 shown in Figure , I. Th frst, less utize routs (Figure 8, la), invoves oxidation of the carbon concomitant wih decarboryaion (oxidative decarbosyation) to form an ad chain lengih botansformaton. perforated carbonic add, which The second observed fot of oxidation does nol (Figure 8, undergo further lh) is the mare common routeo fatty acid oxidation, 3s evidenced by the formation of detectable - turmanbseierntcaB-tobxoindacthioanipoelnyfgihucorairnabtoendifcattayciadcsid intermediates and the more abundant even 3 Amilchroobuigahl dtexgircatdyatoifonthoebseelrmveedrhaerecolverwtahs enont tadioefstsheed16a-sdapyarsttuodfyhsiusggseust,s hhte ) microorganisms concentration of preset in the he test materia sudge were not signfcanty nbd by the tested } : PSache PDroeutrAOSR.aSnadeLgaodea ion Sree Page Stuy 27 for of 38. Toomer Aono Revision Weanessa.Ravana3 3003 11 Sorvees Go 000029 - 80 Literature Cited - 1.Final Reports for Pace Project CAOS8 (3M # E00-2252), 2-Week N-EIFOSE Alcohol Biodegradation Screen Study Report" and "Aerobic Biodegradation of _ N-EIFOSE alcohol Study Report". Author: Cleston C. Lange, Ph.0. 2.Final report for Pace Project CAOS7 (3M # EO1-0415), "The 18-Day Aerobic - Biodegradation Study of Perfluorooctanesulfonyi-based Chemistries." Author: Cleston C. Lange, Ph.D. 3.Final report for Pace Project CA104 (3M # E01-0444), "The 35-Day Aerobic, _ Biodegradation Study of Perfluorooctanesulfonate (PFOS)." Author: Cleston C. Lange. Ph.D. - 4.Final Report for Pace Project CA105 (3M # E01.0683), * Fluorochemical Adipate (L15488) Biodegradation Screen Study." Final report issued July 10, 2001 = Study Director: Cleston C. Lange, Ph.D. } 5.Final Report for Pace Project CAT32 (3M # E01.0682), "The Aerobic: Biodegradation Study of the Fiuorochemical FCB07-Diester". Final report issued May 29, 2001. Study Director: Cleston C. Lange, Ph.D. 6.Pace method CAG-SP-03. "Culture Preparation for Assessment of Asrobic . Biodegradabilty of Fluorochemicals Using Municipalo Industral Siudge as Microbial Inoculum." | 7.Pace method CAG-SP-04. "C18 Salid Phase Extraction Procedure for Fiuorochemicals Recovery from Aqueous/Sludge Matrices." 8.Pace method CAG-ORG-23 "Quantitative AnalysisofFluorochemicais by High . Performance Liquid Chromatography with Mass Spectrometric Detection." Page 28 of 38 PStaucdeyPDriorjeecctotr:CACDlSeSs:to"nBiC.odeLgarnagdea.tiPohn.OScreen Stuy for Telomer Alcohols ~Revision 1 WPeadcneeAsndalayyt,icaNlovSeermvbiecers,06,Bio2-0A0n2alyical Services Group 000030 _ 9.0 Sample and Data Retention Ata minimum, one copy of all pertinent raw data and one copy of the signed final report - will be retained in the Pace Analytical-Tier 2 dala archives for a minimum period of 2 years after completion of the project. The remaining sample extracts wil be retained at - the Pace Analytical faci for a period of 2 years after completion of the project at 4C in the Carroll walk-in cooler (Pace ID 0140) located in the Pace Analytical-Tier 2 faciy. The following wil be provided to the sponsor (3M): - + The original signed analytical report and one copy of the signed original. The final scanned report (read only) and pertinent electronic data on a CD. - * Al original Data, comespondence, chromatograms, sample & standards prep sheets, etc. _ * Upon request before 2 years, the stored samples may be sent to the sponsor. All electronic copies of the instrumental raw data will be archived onto CD disks and one. - `copy provided to 3M and one copy retained at Pace. - Facilty data wil be retained for a period of 10 years. Facilty data is available for inspection and includes the following records: Training records. * Controlled storage temperature logs . + Standard preparation logs. + Calibration and maintenance logs - + Chemical and solvent traceabilly logs + Standard Operating Procedures . * Methods pertaining to the conduct of his project : Page 29 0f 38 . SPtaucdeyDPirroejcecttorCCAlSeSs:to"nBCi.odeLagnrgaed,atiPho.nD.Screen Study for Telomer Alcohols" --Revision 1 PWaecdeneAsndalayyt,icaNlovSeermvbiecers0,6B.io2-0A0n2alyical Services Group 000031 } roAmpesnooAns JA - ContractAnalytical Project Number: CA085 Mourad Rehi : FO cava DD _NM7-02. "hi fr } Page 30 0f 38. BS wvosons Wednesday, Novem06b,2e00r2 000032 i Appendix B Final Quantitative Results for PFOA in samples, controls and blanks. Table 1. The measured PFOA concentrations in test cultures during the 16-day screening study i for telomer alcohol aerobic biodegradabiily. ET ------ ---- _-- -- E-- r ---- Ee : Table 2. The measured PFOA concentrations in the abiotic control cultures during the 16-day screening study for telomer alcohol aerobic biodegradabilty. p p C em p e resa e e -- sa ) s -- s -- | p EEmES e EsNT s ------ : Talacbolheol3.aerTohbeicfbiinaoldedgartaadabrieslullyt.s PfoFrOsAluwdages bnloatndkestedcutreidnginthaeny16of-dtahye sslcurdegeenibnlganskts.udy for telomer i C T Er r p -- -- e -- -- -- ) -- -- p prE mmE as m aa anS ----e -- . S EEe E ---- -- } Page 310f38 i PStaucdeyPDriorjeeccttorC:ACBlSos:to"nBiCo.deLagnrgaed,atPiho.nD.Screen Study for Telomer Alcohols" Revision 1 Pace Analyical Services, Bio-Analyical Services Group Wednesday, November 06, 2002 000033 : Appendix C Integrated Chromatogram Peak Area Data for telomer alcohols Taacqbuliere1d. fIrnotmegsraetqeudeLncCe/MrSunpCeAaOkBaSr_e0a92r7a0w1daa.lsaplforasthSeIpRardaelnat. telomer alcohols substrates. Data C Emm= tre To--mamsw-- T ET-- a eT Ta Jcmee TemTaeLIToonS--Toaen Toonw a TeTaTee Tee Te e ae] pT uss agS T | Te BE Te TaTTTo TeTeTa B BET E Ee E TTee e Toe T To oT Tae e TT Te eaeneT T Te a eT TToe eT Toe| EEsNSssLsLlL essa B I r e e reeTeTe e e -- ee e --T [-- e = EEE e ee re P e ee e Tee Te ee Te e eTe Tee]] E TT eTeTeTeTeTw] pTaebalkea2r.eaPaetrciemnetazgeerooffoirnttehgeriatreedsppeecatikvearceualtruermeatiynpiesn.g aNteiafrerceonmtpliemteedpeogirntasdawtiilohnreofspseecvtertalthe f{oerlaolmleerxaciecpotnoClyss (TTAA,)wwhaischowbsaesrv79e.d4i%n.test cultures only. Near 90% remained in conlrol cutures i DDiEy O1bbiooedgergaraddsaitoinonssaammpplleeCCAAGOGEES00802777S5AAG0S3T3..E2Z||01T|o0000m0%%(|010G40.%0%|m|04c050u%||ac0o0ao0Aw%[[l1sc0p0.eA0w%[tlCoouroria|] i DDaayy sTocdseperpadraatodnasatmopsnamp. CCAAUUBBS--GGaa2277ASAs03s7_22|152||0003%% |[3122%% oowwe|| dtoooomn||wsoorwm|[oSrhoomw|| DDaayy T1bdoecpergardaadtatoonnsasmamepl.e,CCARGOSSS-SG08e2277-SSAA-003491..E22 17141|0000%% ||2297%%[[oomme||sooww[|asesamn[[eoaren|| DarecaacaionsageCABSGETSADRETeLoo TamLonLor zoe esr) [DBBoaiyiy02o0no0 ssoluugdegeecccooonnnvvveolrl, CCCAAAOOSBBSS00059322777-S5S2AA.0005%8560.62221 |01|1o0060%T|11000r.s0w%[|s10o0z0% |[1s00z.0%||913a000%%L[iscoooww|| 711025%[0a%[om1c 10100[or3% |son| i [Dax 5isiodgeconvoCOREOsg7-SAg80.2 15 |Baw| boi [wos | sam ses o34%| [oDasy 146noosSousee ccoonnvvo,CCAAGDBS-S005G727T-S5AxG0e6:222 ||1764[|o6r54n%[ebooonn[[evosns|| Tsuoswh[|osososs.[]soiosr|] Page 32 of 38 SPtaucoeyDPirroejcetcotr.CACOlBsSt:o"nBCi.odLeagnrgaed.atiPohnO.Screen Siudy for Telomes AlconolsTM Revision 1 Pace Analyical Services. Bio-Anaytical Services Group Wednesday. November06, 2002 000034 Appendix 0 Intagrated Poak Area Data for Perflucrinated Acid End Products Tsaebqlueen1.ceIrnutnegCrAat0eBdS_L0C9I2M7S01pae.a5kplaraesaSrIaRwddaattaa. for the end products formed. Data acquired from rr aa Em EmEmeE nntee ssee ste =FF ePe ePoP reea ebae Te pees Pe PeToPa aTe a et tn en Bpae ssea=ammane feemmanasls SePa arbab d BS e e a pB BrP aamme smeomm mmeansa ye ttteeemmemmmnmmnssttteSSe P PPo w oe F b Po s Rb P Pueob Po eo p ob mo BPrraamscmemermse tfoossssmeesteab PoeF bs sp PaoH peEpa ro e } Page 33 of 38. PStaucdeyPOrrojeeccitoCrACQlBeSs:toBnioC.deLgarnagdea,tiPohnO.ScreenStudyfor Telomer Alcohols" Revision 1 PWaadcneoAsndaalyyt,icaNlovSeermvbiecers0,5B,i2o0-0An2ayical Services Group 000035 ] Intagratd Pek Ave ta for TraAnpspaetndfioErxmed Poyiorinated Acid Products ) CTablR1e. InTtegratoedo en tn 0 LC/MSpeakarea datafortransient 30 products. Datafrom sequence. | hme rr = se 5 RE : it we ae) a la ESE as ssFrE erE EE EEE EET E mEEEREsEEeEf E E eH e SommsmemmARs Pemsme EPS ooo Siac Lane aS E g ee mEmE e eaeE E TEr e EEE eRRSE TR e PTEeE ee e E e eT e rE ) SaS cee i rinSAScme ag:rBaas tsirnaSSsneeSP siGta3bv4egoTfoe 3l8a.r An -Rovson Wednesday. November 06, 2002 000036 AppendiFx PoTlyyphiucoarlinTaottaald lFoanttCyhArcSoiimdoadPtorogorgdarudacamttssioffnorroCmuSDlIatRuyDr-ae0tStaPoSEhDaoelywu-ia1nt6gof2ToerlHoPmLeCrIAMlcSoAhnoallsyasinsdof miSzIR37o7f, 14777anainodns$:7m7/fzor26p3o,ty3f1l3u,or3i6n3ta,ta4el1do3ma,ac4ir6d3sa,:lc5ao1nn3da,lm5s6i3z,3a2n3,d462133,f5or23,pe6r2f3l,uo7r2i3n,aatnedd 8ac2i3dfso;r ParfuoFratiyAncaidtPeaakds owas Ld Day-147 " HA . OWT pei = Days rile SE Sh - epeeDRE titre: JUS ad ere es pr o2 2 PR rs ow VR ven we A smn : hid - a on [ HEnAeRSJ Img eae TelomerAlcohol Peaks Page35of38. P5Paa1ccee,PABrnroajeeycciotenCaACGSBeSo:nio"sBni,odLBeoag-rgaAdenaatyi0oinc.aScreSeannicSetsuGyrfoourpTelomer Alcohals" Revision Wednesday. Novem0,b2e00r2 000037 ) Appendix SIR dita Showing lons foBrioTceelaormaadraAtlocnohCoultsoartsDSaHy-E0haantdeDay-16 rom Analyse of the mtu nn ri - m. iiJ re rere.Be SS : PS -- Ba Sm RANa ee em ah -- ) Fac Se Pa rjct CAeGSEoSs:nBsrn.deStyaedeoptioonnsa,ScSreoenniScPuassgGeyr3fu6orpoTfo3l8a.r Ach -Ro visn 1 vem 0.5508 000038 SIR data Showing lon firePrBfliuoroindateeAdpFgpateCrtnuyldaAticuHixdrdesSaaPttEDaatiuya:otean2nd Day-16 rom Analysis of GA eee i rrr greener -- a -- me GE oe aa EE 3 eeme re en mses Bn Y ireeommereemrreremmemmeeee Seem GG 3 ---- saree msgs: - ! TR... J-- 3 aww A ee Page 3701 sCBiciemPriarct,cGAdSGeSov.sa"onpn,sSLroaanAgd0mtPnnr.ScSaonnicSuscGrfoorTamer Aechol Rowso1n Wetman.Moves 5, 302 = 000039 i Appendix | CfohrrPoomlaytfoligorraimnasteodf EAxctirdacItnetderimoendsiamt/ezs 3F7o7r,m4e77d,Tarnadns5i7e7natnildnyMBaisosdeSgpreacdtartaiofnorCEualtcuhrePseaakt : 5ay.0, Day-1, Day-2 and Day-5. Suspected p-Oxidation olefinic acid Intermediates. SR / = Bee Te Fon wz | y/ . byt oMr4 i Dayo i | Page 38 0f 38 - PSPaatccueeyAPOrnroajlseycciotcraClAGDeBlnSs:cte"osBin,Co.diLeoga-rnAagndeaa,tyiPlohcnOa.SlcSreeennicSetsudGyrofourpTeomer Alcohols Revision 1 Wednesday. November 06, 2002 000040