Document 3GwZZn6Oq6DbLOZp1e2qnDJx

INTERIM REPORT # 17 - Analysis of Sediment and Water Samples SAmPenaralflllyuMsoiasromoomfcPtaaelnrLeflsiuvuoelfrrooanbnaudtteaSn(mePsFaulOllfSMoM)naaSiomntTnemWiUt(oPaDarlFtiYenSBrge,STrPS)uI,ormToPilLge,UrrESafslmeiundogirmoLehCne/txM,aFnSies/Mshu,SlCfoflnaomarttesh,e(VP3FeMgHeStDa)t,eicoaanntd,ur EPA TSCA GooDd ALaTbAorRatEorQyUPIraRcEtiMceESNtaTndSards 40 CFR 792 JaWWiSPseTih1emsoUts4nht0CDoea0nh:YKeW6SseD1toese0lIasru-Rtr,7otiiP0EnoP1ACn.W-Es3T,1.7a,9OI6ynD31Rc8E.0E INTERIM REPOJuRneT2C9,O2M00P7LETION DATE PERFSOtP3ahR0tEeo5Mxn8CyeIoR:gNlee8lGens1ge4ReaL-,re2AcPs7heBA2aD-Or1c1rR0hi6v38Ae90T1ORY 3MSBTuUi3lMDdiYnCgSo0Pm2Op3a6Nn-S0yO1-RB-10 PShto. nPea:u6l,5M1-7N335-56134744 ExyPgroetnocSotulPdNyRuNOmuJbmEerCb:eTPr:0P0001010311131 Total Pages: 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT UPECSSeetxosardiymnfnigldmgpueaaonernrnLdoSytsC,h,tu4e/iFMd0xsiyasbCSnhNe/Fe,MiusnRCumgSll7fabp9omeef2norrsafrbP,toye0rVtm0hE(eP0eexg1Fdye1Hg3ti3aeMnS1tni),co,RoenDanme,nsteipdSectlalmaiePratdcuaenhlrr"cl.fAelMMunwoaaorilmotnyhoismtcioEstarPailonnAfeLgPsiTuvePSlreffrCrolounAgaoanrrtaGdoembo(S,uoP"mtdFanaOcLleolSasnb)uMdoliufranoacmnttWoeamdrtyaeatleP(fPorrS,raFecSBrt3ouiScMim)le,, PErxiyngciepnaRl Iensveeasrctihgator JWSatiuesdsitmyoDnhaiSrKeocleutsotairorinPs,.EIn.,c.DEE S3MponCsoomr Rpaenpyresentative Exygen Research Page 2 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 QUALITYASSURANCE STATEMENT EVaEe(3aPlncMxxleFctyygioaHtggelDrpeeetSdpadnnei)tln,c,iiPoRcgaaranteiu"tnb,nosArdlecSenEaiMmPparxGcaeloayyhlrolngfsl'IoilisentusMdnovoQrearLRiuosnomataeogfiblsmgcioetatPayaarPtralonreoctAreLroghfssarirl'uyvsnuasluemdofSrProraM"tornanaa.banncacutdednteAtiaaacUSrgl(nedlPmeneFmsSriOateuOtlevapllrfnSneieoMetvd)rnwaaiaaaetinreinmtwdnddesgmeW.tpdoPa(halPtrEAathoeFSxselcrBleey,eSsrgSdfu1Steiu)umnno,wrddeiyleiSsU,nPr,DtegseuStisihrdnerifeynedlgwucsiSNmotpeLotrreuCeureoc.mndh/tMtreye,bedxepPSFaror/finMoorsPethtre0osS,du0ccloC0offtol1nool,ar1dnma3tutanhh1tcsdeeet,, 23. RRIneatvweireiDmwPahtRaaseaepnodrt InsDpaetceted 02/23-24/06 DaItnePvRreinestpciiogparattelodr to DaMteaERnxaeygpgeoemrnteendtto DSattuedRveDpoirretcetdorto 03/09/06 03/10/06 04/03/06 52. FRienvaileIwnterim Report 06/07/07 06/12/07 06/15/07 06/18/07 LQyunaalintyn APosrstuerrance Group Leader, Quality Assurance Unit DateL l t f / o i ra'eNnvaoilteyewt:icoAaflltlrheeipnoi-nlrattebraitminthsrpeeepccotorintocnalnsudswiaoisnlsloocbfieatthededocrsuatuwmdeydn.atteadT. hiins QthAe QstaAtemstaetnetminenvtolfvoers tohnelyfitnhael Exygen Research Page 3 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 CERTIFICATION OF AUTHENTICITY rTehpirsesienntteartiimon roefptohret,rafwordaEtax.ygen Study Number P0001131, is a true and complete Submitted by: E(3S80xt1a5yt48eg)eRC2n7eoRs2lle-eea1sgr0eeca3,hr9PcDAhriv16e801 Principal Investigator, Exygen: DECxhiryaegrcleteonsrR,SAiemsneoaanlryscthical^Laboratory Operations Exygen Research Facility Management: Date Exygen Research Study Diretr, Weston Solutions, Inc. JWaiessimtohnaSKoleustaiorinPs,.EIn.,cD. EE Sponsor Representative, 3M Company: MI/icAhmaelMAa.tSjant0o\ro Director of Regulatory Affairs Exygen Research Date Page 4 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 STUDY IDENTIFICATION SAmPenaralflllyuMsoiasromoomfcPtaaelnrLeflsiuvuoelfrrooanbnaudtteaSn(mePsFaulOllfSMoM)naaiomntnemWit(oPaarlFtienSBrge,SrSP)u,ormoPilge,UrrSafslmeiundogirmoLehCne/txM,aFnSies/Mshu,SlCfoflnaomarttesh,e(VP3FeMgHeStDa)te,icoaanntd,ur PROTOCOL NUMBER: P0001131 EXYGEN STUDY NUMBER: P0001131 TYPE OF STUDY: Residue SAMPLE MATRIX: Sediment and Water TEST SUBSTANCE: PPPeeerrrfffllluuuooorrrooohobcuexttaaannneeesssuuulllfffooonnnaaattteee(((PPPFFFOBHSSS))),, and SPONSOR: 33SMMt. PBCauouimll,dpMianNngy05253164-401-B-10 STUDY DIRECTOR: JWW1a4iee0sssi0mttoWCnhhaeSesKosttoleeunsrta,iWorPinAaPsy,.E1In9.,c3D.80EE STUDY MONITOR: M33SMMt.icPhBCaauoueimlll,dApMian.NngSya05n25t31o64ro-401-B-10 PERFORMING LABORATORY: E3S0txa5yt8egeRCneoRslleeeasgreeca,hrPcDAhriv16e801 ATINMAELTYATBILCEA:L PHASE IIISnnnttttueeedrrryiiimmmInARAitenniapaatolliyyortttniiccCDaaollamTStepte:alrremttiDionnaatteDio:anteD: ate: 01116121////21009195////00005574 Exygen Research Page 5 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 PROJECT PERSONNEL TfinohtleeloriwSmtiunpdgoyrptDeiorisnroeoncfntotehrlefforsotrumdthyiE:sxpyrgoejnecRt eisseaJracishimwheareKaesssaorciiaattedWwesitthonvaSroioluutsiopnhsa,sIensc.of Tthhies Name John Flaherty Charles Simons Karen Risha Eric Edwards Christine Edwards Mark Ammerman Amy Sheehan Mindy Cressley Brittany Kravets Krista Gallant Title Vice President Director, Analytical Laboratory Operations Scientist Sample Custodian Technician Sample Custodian Associate Scientist Technician Technician Technician Exygen Research Page 6 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 TABLE OF CONTENTS PLL4TQCTL2679G358S11........R.0IITIAIE0U0000000O066666666TSSS.UO0R...B.....OATTT1L3582467RDEOCIRSDTJLNEDLRPEUEPIDOOOEQXCAEEOEBIEYnITFreExxCPFFFhFeuSPSMTNLsJNerIttORsTrtaAEEICETUpYcrrCArATFAocCDMnaaEeOFuaRRCRGrLmAIBAtPccLnPrLAmiENGiAEIDTapIttCTttEETOPYaBMiiUPNaSteTtNooIURUtSSiOERiI.tTRTVnLSoo.SonnITOiECo..YRNotCOSgInNI.U.nAEIEIlN..OPNPCEnOriS...OEFTDNSRo..TTd..aorrTA...N.eIaoSoNI.s.p...NMfOIfOAEC.n....OAnOCccL...h...SPLS..s.F..N.ONRdeNeAA.....y.LNFEitr....C...ddTta..F..Y...oE.TACETTi..S.......uunD/DE..v.....c..LM.xS...I...E..A.ErUrd.e.i...P....S.OAee.aE.....t......adR........N.yT...RSTm..S..........rNTSfFu.............I/dH...oA..A....T....Mrp.IoA.A......S......s...r..e........G.EAl..CrL............A..eL..........S.Wa....A..F.....N...NSY.TT.......n........M...C..........o..eI.N.....I.d..TT....aE...........n.....rC...d.a......P.t........IDI.M......s..F....e...lSi.....C.C.E......L...t...c..m...r.......o.r.......e..........EA.uI.SE......u........r.C.....d...T.....e.....t..l.....Nm.A.....S..L....i....i.a.....n...O.Y.......f......m............t.T..M...e.it............M.....iM...c...........n........o..e.................a............P...t.........n.n.....E.P......t..........................iLa....t.............L......To.................n.................E............n...I.........H....d..................A..............S..........................S.......O..........O............N..............................o....................D.....p.............C................l....................e.......u.......................E...............r..............t....................a............i................................ot..S.........................i............................n.n...T...............................................g..s..............A................................................................C...........T.................................................................oE.......................................................n..........M..............................................d................................................E..........i................................t........................N........i.............................o.........................................T...............n...........................................................s............................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................P...........................................................a.................................2222..g.111111111111111.......110013425629354632767e8678571 Exygen Research Page 7 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 LIST OF TABLES Page Table I. Summary of PFBS, PFHS and PFOS in Sediment Samples...........................23 Table II. Summary of PFBS, PFHS and PFOS in Water Samples.................................24 Table III. Matrix Spike Recovery of PFBS, PFHS and PFOS in Sediment Samples ....26 Table IV. Matrix Spike Recovery of PFBS, PFHS and PFOS in Water Samples.......... 28 Table V. Total Percent Solids in Sediment Samples.....................................................34 Exygen Research Page 8 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 LIST OF FIGURES Page Figure 1. Typical Non-Extracted Calibration Curve for PFBS in Methanol.................36 Figure 2. Typical Extracted Calibration Curve for PFBS in Methanol.........................37 Figure 3. Nanodn-0E.5xtnrga/cmteLd,SRtaenspdearcdtisvoelfyP..F..B..S....i.n...M....e.t..h..a.n..o..l.,...0....2...n..g../.m...L............................38 Figure 4. Eanxdtra5c0tendg/SLt,anRdeasrpdescotifvPeFlyB..S...i.n...M....e..t.h..a..n..o..l.,..2..5...n..g../.L.........................................39 Figure 5. RPFeBagSenint S1p%kAAc,eatnicdA4.c0idngR/emaLgeFnot rBtilfainekd,R0e.4agnegn/tmSLpkFoBr,tiRfieedspectively.......40 Figure 6. aPnFdB5S0i0nnWg/aLteFroRrteiafigeedntRBealagnekn,t 5S0pkngB/L, RFeosrptieficetdivReleya.g..e..n..t..S..p..k....A..,................ 41 Figure 7. (CEhxryogmeantoIDgr:aCm0R09e1p5re8s6e,nDtinatgaaSeSte:d1im12e1n0t 5SCam)..p..l.e...A...n..a..l.y..z..e.d....f.o..r..P..F...B..S............42 Figure 8. (CEhxryogmeantoIDgr:aCm0R09e1p6re5s2e,nDtinatgaaSWet:a1te1r2S1a0m5Dpl)e...A...n.a..l.y..z..e..d...f.o..r...P..F..B...S.................43 Figure 9. Typical Non-Extracted Calibration Curve for PFHS in Methanol................ 44 Figure 10. Typical Extracted Calibration Curve for PFHS in Methanol.........................45 Figure 11. Nanodn-0E.5xtnrga/cmteLd,SRtaenspdearcdtisvoelfyP..F..H...S...i.n...M....e..t.h..a..n..o..l.,..0....2...n..g../.m...L.............................46 Figure 12. RExetsrpaecctetdivSeltyan..d..a..r.d..s...o..f...P..F..H...S...i.n...M....e..t.h..a..n..o..l.,..2..5...n..g../.L....a..n..d...5..0...n..g../.L..,.................. 47 Figure 13. PRFeHagSenint S1p%k AAc,eatnicdA4.c0idngR/emaLgeFnotrBtilfaiendk,R0e.4agnegn/tmSLpkFoBr,tiRfieesdpectively.......48 Figure 14. PanFdH5S0i0nnWg/aLteFroRrteiafigeednRt Bealagnekn,t 5S0pkngB/,LRFeosrptiefcietidvReleya.g..e..n..t...S..p..k...A...,................49 Figure 15. C(EhxryogmeantoIDgr:aCm0R09e1p5re8s7e,nDtinatgaaSeSte:d1im12e1n0t5SCam)..p..l.e...A...n..a..l.y..z..e.d....f.o..r..P..F...H..S.............50 Figure 16. C(EhxryogmeantoIDgr:aCm0R09e1p5re4s9e,nDtinatgaaSWet:a1te1r1S4a0m5Cpl)e...A...n.a..l.y..z..e..d...f.o..r...P..F..H...S..................51 Figure 17. Typical Non-Extracted Calibration Curve for PFOS in Methanol................ 52 Figure 18. Typical Extracted Calibration Curve for PFOS in Methanol.........................53 Figure 19. Nanodn-0E.5xtnrga/cmteLd,SRtaenspdearcdtisvoelfyP..F..O...S...i.n...M....e..t.h..a..n..o..l.,..0....2...n..g../.m...L.............................54 Exygen Research Page 9 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 LIST OF FIGURES (continued) Page Figure 20. RExetsrpaecctetidvSeltyan..d..a..r.d..s...o..f..P...F..O...S...i.n...M....e..t.h..a..n..o..l.,..2..5...n..g../.L....a..n..d...5..0...n..g../.L..,...................55 Figure 21. PRFeOagSenint S1p%k AAc,eatnicdA4.c0idngR/emaLgeFnotrBtilfaiendk,R0e.4agnegn/tmSLpkFoBr,tiRfieesdpectively.......56 Figure 22. PanFdO5S0i0nnWg/aLteFroRrteiafigeednRt Bealagnekn,t 5S0pkngB/,LRFeosrptiefcietdivReleya.g..e..n..t...S..p..k...A...,................57 Figure 23. (CEhxryogmeantoIDgr:aCm0R09e1p5re6s6e,nDtinatgaaSSeet:d1i1m2e1n0t5SAa)m..p..l..e..A...n..a..l.y..z..e..d...f.o..r...P..F..O...S............58 Figure 24. (CEhxryogmeantoIDgr:aCm0R09e1p5re5s8e,nDtinatgaaSWeta:1te1r1S4a0m5Cpl)e...A...n..a..l.y..z..e..d...f.o..r..P...F..O...S................. 59 Exygen Research Page 10 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 LIST OF APPENDICES Page Appendix A AStnuadlyytPicraoltoMcoelthPo0d0s0a1n1d31Pr(oEtxoycoglenASmteunddymNeon.tsP010a0n1d1231.)...w...i.t.h................. 60 Exygen Research Page 11 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 1.0 SUMMARY pppEEsaeeexxmrrryyffflllggpuuueeloooennsrrroooMRhboeecuetxtsthaafeanoonanerderecsssVuhuull0lfffeoo0toxhnn0ntea1aratat7teece8t0e((PddP(FAeFatO(pHePnrSpdFSm)eB)ni,anSadnac)aai,tcnxlioyodArznde)pi.dnepgrefsrleofutdlofouirmooEreooxnchpytteegaxrnesfaanelnumseuoMpslrfuloeeolbtsfnhuoaotnftadoeanrteeV(stPhu0Fel0fOo0dn1S(ePa7)ttF8ee2aHrmcScai)onn,rad(dPtiiFonwngBaaStnoet)odr,f LrTeOhpeoQrltipemdroitbvaiodsfeeddquoiasnnadtsirtsyautwmioeinniggfhotwr. ePtFwBeSi,ghPtFvHaSlu,eas,ndanPdFtOheS riensusletsdifmorenthtewaacstu0a.l2snamg/pgl.esTahree The limit of quantitation for PFBS, PFHS, and PFOS in water was 25 ng/L. rAseencdoaimlvyeetrincietaslsfraoemsruptlhlteessaannbadalsyaessdissesoosnfePddFarBycScu,wrPaecFigiHehsSt ,faoarrnedthsePuFamOnmaSlayirsniizsesedodfiimPnFeBnTtSa,sbaPlmeFpHIle.Ss, baFanosdertdPifFiocOnaStwioiennt Ffrtswd9heou%eoesermvrpi,tgPiaeima9fhFncti8tiatacBoirlavaSniytrzeiss,eelo1iyPsdfn4s.oF%uorirHmnef,PcSmaFTPo,nBFavadaBrebSnir8lz,Sdie4eeP,dsPIFPVFfHiF2onO.HS4rST,%StThaa,hi,ennbeardleanweosnPdpavaIFteIelPeOyIcrFr.astSiOilsvslTaSeianmohlyvfeisp.enePlroedaAFwvsigBmenaearaSteraelp,enlyrletPt8risaFscc8avaaeHmemlnSrptrape,1lgrles8eeaesus%cnlopdtab,sveraPere1ascFr0neesi5OddeunsmtSoasnrmsie1newcs9asotr%weasivtezn,aedewdtradeainaeerrcidsidgsncahu8dmTt7reasavaptcraiblieaeenl2tsesd7i2o9aaf%InrorIdesr., wQsaaumtaepnrlteistsaattmihvpaetleaasr,neaPdlyFetHsiicSganlainrteesdounaletssnwwoateterreerpsooabrmtteapdilne(e,NdaRnf)oddruPaeFllOtosSaqmuinapllitetwysoceoxsncetedrpiomtl effaonirtluaPrneFsdB. Stwion wtharteeer 2.0 OBJECTIVE ppT(AeehrrpeffllpuueooonbrrdoojeboixccutttiAaavnn)e.eessuoullfffootnnhaaettee a(PnaFlOy(tPSicF)aBilnS)ps,eadrtimoepnfetrftalhuniodsrowshateutxedraynaecwscuoalrsfdoinntaogtetdoePterro(mtPoiFcnHoelSP)l,e0v0e0l1s1a3no1df Exygen Research Page 12 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 3.0 INTRODUCTION PeATfaonnFhcrtdOiiisdttflSheor(eedrPip,nFtDohO"sereVetAteddd0)reie0mmtite0naeir1nimnWl7ast8itaniut0ohtas:enetirinoMrgboneyfestohtuLPhefleCotPsrda/fFMnlouBaofSolSfytr/h,toMAiePocnSacFaalt.Hna"lmyanSlsoe,yiitssachinsofdAodfrcPoeirFtdnhOtthei(tSePleDFiddnOee,ttwAe"eVrra)mmt0eiii0nrnn0uaaS1tsti7eiioond8nngi2m:tooheMffenPtPeaFtnebhBrayoflSlyduL,toioCcPrfao/FMlAoHmcnSStaea/,MltnyhaosonSiiddc"s Tn20uh0me5bs,etaurnddPy0twh0e0a1sa1ni3na1ilty.itaitcTeadhl etoenramnNailnoyavtteiicmoanlbedsratat0ert5f,do2art0teh0i4fso, irwntthheerisinmitnhrteeeprsoitmrutdwryeapdsoirDretcewtcoearmssbNigeonrve9de,m2p0bro0et5ro.c1o1l, 4.0 ANALYTICAL TEST SAMPLES TaeTaawECnqsvwix0asmduayt0oeiiegl9pcliraFveni1mbarent5silteenaee2prnsmda9seetaktrdpit-srftilaiiiomesneatnCsollseWdnme0n(eia0pnteelst9oescp(at1olriSonubek6fDdnd)le5ate)6hpSnws)elkosacaaalmroctlusemeoematpprdwipmlorpesel-inlepsanysess.lmeit,ereenesapIltdfnAnilfrwneicdiinles.ggsegloeordPaternwpnalneesortdeoeeparlhdtcseivaekeuesesewinstnvtoodhsaoesracratdteaemeietrgadriotptedeehns.lsaudiaa.nnewtmdgtrretpiToittnslphihisintrteecebehgaeseletaselofnaowlonmkbicrmaslapSta,tseilnieteerotepkdrsnditsipessmaw,amvmmebeonbnplrolpaeteult,lnermeskasls2oeul,(ldgsrE2fpogoiax0niefckny0tdeeeogs5rsateswi,mnfnhtriaaatoptiInbvmelaDdeyerl cSoammppoleseiddeonftitfhiecasttiroinng(sIDde)sccoridbeesdabreeloowf:the form DNS-xx-STA0xx-x(x)-05082x and are The first string defines the general sampling area where DNS = Decatur near shore. TwhaeterseacnodndSDstr=insgeddimefeinnet.s the matrix where SW = surface water, IW = interstitial (pore) The third string indicates the sampling station number (1-12). Tduhpelifcoauterthsasmtrpinlegvdoelsucmrieb,eLsSth=e lsoawmpspleikael,iqHuSot=whhigehresp0ik=eparnimd aRrBy =sarminpsleeavteolbulmanek,.DB = The final string is the sample collection date in YYMMDD format. aSsasmocpilaeteldogwiinthatnhdis cinhtaeirnimorfepcourstt.odSytorinagfoermrecaotirodns wisilllobceatkeedptinat EthxeygreanwRdeasetaarcpha.ckage Exygen Research Page 13 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 5.0 REFERENCE MATERIAL ETfJraxhoneymugaaern3nyaM,oly2nt0aiAct,apE2lr0xisl0y,t3ag2n.e3dn,aP2roFd0nOs0,3SM.PaFwyBa,Ss 1p3au,nrdc2h0Pa0Fs5eH.dS,fPrFwoHmerSeFwlsuuakpsaplrCieecoderpibvoyerda3tiMforno. maPnF3dMBwSaawst arEsexcryeegicveeenidvoeandt TwheereasvtaoirleadblferoiznefnoramndatiPoFnOfSorwtahsesrteofreerdenrecferimgeartaetreida.ls is listed below. PFBS and PFHS CoPPPmFFFpHOBoSSSund ExygeSSSnPPP000In000v000e252n476t029o164ry No. Lot # 43S0E110081306-1 Pur199i0t68y1..76.(2% ) Exp111ir020a///t301i148o///n000766Date The molecular structures of PFBS, PFHS and PFOS are given below: PFBS MTCStrhroaeunlmcestciuitucirlaoeal:nrNsWMameoiegn:hitto:Pree3rd3f:l8uo2sru9op9bpu--lit>eadn9e9assultfhoenpaotetassium salt (C4F9SO3IC) F F F F F SO3 FFFF PFHS MTCStrhroauenlmcestciuitucirloaeal:nrNsWMameoiegn:hitto:Pree4rd3f:l8uo3sru9op9hpe-lxiead8n0eassutlhfeonpaotteassium salt (CFnSOa'K"1") F F F F FF F SO 3 F F FF FF Exygen Research Page 14 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 PFOS MChoelmeciuclaalrNWameieg:ht:Pe5rf3l8uosruopopcltiaendeassultfhoenpaotetassium salt (CgFnSCb'K*) TStrrauncstiutiroe:ns Monitored: 499 -- 80 F FF FF FFF F SO 3 FFFFFFFF 6.0 DESCRIPTION OF ANALYTICAL METHOD PALTeChnrea/fMllyuasoSnir/saoMloySfctoit"caranwlotehirmceeeAutDhsceioedddtes(frPomrF"iVtOnh0aAist0i)0osit1nnu7d8Syo2e.f:dimPMeeernfttlhuobodyroLooCcft/aMAnSonia/cMlysSAi"scaifdnodr("PtVhF0eO0A0D1)e7t8ein0rm: MiWnaeatttiheoorndbooyff 6.1. Extraction Procedure For Sediment vBtmmafcTuoloiwelgbLirnolfeoelo~dwio.rnrli2oifeett0tiyudeo1Tstrmn%mhthloyeceeiidenalsslnucsshhiwtleataaarietmmitkikisfetceiuhpprnasgllaotgeeec1no~ssi0tdtfh3auwwe0mmbiwn0eeecLer0rroweteifhsnrooatpatvwrftanmaeoiteomcrnhr.itltegewieeorThwxta.ehnehesxaaddeAstsnarhadsoafaa5ucodldnktpreddigeaeoderrntrnadhandtf.moeloaltro2totAaeph0w~nxtoef6hetttmrer0destwariaLocmsmatftneosoiiopdonrtofltinshueimfh,fwetsieasn.ectaesknha.tdleTteteoiirhymToas.oeadnhnewmeesnTodaeaptsfhmrloaweeeawmnpspatrelmpspoeillsrelsuiweoatxfawstpeetCaerdiwiecjignrgatewheittotrheeShaevndoPsesorcEairosedcnmthuneeictsaogxtnpacrkherltiaaeretfdlrirsuyrfidf,dugaieffn3bggeodt5ddyyeer. mhLaTcamgshnonurCheeaaoldnetlt/dkthdehMhuica2rasneaetnSun0gretdopo/edM3lldeam.m0nrSinwLdnimT1alatel5tlohsioialolneenifacutmaatdewttwdreerLwodas5dsasse.0taphedoo0psrrnTfoaw.tttlmyhoohwya.eespLTaatnrshtnchoeaeNooeplrmoltyalhcapweelleadgelclraeruneettsseadnerCditewde|agogcdiennBewedrSte.noeotPathdtscrEteilteFfehtdnuciso.evitgaasrermeictstfhraTauemiermtdhgdubigeeeelbbodeldeoitcl.tC.tcailotteiogleglenaurAsaisdmE.snSpiatPnotpfichEoofTrheonwrhexecee~alsidalusm2auras0mwtaatarwteitmpeimdeta.lhlgwpesiynehlAa1eu.esw0fstpdievapcmsTwserwohoLaelielxtrmateeohi~ncmiefal3tlmleal4mu0iydltai0iezexttm0leeteieyhndrdLrsatwptonwmbbaooooyysal.ff Exygen Research Page 15 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 6.2 Percent Solids Procedure For Sediment PVta~phte1e0err510ccs00eema4n0nmit4tnp2ssu2loo7teell.iispdCd.Als.ufpEoswTpraterhhceroeheaexncpsdihmataehmnstaeeatpwrmesmllaeapymislwne2rpea0ewldscegoasutrshrswadeitmnehendgresew.ntohteTcfreiaaghsnlpahecsmreufosdelpacarmleatreedgepdduwal.irenatesos,SiweiawnneeddecriTielecgusaadhtstbhiieecnelddeagntiiVonntdhrtrEoefiaeoxnwadrydppeginiaaeegnlrnlahc.ontemwnTooetehfvtdsehteohontwleodiodepcvsiaogenrohre.nltsifuoTgolfhhtrste. 6.3 Extraction Procedure for Water AAfcewoolauprnst4apdit0rfaeioitncixmwaaoiltnmaLyioeszadneactedlowoillqyblfiueytfahcoiLptvtepCe1dor0/omfMipnmitrtSlhoilLa/ieMltaietoweSgfsrarsaeamtmeldoereupcftahtlsmretaaeosmends,tophpt1lrhala5eenayomn.wsldaLawms5paposumllesaysLedpddrwooefepfdoryerwtloeatlnhotteeheared.ceeecxdnaTttrrorthairnecfiudttoiggeoleenau.atuCptFebriigoevw.ceSeaEdPmsuEairdclelhici.slacisrtaateArrmrdisdfpetgodeleer.f 6.4 Preparation of Standards and Fortification Solutions Apcbmmmsmpppptourggggaiiiiinr//xxx//xnnimmmmmceeeetgdyeddddiLLLLiannxra0t0gde11nrffff..d.0a0oooo1d0tst1rrrrhiopttttspposeiiiilgapfffftggnuoliiii/vg//ttccccmcmmoio/aaaacokmLflttttLoLnuiiiiLoooon1mfsffnnnn0wtoteoofe0aroanrrn0sssstttsrtiuttttdii)tfaaaaffppiiapiinnnnfcigccrinrddddatdcaa/eotamaaaattmpiirirrrtoaooddddLsi1enoronn0tenhlabaaa0dsussnnannytstttamnddddaaitbdonaonnyLnindbbbbldds.darrrraastwiiiiraaorrnnonnFddrdkilggggfdrtviohwiiiiwnwinnnnwmPngaagagggmgFassstsB1etttthp1pphhhh0tpSih0rrreeeesreee,amempppnsvvvvpaLPoaaogooooarrrlFlllrlleeue.uuuuooeHdddtdmmmmiff...oSB.eteeeen,yhaBBB,ceuuuuyyyatahappppnattktmdoaaapttttikkkfoooopnixiiirtPgnnnheo1111Fgggdep00001Or00000s111i1Sat0000ammmmmt0enmmmLwLLLLdpLLsaLagtorwwwwso/doofomciiiisffttttkftphhhhLhtt(rthhechesmmmmeefoopeomrlaeemmeerrumrtttitteteihhxhhiicifixxdoaaaaexiteecennnndneddadoooodatall1llti0f1,,,,on01o..1nd000aaaaar mpAreestpheaatrneoodfl: nasons-peexctirfaicetdedinsEtaxnydgaerndsmceothnotadinVin0g00P1F7B80S., PTFhHe SfolalnodwiPnFgOcSonwceanstrpartieopnasrewderine Co(Snnoeg,11l/u0m0ot00ifLoFn)1o_r_t____V(Fom51ol.u0Lr0tm)_e____F_o_r_Vt_iofil(eu11mdm00L00Se)a_om_f_p_le____C_F_ainli(abnlrgaC51/t.mi0o0onLne),Sotdf. Exygen Research Page 16 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 1of PF1251B0..0000S, PFHS and2111P.0000FOS 111100000000 2001....5200 The stock standard solution and all fortification and calibration standard solutions were pstroerpeadratiinonaisrelofrciagteerdationrth(e4raw d2atCa )pawckhaegne ansostociinateudsew. ithDthoicsuimnteenrtiamtiorenpoortf. standard 6.5 Chromatography TmQsahuimneaspn,rlteeirtfseeiscncpaoteitroicrontenisvpteoiomlfynPe.dsFinPBogeSfat,PkoPsFtFhBaHebSSoa, vnPaeanFlytdHhteeSPFrLeaOOtneSdQntwPiwoFanesOrtaeSimcncweoo.emt rdepelit~se0hc.et5eddmbiyinnLsa,Cny-/M8o.4Sf /tMhmeSinrsee,alegacentnrdot s-bp1lra1an.y0k. 6.6 Instrument Sensitivity Tcohnecensmtraatliloenstofs0ta.2ndnagr/dmLamofoPuFntBSi,nPjeFcHteSd anddurPinFgOSt.he chromatographic run had a 6.7 Description of LC/MS/MS Instrument and Operating Conditions Instrument: Interface: Computer: Software: HPLC: API 4000 Biomolecular Mass Analyzer Turbo Ion Spray Liquid Introduction Interface DELL OptiPlex GX400 Windows NT, Analyst 1.4.1 HewlettHHHHPPPPPacAVCQkouauacltaruodutmsuP(amHnummPOpD)plveeSegrenarsiseesr1100 HMIMCnoPjooelLbbucCiimtllieeonCnPPoThhVleaauomssmiee.pn:((.::AB15T))-::h3pe0L2rMmmeoCtMhFaluAnoomplhmasoenRiuPm, 5A0cmemtatxe2i.n1wmamter Exygen Research Page 17 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 Total run timTei:m111~0e81018...1(000...m0008imn'i)n Flow Rate: 0.3 mL/min %226666555555A %337733555555B Ions monitored: Analvte PFBS PPFFHOSS Mode negative negative negative MT29roa9nn-ist*iotir9oe9nd 439999 -> 8800 RAet-pe0pn(.rtm5oioximnnim)iTna.itmee -~181..40mmiinn.. 6.8 Quantitation and Example Calculation tpFcETlhiorinqheofneeutgceaasrpeertateinnamoatrnnrekm.dagatia1ricrroedrecsnoaaslliwciwcotueuanarrsl)vssaedtmbeoedyt(efelaistrnshAamueemnraineaaprdlemlyedrasoetnofgurdrrnosetmcotshafosetlitfwiohbsaneatrnarafaentpoildaoylluarntoaerswdmisnftioceganutungerndrsvedsaiexq)r(duwigancawetoisnnonegegnrcre/eesma.nntieeLtnrrdj,aaettbcibeoatdyensdes(tduhisnoeoitnfnogAsptnth1eaae/anxlkdyLfasiaCtrtrd/ewMssao.e)Sfitgu/wMhsTiatnheSrgdee. Equation 1: Where: DF = Analyte found (ng/mL) = (Peak Dilution Factor, factor by which the afisrneloaalp-evionltuemrceepwt)asxdDilFuted, if necessary. FEoqruastaimonpl2eswfaosrtuifsieeddtwo ictahlckunloawtenthaempoeurnctesntorfePcFovBeSr,y.PFHS and PFOS prior to extraction, ERqecuoavtieorny2(:%) = (analyte found (anmg/omuLn)t a-dadneadly(tnegi/nmcLo)ntrol (ng/mL)) xl00% Nuwnoasttpee:irkaFendodraotlhnieqnuPgoF/tgBo(Sfp,pthPbe)FHfporSri,mseaadnridymPfeiFneOtl.dSsraemcoplvee.ryRceaclcouvleartyioniss,btahseed"coonntnrogl/"miLs tfhoer Exygen Research Page 18 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 Engq/uga(tipopnb)3. was used to convert the amount of PFBS, PFHS and PFOS found in ng/mL to Equation 3: Analyte found (ppb) = fAnalyte fosuanmdp(lengw/meiLgh) tx(5finga)l volume (5 mL)l Ebaqsueadtioonn d4rywawsetihgehnt. used to calculate the amount of PFBS, PFHS and PFOS found in ppb EAqnuaalytitoenfo4u:nd (ppb) dry weight = Analyte found (ppb) x [100% / total solids(%)] AonnlywSe):exitdahimmwpehlneepidntsafstrnoilamegieonosttlma/:eauaafptgkrlltmpeiciaPnslvoaepeoFronpcclelweHlatioaufdlareSmEcsrciuexgte(also%yhdapr(gttdmo)ie(eongnLdnd))iI(nuDfogs:irntsCagl0mev0ape9nll1e)a5c8t2uaSl pskamEp45305551(lS.634e14e.07990tf0601:1o20l160lo1w21s05(cBa)l,cufolarttiiofinedisatfo4r PFHS ng/mL From eAqunaaltyiotenf1o:und (ng/L) 15476121690-0033001 x 1 From equation%2:Recovery 4.57 ng/mL (4.57 ng/4gn-g/0g.491 ng/g) x 100% From equatiAonna3l:yte found (ppb) 102% = (4.57 ng/mL x 5 mL) = 4.57 ppb5 g Exygen Research Page 19 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 FromAenqaulyatteiofonu4n:d (ppb) dry weight =4.57 ppb x (100% / 56.01%) = 8.16 ppb NOTE: This value may be slightly different than that of the raw data due to rounding. 7.0 EXPERIMENTAL DESIGN vwPFwlaoFoeeblrHrroueermsSaewa,teddoetaidrrimniygecdhedbfneiPaedtltlfFsoflOaaoirnmerSkeenpbxiwolneetwiresntarhngcedeteciafossoihdinengid.lcpndeepa.dFnetoetdradratwttaoiaosanttkelhanrteboosowaftrhimaneetlpodclsr.eoyasmnmcdTpeehalnseteitrsgriaxsnitanaimsotetpnhpdiekletaeoslssa,bftwhiPoeeeFrlradBetsomaSrfm,yialtpPlrealFidefxtHesbtrSopo,tithktaaleenes2sds,0aPiPm0nFFpBmOtlheSLSes, TTwslaehwabtesooinrscasceoteldolutilsrdmeycceeotddennudtotpanslfiiaenocmreardetpeealafegwicsevhenewrtessebaaremlmexantpperklxlaeetcratsaseniicddttete.es.tdwaionFndorfreooaneugearecnssheetttbssc,iltoaeonn,nkteatswionfofeodrwltaithfbwiioecodrhastwaaotmarykspnalmoerwaestin-rteeixxcsot.rsnapOccietnkineoetsnrsa.aatmEinoadpnclshea. EttttTcoosfdSslwhwwaopuonnueahbrrirnoooeetpceekofschtelaerswicsesscwfcasaaoaanoce.otammmamnotsoutrteetrwnpsrpaeSppaytatltlrilealaaeaiemenedottnniseeinsunpsddnnasrdsiiepslitmiciteted.ntfetlflpeweii.uopescsvos,p.rSdl.oaiereSwotete-teetswSeemrdtweaSsetwea.sowoweaotscantwtaeaohesrtrSesvseiirenairxxeaenicerteecegtmeson-ocfxfhreetenoipnotecatxerxtrrflnntoetaatletoacedrttarniciworaanwagnttsicnbsaeeceooeinatptiddttlndenseaeniaisodtdksieneapapan.nendkompoesisdrsbrnodorape,pFelewmrft-oalowepteaenwrpweeprxoneokwrnolsatardeee,rrwiettalteaaeesaecttasccrewrebhstowithttwrtoeriflsaaeaeoalor,rfnotenseatnoerdfaciedttrtrowontrtetoewrsrrtsrdfofdewufiyouaoopoa.rprrnsonrfmgofuoadesbaoeoFfsrrcaaclnnfenasoeamttewaetymraenrciwwmppwhxkepseselbiasaapealaacstelwmtmtlcehpaoeweeshrnasirpfprikiittscklflttefsewefieooeeshotisorsestnrarresswatttfiataieohafootnwtannoeerienenrdnn,d,drroieeedteatrasiswssdnssfsuuautuaiaaadoyslrresrrmmmrsffapfdfseoaasmaaseoppp-uccaccecellelepremeoeeaexixflaatftetwwwwpstsrsrc,ewlaiaiieaaaaetttdkccaeeettatteettneew.s.tsierrfferrooidiiaatrwSSeneffffe.t,noooolleseesndddar.rrrr.tt 8.0 RESULTS rAseencdoaimlvyeetrinicetaslsfraoemrsuptlhlteessaannbadalsyaessdissesoosnfePddFarBycScu,wrPaeFcigiHehsSt ,faoarrnedthsePuFmaOnmaSlayirsniizsesedodfiimPnFeBnTtSas,baPlmeFpHIl.eSs, baFanosdertdPifFiocOnaStwioiennt Exygen Research Page 20 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 wdeevigiahttioanrse fsourmPmFBarSiz, ePdFHinST, aanbdlePIFIOI.S Tinheseodvimereanllt asvaemrapgleespbearsceedntonrewcoevtewrieeisght satraen7d9ard FtrPt9fshweou%oFesrmorBp,tPaeimSw9fFnci8aatacBiirntlaveiSytzertis,ehlo1isyPsdrn4a.eF%moeirHne,fpwQcSalTPoeanu,FavstdaaeBbentnrrh8lStdiei4ase,tatasPImPtaVFifFrv2po.OeHe4lreSd%StsTaeh,,i,nsehnPiaaregeFalnwnysnodHpataavitSetclPeeeycadrFrtsilainOiavslsrlaseSoemolansnyvfueiop.nelPlttwreAsFarwseagBnawptaeeaStoreerl,epryrrtseePte8airsFdc8cmaoaHem(blpnNStplrtaee,R1li,resn8ae)usa%encdlnddotau,dsrvePef1eaPoF0trnFsori5OdeuOsqamSaSluslmasi1inelsna9sisattr%wysatmiwezn,acdepdotaodelaanenrsrcsdtidesrncdoaue8dimlTxr7meafcavpaceebiliinapelel2uttstesi2raoeaff%InnoosrIdes.rr., Acomaroaeoccbfcasccntfcutuautarulrirriitnoarxalsaeeclccdydaysfiv..paelafaiisokliFbuleraoowrberrerae,laeterthocceaehfnoroydvawrsesoararsereymteaecfscpriosrheleevslesddeasuarmsislmfeitpptossepilrk.elaaeesvse,srasaFteoiichotlccheearoirbavtetlhsteheeaawredmtifeeowpswsrree,leiedttrtehhiewmaebcouoyehsstnahleatrmedsebarsvpotmaoilsreemappwsatilpoksewilrseneyeesgtsshr,asewanttthtnehdheroeweettrweeiarurnoeceswdpcefiuodeuvirerrsiateldteedcouddytasw.adpltsoouisIkQeneealsCtassiorbnseesosqcrtsareouasamsnvatuolceptilherrtltyyyeess 9.0 CONCLUSIONS EaVqnux0aac0lleyi0tpz1yte7oda8rs0fi,nonortreteegPsdrpFietBaycbStoio,vvfePetlhF,yeH.thdSeaTtsaahe.neddriemPewFnOetrSeannadcowcocaritdrecirnugsmasmttoapnlaecnseaswlytethircaeatlswumcoceuetlhsdsofduhslalvyVee0x0atf0rf1aec7ct8tee2dd aatnnhdde 10.0 RETENTION OF DATAAND SAMPLES RWdrsGeiaegothtasnaoeeiednnasderuLtcdcchahohebipnwoafyritisanhloltaeiofnblrtEseyahtxnresPuyahfrmlgiiaypnecetpnaitnceiltcaRdaelonetrroSsaeelttpytaaehotnrmiecrdcthpaasieltrsaudrrradscectyopuh4mori0dveripetCrllesaoeFcntgfRtedoos,r.r7a.atl9lhlEl2Teox.ohrapriiicsgegtirdinincooaaodellpsfoipanefacsopittlieoimitrfnyedc-aslalsupltpadereceagciwfifefiancicdeeirdarlaaittawtiyne,-ddsaEpasbPteyawAc,iEefwTlixlciSylalrgCsaebAwnea Exygen Research Page 21 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 TABLES Exygen Research Page 22 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 Table I. Summary of PFBS, PFHS and PFOS in Sediment Samples ExvqenID C lie n t S am ple ID C4 Sulfonate PFBS C6 Sulfonate PFHS C8 Sulfonate PFOS Perfluorobutanesulfbnate_______Perfluorohexanesutfonate______ Perfluorooctanesuttfonate Analyte Found Assessed Analyte Found Assessed Analyte Found Assessed (ppb, ng/g) Accuracy (ppb, ng/g) Accuracy (ppb, ng/g) Accuracy Dry Weight (+/-% ) Dry Weight <+/-%) Dry Weight <+/-%) C0091547 C0091547 Rep DNS-SD-STA001 -0-050824 DNS-SD-STA001 -0-050824* 0.979 0.787 30 30 1.29 1.42 C0091557 DNS-SD-STA002-0-050824 0.452 30 0.435 C0091557 Rep DNS-SD-STA002-0-050824* 0.494 30 0.427 30 30 30 30 21.1 21.3 NR NR 30 30 _ - C0091566 DNS-SD-STA003-0-050824 0.519 30 ND 30 10.1 30 C0091566 Rep DNS-SD-STA003-0-050824* 0.617 30 ND 30 11.7 30 C0091579 DNS-SD-STA004-0-050825 ND 30 ND C0091579 Rep DNS-SD-STA004-0-050825* ND 30 ND 30 1.22 30 30 1.41 30 C0091580 C0091580 Rep DNS-SD-STA005-0-050825 DNS-SD-STA005-0-050825* ND ND 30 2.08 30 30 2.03 30 88.0 88.0 40 40 C0091581 DNS-SD-STA006-0-050825 ND 30 ND C0091581 Rep DNS-SD-STA006-0-050825* ND 30 ND 30 0.810 30 30 0.810 30 C0091582 DNS-SD-STA007-0-050825 ND 30 0.877 C0091582 Rep DNS-SD-STA007-0-050825* 0.357 30 1.25 30 30 26.1 25.7 30 30 C0091583 C0091583 Rep DNS-SD-STA008-0-050825 DNS-SD-STA008-0-050825* ND ND 30 0.421 30 0.428 30 30 16.4 16.1 40 40 C0091584 DNS-SD-STA009-0-050825 4.13 30 6.20 50 24.7 30 C0091584 Rep DNS-SD-STA009-0-050825* 4.72 30 6.66 50 26.5 30 C0091585 DNS-SD-STA010-0-050825 0.392 30 1.30 30 NR - C0091585 Rep DNS-SD-STA010-0-050825* 0.430 30 1.21 30 NR - C0091586 D N S -S D -S TA 011-0-050825 0.582 30 ND C0091586 Rep D NS-SD-STA011-0-050825* ND 30 0.393 30 30 4.67 4.53 40 40 C0091587 DNS-SD-STA012-0-050825 0.822 30 0.553 C0091587 Rep DNS-SD-STA012-0-050825* 0.743 30 0.905 30 30 15.7 15.6 30 30 'Laboratory Duplicate ND = Not detected at or above the Limit of Quantitation (LOQ) of 0.2 ng/g (wet weight). NR = Not reported due to quality control failure. Exygen Research Page 23 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 Table II. Summary of PFBS, PFHS and PFOS in Water Samples Exygen ID C0091529 C0091533 C0091536 C0091539 C0091539 Rep C0091540 C0091543 C0091543 Rep C0091544 C0091548 C0091549 C0091549 Rep C0091550 C0091553 C0091553 Rep C0091554 C0091558 C0091558 Rep C0091559 C0091562 C0091562 Rep C0091563 C0091588 C0091588 Rep C0091589 C0091592 C0091592 Rep C0091593 C0091596 C0091596 Rep C0091597 C0091600 C0091600 Rep C0091601 C0091604 C0091604 Rep C0091605 Client Sample ID D N S -S W -T R IP 01-0-050824 DNS-SW-TRIP02-0-050824 DNS-SW-TRIP03-0-050824 DNS-IW-STA001 -0-050823 DNS-IW-STA001-0-050823* DNS-IW-STA001 -DB-050823 D N S -S W -S TA 001-0-050823 D N S -S W -S TA 001-0-050823* DNS-SW -STA001-DB-050823 DNS-SD-STA001-RB-050824 DNS-IW-STA002-0-050824 DNS-IW-STA002-0-050824* DNS-IW-STA002-DB-050824 DNS-SW-STA002-0-050825 DNS-SW-STA002-0-050825* DNS-SW-STA002-DB-050825 DNS-IW-STA003-0-050824 DNS-IW-STA003-0-050824* DNS-IW-STA003-DB-050824 DNS -S W -S TA 003-0-050824 DNS-SW-STA003-0-050824* DNS-SW-STA003-DB-050824 DNS-IW-STA004-0-050824 DNS-IW-STA004-0-050824* DNS-IW-STA004-DB-050824 DNS-SW-STA004-0-050824 DNS-SW-STA004-0-050824* DNS-SW-STA004-DB-050824 DNS-IW-STA005-0-050823 DNS-IW-STA005-0-050823* DNS-IW-STA005-DB-050823 DNS-SW-STA005-0-050823 DNS-SW-STA005-0-050823* DNS-SW-STA005-DB-050823 DNS-IW-STA006-0-050824 DNS-IW-STA006-0-050824* DNS-IW-STA006-DB-050824 C4 Sulfonate PFBS C6 Sulfonate PFHS C8 Sulfonate PFOS Perfluorobutaneiulfonate_________ Perfluorohexanesulfonate_________ Perfluorooctanesulfonate Analyte Found (PPt, ng/L) Assessed Accuracy (+/-%) Analyte Found (ppt, ng/L) Assessed Accuracy <+/- %> Analyte Found (ppt, ng/L) Assessed Accuracy (+/-%> ND 30 ND 30 ND 30 ND 30 ND 30 ND 30 ND 30 ND 30 ND 30 682 30 790 50 20000 50 690 30 832 50 19100 50 666 30 793 50 18500 50 111 30 24.0 30 164 30 110 30 22.4 30 148 30 101 30 18.8 30 128 30 ND 30 ND 30 ND 30 364 30 620 40 2860 40 368 30 633 40 3170 40 353 30 632 40 3140 40 140 30 59.2 30 403 30 130 30 48.5 30 346 30 134 30 50.6 30 358 30 76.4 30 51.1 30 645 30 79.2 30 44.4 30 593 30 77.0 30 64.5 30 633 30 58.7 30 42.7 30 180 30 59.5 30 43.1 30 165 30 58.1 30 47.3 30 200 30 75.8 30 45.2 30 184 50 77.1 30 45.5 30 151 50 61.9 30 30.4 30 131 50 134 30 178 30 555 30 133 30 168 30 478 30 131 30 180 30 555 30 86.9 30 21.1 30 167 30 90.1 30 22.8 30 158 30 86.2 30 21.3 30 167 30 64.5 30 16.9 30 124 30 61.4 30 15.8 30 99.8 30 66.9 30 15.8 30 136 30 58.2 30 57.4 30 263 30 56.1 30 57.4 30 249 30 59.6 30 61.0 30 320 30 ` Laboratory Duplicate ND = Not detected at or above the Limit of Quantitation (LOQ) of 25 ng/L. Exygen Research Page 24 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 Table II. Summary of PFBS, PFHS and PFOS in Water Samples Continued Exygen ID C0091608 C0091608 Rep C0091609 C0091612 C0091612 Rep C0091613 C0091615 C0091615 Rep C0091616 C0091619 C0091619 Rep C0091620 C0091623 C0091623 Rep C0091624 C0091627 C0091627 Rep C0091628 C0091631 C0091631 Rep C0091632 C0091635 C0091635 Rep C0091636 C0091638 C0091638 Rep C0091639 C0091642 C0091642 Rep C0091643 C0091644 C0091644 Rep C0091645 C0091648 C0091648 Rep C0091649 C0091652 C0091652 Rep C0091653 C0091656 Client Sample ID DNS-SW-STA006-0-050824 D NS-S W -S TA 006-0-050824* DNS-SW-STA006-DB-050824 DNS-IW-STA007-0-050825 DNS-IW-STA007-0-050825* DNS-IW-STA007-DB-050825 DNS-SW-STA007-0-050825 DNS-SW-STA007-0-050825* DNS-SW-STA007-DB-050825 DNS-IW-STA008-0-050825 DNS-IW-STA008-0-050825* DNS-IW-STA008-DB-050825 DNS-SW-STA008-0-050825 DNS-SW-STA008-0-050825* DNS-SW-STA008-DB-050825 DNS-IW-STA009-0-050826 DNS-IW-STA009-0-050826* DNS-IW-STA009-DB-050826 DNS-SW-STA009-0-050826 DNS-SW-STA009-0-050826* DNS-SW-STA009-DB-050826 D N S -IW -S T A 0 1 0-0-050826 DNS-IW-STA010-0-050826* DNS-IW-STA010-DB-050826 DNS-S W -S T A 0 10-0-050826 DNS -S W -S TA 010-0-050826* DNS-SW-STA010-DB-050826 DNS-IW -STA011-0-050826 D N S -IW -S T A 0 1 1-0-050826* DNS-IW-STA011-DB-050826A DNS-SW -STA011-0-050826 D N S -S W -S T A 011-0-050826* DNS-SW-STA011-DB-050826 D N S -IW -S T A 0 1 2-0-050826 DNS-IW-STA012-0-050826* DNS-IW-STA012-DB-050826 D N S -S W -S TA 012-0-050826 D N S -S W -S TA 0 1 2-0-050826* DNS-SW-STA012-DB-050826 DNS-IW-STA012-RB-050825 C4 Sulfonate PFBS C6 Sulfonate PFHS C8 Sulfonate PFOS Perfluorobutanesulfonate_________ Perfluorohexanesulfonat*_________ Perfluorooctanesulfonate Analyte Found (PPt. ng/L) Assessed Accuracy (+/-%} Analyte Found (ppt, ng/L) Assessed Accuracy (+/-%} Analyte Found (ppt, ng/L) Assessed Accuracy (+/-%} NR 39.6 40 NR NR 36.6 40 NR . NR 41.6 40 NR * 578 30 703 30 16200 30 564 30 678 30 15700 30 500 30 675 30 18100 30 132 30 56.9 30 388 30 132 30 56.1 30 386 30 131 30 54.5 30 387 30 6020 30 5190 30 901 30 5860 30 4960 30 857 30 5700 30 5230 30 1130 30 217 30 158 30 NR 218 30 163 30 NR 220 30 163 30 NR . - - 8580 8650 8710 30 11800 30 30 11900 30 30 12600 30 3260 3090 4220 30 30 30 53.4 30 40.7 30 134 30 48.1 30 36.8 30 107 30 46.0 30 36.2 30 106 30 NR - 846 40 19200 30 NR - 818 40 17700 30 NR - 837 40 21500 30 73.2 40 53.6 30 139 40 73.5 40 52.1 30 122 40 79.2 40 57.5 30 177 40 1880 30 844 30 850 30 1830 30 807 30 756 30 1630 30 750 30 1320 30 NR . 39.4 30 96.4 50 NR - 47.0 30 110 50 NR - 42.4 30 131 50 8730 9140 8850 30 30 30 NR NR NR - 1330 40 - 1220 40 - 1230 40 53.3 30 42.2 30 142 30 56.5 30 40.0 30 144 30 57.3 30 41.4 30 187 30 ND 30 ND 30 ND 30 'Laboratory Duplicate A Sam ple contained only 33 m l of water when received. NO = Not detected at or above the Limit of Quantitation (LO Q ) of 2 5 ng/L. N R = Not reported due to quality control failure. Exygen Research Page 25 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 Table III. Matrix SpikSeedRimeceonvterSyamofpPleFsBS, PFHS and PFOS in S am ple Description Amount Spiked (ng/g) C4 Sulfonate PFBS Wet Weight Am t Found Amount in Sample Recovered Recovery (ng/g) wet wt. (ng/g) w et wt. (%) C6 Sulfonate PFHS W et Weight Amt Found in Sample (ng/g) wet wt. Amount Recovered (ng/g) wet wt. Recovery (%) C8 Sulfonate PFOS W et Weight Am t Found in Sample (ng/q) w et wt. Amount Recovered (ng/g) wet wt. Recovery <%) DNS-SD-STA001-0-050824 (C0091547 Spk C, 4 ppb Spike) DNS-SD-STA001-0-050824 (C0091547 Spk D, 40 ppb Spike) 4 40 0.565 0.565 3.49 22.7 73 0.743 55 0.743 4.95 36.3 105 12.2 89 12.2 34.8 48.8 92 DNS-SD-STA002-0-050824 (COO&1557 Spk E, 4 ppb Spike) DNS-SD-STA002-0-050824 (C0091557 Spk F, 40 ppb Spike) 4 40 0.236 0.236 3.05 27.6 70 0.227 68 0.227 4.03 39.9 95 NR 99 NR NR NR NR NR DNS-SD-STA003-0-050824 (C0091566 Spk G, 4 ppb Spike) DNS-SD-STA003-0-050824 (C0091566 Spk H, 40 ppb Spike) 4 40 0.238 0.238 3.15 31.1 73 ND 77 ND 4.16 38.9 104 4.61 97 4.61 8.31 28.8 93 60 DNS-SD-STA004-0-050825 (C0091579 Spk I, 4 ppb Spike) DNS-SD-STA004-0-Q50825 (C0091579 Spk J, 40 ppb Spike) 4 40 ND ND 2.84 28.7 71 ND 72 ND 4.04 38.1 101 0.855 95 0.855 4.74 34.5 97 84 D N S -S D -S T A 0 0 5 -0-050825 (C0091580 Spk K, 4 ppb Spike) D N S -S D - S T A 0 0 5 -0 -0 5 0 8 2 5 (C0091580 Spk L 40 ppb Spike) 4 40 ND ND 3.33 30.5 83 1.23 76 1.23 5.49 37 8 107 52.0 91 52.0 51.1 77.5 64 DNS-SD-STA006-0-050825 (COO1581 Spk C, 4 ppb Spike) D N S -S D - S T A 0 0 6 -0 -0 5 0 8 2 5 (C00915S1 Spk D, 40 ppb Spike) 4 40 ND ND 3.63 33.6 91 ND 84 ND 4.32 39.4 108 0.584 99 0.584 5.38 36.7 120 90 DNS-SD-STA007-0-050825 (C0091582 Spk E, 4 ppb Spike) DNS-SD-STA007-Q-050825 (C0091582 Spk F, 40 ppb Spike) 4 40 ND ND 3.47 29.8 87 0.491 75 0.491 4.59 38.8 102 14.6 96 14.6 16.1 47.4 82 DNS'S D-STA008-0-050825 (COO01583 Spk G, 4 ppb Spike) D N S 'S D -S T A 0 0 8 -0-050825 (C0091583 Spk H. 40 ppb Spike) 4 40 ND ND 3.40 30.9 85 0.251 77 0.251 4.43 38.4 104 9.77 95 9.77 11.0 37.4 31 69 DNS-SD-STA009-0-050825 (C0091584 Spk I, 4 ppb Spike) DNS'S D-STA009-0-050825 (C0091584 Spk J, 40 ppb Spike) 4 40 2.54 2.54 6.30 37.9 94 3.81 66 3.81 9.64 43.2 146 15.2 98 15.2 34.5 55.3 100 DNS-SD-STA010-0-050825 (COO01565 Spk K, 4 ppb Spike) DNS-SD-STA010-0-050825 (C0091585 Spk L, 40 ppb Spike) 4 40 0.227 0.227 3.81 36.5 90 0.755 91 0.755 4.10 31.3 84 NR 76 NR NR NR NR NR ' Sample residue exceeds the spiking level significantly (3x spiking level); therefore, an accurate recovery value cannot be calculated NO = Not detected at or above the Limit of Quantitation (LOQ) of 0.2 ng/g (wet weight). NR - Not reported due to quality control failure. Note: Since this summary table shows rounded results, recovery values may vary slightly from the values in the raw data. Exygen Research Page 26 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 Table III. Matrix Spike Recovery of PFBS, PFHS and PFOS in Sediment Samples Continued Sample Description Amount Spiked (n g /g ) C4 Sulfonate PFBS W et W e ig h t_________C6 Sulfonate PFHS W et W eight Amt Found in Sample (ng/g) Amount Recovered (ng/g) Recovery (%) Amt Found in Sample (n g /g ) Amount Recovered (ng/g) Recovery (%) C8 Sulfonate PFOS W et Weight Amt Found in Sample (ng/g) Amount Recovered (n g /g ) Recovery (%) D N S -S D -S T A 0 1 1-0 -05 0 82 5 (C0091566 Spk C, 4 ppb Spike) D N S -S D -S T A 0 1 1-0 -05 0 82 5 (COO015S6 Spk D, 40 ppb Spike) 4 40 0.329 0.329 3.52 33.8 80 ND 84 ND 4.03 37.5 101 2.64 94 2.64 8.00 34.6 134 80 DNS-SD-STA012-0-050825 (COO015S7 Spk E, 4 ppb Spike) DNS-SD-STA012-Q-050825 (COO01587 Spk F, 40 ppb Spike) 4 40 0.385 0.385 3.70 27.5 63 0.259 68 0.259 3.81 28.9 89 7.34 72 7.34 10.8 87 33.1 64 Average: Standard Deviation: 79 9 Average: Standard Deviation: 98 14 ` Sample residue exceeds the spiking level significantly (3x spiking level); therefore, an accurate recovery value cannot be calculated ND = Not detected at or above the Limit of Quantitation (LOQ) of 0.2 ng/g (wet weight). Note: Since this summary table shows rounded results, recovery values may vary slightly from the values in the raw data. Average: Standard Deviation: 84 24 Exygen Research Page 27 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 Table IV. Matrix Spike RecovSeraymopflePsFBS, PFHS and PFOS in Water Sample Description D N S -S W -T R IP 01-LS -050824 (C0091530,1.0 ppb Field Spike) D N S -S W -T R IP 01-H S -050824 (C0091531, 10 ppb Field Spike) D N S -S W -T R IP 02-LS -050824 (C0091534,1.0 ppb Field Spike) D N S 'S W -T R IP 0 2 -H S -0 5 0 8 2 4 (C0091535,10 ppb Field Spike) D N S -S W -T R IP 03-LS -050824 (C0091537, 1.0 ppb Field Spike) D N S -S W -T R IP 03-H S -050824 (C0091538,10 ppb Field Spike) D N S -IW -S T A 0 0 1 -0-050823 (C0091539 Spk C, 1.0 ppb Lab Spike) D N S -IW -S T A 001-0-050823 (C0091539 Spk D, 10 ppb Lab Spike) D N S -IW -S TA 0Q 1-LS -050823 (C0091541, 1.0 ppb Field Spike) D N S -IW -S TA 001 -H S -050823 (C0091542, 10 ppb Field Spike) D N S -S W -S TA001 -0-050823 (C0091543 Spk E, 10 ppb Lab Spike) D N S -S W -S T A 001-0-050823 (C0091543 Spk F, 100 ppb Lab Spike) D N S -S W -S TA 001-LS -050823 (C0091545,1.0 ppb Field Spike) D N S -S W -S TA001 -H S-050823 (C0091546, 10 ppb Field Spike) D N S -IW -S T A 002-0-050824 (C0091549 Spk C, 1.0 ppb Lab Spike) D N S -IW -S TA 002- -050824 (C0091549 Spk D, 10 ppb Lab Spike) D N S -IW -S TA 0Q 2-LS -050824 (C0091551, 1.0 ppb Field Spike) D N S -IW -S TA 002-H S -050824 (C0091552.10 ppb Field Spike) C4 Sulfonate PFBS ________ C6 Sulfonate PFHS__________ C8 Sulfonate PFOS Amount Amt Found Amount Amt Found Amount Amt Found Amount Spiked In Sample Recovered Recovery In Sample Recovered Recovery in Sample Recovered Recovery (ng/L) (ng/L) (ng/L) <%) (ng/L) (ng/L) (%) (ng/L) (ng/L) (%> 1000 10000 ND ND 1000 10000 ND ND 1000 10000 ND ND 1000 10000 1000 10000 682 682 682 682 10000 100000 1000 10000 111 111 111 111 1000 10000 1000 10000 364 364 364 364 913 8980 91 90 960 10800 96 108 843 10900 84 109 1710 10200 1410 8520 8550 80600 815 7970 103 95 73 78 84 80 70 79 1120 8690 1120 7740 76 83 76 74 ND ND ND ND ND ND 790 790 790 790 2 4 .0 2 4 .0 2 4 .0 2 4 .0 620 620 620 620 970 11100 97 111 1160 11100 116 111 1090 10800 109 108 2290 13000 2170 12400 9470 90800 924 8610 150 122 138 116 94 91 90 86 1320 9950 1240 8940 70 93 62 83 ND 1200 120 ND 11700 117 ND 1180 118 ND 10500 105 ND ND 20000 20000 20000 20000 164 164 164 164 977 10600 18700 25900 18400 25100 8870 85200 859 7230 98 106 . 59 . 51 87 85 70 71 2860 2860 2860 2860 4230 9740 3970 8850 137 69 111 60 'S am ple residue exceeds the spiking level significantly (3x spiking level); therefore, an accurate recovery value cannot be calculated ND = Not detected at or above the Limit of Quantitation (LOQ) of 25 ng/L. Note: Since this summary table shows rounded results, recovery values may vary slightly from the values in the raw data. Exygen Research Page 28 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 Table IV. Matrix Spike RSaecmopvleersyCoofnPtFinBuSe,dPFHS and PFOS in Water Sample Description D N S -S W -S TA 002-0-050825 (C0091553 Spk E. 1.0 ppb Lab Spike) D N S -S W -S TA 002-0-050825 (C00S1S53 Spk F. 10 ppb Lab Spike) D N S -S W -S TA 002-LS -050825 (C0091555, 1.0 ppb Field Spike) D N S -S W -S TA 002-H S -050825 (C0091556.10 ppb Field Spike) D N S -IW -S T A 003-0-050824 (C0091558 Spk G, 1.0 ppb Lab Spike) D N S -IW -S T A 003-0-050824 (C0091558 Spk H. 10 ppb Lab Spike) D N S -IW -S T A 003-LS -050824 (C0091560, 1.0 ppb Field Spike) D N S -IW -S T A 003-H S -050824 (C0091561, 10 ppb Field Spike) D N S -S W -S TA 003-0-050824 (C0091562 Spk C. 1.0 ppb Lab Spike) D N S -S W -S TA 003-0-050824 (C0091562 Spk D, 10 ppb Lab Spike) D N S -S W -S TA 003-LS -050824 (C0091564,1.0 ppb Field Spike) D N S -S W -S TA 003-H S -050824 (C0091565,10 ppb Field Spike) D N S -IW -S T A 004-0-050824 (C0091588 Spk E. 10 ppb Lab Spike) D N S -IW -S T A 004-0-050824 (C0091588 Spk F, 100 ppb Lab Spike) D N S -IW -S TA 004-LS -050824 (C0091590,1.0 ppb Field Spike) D N S -IW -S TAQ Q 4-H S -050824 (C0091591,10 ppb Field Spike) D N S -S W -S TA 004-0-050824 (C0091S92 Spk G. 1.0 ppb Lab Spike) D N S -S W -S TA 004-0-050824 (C0091592 Spk H, 10 ppb Lab Spike) D N S -S W -S TA 004-LS -050824 (C0091594, 1.0 ppb Field Spike) D N S -S W -S TA 004-H S -050824 (C0091595,10 ppb Field Spike) C4 Sulfonate PFBS______________ C6 Sulfonate PFHS______________ C8 Sulfonate PFOS Amount Amt Found Amount Amt Found Amount Amt Found Amount Spiked in Sample Recovered Recovery in Sample Recovered Recovery in Sample Recovered Recovery (ng/L) (ng/L) (ng/L) (%> (ng/L) (ng/L) (%) (ng/L) (ng/L) (%) 1000 10000 1000 10000 140 140 140 140 1000 10000 1000 10000 1000 10000 1000 10000 76.4 7 6 .4 76.4 76.4 58.7 58.7 58.7 58.7 10000 100000 1000 10000 7 5 .8 7 5 .8 7 5 .8 7 5 .8 1000 10000 1000 10000 134 134 134 134 889 8820 989 7330 913 8130 849 8830 949 9040 860 8850 8140 60900 783 7010 1030 8770 1010 8480 75 87 85 72 84 81 77 88 89 90 80 88 81 61 71 69 90 86 88 83 5 9 .2 59.2 59.2 59.2 51.1 51.1 51.1 51.1 4 2 .7 4 2 .7 4 2 .7 4 2 .7 4 5 .2 4 5 .2 4 5 .2 4 5 .2 178 178 178 178 976 9850 988 7610 92 98 93 76 814 8820 1090 9450 1140 11400 1280 12100 76 88 104 94 110 114 124 121 9630 77100 810 7920 96 77 76 79 1350 11600 1470 12500 117 114 129 123 403 403 403 403 645 645 645 645 180 180 180 180 184 184 184 184 555 555 555 555 1310 9530 1670 8040 91 91 127 76 1510 9080 1880 8350 1050 8950 1380 10900 87 84 124 77 87 88 120 107 9030 73800 764 5710 88 74 58 55 1540 9240 1630 8790 99 87 108 82 ` Sample residue exceeds the spiking level significantly (3x spiking level); therefore, an accurate recovery value cannot be calculated ND = Not detected at or above the Limit of Quantitation (LOQ) of 25 ng/L. Note: Since this summary table shows rounded results, recovery values may vary slightly from the values in the raw data. Exygen Research Page 29 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 Table IV. Matrix Spike RSaecmopvleersyCoofnPtFinBuSe,dPFHS and PFOS in Water Sample Description D N S -IW -S T A 005-0-050823 (C0091596 Spk C. 1.0 ppb Lab Spike) D N S -IW -S T A 005-0-050823 (C0091596 Spk D. 10 ppb Lab Spike) D N S -IW -S T A 005-LS -050823 (C0091S9S, 1.0 ppb Field Spike) D N S -IW -S TA 005-H S -050823 (C0091599,10 ppb Field Spike) D N S -S W -S TA 005-0-050823 (C0091600 Spk E, 1.0 ppb Lab Spike) D N S -S W -S TA 005-0-050823 (C0091600 Spk F, 10 ppb Lab Spike) D N S -S W -S TA 005-LS -050823 (C0091602,1.0 ppb Field Spike) D N S -S W -S TA 005-H S -050823 (C0091603,10 ppb Field Spike) D N S -IW -S T A 006-0-050824 (C0091604 Spk G, 1.0 ppb Lab Spike) D N S -IW -S T A 006-0-050824 (C0091604 Spk H, 10 ppb Lab Spike) D N S -IW -S T A 006-LS -050824 (C0091606, 1.0 ppb Field Spike) D N S-lW -S T A 006-H S -050824 (C0091607, 10 ppb Field Spike) D N S -S W -S TA 006-0-050824 (C0091608 Spk I, 10 ppb Lab Spike) D N S -S W -S TA 006-0-050824 (C0091608 Spk J, 100 ppb Lab Spike) D N S -S W -S TA 006-LS -050824 (C0091610, 1.0 ppb Field Spike) D N S -S W -S TA 006-H S -050824 (C0091611,10 ppb Field Spike) D N S -IW -S T A 007-0-050825 (C0091612 Spk C, 1.0 ppb Lab Spike) D N S -IW -S T A 007-0-050825 (C0091612 Spk D, 10 ppb Lab Spike) D N S -IW -S TA 007-LS -050825 (C0091614,1.0 ppb Field Spike) C4 Sulfonate PFBS C6 Sulfonate PFHS C8 Sulfonate PFOS Amount Amt Found Amount Amt Found Amount Amt Found Amount Spiked in Sample Recovered Recovery In Sample Recovered Recovery in Sample Recovered Recovery (ng/L) (ng/L) (ng/L) (%) (ng/L) (ng/L) (%) (ng/L) (ng/L) (%) 1000 10000 1000 10000 86.9 8 6 .9 8 6 .9 8 6 .9 1000 10000 1000 10000 6 4 .5 6 4 .5 6 4 .5 6 4 .5 1000 10000 1000 10000 58.2 58.2 58.2 58.2 10000 100000 1000 10000 NR NR NR NR 1000 10000 1000 578 578 578 945 9450 987 8470 86 94 90 84 845 9920 909 9870 940 9070 1020 10200 78 99 84 98 88 90 96 101 NR NR NR NR NR NR NR NR 1540 10100 1410 96 95 83 21.1 21.1 21.1 21.1 16.9 16.9 16.9 16.9 5 7 .4 5 7 .4 5 7 .4 5 7 .4 3 9 .6 3 9 .6 3 9 .6 3 9 .6 703 703 703 1120 11900 1270 9370 110 119 125 93 990 11900 1200 13100 1150 11000 1360 12800 97 119 118 131 109 109 130 127 10700 87000 696 9680 107 87 66 96 1720 11800 1670 102 111 97 167 995 83 167 9020 89 167 1010 84 167 9980 98 124 124 124 124 263 263 263 263 777 8800 863 10600 1130 8510 1320 10300 65 87 74 105 87 82 106 100 NR NR NR NR NR NR NR NR NR NR NR NR 16200 16200 16200 18100 26200 17400 100 * 'S am ple residue exceeds the spiking level significantly (3x spiking level); therefore, an accurate recovery value cannot be calculated NO = Not detected at or above the Limit of Quantitation (LOQ) of 25 ng/L. NR = Not reported due to quality control failure. Note: Since this summary table shows rounded results, recovery values may vary slightly from the values in the raw data. Exygen Research Page 30 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 Table IV. Matrix Spike RSaecmopvleersyCoofnPtFinBuSe,dPFHS and PFOS in Water Sample Description D N S -S W -S TA 007-0-050825 (C0091615 Spk E. 1.0 ppb Lab Spike) D N S -S W -S TA 007-Q -050825 (C0091615 Spk F, 10 ppb Lab Spike) D N S -S W -S TA 007-LS -050825 (C0091617, 1.0 ppb Field Spike) D N S -S W -S TA 007-H S -050825 (C0091618,10 ppb Field Spike) D N S -IW -S T A 008-0-050825 (C0091619 Spk G, 1.0 ppb Lab Spike) D N S -IW -S TA 0Q 8-0-05G 825 (C0091619 Spk H. 10 ppb Lab Spike) D N S -IW -S T A 008-LS -050825 (C0091621, 1.0 ppb Field Spike) D N S -IW -S TA 008-H S -050825 (C0091622,10 ppb Field Spike) D N S -S W -S TA 008-0-050825 (C0091623 Spk I, 1.0 ppb Lab Spike) D N S -S W -S TA 008-0-050825 (C0091623 Spk J, 10 ppb Lab Spike) D N S -S W -S TA 008-LS -050825 (C0091625, 1.0 ppb Field Spike) D N S -S W -S TA 008-H S -050825 (C0091626.10 ppb Field Spike) D N S -IW -S T A 009-0-050826 (C0091627 Spk C, 1.0 ppb Lab Spike) D N S -IW -S T A 009-0-050826 (C0091627 Spk D, 10 ppb Lab Spike) D N S -IW -S TA 009-LS -050826 (C0091629,1.0 ppb Field Spike) D N S-lW -S T A 009-H S -050826 (C0091630, 10 ppb Field Spike) D N S -S W -S TA 009-0-050826 (C0091631 Spk E, 1.0 ppb Lab Spike) D N S -S W -S TA 009-0-050826 (C0091631 Spk F. 10 ppb Lab Spike) D N S -S W -S TA 009-LS -050826 (C00915633, 1.0 ppb Field Spike) D N S -S W -S TA 009-H S -050826 (C0091634,10 ppb Field Spike) C4 Sulfonate PFBS______________ C6 Sulfonate PFHS______________ C8 Sulfonate PFOS Amount Amt Found Amount Amt Found Amount Amt Found Amount Spiked In Sample Recovered Recovery in Sample Recovered Recovery in Sample Recovered Recovery (ng/L) (ng/L) (ng/L) (%) (ng/L) (ng/L) (%) (ng/L) (ng/L) (%) 1000 10000 1000 10000 132 132 132 132 1000 10000 1000 10000 6020 6020 6020 6020 1000 10000 1000 10000 217 217 217 217 1000 10000 1000 10000 8580 8580 8580 8580 1000 10000 1000 10000 53.4 53.4 53.4 53.4 979 9300 995 8960 6810 15400 6180 14500 1160 9440 934 8200 11100 22400 11700 19500 909 18700 958 9100 85 92 86 88 * 94 * 85 94 92 72 80 . 138 * 109 86 186 90 90 5 6 .9 5 6 .9 5 6 .9 5 6 .9 5190 5190 5190 5190 158 158 158 158 1100 9920 1280 11000 6930 17100 7310 16300 1320 11900 1090 10600 104 99 122 109 . 119 * 111 116 117 93 104 11800 11800 11800 11800 12800 27600 13500 24700 158 129 4 0 .7 4 0 .7 4 0 .7 4 0 .7 1130 11500 1290 12600 109 115 125 126 388 388 388 388 901 901 901 901 NR NR NR NR 3260 3260 3260 3260 134 134 134 134 986 9060 1290 7570 60 87 90 72 1810 8370 1900 6950 NR NR NR NR 4920 12400 5500 14500 91 75 100 60 NR NR NR NR . 91 112 932 8850 1080 8500 80 87 95 84 ` Sample residue exceeds the spiking level significantly (3x spiking level); therefore, an accurate recovery value cannot be calculated ND = Not detected at or above the Limit of Quantitation (LOQ) of 25 ng/L. NR = Not reported due to quality control failure. Note: Since this summary table shows rounded results, recovery values may vary slightly from the values in the raw data. Exygen Research Page 31 of 127 Interim Report #17- Analysis of Sediment and Water Samples Exygen Study No. : P0001131 Table IV. Matrix Spike RSaecmopvleersyCoofnPtFinBuSe,dPFHS and PFOS in Water Sample Description D N S -IW -S T A 010-0-050826 (C0091635 Spk G, 1.0 ppb Lab Spike) D N S -IW -S T A 010-0-050826 (C0091635 Spk H, 10 ppb Lab Spike) D N S -IW -S TA 010-LS -Q 50826A (C0091637, 1.0 ppb Field Spike) D N S -S W -S T A 010-0-050826 (C0091638 Spk C. 10 ppb Lab Spike) D N S -S W -S TA 010-0-050826 (C0091638 Spk 0.100 ppb Lab Spike) D N S -S W -S TA 010-LS -050826 (C0Q91640, t.Q ppb Field Spike) D N S -S W -S TA 010-H S -050826 (C0091641,10 ppb Field Spike) D N S -IW -S T A 011-0-050826 (C0091642 Spk E, 1.0 ppb Lab Spike) D N S -IW -S T A 011-0-050826 (C0091642 Spk F. 10 ppb Lab Spike) D N S -S W -S T A 0 1 1-0-050826 (C0091644 Spk G, 10 ppb Lab Spike) D N S -S W -S T A 0 1 1-0 -0 5 0 8 2 6 (C0091644 Spk H, 100 ppb Lab Spike) D N S -S W -S T A 011-LS -050826 (C0091646, 1.0 ppb Field Spike) D N S -S W -S T A 0 1 1-H S -050826 (C0091647, 10 ppb Field Spike) D N S -IW -S T A 012-0-050826 (C0091648 Spk C, 1.0 ppb Lab Spike) D N S -IW -S T A 012-0-050826 (C0091648 Spk D, 10 ppb Lab Spike) D N S -IW -S T A 012-LS -050826 (C0091650, 1.0 ppb Field Spike) D N S -IW -S TA Q 12-H S -050826 (C0091651, 10 ppb Field Spike) C4 Sulfonate PFBS______________ C6 Sulfonate PFHS______________ C8 Sulfonate PFOS Amount Amt Found Amount Amt Found Amount Amt Found Amount Spiked in Sample Recovered Recovery in Sample Recovered Recovery in Sample Recovered Recovery (ng/L) (ng/L) (ng/L) <%) (ng/L) (ng/L) <%) (ng/L) (ng/L) (%) 1000 10000 10526 NR NR NR NR NR 646 2130 128 19200 19200 . NR NR 846 12500 117 19200 28800 96 NR NR 846 7900 67 19200 27500 * 10000 100000 1000 10000 73.2 73.2 73.2 73.2 16800 63600 758 6990 167 64 68 69 53.6 5 3 .6 53.6 5 3 .6 12800 86500 1080 10200 127 86 103 101 139 18400 183 139 66500 66 139 832 69 139 6580 64 1000 10000 1880 1880 2840 10700 96 88 844 2020 118 844 11900 111 850 850 1730 7860 88 70 10000 100000 1000 10000 NR NR NR NR 1000 10000 1000 10000 8730 8730 8730 8730 NR NR NR NR 9060 19300 8840 16100 NR NR NR NR . 106 74 3 9 .4 39.4 3 9 .4 3 9 .4 NR NR NR NR 9310 97500 746 6640 NR NR NR NR 93 97 71 66 NR NR NR NR 9 6 .4 9 6 .4 9 6 .4 96.4 1330 1330 1330 1330 7110 78500 607 4700 2090 10800 2030 7650 70 78 51 46 76 95 70 63 'S am ple residue exceeds the spiking level significantly (3x spiking level); therefore, an accurate recovery value cannot be calculated ASample bottle was received with only 19 mL of water. ND = Not detected at or above the Limit of Quantitation (LOQ) of 25 ng/L. NR = Not reported due to quality control failure. Note; Since this summary table shows rounded results, recovery values may vary slightly from the values in the raw data. Exygen Research Page 32 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 Table IV. Matrix Spike RSaecmopvleersyCoofnPtFinBuSe,dPFHS and PFOS in Water Sample Description D N S -S W -S TA 012-0-050826 (C0091652 Spk E, 1.0 ppb Lab Spike) D N S -S W -S TA 012-0-050826 (C0091652 Spk F, 10 ppb Lab Spike) D N S -S W -S TA 012-LS -050826 (C0091654, 1.0 ppb Field Spike) D N S -S W -S TA 012-H S -050826 (C0091655,10 ppb Field Spike) C4 Sulfonate PFBS______________ C6 Sulfonate PFHS______________ C8 Sulfonate PFOS Amount Amt Found Amount Amt Found Amount Amt Found Amount Spiked in Sample Recovered Recovery in Sample Recovered Recovery in Sample Recovered Recovery (ng/L) (ng/L) (ng/L) i% ) (ng/L) (ng/L) (%) (ng/L) (ng/L) <*) 1000 5 3 .3 886 83 4 2 .2 1160 112 142 1000 86 10000 5 3 .3 9030 90 4 2 .2 10500 105 142 7860 77 1000 5 3 .3 870 82 4 2 .2 1100 106 142 971 83 10000 5 3 .3 7620 76 4 2 .2 10300 103 142 7500 74 Average; Standard Deviation: 88 18 Average: Standard Deviation; 105 19 ` Sample residue exceeds the spiking level significantly (3x spiking level); therefore, an accurate recovery value cannot be calculated ND = Not detected at or above the Limit of Quantitation (LOQ) of 25 ng/L. Note; Since this sum m ary table show s rounded results, recovery values may vary slightly from the values in the raw data. Average: Standard Deviation: 87 22 Exygen Research Page 33 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 Table V. Total Percent Solids in Sediment Samples Exygen ID C0091547 C0091557 C0091566 C0091579 C0091580 C0091581 C0091582 C0091583 C0091584 C0091585 C0091586 C0091587 Client Sample ID DNS-SD-STA001 -0-050824 DNS-SD-STA002-0-050824 DNS-SD-STA003-0-050824 DNS-SD-STA004-0-050825 DNS-SD-STA005-0-050825 DNS-SD-STA006-0-050825 DNS-SD-STA007-0-050825 DNS-SD-STA008-0-050825 DNS-SD-STA009-0-050825 DNS-SD-STA010-0-050825 DNS-SD-STA011-0-050825 DNS-SD-STA012-0-050825 Total Percent Solids (%) 57.70 52.23 45.84 70.28 59.06 72.13 56.01 59.64 61.43 57.91 56.54 46.84 Exygen Research Page 34 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 FIGURES Exygen Research Page 35 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 Figure 1. MTyepthicaanloNlon-Extracted Calibration Curve for PFBS in 1 1 2 1 0 5 A S e d im e n t.rd b (P FB S ): "Linear" Regression ("1 /x " w eightin g): y 7 .0 6 e + O O 4 x + 4 .3 3 e + 0 0 3 (r = 0 .9 9 8 8 ) Area, counts Exygen Research Page 36 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 Figure 2. Typical Extracted Calibration Curve for PFBS in Methanol 1 1 1 4 0 5 C P ore W a te r.rd b (P F B S ): '' Linear1' Regression ("1 / * ' w e ig h tin g ): y = 1 5 7 x + 0 .0 0 0 3 8 9 (r - 0 .9 9 8 7 ) Area, counts Exygen Research Page 37 of 127 Interim Report #17- Analysis of Sediment and Water Samples Exygen Study No.: P0001131 Figure 3. Nanodn-0E.5xtnrga/cmteLd, SRteasnpdeacrtdivseloyf PFBS in Methanol, 0.2 ng/mL I SAS0reOaT:081669049PcFoBuSn t(sStHanedigahrtd: ) 29%0/9%0 am f.3fe+O03cp* uRT- s:a0m.5p3l2em1ion f 24 from 112105G.wiff 0.5 3 A rea: 4 0 0 2 3 counts H e ig h t 3 .2 9 e + Q 0 3 cps R T: 0 .5 2 3 m in 0 .5 2 Exygen Research Page 38 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 Figure 4. Engx/tLra, cRteedspSectatinvdealyrds of PFBS in Methanol, 25 ng/L and 50 XACr1e1a2:340957-11 c-PoFunBtSs (HSeta/gnfdtta:r3d.)972e9+9Q.00/929c.p0sfirRmT:-Asa6m07pmlein2 of 44from m i0 5 a w iff 0 .6 1 &Cfl A re a : 8 9 4 8 counts H e ight: 7 .5 2 e + 0 0 2 cps R T: 0 .6 0 0 m in 0.6 0 Exygen Research Page 39 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 Figure 5. PFSpForkBtSiBfi,ienRde1Rs%peeaAcgtecinvetetilcSypAkciAd,Raneadg4e.n0tnBgl/amnkL, F0o.4rtnifgi/emdLReagent I R epaegaenktnCootnfotruonld-)PFBS (Unknown) 299.0/99.0 am u -sam ple O of 24 from 112105C.wiff T im e , m in I R e ag en t Spk A - PFBS (Q C ) 2 9 9 .0 /9 9 .0 a m u -s a m p le Q of 2 4 from 1 121 05 C .w iff A re a: 3 2 0 0 7 counts H e ight: 2 .5 7 e + 0 0 3 cps R T: 0 .5 2 5 m in 0 .5 2 co o. <_> & CcOz a> c I Tim e, min R e ag en t Spk B - PFB S (Q C ) 2 9 9 .0 /9 9 .0 a m u -s a m p le 10 of 2 4 from 1 121 05 C .w iff A rea: 3 0 2 1 7 1 counts Height: 2 .4 8 e + 0 0 4 cps RT: 0 .5 2 4 min 0.5 2 Exygen Research Page 40 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 Figure 6. PSpFkBSA,inanWda5t0e0r Rnge/aLgeFnotrBtilfaiendk,R5e0agnegn/Lt SFpokrtBif,ieRdesRpeeacgtievnetly R epaegaenktnCoot nfotruonld-P) F B S (Unknown) 299.W99.0amn -sam ple 9 o f4 4 fro m ifiiO S ftw iff 1 2 .1 9 T im e , min R eag ent Spk A - PFBS (Q C ) 2 9 9 .0 /9 9 .0 am u - sam ple 10 of 4 4 from 1 12105D .w iff A re a: 8 5 7 S counts H e ig h t: 6 . 7 9 e + 0 0 2 cps R T: 0 .0 1 1 m in 0 .6 1 T im e, min R eag en t Spk B - PFB S (Q C ) 2 9 9 .0 /9 9 .0 am u - sam ple 11 of 4 4 from 112105D,vuiff A rea: 9 3 4 4 4 counts H eight: 8 . 1 5 e + 0 0 3 cps RT: 0 .0 2 5 min 0 .6 2 Exygen Research Page 41 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 Figure 7. fCohrrPoFmBaSto(gErxaymgeRneIpDr:esCen0t0i9n1g5a86S,eDdaimtaenStetS:a1m12p1le05ACn)alyzed Intensity, cps I CO09T586 - P F B S (Unknown) 299.0/99.0 amu -sam pfe 15 o f2 4fro n t i f 2f 05C.w/ff A rea: 271S4counts Height: 1.G9e+Q03cps R T : 0.529min 0.53 1600 1500 1400 - 12001300 11001000- 900 800 - 700 600 500 400 300 200 100 0 2 3 4 5 6 7 8 9 10 11 12 13 1 4 15 16 17 T im e , m in Exygen Research Page 42 of 127 Interim Report #17- Analysis of Sediment and Water Samples Exygen Study No.: P0001131 Figure 8. fCohrrPoFmBaSto(gErxaymgeRneIpDr:esCen0t0i9n1g6a52W, Dataetra SSaemt:p1l1e2A10n5aDly)zed 0A00re9a7:6592132PcFoBuSnt(sUnHkeniogwhtn:)S2.3919.e0+/9090.20capms uRT-s:a0m.6p4l2em2i3no f 44 from 112105D.wiff 0 .6 4 Intensity, cps Exygen Research Page 43 of 127 Interim Report #17- Analysis of Sediment and Water Samples Exygen Study No. : P0001131 Figure 9. TMyepthicaanloNl on-Extracted Calibration Curve for PFHS in 1 1 2 1 0 5 A S e d im e n t.rd b (P F H S ): "L in e a i'1 Regression ("1 / x " w eig h tin g ): y = 1 .2 4 e + 0 0 5 x + 7 7 7 (r = 0 .9 9 9 0 ) Area, counts Exygen Research Page 44 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 Figure 10. Typical Extracted Calibration Curve for PFHS in Methanol !>?m t l O S C P o ie W a te r.rd b (P F H S ): "L in e a i" Regression C'1 w eighting): y = 3 7 6 x + 8 3 9 (r= 0 .9 9 6 3 ) Area, counts Exygen Research Page 45 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 Figure 11. Nanodn-0E.5xtnrga/cmteLd, SRteasnpdeacrtidvseloyf PFHS in Methanol, 0.2 ng/mL Area: 24963 count(sStHanedigahrtd:)319.096Ae)+m0.0i3>capms uRT-s:a8mw3p2lemi1tto f 24 from 112105C.wiff 1000- B 400 - -- ,------ -------- , ------- ,------ --------1 in " i * -- 123456789 10 Tim e, min S S 0 0 0 9 7 4 9 - PFH S (S ta n d a rd )3 9 9 .0 /8 0 .0 am u - sam ple 2 of 2 4 from 1 12105C.uviff A re a : 5 8 1 3 5 counts H e ig h t: 2 .4 3 e + 0 0 3 ops R T : 8 .2 9 m in 12 15 Exygen Research Page 46 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 Figure 12. Engx/tLra, cRteedspSectatinvdealyrds of PFHS in Methanol, 25 ng/L and 50 TM XACr1e1a2:370752-14 c- PouFnHtSs (HSteaignhdta:r3d.6) 53e9+90.00/280c.p0samPTu: -8s.a4m3 mp lien 2 o f 44 from 112105D.wiff 8 .4 3 CO2O. & CO c 0) I A re a : 1 4 8 7 2 counts H e ig h t: 5 .9 0 e + 0 0 2 cps R T : 8 .4 4 m in 8 .4 4 (O Q_ o CO Exygen Research Page 47 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 Figure 13. PFSpForkHtiBSfi,eiRnde1Rs%peeacAgtecinveettilcSypAkciAd, Ranedag4e.n0tnBgl/amnLk,F0o.4rtnifgie/mdLReagent I R epaegaenktnCootnfotruonl d-)PFHS (Unknown) 399.0/30.0 anw -sam ple 8 o f 24 from 1121tS5C.wiff (Cnl <_> I Tim e, min R eag en t Spk A * PFH S (Q C ) 3 9 9 .0 /8 0 .0 am u - sam ple 9 of 2 4 from 1 12105C .w iff A rea: 4 9 0 4 6 counts Height: 2 . 1 6 e + 0 0 3 cps R T: 8 .3 8 min 5 1 0 0 0 L8.38 7 .0 7 . I 8 9 10 11 12 13 1 4 15 16 17 T im e , min I R e a g e n tS p k B - P F H S (Q C ) 3 9 9 .0 /8 0 .0 am u - sam ple 10 of 2 4 from 1 12105C.vuiff A rea: 4 9 7 9 8 3 counts H eight: 2 .0 6 e + 0 0 4 cps R T :8 .3 5 min Exygen Research Page 48 of 127 Interim Report#17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 Figure 14. PSpFkHAS,inanWd a5t0e0r nRge/aLgFenotrtBifliaendkR, 5e0agnegn/tLSFpokrBtif,iRedesRpeeactgievnetly R efargeaenktnCootnfotruonl d')PFHS (Unknown) a399.0/80.0 nta -sanrpfe 9 o f 4 4 front f2 0 5 ftw 'ff 0 .5 9 2.35. v A A 13, 23456789 1 0 .1 2 1 2 .0 3 ^ 12,58 1442 1210 11 13 14 15 16 17 T im e , min R eagent Spk A - PFHS (Q C ) 3 9 9 .0 /8 0 .0 am u - sam ple 10 of 4 4 from 112105D .w iff A re a: 1 5 5 8 0 counts H eig h t: 7 .5 6 e + 0 0 2 ops R T : 8 . 5 5 m in 8 .5 5 co AQ. "cCO c oe I Tim e, min R eag en t Spk B - PFHS (Q C ) 3 9 9 .0 /8 0 .0 am u - sam ple 11 of 4 4 from 1 12105D .w iff A rea: 1 4 3 7 2 8 counts Height: 6 .7 8 e + 0 0 3 cps R T: 8 .5 8 m in 8 .5 8 Exygen Research Page 49 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 Figure 15. fCohrrPoFmHaSto(gEraxmygeRnepIDre:sCen0t0i9n1g5a87S,eDdiamtaenStetS:a1m1p2l1e0A5Cn)alyzed I CA0r0e9a1:539273-P62FcHoSun(UtsnHkneoigwhnt:)329.290.<V+8000.30acmpsa R-sTa:m8.p4l5em2i1no f 24 from 112105C.wiff Intensity, cps Exygen Research Page 50 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 Figure 16. PCFhHroSm(aEtxoyggreanmIRD:epCr0e0se9n1t5i4n9g, aDWataatSeert:Sa1m11p4l0e5ACn)alyzed for Intensity, cps I CA0rOe9aT:542934-3P69FHcoSu(nUtsnkHnoeiwgnh)t:39?9.L805/e80+LOO0a4cmpu* -RsTa:m1p0l.2e mT6itot f 38 from 1 .8 e 4 - 1 0 .1 7 1 .7 e 4 - 1 .6 e 4 - 1 .5 e 4 - 1 .4e 4 - 1,3e 4 - 1 .2 e 4 - 1 .1 e 4 - 1.0e 4 - 9 00 0.0 - 8 0 0 0 .0 7 00 0.0 - 6 00 0.0 - 6 00 0.0 - 4 00 0.0 - 3000.0 - 2000.0 - 1000.0 - 0.S 4 0 .0 1 2 3 4 6 6 7 8 9 10 11 12 13 1 4 1 6 16 17 T im e , min Exygen Research Page 51 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 Figure 17. MTyepthicaanloNlon-Extracted Calibration Curve for PFOS in 1 1 2 1 0 5 A S e d im e n t.rd b (P F O S ): "L in e a i" Regression ("1 / x " w e ig h tin g ): y = 1 .1 5 e + 0 0 5 x + S .6 e + 0 0 3 ( r = 0 .9 0 9 1 ) Area, counts Exygen Research Page 52 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 Figure 18. Typical Extracted Calibration Curve for PFOS in Methanol 1 1 1 4 0 5 C Pore W ater.rdb (P F O S ): "Linear" Regression ('1 / * ' w eighting): y = 3 0 9 x + 5 8 7 (r = 0 .9 9 8 0 ) Area, counts Exygen Research Page 53 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 Figure 19. Nanodn-0E.5xtnrga/cmteLd, SRteasnpdeacrtidvseloyf PFOS in Methanol, 0.2 ng/mL I SSAQreQa1:09264958- 7PcFoOuSn t(sS tHa ne idgahrtd: ) 499M 0.0 am 15te+003cp* nRT-s: a1m1 .p0/me i1no f 24 from 112105C.wiff 11.02 I S S 0 0 0 9 7 4 9 - PFO S (S tandard) 4 9 9 .0 /8 0 .0 a m u -s a m p le 2 of 2 4 from 1 12105C .w iff A re a: 5 9 3 9 9 counts H e ight: 3 .8 4 e + 0 0 3 cps R T: 1 1 .0 m in 11.01 Exygen Research Page 54 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 Figure 20. Engx/tLra, cRteedspSectatinvdealyrds of PFOS in Methanol, 25 ng/L and 50 I .XACr1e1a2:39025-913-cPoFunOtSs (HSetaignhdta: r5dL)3459e+90.MO2ttctp.QffaRmTu: -1s0a.9mmpliea 2 o f 44 from 112105tXwiff CL O & CO c a> c I X C 1 1 23 05 *2 P FO S (S ta n d a rd )4 9 9 .0 /8 0 .0 am u sam ple 3 of 4 4 from 112 10 5 D .w iff A re a : 1 5 2 9 1 counts H e ight: 9 .7 3 e + 0 0 2 ops R T: 1 1 .0 m in 1 0 .0 5 Exygen Research Page 55 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 Figure 21. PFSpForkOtiBSfi,eiRnde1Rs%peeacAgtecinveettilScypAkcAid, Ranedag4e.n0tnBgl/amnLk,F0o.4rtnifgie/mdLReagent I RAeraegae:n9t3C5ocnoturnotts- PHFeOigSftt(:UXn1k3neo+wQnQ)14c9p9sUVR9TA:01a1n.0tnm-itstam ple 9 o f 24 from 112105C.wiff 1 1 .0 5 T im e , min R e ag en t Spk A - P FO S (0 0 )4 9 9 .0 /8 0 .0 am u - sam ple 9 of 2 4 from 1 121 05 C .w iff A re a: 4 7 4 0 6 counts H eig h t: 3 .1 3 e + 0 0 3 cps R T: 11.1 m in 3000 co o3. 2000 - <s> 1000 - 0 J---- -----------------1---- ------1---- ------.---- ------.---- ------.---- ------.---- ------r 123 45 6 78 Tim e, min R e ag en t Spk B - P FO S (Q 0 )4 0 9 .0 /8 0 .0 am u - sam ple 10 of 2 4 from 1 12 10 5 C .w iff A re a : 4 5 8 0 7 6 counts H e ight: 2 .9 5 e + 0 0 4 cps R T: 1 1 .0 m in 1 1 .0 7 1 1 .0 5 Exygen Research Page 56 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 Figure 22. PSpFkOAS,inanWd a5t0e0r nRge/aLgFenotrtBifliaendkR, 5e0agnegn/tLSFpokrBtif,iRedesRpeeactgievnetly R e(apgeeanktnCootnfotruonfd-)PFOS (Unknown) 499M 0.0 am u -sam ple 9 o f 44 from 112105D,wfF 1 1 .0 5 T im e , min R e ag en t Spk A - P FO S (0 0 ) 4 0 9 .0 /8 0 .0 am u - sam ple 10 of 4 4 from 112105D.iAiiff A rea: 1 6 0 8 5 counts Height: 1 .0 6 e + 0 0 3 cps R T: 1 1 .0 min T im e , min R e a g e n t Spk 8 - P F O S ( Q 0 ) 4 0 9 . 0 / 8 0 . 0 am u - s a m p le 11 of 4 4 from 1 1 21 05 D .w iiff A re a: 1 6 0 0 5 7 counts H e ight: 9 .7 1 e + 0 0 3 cps R T: 1 1 .0 m in 1 1 .0 4 Exygen Research Page 57 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 Figure 23. fCohrrPoFmOaSto(gEraxmygeRnepIDre:sCen0t0in91g5a66S,eDdiamtaenStetS:a1m1p2l1e0A5An)alyzed Intensity, cps I CAOr0e9a7:565637-8P6F9OcoSu(nUtns kHnoewignh)t:4939.9.02/e8+0.00$a4mcpns -RsaTm: 1p1l.e1 2m4ino f 37 from 112105A.witf 11.11 3 .8 e 4 - 3 .6 e 4 - 3 .4 e 4 - 3 .2 e 4 - 3 .0 e 4 - 2 ,8 e 4 - 2 .0 e 4 2 .4 e 4 - 2 .2 e 4 - 2 .0 e 4 - 1 .8 e 4 - 1.6e4 - 1 ,4e4 - 1 .2e4 - 1 0e4 - 8 0 0 0 .0 - 10.60 6 00 0.0 - 4000.0 2000.0 - 0 .0 ------ ------- .------ -------- .-------------- ---------------.------ -------- .------ -------- 1------ --------,------ .-------r 123 45 67 8 Time, min Exygen Research Page 58 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 Figure 24. PCFhOroSm(aEtxoyggreanmIRD:epCr0e0se9n1t5i5n8g, aDWataatSeert:Sa1m11p4l0e5ACn)alyzed for Intensity, cps m CA0r0e9a7:551899-5P4F4cOoSu(nUtsnkHneoiwghnt):419.92.d0e/+M0004acmpus RsTa:m7p2l.3e m32ino f 38 from 111405C*wiff 1 .2 0 *4 - 12.28 1 .1 0 *4 - 1 .0 0 e 4 - Q00Q.00- 8000.00 - 7 000.00 - 6 000.00 5 000.00 - 11.84s* 4 000.00 - 3 000.00 - 2000.00 - 1000.00- 0.00 J-- *=-- -------------------------------------------. -----.---- ------.---- ------r 12 3 4 5 6 7 8 T im e , m in Exygen Research Page 59 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 APPENDIX A (EPxyrgoetonAcoSnSltauAtludyPmdyt0iyec0Nana0Pnold1rd.Mm1oP3tee0o1nt0chto0sol1d11s3a1n)dw2ith Exygen Research Page 60 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 STUDY PROTOCOL Study Title: PFeMirsfhalu,moCAmrlnoaaaomPllcyesStrsa,eifnVsrlueueosomgfurePlotfMUaheorstneoiifxoannlauntgietno,oLer(SrPsoCmiubnF/laugMOflotlPSanSMrn)a/MoetiangesmSur(WalPmffmooFaarntHleatrhSLte,e)i,Sv(3aePoMrniFld,aBDSnSede)dc,Saimmtuearnllt, Exygen Protocol Number: P0001131 Performing Laboratory: Exygen Research 3058 Research Drive State College, PA 16801 Phone: (814) 272-1039 Sponsor Representative: Michael A. Santoro Director of Regulatory Affairs 3M Building 0236-01-B-10 St. Paul, MN 55144 Phone: (651) 733-6374 Page / oj 65 Exygen Research Page 61 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 DISTRIBUTION: 1) Jaisimha Kesari, Study Director, Weston Solutions 2) John M. Flaherty, Principal Investigator, Exygen Research 3) Michael A. Santoro, Sponsor Representative, 3M Company 4) Exygen Research Quality Assurance Unit Exygen Research Page 2 of 65 Page 62 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 PROTOCOL APPROVAL Study Title: AUF(PinssFiahnHl,ygSCsL)il,saCamo/nMfsd,PSPVe/reMefrlgfuSleuotaforootrirobooutnhct,eatSan3nmeMesauslulDlfloMfeoncnaaamatteutemr(PM(aPFlFoBLnOSiivSt)oe,)rrPsiinneagrnWfldPuaroSotermgorr,haaSlemlxoMailn,aeSmseumdlfiaomlneSanetter,um Exygen Protocol Number: P0001131 APPROVALS JWaiessitmonhaSoCleuitionn,sStudy Director M3MichCaoeml pAa.riSyantoro, Sponsor Representative EbxhyngeMn.RFelasehaerrctyh, Principal Investigator y ' ERxicyhgaernd RAe. sGeraarczh,ni, Pn^sident, Facility Management Date 2P?-Ocl- Date ,ead, Quality Assurance Unit Page 3 of 65 Exygen Research Page 63 of 127 Interim Report #17- Analysis of Sediment and Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 TABLE OF CONTENTS TITLE PAGE.................................................................................................. DISTRIBUTION............................................................................................. PROTOCOL APPROVAL............................................................................. TABLE OF CONTENTS............................................................................... INTRODUCTION........................................................................................... TEST MATERIALS....................................................................................... OBJECTIVE................................................................................................... TESTING FACILITY..................................................................................... STUDY DIRECTOR....................................................................................... SPONSOR REPRESENTATIVE................................................................... PRINCIPAL INVESTIGATOR..................................................................... PROPOSED EXPERIMENTAL START AND TERMINATION DATES IDENTIFICATION AND JUSTIFICATION OF THE TEST SYSTEM .... SAMPLE PROCUREMENT, RECEIPT AND RETENTION.................... SAMPLE IDENTIFICATION....................................................................... ANALYTICAL PROCEDURE SUMMARY............................................... VERIFICATION OF ANALYTICAL PROCEDURE................................. METHOD FOR CONTROL OF BIAS......................................................... STATISTICAL METHODS.......................................................................... GLP STATEMENT........................................................................................ REPORT......................................................................................................... SAFETY AND HEALTH.............................................................................. AMENDMENTS TO PROTOCOL............................................................... DATA RECORD KEEPING......................................................................... QUALITY ASSURANCE............................................................................. RETENTION OF DATA AND ARCHIVING............................................. APPENDIX I, ANALYTICAL METHODS................................................. 1 32 4 5 5 6 b 7 7 7 7 8 8 9 9 91111 II 11111233 14 1514 Page 4 of 65 Exygen Research Page 64 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 INTRODUCTION T(PhFeBpSu)r,popseeroflfuothrioshsetxuadnyesiusltfoonpaetrefor(mPFaHnaSl)ysiasnfdor ppeerrfflluuoorrooboucttaanneessuullffoonnaattee (aPPnroFdgOsrSamm)ai.lnl mwaamterm, asloisle, rsuemdimuseinntg, fLisCh/,MclSa/mMsS, vfeogretthaetio3nM, smDealclamtuarmMmoanlitloivrienrgs The study will be audited for compliance with EPA TSCA Good Laboratory PRreascetaircceh.Standards 40 CFR 792 by the Quality Assurance Unit of Exygen TEST MATERIALS Tpehrefluorotheesxt anesumlfaotneraitaels(PFHaSr)e and ppeerrfflluuoorrooobcuttaanneessuullffoonnaattee (PFO(PSF)BaSn)d, are all supplied by 3M. PFBS MLCohotelNmecuiucmlaalbrNeWra:mei1eg0:h1tP: e3rf3l8uosruopbpuliteandeassultfhoenpaotetassium salt (C4F9SO3TC) Purity: 96.7% Transitions Monitored: 299 - 99 Structure: F F F F F S03 FF FF PFHMCLSohotelNmecuiucmlaalbrNeWra:meiSgeE:h0t:P3e64r3fl8uosruophpelxieadneassutlhfeonpaotteassium salt (CtF] jSOj'KT) PTSutrraruintcsytiu:tiroe9:n8s.6M%onitored: 399 - 80 Page 5 of 65 Exygen Research Page 65 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P000111 PFOLMCSohotelmNecuiucmlaalbrNeWra:mei2eg1:h7t:Pe5r3fl8uosruopopcltiaendeassultfhoenpaotetassium salt (C8F17SO3 IO Purity: 86.9% TStrrauncstiutrioe:ns Monitored: 499 -> 99 OBJECTIVE The purpose of this study is to perform analysis for perfluorobutanesulfonate ((acPPnuFFrdrBOesnSSmt))a,vilenl rpmsweioarafmntlesumro,orasfolothhiselee,xrfsuaoenmdleliosmfuwolerifnnottghn, eaEftie3xshMy,g(cePDlnaFemaHcnasSat,u)lyvrteiMagcneaodtlanmtiitopeonetrhir,nofslgdumsoP:arrloologmcrtaaammnemusuasillnfolginvtaehtrees V0001780: V0001781: V0001782: AAA"""MMMccciiieeedddttthhh(((PPPooodddFFFOOOoooAAAfff AAA))) iiinnnnnnaaalSllWSyyyeosssadiiiiltsssiembrfffyoooebnrrrLyttttChhhLbe/eeyCMDDDL/MSeeCe/tttMS/eeeM/rrrMSmmmS"iSii/nnnM"aaatttSiiiooo"nnn of of of Perfluorooctanoic Perfluorooctanoic Perfluorooctanoic V0001783: V0001784: V0001785: V0001786: AA""AA""MMMMcccciiiieeeeddddtttthhhh((((PPPPooooddddFFFFOOOOooooAAAAffff AAAA)))) iiiinnnnnnnnaaaaFllllVSSyyyyimmesssssiiiighaassssellalltffffanooooMMtdrrrrioaattttChnhmhhmleeeeabmmmyDDDDaaseeeellLttttbLSCeeeeyerrrri/vmmmmMrLueiiiimrSCnnnnb/aaaa/MMbyttttiiiiyooooSLSnnnnL"/CMCoooo/Mffff/SMPPPP"SeeeeS/rrrrM/ffffMlllluuuSuSoooo"rrr"rooooooooccccttttaaaannnnooooiiiicccc TESTING FACILITY E3PS0htxa5oyt8negeeCR:neo(Rs8lle1eea4gsre)eca,h2rP7cDA2hr-i1v106e38901 Page 6 of 65 Exygen Research Page 66 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 STUDY DIRECTOR jJWWPF1.kah4aieeeo0xssssin0:attm(eor6WC:inh1@h(ae0S6eswK)o1st7tol0eeeu0n)ssrt1ta,i7oWo-rP07nin1Aas4Ps-yo,0.3El1I17un9.6t,c3i1D.o8n0EsE.com SPONSOR REPRESENTATIVE M3D3SPMMthi.irocePnhCBcaeatuou:oelmilr(,l6dAopM5iafn.1NngRS)yae075ng235tu331ol6-4ra6o-4t03o17ry-4BA-f1f0airs PRINCIPAL INVESTIGATOR JE3So0txah5ytn8egeMRCneo.RslFleeelasagrehecae,hrrPctDyAhri1v6e801 jPohhonn.fela:(h8e1rt4y)@27e2x-y1g0e3n9.com PDRAOTEPOS SED EXPERIMENTAL START AND TERMINATION IOtetcrmitsoibnpearrotip0oo1n,sde2ad0te0tsh4awttoiltlhDbeeecaiennmcallbyuetdirecda3l1i,np2oth0ret0i5of.innaTol fhreetphoaiscrtt.usatul deyxpbeerimcoenndtaulctsetdartfraonmd Page 7 of 65 Exygen Research Page 67 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 Exygen Protocol Number; P0001131 IDENTIFICATION AND JUSTIFICATION OF THE TEST SYSTEM The following are the test systems for this study: Water (groundwater and surface water) Soil FSiesdhiment VCleagmetsation Small Mammal Liver Small Mammal Serum bTdeehteapiuslsarcmfhoparlseetshdewacinloldnbtperorcelopslalaremecdtpelbdeysbywthiWell tebesestotiinnngcSloufalducetidiloitnyins..thPTeuhrfecinhcaaolsnerteroaplnodrstapmarsposlcoeecssisawitneildgl with this study. The test systems were chosen to access the environmental impact of PFBS, PFHS and PFOS in the Decatur, Alabama area. SAMPLE PROCUREMENT, RECEIPT AND RETENTION Water, soil, sediment, fish, clam, vegetation, small mammal liver and small mS3Moalmutwmiooanrlsk.sepTrluhamen deesnatatmiitlplseldoesf"swPamhilalpslebeep2rroeWccueoirvrekemdePnalattnfEorfxoythrgiesSnasmtudpdirlyeincatgrleyEofnurvtolimirnoenWdmieensntttohanel Mafsotvureaddieyliaa.abc."hilitmyTahitnerixtnhuewmfilibleeldbr.eandTdohcetuymtpoeetasnltoendfumsinabmethpreloesffisncaaomlllpreecleptesodrrtewcaesilislvoecvdiaaratyendddewapnietahnlydtzihneidgs pFaWcrisocahocte,errds,csilnisangomgil,t,aotvaeEntghdxeeytsgaeatedipnoip.mnroeTapnnhrtideasstaesemmsaapanmllelapslmylewtaiscimlawllmiblamlelbeueltihsveosedtdor raes(ssademrereepcflAereiispvgpeewdreaniltdwleidixbtheaot1p)u2.rtoCfcTu-e8rshtsehCesede.r ssfraaammctppilloeenss wwwiiillllllbbbeeeceusnstetordrifeudfgoerfdrtohizneentshteuadtfyie<.ld-Sa1tm0thaCle.l timmSaemmoamlflaclomlslaeemrcutmimoanlwawnildlhotbhleee ssbteolroruoemdd frozen at < -10C. rTehpeorrteacnediprtawanddaptaroacsessosciinagtedofwtihthe tshaemsptuledsy.will be documented in the final Page S of 65 Exygen Research Page 68 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P000U31 SAMPLE IDENTIFICATION Pnurimorbetor. anTalhyesisr,efeearcehncseamnpulme bweirllwbiellasbseigunneidquaelaabnodratwoirlyl sdaimstipnlgeuriesfhereenacche Cnlauhbmroobrmaetrao.troygrsaapmhipcledatthaatwiilsl bperoicdeesnsetidfietdhrboyugthhoeultabtoherataonryalystaimcaplleprroefceerdeunrcee. sStaumdyp.le storage conditions and locations will be documented throughout the ANALYTICAL PROCEDURE SUMMARY RVe0f0e0re1n7c8e0s: V0001781 V0001782 V0001783 V0001784 V0001785 V0001786 ""A""AAMMMMccciiieeeedddtttthhhh(((PoooPoPddddFFFOOOooooAAAffff AAAA))) iiinnnnnnnaaaallllWSSyyyyeossssadiiiiilsssstiembrffffyooooebrrnrrLytttttChhhhLbee/eeyCMDDDD/LMSeeeeC/ttttSM/eeeeM/rrrrMSmmmmS"Siiii/nnnnM"aaaattttSiiiioooo"nnnn of of of of Perfluorooctanoic Perfluorooctanoic Perfluorooctanoic Perfluorooctanoic "A"A"AAMMMcccciiiieeeddddttthhh((((ooPPPPodddFFFFOOOOoooAAAAfff AAA)))) iiiinnnnnnnaaalllFVSSyyymmiessssiiighaasssellalltfffanoooMMtdrrrioaatttCnmhhhmleeebammmyDDDaaseleelLtttbLSCeeeyerrri/mmmvMrLueiiimrSCnnnb/aaa/MMbytttiiiyoooSLSnnnL"/CMCooo/Mfff/SMPPP"SeeeS/rrrM/fffMllluuuSSooo""rrrooooooccctttaaannnoooiiiccc TissuhomemmearebsdoovinfetPoFmtBoetStah,loPPdFFsHBuSSs,eatnotdaanlPaPFlyFOtHiScS.a,lTacnhodentfdoinittaailol nPresFpOocSratpfwaobuillneldi.nocflusdeeptahraetiinsogmtehres VERIFICATION OF ANALYTICAL PROCEDURE Acmoentltharbooodl r,saaatnomdrpyaledcsdo.endtTrtoohlethstaeemsmtpaslteuribcwsetiaslnlvcibeaeawumilsliecdbroefpomipraedtthteee.inptroepsaorlauttiioonnsoafsfopretrifitehde Fbbooottrttllweessa.tewrTilshlaembseeplib5no0gt0,tlEemsxyLhgaevpnerewcblieelelannsuerpdopulStyicnoie/nSlyepebucosettPdlerefpmoerirefsrlaumworpiodlceehcemomloliecucathtledsH.amDTpPhlEee Page 9 of65 Exygen Research Page 69 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 collection at the testing facility and have been shown to be free of PFBS, PbPecicl1ln3iooalFF.nCac2clnBeHnll-hteu1kPaSSacd3iFt,stne(CeaaO2cdPdnmPo0F.AdiFpn0pnHeBtPlrwmertSFoShfAillLO,ewluatl.wPSonsinlFor.datlAoohlHtueoStbPeSrtcfaeFib)itomeOetaaaneclnnpdnSsosrdoldteleiduilpnPacsesutotgwcFepwrtdaOaloetiicedfcltSildalof.adocibtawesiieesnllp(,Tid1tiwaaay3htkbdhCn,eeellddiloaslteelnhPwhdoaaskeFiwstcgtufOshospihdaepAeayemlirndlw.ak)fep.dclevasulhsEepteoheslixc(rPir)k.cogoyFeoohnfgTsOnocteaahatrtnnfAmraeiiodneenpwlrveloeadlarewieisnlcrthuldysaoilapt(gsteaceistuhlkwrix3pvdfeCcffeopoloi(nrlulebPryutPldPtoyFdmiFitFfonOtiOsesnOlegeapAtedArAsmii)kcwffpewiiaaeaelifnnetiloirllhlddddesllf dptfoourorpctcihlfeieicsecasdkteedfsaottrahanrbodilauosgfw.ihelatdhnedspdhiekisgechsr)ibcwoednilclperbnoetcraeetdxiuotrrneacwttoeitddheitPneFrdmBuSipn,leicPmaFteHethSaondadnadwcciPlulFrOaaclSysoaannbdde twbdFpafoieoroeizetlttlrielhchdprl.esa-meaoslasseiiAnoallnao,metlebwdsbptemehaSldafeeegcniot.mihdltr/oetSoeAischfdpntailiieteltgna,cidhoccgcPocnallfu.narteaermtavcaamSeiickslalni,yinmeteayoraarnp,swnnwdl/ddeebniasdpatvccogerwehoosgmcnicluhseecloasteseaubmecsntdteekithpodralfafnHeodott,ihrrdDowrEebsnoPidaixluaEmwlystgogb.ipbhteelhoeenttahePtcclxweFehotBilrdpllcaleeSeoccsr,tnscteusPitrdoaapiFbinmpnHielnewpdySrldielpooaulcrrnnpoboedblcelialceP5egsadFc0htute0iOienpredSpamtnehotaLdodert bdtwFcnfeoeeoeoisrntetlrtehlditrnrealmisaegalfmsuciholfnhgoaaoeelbwcllsdiemtlaueiamfmtebrnyosteahdp,r.mtaoleienhfdaFmidiceogachadnhrcoleclsnaileumttveldarivaeavailreneleckllsiraynn,,mnaogatndarwnEdamdssxnneepmystrrcorfguuoaoeemmclnrcnhecspeseseisacsprwneuemketpidrmtlapfatleo,trltiesharEos,trbnuwoxoaipryuaniwplgsgdlt.liehuytbpnhbeotewhPalesyeFixplBt5l(drrr0Soeaseuscp,dctpymPerplidtFeLbolyHnepiendS)ap,tdocpuauovlrnbypolaedlplccesirue.PccodttaFpaiutAoOiyenrntleSeeatrnknhtasiodeet,t Low and high spiking levels for each matrix are defined below: MWaattreirx Low S50p0ikning/gLLevel High5S0p0i0kinngg/LLevel SedSiomilent CFlaismhs Vegetation SSmmaallllMMaammmmaallSLeirvuemr 4 ng/g 10111140000nnnnnngggggg//m////gggggL 40 ng/g 40 ng/g 110111 00000 0000nnnnnggggg/m////ggggL Page 10 of65 Exygen Research Page 70 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Ralenevaceloylsvs;ieshrioeowsfeatvhreeer,aqtnuhtaeicliietpxyaatccetodnpttrroeoclbisesioanbmepatwlnedeseandcecs7uc0rr%aibcyeadwndailblo1bv3ee0.%detAoerfmstthianeteedmfoebrntyitfitehodef accuracy will be included in the final report. METHOD FOR CONTROL OF BIAS hCanooamnltyorzogilennogefaobtuilsaesamswitxitltwluobreeleoavfdedelsraecoshsfemfdoarbttiryfiixctaafktriioonmngs.ruenptrreesaetendtactiovnetrsoulbs-saammppleless, afnrodmbya STATISTICAL METHODS cSatalctiusltaictisonwoilfl abveerlaimgeiteredcotovethrioesse, asspaepcipfliiecdabilne.the subject methods and to the GLP STATEMENT All aspects of this study shall be performed and reported in compliance with EPA TSCA Good Laboratory Practice Standards 40 CFR 792. The final report or data package (supplied to the Sponsor) shall contain a statement that tshtaendsaturddsy awnadswciollndouuctltiende ainnycodmevpilaitainocnes iwnitthhecustrurednyt fraonmd athpopsleicasbtalnedaGrdLsP TRhepisressetnattaetmiveen.t will be signed by the Study Director and Sponsor REPORT tAaot,tfthihneeaclfoornellcpolowursitinowgn:ilol fbteheprsetpuadrye.d Tbhyethreepporritnwciipllalinicnlvuedset,igbauttowr oilrl tnhoetirbedelsimiginteede Tofhtehneatmesetinagndfaacdilditrye.ss of the Study Director, Sponsor Representative, and Achasitna-toefm-ceunsttoodfy GreLcoPrdcso, mmpulsitanbceein(athneystruedlayterdecodrodcsu).mentation, such as Page I / o f 65 Exygen Research Page 71 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 wTAhesesreurrsaeinpgconeretdeUdnationt dtrheegdaaSrttdeuiddnygsDdtaaitrteeemcsteoonrftasnbtduydMytahinenasgpeEemxctyeignoetn.ns aRnedsedaartcehs fQinudainligtys A description of the exact analytical conditions employed in the study. If tcinhnouecpmoysrbupoeborfjreaatchntteeddmpaineantgahtleoyontdtihucwemalarbsmeeprfeootmrhlltouo. dswt.IebfdAetheniexnycamclmuteldoytehd,doiifdtoicniiassteniapoechnchosetsptosoa-agrrteeyhd.iustocmeidne,cthltuohddeewporinollljyebceat Description of the instrumentation used and operating conditions. Ailedvlelenlrt.eifsiueldtsanfrdomthealdlasteatstaabnlealwyzielld.incCluodnetrsoalmapnlde fnourmtifbieedr asnadmpfolerstifwicialltiobne cRaidheefrpnoortrmeitfisaifeeitncodatgaitrnitaoivmanelsllcceohhvfreroaol.mmstaaaTttnoohdggearrraadlmmoacssna. fdtiooarnceoaocnfhtrtohalensaaalymntaeplylietn,eaepnaedcahakmschawrtoriilmxl ,abtieongccrlaulemdairnlagyt Adaltlacwiricllumbestdaonccuems ethnatetdminaythhearveepoarft.fected the quality or integrity of the Locations where raw data and the final report are to be archived. aAmdednitdiomnesntorsigconrerdecbtiyonthsetoSttuhdey fDiniarelcrtoepr.ortThsheaallmbeendimn etnhteshfoarlml cloefaralyn Diadliterenertacittfioyorntahsn.adt ptThaehrteSopfaomtnhseeonrrdeRmpeoeprntrtetshweanitltlaistibbveeei.nsiggnaeltdereadndanddattheed rebaysotnhse foSrtutdhye All applicable requirements for reporting of study results as per 40 CFR 792.185. SAFETY AND HEALTH Laboratory personnel will practice good sanitation and health habits. esEuxvbpesortysaunrcreee(asos)of.npaebrlseonpnreelcaauntidontheshaelnlvibroenmtaeknetntotothperetveestntorinraedfveerertnecnet Page 12 of 65 Exygen Research Page 72 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 AMENDMENTS TO PROTOCOL psARtfeehouflxelafelplpplenopricrsewoestcsisisrhvuegsteaiedeennnnditgdgfoawiaetifciint.davteahnoewHwtc.oaurofriAcmiwtwtthihtmneeaeregvnnniet,etgtnwaeraedst,nrmsiimiogwtetannetehnmoenneddtsnemsiatnghaumeadnensnmceeudthnd,aaedaldntnlnhmayataagtlneeywtaedndstciiitlchslbsdawaorybalneoietlgqentlpiehdusrbeiaosmresuteboSaedytpctydhuoowedptsblhisyredteiihoaobeDoutrlefeuStiftl.rtoetioeuncsfcidetunIteydtnofidhftriietcaDvhhaitdeeeiiniosrrrondebenctcacutoStalwmiolsmpfyrieeols,elwntnuattsifendhbtoodhdyeerr smRcothreueiapgdtnhriygneoseadpe.l lnasatCstnauoptdiprcvyooieevpp.silidaeoensf.d. thwAeTinlhslyiebgdenoeedrvdioigacaitnumioamenl nesandfmtmreoedemnnadtntsmhdweerniselttlpuobwdreytielalpdplabppneeronommdreapfdirtnoltytmoatioantlheltedhdeaiswnStaripltiyhbotunitsctehoaderl DATA RECORD KEEPING Records to be maintained include the following (as appropriate): SADAHISSnaaalenislmsmmsattrcclopppuyhrrlllimtryeeepiocteearmtianreonxlactandtcdretrkoapoiuapcgitrnfnatteriagasprotnephaansycbherhoaealimeecnrttesisddoota,s(dnnb,said)ensfonitaicfoclnayrshsattst-iagirosnsuehndmdeshaaeierttstdonestsosttoarh(lrysec,tlooomacgnneksddt,ihtcfoiohodrantisinfiscaotfiocnu,stcoadliybration) AAChncryoomontophlleoergtedicolaiclsutpimnregesneotnafttsaitotuinodnyneopceferasslsolansrntyuefdlo,yrscitghonerraertesucproeosnnsdatnerundccitenioitniaolsf the study Chromatograms- All chromatograms will contain the following: oSafminptelereisdt,eanntidficinajteiocnti,oinnnjeucmtiobnerdcaoterr,easrproonwdionrgottohethreinrudnic. ation of the area tAhdedsiatimonpalellyn,umfobrteirfiocfattihoenssawmipllleinthcalut dweatshfeoratmifioeudn. t of analyte added and Aconnacleyntitcraaltionst(aen.gd.a,rpdg/mchLr)o.matograms will additionally include the Page U of 65 Exygen Research Page 73 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 cAainonssnatpldryuaittmriitcoeaonnlsft.sthectoe:ndadoirctuiuonmnssehnsethaeettieoltinsdtteihnsegcrftiohbleilnogswamtihnpegleisnshsetteortusbmweeinrlultnbtyeipnientchalenuddseeodt,pieanrnadetaincangh QUALITY ASSURANCE tTSothuaedsyQsuADreirUceonctmiot rpo,lfiEaExnxycygegewenniMthRaGensaLegaPer'mcsh,eanwnti,dllawnindilsltphreeecptSotprhotenthssoeturfdRiynedapitrnegisnsetenortfvataailuvsdea.itdsetqouathtee RETENTION OF DATA AND ARCHIVING wAwcinhliifrlttohhormmthhtaahaetretodidogaonrtracaifomgrppoisanym,caklwtrahaogsewtreukSdsspuhydobeanemptsatlosi,at,rnt.e,cidnocartlmoruedteshpnineodgnmS,dteuenbndtucyste,D, finiarnoenatcdltolrrriee.mspuiTotlerhttsde, sseahtnowad,lilllatbhlbleee painoerccrrtihliguniivdeneenaddtl Te7q9hu2ei.p1tm9e5set.nintA,g nafnaedcxialsicttytorcasohgpaeyllolokfgetsehpefomarllattheeerleiacpltserorsinouidbcmoriatfwtetdimdtaoetatshpaeencsditfuiadenydydiiinnresc4trt0uomrCweFniRtl,t also be kept at Exygen Research. Erextuyrgneinngwsialml opbletsa.in permission from the study director before discarding or Exygen Research Page 14 of 65 Page 74 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 APPENDIX I ANALYTICAL METHODS V0001780 V0001781 V0001782 V0001783 V0001784 V0001785 V0001786 "Method of Analysis for the Determination of Perfluorooctanoic A""A"A""AAMMMMMccccciiiiieeeeedddddttttthhhhh(((((PoPoPooPPoddddFFdFFFOOOOOoooooAAAAAfffff AAAAA))))) iiiiinnnnnnnnnnaaaaalllVFllSSWyyyyyieoessssssadiiiiigihlssstssieembatfrffffaynoooooebtdrrrrnriLyottttttCChhnhhhLbeleee/eyCbaMmDyDDDD/LMSseeeeCLe/tttttSMbC/eeeeeM/yrrrrr/MSmmmmmMSL"iSiiii/SCnnnnnM"/aaaaa/MMtttttSiiiiiooooo"SSnnnnn"/MooooofffffSPPPPP"eeeeerrrrrfffffllllluuuuuooooorrrrrooooooooooccccctttttaaaaannnnnoooooiiiiiccccc A"AMcciieddth((PoPdFFOOoAAf A)) iinnnalSSymmsiaasllllfoMMr aathmmemmDaaell tLSeerivmrueimrnbabytiyoLnLCCo/Mf/MPSeS/rM/fMlSuSo"r"ooctanoic Exygen Research Page IS of65 Page 75 of 127 Interim Report #17- Analysis of Sediment and Water Samples Exygen Study No. : P0001131 Exygen Protocol Number. P0001131 ANALYTICAL METHOD Method Number: V0001780 M e th o d o f A n aly sis f o r th e D e te rm in a tio n o f P e rflu o ro o c ta n o ic A cid (P F O A ) in Water by LC/M S/M S Analytical Testing Facility: Exygen Research 3058 Research Drive State College, PA 16801 Approved By: * C - ______________________ Paul Connolly ' Technical Leader, LC-MS, Exygen Research /J oJohhnn Flaherty / ' Vice President, Operations, Exygen Research *0 |t.uAW Date /yy f ji, Date Exygen Research Total Pages: 7 Page !6 o f 65 Page 76 of 127 Interim Report #17- Analysis of Sediment and Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Research Method Number V000 P 8 0 a n a l y t ic a l m e t h o d Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Water by LC/MS/MS 1.0 Scope This method is to be employed for the isolation and quantitation o f perfluorooctanoic acid by High Performance Liquid Chromatography coupled to a tandem Mass Spcctrometric Detector (LC/MS/MS) in water. 2.0 Safety 2.1 Always observe safe laboratory practices. 2.2 Consult the appropriate MSDS before handling any chemical for proper safety precautions. 3.0 Sample Requirement 3.1 At least 40 m L o f test sample for extraction. 3.2 No sample processing is needed for water samples. 3.3 Samples stored refrigerated should be allowed to equilibrate to room temperature. 3.4 A ll samples must be thoroughly mixed before being sampled for extraction 3.5 Any samples containing particles should be centrifuged at -3000 rpm for -5 minutes and the supernatant used for the extraction. 3.6 Sample collection procedures w ill be specified in the sampling plan for this project. 4.0 Reagents and Standards 4.1 W ater-HPLC grade 4.2 Methanol - HPLC grade 4.3 Ammonium Acetate-A.C.S. Reagent Grade 4.4 Perfluorooctanoic Acid - Sigma-Aldrich 5.0 Instrument and Equipment 5.1 A high performance liquid chromatograph capable o f pumping up to 2 solvents equipped with a variable volume injector capable o f injecting 5-200 pL connected to a tandem Mass Spectrometer (LC/MS/MS). 5.2 A device to collect raw data for peak integration and quantitation. 5.3 Analytical balance capable o f reading to 0.00001 g. 5.4 50 mL disposable polypropylene centrifuge tubes. 5.5 15 mL disposable polypropylene centrifuge tubes. 5.6 Disposable micropipets (50-lOOuL, 100-200uL). 5.7 125-raL LDPE narrow-mouth bottles. 5.8 2 mL clear HPLC vial kit. 5.9 Disposable pipettes. 5.10 Autopipettes (100-1000 pL and 10-100 pL), with disposable tips. 5.11 Waters Sep Pak Vac 6 cc (Ig ) tC18 SPE cartridges. Pag 2 o f 7 Page 17 of65 Exygen Research Page 77 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: POOO! 131 [Exygen Research AN ALYTICAL m e t h o d Method Number VQOQ1780 Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Water by LC/MS/MS 5 12 SPE vacuum manifold. 5.13 Centriftige capable o f spinning 50 mL polypropylene tubes at 3000 rpm. 6.0 Chromatographic System 6.1 Analytical Column: Fluophase RP (Keystone Scientific), 2.1 mm x 50 mm, 5u (P/N: 82505-052130) 6.2 Temperature: 3QC 6.3 Mobile Phase (A) : 2 mM Ammonium Acetate in Water 6.4 Mobile Phase (B) : Methanol 6.5 Gradient Program: Time (min) 0.0 1.0 8.0 20.0 22.5 65 65 25 25 65 Flow Rate % B ImUmin) 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 Injection Volume: 15 pL (can be increased to as much as 50 pL). 6.7 Quantitation: Peak Area - external standard calibration curve. 6.8 Run Time: ~ 23 minutes. The above conditions are intended as 8 guide and may be changed in order to optimize the HPLC system. 7.0 MS/MS System 7.1 Mode: Electrospray Negative MRM mode, monitoring 413 369 m/z. The above conditions are intended as a guide and may be changed in order to optimize the MSMS system. 8.0 Preparation o f Solutions 8.1 Mobile Phase 8.1.1 2 mM ammonium acetate in water is prepared by adding 0.154 g o f ammonium acetate to 1000 mL o f water. Alternate volumes may be prepared. Page 3 <. Page 8 of 65 Exygen Research Page 78 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Research ANALYTICAL M ETHO D Method Number VOOO1780 Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Water by LC/MS/MS 9.0 Standard Preparation 9.1 Standard Stock/Fortification Solution 9.1.1 Prepare a stock solution o f -100 pg/mL o f PFOA by weighing 10 mg o f analytical standard (corrected for purity) and dilute to 100 mL with methanol in a 125-mL LDPE bottle. 9.1.2 A 10 pg/mL fortification solution o f PFOA is prepared by bringing )U m L o f the 100 pg/mL solution to a final volume o f 100 with methanol in a 125 mL LDPE bottle. 9.1.3 A 1.0 pg/mL fortification solution o f PFOA is prepared by bringing in mL o f the 10 pg/mL solution to a final volume o f 100 with methanol in a 125 mL LDPE bottle. 9.1.4 A 0.1 pg/mL fortification solution o f PFOA is prepared by bringing 10 mL o f the 1.0 pg/mL solution to a final volume o f 100 with methanol in a 125 m L LDPE bottle. 9.1.5 A 0.01 pg/mL fortification solution o f PFOA is prepared by bringing 10 mL o f the 0.1 pg/mL solution to a final volume o f 100 with methanol in a 125 mL LDPE bottle. 9.1.6 The stock and fortification solutions are to be stored in a refrigerator at approximately 4C and are stable for a maximum period o f 6 months from the date o f preparation. 9.2 Standard Calibration Solutions 9.2.1 9.2.2 LC/MS/MS calibration standards are prepared in HPLC water. The calibration standards are processed through the extraction procedure, identical to samples. The following is a typical example: additional concentrations may be prepared as needed. Final Concentration Fortification Volume of Concentration of Calibration of Fortification Volume Fortified Control Calibration Standard ID Solution (ppb) 0 10 10 10 100 100 100 (PL) 0 100 200 400 100 200 400 Sample (mL) 40 40 40 40 40 40 40 Standard (put)4 0 25 50 100 250 500 1000 (example) XCmmddyy-0 XCmmddyy-1 XCmmddyy-2 XCmmddyyO XCmmddyv-4 XCmmddyy-S XCmmddw-6 * The extracted concentration o f the calibration standard is equal to 8* its initial concentration, due to the concentration o f the standard during the extraction (SPE) XC " extracted calibration standard. Page 4 o f * Page 19 of65 Exygen Research Page 79 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Research Method Number V00017SO ANALYTICAL M ETHO D M ethod o f Analysis for the Determination ofPerfluoroootanoic Acid (PFOA) in W ater by LC/M S/M S 9.2.3 A zero standard solution (reagent blank) m ust be prepared with each set o f standards extracted. 9.2.4 Store all extracted calibration standards in 15-mL polypropylene tubes at 2C to 6C, up to two weeks. 9.2.5 Alternate volum es and concentrations o f standards m ay be prepared as needed. 10.0 Batch Set Up 10.1 Each batch o f sam ples extracted (typically 20 o r less) m ust include ai least one reagent control (m ethod blank using HPLC water) and two reugcm controls fortified at known concentrations (lab control spike) to verify procedural recovery for the batch. 10.2 Requirem ents for field and laboratory duplicates and spikes w ill be specified in the quality assurance plan for this project. 11.0 Sam ple Extraction 11.1 M easure 40 m L o f sam ple or a portion o f sam ple diluted to 40 m L with water into 50 m L polypropylene centriftige tubes (fortify as needed, replace lid and m ix well). 11.2 C ondition the C n SPE cartridges (1 g, 6 m L) by passing 10 m L m ethano; followed b y 5 m L o f H PLC w ater ( - 2 drop/sec). Do not let colum n run dry 11.3 Load sam ple on conditioned C | i SPE cartridge. D iscard eluate. 11.4 Elute with - 5 m L 100% m ethanol. C ollect 5 m L o f eluate into graduated 15 m L polypropylene centriftige tub es (final v olum e " S m L). 11.5 Analyze sam ples using electrospray LC /M S/M S. 12.0 Chrom atography 12.1 Inject the sam e am ount o f each standard, sam ple and fortified sam ple into the LC/M S/M S system . A calibration standard m ust precede and follow all analyzed samples. 12.2 Standards o f PFOA corresponding to at least Eve or m ore concentration levels must be included in an analytical set. 12.3 A n entire set o f extracted calibration standards m ust be included ai ihc beginning and at the end o f a sam ple set. E xtracted standards m ust hi. interspersed between every 5-10 samples. As an alternative, an entire set of extracted calibration standards m ay be injected at the beginning o f a sci followed by extracted calibration standards interspersed every 5-10 samples (to account for a second set o f extracted standards). In either case, extracted calibration standards m ust b e the first and last injection in a sam ple set. 12.4 U se linear standard curves for quantitation. Linear standard curves are generated for the analyte by linear regression using 1/x w eighting o f peak area iPage of ? Page 20 of65 Exygen Research Page 80 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Research Method Number V0001780 A N A L Y T IC A L M E T H O D ________________________________ M ethod o f Analysis for the Determ ination o f Perfluorooctanoic Acid (PFO A) in W ater by IC/M S/M S versus calibration standard concentration using M assLynx 3.3 (or equivalent) software system. 12.S Sam ple response should not exceed standard responses. A ny sam ples that exceed standard responses should be further diluted and reanalyzed. 13.0 Acceptance Criteria 13.1 C hrom atogram m ust show a peak o f a d au gh ter io n at 369 am u from a parent o f 413 am u. The 413 am u parent corresponds to the PFO A anion, while the daughter ion (369 amu) represents the loss o f carbon dioxide. 13.2 M ethod blanks m ust not contain PFO A at levels greater than the LOQ. If a blank contains PFOA at levels greater than 50 ng/L, then a new blank sample must be obtained and the entire set must be re-extracted. 13.3 R ecoveries o f control spikes and m atrix spikes m ust be betw een 70-130% of tbeir known values. If a control spike falls outside the acceptable limits, the entire set o f samples should be re-extracted. Any matrix spike outside 70 130% should be evaluated by the analyst to determ ine if re-extraction is warranted. 13.4 A ny calibration standard found to be a statistical outlier by using the Huge Error Test, may be excluded from the calculation of the calibration curve However, the total num ber o f extracted calibration standards that could be excluded must not exceed 20% o f the total num ber o f extracted standards injected. The correlation coefficient (R) for calibration curves generated must be 0.992 (R2 0.985). If calibration results fall outside these limits, then appropriate steps m ust be taken to adjust instrum ent operation, and the standards or the relevant set o f sam ples should be reanalyzed. 13.6 Retention tim es between standards and sam ples must not drift m ore than 4 % w ithin an analytical nin. If retention tim e drift exceeds this limit within an analytical run then the set m ust be reanalyzed. 14.0 Calculations 14.1 U se the follow ing equation to calculate the am ount o f PFO A found (in ng/L. based on peak area) using the standard curve (linear regression parametersj generated by the M ass Lynx software program: PFOA found (ng/L) slope x DF DF factor by which the final volum e was diluted, if necessary. Page 6 of? Page 2 of65 Exygen Research Page 81 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 Exygen Protocol Number. P0001131 Exygeo Research Method Number VOOQ1780 | AW ALVTICAL M ETH O D ' M ethod o f Analysis for the Determination o f Perfluorooctanoic Acid (PFO A) in W ater by LC/M S/M S 14.2 For sam ples fortified w ith known am ounts o f PFO A prior to extraction, use the following equation to calculate the percent recovery. Recovery (%) [ total analyte found (ng/L) - analyte found in control (ng/L)] ^ ^ analyte added (ng/L) Exygen Research Page 7 o f " Page 22 of65 Page 82 of 127 Interim Report #17- Analysis of Sediment and Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 ANALYTICAL METHOD M ethod Number. V0Q01781 M ethod o f A nalysis fo r the D eterm ination o f P erfluorooctanoic Acid (PFO A ) in Soil by LC/M S/M S Analytical Testing Facility: Exygen Research 3058 Research Drive State College, PA 16801 T>_1LiApproved By: Paul Connolly * Technical Leader, LC-M S, Exygen Research mln/<W D ate D ate Exygen Research Total Pages: 7 Page 23 of65 Page 83 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygco Research Method Number VOOOl781 I ANALYTICAL M ETHO D M ethod o f Analysis for the D eterm ination o f Perfluorooctanoic Acid (PFO A) in Soil by LC/M S/M S 1.0 Scope This method is to be em ployed for the isolation and quantitation o f perfluorooctanoic acid by High Performance Liquid Chrom atography coupled to a tandem Mass Spectrom etric Detector (LC/M S/M S) in soil. 2.0 Safety 2.1 A lw ays observe safe laboratory practices. 2.2 Consult the appropriate M SDS before handling any chem ical for proper safety precautions. 3.0 Sam ple Requirem ent 3.1 A tle a s t 15 g o f test sam p le for extraction. 3.2 No sam ple processing is needed for soil samples. 3.3 Sam ples stored refrigerated should be allow ed to equilibrate to room tem p eratu re. 3.4 All sam ples m ust be thoroughly m ixed before being sam pled for extraction. 3.5 Sam ple collection procedures w ill be specified in the sam pling plan for this project. 4.0 Reagents and Standards 4.1 W ater -H P L C grade 4.2 M ethanol - HPLC grade 4.3 Am m onium A cetate - A.C.S. R eagent Grade 4.4 Perfluorooctanoic Acid - Sigm a-Aldrich 5.0 Instrum ent and Equipm ent 5.1 A high perform ance liquid chrom atograph capable o f pum ping up to 2 solvents equipped with a variable volum e injector capable o f injecting 5-200 pL connected to a tandem M ass Spectrometer (LC/M S/M S). 5.2 A device to collect raw data for peak integration and quantitation. 5.3 A nalytical balance capable ofreading to 0.00001 g. 5.4 50 m L disposable polypropylene centrifuge tubes. 5.5 15 m L disposable polypropylene centrifuge tubes. 5.6 D isposable m icropipets (50-1 QOuL, 100-200uL). 5.7 125-m L L D PE nairow -m outhbottles. 5.8 2 m L clear H PLC vial kit. 5.9 Disposable pipettes. 5.10 Autopipettes (100-1000 p L an d 10-100 pL), with disposable tips. 5.11 W aters Sep Pak Vac 6 cc (lg ) tC l8 SP E cartridges. 5.12 SPE vacuum manifold. 5.13 U ltrasonic bath. Page 2tfl'7 Page 24 of 65 Exygen Research Page 84 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Research Method Number V 0001781 | ANALYTICAL M ETHO D M ethod o f Analysis for the Determ ination o f Perfluorooctanoic A cid (PFO A) in Soil by L C /M S7M S 5.14 W rist-action shaker. 5.15 C entrifuge capable o f spinning 50 raL polypropylene tubes ai 5000 rpm. 6.0 Chrom atographic System 6.1 A nalytical C olum n: F luophase R P (K eystone Scientific), 2.1 m m x 50 m m . 5p (P/N: 82505-052130) 6.2 Tem perature: 30C 6.3 M obile Phase (A) : 2 m M Am m onium A cetate in W ater M obile Phase (B) ; M ethanol Gradient Program: Tune (mini 0.0 1.0 8.0 20.0 22.5 %A 65 65 25 25 65 Flow Rate % B fmlVinin) 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6.6 Injection Volum e: 15 pL (can be increased to as m uch as 50 pL). 6.7 Quantitation: Peak A rea - external standard calibration curve. 6.8 R unT im e: - 2 3 minutes. The above conditions are intended as a guide and m ay be changed in order to optim ize the HPLC system. 7.0 M S/M S System 7.1 M ode: E lectrospray N eg ative M R M m o de, m o n ito rin g 413 -- 369 mfz for PFOA. The above conditions are intended as a guide and m ay be changed in order to optim ize the M SM S system. 8.0 Preparation o f Solutions 8.1 M obile Phase 8.1.1 2 m M am m onium acetate in w ater is prep ared by adding 0 .154 g o f ammonium acetate to 1000 m L o f water. Alternate volum es m ay be prepared. Page 3 of 7 Page 25 o f 65 Exygen Research Page 85 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Research Method Number VQOOI78I A N A L Y T IC A L M E T H O D _______________________________ M ethod o f Analysis for the Determ ination o f Perfluorooctanoic Acid (PFOA) in Soil by LC /M S/M S 9.0 Standard Preparation 9.1 Standard Stock/Fortification Solution 9.1.1 P repare a stock solution o f '100 p g /m L o f P F O A by w eighing 10 m g o f analytical standard (corrected for purity) and dilute to 100 mL with methanol in a 125>mL LDPE bottle. 9.1.2 A 10 p g /raL fortification solution o f P F O A is prepared by bringing 10 m l. o f the 100 pg/m L solution to a final v olum e o f 100 w ith methanol in a 123 m L LDPE bottle. 9.1.3 A 1.0 p g/m L fortification solution o f PF O A is prepared by bringing 10 m L o f the 10 pg/m L solution to a final volum e o f 100 w ith methanol m a 123 mL LDPE bottle. 9.1.4 A 0.1 p g /m L fortification solution o f P F O A is prepared by bringing 10 m L o f (he 1.0 p g /m L solutio n to a final v olum e o f 100 w ith methanol in a 123 m L LDPE bottle. 9.1.3 A 0.01 pg/m L fortification solution o f PFO A is prepared by bringing 10 m L o f the 0.1 p g 'm L solution to a final volum e o f 100 w ith methanol in a 125 m L LD PE bottle. 9.1.6 The stock and fortification solutions are to be stored in a refrigerator at approximately 4C and are stable for a m axim um period o f 6 months from the date o f preparation. 9.2 Standard Calibration Solutions 9.2.1 LC/M S/M S calibration standards are prepared in H PLC water The calibration standards arc processed through the extraction procedure, identical to samples. 9.2.2 The following is a typical exam ple: additional concentrations m ay be prepared as needed. Final Concentration Fortification Volum e o f Concentration o f Calibration o f Fortification Volume Fortified Control Calibration Standard ID Solution (ppb) (HL) Sample (mL) Standard (poll* ( e x a m p le ) 0 0 40 0 XCmmddyy-0 ttoo10 100 40 200 40 400 40 25 X C m m d d y y -l 50 XC m m d d yy-2 100 XCm m ddyy-3 100 100 40 250 XCm m ddyy-4 100 200 40 500 XCmmddyy-5 100 400 40 1000 XCm m ddw-6 * The extracted concentration o f die calibration standard is equal to 8* its initial concentration, due to the concentration o f the standard during the extraction (SPE). XC * extracted calibration standard. Page 4 o f7 Page 26 oj 65 Exygen Research Page 86 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Reward Method Number V 000178) _______________________________A N A L Y T IC A L M E T H O D _______________________________ M ethod o f Analysis for the Determ ination o f Perfluorooctanoic Acid (PFO A) in Soil by IC/M S/M S 9.2.3 A zero standard solution (reagent blank) m ust be prepared with each set o f standards extracted. 9.2.4 Store all extracted calibration standards in 15-m L polypropylene lubes at 2C to 6C, up to tw o w eeks. 9.2.5 A lternate volum es and concentrations o f standards m ay be prepared as needed. 10.0 Batch Set Up 10. t Each batch o f sam ples extracted (typically 20 o r less) m ust include at least one reagent control (method blank using S m L o f m ethanol) and two reagent controls fortified at known concentrations (lab control spike) to verify procedural recovery for the batch. 10.2 Requirem ents for field and laboratory duplicates and spikes will be specified in the quality assurance plan for this project. 11.0 Sam ple Extraction 11.1 W eigh 5 g o f sam ple into 50 m L polyp rop ylen e centrifu ge tubes (fortify as needed, replace lid and m ix well). 11.2 A dd S m L o f m eth ano l and shake o n a w rist action sh a k e r fo r ~ l 5 m inutes 11.3 Transfer the tubes to an ultrasonic bath and sonicate for ~ 1 5 minutes. 11.4 Bring the volum e up to 40 m L with w ater in the 50 m L polypropylene centrifuge tube. 11.5 C entrifuge for ~ 10 m inutes at ->3000 rpm. 11.6 C ondition the C u S P E cartridges (1 g , 6 m L ) b y p assin g 10 m L m ethanol follow ed b y 5 m L o f H P L C w ater (> 2 drop/sec). D o n ot let colum n run dry 11.7 Load (decant) the sam ple on the conditioned C u SPE cartridge Discard eluate. ] 1.8 E lute w ith ->5 m L 100% m ethanol. C ollect 5 m L o f eluate into graduated 15 m L polypropylene centrifuge tubes (final volum e * 5 mL). 11.9 A nalyze sam ples using electrospray LC/M S/M S. 12.0 Chrom atography 12.1 Inject the sam e am ount o f each standard, sam ple and fortified sam ple into the LC/M S/M S system . A calibration standard m ust precede and follow all analyzed samples. 12.2 Standards o f PFO A corresponding to at least five or m ore concentration levels must be included in an analytical set. 12.3 A n entire set o f extracted calibration standards m ust be included at the beginning and at the end o f a sam ple set. Extracted standards must be interspersed between every 5-10 samples. As an alternative, an entire set o f Page 5 o f 7 Page 27 of 65 Exygen Research Page 87 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: POOO1131 Exygen Research 1Method Number V000178 ANALYTICAL M ETHO D M ethod o f Analysis for the D eterm ination o f Perfluorooctanoic Acid fPFO A) tn Soil by LC/M S/M S extracted calibration standards m ay be injected at the beginning of a set followed by extracted calibration standards interspersed every 5-10 samples (to account for a second set o f extracted standards). In either case, extracted calibration standards m ust be the first and last injection in a sample set. 12.4 Use linear standard curves for quantitation. Linear standard curves arc generated for the analyte by linear regression using l/x weighting o f peak area versus calibration standard concentration using M assLynx 3.3 (or equivalent) software system. 12.5 Sam ple response should not exceed standard responses. Any sam ples that exceed standard responses should be further diluted and reanalyzed. 13.0 Acceptance Criteria 13.1 Chrom atogram m ust show a peak o f a daughter ion at 369 am u from a parent o f 413 amu. The 413 am u parent corresponds to the PFOA anion, while the daughter ion (369 am u) represents the loss o f carbon dioxide. 13.2 M ethod blanks m ust not contain PFO A at levels greater than the LOQ. If a blank contains PFOA at levels greater than 50 ng/L, then a new blank sample must be obtained and the entire set m ust be re-extracted. 13.3 R ecoveries o f control spikes and m atrix spikes m ust be betw een 70-130% of their know n values. If a control spike falls outside the acceptable limits, the entire set o f samples should be re-extracted. Any matrix spike outside 70 130% should be evaluated by the analyst to determ ine if re-extraction is warranted. 13.4 Any calibration standard found to be a statistical outlier by using the Huge Error Test, m ay be excluded from the calculation o f the calibration curve. However, the total num ber o f extracted calibration standards that could be exclud ed m u st not exceed 20% o f the total n u m b er o f extracted standards injected. 13.5 T he correlation coefficien t (R ) for calibratio n curv es generated must he 0 .9 9 2 (R 2 0.9 85 ). I f calibratio n resu lts fall o utside these limns, then app rop riate steps m ust be taken to adjust instru m en t o peration , and the standards or the relevant set o f sam ples should be reanalyzed. 13.6 Retention tim es betw een standards and sam ples m ust not drift m ore than 4 % within an analytical run. If retention tim e drift exceeds this limit within an analytical run then foe set m ust be reanalyzed. Page 6 of 7 Page 28 of 65 Exygen Research Page 88 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Research Method Number V000178! ANALYTICAL M ET H O P M ethod o f Analysis for the Determ ination o f Perfluorooctanoic Acid (PFO A) in Soil bv LC/M S/M S ' 1J 14.0 Calculations 14.1 U se the follow ing equation to calculate the am ount o f PFO A found (in ng/L, based on peak area) using the standard curve (linear regression parameters) generated by the M ass Lynx software program: PFOA found (ng/L) - (Peak area - intercept) x DF slope DF factor by w hich the final volume was diluted, if necessary. 14.2 For sam ples fortified w ith know n am ounts o f PFO A prior to extraction, use the following equation to calculate the percent recovery. Recovery (%) - [ total analyte found (ng/L) - analyte found in control (ng/L)] ^ ^ analyte added (ng/L) 14.3 U se the follow ing equation to convert the am ount o f PFO A found in ng/L to ng/g (ppb). PFOA found (ppb) - (PFOA found fnc/L) x volume extracted (0.04D1 sam ple weight (5 g) 14.4 U se the follow ing equation to calcu late the am ou nt o f PFO A found in ppb based on dry weight. PFOA found (ppb) dry weight " PFOA found (ppb) x [ 100% / total solids(% )] Exygen Research Page 29 of 65 Page 89 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P00Q1131 ANALYTICAL METHOD M ethod N um ber V0001782 M ethod o f A nalysis fo r th e D eterm in ation o f P erflu o ro o ctsn o ic Acid (P F O A ) in Sedim ent by LC/M S/M S Analytical Testing Facility: Exygen Research 3055 Research Drive State College, PA 16801 Approved By: C-jL Paul Connolly ( Technical Leader, LC-M S, Exygen Research / Jaootha'Jn/F nl a h e / rty ( J./ / Vicie P resident, O perations, E xygen R esearch __ IollbM D ate D ate Exygen Research Total Pages: 7 Page 30 of65 Page 90 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Research Method Number VOOOI782 ANALYTICAL M ETHOD M ethod o f Analysis for the Determ ination o f Perfluorooctanoic Acid (PFO A) in Sediment by LC/M S/M S 1.0 Scope This m ethod is to be em ployed for the isolation and quantitation o f perfluorooctanoic acid by High Perform ance Liquid Chrom atography coupled to a tandem Mass Spectrometric Detector (LC/M S/M S) in sediment. 2.0 Safety 2.1 Always observe safe laboratory practices. 2.2 Consult the appropriate M SDS before handling any chem ical for proper safety precautions. 3.0 Sam ple Requirement 3.1 A t least 30 g o f test sam ple for extraction. 3.2 No sam ple processing is needed for sedim ent sam ples. 3.3 Samples stored tem perature. refrigerated should be allow ed to equilibrate to room 3.4 All sam ples m ust be thoroughly m ixed before being sam pled for extraction. 3.5 Sam ple collection procedures w ill be specified in the sam pling plan for tins project 4.0 Reagents and Standards 4.1 W ater-H P L C grade 4.2 M ethanol - HPLC grade 4.3 A cetic A cid **R eagent grade 4.4 Amm onium Acetate - A.C.S. Reagent Grade 4.5 Perfluorooctanoic Acid - Sigma*Aldrich 5.0 Instrum ent and Equipm ent 5.1 A high perform ance liquid chrom atograph capable o f pum ping up to 2 solvents equipped w ith a variable volum e injector capable o f injecting 5-200 pL connected to a tandem M ass Spectrom eter (LC/M S/M S). 5.2 A device to collect raw data for peak integration and quantitation. 5.3 A nalytical balance capable o f reading to 0.00001 g. 5.4 50 m L disposable polypropylene centrifuge tubes. 5.5 15 m L disposable polypropylene centrifuge tubes. 5.6 D isposable m icro pipets (50-lQ O uL, 100-200uL). 5.7 125-mL LDPE narrow-m outh bottles. 5.8 2 m L clear H PLC vial kit. 5.9 Disposable pipettes. $.10 Autopipettes (100-1000 pL and 10-100 pL ). w ith disposable tips. 5.11 W aters Sep Pak Vac 6 cc (Ig ) tC l8 SPE cartridges. 5.12 SPE vacuum manifold. Page 2 of 7 Page 31 of 65 Exygen Research Page 91 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Reieareb Method Number VOGO 1782 I a n a l y t ic a l m e t h o d M ethod o f Analysis for the D eterm ination o f Perfluorooctanoic Acid (PFO A ) in Sediment by LC/M S/M S 5.13 Vortcxer. 5.14 W rist-action shaker. 5.15 C entrifuge capable o f spinning 50 raL polypropylene tubes at 3000 rpm. 6.0 Chrom atographic System 6.1 A nalytical C olum n: F luophase R P (K eystone S cientific), 2.1 m m x 50 m m . 5p (P/N: 82505-052130) 6.2 Tem perature: 30C 6.3 M obile Phase (A ) : 2 m M Am m onium A cetate in W ater 6.4 M obile Phase (B ): M ethanol 6.5 Gradient Program: I t o (m ) 0.0 1.0 8.0 20.0 22.5 %A 65 65 25 25 65 Flow Rate /.B im L/m m l 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6.6 Injection Volume: 15 pL (can be increased to as m uch as 50 pL). 6.7 Quantitation: Peak Area - external standard calibration curve. 6.8 R unT im e: - 2 3 minutes. The above conditions are intended as a guide and m ay be changed in order to optim ize the HPLC system. 7.0 M S/M S System 7.1 M ode: E lectrospray N egative M R M m ode, m o nito rin g 413 -> 369 m /z for PFOA. The above conditions are intended as a guide and m ay be changed in order to optim ize the M SM S system. 8.0 Preparation o f Solutions 8.1 M obile Phase 8.1.1 2 m M am m onium acetate in w ater is prepared by adding 0.154 g of ammonium acetate to 1000 m L o f water. Page 3 o f 7 Page 32 of65 Exygen Research Page 92 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Reieirch Method Number VQGOI782 | ANALYTICAL M ETHO D | M ethod o f A nalysis for the D eterm ination ofP erfluorooctanoic Acid (PFO A) in Sedim ent bv LC/M S/M S ' 8.2 Extraction Solutions 8.2.1 \% acetic acid in w ater is prepared b y add in g 10 m L o f acetic acid to 1000 m L o f water. Alternate volum es m ay be prepared. 9.0 Standard Preparation 9.1 Standard S tock/F ortification S olution 9.1.1 Prepare a stock solution o f ->100 pg/m L o f PFO A by w eighing 10 tug o f analytical standard (corrected for purity) and dilute to 100 mL with methanol in a !2$>mL LD PE bottle. 9.1.2 A 10 p g /m L fortification solution o f P F O A is prepared by bringing 10 m L o f the 100 pg/raL solution to a final volum e o f 100 w ith methanol in a 125 mL LDPE bottle. 9.1.3 A 1.0 pg/m L fortification solutio n o f PF O A is prepared by bringing 10 m L o f the 10 p g fa L solution to a final volum e o f )00 with methanol in a 125 m L LD PE bottle. 9.1.4 A 0 .1 pg/m L fortification solution o f P F O A is prep ared by bringing 10 m L o fth e 1.0 pg/m L solution to a final volum e o f 100 w ith methane) in a 125 m L LDPE bottle. 9.1.5 A 0.01 pg/m L fortification solution o f PFO A is prepared by bringing 10 m L o f tbe 0.1 p g/m L solutio n to a final volum e o f 100 with methanol in a 125 m L LD PE bottle. 9.1.6 The stock and fortification solutions are to be stored in a refrigerator at approximately 4C and are stable for a m axim um period o f 6 months from the date o f preparation. 9.2 Standard Calibration Solutions 9.2.1 LC/M S/M S calibration standards are prepared in m ethanol via dilution o f tbe 0.1 pg/m L fortification solution. 9.2.2 The following is a typical exam ple: additional concentrations may be Concentration o f Fortification Solution (nn/raL) 100 100 100 10 5 2 Volum e (mL) 10 5 2 10 10 10 D ilu ted to (mL) 100 too 100 100 100 100 Final Concentration (ng/mL) 10.0 5.0 20 1.0 0.5 0.2 Page 4 a f 7 Page 33 of65 Exygen Research Page 93 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Research Method Number VOOOI782 I ANALYTICAL M ETH O D | M ethod o f Analysis for the Determ ination o f Perfluorooctanoic Acid (PFO A) in Sedimeni by LC/M S/M S 9.2.3 Store all calibration standards in 125-m L LD PE narrow -m outh bottles at 2C to 6C, up to six months. 9.2.4 A lternate volum es and concentrations o f stand ards m ay be prepared as needed. 10.0 Batch Set Up 10.1 Each batch o f sam ples extracted (typically 20 o r less) m ust include at least one untreated control and two untreated controls fortified at known concentrations (lab control spike) to verify procedural recovery for the batch. 10.2 Requirem ents for field and laboratory duplicates and spikes will be specified in the quality assurance plan for this project. 11.0 Sam ple Extraction 11.1 W eigh 5 g o f sam ple into 50 m L polypropylene centrifuge tubes (fortify as needed, replace lid and mix well). 11.2 Add 35 m L o f 1% acetic acid, cap, vortex and shake on a wrist action shaker for ~60 minutes. 11.3 Centrifuge the tubes at -3000 ipm for -2 0 minutes. 11.4 C ondition the C ii SPE cartridges (1 g, 6 m L) by passing 10 m L m ethanol followed by 20 m L o f HPLC w ater ( - 2 drop/sec). Do not let colum n run dry 11.5 Load (decant) the sam ple on the conditioned Cia SPE cartridge. Discard eluate. 11.6 Add 20 m L o f m ethanol to the sedim ent left in the bottom o f the 50 mL centrifuge tube. Cap, vortex and shake on a wrist action shaker for -30 m inutes. 11.7 Ccntriftige the tubes at -3 0 0 0 rpra for - 2 0 m inutes. 11.8 Decant the m ethanol onto the sam e SPE cartridge. Collect the eluate. 11.9 W ash the colum n w ith 4 m L o f m ethanol. C ollect the eluate and add it to the eluate collected in step 11.8. 11.10 C ondition a second C u SPE cartridge (1 g, 6 m L ) by passing 10 m L m ethanol followed by 20 raL o f HPLC w ater ( - 2 drop/sec). D o not let colum n run dry 11.11 Add the m ethanol to -2 0 0 m L o f w ater and load on the second conditioned SPE cartridge. 11.12 Elute with -5 m L 100% m ethanol. C ollect 5 m L o f eluate into graduated 15 m L polypropylene centrifuge tubes (final volum e * 5 mL). 11.13 Analyze sam ples using eiectrospray LC/M S/M S. Page 5 Page 34 of65 Exygen Research Page 94 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Research Method Number VD001782 | ANALYTICAL M ETHO D \ M ethod o f A nalysis for the D eterm ination o f Perfluorooctanoic A cid (PFO A ) in Sedim ent bv LC/M S/M S ' 12.0 Chrom atography 12.1 Inject the sam e am ount o f each standard, sam ple and fortified sam ple into the LC/M S/M S system . A calibration standard m ust precede and follow all analyzed samples. 12.2 Standards o f PFO A corresponding to at least five or m ore concentration levels must be included m an analytical set. 12.3 A n entire set o f extracted calibratio n stand ards m u st be included at the beginning and at the end o f a sample set. Standards must be interspersed between every 3-10 samples. As an alternative, an entire set o f calibration standards m ay be injected at the beginning o f a set followed by calibration standards interspersed every 3-10 sam ples (to account for a second set of standards). In either ease, calibration standards m ust be the first and last injection in a sam ple set. 12.4 U se linear standard curves for quantitation. Linear standard curves arc generated for the analyte by linear regression using 1/x w eighting o f peak area versus calibration standard concentration using M assLynx 3.3 (or equivalent) software system. 12.3 Sam p le resp on se should n ot exceed stan d ard resp on ses. A ny sam ples that exceed standard responses should be further diluted and reanalyzed. 13.0 Acceptance Criteria 13.1 Chrom atogram m ust show a peak o f a daughter ion at 369 am u from a parent o f 413 amu. The 413 am u parent corresponds to the PFOA anion, while the daughter ion (369 am u) represents the loss o f carbon dioxide. 13.2 M ethod blanks m ust not contain PFO A at levels greater than the LOQ. If a blank contains PFO A at levels greater than 0.2 ng/m L, then a new blank sample m ust be obtained and the entire set must be re-extracted. 13.3 Recoveries o f control spikes and m atrix spikes m ust be betw een 70-130% of their known values. If a control spike falls outside the acceptable limits, the entire set o f samples should be rc-cxtracted. Any matrix spike outside 70. 130% should be evaluated by the analyst to determ ine if re-extraction is w arran ted . 13.4 Any calibration standard found to be a statistical outlier by using the Huge Error Test, m ay be excluded from the calculation o f the calibration curve However, the total num ber o f extracted calibration standards that could be excluded m ust not exceed 20% o f the total num ber o f extracted standards injectod. 13.3 The correlation coefficient (R) for calibration curves generated must be 0.992 (R2 0.983). If calibration results fall outside these limits, then appropriate steps m ust be taken to adjust instrum ent operation, and the standards or the relevant set o f sam ples should be reanalyzed. Pge 6 ot' ' Page 35 of 65 Exygen Research Page 95 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Researcb Method Number VOOOI782 I ANALYTICAL M ETH O D | M ethod o f Analysis for the Determ ination o f Pcrfluorooctanoic Acid (PFO A ) in Sediment by LC/M S/M S 13.6 Retention tim es betw een standards and sam ples m ust not drift more than 4 % within an analytical run. If retention tim e drift exceeds this limit within an analytical run then the set m ust be reanalyzed. 14.0 Calculations 14.1 U se the follow ing equation to calculate the am ount o f PFO A found (in ng/mL, based on peak area) using the standard curve (linear regression parameters) generated by the M ass Lynx software program: PFOA found (ng/raL) " (Peak are a intercept) x DF slope DF - factor by which the final volum e w as diluted, if necessary. 14.2 For sam ples fortified w ith known am ounts o f PFO A prior to extraction, use the following equation to calculate the percent recovery. Recovery (% ) [ total analyte found (ng/mL) - analyte found in control (ng/mL)] analyte added (ng/mL) 100 14.3 U se die follow ing equation to convert the am ount o f PFO A found in ng/mL to ng/g (ppb). PFO A found (ppb) * [PFO A found (ng/m L ) x final volum e (S m Lli sam ple weight (5 g) 14.4 U se the following equation (if necessary) to calculate the am ount o f PFOA found in ppb based on dry weight. PFO A found (ppb) dry w eight PFO A found (ppb) x [100% / total soltds(% )] Pag 7 of 7 Page 36 of65 Exygen Research Page 96 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 ANALYTICAL METHOD M ethod Number: V0001783 Method of Analysis for the Determination of Perfluorooctaoolc Acid (PFOA) in Fish sod Clan by LC/MS/MS Analytical Testing Facility: Exygen Research 3058 Research Drive State College, PA 16801 Approved By: vt-V C-jL.____ Paul Connolly Technical Leader, LC-M S, Exygen Research /u y Vohn Flaherty 'V i c e President, Operations, Exygen Research D ate Z 0 /4 4 - Date Exygen Research Total Pages: 8 Page 37 of 65 Page 97 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Reiearch Method Number VOOO1783 AN ALYTICAL m e t h o d M ethod o f Analysis for the Determ ination o f Perfluorooctanoic Acid (PFO A) in Fish and Clams by LC/M S/M S 1.0 Scope This method is to be employed for the isolation and quantitation o f perfluorooctanoic acid by High Perform ance Liquid Chrom atography coupled to a tandem Mass Spectrom etric Detector (LC/M S/M S) in fish and clams. 2.0 Safety 2.1 A lw ays observe safe laboratory practices. 2.2 Consult the appropriate M SDS before handling any chem ical for proper safety precautions. 3.0 Sam ple Requirem ent 3.1 A t least 20 g o f test sam ple for extraction. 3.2 Sam ples should be processed before extraction. Place the frozen sam ple in a food processor and hom ogenize w ith dry ice. Place the sam ples in containers and leave open in frozen storage overnight to allow for carbon dioxide sublimation. Seal and place the sam ples in frozen storage until time of analysis. 3.3 Sam ple collection procedures w ill be specified in the sam pling plan for this project. 4.0 Reagents and Standards 4.1 W ater - H PLC grade /* * * 4.2 A cetonitrile - H PLC grade 4.3 Carbon (120-400 m esh) - Reagent grade 4.4 M ethanol - HPLC grade 4.5 Silica gel (60-200 m esh) - R eagent grade 4.6 Florisil (60*100 m esh) - Reagent grade 4.7 Superclean LC*NHj - Reagent grade 4.8 1-O ctanol - H PLC grade 4.9 L*Ascorbic acid - Reagent grade 4.10 D im ethyldichlorosilane - Reagent grade 4.11 Toluene - Reagent grade 4.12 Am m onium A cetate - A.C.S. R eagent G rade 4.13 Perfluorooctanoic Acid - Sigm a-Aldrich 5.0 Instrum ent and Equipm ent 5.1 A high perform ance liquid chrom atograph capable o f pum ping up to 2 solvents equipped with a variable volum e injector capable o f injecting 5-200 pL connected to a tandem M ass Spectrometer (LC/M S/M S). 5.2 A device to collect raw data for peak integration and quantitation. 5.3 Analytical balance capable o f reading to 0.00001 g. Page 2 of 8 Page 38 o f 65 Exygen Research Page 98 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Research Method Number V00178) ANALYTICAL M ETHO D M ethod o f Analysis for the Determ ination o f Perfluorooctanoic Acid (PFO A) in Fish and Clams by LC/M S/M S 5.4 R otary evaporator. 5.5 Tissum izer. 5.6 125 m L pear-shaped flasks. 5.7 50 m L disposable polypropylene centrifuge tubes. 5.8 15 m L disposable polypropylene centrifuge tubes. 5.9 D isposable m icropipets (5 0 -!OOuL, 100-200uL). 5.10 125-mL LD PE narrow-m outh bottles. 5.11 2 m L clear H PLC vial kit. 5.12 D isposable pipettes. 5.13 Autopipettes (100*1000 pL and 10-100 pL ), with disposable tips. 5.14 SPE tubes (20m L) (Supclco cat. no. N057177). 5.15 W rist action shaker. 5.16 Centrifuge capable o f spinning 50 m L polypropylene tubes at 2000 rpni. 6.0 Chrom atographic System 6 .1 A nalytical Colum n: Fluophase R P (K eystone Scientific), 2.1 mm x 50 mm. $m (P/N: 82505-052130) 6.2 Tem perature: 30*C 6.3 M obile Phase ( A ) : 2 tuM A m m onium A cetate in W ater 6.4 M obile Phase ( B ): M ethanol 6.5 G radient Program : Time (mini 0.0 1.0 8.0 20.0 22.5 %A 65 65 25 25 65 Flow Rate % B fmL/min) 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6.6 Injection Volume: 15 pL (can be increased to as m uch as 50 pL). 6.7 Quantitation: Peak Area - external standard calibration curve. 6.8 Run Time: - 23 minutes. The above conditions are intended as a guide and m ay be changed in order to optim ize the HPLC system. Page 3 o f 8 Page 39 of65 Exygen Research Page 99 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Reteirch Method Number V000! 783 ANALYTICAL M ETHO D M ethod o f Analysis for the Detennination o f Perfluorooctanoic Acid (PFO A) in Fish and G am s by LC/M S/M S 7,0 M S/M S System 7 .1 M ode: E lectrospray N egative M R M m o de, m o nito rin g 4 1 3 -- 369 m /z for PFOA. The above conditions are intended as a guide and m ay be changed in order to optim ize the M SM S system. 8.0 Preparation o f Solutions 8.1 M obile Phase 8.1.1 2 m M am m onium acetate in w ater is prepared by adding 0.IS4 g o f am m onium acetate to 1000 m L o f water. 8.2 Extraction Solutions 8.2.1 2% ascorbic acid in m eth ano l is prep ared by d isso lv in g 2 g o f ascorbic acid in 100 m L o f methanol. 8.2.2 30% D im ethyldichlorosilane in toluene is prep ared by b ringing 3 mL. o f dim ethyldichlorosilane to a final volum e o f 10 m L with toluene. Alternate volumes m ay be prepared. 9.0 Standard Preparation 9 .1 Standard Stock/Fortification Solution 9.1.1 P repare a stock solution o f --100 p g /m L o f PFO A by w eighing 10 mg o f analytical standard (corrected for purity) and dilute to 100 mL with methanol in a 125-mL LOPE bottle. 9.1.2 A 1.0 pg/m L fortification solution o f PFO A is prepared by bringing 1 m L o f the 100 pg/m L solution to a final volum e o f 100 with methanol in a 125 m L LOPE bottle. 9.1.3 A 0.1 p g /m L fortification so lu tio n o f PF O A is p rep ared by b ringing 10 m L o fth e 1.0 pg/m L solution to a final volum e o f 100 with methanol in a 125 mL LOPE bottle. 9.1.4 A 0.01 pg/m L fortification solution o f PFO A is prepared by bringing 10 m L o f the 0.1 pg/m L solution to a final volum e o f 100 with methanol in a 125 m L LD PE bottle. 9.1.5 The stock and fortification solutions are to be stored in a refrigerator at approximately 4C and are stable for a m axim um period o f 6 months from the date o f preparation. 01Page 4 '8 Page 40 of65 Exygen Research Page 100 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Research Method Number VQOO! 783 ANALYTICAL M ETHO D M ethod o f Analysis for the Determ ination o f Perfluorooctanoic Acid (PFO A) in Fish and Clams by LC/M S/M S 9.2 Standard Calibration Solutions 9.2.1 LC /M S/M S calibration standards are prepared in m ethanol via dilution o f the 1.0 pg/m L fortification solution. 9.2.2 The following is a typical exam ple: additional concentrations may be prepared as needed. Concentration Final of Fortification Volume Diluted to Concentration Solution (ua/mL) (mL) (mL) (u^m L ) 1.0 5.0 100 0.05 1.0 2.5 100 0.025 1.0 0.05 1.0 10 100 100 0.01 0.005 0.025 10 100 0.0025 0.1 10 100 0.001 0.005 10 100 0.0005 9.2.3 Store ail calibration standards in 125-m L LD PE narrow-m outh bottles at 2C to 6C, up to six months. 9.2.4 Alternate volum es and concentrations o f standards m ay be prepared as needed. 10.0 Batch Set Up 10.1 Bach batch o f sam ples extracted (ty pically 20 o r less) m u st include at least one untreated control and two untreated controls fortified at known concentrations (lab control spike) to verify procedural recovery for the batch 10.2 Requirem ents for field and laboratory duplicates and spikes will be specified in the quality assurance plan for this project. 11.0 Sam ple Extraction 11.1 W eigh 5 g o f frozen sam ple into SO m L polyp rop ylen e centrifuge luces (fortify as needed, replace Ud and m ix well). 11.2 Add 30 m L o f acetonitrile and shake on a w rist action shaker for -15 minutes 11.3 Place the tubes in a freezer for ~ 1 hour. 11.4 Pack and condition the SPE tubes and silanize the pear-shaped flasks. 11.5 Pack the 20 m L SPE tubes in sequence w ith 2 g florisil, 2 g silica gel, 2 g carbon, and I g LC-NHj. Condition the colum ns w ith 20 mL o f methanol, then 20 m L o f acetonitrile. Discard all washes. Do not allow the column to dry. 11.6 Silanize the 125 m L pear-shaped flasks by rinsing with the 30% dim etbyldichlororilane in toluene solution. Rinse the flask with toluene once, followed by m ethanol (three tim es). D ry the flasks com pletely before use, either by air-drying or w ith a stream o f nitrogen. Page 4! of65 Exygen Research Page 101 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Rewsrch Method Number VOOOI783 ANALYTICAL M ETHO D M ethod o f Analysis forthe Determ ination o f Perfluorooctanoic Acid (PFO A) in Fish and Clams by LC/M S/M S 11.7 C entrifuge the 50 m L polypropylene tubes containing sam ple at -2000 rpm fo r-1 0 minutes. 11.8 Decant the extract on to a conditioned SPE colum n fitted inside the mouth o f the pear-shaped flask. Collect the eluate in the 125 m L silam zed pear-shape flask. 11.9 Add 10 m L o f acetonitrile to the sam ple in the 50 m L centrifuge lube. H om ogenize the frozen flu phase using a tissum izer for -3 0 seconds and rinse the tissum izer w ith -1 0 m L o f acetonitrile into the tube. 11.10 Shake die sam ple again fo r-1 0 m inutes on a wrist-action shaker. 11.11 Place the tubes in a freezer for - l hour m ore. 11.12 Centrifuge the 50 m L polypropylene tubes containing sam ple at -2000 rpm fo r-1 0 minutes. 11.13 Decant the extract onto the sam e SPE colum n. Collect the eluate into the sam e pear-shaped flask and com bine w ith the eluent from the initial extraction. 11.14 Pass 20 m L o f acetonitrile through the SPE colum n and com bine the eluate m the sam e pear-shaped flask. 11.15 A dd 3-4 drops o f 1-octanol to the extract in the pear-shaped flask and evaporate at reduced pressure using a rotary evaporator (at < 40C). 11.16 M ake the final volum e, by adding 2 m L o f 2% ascorbic acid in methanol to the pear-shaped flask and swirl to mix/dissolve. 11.17 Transfer the extracts to HPLC vials using disposable pipets. 11.18 Analyze sam ples using electrospray LC/M S/M S. 12.0 Chrom atography 12.1 Inject the sam e am ount o f each standard, sam ple and fortified sam ple into the LC/M S/M S system . A calibration standard m ust precede and follow all analyzed samples. 12.2 Standards o f PFO A corresponding to at least five or m ore concentration levels m ust be included in an analytical set. 12.3 A n entire set o f calibration standards m ust be included at the beginning and at the end o f a sam ple set. Standards m ust be interspersed between every 5-10 samples. As an alternative, an entire set o f calibration standards may be injected at the beginning o f a set followed by calibration standards interspersed every 5 -10 sam ples (to accou nt fo r a seco nd set o f standards). In either case, calibration standards m ust be the first and last injection in a sample set. 12.4 Use linear standard curves for quantitation. Linear standard curves are generated for the analyte by linear regression using 1/x w eighting o f peak area versus calibration standard concentration using M assLynx 3 3 (or equivalent) software system. Pge 6 of 8 Page 42 of 65 Exygen Research Page 102 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 E x y g en P ro to co l N u m b er: P 0 0 0 1 131 Exygen Research Method Number VOOQ1783 I a n a l y t ic a l m e t h o d M ethod o f Analysis for the Determ ination o f Perfluorooctanoic Acid (PFO A) in Fish and Clam s by LC/M S/M S 12.5 Sam ple response should not exceed standard responses. Any sam ples that exceed standard responses should be further diluted and reanalyzed 13.0 Acceptance Criteria 13.1 C hrom atogram m ust show a peak o f a d au gh ter ion at 369 am u from a parem o f 413 amu. The 413 am u parent corresponds to the PFOA anion, while the daughter ion (369 am u) represents the loss o f carbon dioxide. 13.2 M ethod blanks m ust not contain PFO A at levels greater than the LOQ. If a blank contains PFO A at levels greater than 0.5 ppb, then a new blank sample must be obtained and the entire set m ust be re-extracted. 13.3 Recoveries o f control spikes and m atrix spikes m ust be between 70-130% of their know n values. If a control spike falls outside the acceptable limits, the entire set o f samples should be re-extracted. 13.4 A ny calibration standard found to be a statistical outlier by using the Huge Error Test, m ay be excluded from the calculation o f the calibration curve However, the total num ber o f calibration standards that could be excluded must not exceed 20% o f the total num ber o f standards injected. 13.5 The correlation coefficient (R ) for calibration curves generated must be 0.992 (R2 0.985). If calibration results fall outside these limits, then appropriate steps m ust be taken to adjust instrum ent operation, and the standards or the relevant set o f sam ples should be reanalyzed. 13.6 Retention tim es between standards and sam ples m ust not drift m ore than 4 % w ithin an analytical run. If retention tim e drift exceeds this limit within an analytical run then the set must be reanalyzed. 14.0 Calculations 14.1 U se the follow ing equation to calculate the am ount o f PFO A found (in n g /m l, based on peak area) using the standard curve (linear regression parameters) generated by the M ass Lynx software program: PFOA found (ng/mL) - (Peak area - intercept) slope 14.2 U se the follow ing equation to convert the am ount o f PFO A found in ng/m L to ng/g (ppb). p f o a found (ppb) - iPFQAtfflfflil (ng/m U final v oium tim L ) n PF1 sample weight (g) DF " factor by w hich the final volum e was diluted, if necessary. Pige 7 of 8 Page 43 of65 Exygen Research Page 103 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Research Method Number V000178.1 I ...................... ......................... " " a N a LY TIC A JL M E T H O D ... M ethod o f Analysis for the Determ ination o f Perfluorooctanoic Acid (PFOA) in Fish and Clams by LC/M S/M S 14.3 For sam ples fortified with known am ounts o f PFO A prior to extraction, use the following equation to calculate the percent recovery. Recovery (% ) [ total analyte found (ng/g) analyte found in con tro l (ng/g)] r 1Qj} analyte added (ng/g) Exygen Research Page 8 ofS Page 44 of65 Page 104 of 127 Interim Report #17- Analysis of Sediment and Water Samples Exygen Study No. : P0001131 Exygen Protocol Number: P0001131 ANALYTICAL METHOD M ethod Number: V00Q1784 M ethod o f A nalysis for th e D eterm in ation o f P erflu oro octano ic Acid (P F O A ) in Vegetation by LC/M S/M S Analytical Testing Facility: Exygen Research 3058 Research Drive State College, PA 16801 Approved By: c j iL ___ Paul Connolly ' Technical Leader, LC-M S, Exygen Research __lajM Date q/m / JVVoihacne Flaherty Presiden t, O perations, E xygen Research Exygen Research Total Pages: 7 Page 45 of65 Page 105 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Research Method Number V0(il>t ANALYTICAL M ETHOD M ethod o f A nalysis for the D eterm ination o f Perfluorooctanoic A cid (PFO A ) in Vegetation by LC/M S/M S 1.0 Scope This method is to be employed for the isolation and quantitation o f perfluorooctanoic acid by High Performance Liquid Chrom atography coupled to a tandem Mass Spectrom etric Detector (LC/M S/M S) in vegetation. 2.0 Safety 2.1 A lw ays observe safe laboratory practices. 2.2 C onsult the appropriate M SDS before handling any chem ical for proper safety precautions. 3.0 Sam ple Requirem ent 3.1 A t least 20 g o f test sam p le for extraction. 3.2 Samples should be processed before extraction. Place the frozen sample in a food processor and hom ogenize w ith dry ice. Place the sam ples in containers and leave open in frozen storage overnight to allow for carbon dioxide sublim ation. Seal and place the sam ples in frozen storage until time of analysis. 3.3 Sam ple collection procedures w ill be specified in the sam pling plan for this project. 4.0 Reagents and Standards 4.1 W ater - H P L C grade 4.2 Acetonitrile - H PLC grade 4.3 Carbon (120*400 m esh) - Reagent grade 4.4 M ethanol - HPLC grade 4.3 Silica gel (60-200 m esh) - Reagent grade 4.6 Florisil (60-100 m esh) - Reagent grade 4.7 Superclean LC-NH2 - Reagent grade 4.8 l -Octanol - HPLC grade 4.9 L-Ascorbic acid - Reagent grade 4.10 Dim ethyldichlororilane - Reagent grade 4.11 Toluene - Reagent grade 4.12 Am m onium A cetate - A .C.S. Reagent Grade 4.13 Perfluorooctanoic Acid - Sigm a-Aldrich 5.0 Instrum ent and Equipm ent 5.1 A high perform ance liquid chrom atograph capable o f pum ping up to 2 solvents equipped with a variable volum e injector capable of injecting 3-200 pL connected to tandem M ass Spectrometer (LC/M S/M S). 5.2 A device to collect raw data for peak integration and quantitation. 5.3 Analytical balance capable o f reading to 0.00001 g. Page 2 of 7 Page 46 of65 Exygen Research Page 106 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Research Method Number VOOOi784 ________________________________ A N A L Y T IC A L M E T H O D M ethod o f Analysis for the Determ ination o f Perfluorooctanoic Acid (PFO A) in Vegetation by LC/MS/MS 5.4 Rotary evaporator. 5.5 125 m L pear-shaped flasks. 5.6 50 m L disposable polypropylene centrifuge tubes. 5.7 15 m L disposable polypropylene centrifuge tubes. 5.6 Disposable m icropipets (50-100uL, 100-200uL). 5.9 123-mL LDPE narrow-m outh bottles. 5.10 2 m L clear HPLC vial kit. 5.11 D isposable pipettes. 5.12 Autopipettes (100-1000 \iL and 10-100 pL), w ith disposable tips. 5.13 SPE tubes (20m L) (Supelco cat. no.N 057177). 5.14 W rist action shaker. 5.15 Centrifuge capable o f spinning 50 m L polypropylene tubes at 2000 rpm 6.0 Chrom atographic System 6 .1 A nalytical C olum n: F luophasc R P (K ey ston e Scientific}, 2.1 m m x 50 m m . 5p (P/N: 82505-052130) 6.2 Tem perature: 30C 6.3 M obile Phase (A) : 2 m M A m m onium A cetate in W ater M obile Phase (B) : M ethanol Gradient Program: Time tain ) 0.0 1.0 8.0 20.0 22.5 %A 65 65 25 25 65 Flow Rate % B (m Um in) 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6.6 Injection Volum e: 15 pL (can be increased to as m uch as 50 pL). 6.7 Quantitation: Peak A rea - external standard calibration curve. 6.8 R unT im e: - 2 3 minutes. The above conditions are intended as a guide and m ay be changed in order to optim ize the HPLC system. 7.0 M S/M S System 7.1 M ode: E lectrospray N eg ative M R M m ode, m o nito rin g 413 - 369 m /z for PFOA. Page 3 of 7 Page 47 of 65 Exygen Research Page 107 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P000131 Exygen Research Method Number V0OOI7H4 ANALYTICAL M ETHOD M ethod o f Analysis for the Determ ination o f Perfluorooctanoic A cid (PFO A) in Vegetation by LC/MS7MS The above conditions are intended as a guide and m ay be changed in order to optim ize the M SM S system. 8.0 Preparation o f Solutions 8.1 M obile P hase 8.1.1 2 m M am m onium acetate in w ater is prepared by adding 0.154 g o f am m onium acetate to 1000 m L o f water. 8.2 Extraction Solutions 8.2.1 2% ascorbic acid in m ethanol is prepared by dissolving 2 g o f ascorbic acid in 100 m L o f methanol. 8.2.2 30% Dim ethyldichlorosilane in toluene is prepared by bringing 3 mL o f dimethyldichlocosilane to a final volum e o f 10 m L w ith toluene. Alternate volumes m ay be prepared. 9.0 Standard Preparation 9.1 Standard Slock/Fortificaiion Solution 9.1.1 Prepare a stock solution o f ~ 100 pg/m L o f PFO A by w eighing l mg o f analytical standard (corrected for purity) and dilute to 100 mL with methanol in a 125-mL LDPE bottle. 9.1.2 A 1.0 pg/m L fortification solution o f PFO A is prepared by bringing I m L o f the 100 pg/m L solution to a Anal volum e o f 100 w ith methanol in a 125 m L LDPE bottle. 9.1.3 A 0.1 p g/m L fortification solution o f P F O A is prepared by bringing 10 m L o f the 1.0 p g/m L solution to a final v olum e o f 100 w ith methanol in a 125 m L LDPE bottle. 9.1.4 A 0.01 pg/m L fortification solution o f PFO A is prepared by bringing 10 raL o f the 0.1 pg/m L solution to a final volum e o f 100 with methanol in a 125 m L L D PE bottle. 9.1.5 The stock and fortification solutions are to be stored in a refrigerator ai approximately 4C and are stable for a m axim um period o f 6 months from the date o f preparation. 9.2 Standard Calibration Solutions 9.2.1 LC/M S/M S calibration standards are prepared in m ethanol via dilution o f the 1.0 pg/m L fortification solution. 4 t Page 48 of 65 Exygen Research Page 108 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 IExygen Rctewcb ANa L Y I IC A i. M ET H O D Method Number V0001784 M ethod o f Analysis for the Determ ination o f Perfluorooctanoic Acid (PFO A) in Vegetation by LC/M S/M S 9.2.2 The following is a typical exam ple: additional concentrations m ay be prepared as needed. Concentration Final o f Fortification Volume Solution Cua/mLl (mL) Diluted to (mL) Concentration (lis/m L ) 1.0 5.0 100 0.05 1.0 1.0 2.5 1.0 100 100 0.025 0.01 0.05 10 0.025 10 100 100 0,005 0.0025 0.1 10 100 0.001 0.005 10 100 0.0005 9.2.3 Store all calibration standards in 125-m L LD PE narrow-m outh bottles at 2C to 6*C, up to six months. 9.2.4 A lternate volum es and concentrations o f standards may be prepared as needed. 10.0 Batch Set Up 10.1 Each batch o f sam ples extracted (typically 20 o r less) m ust include at least one untreated control and tw o untreated controls fortified at known concentrations (lab control spike) to verify procedural recovery for the batch 10.2 Requirem ents for field and laboratory duplicates and spikes will be specified in the quality assurance plan for this project. 11.0 Sam ple Extraction 11.1 W eigh 5 g o f frozen sam ple into 50 m L polypropylene centrifuge tubes (fortify as needed, replace lid and mix well). 11.2 Add 30 m L o f acetonitrile and shake on a w rist action shaker for ~ l 5 m inutes. 11.3 Centrifuge the 50 m L polypropylene tubes containing sam ple at -2000 rpm fo r-1 0 minutes. 11.4 Pack and condition the SPE tubes and silanize the pear-shaped flasks. 11.5 Pack the 20 m L SPE tubes in sequence w ith 2 g florisil, 2 g silica gel. 2 g carbon, and 1 g LC-N Hj. Condition the colum ns w ith 20 m L o f methanol, then 20 m L o f acetonitrile. D iscard all w ashes. D o n ot allow the colum n lo dry. 11.6 Silanize the 125 m L pear-shaped flasks by rinsing with the 30% dim ethyldichlorosilane in toluene solution. Rinse the flask with toluene once, followed by methanol (three tim es). Dry the flasks com pletely before use, either by air-drying or w ith a stream o f nitrogen. 11.7 Decant the extract on to a conditioned SPE colum n fitted inside the mouth of the pear-shaped flask. Collect the eluate in the 125 m L silanized pear-shape flask. Page Page 49 of65 Exygen Research Page 109 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Research Method Number VOOOI'84 AN ALYTICAL m e t h o d M ethod o f A nalysis for the D eterm ination o f Perfluorooctanoic A cid (PFO A ) in Vegetation by LC/M S/M S 11.8 Add 20 m L o f acetonitrile to the sam ple in the 50 raL centrifuge tube. 11.9 Shake the sam ple again for -1 0 m inutes on a w rist-action shaker. 11.10 Centrifuge the 50 m L polypropylene tubes containing sam ple at -2000 rpm for -5 minutes. 11.11 Decant the extract onto the sam e SPE colum n. C ollect the eluate into the sam e pear-shaped flask and com bine with the eluent from the initial extraction. 11.12 R epeat steps 11.8 through 11.11 again. 11.13 Add 3-4 drops o f 1-octanol to the extract in the pear-shaped flask and evaporate at reduced pressure using a rotary evaporator (at < 40C). 11.14 M ake the final volum e, by adding 2 m L o f 2% ascorbic acid in m ethanol to the pear-shaped flask and swirl to mix/dissolve. 11.15 Transfer the extracts to HPLC vials using disposable pipets. 11.16 Analyze sam ples using electrospray LC/M S/M S. 12.0 Chrom atography 12.1 Inject the sam e am ount o f each standard, sam p le and fortified sam ple into the L C /M S/M S system . A calibration stand ard m u st precede and follow all analyzed samples. 12.2 Standards o f PFO A corresponding to at least five or m ore concentration levels must be included in an analytical set. 12.3 An entire set o f extracted calibration standards m ust be included at the beginning and at the end o f a sam ple set. Extracted standards must be interspersed between every 5-10 samples. As an alternative, an entire set of extracted calibration standards m ay be injected at the beginning o f a set followed by extracted calibration standards interspersed every 5-10 samples (to account for a second set o f extracted standards). In either case, extracted calibration standards m ust be the first and last injection in a sam ple set. 12.4 U se linear standard curves for quantitation. Linear standard curves are generated for the analyte by linear regression using l/x weighting o f peak area versus calibration standard concentration using M assLynx 3 3 (or equivalent software system. 12.5 Sam ple response should not exceed standard responses. A ny sam ples that exceed standard responses should be further diluted and reanalyzed 13.0 A cceptance Criteria 13.1 Chrom atogram m ust show a peak o f a daughter ion at 369 am u from a parent o f 413 amu. The 413 am u parent corresponds to the PFO A anion, while the daughter ion (369 amu) represents the loss o f carbon dioxide. Pag 6 of Page 50 of65 Exygen Research Page 110 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Research Method Number VOGO 1784 A N A L Y T IC A L m e t h o d _________________________________ M ethod o f Analysis for the Determ ination ofPerfluorooctanoic Acid (PFO A) in Vegetation by LC/M S/M S 13.2 M ethod blanks m ust not contain PFO A at levels greater than the LOQ. If a blank contains PFOA at levels greater than 0.5 ppb, then a new blank sample must be obtained and the entire set m ust be re-extracted. 13.3 Recoveries o f control spikes and m atrix spikes m ust be betw een 70-130% of their known values. If a control spike falls outside the acceptable limits, the entire set o f samples should be re-extracted. 13.4 A ny calibration standard found to be a statistical outlier by using the Huge Error Test, m ay be excluded from the calculation o f the calibration curve. However, the total num ber o f calibration standards that could be excluded must not exceed 20% o f the total num ber o f standards injected. 13.5 The correlation coefficient (R ) for calibration curves generated must be 0.992 (RJ 0.985). If calibration results fall outside these limits, then appropriate steps m ust be taken to adjust instrum ent operation, and the standards or the relevant set o f samples should be reanalyzed. 13.6 Retention tim es between standards and sam ples m ust not drift m ore than 4% within an analytical run. If retention tim e drift exceeds this limit within an analytical run then the set m ust be reanalyzed. 14.0 Calculations 14.1 U se the follow ing equation to calculate the am ount o f PFO A found (in ng/mL, b ased on p eak area) using th e stan d ard cu rv e (lin e a r regression parameters) generated by the M ass Lynx software program: PFOA found (ng/mL) - (Peak area - intercept) slope 14.2 U se die following equation to convert the am ount o f PFO A found in ng/mL to n g 'g fo p b ). PFO A found (ppb) = (PFO A found (ng/m L) x final volum e (m Ll x DF1 sample weight (g) DF factor by which the final volum e was diluted, if necessary. 14.3 For sam ples fortified w ith know n am ounts o f PFO A prior to extraction, use the following equation to calculate the percent recovery. Recovery (%) * [ total analyte found (ng/g) analyte found in control (ng/g)] ^ analyte added (ng/g) Page ' oi 7 Page 51 of 65 Exygen Research Page 111 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 E xygen P rotocol N um ber: P 0 0 0 1131 ANALYTICAL METHOD M ethod N um ber. VOOOI78S M ethod of Analysis for the D eterm ination o f Perfluorooctanolc Acid (PFO A ) in Small M am m al Liver by LC/M S/M S Analytical Testing Facility: Exygen Research 3058 Research Drive State College, PA 16801 Approved By: C-JIL Paul Connolly I Technical Leader, LC-M S, Exygen Research Date .John Flaherty / V ice P resident., O perationi s, Exygen R esearch D ate Exygen Research Total Pages: 7 Page 52 of 65 Page 112 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Reiearch Method Number VOOOI785 I A iN A lY TICA L M ETH O D M ethod o f A nalysis for the D eterm ination o f Perfluorooctanoic A cid (PFO A) in Small Mamma] Liver by LC/M S/M S 1.0 Scope This m ethod is to be employed for the isolation and quantitation o f perfluorooctanoic acid by High Perform ance Liquid C hrom atography coupled lo a tandem Mass Spectrometric Detector (LC/M S/M S) in sm all m am m al liver. 2.0 Safety 2.1 A lw ays observe safe laboratory practices. 2.2 Consult the appropriate M SD S before handling any chem ical for proper safety precautions. 3.0 Sam ple Requirement 3.1 A t least 5 g o f test sam ple for extraction. 3.2 S am ples sho uld b e p ro cessed b efo re extraction . P lace th e frozen sam ple in a food processor and hom ogenize w ith dry ice. Place the sam ples in containers and leave open in frozen storage overnight to allow for carbon dioxide sublim ation. Seal and place the sam ples in frozen storage until time of analysis. A ltern ately, i f there is an insufficient a m o u n t o f sam p le {--less than 5 g), then no processing is necessary and the sam ple can be used as supplied 3.3 Sam ple collection procedures w ill be specified in the sam pling plan for this project. 4.0 Reagents and Standards 4.1 W ater -H P L C grade 4.2 M ethanol - HPLC grade 4.3 Acetonitrile - HPLC grade 4.4 Am m onium Acetate - A.C.S. Reagent Grade 4.5 Perfluorooctanoic Acid - Sigma*Aldrich 5.0 Instrum ent and Equipm ent 5.1 A high perform ance liquid chrom atograph capable o f pum ping up to 2 solvents equipped with a variable volum e injector capable of injecting 5-200 pL connected to a tandem Mass Spectrometer (LC/M S/M S). 5.2 A device to collect raw data for peak integration and quantitation 5.3 A nalytical balance capable o f reading to 0.00001 g. 5.4 50 m L disposable polypropylene centrifuge tubes. 5.5 15 m L disposable polypropylene centrifuge tubes. 5.6 Disposable m icropipets (50-100uL, 100200uL). 5.7 125-m L LD PE narrow -m outh bottles. 5.8 2 m L clear H PLC vial kit. Page 2 u p Page 53 of65 Exygen Research Page 113 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No. : P0001131 Exygen Protocol Number: P0001131 Exygen Research Method Number VOQOI785 | ........... ...... ANALYTICAL M ETHO D .......................... M ethod o f Analysis for the Determ ination o f Peifluorooctanoic Acid (PFO A) in Small Mamma] Liver by LC/M S/M S 5.9 Disposable pipettes. 5.10 Autopipettes (100-1000 pL and 10-100 pL ), with disposable tips. 5.11 W aters Sep Pile Vac 6 cc (lg ) tC 18 SPE cartridges. 5.12 SPE vacuum manifold. 5.13 Tissuem izer. 5.14 W rist-action shaker 5.15 C entrifuge capable o f spinning 15 m L polypropylene tubes at 3000 rpm. 6.0 Chrom atographic System 6.1 A nalytical C olum n: Fluophase R P (K eystone Scientific), 2.1 mm x 50 mm , 5p (P/N: 82505-052130) 6.2 Tem perature: 30C 6.3 M obile Phase (A) : 2 m M Am m onium A cetate in W ater 6.4 M obile Phase (B) : M ethanol 6.5 Gradient Program: Time imin) 0.0 1.0 8.0 20.0 22.5 %A 65 65 25 25 65 Flow Rate % B (m Um in) 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6.6 Injection V olum e: 15 p L (can b e increased to as m u ch as 50 pL). 6.7 Quantitation: Peak A rea - external standard calibration curve. 6.8 R unT im e: - 2 3 minutes. The above conditions are intended as a guide and m ay b e changed in order to optimize the HPLC system. 7.0 M S/M S System 7.1 M ode: Electrospray N egative M R M m ode, m onitoring 413 -> 369 m /z for PFOA. T h e abo ve con dition s are intended as a guide and m ay b e chan ged in order to optim ize the M SM S system. Page 1 o f ' Page 54 o f 65 Exygen Research Page 114 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Rewards Method Number VOOO1785 I A N /vL Y T IC A l ' m E T H O D M ethod o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small M ammal Liver by LC/M S/M S 8.0 Preparation o f Solutions 8.1 M obile Phase 8.1.1 2 m M am m onium acetate in w ater is prepared by adding 0.154 g of ammonium acetate to 1000 m L o f water. Alternate volumes m ay be prepared. 9.0 Standard Preparation 9.1 Standard S tock/F ortification S olution 9.1.1 Prepare a stock solution o f -1 0 0 pg/m L o f PFO A by w eighing lO m g o f analytical standard (corrected for purity) and dilute to 100 mL with methanol in a 125-mL LDPE bottle. 9.1.2 A 1.0 pg/m L fortification solution o f PFO A is prepared by bringing I m L o f the 100 pg/m L solution to a final volum e o f 100 with methanol in a 125 m L LDPE bottle. 9.1.3 A 0 .1 p g/m L fortification solution o f PF O A is prepared by bringing 10 m L o fth e 1.0 pg/m L solution to a final volum e o f 100 w ith methanol in a 125 m L LDPE bottle. 9.1.4 The stock and fortification solutions are to be stored in a refrigerator at approximately 4C and are stable for a m axim um period o f 6 months from the date o f preparation. 9.2 Standard Calibration Solutions 9.2.1 LC /M S/M S calibration standards are prepared in m ethanol via dilution o f the 0.1 pg/m L fortification solution. 9.2.2 T he following is a typical exam ple: additional concentrations may be prepared as needed. Concentration Final of Fortification Volume Diluted to Concentration Solution (na/mL) (mL) (mL) (na/m L ) 100 5.0 100 5.0 100 2.0 100 2.0 100 1.0 100 1.0 5.0 10 100 0.5 2.0 10 100 0.2 1.0 to 100 0.1 9.2.3 Store all calibration standards in 125-m L LD PE narrow-m outh bottles at 2C to 6C, up to six months. 9.2.4 Alternate volum es and concentrations o f standards may be prepared as needed. Page 4 of 7 P a g e 5 5 o f 65 Exygen Research Page 115 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Research Method Number VOOQ1785 | AN-U-ynCALMETHOP M ethod o f A nalysis for the D eterm ination o f P erfluorooctanoic A cid (PFO A ) in Sm all Mammal Liver by LC/M S/M S 10.0 Batch Set Up 10.1 E ach batch o f sam ples extracted (typically 2 0 o r less) m ust include at learn one untreated control and two untreated controls fortified at known concentrations (lab control spike) to verify procedural recovery for the batch. 10.2 Requirem ents for field and laboratory duplicates and spikes will be specified in the quality assurance plan for this project. 11.0 Sam ple Extraction 11.1 W eigh 1 g o f sam ple into a 50 m L polypropylene centrifuge tubes (fortify as needed, replace lid and mix well). N ote that alternate weights o f liver may be measured depending on the sample size available for use. 11.2 Add w ater to the sam ple for a final volum e o f 10 m L. 11.3 Hom ogenize sam ple using a tissuem izer fo r-1 m inute. 11.4 T ransfer I m L o f the sam p le using a dispo sab le p ip ette into a 15 m L disposable centrifuge tube. 11.5 Add 5 m L o f acetonitrile and shake for "2 0 m inutes on a wrist-action shaker 11 6 C entrifuge the tubes at - 3 0 0 0 rpm for - 5 m inutes. 11.7 Decant the supernatant into a 50 m L disposable centrifuge tube and add 35 m L o f water. 11.8 Condition the C u SPE cartridges (1 g, 6 m L) by passing 10 m L m ethanol follow ed b y 5 m L o f K P L C w ater (-* 2 drop/sec). D o n ot let colum n run dry 11.9 Load the sam ple on conditioned C u SPE cartridge. Discard eluate. 11.10 Elute with ~2 m L o f methanol. Collect 2 m L o f eluate into a graduated 15 m L polypropylene centrifuge tube (final volum e - 2 mL). 11.11 A nalyze sam ples using electrospray LC/M S/M S. 12.0 Chrom atography 12.1 Inject the sam e am ount o f each standard, sam ple and fortified sam ple into the LC/M S/M S system . A calibration standard m ust precede and follow all analyzed samples. 12.2 Standards o f PFO A corresponding to at least five or m ore concentration levels m ust be included in an analytical set. 12.3 A n entire set o f calibration standards m ust be included at the beginning and at the end o f a sam ple set. S tandards m ust b e interspersed betw een every 5-1 o samples. As an alternative, an entire set o f calibration standards may he injected at the beginning o f a set followed by calibration standards interspersed every 5-10 sam ples (to account for a second set o f standards) In either case, calibration standards m ust be the first and last injection in a sample set. 12.4 U se linear standard curves for quantitation. Linear standard curves are generated for the analyte by linear regression using l/x weighting o f peak area Page 5 of 7 Page 56 o f 65 Exygen Research Page 116 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 ExyganReMtrcti Method Number V000178S I ANALYTICAL M ETHO D M ethod o f Analysis for the Determ ination o f Perfluorooctanoic Acid (PFO A) in Small M ammal Liver by LC/M S/M S versus calibration standard concentration using M assLynx 3.3 (or equivalent) software system. 12.5 Sam ple response should not exceed standard responses. Any sam ples that exceed standard responses should be further diluted and reanalyzed. 13.0 Acceptance Criteria 13.1 Chrom atogram m ust show a peak o f a daughter ion at 369 am u from a parent o f 413 amu. The 413 am u parent corresponds to the PFOA anion, while the daughter ion (369 am u) represents the loss o f carbon dioxide. 13.2 M ethod blanks m ust not contain PFO A at levels greater than the LOQ. If a blank contains PFOA at levels greater than 10 ng/g, then a new blank sample m ust be obtained and the entire set must be re-extracted. 13.3 Recoveries o f control spikes and m atrix spikes m ust be between 70-130% of their know n values. If a control spike falls outside the acceptable limits, the entire set o f samples should be re-extracted. Any matrix spike outside 70 130% should be evaluated by the analyst to determ ine if re-extraction is warranted. 13.4 A ny calibration standard found to be a statistical outlier by using the Huge Error Test, m ay be excluded from the calculation o f the calibration curve However, the total num ber o f calibration standards that could be excluded must not exceed 20% o f the total num ber o f standards injected. 13.5 The correlation coefficient (R) for calibration curves generated must be 20.992 (RJ 20.985). If calibration results fall outside these limits, then appropriate steps m ust be taken to adjust instrum ent operation, and the standards or the relevant set o f sam ples should be reanalyzed. 13.6 Retention tim es between standards and sam ples m ust not drift m ore than 4 % within an analytical run. If retention tim e drift exceeds this limit within an analytical run then the set m ust be reanalyzed. 14.0 Calculations 14.1 U se the follow ing equation to calculate the am ount o f PFO A found (in ng/mL, based on peak area) using the standard curve (linear regression parameters) generated by the M ass Lynx software program: P F O A found (n g /m L ) (P ^ak area - intercept-) x D F x aliquot factor slope D F " factor by which the final volum e was diluted, if necessary. A liquot factor - 10 7Page 6 o f Page 57 o f 65 Exygen Research Page 117 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 ExygenResearch Method Number voooi?S5 I a w a i/y i IC a L m e t h o d M eth o d o f A n aly sis for th e D eterm in ation o f P erflu oro octano ic A cid (P F O A ) in Small M ammal Liver by LC/M S/M S 14.2 F o r sam p les fortified w ith kno w n am o u n ts o f P F O A p rio r to extraction, use the following equation to calculate the percent recovery. Recovery (%) " [ total analyte found (ng/mL) - analyte found in control (ng/mL)) analyte added (ng/mL) 14.3 U se the follow ing equation to convert the am ount o f PFO A found in ng/mL iu ng/g (ppb). PFO A found (ppb) = FPFOA found (na/mD x final volum e (m D l sample weight (g) Exygen Research Page 7 o f? Page 5S o f 65 Page 118 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P00Q1131 ANALYTICAL METHOD M ethod N um ber: VOOOI786 M ethod o f A nalysis for the D eterm ination of P erfluoroocU ooic Acid (PFO A ) in Sm all M am m al Serum by LC/M S/M S Analytical Testing Facility: Exygen Research 3058 Research Drive State College, PA 16801 Approved By: Paul Connolly Technical Leader, LC-M S, Exygeo Research D ate Joh n Flaherty / Vice President, Operations, Exygen Research D ate Exygen Research Total Pages: 7 Page 59 o f 65 Page 119 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 E x y g e n P ro to c o l N u m b er: P 0 0 0 1 131 Exygen Retouch Method Number V0001786 | a n a l y t ic a l m e t h o d M ethod o f Analysis for the Determ ination o f Perfluorooctanoic Acid (PFOA) in Small M ammal Serum by LC/M S/M S 1.0 Scope This m ethod is to be em ployed for the isolation and quantitation o f perfluorooctanoic acid by High Perform ance Liquid Chrom atography coupled to a tandem Mass Spectrometric Detector (LC/M S/M S) in small mam m al serum. 2.0 Safety 2.1 A lw ays observe safe laboratory practices. 2.2 Consult tbe appropriate M SD S before handling any chem ical for proper safety precautions. 3.0 Sam ple Requirem ent 3.1 A t least 1 raL o f test sam ple for extraction. 3.2 No sam ple processing is needed for serum sam ples. How ever, frozen serum sam ples m ust to allowed to com pletely thaw to room tem perature before use. 3.3 Sam ple collection procedures will be specified in the sam pling plan for this project. 4.0 Reagents and Standards 4.1 W ater - H PLC grade 4.2 M ethanol - HPLC grade 4.3 Acetonitrile - H PLC grade 4.4 Am m onium Acetate - A .C S. Reagent Grade 4.5 Perfluorooctanoic Acid - Sigm a-Aldrich 5.0 Instrum ent and Equipm ent 5.1 A high p erform ance liquid chrom atograph capab le o f pum ping up to 2 solvents equipped with a variable volum e injector capable of injecting 5-200 pL connected to a tandem M ass Spectrom eter (LC/M S/M S) 5.2 A device to collect raw data for peak integration and quantitation 5.3 A nalytical balance capable o f reading to 0.00001 g. 5.4 50 m L disposable polypropylene centrifuge tubes. 5.5 15 m L disposable polypropylene centrifuge tubes. 5.6 D isposable m icropipets (50-lOOuL, 100-200uL). 5.7 12 5-m L L D PE n an o w -m o u th bottles. 5.8 2 m L clear H PLC vial kit. 5.9 Disposable pipettes. 5.10 Autopipettes (100-1000 pL and 10-100 jiL), with disposable tips. 5.11 W aters Sep Pak V ac 6 cc (Ig ) tC 18 SPE cartridges. 5 .12 S P E v acu um m anifold. 5.13 Vortexer. 1Page o f ' Page 60 o f 65 Exygen Research Page 120 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No. : P0001131 Exygen Protocol Number: POOO1131 Exygea Research Method Number VOOOI786 | ......... a iW y h c a l m e t h o d M ethod o f Analysis for the Determ ination o f Perfluorooctanoic Acid (PFO A) in Small M ammal Serum by LC'M S/M S 5.14 W rist-action shaker. 5.15 Centrifuge capable o f spinning 15 m L polypropylene tubes at 3000 rpm 6,0 Chrom atographic System 6.1 A nalytical Colum n: Fluophase R P (K eystone Scientific), 2.1 m m x 50 mm. 5p (P/N: 82505-052130) 6.2 T em perature: 3 0 9C 6.3 M obile Phase (A) : 2 m M A m m onium A cetate in W ater 6.4 M obile Phase (B) : M ethanol 6.5 Gradient Program: Tim e (min) 0.0 1.0 8.0 20.0 22.5 %A 65 65 25 25 65 Flow Rate % B fm L/m inl 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6.6 Injection Volum e: 15 pL (can be increased to as m uch as 50 pL). 6.7 Quantitation: Peak A rea - external standard calibration curve. 6.8 R unT im e: - 2 3 minutes. T he above conditions are intended as a guide and m ay be changed in order to optim ize the HPLC system. 7.0 M S/M S System 7.1 M ode: Electrospray N egative M R M m ode, m o nito rin g 4 23 -+ 369 m/z for PFOA. The above conditions are intended as a guide and m ay be changed in order to optim ize the M SM S system. 8.0 Preparation o f Solutions 8.1 M obile Phase 8.1.1 2 m M am m onium acetate in w ater is prepared by adding 0.154 g of am m onium acetate to 1000 m L o f water. Alternate volumes m ay be prepared. Page 3 of 7 Page 6 / o f 63 Exygen Research Page 121 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygeo Research Method Number VQQOI786 | ......... A N A L Y T IC A L M E T H O D M ethod o f Analysis for the Determ ination o fPerfluorooctanoic Acid fPFO A) in Small M ammal Serum by LC/M S/M S 9.0 Standard Preparation 9.1 Standard Stock/Fortification Solution 9.1.1 Prepare a stock solution o f- 1 0 0 pg/m L o f PFO A by w eighing 10 mg o f analytical stand ard (corrected for purity) and d ilute to 100 mL with methanol in a 125-mL LDPE bottle. 9.1.2 A 1.0 pg/m L fortification solution o f PFO A is prepared by bringing 1 m L o f the 100 pg/raL solution to a final volum e o f 100 with methanol in a 125 mL LDPE bottle. 9.1.3 A 0 .1 p g /m L fortification solution o f P F O A is prep ared by bringing 10 m L o fth e 1.0 pg/m L solution to a final volum e o f 100 w ith methanol in a 125 m L LD PE bottle. 9.1.4 The stock and fortification solutions are to be stored in a refrigerator at approximately 4C and are stable for a m axim um period of 6 months from the date o f preparation. 9.2 Standard Calibration Solutions 9.2.1 LC/M S/M S calibration standards are prepared in m ethanol via dilution o f the 0.1 pg/m L fortification solution. 9.2.2 The following is a typical exam ple: additional concentrations may be prepared as needed. Concentration Final o f Fortification Volum e Diluted to Concentration Solution (njt/mL) (m L) (mL) (ng/mL) 100 5.0 100 5.0 too100 2.0 100 1.0 100 21..00 5.0 10 100 0.5 1.02.0 10 100 10 100 00..12 9.2.3 Store all calibration standards in 125-mL LDPE narrow-m outh bottles at 2C to 6C, up to six months. 9.2.4 A lternate volum es and concentrations o f standards m ay be prepared as needed. 10.0 Batch Set Up 10.1 Each batch o f sam ples extracted (typically 20 o r less) m ust include at least one untreated control and two untreated controls fortified at known concentrations (lab control spike) to verify procedural recovery for the batch 10.2 Requirem ents for field and laboratory duplicates and spikes will be specified in the quality assurance plan for this project. Page 4 of? Page 62 of65 Exygen Research Page 122 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Research Method Number V0001786 A N A L Y T IC A L m e t h o d ______________________________ M ethod o f Analysis for the Determ ination o f Perfluorooctanoic Acid (PFO A ) in Small Mammal Serum by LC/M S/M S 11.0 Sam ple Extraction 11.1 M easure 1 m L o f sam p le into a SO m L polyp rop ylen e centrifuge tubes (fortify as needed, replace lid and mix well). Note that alternate volumes of serum m ay be m easured depending on the sam ple size available for use. 11.2 Add w ater to the sam ple for a final volum e o f 20 m L. Cap tightly 11.3 V ortex fo r-1 minute. 11.4 T ran sfer 1 m L o f the sam ple using a d ispo sab le pipette into a 15 m L disposable centrifuge tube. 11.5 Add 5 m L o f acetonitrile and shake for -2 0 m inutes on a wrist-action shaker. 11.6 Centrifuge the tubes at -3 0 0 0 rpm for -S m inutes. 11.7 D ecant the supernatant into a SO m L dispo sab le centrifuge tube and add 35 m L o f water. 11.8 Condition the C u SPE cartridges (1 g, 6 m L) by passing 10 m L methanol followed b y 5 m L o f HPLC w ater ( - 2 drop/sec). D o not let colum n run dry 11.9 Load the sam ple on conditioned Cig SPE cartridge. Discard eluate. 11.10 Elute with - 2 m L o f m ethanol. Collect 2 m L o f eluate into a graduated 15 m L polypropylene centrifU ge tube (final v olum e = 2 m L). 11.11 Analyze sam ples using electrospray LC/M S/M S. 12.0 Chrom atography 12.1 Inject the sam e am ount o f each standard, sam ple and fortified sam ple into the LC/M S/M S system. A calibration standard m ust precede and follow all analyzed samples. 12.2 Standards o f PFO A corresponding to at least five or m ore concentration levels m ust be included in an analytical set. 12.3 A n entire set o f calibration standards m ust be included at the beginning and at the end o f a sam ple set. S tandards m u st be interspersed betw een every 5 -1o samples. As an alternative, an entire set of calibration standards may be injected at the beginning o f a set followed by calibration standards interspersed every 5-10 sam ples (to account for a second set o f standards), in either case, calibration standards m ust be the first and last injection in a sam ple set. 12.4 Use linear standard curves for quantitation. Linear standard curves are generated for the analyte by linear regression using 1/x w eighting of peak area versus calibration standard concentration using M assLynx 3.3 (or equivalent) software system. 12.3 Sam ple response should not exceed standard responses. A ny sam ples that exceed standard responses should be further diluted and reanalyzed. Page 5 o Page 63 of65 Exygen Research Page 123 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Research Method Number VOOO1786 | ANALYTICAL M ETHO D M ethod o f A nalysis for the D eterm ination o f Perfluorooctanoic Acid (PFO A ) in Small Mammal Serum by LC/M S/M S | 13.0 A cceptance C riteria 13.1 C hrom atogram m ust show a peak o f a daughter ion at 369 am u from a parent o f 413 amu. The 413 am u parent corresponds to the PFOA anion, while the daughter ion (369 am u) represents the loss o f carbon dioxide. 13.2 M ethod blanks roust not contain PFOA at levels greater than the LOQ. if a blank contains PFOA at levels greater than 10 ng/m L, then a new blank sample m ust be obtained and the entire set must be re-extracted. 13.3 Recoveries o f control spikes and m atrix spikes m ust be betw een 70-130% of their known values. If a control spike falls outside the acceptable limits, the entire set o f samples should be re-extracted. Any matrix spike outside 70 130% should b e evaluated b y th e analyst to d eterm in e if re-extraction is warranted. 13.4 A ny calibration standard found to be a statistical outlier by using the Huge Error Test, m ay be excluded from the calculation o f the calibration curve However, the total num ber o f calibration standards that could be excluded m ust not exceed 20% o f the total num ber o f standards injected. 13.5 The correlation coefficient (R) for calibration curves generated must be 0.992 (R2 0.985). If calibration results fall outside these limits, then appropriate steps m ust be taken to adjust instrum ent operation, and the standards or the relevant set o f samples should be reanalyzed. 13.6 R eten tion tim es b etw een stand ards and sam p les m u st not drift more than 4 % w ithin an analytical run. If retention tim e drift exceeds this lim it within an analytical run then the set m ust be reanalyzed. 14.0 Calculations 14.1 U se the follow ing equation to calculate the am ount o f PFO A found (in ng/m L based on peak area) using the standard curve (linear regression parameters) generated by the M ass Lynx software program: PFOA found (ng/m L) * (Peak area - intercept) x DF x aliquot factor slope DF = factor by which the final volum e w as diluted, if necessary. Aliquot factor ~ 20 14.2 For sam ples fortified w ith know n am ounts o f PFO A prior to extraction, use the following equation to calculate the percent recovery. Recovery (%) * [ total analyte found (ng/mL) - analyte found in control (ng/mL)] ^ ^ ______________________ analyte added (ng/m L )___________________________P_a_g_e__6__o_f_7 Page 64 of65 Exygen Research Page 124 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: POOO! 131 Exygen Research Method Number VOOQ1786 | ................ a n a L v t ic a i. m e t h o d M eth o d o f A n alysis for th e D oterm inatioo o f P erflu oro octano ic A cid (PFO A ) in Small M ammal Serum by LC/M S/M S 14.3 U se the follow ing equation to convert the am ou nt o f P F O A found in ng/m L io ppb. PFOA found (ppb) - (PFOA found (ng/m L) x final volum e (m U l sample volum e (mL) Exygen Research Page 7 ot 7 Page 65 of65 Page 125 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 3058 Research Drive Phone: 814-272-1039 State College, PA 16801 Fax: 814-231-1580 PROTOCOL AMENDMENT Amendment N um berJ__ Effective Date: 01/19/05 Exygen Study Number P0001131 Client Study Number Page 1 of 1 DESCRIPTION OF AMENDED SECTION 1) Analytical Procedure Summary V0001780:Section 9.1 2) Verification of Analytical Procedure None AMENDED TO 1) Add to Section 9.1: Section 9.1.6, Alternate weights of standards may be used to prepare alternate concentrations of stock solutions as necessary. Alternate levels of fortification solutions may also be prepared. 2) Low and high spiking levels of the analytes for each matrix may be altered depending on sample size available for extraction and/or to cover analyte concentrations expected in the samples. RATIONALE 1) Higher concentrations of standards need to be prepared in order to spike the sample bottles at higher levels. 2) The sample size available for small mammal liver and serum was smaller than expected. Spiking at the pre-determined levels in the protocol puts the spiked concentration lower than the detection limit. Also, the analyte levels in the ground water samples are expected to greatly exceed the pre-determined spiking levels listed in the protocol. When the levels in the samples greatly exceed the spiking levels, an accurate recovery value cannot be calculated for the QC sample. Higher spiking levels in the bottles will cover the analyte concentrations expected in the water samples. IMPACT ON STUDY The LOQ Is 100 ng/g for a 0.1 g sample of small mammal liver and is 1000 ng/mL for a 0.01 mL sample of small mammal serum. Higher levels of spiking for the water samples will ensure that more QC recovery data can be used. /Principal Investigator Signature ament Slgnati is.Date uiipd) Data/ I $ 5Exygen QAU Review o d ili It" - f. LIBRARY ID: W 0001225-6; ' ADMINISTRATIVE FORM Exygen Research Page 126 of 127 Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: P0001131 3058 Research Drive Phone: 814-272-1039 State College, PA 16801 Fax: 814-231-1580 Amendment Number Effective Date: Exygen Study Number PROTOCOL AMENDMENT 2 03/07/05 P0001131 Client Study Number: Page 1 of 1 None DESCRIPTION OF AMENDED SECTION 1) Report, page 11 of 65 2) Test Materials, page 6 of 65: PFOS transition monitored 499 -> 99. AMENDED TO 1) Instead of one final report, interim reports will be issued. 2) PFOS transition monitored may also be 499 -> 80. RATIONALE csfa/ts 'MX1) Due to the excessive sizes of the data sets, interim reports will be issued to allow the client to receive data In a timelier reaner; "m/k *' 2) The API 4000 LC/MS/MS systems detect the 499 -> 80 PFOS transition with greater sensitivity than the 499 -> 99 transition. IMPACT ON STUDY 1) The client will be able to receive and review the data more quickly. 2) The 499 -> 80 transition can be detected with greater sensitivity; therefore, giving better chromatography. LIBRARY ID: V0001226-- Exygen Research ADMINISTRATIVE FORM Page 127 of 127