Document V35mVyBqG9KDN3191K8Ggb1po

COMPOSITE ANALYTICAL LABORATORY REPORT ON THE QuantitativeAnalysisof Fluorochemicalsin Environmental Samples REPORT No. FACT GEN-021, GEN-024, GEN-030, GEN-033 LRN-W2491, W2845, W31979 EOO-1386 ANALYTICAL STUDY INITIATION GEN021: 08/25/99 GEN024:10/12/99 GEN030:12/13/99 GEN033: 03/14/00 3M EnvironmentalLaboratory Report No. Gen-021, Gen-024, Gen-030, Gen-033 TABLE OF CONTENTS AnalyticalStudy Personnel and Contributors..............................................j............. Introductio.n...................................................................2........... Purpose....................................................................2............ Testand ControlArticl.e.......................................................2............ Sample Collectioannd Analysi.s.................................................3............ Sample Receiptand Maintenance..................................................3............ Chemical Characterizati.o.n......................................................3............ Procurement.................................................................3........... Method Summaries ..............................................................3............ Preparatoryand AnalyticaMlethods .............................................4............ AnalyticaElquipment.......................................................5............ Data Summary, Analyses,and Results...............................................6............ Summary ofQualityControlAnalysesResults......................................6............ Summary ofSample Results....................................................7........... Data QualityObjective.s.........................................................8............ Statementof Conclusion..........................................................9............ References ..................:........................................................9............. Attachments....................................................................1.0........... ReportSignature................................................................1.0.......... LIST OF TABLES Table 1.DescriptioonfSamples, by Study ............................................2............ Table2.ProcurementInformatiofnorReferenceMaterialisntheAnalysisofEnvironmental Samples ...............................................................3........... Table 3.IonsMonitoredintheAnalysesofExtractsofGroundwater........................6............ Table 4.Range ofLOQs forSera,by Study...........................................8............ Table5.Range ofLOQs forLiverand OtherTissues,by Study............................8........... Proptietary and Confidendal Page if 3M EnvironmentalLaboratory ReportNo.Gen-021,Cen-024,Gen-=, Gen-= ANALYTICAL STUDY PERSONNEL AND CONTRIBUTORS AnalyticaClhemistryLaboratories 3M EnvironmentalTechnology and SafetyServices(ET&SS) 3M EnvironmentalLaboratory FluorineAnalyticaClhemistryTeam (FACT) 2-3E-09 935 Bush Avenue St.Paul,MN 55106 KrisHansen, Ph.D.,PAI HaroldJohnson,AnalyticaClhemist LisaA. Clemen, AnalyticaClhemist Mark EllefsonA,na"cal Chemist Sponsor 3M ET&SS Buildt6g2-3E-09 St.Paul,MN 55133 Dale Bacon, Sponsor Representative Page 1 of10 3M EnvironmentalLaboratory Report No. Gen-021, Gen-024, Gen-030, Gen-033 INTRODUCTION Purpose The purposeofthiscompositereportistoprovidea summary oftheanalyticdaaltacollectefdor 3M studiesGen-021, Gen-024, Gen-030, and Gen-033. Allofthesamples includedinthese studiesaretissuesamples collectedfrom fish,birds,mammals, and amphibians;Dr.John Geisy of Michigan StateUniversityhas suppliedallsamples to3M. These analyseshave been conducted to supportstudiesdesigned by Dr.Geisy. The targetanalytesforthesefourstudieswere perfluorooctanseulfonate(PFOS; CAS# 2795-393),perfluorooctanesulfonylami(dPeFOSA; CAS# 754-91-6),perfluorooctanoa(tPeFOA orPOAA; CAS# 3825-26-1),and peIrfluorohexanesulfonate(PFHS; no CAS# available). Due to thevarietyof matricesanalyzed(withrespectto bothspeciesand tissues)a,nd due to evolvinganalyticamlethods, some analyticadlata qualityobjectivess,uch as thelimiotf quantitatio(nLOQ) were quitevariableA. summary ofthe achievedLOQ (byspecie,tissueand studynumber) ispresentedinTable 2 ofthisreportT.he stateddataqualityisbased on resultosf data collectioqnualitycontrolss,ample prepqualitcyontrolsa,nd recoveryoftargetanalytesfrom prepared matrixspikesamples. More specifidcataqualityobjectiveasnd parametersforthese analyticasltudiesare outlinedlaterinthisreport. Test and Control Article The testarticlefsoreach studyconsistedof varioustissuesfrom variousspeciesand are listed below,inTable 1.For allstudies,the controlarticlceonsistedof rabbitsera and rabbitlivera,s appropriateR.abbittissueswere chosen as thecontrolarticlebsecause previousstudieshave indicatedverylow levelsof endogenous fluorochemicalisnthese matrices.Samples ofthe controlarticlewsere providedby the3M EnvironmentalLaboratory. This reportdoes notincludedetailsforthecollectioonfthetestarticletsh;esedetailshouldbe obtainedfrom Dr.Geisy. Table1.DescriptioonfSamples,by Study G7en-0Ge2n1-021 Nwivmit@@ Cormorant Blood, Caspian Seal Blood,Sea OtterBk)od I.- CaUforrgaSea Uon, ElephantSeal, Harbor Seal,Gozzi,Mink, River Ofter,Sea Otter,Turtle ex- Sea OtterBrain,Sea OtterKxiney Gen-024 AJbatromsera,Alb&m plasm. Cmwrarit plasm, Hw" Gul Plasma, Bald Eagleplasm. CwTffard bk)od,Hwing GLA bk)od Loon,Bmvm PelicanA,lbaftm AJbahm Iddrwy,CormorantydK Gul yolk Gen-030 NorthernFur Sealbk)odauvenle,subadulta,duk),PolarBear blood,Stdw Sea Lionbk)od Norftm Fur Seal,PolarBear,MnK Map TurtleT,errapin,Tuna, Green Frog,Chinook SWmon, Lake Whkefth, Brown TroLit Carp body,Frog muscle,Frog body, Green Fmg eggs, LakeWHMW eggs, Bmwn TrotAeggs, Carp muscle,Chhook Sahm muscle,Lake VVhbfish muscle, Bmwn TrotAmuscle Gen-033 None Submitted MinK BaikalSeal,GarKies Dolphin, Cwmmrt (adutandit,%wile), BottlenoseDolphin,smped Dolphin, Weddel Seal,Svmmfth, Tuna, BhwJftledGull None Submitted Page 2 of 10 3M EnvironmentalLaboratory Report No. Gen-021, Gen-024, Gen-030, Gen-033 Followinganalysis,extractsgenerated from these samples have been retainedincoldstorage, Sample Collection and Ana"is Tissue samples were submittedtothe EnvironmentalLaboratory-FluorineAnalyticalChemistry Team by Kurunthachalan Kannan of MichiganStateUniversityD.etailsofthe sample receiptare documented on the chain of custodyforms locatedinappendices ofthisreport. SAMPLERECEIPT AND MAINTENANCE Samples were receivedinthe EnvironmentalLab coldor frozenon the followingdates:Gen-021 (8/24/99)G.en-024 (10/11/99),Gen-030 (12/13/99)a,nd Gen-033 (3/13/00)S.ample receipt, identificatioann,d chain ofcustody informationare locatedinthe study folderforeach report;the foldersare locatedinthe 3M archives. The sample extractswillbe maintainedincoldstorageatthe 3M EnvironmentalLaboratoryuntil the qualityof preparationno longeraffordspreservation. CHElilCAL CHARACTERIZATION The targetanalytescharacterizedinthe samples includePFOS, PFOSA, PFOA, and PFHS. Procurement detailsof the referencestandardsused foranalysisare summarized below. Procurement Table2.ProcurementInformatiofnorReferenceMaterialisntheAnalysisofEnvironmentalSamples . kvirivin: PFOS (potassiumsaft) PFOSA rs (:S, PFHS (potassiumsalt) PFOA (ammonium saft) I RK*Imilqlll 171 Gen-021: L-2353; allothers:L-15709 NBI16638-16 Gen-024: 245; allothersc:ommercial 3M ICP/PCP Division 3M SpeciaftCyhemicals(R.Buckanin) 3M SpecialtyChemicals.(G.Moore) Gen-024: 3M SpecialtCyhemicals; allothers:AJddch Fullchemical characterizatiosntudies,includingpurityand stabilitdyeterminationh,ave notbeen completed atthistime.Upon completionofthese studies,a reportwillbe archivedinthe 3M Environmental Lab. METHOD SUMMARIES Followingisa briefdescriptionofthe methods used duringthisanalyticasltudy by the 3M Environmental Laboratory.Copies ofthe actualmethods used forthese studiesare locatedin attachment H. Page 3 of10 3M EnvironmentalLaboratory ReportNo. Gen-021, Gen-024, Gen-030, Gen-033 PREPARATORYAND AMALYMAL METHODS ETS-8-004.1, "Extractionof PFOS or Other FluorochemicalCompounds from Serum for AnalysisusingHPLC-ElectrosprayMass Spectrometry"withsome modificationsd,escribed below. Because the matriceswere so variableand sample sizeextremelylimitedi,twas not possibleto prepareextractedstandardcurves.Allextractswere evaluatedversus unextractedstandardcurves.When sample sizepermittedt,wo matdx spikeswere prepared ineach tissuesample from each specietestedto providesome levelofextractioenfficiency determination. For some samples,lessthan I mL of sample was availableF.or thesesamples,the availablevolume was extractedaccordingto themethod withthe exceptionthatthe final volume ofextractiosnolventwas adjustedtomatch thevolume ofthe initisaalmple. This method was used forthe extractioonfsera,plasma,and whole bloodsamples. ETS-8-005.1,'AnalysisofPFOS or Other FluorochemicalCompounds inSerum Extracts Using HPLC-ElectrosprayMass Spectrometry'withsome modificationsd,escribedbelow. Because the matriceswere so variableand sample sizeextremelylimitedi,twas not possibleto prepareextractedstandardcurves.Allextractswere evaluatedversus unextractedstandardcurves;as a resulta,llsample concentrationwsere adjustedby a factor of 1.25to adjustforthe removal of4/5oftheMTBE from theextractT.he factoris unnecessarywhen an extractedcurve isused forevaluation. ETS-8-006,"Analysisof PFOS or Other FluorochemicalCompounds inLiverExtractsusing HPLC-ElectrosprayMass Spectrometry*withsome modificationdse,scribedbelow. Because thematriceswere so variableand sample sizeextremelylimitedi,tw as not possibleto prepareextractedstandardcurves.Allextractswere evaluatedversus unextractedstandardcurv.esW.hen sample sizepermittedt,wo matrixspikeswere prepared ineach tissuesample from each specietestedto providesome levelofextractioenfficiency determination. For some samples,lessthan 1 g of sample (ascalledforinthe method) was availableF.or these samples,theavailablemass oftissuewas extractedaccordingto themethod. Samples of kidney,brain,egg,and muscle were extractedby thismethod. ETS-8-007, 'Extractioonf PFOS or Other FluorochemicalCompounds from Liverfor AnalysisusingHPLC-ElectrosprayMass Spectrometry*vathsome modificationdse,scribed below. Because the matriceswere so variableand sample sizeextremelylimitedi,twas not possibleto prepareextractedstandardcurves.Allextractwsere evaluatedversus unextractedstandardcurves;as a resulta,llsample concentrationwsere adjustedby a factor of 1.25to adjustfortheremoval of 4/5of theMTBE from the extractT.he factoris unnecessarywhen an extractedcurve isused forevaluation. For Gen-030 and Gen-033 only:Due tothelackof excesstestmaterialformethod development,allsamples determinedto containgreaterthan 0.015 pg/gof PFOS were subjecttoan additionaPlFOS verificatipornocess.Each sample was analyzedseparately withrespectto the499 -) 99 transitioand the499 4 80 transitionTsh.e quantitativreesults Page 4 of10 3M EnvironmentalLaboratory ReportNo. Gen-021, Gen-024, Gen-030, Gen-033 obtained from each transitionanalysiswere compared. When these resultsagreed to with 30%, the identityof PFOS was confirmed (see Reference 1).Those samples where the identityof PFOS could not be confirmed are noted inthe data table. In Gen-021, no PFHS standard was available.Inthese samples, qualitativedetermination of PFHS was conducted based on reasonable retentiontime and a known PFHS transition(399 499). Specific instrumental parameters are available inappendix I-Lof this report,stored inthe 3M Environmental Lab archives. AMALYRCAL EQwmENT For HPLC-Electrospray Tandem Mass Liquid Chromatograph: Howleff-Packae Analyticalcolumn: 1 x30 mm Cl 8 BetasilTm Column temperature: 30 degrees C Cycle Time: 10 minutes Spectrometry: Series 1100- Liquid Chromatograph* system Mobile,phase components: -Component A: 2mM ammoniu m Component B: Methyl alcohol ' Flow rate:300 pumin acetate Injectionvolume: 10 pL Solvent Gradient: Time (min) %B 0 10 1 10 5.5 95 7.5 95 8 10 10 10 For Detection: Mass Spectrometer Micromas? API/Mass Spectrometer Quattro Ultima TripleQuadrapole system or Micromas? APVMass Spectrometer Quattro 11Triple Quadrapole system AcquisitionMode: MRM (referto Table 3) Software: Mass LynxT" 3.3 Mode: Electrospray Negative Source Block Temperature: 125-1500C Source: Z-spray Page 5 of 10 3M EnvironmentalLaboratory Report No. Gen-021, Gen-024, Gen-030, Gen-033 Table 3.Ions Monitored in the Analyses of Extracts of Groundwater wil:T-ei:a . . . . .. 0 . I P7F SA7 PFO0S PFOS PFOSA PFOA 499.0 498 413 80,9978 169 PFHS 399 99 * Indicates the ion used for quantitation Refer to the analyticalmethods and equipment logs found inthe raw data for detailson the actual analyticalequipment settingsused inthe present study. These settingsmay have varied somewhat during actualdata collection.However, slightvariationsin the instrument settingswill not adversely affectthe qualityof the data. Exact settingsduring allphases of data collectionare recorded and presented in the appendix of thisreport. DATA SUMMARY, ANALYSES, AND RESULTS Summary of Quality Control Analy ses Results Standard Curves: The coefficienotf determination(r,2f)or all1/X weighted curves bracketing useable data was > 0.982. High or low curve points may have been excluded to provide a betterfitover the linearrange appropriate to the data. High or low curve points were deactivated ifthe calculated concentration varied from the theoreticalconcentration by more than 30%. Acceptable data was evaluated versus a standard curve containing at least 5 points.Allactions are acceptable and are documented in specificdata sets.All standard curves used to evaluate quantitativedata were acceptable. Continuing Calibration Verifications:On average, one calibrationcheck isanalyzed for every fivesamples. Acceptable data isbracketed by calibrationchecks quantitated to be within 30% of the theoreticalvalue, evaluated at least every ten samples. Allquantitative data isbracketed by acceptable calibrationchecks, as required. Blanks: Extraction blanks were compliant ifno target analyte was detected above the limit of quantitation(LOQ) fora specificanalyte.Inthis study, extractionblanks were often higher than low curve points.Because analyte levels inthe blank are used to determine the LOQ, by default,allblanks were determined to be below the limitof quantitationfor the compounds of interest. Internal Standards: Internalstandard response was monitored inGen-030 and Gen-033 only. Internalstandard response was required to be within *50% of the theoreticalvalue. If samples showed an internalstandard response that deviated more than :L50%, the samples were reanalyzed. Ifthe deviant IS response was confirmed, the analyte data was reported, but noted inthe data table. Page 6 of 10 3M EnvironmentalLaboratory Report No. Gen-021, Gen-024, Gen-030, Gen-033 Summary ofSample Results GEN-021: PFOS was detected inatleastone sample from the followingmatrices:CaliforniSaea Lion liverH,arbor Seal liverG,ozzi liverM,ink liverR,iverOtterliverT,urtleliver, Cormorant blood,Otterblood,and Caspian Seal blood. PFOSA was tentativeliydentifieidnatleastone sample from the followingmatrices: CaliforniSaea Lion liverR,iverOtterliverS,ea OtterliverS,ea Otterbrain,and Otter blood. PFOA was tentativeliydentifieidnat leastone sample from the followingmatrices: CaliforniSaea Lion liverand Caspian Seal blood. PFHS was tentativeliydentifieidnatleastone sample from the followingmatrices: CaliforniaSea Lipn liverG,ozzi liverM,ink liverR,iverOtterliverS,ea OtterliverT,urtle liver,Cormorant blood,Caspian Seal blood,and Otterblood. GEN-024: PFOS was detectedidentifieidnatleastone sample from the followingmatrices: Albatrossplasma, Albatrosssera,Cormorant plasma,Cormorant blood,HerringGull plasma, HerringGull blood,Bald Eagle plasma, Loon liverA,lbatrossliverB,rown PelicanliverA,lbatrosskidney,Cormorant yolk,and Gullyolk. PFOSA was tentativeliydentifieidnatleastone sample from the followingmatrices: Cormorant blood,Bald Eagle plasma, Loon liverB,rown Pelicanlivera,nd Albatross liver. PFOA was tentativeliydentifieidnatleastone sample from the followingmatrices: Cormorant blood,AlbatrossliverC,ormorant yolk,and Gullyolk. PFHS was tentativeliydentifieidnat leastone sample from the followingmatrices: HerringGull plasma and Bald Eagle plasma, Loon liverA,lbatrossliverB,rown Pelican liverA,lbatrosskidney,Cormorant yolk,and Gull yolk. GEN-030: PFOS was detected inat leastone sample from the followingmatrices:Polar Bear blood,PolarBear liverM,ink liverN,orthern Fur Seal liverM,ap TurtleliverT,una liver, Green Frog liverC,hinook Salmon liverL,ake WhitefishliverB,rown TroutliverW,hole Carp, Frog muscle, Lake Whitefisheggs, Brown Trout eggs, Carp muscle,Chinook Salmon muscle, Lake Whitefishmuscle, and Brown Trout muscle. PFOSA was tentativeliydentifieidnat leastone sample from the followingmatrices: Mink liver. PFOA was nottentativeliydentifieidnany sample analyzed. PFHS was not tentativeliydentifieidnany sample analyzed. GEN-033: PFOS was detected inatleastone sample from the followingmatrices:Mink liverB,aikal Seal liverC,ormorant liverB,ottleNosed DolphinliverG,anges DolphinliverS,triped DolphinliverS,wordfish Liver,Tuna livera,nd BlackTailedGullliver. PFOSA was tentativeliydentifieidnat leastone sample from the followingmatrices: Mink liverC,ormorant livera,nd BottleNosed Dolphinliver. Page 7 of10 3M EnvironmentalLaboratory Report No. Gen-021, Gen-024, Gen-030, Gen-033 PFOA was tentativeliydentifieidnat leastone sample from the followingmatrices: Cormorant liver. PFHS was tentativeliydentifieidnatleastone sample from the followingmatrices:Mink liverS,tripedDolphin livera,nd Swordfish Liver. Appendices containdata summary tables. DATA QUALITY OBJECTIVES No circumstances existedduring the presentstudy thatwould have affectedthe qualityor integritoyf the data.The data qualityobjectives(DQOS) followedduringthe presentare indicatedbelow. Linearity:The coeffrcienotfdetermination(?)ofthe standardcurve was equal toor greater than 0.985 with at least5 activepointsusing a linearregressioncurve with 1/xweighting. Instrument Quantitation Limit (IQL):The IQL isequal tothe lowestacceptablestandardin the calibratiocnurve (acceptablestandard isdefinedas a standardwithin30% ofthe theoreticavlalue).As thisvalue isnot usefulinconsideratioonfthe sample data,the IQL was not specificalldyetermined or statedforevery study. 0 Limits of Quantitation (LOQ): The LOQ is equal to the lowest acceptable standard in the calibratiocnurve (definedas a standardwithin30% ofthe theoreticavlalue),and isatleast two times the analytepeak area detectedinthe extractionblanks.The LOQ may vary due to the amount ofsample availableforanalysis(particularfloyrsamples extractedaccordingto ETS-8-006) or to day-to-dayvariationsinthe analyticaslystem.The ranges of LOQs for varioustissuesare listedinTable 4 (sera,plasma, and blood)and Table 5 (liverk,idney, muscle, egg, and brain). Table4. Range of LOQs forSera,by Study I -- PF0OS 0.0116 pg/mL 0.00116 pWmL 0.0029-0.0579pWmL NA PFOS P7F PFOSSAA 0.00625pg/mL 0.00626 pWmL 0.000625 pg/mL NA PFHS NA 0.00114 pg/mL 0.00114 pg/mL NA PFOA 0.00599 pg/mL 0.0299 pg/mL 0.00240-0.00958 pWmL NA Table S.Range of LOQs forLiverand Other Tissues,by Study P7F SA PF0OS PFOS PFOSA PFHS PFOA 0.0348 pg/g 0.0375 pg/g NA 0.0359 pg/g NA = not applicable 0.0348 pglg 0.00750 pg/g 0.00683 pg/g 0.180 pg/g 0.00696-0.0696pg/g 0.0188 pg/g 0.00683-0.0342pg/g 0.0180-0-0719pg/g 0.00696-0.0694pg/g 0.0376 pgtg 0.00683 pg/g 0.00719-0.0718pglg Page 8 of 10 3M EnvironmentalLaboratory Report No. Gen-021, Gen-024, Gen-030, Gen-033 Duplicatelacceptableprecision(extraction)S:pikesconductedon samples of control tissueswere reproducibletowithin15% Quality Control Response: A continuingcalibratiovnerificatio(nCCV) was analyzed every 5-10 samples. Acceptable CCV response was within:t30% ofthetheoreticavlalue.No more than 10 samples were analyzed between acceptableCCVS. Spike/acceptable recoveries:Due to the number ofdifferenmtatricesanalyzed,therewas greatvariabiliitnyspikerecoveries.For any givenmatrix(specieand tissue)s,pike recoverieswithin70-130% ofthe expected concentratioinndicatequantitativdeata (good to 30%); spikerecoveriesbetween 50-150% indicatseemi-quantitativdeata forthatmatrix (good to 50%). Spike recoveriesoutsideofthisrange indicattehatsample data should be used forqualitativpeurposes only.Due tosample limitationmsa,trixspikestudieswere not conducted forallmatrices.For PFOS analyses,sample data thatisnotsupported by matrix spike studiesshould be consideredforqualitativpeurposes only.Since no identity verificatioenxperiments were performed forPFOA, PFHS, and PFOSA, forthese analytes, allanalysesthatare notsupported by matrixspikestudiesshould be consideredto provide unconfirmed qualitativdeata only. Use of InternalStandards: Tetrahydro-perfluorooctsaunlefonateCRHPFOS) was spiked intothe extractspost-extractioannd used as an internasltandardforsamples inGen-030 and Gen-033. For allsamples inthese studies,THPFOS levelswere monitoredto verifythe analyticasloundness of the data.THPFOS levelsthatwere determined tobe deviantfrom expected values by more than 50% were reanalyzed.Ifthe deviantTHPFOS levelswere confirmed,analytelevelswere reportedbut are noted inthe resultstable. Use of confiffnatorymethods: Given the selectiviotfythe analyticatloolused (HPLCESMSMS) and lackof a viablealternativfeoranalysisn,o confirmatorymethods were used. Demonstration of specificityS:pecificitwyas demonstrated by chromatographicretention time (matched to standardstowithin3%) and the responseof atleastone characteristic product ionarisingfrom collisionosfan analyte-speeifpiacrention. Assuming spikerecovery studiesform a suitableindicatioonf endogenous analyterecovery, matrixspikestudieshave been used as an indicatoorf dataquality(seeabove).The validitoyf thisassumption has not been verifiedby othertechniques. STATEMENT OF CONCLUSION Under the conditionsofthe presentstudies,the presence offluorochemicalwsas observed inthe quantitativaenalysisof a selectionof environmentalmatrices. REFERENCES 1) "Acceptance CriterifaorUltratraceHPLC-Tandem Mass Spectrometry:Quantitativeand QualitativeDeterminationofSulfonylureaHerbicidesinSoil";Li,L.Y.;Campbell, D.A.;Bennet, P.K.;Henion, J.;Anal. Chem., 68 (19),3397-3404,1996 Page 9 of10 3M EnvironmentalLaboratory Report No. Gen-021, Gen-024, Gen-030, Gen-033 ATTACHMENTS Attachment A: Gen-021 Sera/Plasma/Blood Results Attachment B: Gen-021 Liver/Miscellaneous Results Attachment C: Gen-024 Sera/Plasma/Blood Results Attachment D: Gen-024 Liver/Miscellaneous Resufts Attachment E: Gen-030 Sera/Plasma/Blood Results Attachment F: Gen'-930 Liver/Miscellaneous Results Attachment G: Gen-033 Liver Results Attachment H: AnalyticalMethods Attachment I- L (additionalbound document availableinthe 3M Environmental Lab arrhives):AnalyticalDetails for Gen-021, Gen-024. Gen-030, and Gen-033 REPORT SIGNATURE &.46, Msten J. Hansen, Ph.D., PrintipalAnalyticalInvestigator 5-110100 Date Ta/e L. Bacon, Spinsor Rep -resentative Date Page 10of10 I @l., A , I I 0 WifteMons- OvicRkatefeInnedaeS.y(stem '0199W1ilsJoonnCeosmpany FACT-GEN-021 Study. ProdadNumba(rem Substmce): Mabriz: Medwd/Rovision: A-b,-a E"pn-i Sy- N-nbcr lasuvnww Softwwetvcnion: Filenum: R-Squefed Vahm: Slope: Y-Wwccpt: Dan of ExmwdWAaMyst Dan ofAnalysis/Aneyst Dam ofData Reducdon/Analyst: Sample Data BLOOD Group Dose smvle 0 Method Bik H20 Rik-I&?SM H20 Blk-2 Lr25i99 H20 Bik-3 &75M H20 Bik-4 W25M Caspim Sad Blood W249140.J 33 W2491-41.1 11 W2491-42,J 46 - W2491-43.J 17 W249144J 13 W249145,1 8 W2491-46J t2 W249147.19 W249148,J 14 W249149J 18 W2491-SOJ S2 W2491-$IJSS W2491-52,J 10 W2491-S3j 15 No anab=d forPFHS. PFDS. PFHS bmd an PFOS No PFOS q=Hbtive cmfinmw= - 6 * ldcod&sdm m pmhmkwy. DeviamSunopto lmb m " notedmd wem wt eudbnw& DaftEntmeny. 08127/990.mv99, tv3oi99o.traft ovi4= lAc DateVaifie& By- 02r2M NM GENo2i Vmious mavicesfromMSU None Blood EM&4.1 & ETS-9-@I.uing mcxnewd mwrva Amlia 062490 Manlynx 3.2 See listtoright See Anwhumb See Anachmenti See Amwhmmts O&r25M MCHffdUSALJSEE OLIX" 08/27/9O9L,'2&% 12MM @M 08/27199,08/30199,OV31/99,12110",01120=MEE/lAs coacm&adon mean ofPFOS PPOS agiml givml <LOQ(0.0116) <LOQ(0.0116) <LOQ(0.0116) <LOQ(0.0116) <LOQ 0.0180 <LOQ(0.0116) <LOQ(0.01163- 0.0166 <LOQ(0.0116) 0.0131 <WQ (0.0116) CLOQ(0.0116) <.OQ(o.otl6) <LOQ(0.0116) <LOQ(0.0116) <LOQ(0.0116) --LOQ(0.0116) <LOQ(0.0116) LOQ -LkaftOfQuwdtafim <LOQ - 3OWU- NA - Not AppH=ble RSD - RelatFmStndwd DrAdw ND - Not Deteewd D - Detwwd RSD Std.Dev. blA NA MA NA Co@tndoo Mean RSD OFPFOSA EW-L .' PFOSA std.Dev. <LOQ (0.00625) <LOQ (0.00625) <LOQ (0.00625) NA <.OQ(0.00625) <LOQ NA <LOQ(0.0062S) <.OQ (0.00625) <-OQ (0.00625) <LOQ(O.Ooas) <.OQ(0.00625) <LOQ (0.00625) <LOQ (0.00625) <LOQ (0.00625) <LOQ (0.00625) <LOQ(0.00625) <LOQ (O.OOU5) <LOQ (0.00625) <LOQ (0.00625) NA I <OQ (0.00625)1 <LOQ I NA PFOS - Perflooroocumcmlfmmk PFOSA - lfoomide POAA - Pagoorooconage PFHS - ETS-8-5.1 Emi Vasiam 5195 GEN-021-sam-,tb silo= FACT-GEN-021 Study-. hoduct Numbergest Substance): Mabil: MedxA(Ravisim: AnalydW Equorem SyMm Number. Inibmmmt Softwwr4Vmion: File@: R-Squwcd Vabjc-. Slope: Y-lowcew. DatesofExuictioWAnityst Datesof Analy*WAnslyst: Date *fdas PodacdonfAmalyst: Simple Data BLOOD Group Domi Semple N Method Bik H20 Bik-ILi" HZD Btk-2 V25M H20 8&-3 LPASM H20 Blk-4L75M Cas&s sew Blood VrA491-4V 53 w249i.4ij 11 W249t42J.46 W2491-43) t7 VfMI-44.F 13 vrml4sj a Vrh49l-46J 12 W2491-47.1 9 W24914$j t4 Vr2491419.1 18 VrMI-XJ$2 W2491-Slj 55 Vr2491-Rj 10 W2491-53J IS No cum analyxwforPRM PFDS. PLFHSbasedon PFOS - No PFOS quaHatWo coafiruntimp- I ,' ldendficadamms pwlimhmy. DevimaSurrogateL-wb we no notedand wao to a I Dm ExUreNSr. 0&271".OWOV99,12rJOI".OV2*)W.OV14= LAC DaftVairio& By- OV22/00 MEE GEN021 Vwious Matrim ftm MSU None Blood ETS-&4.1 & ETS@9-5.1 using mazeacted cm@es AmeHa 062499 Masslynx 3.2 See Ha toright See Auachments See Atbwbments See AtMcbments OL25/" MCIVKK/SAL/SEE Ota&". 08/27/99.08/2L". 12MW" KWAAS 08/27199O,LWM. M31)99, IVIO199,01/20100MEE4M Concem&atioa Mma RSD Co@tntion Mean RSD ofPOAA POAA SOL Dev. ofPFHS PFHS ShL Dev. agiml mo@ML mg/mL U91ML 0.00629 ND <LOQ (0.005") ND <LOQ(0.00599) NA ND <LOQ (0.00599) <.OQ - IOutlier NA ND NA ND NA CLOQ (0.00599) D 0.00759 D <LOQ(0.00599)1- ND <.OQ (0.00599) D :LOQ(ow599) ND <LOQ(O.Owm ND 14.OQ (O-OOSM ND 4.OQ (0-005") ND 0.00728 ND <LOQ (O-OOS99) ND 0.0108 ND 0.0234 ND <LOQ(O.OOS") NA <LOQ 0.00599) <LDQ -4 Oudien NA ND NA ND ND -3 Oudias I NA LOQ-LkokofQ=ndtodon PFOS - PufluorooctonessUbuto NA - Not Applicabk PFOSA - Perflooreoctmesutfounaide RSD - RelsfinStandardDeviatim POAA - Perejorooctanooft ND - NotDebmW PFHS PatlooraboxantrAfenate D Deacted ETS41-5.1 Evxi Vasion S195 GEN-021-susids 5/10/00 so*: Pmdm Nwnber(Ten Submwoe).- maoi3L M9606T.ew6m A-ly@ E"p-t S)-- N-Ykb- bwwwmd SaftwwwVersion: Pi R-Squwed VWw. swpc Y-inumept Do= of ExwacWWAnalyst Dow ofAnaiysWAnslyst Due of Dm" Re&wdoWAn@dyst Sample DaU BLOOD QC Gmup Dm Smpb 0 Madood Blk Caspim Smi Bi"d HZO Blk-I L25M HZO Blk-2 LPISM H20 Bik-3 LP25M H20 Btk-4 LP15199 W2491-40J 53-bM W249141,J II-MS W2491-42J 4640 W2491-43J 17-M W249144J L3@.WS W249143,X&M W2491-46J 12-ba W2491-47J 940 W2491-4$j 1440 W2491-49J 111-NS W2"1-MJ52-M W2491-51,155.US W2"1-nj 10-M vrmi-ni mms No cw" wAly3" thr MIS. 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IZOM LAS 08/30/".12/1(W" IAS arves Commustim Man O(PPM PFM wWg or % RaL Rece-y MA NA NA NA NA 91% 78% 6VA 45% 31% 64% 63% 105% 77'A 51% 44% 33% a% 35% 31% 50% 55% s7m% MA NR NR NR NR NR NR 49% 38% 37% 41% 42% 61% 44% 27% 34% 69% 36% 65% 47% 37% 35% 51% 41% 42% 46% 44% 61% 75% 24% 53% 63% 69% IIS% 22% lhmk ofQ m RSD Rda" Sto3dwd Dvvkwim NA - NotAnficibb NS - Not q" RSD St& Dev. MA NA 35% 22% 47% 29% 42% 23% NA MA MA 13% 6% 33% 16% 20% 2% 7% 3% 49% 26% 34% 22% Coomwadon OFFFOSA PFOGA aft or% Rw- NA NA NA M NA' 66% 52% 49% 30% 15% 43% 42% 79% 6s% 35% 11% 29% 45% 42% 79% 42% 54% 63% MA NR NR NR NR NR NR 47% 34% 29% 36% 11% 50% 35% 16% 22% 44% 26% 33% 3(PA 42% 51% 3"6 44% -3% 27% 12% "% 57% 14% 46% 65% 64% 104% Tl% p PPOSA-?CrgltlCgCOCtlrlggtltibrlMFAi& POAA - PadomeocamoM PFHS PwOoombccmesWGxuw RSD Std.Dev. NA NA 42% 11% 56% 25% 39% 21% NA MA MA 26% "6 45% 13% 15% 6% 171% 21% 61% 28% 29% 23% ETS41-7.0 excd Vesim 519s GEN-M-&wxb SA0100 6:07AM FACT-GEN-021 sh*: Pmdud Number(Tat Subsumm): Maoix: MCL%Dd(Rcvwiork: A-IYUW B"p- Sysim Nm*w WBOUMCK softwwdverwiow DWAO(ExtubWAmt"t Dateo(Anslysti/Anal"t Dam ofdam Rc&KdowAnalyst Sample Data LIVERrArHOLE BLOOD Gr-p Dan QC Smpk 0 Mediod Bik H20 Sik-i L12SM H20 Olk-2 &425M H20 B&-3 &75M Camfwuis Sea tA= uvw H20B&-4 L125M W2491-3.CSL 344$.MS W24914.CSL 339S-bM W2491-CCSL3020.MS W2491-7=L21WMS V/2491-9j=2139.M W2491-InCM 2M7-NO gkpbamt Saw Lhw W249l.2jS iSO04-0 ,W2491-SJS ISS2-bG -W2491-IIJSSOI-MS W2491-12JS 7724WS Habor Sod U"w V401-14JS 78240 W24914UM44 W2491-13.M Ilgt-bd W2491-ISJM 11994B k@4 CARD LKW busk Ll- W2491-IMS 100.MS W2491-34.DI(00USr*'&M W2491-3SJ)l146 USPVIS-M 11; Rlvw Onw Lim W2491-36j)l IS&USFWS4AS W2491-29jtAO0664a. VM91-30JLAGO28-bW., W2491-31jtAG 14845 W2491-32jtAG 2304M W2491-33jtAG 2374& Sm OttmrLh-or W2491-16.SO 12593-001-MS W2491-19.SO 11494@MI-M W2491-Z2.S1O1940-001.MS W2491-24.SO 11309.WI-WS W2491-25.SO 12797-001-M W2491-26.SO 13110401-NIS W249l-Z7.SO 12679-0014oM Tank Lkvw W2491-2&W 12?W@W14WS W2491-37Mak Turde (.2A.M W2491-3&Mak Tmrde (LirpM W2491-39Ycmk Torde(-3.*MS seaomw arsk W2491-ILSO 12593-WI40 W2491-2i.SO 11494-001-M S" oner mmw W?491-17.30 12593-0014AS W2491-20.SO 11494-001-M W2491-23" 11%0401-M *%Oilmw Vn491-UX"mar=tD= LCMrityM wugl-ss.cwmgms D= klymW" um U;@.ms WMI -*OWD= MMLAMM h L "Mbmd=FKM NoPFOSqmWAd"mamodon- 0 - NR - Not W@ dw spin a dmcmk ampdboiiwy km emb*@ kvds. DaftBmov&Anabu Dw Vai&&Amb-st ocinGM02n7iW LAC 2WI2000 16M CiENMi Vim Mawa ftm MSU None vmimmwka M"O A ETS-3-7.0mirg umcrwAcW Amdis 0624" Man@ym 3.2.3.3 OtaSM MCH/KK/SALISEE OV2L". 12MM LAS 01130t".12JIO/99LkS oxvcs c@a2tim ofPOAA so or% ROL MA MA NA NA IOVA Li% 69% 45% 20% "% 100% IVA 44% 31% M% 49% 94% Sg% 91% NR NR NR 21% "% 69% 52% 70% 47% "% 47% 22% 3VA 13% 44% "% 43% 71% 42% 41% sm 94% 95% 9% IV% 2M In% man FOAA MA RSD St4L DIV. NA NA 47% 65% 31% 52% Sa% 30% 37'A 67% 25% KA MA NA NR NA 39% SrA 20% 39% 50% 20% 29% S4% 15% 13% 46% 75% "% 49% 2% 121% 3% LA)Q-LArAO(Qomwudm RW - RdatkeSWdwd DcvW= MA - Not Appficobk NS-Not*&od Coamtratwm ofFM oft or% Rec NS NS NS NS NS NS NS NS NS NS NS NS m m m NS NS NS NS m NS NS NS NS NS NS NS NS NS NS m NS NS NS NS NS NS m NS NS NS NS NS NS mun PFFE NS m NS m NS NS NS NS NS NS m NS ryw-- 1 NS "OSA - -onwikmwi& POAA - Patuaroocanoft PPM - Paflumdxxmmsu[Exmft RSD St(L OCY. NA NA MA MA MA MA MA MA NA NA KA RA NA NA MA NA NA NA NA NA MA Mg-7:0 E%cdVmim SM GEN-Ml.iivaxis silow 6:07AM 1 c Wilsoni.onoeusiRcekfetIenndSceyer7tem m 4 C,199W1IsJoonnCeosmpanv FACT-GE,*4-024 IL k ;:, Study: PmductNwrbcr('resStubst@): maww Modmdlt@: A-1yoW E"p- Sy-n N-rb- bwoument Softwmefversim: FUCUMM @ R.Squwed Vdw. SIW. Y.' , ,p Dou a(Emm2kWA=Iyst Data ofAmilysio/Aa&lyst: Due ofD= RoducWWAnslyst Sample Data SERA G-p DO" Sample 0 Mgdwd Bik MatrixBlk N&MD 250 ppb ARmt@ comamt "wrtme said zagk DirdIO129-"Ik-S-1 NE NE Bird 040-Alb sat.MS.ZSO ppb.$-l Bird 040-Alb swo-MSD.250 ppt@-5.1 Bird 060.9 phunb.MS-250 ppb.5-2 Dir4060-E phym44SZ).2SOppb-S-2 BUd 054-C bkod-MS-250 ppb.$ Bird 054-C bloo&MSD-2SO Mb.5 BW 034-Abomm CM* @ BiF4035-Abona pb@ Bird 036-Absom nit Biid 037- Pit- Bird03&Absftw me Bild039-AAMOM Un Bird040-Abown DirdG$I-Aboomms Bird002-Absom @ Bird063-Abw@ am Bbd 044-Absum @ Bird045-AbWm @ SiFd04&AFAUM Ph- Bird%7-C@ o@ Bird OU4COVWMU pW= Bird049-C@ Bird050-C@ Bird051-Comw= pb@ bimd blood Bird 052-Comormotbiwd BirdOS3.C@blood Bird 0544ComormM blood BirdOSS-Comorwtbt=d Bird05&Haring "pitBird057-Harin GA pb@ Bird059-Harin CMU blood Bird 059@Harii&g Call Mood BiFd 060.Bdd Eqk Bird 061-BoldBe& Bird 062-BaldEa& pb@ pbsm Bird06"W Enk pW= Sifd064-ftM So& - Bud OGS-B&M Es& pbom Bad 066-B&M Baeo pWm Bwd 067-Bdd Ea& pb@ Bird W"&M Er4on pk@ Bird069L@ Be& ph@ BirdW04UN Ea& pW= BirdOll-BoldBe& pl@ Bird072-BaldBMW ph@ Bimd0734MM Boos ph@ Bud Cr7"W BirdO7S4MM Bird07"W Bird077-SW Biid WX41&M EMW pima BMW pb@ Be& ph@ Embpbm Eqk ph@ Bird om4ww go* pb@ Bud owasm ES& pwm Bird081-Bald Em&W ph@ Bird09243aW Be& pb@ Bird083-BaldBqk pb@ Bud M4.8&M Be& ph@ 14aPFOS *Whadn ;E@' pa&mu& Bidend5cadow m App@ io@M hm bow gawl LAC 10(19/" Dow Eamwer. DeftVai&& Br. IWIIV". lO/2W" LAC IV30/" MUH 2/17100 nw& GEN024 MSUEnvk=nMWSwMki vuiow vwiow ETS-11-4A.1STS-9-5I.wing mmmatud @a Analis 0624" Mmb= 3.3 SmA ' See Aftcbmuft See A ' S" AmKbmma 10([Z@" SALIKK 10/ISM. IWIW" LAS/MMH. 12/13." L@kS 10/18/". 10flOt" HOJ. l2/l4d99Mb(H co@&adm bk= RSD efnpos mpos stc Dow. or % Ra <ZQ(0.00116.&*C) NE 14.OQ RPWMLVMD <A)Q NE NE NE 21% 96% 89% 17% 11% 23% 17% 1 69% 64% -46% 19% 671% 4LO3" o@0256 (Locryss 0.01% GLOW" GLOOM 0,00621 CAOM 47.0 0.004M molso 0.00947 aocs" OLOOM toom Sao its OLOOG" O.OOM 060107 PLul 0.242 1.9 '0.110 aut ,,i6l 060M 0.218 aIS3 <LOQ (0.0011n6o@IL) OL 144 44.S 0.273 Q@172 - I Omdkr 0.0764 &453 34.1 oxn o_ms 0.125 0607" OL0664 0.0727 mz 0.00938 M449 0.316 O.ISS 0.167 0.0402 &Ot47 0.122. O.OUS M7 235 0.0324 0609% 0.217 OLIGO &163 0609n 6=9 aosn 4m (0.00116u*4mL) Q2" amt 0.649 CL335 &0712 060391 193 Coam&ad@ ofpvhs vWmL or % Roe @LOQ(0.001149WWL) M@ PFUS n NE NE 69% 91% 75% 59% 65% 62% 76% 14% 45% <LOQ (0.00114VWML) <DQ (MODI 14 aWmL) <LOQ (0.00114v&mL) <LOQ(O.Wlt4vgftL) 4.OQ(0.00114v&,=L) -LOQ(0.00114w&'WL) <LOQ(0.00114*&'nL) <LOQ (0.00114aWmL) <LOQ (&Wl 14u&tL) <LOQ (600114 Wi.L) <LOQ(0.00114vpmL) <LOQ (0.00114v&mL) @LOQ (0.00114 1&%IL) <LOQ (0.00114*mL) <LOQ(0.00114vgAmL) <LOQ(O.OOll4v&4uL) <LOQ (0.00114coml) Plop= <LOQ Sao <LOQ <LOQ <LOQ (0.00114v&L) <LOQ (0@ODI14 vg%L) <LOQ (AOOI14v&*L) <LOQ (0.00114 vWmL) <LOQ (0.00114vofml) O.OM43 <LOQ(O.ODll4oWmL) <LOQ(&00114agmL) <LOQ (0.00114ugfml) <LOQ (0.00114Y&'mL) <LOQ(0.00114v&%L) 0.00142 <LDQ NA <LOQ <LOQ (0.001[4votml) <LOQ (0.00114u*wL) <OQ(MOCI14ugfmL) <LOQ(0.00114volmL) <LOQ (MODI 14 aWmL) <LOQ (0.00114vgftl) <LOQ (0.01)11v4&mL) 4MQ (0.001t4 W&4ML) <LOQ <LOQ (0.00114q&L) (&00114 vg'ml) 0.00131 <LOQ (0.00114v&iml) <LOQ (0.00114ughol) <LOQ (0.00114ve0@nL) <LOQ (0.00114 W@PML) <LOQ(0.00114u&'=L) <LOQ(&00114ughmL) -LOO (0,00114ve@ml) MODG17 <LOQ (0.00114v&=L) <LOQ(O.Wtl4v&mL) <LOQ (0.00114u&hol) ITSD S$iLDw. APD-N$/MD NE 17% 11% 139% MA MA NA NA NA NA NA: I" NA NA NA.MA NA' NE - Not ct&ackd NA - Not AppH=blt NS - Not Spawd LOQ -LimoiftQw=mm RSD - Rdadvt Sundmd Deindon "D - Rebd" Paum Diffacem df=M Ppm Pwomwobgumuwkmft FOAA Patmorooemmma ppm.-.- 87341-5.1 mud Vffd= sm GEN-024-@K&Ak 5/10ow FACT-GEN-024 Study: Pm4M Nuffdm(Ten meow MedbodntgvisimL Subotmm): Anlydcal Bqwpnmm Syston howugwm SoftwwelvmimL Pik"WW R,Squavd Vahm skprY-bwmept Dow of ExazcdoWAnelySt Dams ofAnlysWAnsirt Dm*fDauRc&wdoWAaWyot SERA Sompk Data Gmp D@ smwie 8 Medbod Sik Maui, alk NMIMO 2SO ppb Albot@ ip Comamt Hwrieg NE NE Bird 040-Ara @MS-250 ppb.Sl Bird040-Ab sera-MSD-ZM p*4-1 Bud 060-E pim@MF@250 ppb.5-2 Bird 060-E pbxwMSD-250 ppb-S-2 Bird 0$4.C bWod-MS-250 ppb-5 BirdW4-C bloo&MSD-250 Wa-5 Bird034-ARmom Cbkk @ DiFd 035-A&SUM ph@ Bird036-AAmom som Bird 037-Abumm pk@ Bird031@-Almmo @ Bird039-Abamn @ Btr404(@-Abo@ @ Bird041-Abomm =a Bird 042-Abomm @ Bird 043-Abomm @ Bbd 044-Abd@ @ Bird04S-Abofto @ Bird046-Abotma ph@ Bbd 047-Commo pb@ Bir0d46-COMWAPRh@ Bird0494-@ pb@ Bitd050-C@ blood Difd 05 I-Comorme blood Bird052-Corawmit blood Bird053-Comorm blood BiFd054-Comaram blood Bird OSS-Comount blood Bird056-HaringGA ph@ Bird 0574krr6a GeR ph@ Bird05 11-HaringGA blood Bird 059-Haiiag GoN blood Bald Rogle BirdOW%W Eqk pb@ Bird061-gaidEnkpk@ Bird062-BaW Eve* pb@ Bird063.%W Be& -Bird064-Bald EMU ph@ Bird O6S4MM E2& pb@ Bird 066-gald Be& -I Bird 067.%M Eaoe pb@ Bird061@.BddEqkpb@ Bird069@BaldEsee pb@ Bud 070.Brhd Ba& pham Bird071-Sidd B@SW ph@ BirdM-ftm as& pb@ Bird03-Bald EmOspb@ Died 074-IMM Et& pb@ Bird 0754WW Zqoe ph@ BW 076-@ goes ph@ Bird on-um amb pb@ Bird 07$.BaldPa& pb@ Bird079-B@W Be& ph@ BirdOO-@Eseepb@ Bird091-BaW Enkpb@ Bird082-BaldBulk ph@ Bird 003-DaidEm&pb@ Biyd094-UM gftk pbom Bird 085-SiddEask pb@ 140PPOS qwdibdn cmdmmdm g I- ' bkntigcwm App@ tonahe"be=mumiLAC10/IW99 m p -9 Deftgua"gr. DaftVai" Dr. 10/18/".lOt2W" LAC IV3(V" MMH V17/oonw1h GEN024 MSU E-i-n=W Voi@ SwWks Vuiow M-94.1 & STS4&.S,l owft @oacted @u Anwlia 0624" M-ly- 3.3 Sm A Sm Amwhnmu See Aawhnu= Sm AUKhmeM ICY12/99 SALXK 10/15/99,10/19M LAS/fAMFL IV13M IAS 10/IV". IGFM" HOJ. 12/14/" MMH Cm ORPOAA mp'ml w % Rat @LOQ (0-02"4-L) NE ME tm 105% 77% 64% W% 23% <LOQ (0-02" ushOL4 <t.O(QaamgoiEL) 4.OQ (am" VOW4 <.OQ (OL02" W&IML) <LOQ <LOQ <A)Q <LOQ (O-OZ" vo*ML) (0,OM "OWQ (0-02" mo,=L) (0-02" mo*AL) <LOQ (0-02" IWML) <LOQ(OAMNWNL) <LOQ (0-Mw"oML) <LOQ (0-02" wpOmL) IPOAA mol-L <LOQ ME 97% 80% SG% Ph@ 4.OQ sem <LOQ RSD SOIL Off. RIPD401MD <LOQ NE 17% 9-A 119% MA MA MA I MA <LOQ(OLQMVVWL) <LOQ (0-am MWWL) <LOQ (0-02" walML) <LOQ(OAMVR%L) 4.OQ (0-02"votAL) <LOQ(Olom40%0 ILOQ (0.02"ugf-L) OLOM <LOQ (OLOM QWML) <LOQ (0-M" OWOL) ICLOQ (0-cmWWOL) <LOQ (OAM *%L) <LOQ <LOQ <LOQ <LOQ @LOQ (0-02" IWML) (06M" oW=L) (OLM"VWML) (O-OM ROFWL) ((LM" oWmL) <LOQ <LOQ <LOQ AMQ <LOQ <LOQ (O-OZ" wa@OL) (O-OM .6,ML) (0-mg vow (0-02" welML) (am" "h"L) (MO2" ushOL) <LOQ <.OQ 4@OQ <OQ <LOQ (ao2" owiML) (0-02" owkpl) MM" waiML) (OAM 10%NW PAM VWMQ <LA)Q(OM" vow 4@OQMA"V&IWL) <LOQ (am" 90%0 <LOQ (O-OM VOUL) 4,OQ(MomqwmL) <LOQ (OA"i9tNL) <.OQ (*-am 9#NQ <LOQ (MOM wo'ML) <LOQ ((LO2" vo%L) <LOQ (aam oo%L) MA 4.OQ NA NA 4.OQ - Iowmw XA MA am MA MA <LOQ MA NA <.OQ- NA NE - Not concood MA - Not AnOcsbk NS - Not Sp&od LOQ-LWAQfQ=dbdm RSD - Rekti" Susdwd Delatim RFD - Rdidwe Pa Diffumm Coocm&adoa of?FOSA *WmL or % Rat ILOQ (0.00625qW-L) NE Mum PFOSA uSfmL < NE NIIBI 70% 80% 7S% 59% 65% 62% TM 14% 45% <LOQ (CLMU vv%L) *.OQ (O-OM oshnl) <LOQ (O-OMS oefml) ILOQ (O-OOC volML) <.OQ (06MM W&'ML) 44Q (0.0062SvWmL) <LOQ (O-OMSvsfml) <LOQ (0-M" goML) <.OQ (MOMS goML) <ZQ (0-MU sop-L) <,OQ (10062 9&inL) Pl@ V)Q <LOQ (O-OOM 0&%L) = ((O.== <LOQ (O-OM VWML) swo <LOQ ILOQ (O-OOM VafnL) <LOQ (O-OOW valML) 0.0145 0.00971 <m (DOMS U&IUL) <LOQ (0.0062 uW-L) 0.0426 O.MI9.3 Oudim <LOQ (0-0062Saw%L) <LOQ (O-OMSwaiML) <LOQ (0-00625vWmL) 4A)Q (0 W625 WWWL) <LOQ (O-OMS vo@IL) <LOQ (0-00625ushrl) <A)Q (O-OW5 Uv%t) <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ (0-000 U&IML) (0-00625VOIWL) (0-00625aWmL) (0.00625v&*C) (O.W62S %hrA.) M07SI QLO"6 IZLM (0-MM ua%L) 4-OQ (0-MM VWML) <LOQ (0.0062VSWUAL) 4.OQ (O-OMS owaIL) <M (060M VfkAL) 4.OQ 4.OQ 44Q <@OQ <LOQ (A-OMS *IOL) (0-00M MVML) (O-WW NWML) (O-ODW vo*L) (0-0062 VWML) 4.OQ (&@ u@) <LOQ (0.00625vairl) <LOQ (CLOO62Smohvl) CLOQ (0-MM VWML) <,OQ (0@00625 ow'=L) <LOQ (D.OD625 <LOQ -2 Oudim ?FOS - = FMS - Perduarobeummendloom POAA - P "OSA Pat F=NUWI RSD SIL Dgw. aPD4=AWSD <LOQ WE 14% 9% 140-A NA MA MA MA NA MA ZL7 motsi NA MA MA MA NA NA vrs-&S.i bed Vaske Si" GEN-424-mLxb $/low D 0 Wilsookm - OvicftielefILn-dnaStiyLs-tem (: 1991 WilsonJonesCompany FACT-GEN-024 Study. Pmdua Nwoba(Ten SubrUm): mauix-. mcdw"rasion: AndytlW E"pnwA Sys%= N=6w 1100UMCrA SORWWVVWgiolL Dta ofEinaioWAnlya DaftofAn*/sWAmW Dswo(DauRe&ctiowAraW Sample Data LIVEK KIDNEY, Group Dow YOLK sampw 0 Metbod Bik BkdIO129-wbik-5.1 QC 230 ngfg BirdIO129-wbk-6.1 - BWWI.LoooLw4AS BkdDOI-Low Lvrb= BudD23-Aftom Kdmy-MS Bit(=-AM@&= X&W4AD BwdWO.r@Ydk-MS BWWO-C@Ydk4W !;Wl-um abdom-Low BidM3-L@ BWW4.Lom, BkdDOS-Lace DidDO6-Lom BkdW74Am DkdmLem LIVW BkdOOP.-@ hs= BiMO104kom Pdkm LKW aboll-Almom BkWI2-Abom . DinMI3-Almom -4W BkWI4.Aflmmn %i Is BinSIS-AM@um BiMIG-Abown BkdMO-Almom Bk=I-AJbom BbdM2-Alown BkW17.AVAmm BkOll-ARxI@ BkWI9.Abwm BkdM3-Albwm BirdM4.Abom BbdD25-Ageom DidM&Aboftu Yolk BwdM7-Cmmrja BkdM$4mmrjmg BkdM94oamnot BirdD3OrAmoraK Yolk ok4wl." BWM2." BWM34d No "OS 4w@ta6m patrnmxl ldenuficat@we pmbmbmy, LOQ - Ljmk O(QONdmdoo RSD - Rcb*n Stmdwd Devieks RPD - Rebdve Pa Diffiwmw DoleEmwe&Andyst IWA&" LAC. 12JLI" GBCL, 12M" MMH DoeVoig@&Amtya V17M M)AH rM4M4 MSU En@woomenW SuMics vxious vxiow EM&6.0 & M-9-7.0 tw4 unavictedcwvcs Annfis0624" Man@,= 3.3 10(12199SALJKX 10/14199,12JI3/99HOJ/LAS 10/15/991.til5M, 12114M HOJ/MMH coomtmtim orpnx qft or% R*L <LOQ (MO348 ugfg) <LOQ (0,0348 21% 28% 71% m 121% 134% &345 0."9 miss &I" 0.2m alos 'IM (OL0342vgfj) <M (MO348 vsfs) &0460 Q294 0.617 <LA)Q (1)-MaUWB) <LOQ (0-0343u" <LOQ (&0349uft <koQ (003" V" <.OQ (0OM UWI) <M (aO349 uWg) <LOQ (aO349oWS) <-OQ (0.0349ugW <M (00348 vWS) <LOQ (OL0349vglg) <LOQ (0.034v3Wg) <LOQ (00348uWl) <-OQ (aO348uWg) <.OQ (0.034U2WS) <.OQ (0.0349MM <LOQ (0.0349UWX) 0.134 0.317 &254 0.146 <LOQ (00348vWS) O@0541 MA NotApokabk Mftm PPM gwo <U)Q 28% 79% 128% RSD StiLDgv. btSAWMRPD NA 3% 20% to% .- . O.M -2 Oudiem OL170 73.6 OL212 103 Q175 Conmtratim or PPM -.M(Oro%P0AILM,U 1) <LOQ (0.0693uWg) 54% 65% 63% 77% 71% 75% <LOQ (&OU3 uWg) <LOQ (&0683 v#g) 4@OQ (MOU3 "W <-OQ (OL0693 uWg) <LOQ (0-0693uWg) <LOQ (ao6B3 VWB) <LOQ (aO6g3 vWj) <LOQ (OL0693MM <-OQ (0-0693 vWl) <LOQ (0.0693vgW Mun FoFwMl 4ZQ 6m 10.1 73% <.OQ <.OQ <LOQ (0.0683vWl) <LOQ(0ow Usfs) <LOQ (MOM mw <,OQ (0-069g3WI) <LOQ (0.0693WV <LOQ-lOuWa <-OQ (00683MW <LOQ(MOGO"W MA <.OQ (MOU3 vWg) <LOQ <LOQ (0-0683vW$) <LOQ (OL0693u" <LOQ (006IL3uwjl) <LOQ (0.069u3&fg) <.OQ (&0693vWS) <LOQ (00693UWS) <LOQ MA <LOQ (aO6g3 vWg) <LDQ <.OQ (0060 VWS) - <LOQ (00693 ugfg) 39.5 <LOQ (0.0693ugW &23$ - I Oudier OL0930 - - <LA)Q 64.8 &0999 -I Oudier Pos .01u; = ((Ol= U*g,99)) <LOQ (0-0"3 uwg) -(aomog) I 'am PPM - Ptrfluorobexan=Wfoagg PC)AA - PcrOuorooctir@w PFOSA PeOuorooctm offono" RSD S.t& Dow. hiS/MSD RPD IA 2D% 5% MA NA MA, NA NA MA RTS4.7.0 Exed vwsion 5/" GEN-024-Hmxb snoow 617 AM FACT-GEN-024 Saidy-. Pmdua Nmtber(Tee Subgtww@ Mauix: MCON"Cvigio,L A-lybW E*upram Syftm N-nbhEMUMerA SoftwWdVersion: DawofExnedoWA=bvLDm of An*fsWA=dyst Dateofdm Re&wkWAnabpst Sample Data LIVEP, IUDNEY, Group Do" YOLK Smpk 0 Mcdwd B& QC 250 nWg Lkvw Lhw LJVW BirdIO129-wblk-S4 BirdIO129.wbik-&i Bidmi-LDMLVf-ms BK"I-LQM Lyr-Nm BkdM3-ARnma Kday-MS BidM-Ab@Wm = BkdO3O4onmut Yak-US BkdO3O,Cmuorxmt Yo&4= SK"I-LON BkdOD2-Lem BidOWLAm BkdDD4-LA= BkdODS-L4= Bbdw&LOM BkdW7-Low BbdW&Lom BadW$4kuvm BkOI04ka@ PWkn Bball-Abefto BbOIZAftwo DiniOMAbom BbWI4-Almem BbWI6-Abo@ BkW2O-Alafto OWMI-Abd@ DidC27,Abm@ KWhuy B;EI74A@W-u BinVIS-Agnma BiWISLAInam BbW23-ANano DirdM4-Almum BkdMS-Ar4onu BkdM6-Agofto Yolk BWM7-Comonmt Diams-comorm BkdO294Comorad BbdWOAComorxit Yolk Bkdo3l." BWW2-" BirdM.'@." I LOQ LimkOfQowf"m RSD-RdatkvS' I Dgvimdm RPD - Rdsdve Pa Diffavoce DiftEmr*&Ambvt IQQ3/99LAC 12/IA9GBCL.12P," MM DaftVai&&Ambst Zq7=MMH GEN024 MSU enveonmemw S=npla vwiow various MI-6.0 A ETS-&7.0 usingworneted arm Ameba 0624" Mmlra 3.3 [Oft2l"SALXK ICY14M. 17JI31" HOMM 1&15199.1I/ISM, 17Jl4M HOJ/MMH Coomtimtka OFPOAA oft or% RaL <LOQ(0.1909wx) <LOQ(0.180uwl) 101% 101% 113% 121% 105% 130% --Zw(&-Isovw- <LOQ(O-l$DV" <LJOQ(&ISDV" <.OQ(Ol$DVWO <MQ(OLIIOV" 4-OQ(OLIIDV" 4.OQ(OLIIOVW a.OQ(0.1304N <LJOQ(MIBOVW lam(O.Isoqm lam(O-18ov" <.OQ(QLIIOV" <LOQ(0-1809" <LOQ(0.1300" <UV(OLIIOVW 4-00(allovow <LJOQ,(alsovw <L.OQ(0.1300" al$2 <LOQ (a1$0Ves) <LOQ(0.1809" <LOQ(&Isowl) <LOQ(MIIOWS) <LOQ(Ol&OWI) <L.OQ(0-1800" <LOQ(O@ISOWI) <LJOQ(OLISONW M24S <LOQ(O-l$OUW OL192 OL197 <LOQ(Ol$OM" &196. man POAA <LOQ 104% 117% 1m -CLOQ < LOQ < LDQ. I Oudia -cEADQ OL218.2 oudias al96 -1Owier NA NotAnik" RSD Sid.Div. MSAM RFD NA 6% 7% zm MA MA MA MA MA MA NA MA 17.2 aO374 0.528 0.00104 Concen6ition ofPIMA wWg or % R*L <LOQ (&007504g)@ PIMA oft <LOQ (0.00750ugtg) <LOQ 53% 51% 52% 56% 58% 57% 62% 72% 67% 0.0153 <.OQ (000750 vWS) M0147 OL0262 O@0213 &0242 <LOQ (O.W730vg(X) <LOQ (QLW75w0v <LOQ(&00750MM OL179 &0204 - 3 Outfim Q 179 - I Oudier OL527 <LOQ (0-00750v" <M (0007$0MM <LOQ(OLW750 qffl <LOQ (o-w75oam <M (000750uWa) <LOQ (OLW750 ug(S) <LOQ (MW750 uWj) CLOQ (aoom U" <LOQ (OLOM VWI) <LOQ (000750a" <LOQ (0-00750WS) <LOQ <LOQ (MW7SO WS) 4M (0.0075W0S) <M (CLOO750WS) <LOQ (o-w75oWI) <M <LOQ (0-0075u0" <LOQ (0-007"Wg) <M (0@00750tWg) <LOQ (OLW7$0 "W <LOQ <LOQ (000750" <LOQ (0w7so WI) <LOQ (OLW730 Wz) <LOQ PFOS - PerDLiorooctmoemtdfa:,mft PFHS - PerfhicrabOXMIAMdfOrAN POAA - Pafluomoctmosto PFOSA - PefloomoctimsWfmxW& RSD StiL Dew. MLIMD RPD MA .4% 5% 15% .125.5 OLOO520 MA MA NA MA ETS4.7.0 Exed Vwsion "S GEN-024-UM.Xll 5/10= 6:07AM E m WilSOnJOnN - GU(CKRetefei7C/e,7deSrystem c 1991WilsonJonesCompany Study: ProductNun6er(Tem SubKmncc): Mairix: Nkdwd4tevioam: A-b@dW E*4- Sy- N-mblmbwrWrA SoftwuMVaSiorr. fralounw. R-Squwad Value: Slope: Y-Intacept Do= ofExblc&WAnalyst Data *fAndysWAradM Deft of Data Re&wdontAnalyst Sample BLOOD Data r.mvp Do" Smpk 0 Medbod Bik h=12129-H20bik akfibered5.3 PASUI 2129.H2Oblk ffiftre5d-4 MatrixBik HM3312129.@bik--'Ll EMB 121294Aood bik-5-2 HMB121294iondbik-5-3 MO]21294coidbik-S-4 H)WB1212944ocidb&.$.S HM121V-bloodb&-5-4* HMB 1212%Uood b&-5-7 HbOM121294AOod bik-S-V HM33121294*ood b&-" QC PBI@.WS.250&99-9-1-2 PBB-6253-250&CW65-1-2 PSB,SOW~ IMAS,*1@-2 FSBMW~MSD-'-LI-2 SSBSSL49L~MS-5.1-2 v SSO-SSL49- bal)65.1-2 MM&FE52IM250 bti-S-1-2 Hbg@-PMIM250 MSI@6'-LI-2 Bi"d P205 Wardmm Pw SeW P206 rum m P208 P209 P210 P2[1 ?212 P215 P217 P219 P= P221 P= P223 P224 P226 P229 nm Blood NordiereFur Seol MIO-4 mias Adak Fanda M106 M107 mi 12 mits mi 16 mitt M119 M122 Blood scoi Nor*Am Pw Sod S002 Subo&k M" 3003 S006 SW7 Soo@ Blood NorthernPw SW S009 P2NB P23&A 36$C P406 P411 93 CUKB 3* NA2 EDTA Sunopft 2-50%dramma. am coo&nud 98 CLMD 7 " CUKB 9 I- May needtor@ idtswgl= forPFOSA, Wafemot, inboth --PFOS NOT @lbned, NS bwwhiom vubtlos > 30% Dow Eowre&By- 01/21/00 LAC FACT-GEN-030 GEN030 MSU EnvironmentalSaMles Various VwiousBlood ETS-94.1 & EMS-5. Iusinguwxq&cw cwm Smo2Ot99 Mmlynx 3.3 See Amachments See Auwhmcnts See Anwhnnnts See Anschments 12/14/99SAIJSRPIKK 01/06100,01/07/0M0MH/IAS 01107100,01/10/00LASIbOOf Coacmtmdou of?FOS mg/mL or % Po@- mas PFOS ushal MD So& Day. MSAKM RFD <LOQ (00.0290 uW-L) NA <LOQ (0.00290ugtml) <LJDQ NA 0.0253 0.0248 0.0262 0.0243 <LOQ (0.00579ugft&) <LOQ (0.005W ughnl) <LOQ (0-005" UWML) <.OQ (0.00579ueml) <LA3,Q (0.00579u@tml) -:LM -4 oudiers MA 0.000790 74% 91% 82% 21% -6% .5% -6% 25% 77% 65% 71% 17% -1% 77% 38% 208% <LA)Q (@.00579uWmL) <LOQ (0.00579ug(ml) <LOQ (c005" vo%L) <LOQ (0.00579ug/mL) <LOQ (0-005"D&L) <LOQ (0.005"up*L) <LOQ (0.00579ugk&) <LOQ (0.00579ushRL) <LOQ (0-005"ugfml) <LOQ (0.00579ugfml) <LOQ (0-00579ugknl) <LOQ (0.00579u&l") <LOQ (0.00579ughnl) <LOQ (0-005" uw@nl) <LOQ (0.00579ugfml) <LOQ (0.00579ugftnl) <LOQ (0.00579ugfml) <LOQ (0-00579ushnl) NA <LOQ (0.00379ughnl) <-OQ NA <LOQ (o.oos"uwml) <LOQ (O.OOM ugAmL) <LOQ (0-005"usmIL) <tDQ (O-OOSW volml) <LOQ (0005" vf%L) <LOQ (0-005"uwml) <LOQ (0OOM upinl) <LOQ (0OOM ug%nl) <LOQ (O-OOS" uwinl) NA <ZQ (0005" uofml) IMM NA <LIOQ (O-OOS" uw,=L) <LOQ (0-005"a&,-L) <LOQ (O.OOS79u&PmL) <LOQ (0-00579upNnL) <LOQ (0.00579ughnl) <LOQ (O.OOS79ug/mL) NA <LOQ (0.00579ughnl) <LADQ NA <LOQ (0.005"u$hul) <LOQ (O.ODM up'cnl) <LOQ (O.ODM ul#nl) <LOQ (O.OOS79ug'nl) <LOQ (0.00579ughnl) <LOQ (0.00579ug'ml) <LOQ (0.00579uW") NA 1 <LOQ (0.00579uWmL) I <.OQ I NA NE Not Exuactod B - Loa &ring cmudm NA - Not Applic@Me LOQ - LimitofQumdtadm Comuntmues of PFHS Rw- me" PFM OQ (1 11 UW-L) <LOQ (0.00114up'tnl) <LOQ (0.00114 ughnl) <LOQ <LOQ (0.00114uWtnL) <LOQ (0.00114ugfrnl) <LOQ (0.00114ug/mL) <LOQ (0.0114ughnl) <LOQ (0.0114ugAmL) <LOQ(0.0114ugfmL) <LOQ(0.0114u&'mL) <A)Q (0.0114ugkl) 76% <LOQ 77% 76% -1% -1% -1% 83% 7?% 80% 1% 74% 37% <LOQ (0.0114ur'ml) <LOQ (0.0114ugMiL) <LOQ(0.0114uW@nL) <LOQ (0.0114u$fml) <LOQ (0.0114ughnl) <LOQ (0.0114u&'mL) <LOQ(0.01[4ugimL) <LOQ(0.0114ugOmL) <LOQ (O.Ot14 ug/mL) <LOQ (0.0114ugfinl) <LOQ(0.0114uptmL) <LOQ(0.0114u&ftnL) <LOQ(0.0114u&hnL) <LOQ(0.0114ughnL) <LOQ (0.0114ug/mL) <LOQ (0.0114ughnl) <LOQ (0.0114ughnl) <LOQ(0.0114u&/mL) <LOQ (0.0114uglinl) <LOQ (0.0114u&4nL) <LOQ(0.0114ug/mL) <LOQ <LOQ (0.0114up%L) <LOQ (0.0114upfinl) <LOQ(0.0114ugiuL) <LOQ (0.0114up-ml) <LOQ (0.0114vg%L) <A)Q (0.0114ugfml) <LOQ (0.0114uWmL) <LA)Q (0.0114vgknl) <LOQ(0.0114ughnL) <LOQ <LOQ (0.0114ugfml) <LOQ(0.0114uStinL) <LOQ (0.0114ughnl) <LOQ(0.0114u#/mL) <LOQ (0@0114UWWL) <LOQ (0.0114 u@tml) <LOQ (0.0114uofml) <L.OQ <LOQ(0.0114vlhnL) <LOQ (0.0114ugfml) <LOQ (0.0114 ughnl) <LOQ (O.Ot14 ug%d.) <LOQ (0.0114ugftnl) <LOQ (0.0114uWtnL) I <LOQ(0.0114uglfaL)I <LM PFOS - pan room PFOSA - Pefluamoctrm sulfomwd& FFHS PerfluorohcxmaWfonge POAA Pcr(lumonsloaft RSI) St& Dow. MS4@M RFD NA MA MA 2% 20% 9% 196% NA NA NA NA NA NA NA I NA UrS41-5.t Excd Vwsim 5n5 GEN-0304wLik &29AM FACT-GEN-030 Study: ProductNumber(T=t Subamee): Matrix: hicthodntevition: Amlyticel EquiF - SystemNumber. instrumentSoftware/Vasion: Filenww. R-Squared Value: siqw Y-IntercepL Dea of Exbw:ticWAnalyst DonafAnalysWArWysL Date of Data Rc&wtioWAradyst Sample Data BLOOD G-p Don s@pk 0 Mcdiod Bik MSU l2l2Sl.H20bikummund 5.3 MatrixBlk MSU12129-ICHM Mkred S-4 HNM 12129-bloodb&-54 HMB 121294"W bik-S-2 HMB1212"AaWbik--1-3 KMB]2129-bloodbik-S-4 HM]31212"Aocd bOt-5-3 HMB12129-bioadbik-5.6- HMB121"4aedbffic-5.7 HM 1212%@bbW b&-54 HMB12129.blood bik-S-9 QC ?984255-250bgS.5-1-2 PB34i2S3-250&CW,S.1-2 FSB,SWSL~ bS,-@1-2 FSB,9WSL~ MSD-.-@1-2 SSS,&U,49-~ SSBeSL49-~ MSI)63-1-2 HMB-FES2lW250 hIS@.5-1-2 HKBffMIM250 Blood P205 NorthernFor SaW P206 pwg P2Cr7 PM rZD9 P210 nil nl2 nis P2]7 n]9 P220 P221 P222 pn3 P224 P226 pm Blood P230 M104 Nordwra Fw Sod mios Adak Famks mios M107 MI 12 miis mi 16 mi Is M119 M122 Bind 3001 Nordicm Pur Sed S002 Sabodah b(do sm] S006 S007 soot S009 mood Nordmn For Sed P2ftS P236A 3UC P406 P411 98 CUXB 3* 99 CUKB 7 NA22VTA 99 CLTKB 9 swropto >50% de"afim fbmod I- May rAW tofem ellarnoo forPFOSA, imerfcr=t inboth wwysm ** PPOS NC)T @rwmed, MS &amftbu varbtim > 30% Dm PAtwe&Br 01121= LAC GEN030 MSU Eaviramnmtal Sampl= Various VariousBlood ETS-8-4.1 & STS41-5.1 usinguncxu=W soupo2olgg Mmlynx 3.3 See Attwbmcnts See Anwhments See Anuhments See AawhTnents 12/14/99SAIJSRP/XK OIA)6100.01/07/00MMWIAS 01/07/00,01/10/00LkS/Ngoffl curves Coacmautim of FOAA m@ IPOAA pm StL Day. ,LrL(oo ;"% Km aWmL MS mm RPD MA <LOQ (0-00240ughnl) <LOQ NA <LOQ (0.00240me-L) <LOQ (O-OMO uwinl) <LOQ (O-OOM vofml) <LOQ (O-OOM UW%L) <LOQ (O-OOM uw%&L) <.OQ (0OOM owtOL) <LOQ (O-WM useenl) <LOQ (0-0092 GWML) NA <LOQ (O-ODM ontml) <LOQ NA 76% 7S% 76% 1% 0% 1% I%- '16% 71% 74% 7% 1% 70% 33% 19s% <LOQ (O-OOM U&%L) <LOQ (O-OOM uvtnl) <LOQ (0.00958UWML) <LOQ (0OOM oo%L) <LOQ (0.00953wa'ml) <LOQ (0.00953usouIL) <LOQ (0.00959vg(-L) <LOQ (0-009Mov%L) <LOQ (O-ODM us%L) <LOQ (0-009saUgfmL) <LOQ (0.00958arML) <LOQ (O.Wgso oofml) <LOQ (O.WM MOML) <LOQ (0-MM uw@ML) <LOQ (0.00952UGPML) <LOQ (0.00953uo'ml) <LOQ (0-00953gohnl) <LOQ (O-ODM UWML) MA <LOQ (0-00958usfml) a.OQ NA <LOQ (O-OOM UWML) <LOQ (O-OOM go,-L) <LOQ (O-OOM vwiol) <LOQ (0-00959UWML) <LOQ (0OOM 4*4 <LOQ (0OOM we*-L) <LOQ (0OOM VO#ML) <LOQ (0OOM voiEL) <LOQ (O-OOM voisl) NA <LOQ (0-00M usfml) <MQ NA <LOQ (o.w958 UWML) <LOQ (O.WM vehOL) <LOQ (0 009= UWML) <LOQ (OWM UWML) <LOQ (O.WM up'ml) <LOQ (0.00958aWmL) MA <LOQ (0.00952se%L) <LOQ MA <LOQ (0-00959go,-L) <LOQ (O-OOM 99PML) <LOQ (0-009soOW-L) <LOQ (O.WM ushOL) <LOQ (O.WM mofml) <LOQ (0.0095aUWML) <LOQ (O-ODM VOIML) NA <LOQ (O-ODM giftl) I <.OQ NA NE - Not iIt,@ a - L" &wing c=wdm MA - Not Applicabk LA)Q - Limitofqumdtodon ef?FOSA volsowl % go <LDQ (0.000625no <LOQ (O.OW625 oWmL) 0.00195 0.00190 0.00225 0.00129 I I I I I 3VA 47% 1 =@SLA L itSD StL Dm NMAKSD RFD '<L NA NA I NA 49% 6% NA 1 1 NA MA NK I NA NA I NA - I NA I I NA I NA I NA FFM PFOSA Pdlwrooctme rafomw" PFHS - PanuorchaAr=Wfoam POAA Pwfl ETS-8-S.I axed version$195 GEN-03049mxb silo= C-29AM FACT-GEN-030 Study: Pfodua Nwnba(Tem Substance): Manix: medm)&?Avwm: AnalyticalEqLLipnentSystem Number. toguwrmt Softwwe/Vcmion: Fil GEM30 MSU EiMrannunW Sw*es Vwiom VwiousBlood ETS-84.1 & ETS-8-5.1usingwmwacte&curves smpml" Mmdynx 3.3 Sea Amwhnienb R-Squwed Val%w. Slopc See Anwhments See Aawbrentg Y-tatovept See AUwJmmts Duo of ExwwdoWAnalyst 12/14A9 SALJSRP/XXX Dun of AnalynalAnalyst Due of DataRWucdordAnslysL 0 1/0610D0.1107= hO4HAM otic7loo0,1/10" Lts/how(H Sample Data BLOOD Gr-p Dow Sompk 0 rpm coamtradow m@ RSD verom OFFFOS "M ShL DOV. h(edw Bik Monx glk MSU12129-HI20bik mMUred 5.3 MA M= I2129-H20blkffitui5d.4 NA HMBt2l29-blood bik-3.1 NA aWaL or % Rw- <LOQ (0-00"0UptdL) <LOQ (O-Wi" UWWL) 0.0253 oviol <LOQ btSI&M RPD NA NA HMB12129-blood b&--1-2 MA HbM121294Aoodbik-S-3 NA 0.0249 0.0262 HMB12129-blood bik-5.4 MA 0.0243 HMB1212940oodb&,S-S NA <A)Q (0-05"Uw%,L) HMB12129-bloodblk-"O HMB12129-bloodbik-5-7 MA <LOQ (&05" up,") MA <LOQ (0-05"9viUL) HMB121294Amdbl,.54 MA W0121294dood bik--'@9 NA <LOQ (0On goML) <LOQ (O-OS" VWML) <.OQ -4 audiem NA 0.000790 QC PBB-M-9-2"MS-34-2 MA 74% PBB.6255.250btM,5-1-2 MA 91% 12% 21% FSB-MW~ bts-5.1-2 MA -6% FSB.SOW2SOppb bCI)65.1-2 XA -S% .6% 25% SSB-SS"9.~ MS-5-1-2 MA 77% SSS-SSL49-~ MSD.5.1-2 MA 63% 71% Blood POIW BCW HMB-FE521 W230 MS-5-1-2 MA MO@FMIM250 MSD.5.1-2 NA 6255 Okporim) x 204M (NA2 EUTA) x 20"7 (NA2 1EVrA) x -1% 77% 0.0518 0.0381 0.0358 31% 209% 204" (EDTA) x 0.0315 20470 .x 0.0281 20472 x 0.0327 20473- x 0.0309 20474OW EDTA) x 0.0256 20475 (NA2 EURA) 20476 (NA2 EDrrA) 2047'7 2OUS (Hepwin) x <LOQ (0 OOM VWML) x 0.0345 x 0.0272 x 0.0429 Blood stenwScalion 2o4g6 Mcpwim) 20497 (Hepwin) SSLA9 (7.2fng K2 ElYrA) SSLSO (7-2mg K2 EDTA) SSL51 (7.2nig K2 EIYTA) SSL52 (7.2ng K2 EDTA) x &0316 0.214 x <LOQ (0OOM US(ML) 0.0342- 2 outlien 0.00731 NA <LOQ (O.OOS79up*-L) NA <LOQ (0 005W V&NNL) MA <LOQ (0-0()Sg"af-L) NA <LOQ (0-00579ug%&L) SSL53 (7.2nigX2 EEFTA) NA <ZQ (0.00579vgknl) SSL$4(7.2niSK2EUTA) NA <LOQ (000" Vv%,L) SSLSS (5.4mg K2 EVRA) MA <LOQ (0ON" UWIML) SSLJ6 (S.4nigX2 E[YrA)-O MA <.OQ (0005" vvinl) SSL$7 (5.4wg K2 EDTA) NA <.OQ (000" VWML) SSL58 (5.4a% K2 EDTAR MA <LOQ (O-OOM opiML) S@ (5-4mg X2 WrA) MA <LOQ (O-ODMUWML) NA NA <.OQ (O.OOS" alAOL) <LOQ MA t May needtomm @Hsampksfor#tEz== - Swmpft >50% &vmdan cm&md NE-Nmw"rumd &wmpm >50% 4rAadam. am coo&wad PM NOT cmfl hO onowdcm vwmon > 30% 8 - 140 &a* awaosm MA - NotApok" DiftEaroW. 11131M.01/21/00b8Gt4AC NV - NcitVerffiod Dow Venfoo& By- 02/IWW lgh LOQ Lbritof"ndmem coucentmdom NUSU of?FRS PYRS of/=L or% Rw. "L <LOQ (0.00114ugtml), <LOQ (0.00114ughnl) <LOQ <LOQ (0.0011U4SAUL) <LOQ (0.00114ugftnl) <LOQ (0.00114 ughnl) <LOQ (0.00114 uW") <LOQ (0.0114%AnL) <LOQ(O.Oll4uWmL) <LOQ(0.0114ughnL) <LOQ (0.0114uWinL) <= (0.011u4ghnl) 76% <.OQ 77% 76% -1% -1% -1% 93% 77% 30% 1% 74% 37% <LOQ (0.00114 uWvnL) <LOQ (0.00114 mp%il) <LOQ (0.00114 uWmL) <LOQ(O.OOll4uWmL) <LOQ(O.OOll4uWmL) <LOQ(0.00114u&'mL) <LOQ (0.00114u&mL) <LOQ (0.00114ughnl) <LOQ (0.00114ugtfnl) <LOQ (0.00114ulftnl) <LOQ (0.00114t*mL) <LOQ(0.00114ugImL) <LOQ(0.00114ughnL) <LOQ (0.00114uWmL) <LA)Q (0.0114ugftnl) <LOQ <LOQ (0.0114uWmL) <LOQ (0.0114ulhnl) <LOQ (0.0114 ugMiL) <LOQ(0.0114uglinL) <LOQ (0.0114uglinl) <LOQ (0.0114ugImL) <LOQ(0.0114ugfmL) <LOQ (0.0114ughnl) <LOQ(0.0114u@) <LOQ (0.0114uWmL) P=(0.0114optnL)_. <.OQ Pernoorcoctmkwdrow" PFOSA - Pgfluorooctmeodfoomtd& PFHS - "uorobcmcmdfaom POAA - Patuogooctmmft RSD Std. Dow. MS/NW ITPD MA NA MA MA 2% 20% 9% 196% NA NA NA MA M41-5.1 Bud valion 5193 GEN-030-nmxls Sim 5:17 PM FACr-GEN-030 Subdy! Pro&ict Number(Test Subnanec): Mmix: ModiodfRcvwm: AaWyticW EqWpywm System Number lnwwnad Softwwervvi.: Pikoa"W. R-Squwed Value. Si"-. Y-Intacept Dan of ExuactioWAnalysL Duo of Anslysts(AnWysL Due o(Di" Rc&"oWAnalyst Sample Data BLOOD GEN030 MSU EnvwonmcntW Samples Vwious Vwicm Blood ETS41-4.1 & LITS41-5.1 usingunexuwte& SoupO2Ol99 Manlynx 3.1 See Anacbments See Amachments See Amwlunents See Auawhnmits 12/14199 SALISRPFKICK 01/06=. 01/07= N04HIIAS 01/07/00, 01/IOMO LAS/MM Group Dow Sanpk 0 hiediod Bik MSU 12129-H20bik MSU]2129-H2Oblk unfihemd 5.3 filteredS-4 Manix Bik HMB12129-blwdbik-5-1 HMBI 2129-blood blk-S-2 MAB I2129-blood blk-S-3 HbM 12129-bb)od bik-S-4 HMB t2129-blood bik.-g-5 HMB121294Aoodb&.3.60 ED6(B121294Aood brA-5.7 HMB 12129-biood bik-34 HMB I2129-blood bik-!@.9 PBB-MS-L4MS-5-1.2 PBB4MSS-25ObC5D-S-1-2 FSB-VWZSOppb MS-S-1-2 FSB-SO09-2MIVb)AMD-3-1-2 SSB-SSIA9-250ppb MS-5-1-2 SSB-SSL49-2"ppb HMB-FES2199-M MMO@-S-1.2 bG-3.1.2 IDAD-FMIM250MM-'-1-2 ,A ob" -6255 Okpwin) POW uw 204M (NA2 =A) 2NO (?W MnA) 2NU Q=A) 20470 20472 204730 20474 (NAZ EDTA) 20475 aW EDTA) 20476 (NA2 WrA) 20477 20"5 (Hq-io) 204W Mqmin) 20487 Mepwin) Blood swtw sewica SSIA9 (7.2nig K2 EDTA) SSLSO (7.2 rng X2 EDTA) SSLS 1 (7.2 wg K2 EDTA) SSL52 (7.2nigK2 IMA) SSL53 (7.2ng K2 EDTA) SSL54 (7.2 mg K2 EURA) SSL55 (5.4ag X2 EDrrA) SSL56 (5.4nigX2 IMA)-* SSL57 (S.4mg K2 EWA) SSLSB (3.4 ag K2 EDRTA)* SSL59 (5.4nig K2 WrA) (1*4= M)" I May and torerakasumon forPFOSA,=, 00Swrqpft >3^ devalm o=N Swrople >30% &madam. am awfinned 0* PPOS NOT cmfi bO variation> 30% DoW EftvWW. Doe Vaifie&B)r 1/13100O,tt2l= MMRILAC 02/IWW lgh Coacmtratbe otPOAA sWuL w % Bm <LOQ (0.00240ogAnL) <LOQ (0.00240up-ml) <LC)Q (0.00240 v@O@ML) <LOQ (0OOM VOIUL) <LA:)Q (0 00240 vgfml) 4.OQ (O.WM vf%L) <LJOQ (0.00958 vo%L) <LOQ (O.ODM gof=L) <LOQ (0OOM VWML) <LOQ (O-ODMgw,=L) <LOQ (0-00958 OWOL) 76% 7PA (PA 1% 76% 71% 1% 70% <.OQ (0OOM IWML) <U)Q(O.OMG*ML) <WQ (O.Wlio4ML) <-OQ (O-WIQ*") <LOQ (0.0=40 VWML) <LOQ (0 00240 vWinL) <LOQ (0OOM mo@OL) <LOQ (000240 vWknL) -CLOQ (0.00240vg%nl) <LOQ <LOQ <.OQ <-OQ (000240 vWgmL) (O.WM vo%L) (O.ODM NDIML) (0-00240aWknL) <LOQ <LOQ <LOQ <LOQ <LOQ <.OQ (0MM NWML) (O-OOM WONOL) (0.00952grUL) (0.00958apiEL) (0OOM VV%L) (O_OOM V04&L) <LOQ (O.WgsgVWML) <LOQ (0-00959VehOL) <LOQ (0OOM asoknl) <-OQ (000958 voWL) <LoQ (0-009so gw@OL) <LOQ (0W9sg vo%L) (0-00958 vo%L) mum FOAA fti-L <LOQ <LOQ 76% 1% 74% 35% <LOQ 4.OQ RSD StiL Dov. MS940 RFD NA NA NA MA 1% 3&A 7*A 1 193% MA NA NA MA M Not Exua*W B - Loa &missamwm MA - Not Amgkabk NV-NotVaifeed LOQ - Lhaitof"Nwudm Comm&mdbe OfFY06A alfal w % Rm <LOQ (0-000=5 <LOQ (O-OOMGSWML) <LOQ (0 000625 a&%nL) <LOQ (0 W0625 OWML) <LOQ (O-OOOCS GWML) <.OQ (0.000625 aWmL) Man PFOSA <Log I I 50% 47% I I 1 I 1 1 <.OQ (O-OON" nhOL) <U)Q (0ODOM *WL) a.OQ (O-OOOM %%L) <LOQ (O-ODOW 4") <LOQ (0 000625 IW-L) <.OQ (0 000625 aWraL) <LOQ (0MOM UWML) <JDQ (0.000625upinl) <.OQ (0.000625owinl) <LOQ(OONWWML) CLOQ (0OOOQS u@) <.OQ (0.00= O&NOL) <LOQ (0-00005 askUL) <@OQ (0.000625 oghnl) I 49% I I 1 <.OQ RSD SUL Dev. bIS4W[SD RFD NA NA MA NA 6% MA MA NA NA I I t I I MA I NA PPOS Pafloomomoemdfame MK)SA - Pdhxwoocum sdfanamide "M - PadoombomerAfoome POAA Palloorcoctanomto azew Vasion 995 GM4.030.mLxls SfAw 5:37*iM 6' Wilsonj-onaeus,ck qffvl ,, m c 1991WilsoJnoneCsOMIldnv FACT-GEN-030 MSU Environmentzi Samples Study ProductNumbel@Ten Substancc): Manx: Method(Rcvision: AnalyticaPlquip@t SystemN@mb@ Insuwnent Sortware(Venion: Date of Exwaaion/Analyst Date of Analysis/Analyst: Date of Data ReductiontAnalyst Sample Data GEN030 MSU Various Various En@irannxmtalSampim ETS-9-6.o& ETs-s-i.o Soup 020199,A@lia 062498 Masslynx 3 3 12/12199 SALIKKISPP/CSH 01/10/00NUui 01/12= LAS ?4USCLE Group Dow Sample ?FOS Verified Method Sik Unfilteredwater Method Bik Filteredwater MatrixBik FishLiver QC 250 nglg M=de Carp MSU 12149-H20unfil5-1 MSU I2149-H20mfil 5-2?4SU12149.H20fil 5-11 MSU12149-H20fit 5-2- MSU I2149-Fishbik5.1 MSU 12149-Fishbik5-2 MSU12149-Fishbik 5-3. CPM-BLN7-ms 5-1-2 CPM-B2N6-MS 5-1-2 CSM-1999030-03-01-MS S-1.2 CSM-199903"3-01-?ZD ---t-2 LWM-1999029-16-MS 5-1-2 LWM-1999029-11-MS 5.1.3 BTM-1999040-WhO 5.1-2 BTM-1999OW10-NE 5-1-2 BINI 8iN2 BIN7 SINIO B2N2 B2N6 B2N8 B2NIO "IP2. Chinook Salmon 1999030-01 1999030-02 19990304rl-Ol 1999030-02-04 19990306-0_1@ol 1999030-03-04 Masd* 1999029-11- Lake Whittrub 1999029-12- 199902SL13 1999029-14 Muscle 199902-9-16 1999040-01 Brown Trom 1999040-02 -- 1999040-03 1999040-04 1999040-05 1999040-06 19990404)7 199904049 1999040-09 1999040-10 ffigh(>50%) suncgm devindons ** PFOS NOT coaftnaw; MS b2jwdoas variatwa> 3*% Doe Ent=&Anabst Olf24=,01/2M,01/2&VO LAC Date Verifie&Anabpst OVIG= igh NA NA WA NA NA NA NA NA NA NA NA NA MA MA MA x x x x x x x x x x NA NA x x x x x x x x x x X.. NA. NA x x NA x NA NA Cosentiration of?FOS utlgor % Rm 0.0163 <LOQ 0-00696) <LOQ 0.00696) O@M7 OOD943 0.00"5 0.00454 59% 31% 160% 127% 67% 122% 148% 166% 0.101 0.0794 0.090 O.Ors 0.103 O-OM 0.05% O.Oi3g 0.297 0.243 0.189 0.126 <LOQ ( 0.00696) 0.113 0.0514 0.0573 0.169 0.130 0.0967 0.0933 0.1659 <LOQ ( 0-00696) <LA)Q(0.00696) <LOQ ( 0-00696) <LOQ ( 0.00696) <LOQ ( 0.00696) <LOQ ( 0-00696) <LOQ ( 0-00696) 0.0460 <LOQ(O.OOW <LOQ(0.00696) Mean ?FOS NA NA 0.00796 45% 143% 95% 157% 0.124 0.107 - I oudier 0.132 <LOQ I mdicr RSD Std.Day. msnwm RPD NA --NA NA NA 37.2 0.00296 63% 23% 59% 11% Concentratiom orpvhs % 0,1L@4r(O.034.) <LOQ(0.034 ) <LOQ(0.0342) LOQ(O@0342) -<LOQ (0.0342) <LOQ (0.0342) <LOQ (0.0342) 60% 45% 149% 183% 129% 130% 1"% 169% ELOQ ( F03-42) Mes P= welt <LOQ <@OQ <LOQ 53% 166% 130% 156% RSD Std. Dew. MSNKSD RPD NA NA NA NA NA NA 29% 21-.@ 0% 15% <LOQ ( 0.0342) <LOQ(0.0342) <LOQ ( 0.0342) <LOQ ( 0.0342) <LOQ(0.0342) <LOQ(0.0342) 64.2 0.0793 <LOQ(0.0342) <LOQ(0.0342) NA <LOQ(0.0342) <LOQ NA <LOQ(0.0342) <LOQ(0.0342) 0.524 0.0562 <LOQ (0-0342) <LOQ(0.0342) <LOQ <LOQ ( 0.0342) <LOQ ( 0.0342) NA NA - <LOQ(0.0342) 26.4 0.0349 <LOQ(0.0342) <LOQ (0.0342) <LOQ(6.0342) iA <LOQ(0.0342) <LOQ NA- <LOQ(0.0342) <LOQ (0.0342) <LOQ (0.0342) <LOQ ( 0.0342) <LOQ(0.0342) <LOQ ( 0.0342) <LOQ ( 0.0342) <LOQ (0.0342) MA <LOQ(0.0342) NA MA NE Not Exwacted 0342) am(-O. <LOQ NA PFOS - F-ftom@ B - L@m duringexbiwdon PFOSA - Fafluoroocom swfonamide NA - Not Appik" PFHS - PaflumobamesWfoom LOQ - Liitiotf Quandtodon POAA Peffluorcoctuiome X VerifiedPFM concentration ETS-8-7.0 E.Cel Vcmim 5195 GEN-030-li@..b 4:46 PM FACT-GEN-030 MSU Environmental Samples Study: ProdtAcNtumber(Tcst Substance), Matrix: MethodtRcvision: AnalyticalEquipmmt System Number Instmment SoftwareJVmion Date of Exu@tiorLIAnalyst Date of Analysis/AnaJyst Date of Data Rcducdon/Analyst Sample Data GEN030 MSU En@ira@tal various Various ETS-9-6.0& LrTS.8-70 Soup 020199,Amelia 062498 Manlynx 3.3 12112/99 SALqUC/SRP/CSH 01110/00 mmli 01/12100 [AS Sunpim MUSCLE Group Dan S@ple 0 Method Blk Unfilteredwater MSU]2149-H20unfl MSU]2149-H20unril -'-2* Method Blk MSU12149-H20AF5-1-- Filteredwater MSU 12149-H2051 5-2- Mauix Blk Fish Liver MSU12 ITi:F-i@s-hIbik MSU12149-FrL%hbik 5-2 MSU12149-Fahbik 5.3- QC 250 ngtg CPM-B Ibr7-MS 5.1-2 CPM-82N6-NO 5.1-2 CSM-1999030-03-01 5-1-2 CSM-1999030-OMi-?AM 5-1-2 LWM-1999029-16-US 5-1-2 LWM.1999029LIt-bG 5.1-3 13TM-199904049-bU 5.1-2 muscle BTM-1999040-10-hG 5.1.2 BINI CNP BIM BIN7 SINIO B2N2 B2N6 B2N8 B2NIO carplo ca'a. Muscle 1999030-01 Chinook Salmon 199900-02 199W30-02-01 19M30-02-04 1999030-03-01 Muscle 1999030-03-04 1999029-11, Lake Whitefish 1999029-12- 1999029-13 19M29-14 1999029-16 Muscle 1999040-01 Brown Trout 1999040-02 -- 1999040-03 1999040-04 1999040-05 1999040-06 1999040-07 1999040-03 1999040-09 1999040-10 ifigh(>30%)mffogm dmadom ** PFOS NOT coaflrmed;MS &aaxidow Y=iatwn > 30% D- Entcre&Analyst 01124/00. OIMM LAC Due Verifie&AnalysL 02110/00 kjh Concmtratioa arpoAA at' t *,RI0.0359) 59) :LL(" ( 00@o.,@59) <LOQ (0.0359) -<LOQ (0.0359) <LOQ (0.0359) <LOQ(0.0359) 6% 39% 139% 178% 146% 105% 147% IS2% <LGQ(0.0359)-<LOQ(0.0339) <LOQ(0.0359) <LDQ(0.0359) <LGQ ( 0.0359) <LOQ(0.0359) <LOQ(0.0359) <LOQ ( 0.0359) <LOQ ( 0.6359) <LOQ ( 0.0359) <LOQ (0.0359) <LOQ (0.0359) <LOQ (0.0359) <LOQ(0.0359) <LOQ ( 0.0359) <LOQ ( 0.0359) <LOQ ( 0.0359) <LOQ(0.0359) <LOQ ( 0.0359) <LOQ ( 0.0359) -<LOQ(0.0359) <LOQ(0.0359) <LOQ(0.0359) <LOQ ( 0.0359). <LOQ (0.0359) <LOQ(0.0359) <LOQ(0.0359) <LOQ ( 0.0359) <LOQ (0.0359) <LOQ(0.0359) Meno POAA oVg <LOQ <LOQ <LOQ 23% 159% 125% 130% <LO() <LOQ <LOQ <.OQ RSI) Sid.Dev. MS/MSD RPD NA NA NA NA NA -- NA 148% 25% 33% 3% NA NA NA NA NA NA NA NA NE Not Exuacted E - Loa &Kingcm=(m MA - Not Applicable LOQ - LimitofQumtitadon X Vaifed PEOS cwmu2fion *FPFOSA ogilor % Per- PFOSA mgtg ('-C0LOQ 0 1U) <LOQ(0.0193) <LOQ (--oLOQ 0193) <LOQ(0.01 <LOQ <LOQ(O.Olw)<LOQ(0.0158) <LOQ(O.Olgg) 11% -I-A <LOQ 77% RSD ShL Dev. MSNKM RPD IIA NA NA MA NA -MA 1"6 69% 73% 11% 61% 76% 68% 22*A sm V% 83% 8% -@@( 0.0,U)@ <LOQ(O.Olgp <LOQ (0@O[U) <U)Q(O.Olu) <LOQ(O.OIU) <LOQ(0.0188) <LOQ(O.OIU) <LOQ(0.0188) <LOQ(0.0188) MA <LOQ(O.Olm <LOQ MA <LOQ ( 0.0138)- <LOQ(O.OIU) <LOQ (0.01n) <LOQ(O.Oln) <LOQ (O.OIM MA <LOQ MA @Lc"" (('.@0a0,.$) <LOQ(O.OIU) <LOQ(O.OIM. <LOQ(0.013&) KA <LOQ(O.OIU) <LOQ MA <LOQ(O.OISB) <LOQ(0.0188) <LOQ(O.Olw <LOQ(O.OIU) <LOQ(O.Olm <LOQ(O.Olu) <LOQ(O.Olm <LOQ(0.0183) <LOQ(O.Olm MA I <LOQ(0.01 MA ?FOS - Paflum:=cm:m<WloftQM@ PFOSA - Pdhwmoctme swfoanniide PFM Pernuarobexamswfcaaft POAA Perflocroactmoge :1 ETS-9.7 0 Pxcel version5/95 GEN-0304ivaxls SM" 4:46 PM l@ A 18 FACT-GEN-030 MSU EnvironMeU(21 SZMPle-I Study: PmductNumbei(TatSLibitanm): Matrix: M@d.,&R"isim: AnalyticEaqluonew SyittmN=tb@. tmuunum Saftware(Vmim: Daft@fEbctioWAnalyt DateofAsWysis/Aairt DaicO(DOM ReductioWAnWyst Sample Data GEN030 MSU E-i@tj Vari@w Vio.4 ElS-l-&OA M-9-7.0 Amlia 062491 MMF/.x 3.3 12112M SALtKK/S"/CSH 01/toftmmm 01/11= Lks Smvl. MUSCLE and EG4GS G@p Dose Mcdwd Blk wa. MatrixBik Egg QC m OWS C-P frogMOWJE Wbddmdy Gram FroggMp LAke Wkitdbh tep Bnmo TroutZgp High(>50%) **?FOS t4OTcoofl DateEnwr*&Analyst DaftVaiSed/Andyst Sangle 0 MSU12159-K2OBlk@mflitcred-S-9 MSU121S9-H20BIk-filt..d-5.9 EggBIk-S-1 BUBIh-S-2 EuB[k-S-3 FGW-AL.SWC]t-?92M&-2S5-/59-81 -2 FGW-ALSWCR-TD2 &12SVS.MSD-5-1.2 PGE-*,Ll19-HP26n"S.MS-S- 1-2 PGP-ALA I9-HP26r2S/".MSD.S.1-2 LWE-19999029-13-MS-5-1-2 LWII-19999029-13-MSD-5-1-2 BTF,19999040.01-MS.S-1.2 SM19999040-01-MSD-S4-2 DilI(gp5)-- Dktt(Cm" Dim 3 (Gop4) Did 4 AL-111@-YOY OIV2&98 ALSWCR-TD 06"8 ALSWCR-TDZ 06125M KZCXDM-A)V O&RMS KZCRDM-TD 0610"8 KZCXDM-TD-2 06C"$ SJOD02-TD-2OGM"L SMM-TD-2 06rl"o AL-119-HPSO W/2S/99 AL-118@HP94 0&2S/93 SIODOIO&WM -a SIODDI OGOMS L999029-13 1999029-L4 1999040-01 1999040-04 199904046 MS tr---donvadadoa> 30% 01/21/MOI124/00LAC OV 10/00kib Coacemtmdm Me" ofPOAA POAA utfgw % RaL owz <LOQ((LO3S9) <LOQ(O@03S9) <LOQ <LOQ(O@0359) <LOQ (0.03S9) -<LOQ(0,0359) <LOQ 34% 33% 34% 22% 26% 24% 90% 93% 36% 98% 112% IOS% <LOQ(0-0359) <LOQ(MO359) <-OQ(0.0359) <LOQ(0.0359) U)Q --ZLOQ((L-0110) <LOQ(O.Olsm <LOQ(0.018% <LOQ(O.OIW) <.OQ(0.018% <LOQ(0.0190) <.OQ(O.Olgo) <LOQ(O.Ollm <LOQ :<LOQ(0.0130) <LOQ (0.01" <LOQ(0.0180 <.OQ(0.01$0) ILOQ <LOQ(-O.C)lso) <ZQ(0.01110) <L.OQ <LOQ(ooig)-<LOQ(MOIBM a-OQ RSD St4L D@. MSIMSD "D NA NA NA NA 4% 15% Coamtrad@ ofPF%0OS.Ai0t1m") otfgor @LOQ ( '= ((0.-001.18,1-) < @LOQ(0.0184) <LOQ(O.Oiu) 49% 49% 46% 54% PPOSA <LOQ 49% 50% RSD Std.Dtv. MS/FASD RFD NA NA NA 1% 16% 9% 64% 66% 6% -10-4%- 13% 106% 106% 4% <LOQ((LOIU) <LOQ(MOIU) NA <.OQ(CLOIM NA NA <.OQ(0.01") <LOQ NA <LOQ(CLOIU) <LOQ(MOISX) @LOQ(O.Olu) <LOQ(O.OIU) <LOQ(0.01") <LOQ(O.Oln) NA <LOQ(0.0128) NA NA <LOQ(O.Olu) <LOQ NA ILOQ(0.0199) ILOQ((Loin) NA <LOQ((LOISS) NA NA <LOQ(QOIU) <LOQ NA NA NA NA <LOQ NA :@oNq NA <LOQ(0.018B) NA <LOQ(0.01") NE - Notzxwocova E - Laffdturingcumcbou MA-NotAppficable LOQ -Lirrdotfqimtibdm X Vai6w PFOS co@,ccnmdm NA <LOQ I NA PFOS - Pcruuoroocunmdfbam PFOSA - Pefluemocum sWfonmrA& PFHS - PwfluorohcxnmsWfimmo POAA - Pcguorooctmnoate EMI-7.0 E.cdvmim 5/95 GEN-030-Hvcrxlz S/IOMO 6:46AM FACT-GEN-030 MSU Environmental Samples Study ProductN@bcT(Test Substance) Matrix: McthodIRe@isi-: AnalyticalEquipmmt System Number InstrumenStoft-arcfV=ion Date of ExtractiotVAnalyst. Dateof Analysis/Analyst: Dateof Data Reductiort/Analyst Sample Data GEN030 MSU En"ronmental Samples Various Various ETS.".O & ETS-3-70 Soup 020199, Amelia 062498 Masslynx 3 3 12/12t99SALIKK/SILP/CSH 12/17M. 12/2Qt99,12/2&V9.12129t99,01103/00,0110610[0Asimmli 12120/99,12121/99,1V22/99. 12t3O/99.01/03/00,01/05/00,01107/00WVGMAS Miscellaneous Liver Group D@ Sample 0 Method Blk Unriltered ater Method Sik Filtered water MSU[2129-H20BIk-mrsitere&S-l MSU[2129-R2OBlk-unfiltatd-5-2 .MSU12129-H20BIk-mfiltat&5-3 MSU 12129-H@IOBIk-unfiltered-54 MSU12129-R2OBlk-unriltcrc&5-5 MSUI MSUI 2129-R20BIk-unflltere&5-6 2129-H20BIk-mfiltcred-5-7 MSU12129-H20BIk-unfiltac&5-8 MSU 12129-H2OBtk-fittered-5-1 MSU12129-H20BIk-Ritae&5-2 Matrix Bik Fish Liver Minix glk Rabbit Liver QC 250 ngtg MSU12129-H2081k.filterc&5-3 MSU[2129-H2OBlk-filUre&54 MSU[2129-H20BIk-fiitcre&5-5 MSU12129-H20BIk-filtere&5-6 MSU12129-H2OBlk-fihm@d-S-7 MSU12129-H20BIk-ffhmv&5-8 FSH12129-LvrB[k-S-1 FSH[2129-LvrB[k-5.2 FSH12129-LvrBIk-S-3 RBL12129-LvrBlk--'Ll PL12129-LwBik-S-2 RI)L I2129-LvrB[k-5-3 PBL[2129-LvrBlk-5.4 FLBL12129-LwBik-5-5RBL12129-LwBik-5-6* Mink Liver. D530, MS-5-1-1 Mink Liver, D530, MSD-5-1-2* CSL-1999030-03-01.MS-5-1-2* CSL-1999030-03-01-MSD-5-1-2* LWL-1999029-i2-MS-5-1-2* LWL-1999029-12-MSI).5-1-2* BTL-1999040-01.MS-5-1-2* BTL.1999040-01-MSD-54-21 TNL-TU54-MS-5-1-2 TNL.TU54-MSD-5-1.2 FSL-P295-MS-5-1 FSL.P295-MSD-5-1 PBL-M390LB-MS-5-1 PBL-990390LB-MSI)@5-2* OFL.KZCKDM-DI-M&5.1-2 OFL,KZCKDM-DL-MSD-5-1TTL,LCPTR99503C-MS-5.1-2* TTL-LCPTR99503C-MSD-5-1-2 MTL. IOVanclcrit98-MS-5-1-2 LATW MTL-IOVandeavc9g-MSD-S.-I-2 1999030-01 Chinook Salmon 1999030-02 1999030-02-01 19M30-02-04 1999030-03-01 Liver Like Whitertsh 1999030-03-04 1999029-11 1999029-12 1999029-13 1999029-14 1999029-16 Uver Brown Trout 1999040-01 1999040-02 1999040-03 1999040-04 1999040-05 1999040-06 1999040-07 1999040-08 1999040-09 High (>50%) surrogate deviations 1999040-10 Date Entered/Analyst: Date Vc@iF%ed/Analyst: 12/22/99.12r&199,12/29199.i2f3O/", 01/12/00,01/17/00,01/1&00 LAC 0 ?FOS Verifkd NA NA NA NA NA NA NA NA NA MA NA NA MA NA MA MA NA MA HA NA MA NA NA NA NA NA NA NA NA NA NA NA NA NA MA NA NA NA MA MA NA NA MA NA MA x x NA MA MA KA NA NA x x NA NA KA MA MA NA NA NA NA MA NA Concestradon ofppos ag/Cor% Rm NA NA <LOQ (0.0347) <LOQ (0.0347) <LOQ E (0.0696) <LOQ (0-0696) <LOQ (0.0696) MA NA E <LOQ (0.0347) <LOQ (0-0347) <LOQ (0-0696) <LOQ (0-0696) <LOQ (0-000 0.0305 0.0331 <-LOQ 0.0170 (0.0347) <LOQ (0.0347) <LOQ (0-0696) <LOQ (0-0696) <LOQ (0.0696) <LOQ (0.0696) 145% 539% 196% 140% 321% 2lt7% 133% 132% 68% B 91% 36% 19% 17"6 93% IOS% 90% 100% 79% sm 0.109 0.169 0.0329 0.126 0.173 0.0405 0.0679 0.0812 0.0733 0.0329 0.0778 <LOQ(0.0174) <LOQ (0.0174) <LOQ(0.0174) <LOQ(0.0174) 0.0255 <LOQ(0.0174) <LOQ(0.0174) <LOQ(0.0174) <LOQ(0.0174) <LOQ(0.0174) Mean PFOS net RSD Std. Dg,. MSIMSD RPD NA <LOQ NA <LA)Q 0.0269 <LOQ 342% 169% 304% 13S% 69% 38% 99*A 99% 95% 94% 0.108 0.0667 NA NA 32-1 0.00962 NA NA 115% 33% 11% 5% NA 6% 163% 12% 11% 11% 56.1 0.0608 29.3 0.0195 NA <LOQ - I outlier NA NE Not FAU%IW . B - Lost during exa-wdon NA - Not Applicable LOQ - Lirrdtof Qwmtitsfion X - Verified PEOS conccnuafim Concmftatioo of?FHS ag/tor% Rm NA NA <LOQ (0.00683) <LOQ (0.00683) <LOQ <LOQ E (0.0171) (0.0171) <LOQ (0.0171) NA MA E <LOQ (0.00693) <LOQ (0.00693) <LOQ (0.0171) <LOQ (0.0171) <LOQ (0.0171) <LOQ(0.0171) <LOQ(0.0171) <LOQ(0-0 71) <LOQ (0.00693) <LOQ (0.00683) <LOQ (0.0171) <LOQ (0.0171) <LOQ(0.017i) <LOQ(0.0171) 61% 50% Mean PFRS ogtg <LOQ <LOQ <LOQ <LOQ 55% 116% 11% S7% 69% 91% 34% 55% E 97% 94% 70% 76% 92% 99% 66% 72% 74% 94% <LOQ (0.0171) <LOQ(0.0171) <LOQ (0.0171) <L4DQ (0.0171) <LOQ (0.0171) <LOQ(0.0171) <LOQ(0.0171) <LOQ(0.0171) <LOQ(0.0171) <LOQ(0.0171) <LOQ(0.0171) <LOQ 0. 171) <LOQ(0.10171) <LOQ(O.Ot7l) <LOQ(0.0171) <LOQ(0.0171) 114% 63% 83% 55% 90% 73% 96% 79% <LOQ <LOQ <LOQ(0.0171) <LOQ(0.0171) <LOQ(0.0171) <LOQ(0.0171) PFOS <LOQ(0.0171) <LOQ - Perfluorooctonesulfonate PFOSA - Pefluarcoctane swfonmnide PFHS Pafluorchexamsulfonate POAA PerfluoroocUnoste RSD Sid. Dgv. MSFMSD RPD NA NA NA NA NA NA NA NA 21% 4% IBV. 4% NA 15% 9% 9% 13% NA MA NA NA NA NA ETS-8-7.0 ExceVlenion Y95 GEN-03(@-liverxls "/00 4:46 PM FACT-GEN-030 MSU Environmental Samples St.dy: Pmduct NmbcT(Teso Subumce) Matrix: McdxKi(Rcvision: AFtalyficaElquipment System Number tnsuummt Soft-are/Vmim Date or Fxb-&,tiori/Anadyst: Date orAnalysis/Analyst: Date of Doz Reduction/AnaJyst: Sample Dat2 GEN030 MSU EnvironmentalSmplm Various Various ETS-9-6,0& ETS-8-7.0 Soup O2Ot99, A@lis 062499 Manlynx 3.3 12/12199SALTJrJSRP/CSH 12117199.12/2W". 12/2at99,12129/99.01/03/00,01/06/I0A0SIMMH 12120/99.12121/99,12/22/99,12/30199.01/03100,01/05100,Ot/O7/OO,,,4mH/LAS ,Nfiscellaneous Liver Group Dose Sampit 0 Method Bik Unfilterewdatff MSUI 2129-H.'OB]k-unfihcrc&5-1 MSUI 2129-H-10BIk-mfihered-S-2 KSUI 2129-ECOBik-umfihacd-5.3 MSU12129-H20BIk-umfiherai-5-4 MSU]2129-H20BIk-mfnure&5-5 MSU I2129-H20BIk-=Mkred-.1@.6 Method Bik Filteredwater MSU12129-H208tk-=Ghcmd-S-7 MSU12129-H2OBfk-mfihenDd." MSU I2129-H20SIk-fib..&S-I MSUI 2129-H20RIk-Mbnv&5-2 WU12129-H20BIk-Mmrad-S-3 MSU12129-H20BDt-Gbme&5-4 MSU12129-H208&-do..i.--@S MSU12129-H20SIk-fibamd-5-6 MSU12129-H208&-fibmai-5-7 USU12129-H20BIkfihmxl.M MatrixBik FishLiver FSH]2129-LvrB&-S-l FSH12125LLwBlk-S-2 MatrixBlk P."t Liver FSH12129-LvrBIk-S-1 FtBL12129-LvrBik-5-1 ILBLI2129-LvrB]k-5-2 PBL12129-LvrB&-5.3 ftBLI2129-LvrB&-5-4 QC 250 ng/S RBL]2129.Lvrglk-5.5PBL12MLvrBlk-5-6- Mnk Liver.D530. M&S-1-1 MiAk Liver,D530. MSD-5-1-2* CSL-L 0-03-01-M&-'-1-2- CSL-1999030-03-01-MSI),5.1.2* LWL-1999029.tZ-MS--1.1-2LWL-1999029-12-NEI),5-1-2- BTL. 199904"1-MS-5.1-2BTL-1999040-01-MSD-5-1-2- TNL-TU54-MS.-5-1-2 TNL-TU54.MSD-5-1-2 FSL-P295-MS-5-1 FSL-?295-MSD-5-1 PBL,98039OLS-MS-54 PBL-M390U&.MSD.S.2- GFI,XZCKDM-DI-MS-5-t-2 GFL-KZCKDM-DI-MSD--'-ITTL-LCPTR99503C-MS-5-1-2* TrL-LCPTR-99503C-MSD-5-1-2 MTL-IOV=dcavcWMS-5-1-2 UTer ChirA)oSkabm MTL-IOVandcavc9g-hSD-5-1-2 1999030-01 1999030-02 19M30-02-01 19M304244 1999030-03-01 1999030-03-04 Uver Lake Whitermh 1999029-11 199902S'.12 1999029-13 1999029-14 1999029-16 Uver Brown Trout 1999040-01 1999040-02 1999040-03 1999040-04 199904045 1999040-06 1999040-07 1999040M 1999040-09 High(>50%) sum)gatedc"atiou 1999040-10 Date Entcrc&Analyst: Date Veriried/Analyst: l2/22J99.l2f2&M,12/29199,12130M. 0It12,'000.1/17100,0 111&W LAC 0 Concentradon ofPOAA uvg or % P"NA NA <LOQ (0.0719) <LOQ (0.0719) s <LOQ (0.0719) <LOQ (0.0719) <LOQ (0.0719) NA MA B <LOQ (0.0719) <LOQ (0.0719) <LOQ (0.0719) <LOQ (0.0719) <LOQ (0.0719) <LOQ (0.0719) <LOQ (0.0719) ((.0@*719)) Mean POAA a <LOQ <LOQ 1, RSD Std.Dev. m D RFD NA NA NA NA NA NA <LOQ (0.0719) <LOQ (0.0719) <LOQ (0.0719) <LOQ (0.0719) NA <LOQ (0.0719) <LOQ NA 96% 94% 85% 3% 131% 111% 121% 17% 133% 145% 139% 9% t2o% 84% E 72% 90% 95% 129% 92%. 103% 73% 64% 100% 75% <LOQ (0.0719) <LOQ (Oa7l9) <.OQ (0-0719) <LOQ (0-0719) <.OQ (0.0719) <LOQ (0-0719) <LOQ (0.0719) <LOQ (0.0719) <LOQ (0.(1719) <LOQ (0.0719) <L (0.0719) <LOQ (0.0719) <LOQ (0.0719) <LOQ (0.0719) <LOQ (0.0719) <LOQ (O.Or7l9) <LOQ (0.0719) <LOQ (0.0719) <LOQ (O.Or7l9) <LOQ (0.0719) <LOQ (0.0719) 124% 6% 94% NA 76% 10% 107% 41% 92% 23% 69% 13% 99% 29% <LOQ NA NA CLOQ NA NA <LOQ NA NE - Not E - Lou duringextraction NA - Not ApplicWAc LOQ - Limitof Quanfitadon X - VerifiedPFOS concenum6on Concentration of PFOSA uWt or % Rec. -WA- Mean PPOSA . ugtg RSD Std.Dev. ___@@D RPLD@ NA <LOQ (0.0188) <LOQ (0.0189) a <LOQ (0@0188) <LOQ (0.0189) NA <LOQ (0.0188) <LOQ NA NA NA B <LOQ (0.0tog) <LOQ (0.0188) <LOQ(0.0188) <LOQ (0.0182) NA <LOQ (0.01Be) <LOQ NA <LOQ (0.0188) <LOQ (0.0a1s) NA <LOQ (0.0198) <LOQ NA <LOQ (0.0188) <LOQ (0.0188) <LOQ (0.0188) <LOQ(O.Olgs) <LOQ (0.0189) NA LOQ (0.0189) <LOQ NA 43% 91% 62% .@60% 64% 52% 58% 21% 77% 83% 80% 7% 94% 92% 93% 3% 71% E 71% NA 87% 92% 95% 7% 71% 65% 69% 9,Y. 92% 93% 99% 12% 79% 71% 74% 10% 74% 74% 74% 0% -LOQ(0-0198) <LOQ (0.0188) <LOQ(O.Olsg) <LOQ (0.0188) <LOQ(O.OIU) NA <LOQ(0.0188) <LOQ NA <LOQ(0.0188) <LOQ(O.Olgp <LOQ (0.01es) <LOQ(O.OIU) NA <LOQ (0.0iss) <LOQ NA <LOQ (0-0193) <LOQ(0.0188) <LOQ(0.0188) <LOQ (0.0138) <LOQ(0.0189) <LOQ(0.0188) <LOQ(0.0188) <LOQ(0.0138) <LOQ(0.0138) NA <LOQ ( <LOQ NA PFOB - rern=CS-Wifonatc PFOSA - Pefluomoctme gwfonamide FFHS Paflwrahexenmulfonme POAA Perfluorooctanout ETS-9-7 0 ExceiVersion-"5 GEN-0304iver.xb t 4@46 PM FACT-CEN-030 MSU En@ima@t2l Samples ShWr Eq.M- 3r-,4sftw..V0-.(E...IkWA-ty& Sa.ple Data (EM)o MSU E,@ 3www, V_ ETS4-6.0a M-9-7 a S-WO201",AmWLI@24% v@Wy- 3.3 12JIV" SAL@KKisRpxsH l2/l7m9.tv2@. tzrLI".32r-l@,01'03=.oimsm, aim&=. 01@ L%SMTAH Milcelt"eous U@ Grwp D- M..b@dSik W(li-W S-pk a VSLT212%4008&-4ndbmm&$-l VSLI Ni.d.d80. %Ml 21204CK)Ift-..Mb..&" I'mcizi MSLI 21294C$)Mdb..&S-2 aft Fi.6L.Fm@ Bik RabbilLi@ QC 230 wg MsLq2l2o4cxna.db..&S-7 RBL[212D.L"Bksi RBLI2129-L."M12121@.Lvlmm SK1212SILvdNk44 PJLln2l6LvdM@W RXJ212WL@dft-54- CSL-19P)0304"1-M&$4-P CSL-L I I. LWL-I 12445-54.r LWLlvjmm@-124UD.3.1.r 7%'LIX344AM@15-1-2 FSL4--'M4AS-54 P&4'B$443Z).S.l POL48039OL84fs.s@l GFL4UEXEM4@l-M&3-1-2 (FL4CDC3MM.Dl4AM654TTL4jCPTRP"03C4dS,@l-r TM4.CFnt"M3C4A31)-g-1-2 LA'kalk- P. Sd I L'". P.L.B- Pn3 P2".. r-." W7 CU G2 nCUKDO2 NCUKB03 NCUKBO7 MCUKDO9 98 cu Ke 10 go CU KB II 98 cu KD 12 95 CU KO 13 ggoocu KB 14 CUXB ii vmlz VPMI 2w34l 69-,4t=4m3 ovairyLB gm3sxa 980],OLJP. Mi.k 9BD3MtA 90136$LB MIIZLB glas9n.^ 90*"La gnso" 990do" 9MIOLS 99OU2LC 9906"" D0330 DDSW DD$90 DOGIA D06". DN". DIODO 01=4 D1030 D1092 Dillo. 01134 IGGH (>50%) 8 d@.- -- po,i- -ay% -Wk..d- - - .6. DIISOX- DIIN DIIN Dil" DIU$ D16W. -44" D@ ENW.&Amky@ IV22@9. 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J.@@ -6@-4. -- posid@ mgyft oma@m @ @ hw,,4 @d 4" @ 99 D- E.W.&Ady. D-V-fi.VA.Wy. 12r2M. lir)GM, Oldt!MO.OtlllA)0.011IL4CO.01119100.QirQW LAC 0 <.OQ -LOQ (00719) (0 0719) (0,0719@ NA <OQ NA NE - t4.9E..-.d a - L.4 &.im @ NA - Nm Appbc" LOQ-Lhi..(Q-iX V-BW PFOS 4.M(G,oin) 0.0345 0.0611 0.0442-$GoUm PFOG . PPOILA P. pm. P()M ols 0.0201 h@ h@ 6134-70 a.d v- IM 4-.4phi FACT-GEN-030 MSU Environmen(al Samples Saxty: Pmduct Nun*ci(Tat Subst@): Matn. mcdwdm@ision: AAaly6caPlquip@t Sysim Number lmaumentSo(twarc(Vmion: Data.(E.U-6@,dA..Iyst Dow ofAmlysis/A.Ayst Due o(DwA Reduction/Anairt: Sample Data GEN030 PASU EnvitonnuraW SuMi@ vwi@ vwi@ ETS-4-6A.0LITS-9-7.0 Soup0201". A=Iia0624" Mmlym 3.3 IVIZM SALJXKISFLPICSH IV17,99.l2t2O/"1,2121V".l2n9/99.01/03/00.01/0W6G=I.M V-S(MMWMEE 12/2(Y99.1212lt99.12r22)1"2./30199O,IIOUW. 01105100O.IJ07/00O.V03iOOMMIVIAS(MEE .MisceRaneonsLiver G@p Do" S"ke ,4 ;r. 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MSU[2129.H20BIk-Mtac&54 MSU12129.H20BIk-MW*&5-2 MSU12129-H20DIk-6kwrc&5-3 MSU12129.H208&.E*roi-5-4 MSU12129-H2OBtk-Mkre&5-5 MSUt2l29-H20BIk-Ohmad-S-6 MSU12129-H20BIk-Mbmt&5-7 MSU12129-H20BIk-MUFO&" PSH12129-LvMlk-S-1 PSH12129-L"131k.S-2 FSHL2129-L"Bik-S-3 RSL]2129-LvrOlk-S-1 RBLI2129-L"Blk-S-2 PBLt2l29-L"Slk-S-3 RBL12129-LvrBlk-54 RDLI2129-LvrSlk-S-S* RBL12129-L"Blk-5.6Mink LiverD530,MS-".l Mink LiverD,$30.USD-5-1-2* CSL.1999030-03-01.MS-5.1-2CSL-19"030.40141.MSD-5-1-2* LV&-1999M942-MS-5.1-2- LWL-1999029-12-MSD-5-1-21 BTL-L999040-014AS.S4-2- BTL-1999040-014(SD.54-2- INL-TV544M5-1-2 TNL-TUS4-USD-S-t--2 PSL-P295-MS-S-1 PSL-P295-USD-S-t PBL-990390LB-MS-5-1 PBL-980390LB-MSI)-5.2- GFL-KZCKDM-DI.MS-S-1-2 GFL-KZCKDM-DI-MSD-5-1- TTL-LCPTR99503C4AS-S-1-2* TTL-LCPTR99503C44SD-S-1-2 MTL-LOV-wlcff,c9t-MS-5-1-2 .MTL-IOV=kzve9S.MSI)@5-1-2 Uvw Map Tmrtic Liver Tarwin Uver TUU P,,*IOV - " P, #09, Vmckavc 92 F,NM LedwfM. 99 P. M6.Lakrime (.1) P.(89. "12) LC7M "03C LCRM 95W LCM "OSC TU25. 71J.@4. TL%I* TU4$* TIM9 TU54' TUSS Tm. Liver G@ Pmg TU" TU$4 TUU TIM KZCKDM-ot KZCKDM-D2 Poci at 4 High(>50%)surmgatdcc@iefions siowi PFOS V@dfld NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA MA MA NA MA NA KA MA NA NA MA KA NA MA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 14A NA x x x x NA x MA x x NA x I NA NA x NA x Date E@tc,.&A.Wyst Dm Vaific&ArWyit: 12122/99,12/29/99.12rlW". 12f3QV9. 01/12100,01ii7/00,0111VM02104= LAC Cm@t-dofPFOS NA NA <LOQ (0.0347) @LOQ (0@0347) B <LOQ (0.06%) @LOQ (OL0696) <LOQ (0.06%) MA NA a :LOQ (060347) <LOQ (0.0347) <LOQ (0-0696) <LDQ (0-0696) <LOQ(&06W CLO305 CAMI 0.0170 <LOQ (OL0347) <.OQ (0.0347) <LOQ (0.0696) <LOQ (&06" @LOQ (0-06" <LA)Q(0.0696) 14s% S39% 196% 140% 321% 297% 139% 132% 69% E 91% "% 19% 179% 93% 105% 90% 100% 79% "% (Logot 0.0514 0.0739 M03% 0.703 06179 <LOQ(0.0347) *-OQ ((XO347) <LOQ(OM47) <LOQ(0.06%) <LOQ(O.OM <LOQ(0.06" <LOQ(CL06" <.OQ(0.06W <LOQ(0-0696) <LOQ(0-0696) <LOQ(0.06%) aoos9t <LOQ(0.0696) <LOQ (0.00696) <LOQ (0-0696) <LOQ(0.034 0.2$5 <LOQ(o-w47) <LOQ(0.03.47) Mean RSD PFOS Std.D@. NT-VMD RPD NA <LOQ NA 4= 0.0269 NA NA 3LI 0.00862 <LOQ 342% 1"% NM% 135% 1 68% a% 99% 99-A 95% 84% NA NA 115% 33% 11% 5% NA 6% 16.1% 12% 11% 11% O.IU -3oW@n 0.730 0157 NA -LOQ - Io@Ma NA NA <LOQ - Ioudier NA NE NotExoicW 9 -Lee&winsmuwdm NA - Not Applimbic LOQ - Liadtof Qumtiu6,m X - Vaified?FOS @t@d- Cm@tmdm ofPlrHS w % RWNA NA @LOQ (O.W683) <LOQ (0.00683) E <LOQ(0.0171) <LOQ(0.0171) <LOQ(0.0171) NA NA a <LOQ (0-006&3) <LOQ (0.006&3) <LOQ(&0171) <LOQ(0.0171) <LOQ(MO171) <LOQ(0.0171) <LOQ(0.0171) <LOQ(0.0171) <LOQ (O-ODW) <LOQ (0.00693) <LOQ(0.0171) <LOQ(0.017t) <LOQ(0.0171) <LOQ(0.0171) 61% 50% 116% 112% 57% "% 81% 94% 55% B 97% 94% 70% 76% 92% 99% 66% 72% 74% 54% <LOQ (O-ODW) <LOQ(0.00"3) @LOQ(0.00693) <LOQ (O-OOU3) <LOQ(0.00683) <LIOQ(O-ODW) <LoOQ(O.OOU3) <LOQ (0.00693) 4.OQ(O.OOU3) <LOQ(0.0171) <LOQ(0.0171) <LOQ(0.0171) <LOQ(0.0171) <LOQ(0.0171) <LOQ(0.0171) <LOQ(0.0171) <LOQ(0.0171) <LOQ(0.0171) <LOQ(0.0171) <LOQ(0.0171) <LOQ(0.0171) <LOQ (O.ODM) <LOQ(O.ODM) <LOQ(O.OOU3) <LOQ(O.ODU3) P4@ RSD PFHS StL Dew. NtS/M" RPD MA <LOQ I MA NA <L.OQ NA 14A <LOQ MA MA <LOQ MA SS% 21% -114% 436 1 63% 83% 4% 55% MA 90% 15% 73% 9% 96% 9% 69% 8% 79% 13% tm <.OQ MA KA <LOQ NA NA "Q NA PFOS - Paftooroactm=Wfooeft PPOSA -h&wmocbu adbmui& PFHS - PcrduorabcxmoaWfomft POAA Pwducroocanonk ETS-9-7.0 End vcmm 5/" GEN-030-d@ids 4.46 PM FACT-GEN-030 MSU Environmental Samples Study: Pmdwt Numbcv(Tat Subst@): M&tri.: Mcthod(R"ision: Amlytic4iEquq@t System Nuniber in.v@t Daw fExUwdoWA..Iyst: DateofAndMi'An*uDue of Dam ReductiontAmlyst: Sample Data GEN030 PASU Emiremn=W vw@m vui@ ETS4@-&O & ETS-8-7.0 Soup 020199. Amlia 0624" Sampla MLwiym 3-3 IV12199 SALAWSRP/CSH 12/17J99.1212(Y". [2t2V". 12129t99.01103100.01/06100,OVOIM V--WMMWMEE 1212&". 12/21199,12122/99.12130/",01103100.01/05JO0,01/07/00,02/03/W MMH/LkS(MEE MiscellaneousLiver Group Dwe Sanipk0 Mcdwd Bik MSU12t29-HZOB[k@Mftrt&5-1 Unfiltered MSU12129-H20SIk-Mftre&5-2 h4SU12129-H2061k@nfilocrc&5-3 MSU 12129-14200lk-mfiltcmd-54 MSU 12129-H2OBrk-=Blured.5-5 MSU 12129-H2OB[k-wafflumci-S-6 MSU I2129-H20BIk-unfflftroi-5.7 Method Btk MSU12129-H2OBtk-unfflkrcd-S-8 MSU 12129@-R2OBlk-Mwm&5.1 Filterewdum MSU12129-H20BIk-Mkrc&5-2 PASU12129-H20SIk-Mftre&5-3 MSU12l29-H20BIk-Mwe&S-4 MSU 12129@H20RIk-Maxed-S-S MSU12129.H20BO@Mave&S-6 MSU1212.9-H20BIk-Mkv.&S-7 MatrixOlk FishLiver )o(SU12129-H2OB&-Mkore&S4 FSH12129-Lrglk.-I-l FSHt2l29-Lvrglk-S-2 FSH12129-Lvrglk.5.3 MatrixDik RabbitLiver RBL12129LLvrBik-S-1 RBL12129-LwBlk-S-2 RDL12129-LvrOlk-S-3 RBLI2129-LdM-S4 RBL12129-LwBlk-S-S- RBLI2129@LvrOlk-S-e QC Mink LiverD.53% MS-S-1-1 250 mg/g Mink Livw.D530.MSD-S-1-2- CSL-19"(k3O-O3-01-MS-5-1-2- CSL-19990304341-MSD-5-1-2- LWL-1999029-t2-MS.S.1-2- LWL-1999029-12-MSD,S-1-2- BTL-1"9040-01-MS-S-1-2- BTL-19"040-01-MSD-5-1-2- TNL-TUS4-MS-S-1-2 TNL-TU54-MSD.S@-1.2 PSL-PZ9544S.S-t FSL-P2"-MSI)-5-1 PBL-990390LB.MS-S-1 PBL-980390LB-MSD.S-2- GFL-KZCKDM-DI-MS-5-1-2 GPL-XZCXDM-DI-MSD-5-1- rrL-LCPTR99503C-MS-54-2- TTL-LCPTR"503C-MSD-S4-2 ?4TL-IOVanduvcWMS-S-I.'2 MTL-IOVandeave9g-MSO-S-1-2 Liver F.#Iov-..,-" Map Turtle P.009.Vowkwe " F.#M LockrAk" P.#K Leckm-nc99 M.(.I) P,(99.S912) LA,w LCRM 9503C Tarvin LCFTR 9sw Liver Tuna LC3,m 95= T=. TU34. TU41* TU4$- TU49 TU54TUSS TU631 TU66 TU$4 TUSS Liver TU90 KZCKDM-DI Gn= Fmg KZCKDM-DZ Podo(4 sioool High(>50%) sunopgc dc@i&ciom DateEntere&Analyst Dm VrifieNA=I"t: 12/2V",121291".12/3(Y", 01/12iOO,Ol/l7=.Ol/iMO2/OVODLAC Contaktradon M@ ofPOAA POAA Sftw % Rw- vt NA MA @LOQ (0.0719) @LOQ (0.0719) E <LOQ (0.0719) <LOQ(OLIY719) @LOQ (MO719) <LOQ MA MA a <-OQ(D-G7i9) <.OQ (0.0719) 4.OQ (0.0719) <LOQ (tLO719) <MQ (0.0719) <LOQ 4DQ (0-0719) <LOQ (&0719) <LOQ (OLCV719) <LOQ ILOQ (0.0719) <LOQ (MO719) <L()Q(0-0719) 4-OQ (OL0719) <LOQ(&0719) -OQ(0.07i9) "% 84% <A)Q as%- 131% 111% 121% 133% 145% 139% IX?% 120% 124% 94% a 84% 72% 30% 76% as% 129% IU7% 82% 103% 92% 73% 64% 69% 100% 75% U% <LOQ (&(F719) <LOQ (M*719) <LOQ (aD7l9) <LOQ (0.0719) <LOQ(I)-0719) <LOQ (0.0719) <LOQ(0.0719) <LOQ (qLO719) 4m(qLG719) LOQ <LM ((LO719) 4.M (0.0719) <LOQ(0.0719) <LOQ (OLD719) <L.OQ (OL0719) 4@OQ (0.0719) @L@ (0.0719) <LOQ(&07i9) <LOQ (0.0719) <LOQ (0-0719) 4@OQ(0.0719) <LOQ(&0719 ILOQ (0-0719) <LOQ ((LO719) <LM (0-0719) <LOQ (OL0719) <LOQ RSD StdLDw. MS/MSD RPD Cmmtrad" ofPFOSA ugfgw % P@ NA NA @LOQ(0.0199) Mno PFOSA aivg RSD St& Dev. MSD RFD @LOQ(O.OIU) B @LOQ(0.0188) NA <LOQ(0.0113) NA NA <LOQ(C-O[U) I <LOQ NA NA MA a <LOQ(O.Olm <LOQ(MOIM <.OQ(O.OIU) NA <LOQ(CLOISS) NA NA <.OQ((Laigg) 4DQ MA <LOQ 0.0ta) NA <LOQ((O.Olm MA NA <LOQ(O.Olu) I <LOQ MA <LOQ(O.OIU) <LOQ (0.0133) <LOQ(&OIU) <LOQ(O.Olss) NA <LOQ(O.Olu) NA NA <LOQ(0.0188) <IOQ NA 43% 3% 11% 62% GM 64% 17% 52% St% 21% 77% 9-A 93% go%-- 7% 94% 6% 92% 93% 3% 71% NA B 71% NA FT% 10% 82% SS% 7% 71% - 41% 6s% Gt% 9% 92% 23% "% 89% 12% 79% 1 13% 71% 74% 10% 74% 29% 74% 74% 0% <LOQ(OLQIII) <LOQ(OOiu) <LOQ(G.Olu) <LOQ (0-01$S) <.OQ(O.Gtm <LOQ(O.Olm 144Q (OLOIM NA 14ZQ(Utgs) NA @u 4@(0-0190 MA <LOQ((LOISS) 4,OQ(O.Olu) <LOQ(10188) NA MA N@ I ME Not Exuactod B - Loffdtw* mumdon MA - NotAppikable LOQ - LirootfQ@titi6m X -VaifiePdFOS@nadw -LOQ("Im <LOQ(0.0182) <LOQ(0.01$S) <LOQ(O.OIU) <LOQ(O.OIBS) <LOQ(O.OIBS) <LOQ((Lolll) <LOQ(O.Olsg) NA <LOQ(O-Olw <.OQ NA <LOQ(0.0188) <LOQ(0.0188) ILOQ(Ckolts) NA <LOQ(0.0188) NA PFOSA - Pcdumomm swkmni& PPHS - Fafluon*Axane&Wfonm POAA - Patuoroocbnode ETS-11-7.0 E=l versio"nS GEN-0304iva..b 4:46PM G o Wilsonjone-s OuicRieterenIcnedeSxystem C 199W1ilsoJnoneCsompany FACT-GEN-033 Stodr. Pmd"a NWMGKTON Ma&i3L medwdnt4@wim, A@Wydcal 0 1 'l Swwtmm@ Sy-n Num6-. bwjwnm softworefvasim. DwA of ExuacdonfAmlyst Dan o(AndysWAAmlyst Dek A(DWA R**AOWAnalyst Sample Data Varim tjven G-" D@ hunk tivw Be" Sam liver I I r-M Dow" LAW- IVA- t4o AnUci6k suwk 0 C23 (45) C26 (44) C27 (49) C33 (46) C3S (47) C37 (48) C42 (43) C44 (S4) DIO(SO) ris ($S) Fig(sd) F21 (S7) F24 (58) FOI-(60) P& (64) Po (61) SII(37) SIS(41) SIB(40) sig(sq) sum S30(346) S3$(42) S39(39) TOI(SI) T04 (53) To ($2) V12 (62) V03 (65) Vol (63) JOB(zi) jog(n JIO(so J12(19) Jig (94) 120 (U) J7A(82) )27(93) J36(85) J37(20) R04 (69) R13 (79) R14 (74) R[6(71) R29 (72) R42 (66) lt43(73) R45 (79) R" (77) R47 (M P$4 (67) RSS (75) 257 CM a" (st) L04(PI) Los (OM DeftEmmo&Amkyst DaftVai&&Analpt OVUM OVOSM 04107/M OSM/00 BDOVLAC GEN033. MSU -Liver Smrpko NA Vwioto Ifws -thm&acud Curves Folea@. BTS-"O Md urs44.0 Davey0707". AmeHa 06249t R-Squwed Val= Slope: Mmlym 3.3 Y-inmavt 03/14100SALICSWKKK 03116M 03117A)O3./IWM 03120/OD0.3/21/0(0k3/29/M 04107= LA-&MMH 03nO/OO.03/22/00.03aM 03/24=,040M 04/11/00LAS(MMH SeeAtMchments SeeAnschnuft SeaAnsdonma SeeAdwJmino comm&adm ofPros oWS or % Roe 0.901 0.443 (L145 0.435 0.3SS 0.833 0.420 0.237 1.67 0.5" (L7&3 1.03 &a" 2.16 4.90 OL"l O.M 1.27 I." 2.a a-"9 MI$6 O.M3 0.317 0.633 1.35 O-W 4.27 I.SZ 3.65 0.0127 0.0229 <LOQ (&0347vWg) 0.0141 0.00931 0.0154 <LOQ (0.0347aWg) <LOQ (O-M47uWl) 0.0146 <LOQ (aO347oWS)' <LOQ (&Ck47vWg) 0.0100 @LOQ (0-0347vWg) 0.00609 0.00795 0.0138 0.0156 0.00649 alowm <LOQ (O.OS47MM <LOQ (Olow a" (LOI$3 0.0158 moom <LOQ (CM47 V&V 4LOB13 M@ RSD rym sic DVV. a" MS/MSD ITPD 103 1.23 1 1.27 1 0.01231 NA 33.7 &00416 KA NA Coomwadom *FPDAA agigw % Rec < LOQ (0.0359vWg) @ LOQ (0.0359uws) < LOQ (0.0359uW&) @ LOQ (0.0359Wg) < LOQ (&03S9 ug/g) < LOQ (0.03S9uW&) @ LOQ (0.03S9@WS) < LOQ (0-0359vws) @ LOQ ((LO359 Wg) < LOQ (0-0359vwg) < LOQ (aossgWWI) < LOQ (0-03"VWS) < LOQ (&0339*g) < LOQ (0-0359awls) < LOQ (o-o3sa9" < L.OQ (&0359"W < LOQ (OM59 VWI) < LOQ (0-055v9w < LOQ (143"gwi) < LOQ (OLOSS9VWI) < LOQ (0-0359vow men IPOAA lwg RSO S&L Der. BUVMD RPO c LOQ (0-0359WWI) < LOQ (0-035V9" < LOQ (06059a" < LOQ (0-0359MM < LOQ (aO359vWg) < LOQ (OLOSS9vgfg) < LOQ (OL0359vWg) NA < LOQ (a(13sOgwl) <.OQ NA < LoQ (0-035O9wl) <LOQ(0.0719%Wg) < LOQ (eO7l$vgtg) < LOQ (0-0359vWl) < LOQ (OL03599WIl) <LOQ(MO71&tWg) < LOQ (0.0718tgfg) <LOQ(O.O7lguWg) < LOQ (0-03S9MM < LOQ (Wit WWI) < LOQ (0.071v2afg) < LOQ (O.M$g uWS) <LOQ(0,071$uWg) < LOQ (0-0359aWg) @ LOQ (0.(k3sV9WS) < LOQ (OM59 Viva) < LOQ (0-0359vWg) < LOQ (0-0359VWS) -cLOQ (ae3S9vWg) @ LOQ (aQ7l$a" <LOQ(0.0718u" < LOQ (Mis mm <LOQ""" NA 1 < LA)Q (OiNSt v" I 4DQ I MA <LOQ (M 1:1Wg) LOQ MA <LOQ(OL07 KA "os - p PPOSA - Faflemoocum=Wbmui& MIHS - Pcrdoombcmaw waf=oe POAA Padmaroocomom rrS-9.7.0 tudvu"sm CdR4.W341mzk 4-.41%rM FACT-GEN-033 h Numba(Tat Subsumm): maww- Mcdw&Rcvkkm: AMIY*W gqu*mm Symm Numbcr. bwvwmm Sodwwt(Vasim- DabsOtLUmdi*WA,,Ipt Dam o(An*jsidAndyst Orm o(Dam RWOOdOWAnahme Sampk Data Vario= EJven Group Dan busk EJvw RdiLd Sag Uw GEW DOW- Ui LOQ - um atqwwuom MA -No App*" Sample 0 C23 (45) CZG (44) C27 (49) C33 (46) C35 (47) C37 (48) C42 (43) C44 (S4) 010(m pis(55) Fig(so P21 (S7) P24 (So rot(60) PM (64 pop(41) SII(3-7) SIS(41) SIB(44) sig(so) 325(39) S3004) S3S(42) S" (38) TOI(SI) TOO(s3) TW(M V12(4M vas(GS) VOS (43) in (81) jot OM JIO(26) J12(U) Jig(94) J20(U) J24 (12) J2? (93) J36(Li) J37 (90) R04(" IL13(7$) R14 (74) R16 (71) R29 (72) R42 (66) R43 (73) R4s R46 "7 CM RU (67) ass (?* R57 Lm (9m Dow ftum&A..Iim DaftVai&&Amhmt UV2M O*VM 04W)M OSMAO DOOLILAC GM4033. MSU -Liva SoMko NA VWIMS liVM - UB=MCfAd CWM ffm-$-&o md ETS4.7.0 D-ey 0707".Anw& 0624" Maulym 3.3 03/14;00SALCSWXKK pookmm R-Squamd Vahw. Skpe: Y-hwccpt: sm ati@ Sm AnWJU@ts So Adwluncnts S. Anad@to 03/14'M03/17/M 3/19M 03r"ft 03/21M 03/29/0O046M7/00 TASNMH 03/20/M031221M OU23/00O.Y241006O4M4'M 04111/0I0ASft4MH Coftnwadm ofPFOSA aft or% Ra owa3 < LOQ (0.037u6WS) < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ (0.037v6WS) (CLO37a6WS) (0.037a6WS) (0.037*6a) (0.03o7Wg6) (0.037a6WS) 0.0828 <LOQ (0.037u6Wj) <LOQ (0-037o6WS) 0605" < LOQ (0.037o6WS) CLSSI 0-5" < LA)Q(OM76 oWg) <LOQ (0-037v4W6#) < LOQ (0.0376iWg) 0.0414 0.132 < LOQ (060376MW < LOQ (MI376"W < LOQ (0.0376qffl < LOQ (0.037u6Wg) <LOq(06-.Wg) < LOQ (0-(1v3W7S6) < LOQ (0-0376vws) 0.345 0.(IS% 0.05" < LOQ (0-0376EWS) < LOQ (0-(137o6wl) < LOQ (0-0376VWI) < LOQ (QQ374 vWg) < LOQ (0-037a6WS) < LOQ (0-037v6Wl) < LOQ (0-037v6Wg) < LOQ (0.037w6Wg) < LOQ (0.037u6WS) < LOQ (0.037v6WX) < LOQ (0.037u6Wg) < LOQ (&0376 aWS) < LOQ (C.W76aWg) < LOQ (0-0376OWS) @ LOQ (0-0376vws) < LOQ (0-0376vws) < LOQ (0.076aWg) < LOQ (0.0376oWS) < LOQ (&0376 vWg) -cLOQ (OM76 uWl) -cLOQ (CM76 w*$) -cLOQ (06037w6Wg) c LOQ (OA*76 vWS) < LOQ (0605%vWg) @ LOQ (IL9376W" < LOQ (&C*76oft m@ PFOSA aft 4A)Q - 10Oudim -GA)Q <M RSD StiLDw. 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Word 6/95 ETS-8-4.1 ExtractionofPFOS from Serum Page 1 of 14 2.0 SUMMARY OF METHOD 2.1 This method describesthe procedure forextractingpotassium perfluorooctanesulfonate (PFOS) or otherfluorochernicaslurfactantfsrom serum,or otherfluidsu,sing an ion pairingreagentand methyl-tert-buteytlher(MtBE). In thismethod, seven fluorochemicals were extractedP:FOS, PFOSA, PFOSAA, ETFOSE-OK PFOSEA, M556, and surrogate standard(see3.0 Definitions).An ion pairingreagentisadded to thesample and the analyteionpairispartitioneidntoMTBE. The MTBE extractisremoved and put onto a nitrogenevaporatoruntildry. Each extractisreconstituteidn 1.0niL of methanol,then filteretdhrough a 3 cc plasticsyringeattachedto a 0.2 @Lm nylon filteirntoglassautovials. 2.2 These sample extractsare analyzedfollowingmethod ETS-8-5.1 or otherappropriate method. 3.0 DEFINfflONS 3.1 PFOS: perfluorooctanesulfonate(anion of potassium salt)C&Fl7SO3' 3.2 PFOSA: perfluorooctanesulfonylaniidCegFl7SO2NH2 3.3 -PFOSAA: perfluorooctanesulfonylamido(ethyl)acetatCe&Fl7SO2N(CH2CH3)CH2CO - 2 3.4 ETFOSE-OH: 2(N-ethylperfluor6octanseulfonaniido)-ethayllcohol CSF17SO2N(CH2CH3)CH2CH20H 3.5 PFOSEA: perfluorooctanesulfonylethylamideCBF17SO2N(CH2CH3)H 3.6 M556: CgFl7SO2N(H)(CH2COOH) 3.7 Surrogatestandard:IH- IH-2H-2H perfluorooctanesulfonicacid 4.0 WARNiNGs AND CAuTioNs 4.1 Health and safetywardings 4.1.1 Use universalprecautions,especialllyaboratorycoats,goggles,and gloveswhen handlinganimal tissuew,hich may containpathogens. 5.0 INTERFERENCES 5.1 There areno interferenceksnown atthistime. 6.0 Eouipm[ENr 6.1 The followingequipment isused whileperformingthismethod. Equivalentequipment is acceptable. 6.1.1 Vortex mixer,VWR, Vortex Genie 2 6.1.2 CentrifugeN,fistra1l000 or IEC 6.1.3 Shaker,Eberbach or VWR 6.1.4 NitrogenevaporatorO,rganomation ETS-84.1 ExtractioonfPFOS from Senun Page 2 of14 6.1.5 Balance( 0.100g) 7.0 SUPPLIES AND NIATERIALS 7.1 Gloves 7.2 Eppendorf or disposablepipettes 7.3 Nalgene bottlesc,apableofholding250 niL and I L 7.4 Volumetricflasksg,lass,typeA 7.5 I-C]HEM vialsg,lass,40 niL glass 7.6 Centrifugetubes,polypropylene,15 niL 7.7 Labels 7.8 Oxford Dispenser- 3.0 to 10.0 mL 7.9 Syringes,capableof measuring5 gL to 50 gL 7.10 Graduated pipettes 7.11 Syringes,disposableplastic3,cc 7.12 Syn*ngefilternsy,lon,0.2 gm, 25 nim 7.13 Timer 7.14 Crimp cap autovialasnd caps 7.15 Crimpers Note: Priorto usingglasswareand bottlesr,inse3 timeswithmethanoland 3 timeswith Nfilli-wQaTtmer. Rinse syringesa minimum of 9 timeswithmethanol,3 rinsesfrom 3. separatevials. 8.0 RIEAGENTS AND STANDARDS 8.1 Type I reagentgradewater, or equivalenta;llwater used inthismethod should be Nfilli-Q"'4wataenrd may be providedby a NM-Q TOC Plu@m system 8.2 Sodium hydroxide(NaOH), J.TBaker orequivalent 8.3 Tetrabutylammoniumhydrogensulfate(TBA)K,odak orequivalent 8.4 Sodium carbonate(Na2C03), J.T.Baker or equivalent 8.5 Sodium bicarbonate(NaHC03), J.T.Baker or equivalent 8.6 Methyl-T-ButylEther,Omnisolv,glassdistilloerdBPLC grade 8.7 Methanol,Omnisolv,glassdistilloerdIHPLC grade 8.8 Serum orblood,ftozenftom supplier 8.9 Fluorochemicalstandards 8.9.1 PFOS (3M SpecialtCyhemicalDivision)m,olecularweight= 538 8.9.2 PFOSA (3M SpecialtCyhemicalDivision)m,olecularweight= 499 8.9.3 PFOSAA (3M SpecialtCyhemicalDivision)m,olecularweight= 585 ETS-8-4.1 ExtractioofnPFOS fromSenun Page3 of14 8.9.4 ETFOSE-OH (3M SpecialtCyhenficaDlivision)m,olecularweight= 570 8.9.5 PFOSEA (3M SpecialtCyhemicalDivision)m,olecularweight= 527 8.9.6 M556 (3M SpecialtCyhemicalDivision)m,olecularweight= 557 8.9.7 Surrogatestandard:4-H, perfluorooctanseulfoniaccid(I-H,I-H, 2-H, 2-H C&Fl3SO3H) molecularweight = 428 8.9.8 Other fluorochemicalsa,s appropriate 8.10 Reagent preparation NOTE: When preparinglargervolumes thanlisteidnreagent,standardo,r surrogate preparationa,djustaccordingly. 8.10.1 10 N sodium hydroxide(NaOH): Weigh approximately200 g NAOH. Pour intoa 1000 mL beaker containing500 mL water,mix untilallsolidsare dissolved.Storeina I L Nalgenebottle. 8.10.2 1 N sodium hydroxide(NaOH): Dilute10 N NAOH 1:10. Measure 10 mL of 10 N NAOH solutionintoa 100 niL volumetricflaskand dilutteo volume usingMiUiQlm water. Storeina 125 mL Nalgenebottle. .&10.3 0.5 M tetrabutylammoniumhydrogensulfat(eTBA): Wei approximately169 g ofTBA intoa I L volumetricontainin5g00 mL NfiUi-Q water.AdjusttopH 10 usingapproximately44 io54 mL of 10N NAOH (WhileaddingthelastmL of NAOH, add slowlybecausethepH changesabruptly)D.ilutetovolume withNulliwater. Store-ina I L Nalgene bottle. 8.10.3.1TBA requiresa checkpriorto eachuse to ensurepH = 10. Adjustas needed usingIN NAOH solution. 8.10.4 0.25M sodium carbonate/sodiubmicarbonatbeuffer(Na2C03/Na]HC03): Weigh approximately26.5 g of sodium carbonate(Na2C03) and 21.0 g of sodium bicarbonat(eNaHC03) intoa IL-volumetrifclaskand bringtovolume withMmi- QTm water. Storeina IL Nalgenebottle. 8.11 Standards preparation 8.11.1PreparePFOS standardsforthestandardcurve. 8.11.2 Prepareotherfluorochemicasltandardsa,s appropriateM.ulticomponent fluorochemicalstandardsareacceptable(forexample,one working standard solutioncontaining1.00ppm PFOS, 1.02ppm PFOSA, 0.987ppm PFOSAA, and 1.10 ppm EtFOSE-OH.) 8.11.3 Weigh approximately100 mg of PFO S intoa 100 mL volumetricflaskand record theactualweight. 8.11.4 Bring to volume withmethanolfora stockstandardof approximately1000 ppm (@L9/ML). 8.11.5 Dilutethe stock solutionwith methanol fora working standardI solutionof approximately50 ppm. 8.11.6 Diluteworking standardI withmethanolfora w orkingstandard2 solutionof approx.5.0ppm. ETS-84.1 ExtractioonfPFOS fromSenim Page 4 of14 8.11.7 Diluteworking standardI withmethanolfora working standard3 solutionof approx.0.50 ppm. 8.12 Surrogatestockstandardpreparation 8.12.1 Weigh approximately50-60 mg of surrogatestandardI-H,I-H, 2-H, 2-H, C8Fl3SO3H intoa 50 mL volumetricflaskand recordtheactualweight. 8.12.2 Bringto volume withmethanolfora surrogatestockof approximately1000-1200 PPM- 8.12.3 Preparea surrogateworkingstandard.TransferapproximatelyI mL of surrogate stockto a 10 mL volumetricflaskand bringtovolume withmethanolfora working standardof 100 ppm. Record theactualvolume transferred. 9.0 SAMPLE HANDLING 9.1 Allsamplesarereceivedfrozenand must be keptfrozenuntiltheextractioinsperformed. 9.2 Allow samplesto thaw to room temperaturepriorto extraction. .10.0 OUALITY CONTROL 10.1 Solvent Blanks, Method blank s and matrix blanks 10.1.1 An aliquotof 1.0mL methanol isused as a solventblank. 10.1.2 Extracttwo 1.0niL aliquotsofMlli-e as method blanks. water followingthisprocedure and use 10.1.3 Extracttwo 1.0 niL aliquotsof the serum followingthisprocedure and use as matrix blanks. See 11.1.4. 10.2 10.3 Matrix spikes 10.2.1 Prepare and analyze matrix spikeand matrix spike duplicatesamples to determine the accuracy of the extraction. 10.2.2 Prepare each spikeusing a sample chosen by the analyst,usuallythe controlmatrix received with each sample set. 10.2.3 Expected concentrationswillfallinthe rnid-rangeof the initiaclalibrationcurve. Additionalspikesmay be includedand may fallin thelow-range of the initial calibratiocnurve. 10.2.4 Prepare one matrix spikeand matrix spikeduplicateper 40 samples,with a minimum of 2 matrix spikesper batch. Continuing calibrationchecks 10.3.1 Prepare continuingcalibratiocnheck samples to ensure the accuracy of the initial calibrationcurve. 10.3.2 Prepare, at a minimum, one continuingcheck per group of 10 samples. For example, ifa sample set= 34, four checks are prepared and extracted. 10.3.3 Prepare each continuingcalibratiocnheck from the same matrixused to prepare the initiaclurve. ETS-8-4.1 ExtracfionofPFOS from Senim Page 5 of 14 10.3.4 The expectedconcentrationwsillfallwithinthemid-rangeof theinitiaclalibration curve. Additionalspikesmay be includedthatfallinthelow-rangeof theinitial calibratiocnurve. This isnecessaryifthe analystmust quantitatuesingonly the low end of thecalibratiocnurve (forexample,5 ppb - 100 ppb, ratherthan 5 ppb - 1000 ppb). 11.0 CALIBRATION AND STANDARDIZATION 11.1 Prepare matrix calibrationstandards 11.1.1 Transfer I niL of serum to a 15 mL centrifugetube. 11.1.2 Ifmost sample volumes arelessthan 1.0ML, extractstandardswith matrix volumes equalto the sample volumes. Do not extractlessthan 0.50 niL of matrix. Record each sample volume on the extractionsheet. 11.1.3 V;hileprepaIringa totaloftwenty aliquotsin 15 mL centrifugetubes,mix or shake between aliquots. 11.1.4 Two 1 niL aliquotso,r otherappropriatevolume, serveas matrixblanks. Typically use the standardconcentrationsand spikingamounts listedinTable 1,atthe end of thissection,to spike,induplicatet,wo standardcurves,fora totalof eighteen standards,two matrixblanks,and two method blanks. 11.1.5 Refer to validationreportETS-8-4.0 & ETS-8-5.0-V-1, which fisttshe working rangesand theLinearCalibratioRnange (LCR) forcalibratiocnurves. 11.1.6Use Attachment D as an aidincalculatintgheconcentrationosf theworking standards.See Section13.0 to calculateactualconcentrationsofPFOS,in calibratiosntandards. 11.2 To each standard,blank,or continuingcheck,add appropriateamount of surrogate working standardforthe concentrationto fallw-ithinthe calibratiocnurverange 5 ppb-1000 ppb. 11.3 Extract spiked matrix standardsfollowing 12.6-12.16 of thismethod. Use these standards to establisehach initiaclurve on the mass spectrometer. ETS-8-4.1 ExtractionofPFOS from Serum Page 6 of 14 Table I Approximate spikingamounts forstandardsand spikes Usi g 1.0mL ofmatrix Working standard (approx.conc.) - P-L Approx.finalconc.of analyteinmatrix - Blank 0.500 ppm 10- 0.005 ppm 0.500 ppm 20 0.010 ppm 5.00 ppm 5 0.025 ppm 5.00 ppm 10 0.050 ppm 5.00 20 0.100 ppm 50.0 ppm 5 0.250 ppm 50.0 ppm 10 0.500 ppm 50.0 PPM 15 0.750 m 50.0 ppm 20 1.00 pE)pP:mMM:@ 12.0 PRO@F.DURE 12.1 Obtainfrozensamplesand allowto thaw atroom temperatureor ina lukewarm waterbath. 12.2 Vortex mix for 15 seconds,thentransfer1.0mL or otherappropriatveolume to a 15 mL polypropylenecentrifugteube. 12.3 Return unused samplesto freezerafterextractionamounts have been removed. 12.4 Record theinitivaollume on theextractiownorksheet. 12.5 Labelthetubewith thestudynumber, sample ED, dateand analystinitialsS.ee attached worksheetfordocumentingthe remainingsteps. 12.6 Spikeallsamples,includingblanksand standards,readyforextractiownith surrogate standardas describedin 11.2. 12.7 Spikeeachmatrixwiththeappropriateamount of standardas describedin11.1,or Table 1 inthatsectionf,orthecalibratiocnurve standards.Also preparematrixspikesand continuingcalibratiosntandards. 12.8 Vortex mix thestandardcurvesamples,matrixspikeswnples,and continuingcalibration samplesfor15 seconds. 12.9 Check to ensurethe 0.5 M TBA reagentisatpH 10. Ifnot,adjustaccordingly. 12.10 To each sample,add I mL 0.5M TBA and 2 mL of0.25M sodium carbonate/sodium bicarbonatebuffer. 12.11 Using an Oxford Dispenser,add 5 mL methyl-tert-buteytlher. 12.12 Cap each sample and put on theshakerata settingof 300 rpm, for20 minutes. 12.13 Centrifugefor20 to 25 minutesata settingof 3500 rpm, or untillayersarewellseparated. ETS-8-4.1 ExtractioonfPFOS from Senun Page 7 of 14 12.14 Label a fresh15 mL centrifugteubewiththe same informationas in 12.5. 12.15 Remove 4.0 mL of theorganiclayerto thisclean15 mL centrifugteube. 12.16 Put each sample on theanalyticanlitrogenevaporatoruntildry,approximatelyI to 2 hours. 12.17 Add 1.0mL of methanolto eachcentrifugteubeusinga graduatedpipette. 12.18 Vortex mix for30 seconds. 12.19 Attacha 0.2 gm nylonmesh filtetro a 3 cc syringeand transfetrhesampleto thissyringe. Filteirntoa 1.5 mL glassautovialor low-volume autovialwhen necessary. 12.20 Label theautovialwith thestudynumber,animalnumber and gender,swnpletimepoint, matrix,finalsolvente,xtractiodnate,and analyst(sp)erformingtheextraction. 12.21 Cap and storeextractastroom temperatureor atapproximately4 *C untilanalysis. 12.22 Complete the extraaionworksheet,attachedto thisdocument, and tapeinthestudy notebook or includeinstudybinder,as appropriate. 13.0 DATA ANALYSIS AND CALCULATIONS 13.1 Calculations 13.1.1Calculateactualconcentiationosf PFOS, or otherapplicablfeluorochemicali,n calibratiosntandardsusingthefollowingequation: mL of standardx concentratioonf standard(gg /mLj mL ofstandard+ mL ofsurrogatestandard+ initiamlatrixvolume (mL) FinalConcentration(@Lg/ml-o)f PFOS inmatrix 14.0 METHOD PERFORMANCE 14.1 The method detectiolnirii(iMtDL) isanalyteand matrixspecificR.efertoNML report forspecifiMcDL and limitofquantitatio(nLOQ) values(seeAttachments B and C). 14.2 The followingqualitycontrolsamplesareextractedwitheach batchof samplesto evaluate the qualityofthe extractionand analysis. 14.2.1 Method blanksand matrixblanks. 14.2.2 Matrix spikeand matrixspikeduplicateswnplesto determineaccuracyand precisionofthe extraction. 14.2.3Continuingcalibratiocnheck samplesto determinethe continuedaccuracyof the initiaclalibratiocnurve. 14.3 Referto section14 ofETS-8-5.1 formethod performancecriteria. 15.0 POLLUTION PREVENTION AND WASTE MANAGEMENT 15.1 Sample waste isdisposedinbiohazardcontainersf,lammablesolventwaste isdisposedin highBTU containersa,nd used glasspipettewaste-isdisposedinbroken glasscontainers locatedinthe laboratory. ETS-8-4.1 ExtractioonfPFOS from Senun Page 8 of14 16.0REcoRDs 16.1 Complete the extractiownorksheet attachedto thismethod,and tapeinthestudy notebook or includeinthe 3-ringstudybinder,as appropriate. 17.0 ATTACHMENTS 17.1 AttachmentA, Extractionworksheet 17.2 AttachmentB, MDL/LOQ valuesand summary 17.3 Attachment C, Calibratiosntandardconcentratiownorksheet 18.0 RIEFERENCES 18.1 The validatiornepoiratssociatewdiththismethod isETS-8-4.0& 5.0-V-1. 18.2 FACT-M-3. 1,"AnalysisofSerum orOtherFluidExtractfsorFluorochemicalussing BPLC-ElectrosprayMass Spectrometry" 19.0 AFFECTED DocumiENTs ,;ill 19.1 ErS-8-5.1, "AnalysisofSerum.orOther FluidExtractsforFluorochemicalussingBPLC- ElectrospraMyass Spectrometry" 20.0 REvisioNs Revision Number I Revision Reason For Revision Date Section12.21 Changed to includesamplestorageatroom temperature. 04/02/99 Section12.13 Added the shakerspeed. Section12.17Finalvolume is1.0niL;not adjustedforinitivaollumes less than 1.0mL. ETS-8-4.1 Extmctionof PFOS from Senlin Page 9 of 14 4.1.2 When handlingsamplesor solventswear appropnateprotectivgeloves,eyewear, and clothing. 4.2 Cautions: 4.2.1 Operatethesolventpumps below a back pressureof400 bar(5800 psi).Iftheback pressureexceeds400 bar,theBPI 100 willinitiataeutomaticshutdown. 4.2.2 Do not run solventpumps todryness. 5.0 INTERFERENCES 5.1 To minimizeinterferencwehsen analyzingsamples,Teflonshallnotbe used forsample storageor any partofinstrumentatiotnhatcomes incontactwiththesample or extract. 6.0 EouiopmmNT 6.1 Equipment listebdelow may be modifiedinordertooptimizethesystem.Document any modificationisntheraw dataas method deviations. 6.1 NEcromass QuattroH triplqeuadrupoleMass Spectrometerequippedwithan electrospraiyonizatiosnource. 6.1.2 ET I100 low pulsesolventpumping system,solventdegasser,column compartment, and autosampler 7.0 SUPPLIES AND MATERIALS 7.1 Supplies 7.1.1 Iiighpur-itgyradeairregulatedto approximately100 psi(houseairsystem) 7.1.2 EPLC analyticaclolumn,specifictso be determinedby theanalystand documented intheraw data 7.1.3 Capped autovialosr capped 15 n@ centrifugteubes 8.0 REAGENTS AND STANDARDS 8.1 Reagents 8.1.1 Methanol,BPLC gradeor equivalent 8.1.2 NElli-Qlmwater(ASTM typeI),allwaterused inthismethod shouldbe ATSM type I,or equivalenta,nd be providedby a Mlli-Q TOC Plussystemorothervendor 8.1.3 Ammonium acetater,eagentgradeor equivalent 8.1.3.1 When preparingdfferentamounts thanthoselisteda,djustaccordingly. 8.1.3.2 2.0mM ammonium acetatesolutionW:eigh approximately0.300g ammonium acetateP.our intoa 2000 ml'volumetriccontainercontaining 2000 ffilNEiii-Q"4water,mix untilallsolidsaredissolved.Storeatroom temperature. ETS-8-7.0 AnalysiosfLiverExtractUsingES/MS Page 3 of10 8.2 Standards 8.2.1Typicaltlwyo methodblankst,womatribxlanksa,ndeightemeantrisxtandaradrse preparedduringthe extractiopnrocedure.Referto ETS-8-6.0. 9.0 SANWLE E[ANDLING 9.1 Fresh matrixstandardsarepreparedwith each analysisE.xtractedstandardsand samples arestoredincapped autovialsor capped 15 n-dcentrifugteubesuntilanalysis. 9.2 Ifanalysiswillbe delayed,extractedstandardsand samplesmay be storedatroom temperature,or refrigerateadtapproximately4' C, untilanalysicsan be performed. 10.0 OUALITY CONTROL 10.1 Method Blanks and Matrix Blanks 10.1.1Solvenbtlanksm,ethodblanksa,ndmatribxlankasreprepareadnda,nalyzewdith each batch to determine contaminationor carryover. 10.1.2 Analyze a method blank and a matrix blank priorto each calibratiocnurve. 10.2 Matrix Spikes 10.2.1 Matrix spikesare prepared and analyzedto determinethe matrix effecton the recovery efficiency-. 10.2.2 Matrix spike duplicatesare prepared and analyzed to measure the precisionand the recovery for each analyte. 10.2.3 Analyze a matrix spikeand matrixspike duplicateper fortysamples. With a minimum of 2 spikesper batch. 10.2.4 Matrix spikeand matrix spike duplicateconcentrationswin fallinthe mid-range of the initiaclalibratiocnurve. Additionalspikeconcentrationsmay fallin the lowrange of the initiaclalibratiocnurve. 10.3 Continuing Calibration Checks 10.3.1 Continuing calibratiovnerificationasreanalyzed to verifythe continued accuracy of the calibratiocnurve. 10.3.2 Analyze a mid-range calibratiosntandardevery tenthsample,with a minimum of one per batch. 11.0 CALIBRATION AND STANDARDIZATION 11.1 Analyze the extractedmatrix standardspriorto and followingeach setof sample extracts. The average of two standardcurves willbe plottedby linearregression(y = mx + b), weighted I/x,not forced through the origin,using MassLynx or other suitablesoftware. 11.2 Ifthe curve does not meet requirementsperform routinemaintenance or reextractthe standard curve (ifnecessary)and reanalyze. ETS-8-7.0 AnalysisofLiverExtractUsing ES/N4S Page 4 of 10 11.3 For purposesof accuracywhen quantitatinlgow levelsof analyte,itmay be necessaryto use thelow end of thecalibratiocnurve ratherthanthefullrange of thestandardcurve. Example: when attemptingto quantitataepproximately10 ppb of analyte,generatea calibratiocnurveconsistinogf thestandardsfrom 5 ppb to 100 ppb ratherthanthe full rangeofthecurve(5 ppb to 1000 ppb). Thiswillreduceinaccuracyattributetdolinear regressionweightingof highconcentratiosntandards. 12.0 PROCEDURES 12.1 AcquisitionSet up 12.1.1 Setup the sample list. 12.1.1.1Assigna samplelisftilenameusingMO-DAY-last digitof year-increasing letteorf the alphabetstartinwgith a 12.1.1.2Assigna method (MS filef)oracquiring 12.1.1.3Assignan BPLC program (Inleftile) 12.1.1.4Type insampledescriptionasndvialpositionumbers 12.1.2To createa method clickon method intheAcquisitiocnontrolpanelthenmass spectrometerheadingsand selectSIR (SingleIon Recording)orNIRM (Multiple ReactionMonitoring).SetIonizatioMnode asappropriataend mass to499 or otherappropriatemasses.'A- fullscanisusuallycollecteadlongwiththe SEFS. Save acquisitiomnethod. IfMS/MS instrumentasreemployed,additionaplroduction fragmentationinforinatiomnay be collectedR.eferto NficromassMassLynx GUIDE TO DATA ACQLTISITION foradditionailnformatioannd MRM. 12.1.3Typicalltyheanalyticablatchrunsequencebeginsand endswitha setofextracted matrixstandards. 12.1.4 Samples areanalyzedwith a continuingcalibratiovnerificatiionjectedstandard aftereverytenthsample. Solventblanksshouldbe analyzedperiodicalltyo monitor possibleanalytecarryoverand arenot consideredsamplesbut may be includedas such. 12.2 Using the Autosampler 12.2.1 Set up sample trayaccording to the sample listprepared in Section 12.1.1. 12.2.2 Set-up the BPI I00/autosampler at the followingconditionsor at conditionsthe analystconsidersappropriateforoptimalresponse.Record actualconditionsinthe instrumentlogbook: 12.2.2.1Sample size= 10 p.Linjection 12.2.2.2Inject/sampl=e 1 12.2.2.3Cycletime= 9 minutes ETS-8-7.0 AnalysisofLiverExtractUsingES/MS Page 5 of 10 12.2.2.4Solventramp conditions Time MEOH 0.00 min. 40% 1.0min. 40% 4.5 min. 95% 6.5 min. 95% 7.0 min. 40% 9.0 mi. 40% 2.0 mM Ammonium acetate 60% 60% 5% 5% 60% 60% 12.2.2.5Pressthe "Start"button. 12.3 Instrument Set-up 12.3.1 Referto ETS-9-24.0, "Operationand Maintenance of the NficromassQuattro H TripleQuadrupole Mass SpectrometerFittedwith an Atmospheric Pressure IonizationSource,"formore details. 12.3.2 Check the solventlevelinreservoirasnd refililfnecessary. 12.3.3 Check the stainlesssteelcapillaraytthe end of the probe. Use an eyepieceto check thetip.The tipshouldbe flatwith no jagged edges. Ifthetipisfound to be unsatisfactoryd,isassemblethe probe and replacethe stainlessteelcapillary. 12.3.4 Turn on the nitrogen. 12.3.5 Open the tune page. Clickson operateto initiatseource block and desolvation heaters. 12.3.6 Open the InletEditor. 12.3.6.1Set BPLC pump to "Ow@ 12.3.6.2Set the flow to 10 - 500 uL/min or as appropriate 12.3.6.3Observe dropletscoming out of the tipof the probe. A finemist should be expelledwith no nitrogenleakingaround the tipof the probe. Readjustthe tipof the probe ifno mistisobserved 12.3.6.4Allow to equilibratfeorapproximately10 minutes. 12.3.7 The instrumentusesthese parametersatthe followingsettings.These settingsmay change inorder to optimizethe response: 12.3.7.1Drying gas 250-400 liters/hour 12.3.7.2ESI nebulizinggas 10-15 litersihour 12.3.7.3HPLC constantflow mode flow rate10 - 500 gumin 12.3.7.4Pressure<400 bar (Thisparmneter isnot set,itisa guide to ensurethe IHPLC isoperatingcorrectly.) 12.3.7.5Source block temperature150' 12.3.7.6Desolvationtemperature250' ETS-8-7.0 AnalysisofLiverExtractUsingES/MS Page 6 of10 12.3.8Printthetune page,withitsparameters'a,nd storeitinthe studybinderwith a copy taped intothe instrumentlog. 12.3.9 Clickon startbuttonintheAcquisitioCnontrolPanel(thismay vary among MassLynx versionsr,efertoappropriatMeassLynx User'sGuide).Ensure startand end sample number includesallsamplesto be analyzed. 13.0 DATA ANALYSIS AND CALCULATIONS 13.1 Calculations: 13.1.4 Calculatematrixspikepercentrecoveriesusingthefollowingequation: % Recovery Observed Result-Background Resultx 100 Expected Result 13.1.5 Calculateper@entdifferencuesingthefollowingequation: % Difference Ex@pectedConc. - CalculatedConc. x 100 Expected Conc. 1.3.1.6Calculateactualconcentrationisnmatrix(@tg/g): (ngofPFOS calcf.romstd.Curvex DilutioFnactor) x (InitiWaelightofLiver(9)FinalVolume (mL) I Ug 1000 ng 14.0 METHOD PERFORMANCE 14.1 Method DetectionLimit(MDL) and LimitofQuantitatio(nLOQ) aremethod,analytea,nd matrixspecificR.efertoETS-8-6.0,Attachment B fora listinogfcurrentvalidateNdOL and LOQ values. 14.2 Solvent Blanks, Method Blanks and Matrix Blanks 14.2.1 Solventblanks,method blanks,and matrixblanksmust be below theloweststandard inthecalibratiocnurve. 14.3 CalibrationCurves 14.3.1 The r2 valueforthe calibratiomnust be 0.980or better. 14.4 Matrix Spikes 14.4.1Matrix spikepercentrecoveriemsust be within 30% ofthespikedconcentration. 14.5 Continuing CalibrationVerification 14.5.1Continuingcalibratiovnerificatipoenrcentrecoveriemsust be within 30% ofthe spikedconcentration. 14.6 Ifcriterilaistedinthemethod performancesectionarenotmet,maintenancemay be performedon the systemand samplesreanalyzedor other'actionas determinedby the analyst.Document allactionsintheappropriatelogbook. ETS-8-7.0 AnalysisofLiverExtractUsingES/N4S Page 7 of 10 14.7 Ifdataareto be reportedwhen performancecriterihaave not been met,thedatamust be footnotedon tablesand discussedinthetextofthe report. 15.0 POLLUTION PREVENTION AND WASTE NTANAGEMIENT 15.1 Sample extractwaste and flammablesolventisdisposedinhighBTU containersa,nd glass pipettewaste isdisposedinbroken glasscontainerslocatedinthelaboratory. 16.0 REcoRDs 16.1 Each page generatedfora studymust have thefollowinginformationincludedeitherinthe headeror hand writtenon thepage: studyor projectnumber, acquisitiomnethod, integratiomnethod, samplename, extractiodnate,dilutiofnactor(ifapplicablea)n,d analyst. 16.2 Printthe tunepage,iamplefista,nd acquisitiomnethod from MassLynx to includeinthe appropriatestudyfolder.Copy thesepages and tapeintothe instrumentrunlog. 16.3 Plotthe calibratiocnurveby linearegressionw,eighted I/x,then printthesegraphsand storeinthestudyfolder. 16.4 Printdataintegratiosnummary, integratiomnethod, and chromatogramsfrom MassLynx and storeinthestudyfolder. 16.5 Summarize datausingsuitablseoftware(Excel5.0+)and storeinthestudyfolder,referto Attachment A foran example of a summary spreadsheet. 16.6 Back up electronidcatato appropriatmeedium. Record in studynotebook thefilename and locationofbackup electronidcata. 17.0 TABLES. DiAGRAms, FLowcHARTs. AND VALI]DATIODNATA 17.1 AttachmentA: ETS-8-7.0 Data summary spreadsheet 18.0 REFERIENCES 18.1 FACT-M-2. 1,"Extractionof PotassiumPerfluorooctanesulfonaotreOtherFluorochemical Compounds from LiverforAnalysisUsing BPLC-Electrospray/MassSpectrometry" 18.2 ETS-9-24.0,"Operationand Maintenanceof theNficromassAtmosphericPressure Ionization/MassSpectrometerQuattroH triplqeuadrupoleSysteme' 18.3 The validatiorneportassociatedwith thismethod isETS-8-6.0 & 7.0-V-1 19.0 AFFECTED DOCUMENTS 19.1 ETS-8-6.0,"Extractionof PotassiumPerfluorooctanesulfonaotreOtherFluorochemical Compounds from Liveror FluidforAnalysisUsing BPLC-Electrospray/Mass Spectrometry" ETS-8-7.0 AnalysisofLiverExtractUsingES/MS Page 8 of 10 20.0 REvisiONS Revision Number Reason For Revision Revision Date ETS-8-7.0 AnalysisofLiverExtractUsing ES/MS Page 9 of 10 Laboratory Study Study: TestMaterial: Matrix/FinaSlolvent: Method/Revision: AnalyticaElquipment System Number: InstrumentSoftware/Version: Filename: R-Squared Value: Slope: Y Intercept: Date of Extraction/Analyst: Date of Analysis/Analygt: Group Dose Sample# Concentration Initiawlt Dilution Factor FinalConc. UK/it Slope: Taken from linearregessionequation. Group/Dose: Taken from thestudyfolder. Sample#: Taken from thestudyfolder. Concentration(ngtg):Taken from theMassLynx integratiosnummary. InitiaWlt (g):Taken from thestudyfolder. DilutionFactor: Taken from thestudyfolder. FinalConc. (ugtg):Calculatedby dividingtheinitiavlolume from theconcentration AttachmentA: Summary Spreadsheet ETS-8-7.0 Analysisof LiverExtractUsing ES/MS Page 10 of 10 3M ENVIRONMENTAL LABORATORY METHOD EXTRACTION OF POTASSIUM PERFLUOROOCTANESULFONATE FLUOROCHEMICAL COMPOUNDS FROM LIVER FOR ANALYSIS ELECTROSPRAY/MASS SPECTROMETRY OR OTHER USING HI@LC- Method Number: ETS-8-6.0 Author: L'isaClemen, Robert Wynne Approved By: Adoption Date: RevisionDate: Laboratory Manager Date Group Leader Date TechnicalReviewer Date 1.0 SCOPE AND APPLICATION 1.1Scope: This method isforthe extractioonf potassiumperfluorooctanesulfona(tPeFOS) or otherfluorochemicalcompounds from liver. 1.2Applicable Compounds: Fluorochemicalsurfactantosr otherfluorinatecdompounds. 1.3 Matrices: Rabbit,rat,bovine,and monkey liversor othertissuesas designatedinthe validationreport. Word 6.0/95 ETS-8-6.0 Extractionof PFOS from Liver Page 1 of 14 2.0 SUMMARY OF METHOD 2.1 Thismethod describestheprocedureforextractinpgotassiumperfluorooctanesulfonate (PFOS) orotherfluorochemicaslurfactantfsrom livero,r othertissuesu,singan ionpairing reagentand methyl-tert-buteytlher(MtBE). Inthismethod,sevenfluorocheniicaclasn be extractedP:FOS, PFOSA, PFOSAA, ETFOSE-OH, PFOSEA, M556, and surrogate standard.An ionpairingreagentisadded to the sampleand the analyteionpairis partitioneidntoMTBE. The MTBE extractistransferretdo a centrifugteubeand put onto a nitrogenevaporatoruntildry. Each extractisreconstituteidn1.0ffilmethanolthen filtered througha 3 cc plastiscyringeattachedto a 0.2 gm nylonfilteirntoglassautovials. 2.2 These sampleextractasreanalyzedfollowingmethod ETS-8-7.0 orotherappropriate methods. 3.0 DEFE"IITIONS 3.1 PFOS: perfluorooctanesulfonat(eanion of potassium salt)CgFl7SO3 3.2 PFOSA: perfluorooctanesulfonylamideCgFl7SO2NH2 3.3 PFBSAA: perfluorooctanseulfonylamid(oethyl)acetaCtgeFl7SO2N(CH2CH3)CH2CO2 3.4 ETFOSE-OH: 2(N-ethylperfluorooctasnuelfonamido)-ethayllcohol CgFl7SO2N(CH2CH3)CH2CH20H 3.5 PFOSEA: perfluorooctanseulfonyelthylamideC&Fl7SO2N(CH2CH3)H 3.6 M556: CsFl7SO2N(M(CH2COOH) 3.7 Surrogatestandard:IH-IH-2H-2H perfluorooctanseulfoniaccid 4.0 WARNINGS AND CAUTIONS 4.1 Health and SafetyWarnings: 4.1.1 Use universaplrecautionse,speciallyaboratorcyoats,goggles,and gloveswhen handlinganimaltissuew,hich may containpathogens. 5.0 LNTERFERENCES 5.1 There areno interferenceksnown atthistime. 6.0 EouEpmzNT 6.1 The followingequipmentisused whileperformingthismethod. Equivalentequipment is acceptable. 6.1.1 Ultra-TurraTx25 Grinderforgrindingliversamples 6.1.2 Vortexmixer,VWF,, VortexGenie2 6.1.3 CentrifugeM,stral 1000 orIEC 6.1.4 Shaker,Eberbachor VWR ETS-8-6.0 Extractionof PFOS from Liver Page 2 of 14 6.1.5 NitrogenEvaporator,Organomation 6.1.6 Balance(sensitivtioty0.100g) 7.0 SUPPLIES AND MATERIALS 7.1 Gloves 7.2 Dissectingscalpels 7.3 Eppendorf or disposablepipettes 7.4 Nalgene bottlesc,apableofholding250 mL and I L 7.5 Volumetricflasksg,lass,typeA 7.6 I-CHEM vials4,0 mL glass 7.7 PlasticsampulevialsW,heaton, 6 mL (orappropriatseize) 7.8 Centrifugetubes,polypropylene,15 niL 7.9 Labels 7.10 Oxford Dispensor- 3.0 to 10.0 n-d 7.11 Syringes,capableof measuring 5 gL to 50 gL 7.12 Graduated pipettes 7.13 Syringes,disposableplastic3,cc 7.14 Syringefilternsy,lon,0.2 gm, 25 mm 7.15 Timer 7.16 Crimp cap autovialasnd caps 7.17 Crimpers Note: Priortousingglasswareand bottlesr,inse3 timeswithmethanoland 3 timeswithMM- Ql"4water.Rinse syringesa minimum of9 timeswithmethanol,3 rinsesfrom 3 separate vials. 8.0 REAGENTS AND STANDARDS 8.1 Type I reagqntgradewater,NM-QTM or equivalenta;llwater used inthismethod should be Nfilli-Q"waterand be providedby a Mffi-Q TOC Pluim system 8.2 Sodium hydroxide(NaOH), J.TBaker orequivalent 8.3 Tetrabutylammoniumhydrogensulfate(TBA)K,odak orequivalent 8.4 Sodium carbonate(Na2C03), J.T.Baker or equivalent 8.5 Sodium bicarbonate(NaHC03), J.T.Baker or equivalent 8.6 Methyl-tert-buteytlher,Omnisolv,glassdistilloerdBPLC grade 8.7 Methanol,Onmisolv,glassdistilleodr IHPLC grade 8.8 Liver,frozenfrom supplier 8.9 Dry icefrom supplier 8.10 Fluorochemical standards 8.10.1PFOS (3M SpecialtCyhemicalDivision)m,olecularweight 538 ETS-8-6.0 ExtractioonfPFOS fromLivcr Page 3 of 14 8.10.2PFOSA (3M SpecialCthyemicaDlivisiomno)l,eculwaerigh=t 499 8.10.3 PFOSAA (3M SpecialtCyhemicalDivision)m,olecularweight= 585 8.10.4 ETFOSE-OH (3M SpecialtCyhemicalDivision)m,olecularweight= 570 8.10.5 PFOSEA (3M SpecialtCyhemicalDivision)m,olecularweight= 527 8.10.6 M556 (3M SpecialtCyhemicalDivision)m,olecularweight= 557 8.10.7 Surrogatestandard:4-H, perfluorooctanseulfoniaccid(1-H,I-H, 2-H, 2-H CgFl3SO3H) molecularweight= 428 8.10.8 Other fluorochemicalsa,s appropriate 8.11 Reagent preparation NOTE: When preparinglargervolumes thanlisteidnreagent,standard,or surrogate preparationa,djustaccordingly. 8.11.1 10 N sodium hydroxide(NaOH): Weigh approximately200 g NAOH. Pour intoa 1000 mL beakercontaining500 mL water,mix untilalls.ohds are dissolved.Storeina IL Nalgene bottle. 8.11.2 1 N sodium hydroxide(NaOH): Dilute10 N NAOH 1:10. Measure 10 mL of 10 N NAOH solutionintoa 100 mL volumetricflaskand diluteto volume using Mlli-QTm water. Storeina 125 mL Nalgenebottle. 8.11.3 0.5M tetrabutylammoniuhmydrogensulfat(eTBA): We* approximately169 g ofTBA intoa ILvolumetriccontainin5g00 niLNCM-Q water.Adjustto pH 10 usingapproximately44 to 54 mL of 10 N NAOH (Whileaddingthe lastmL ofNAOK add slowlybecausethepH changesabruptly)D.ilutetovolume with NElli-QwTamter. Storeina IL Nalgene bottle. - 8.11.3.1TBA requiresa check priortoeach useto ensurepH = 10. Adjustas needed using1 N NAOH solution. 8.11.4 0.25M sodium carbonate/sodiubmicarbonatbeuffer(Na2C03/N&HC03): Weigh approximately26.5g of sodium carbonate(Na2C03) and 21.0g ofsodium bicarbonat(eNaHC03) intoa I L volumetrifclaskand bringtovolume withMlhe water.Storeina I L Nalgenebottle. 8.12 Standards preparation 8.12.1PreparePFOS standardfsorthestandardcurve. 8.12.2Prepareotherfluorochemicasltandardsa,sappropriateM.ulticomponent fluorochemicasltandardsareacceptable(forexample,one working standard solutioncontaining1.00ppm PFOS, 1.02ppm PFOSA, 0.987 ppm PFOSAA, and 1.10 ppm EtFOSE-OH.) 8.12.3 Weigh approximately100 mg of PFOS intoa 100 niL volumetricflaskand record theactualweight. 8.12.4 Bring to volume withmethanolfora stockstandardofapproximately1000 ppm (RgtmL). 8.12.5 Dilutethe stocksolutiownithmethanolfora working standardI solutionof approximately50 ppm. ETS-8-6.0 ExtractioonfPFOS fromLiver Page 4 of 14 8.12.6 Dilutethe stocksolutionwithmethanolfora workingstandard2 solutionof approx.5.0ppm. 8.12.7 Dilutethe stocksolutionwithmethanolfora workingstandard3 solutionof approx.0.50 ppm. 8.13 Surrogatestockstandard preparation 8.13.1 Weigh approximately50-60mg of surrogatestandard1-H,I-H,2-H, 2-H, C8Fl3SO3H intoa 50 n@ volumetricflaskand recordtheactualweight. 8.13.2 Bringto volume withmethanolfora surrogatestockofapproximately1000-1200 PPM- 8.13.3 Preparea surrogateworking standard.Transferapproximately1.0ml of surrogate stockto a 10 ml volumetricflaskand bringto volume withmethanolfora working standardof 10-20ppm. Record theactualvolume transferred. 9.0 SAWLE HANDLING 9.1 Allsamplesarereceivedfrozenand must be keptfrozenuntiltheextractioinsperformed. .10.0OliiLyry CONTROL 10.1 Matrix blanksand method blanks 10.1.1An aliquootf 1.0n-LLmethanolisusedasa solvenbtlank. 10.1.2Extracttwo 1.0niL aliquotosfMfli-QTm waterfollowingthisprocedureand use as method blanks. 10.1.3 Extracttwo 1.0niL aliquotsofliverhomogenate followingthisprocedureand use as matrixblanks.Referto 11.1.6. 10.2 Matrix spikes 10.2.1 Prepareand analyzematrixspikeand matrixspikeduplicatseamplesto determine the accuracyof theextraction. 10.2.2 Prepareeach spikeusinga sample chosenby theanalystu,suallya controlliver receivedwith each swnple set. 10.2.3 Expected concentrationwsillfallinthemid-rangeof theinitiaclalibratiocnurve. Additionalspikesmay be includedand may falilnthelow-rangeof theinitial calibratiocnurve. 10.2.4 Prepareone matrixspikeand matrixspikeduplicatpeer40 samples,with a minimum of 2 matrixspikesperbatch. 10.3 Continuing calibrationverirications 10.3.1 Preparecontinuingcalibratiovnerificatisoanmplesto ensuretheaccuracyof the initiaclalibratiocnurve. 10.3.2Prepare,ata minimum, one continuingcalibrativoenrificatisoanmplepergroup of 10 samples. For example,ifa sample set 34,fourverificatioanrsepreparedand extracted. ETS-8-6.0 ExtractioonfPFOS fromLiver Page 5 of 14 10.3.3 Prepareeach continuingcalibratiovnerificatiofnrom the same matrixused to preparetheinitiaclurve. 10.3.4The expectedconcentrationwsillfallwithinthemid-rangeof theiniticaallibration curve.Additionalspikesmay be includedthatfallinthelow-rangeof theinitial calibratiocnurve. Thisisnecessaryiftheanalystmust quantitatuesingonlythe low end ofthecalibraticounrve(forexample,5 ppb - 100 ppb,ratherthan 5 ppb 1000 ppb). 11.0 CALIBRATION AND STANDARDIZATION 11.1 Prepare matrix calibratiosntandards 11.1.1Weigh approximately40 g ofliverintoa 250 mLNalgene bottlecontainin2g00 mLs NEHI-C water.Grindto a homogeneous solution. 11.1.2If40 g isnot ivailableu,se appropriataemounts of liverand waterto ensurea 1:5 ratio. 11.1.3Refer to 13.0to calculatteheactualdensityofliverhomogenate and the concentratioonf solidlivertissuedispersedin 1.0mL of homogenate solution. 1 l.l..A5dd I mL of homogenate to a 15 mL centrifugteube.Re-suspendsolutionby shakingbetween aliquotwshilepreparinga totalof eighteenI mL aliquotsof homogeneous solutionin15 mL centrifugteubes. 11.1.6 Two 1 mL aliquotso,r otherappropriatveolume,serveas matrixblanks. 11.1.7 Typicallyuse thestandardconcentrationasnd spikingamounts listeidnTable 1,at the end of thissectiont,o spike,induplicatet,wo standardcurves,fora totalof eighteensamples,two matrixblanks,and two method blanks. 11.1.8Referto validatiorneportsETS-8-6.0 and ETS-8-7.0-V-1 or Attachment B, which listtsheworking rangesand theLinearCalibratioRnange (LCR) for calibratiocnurves., 11.1.9 Use Attachment C as an aidincalculatintgheconcentrationosftheworking standards.Referto 13.0to calculataectualconcentrationosf PFOS incalibration standards. 11.2 To eachworking standardb,lank,or continuingverificatioand,d appropriataemount of surrogateworking standardforthe concentratiotno falwlithinthecalibratiocnurverange5 ppb -1000ppb. ETS-8-6.0 Extractionof PFOS from Liver Page 6 of 14 11.3 Extractspikedliverhomogenates following12.14-12.25of thismethod. Use these standardsto establisehach initiaclurveon themass spectrometer. Table 1 Approximate Spiking Amounts forCalibrationStandards Working Standard (Approx.Conc.) @Ll Approx. finalconc.of PFOS inliver - - Blank 0.50 PPM 2 0.005 ppm 0.50 ppm 4 0.010 ppm 0.50 ppm 10 0.025 ppm 0.50 ppm 20 0.050 ppm 0.50 ppm 40 0.100 ppm ILI 5.0ppm 10 0.250 ppm 5.0ppm 20 0.500 ppm 5.0ppm 30 0.750 ppm 50 ppm 4 1.00ppm_ 12.0 PROCEDURIE 12.1 Obtainfrozenliversamples. 12.2 Cut approximatelyI g of liveursinga dissectinsgcalpel.Thispartoftheprocedureisbest performedquickly,not allowingthelivetro thaw. 12.3 Weigh thesample directliyntoa taredplastiscampulevial. 12.4 Record theliverweightinthestudynotebook. 12.5 Return unused liverportionsto freezer. 12.6 Add 2.5mLs of water to sainpulevial. 12.7 Grindthe sample.Put thegrinderprobe inthesaznpleand grindforabout 2 n-dnuteso,r untilthe sample ishomogeneous. 12.8 Rinsetheprobe intothesamplewith2.5mLs waterusinga pipette. 12.9 Take thegrinderapartand cleanitwithmethanolaftereach sample.Referto AMDT-EP- 22. 12.10 Cap the sample and vortexfor15 seconds.Label thesampule vialwiththe studynumber, weight,liverIID,dateand analystinitials. ETS-8-6.0 ExtractioonfPFOS fromLiver Page 7 of 14 12.11 Pipette1.0mL, or otherappropriatveolume,of homogenate intoa 15 niL polypropylene centrifugteube.Labelthecentrifugteubewiththeidenticailnformationas thesampulevial. Referto attachedworksheetfordocumenting the remainingsteps. 12.12 Pipettetwo I mL aliquotosf NElli-QT7w*aAter to centrifugteubes.These willserveas method blanks. 12.13 Spikeallsamples,includingblanksand standardsreadyforextractiownith surrogate standardas describedinsection11.2. 12.14 Spikeeach matirxwiththeappropriateamount of standardas describedin 11.1,or,Table I of thatsectionf,orthecalibratiocnurvestandards.Also preparematrixspikesand continuingcalibratiosntandards. 12.15Vortex mix the standardcurve samples,matrixspikesamples,and continuingcalibration samples for15 seconds. 12.16 Check to ensure0.5M TBA reagentisatpH 10. Ifnot,adjustaccordingly. 12.17 To each sample,add I mL 0.5M TBA and 2 mL ofthe0.25M sodium carbonatelsodium bicarbonatebuffer. 12.18 Using an Oxford Dispenser,add 5 mL methyl-tert-buteytlher. 12.19 Cap each sample and put on the shakerata settingof 300 rpm, for20 minutes. 12.20 Centrifugefor20 to 25 minutesata settinogf 3500 rpm, or untillayersarewellseparated. 12.21Label a fresh15 mL centrifugteubewith thesame informationasin 12.10. 12.22 Remove 4.0mL of theorganiclayerto thefresh15 ffilcentrifugteube. 12.23Put each sample on theanalyticanlitrogenevaporatoruntildry,approximately1to 2 hours. 12.24 Add 1.0mL to each centrifugteubeusinga graduatedpipette. 12.25 Vortex mix for30 seconds. 12.26 Attacha 0.2 @Lm nylonmesh filtetro a 3 cc syringeand transfetrhe swnpleto thissyringe. Filteirntoa 1.5ffilglassautovialor low-volume autovialwhen necessary. 12.27 Labeltheautovialwiththe studynumber, animalnumber and gender,sample timepoint, matrix,finalsolvente,xtractiodnate,and analyst(sp)erformingtheextraction. 12.28 Cap and storeextractastroom temperatureor atapproximately4 'C untilanalysis. 12.29 Complete the extractiownorksheet,attachedto thisdocument, and tapein studynotebook or includeinstudybinder,as appropriate. ETS-8-6.0 ExtractioonfPFOS fromLiver Page 8 of 14 13.0DATA ANALYSIS AND CALCULATIONS 13.1 Calculations: 13.1.1 Calculatetheaveragedensityof theliverhomogenate by recordingeach mass of ten separate1.0mL aliquotsof homogenate. Averagedensity(mg/mL) = Averaizemass (mg) ofthealiquots 1.0mL aliquot 13.1.2 Calculatethe amount of liver(mg) per 1.0mL homogenate (orconcentrationof dispersedsolidtissueper mL of homogenate suspension)usingthefollowing equation: %tofLiver x Averagedensiiy*ofhomoizenate(mm"L (g ofLiver+ g ofWater) referto 13.1.1fordetails. 13.@i.3Calculateactualconcentrationosf PFOS and otherfluorochemicalisncalibration standardsusingthe followingequation: UL ofStandardx Concentratio(nRg Lrd) = FinalConcentration(@tg/ogr mg/kg) mg Liver/I mL homogenate* ofPFOS inLiver *referto 13.1.2fordetails. 14.0 MIETHOD PERFORMANCE 14.1 The method detectionlimit(MDL) isanalyteand matrixspecificR.eferto MDL reportfor specifiMcDL and limitofquantitatio(nLOQ) values(refertoAttachments B and C). 14.2 The followingqualitycontrolsamplesareextractewdith each batchof samplesto evaluate thequalityofthe extractionand analysis. 14.2.1 Method blanksand matrixblanks. 14.2.2Matrix spikeand matrixspikeduplicatesamplesto determineaccuracyand precisionofthe extraction. 14.2.3Continuingcalibratiovnerificatisoanmplesto determinethe continuedaccuracyof theinitiaclalibratiocnurve. 14.3 Referto section14 of ETS-8-7.0 formethod performancecriteria. 15.0 POLLUTION PREVENTION AND WASTE MANAGEMENT 15.1 Sample waste isdisposedinbiohazardcontainersf,lammablesolventwaste isdisposedin highBTU containersa,nd used glasspipettewaste isdisposedinbroken glasscontainers locatedin the laboratory. ETS-8-6.0 Extractionof PFOS firomLiver Page 9 of 14 16.0 REcoRDs 16.1 Complete the extractionworksheet attachedto thismethod, and tape in the study notebook or includein the 3-ringstudy binder,as appropriate. 17.0 TABLES. DiAGRAms, FLOWCHARTS, AND VALIDATION 17.1 Attachment A, Extractionworksheet DATA 17.2 Attachment B, MDL/LOQ values and summary 17.3 Attachment C, Calibrationstandardcalculationand concentrationworksheet 18.0 REFERENCES 18.1 The validationreportassociatedwith thismethod isETS-8-6.0 & 7.0-V-1. 18.2 AMDT-EP-22, "Rou-tine Maintenance ofUtra-Turrax T-25" 18.3 FACT-M- 1.1,"Extractionof PFOS or Other Anionic FluorochernicalSurfactantsfrom Liver forAnalysisUsing EPLC-Electrospray/Mass Spectrometry" 19.0 AFFTCTED DocumzNTs 19.1 ETS-8-7.0, "Analysis of Potassium Perfluorooctanesulfonatoer other Fluorochernicalsin Liver Extractsusing BPLC-Electrospray Mass Spectrometry" 20.0 REvisioNs Revision Number. Reason For Revision Revision Date ETS-8-6.0 ExtractionofPFOS from Liver Page 10 of 14 Study # Matrix Box # Wk/Day Date Spiked/Analyst ccv ms MSD SurrogateStd approx. ppm actual ppm FC Mix Std approx.0.5ppm actual ppm FC NEx Std approx.5 ppm actual ppm FC Mix Std approx. 50 ppm actual PPM Comments Blank LiverHomogenate: Std Liveramount LiverExtractionMethod SRike surrmate and Standard mix. Vortex 15 sec. PiWtis I mL ofLiverSolution PipetteI mL oftO.5M TBA, PH 10. PH Std. Pipette2 mL of 0.25Na2CO3/0.25M NaHCO3 Buffer Std. Dispense 5n-dof Methyl-t-ButyElther TN-A- Shake 20 min. Centrifuae20-25 niin. Shaker Speed CentrifugeSRCW Remove a 4 mL aliguotof organiclue-r Put on NitrogenEyARgLatm todryness Add 1.0mL ofMeffimol EnRmM TN-A- TeMDerature Vortex 30 sec. I Filteursinga 3cc B-D svfinpewitha 0.2um SRI filteirntoautoomle vial Cont. Cal.Verificationsused the same matrix as forthe standardcurve. Attachment A: ExtractionWorksheet ETS-8-6.0 Extractionof PFOS from Liver 9 Date & Wtials Page II of 14 MDL/LOQ Compound PFOS PFOSA PFOSAA ETFOSE-OH M556 PFOSEA valuesforrabbitliver MDL LOQ LinearCalibratioRnange (LCR) (ppb) (ppb) Approximateconcentrationtsobe usedforpreparingthe Standard CalibrationCurve 8.45 26.9 30 ppb - 1200 ppb 3.50 11.1 12 ppb - 1200 ppb 24.6 78.3 30 ppb - 1200 ppb 108 345 60 ppb - 900 ppb* 82.3 262 60 ppb - 1200 ppb 33.9 108 1 30 ppb- 1200 ppb MDLALOQ valuesinrat,bovine,and monkey liverwere not statisticadleltyermined.Two curves ineach of thesematriceswere extractedand analyzedwiththerabbitlivercurvesto determine equivalence.Responses intherat,bovine,and monkey Evercurveswere equivalentothe rabbit responses,thereforet,heirMDL and LOQ willbe assumed to be equivalento thosevaluesas determinedfortherabbitliver. RefertoLOQ Summary and MDL studyinETS-8-6.0& 7.0-V-1forfurtheirnformation * EtFOSE-6H estimateosnlyforMDL andLOQ. Didnotmeetcriterifaorvalidation. Compou Liver matrix d: PFOS Prepared rangeof standards (ppb)(ng/mL) .............. Range of .L..-.C-...R..-'.*.f.m.m.@.@..-...@R..a.n..g.e of ........... Range of average ilav low std ..........*........h.i..ghstd curve ......................c..urve .........................c..u.r.ve (ng/rrtL) ................-.(..p.p.b)(ngtml) @@*@tOo@@i.@.@-.-...--.......;.. (ppb) .. (ng/mL) Rabbit 6.19-1237 12-1200 6-300 60-1200 ..................... ... b.l..i.&....... a'.-.@'.".iV.e.*......'... ................ ......*....,....'@.,@.@@.@*@,.@.@@@@..,.@@:@@l ................................... Compou Liver matrix Rabbit d: PFOSA Prepared rangeof standards (Ppb) (ng/mL) 6.19-1237 Range of average curve (Ppb) (ng/mL) 12-1200 ......................R.a.n..g.e of :::.:.a.v.e.:.4.@..m......e..*..*.,.:*.l:.o,w std curve (Ppb) (ng(niL) .... ...... 12-300 ............... .............. Range of ........i..@.1.4D..............................h..i.ghstd ....... ........ curve .. ......... ............. (I)p(bn)giriil) .................................. .................30.,.0*..@@.".-@'..6.'.0.i-1200 .................. . ............. Compou Liver matrix Rabbit d: PFOSAA Prepared rangeof standards (ppb)(ng/mL) 6.16-1232 Range of average curve ........................R..a.n.g..e.of ............low std ......................c.u.rve ............................................R..a.n.g..e..o.f................... ..W..:.................h.i.g..h.s..t.d... .................. curve ....... (ng/mL) (ppb)(n&/mL) ......... (ppb)(ng/ML) 12-1200 ....3.0.:.':,*: 30-900 .........................N../.A.... ............................. AttachmentB:MDL/LOQ Values ETS-8-6.0 ExtractioonfPFOS fromLiver Page 12 of 14 Compou Liver matrix d:ETFOSE-OH Prepared Range of range of average standards curve (ppb) (ng/mL) (ppb (ng/mL) Rabbit 6.17-1235 31-900 ::,-,ILCRfrom,::R:ange of e.@.uM low std curve (ppb) (ng/mL) . .......................... ................... .....3..1......@.@.. 0:.@.:.:...:.. N/A :LCR ftd ..... kd: Range of high std curve kopb.@...(...f..i..W.I .i..i..i..L..)..;..-..@pp@T (nginil) ...................................... ............. N/A high d@d ........ ....... WA, ..... Compou Liver matrix Rabbit d: PFOSEA Prepared range of standards (ng/mL) 6.17-1235 Range of average curve (ppb) (ng/mL) 31-120 0 :L L.C.R......f..r.............R.a.n..ge of ..........................R..a.n.ge of @LCR f..i...O.m....... ....................1......@...........I..................................................................l..c..o....u.w..r...s.v....te....d..............:.:...1..0..'..W..".:................................h.ic..gu.hr.v.s.et.d.... ............'..9.L....@... . . ................................ (ppb) (ng/mL) (ppb) (ng/mL) ............. ......... .................................... N/A . ................... ........... N/A ......... ............ ................................ Compou Liver matrix Rabbit d: M556 Prepared range of standards (Ppb) (ng/mL) 6.17-1235 Range of average curve (Ppb) (ng/tnL) 31-1200 ........................................................R...a...n..g.e..o..f............................R.a..n,9,eof ............................... low std ...............h..i.g.h..s.t.d ....................................... curve ......... ........ curve .........................(.P.p..b.)....(.n.gt.m.l.) ............(.Ppb) (ng/mL) .................. ...........................................................N../.A....... ..................................................N../..A .................... .................................................... ....... ......................-----........ Attachment B: MDL/LOQ Values ETS-8-6.0 ExtractioonfPFOS fromLiver Page 13 of 14 Ion PairStandard Curves - Tissue Prep date(s): Analyte(s): Sample matrix: Methodirevision: Targetanalyte(s): FC mix stdapprox.0.500ppm: FC mix stdapprox.5.00ppm: FC mix stdapprox.50.0ppmSurrogatestdapprox. 100 ppm- Standard number: Equipment number: Finalsolventand TN: Blank liverlidentifier: Actual concentrationosfstaiidardisnthe FC mix PFOS PFOSA PFOSAA ETFOSE PFOSEA Stdconc Std conc Std conc Std conc Std conc ug/mL ug/mL ug/mL ug/mL ug/mL 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 -0.500 0.500 0.500 0.500 0.500 0.500 0.500 1 0.500 0.500 0.500 o.500 0.500 0.500 0.500 5.00 5.00 5.00 5.00 5.00 5.00 5.00 5.00 5.00 5.00 5.00 5.00 5.00' 5.00 50.0 50.0 50.0 50.0 50.0 M556 Std conc ug/mL 0.500 0.500 0.500 0.500 0.500 5.00 5.00 5.00 50.0 Stdconc ug/mL AR All ArWt spiked Density ML 9 0.002 0.167 0.004 0.167 0.010 0.167 0.020 0,167 0.040 0.167 0.010 0.167 0.020 1 0.167 @i@030@ --d1.67 0.004 0.167 Calculated PFOS Final conc ng/g 5.99 12.0 29.9 59.9 120 299 599 898 1198- concentrations of stand ards PFOSA PFOSAA ETFOSE Final Finalconc Final conc nglg conc ngtg ng/g 5.99 5.99 5.99 12.0 12.0 12@O 29.9 29.9 29.9 59.9 59.9 59.9 120 120 120 299 299 299 599 599 599 998 898 998 1198 1198 1198 in the sa PFOSFA Final conc ng/g 5.99 12.0 29.9 59.9 120 299 599 898 1198 ple matrix M55 Final conc ng/g5.99 12.0 29.9 59.9 120 299 599 898 1198 Stdconc ng/9 Surrogate Std conc ng/mL 100 Surrogate Finalronc ng/mL 0.500 All Am't spiked ML 0.005 Validatedran2es- approximateconcentrations Liver PFOS PFOSA PFOSAA Rabbit 5-1000ppb 5-1000ppb 5-1000ppb Bovine Estiimteosnly,userabbitvalues. Rat Estimatesonly,userabbitvalues. Monkey Estimatesonly,userabbitvalues. I ETFOSI-OR I _5-1000ppb POAA 5-1000ppb PFOSEA 5-1000ppb AttachmentC: StandardCalculations ETS-8-6.0 ExtractioonfPFOS from Liver Page 14 of 14 3M ENVIRONMENTAL LABORATORY METHOD ANALYSIS OF POTASSIUM PERFLUOROOCTANESULFONATE OR FLUOROCHEMICALS IN SERUM EXTRACTS USING BPLC-ELECTROSPRAY/MASS SPECTROMETRY OTHER Method Number: ETS-8-5.1 Author:,LisaClemen,Robert Wyme 7 Approved By: Adoption Date: 03/01/99 RevisionDate: LaboratoryManager Group Leader TechnicalReviewer Date Date Date 1.0 SCOPE AND APPLICATION 1.1 Scope: This method describesthe analysisof serum extractsforfluorochemicalsurfactants usingBPLC-electrospray/mass spectrometry. 1.2 Applicable Compounds: Fluorochemicalsurfactantosr otherfluorinatecdompounds, or otherionizablecompounds. 1.3Matrices: Rabbit,rat,bovine,monkey, and human serum, or otherfluidsas designatedinthe validatiorneport. Word 6/95 ETS-8-5.1 AnalysisofSenun ExtractUsingES/MS Page I of9 2.0 SUMMARY OF METHOD 2.1 This method describesthe analysisof fluorochemicalsurfactantesxtractedfrom serum or otherfluidsu,sing HPLC-electrospray/mass spectrometry,or similarsystem asappropriate. The analysisisperformed by monitoringa singleion characteristoifc a particular fluorochernicaslu,ch as the perfluorooctanesulfonat(ePFOS) anion,ni/z-4-99. Additionallys,amples may be analyzedusing a tandem mass spectrometerto furtherverify the identityof a compound by detectingdaughterionsofthe parention. 3.0 DEFMTIONS 3.1 Atmospheric Pressure Ionization(API): The NEcromass Quattro 11triplqeuadrupole systemsallowfor variousmethods of ionizationby utilizinvgarioussources,probes,and interfaces.These includebut arenot limitedto:ElectrosprayIonizatio(nE.SI),Atmospheric PressurechemicalIonization(APcl),Thermospray, etc.The ionizatiopnrocessinthese techniquesoccurs atatmosphericpressure(i.e.n,ot under a vacuum). 3.2 Electrospray Ionization(ES, ESI): a method of ionizatiopnerformed atatmospheric pressure,whereby ionsin solutionare transferredtothe gas phase viatinycharged droplets. These charged dropletsare produced by the applicatioonf a strongelectricaflield. 3.3 Mass Spectrometry, Mass Spectrometer (MS), Tandem Mass Spectrometer (MS/MS): The API Quattro IItriplqeuadrupole systemsare equipped with quadrupolemass selectivedetectors.Ions are selectiveldyiscriminatebdy mass to charge ratio(nVz)and subsequentlydetected.A singleMS may be employed foriondetectionor a series(MS/MS) formore specififcragmentationinformation. 3.4 Conventional vs.Z-spray probe interface:The latesmtodels of NficromassQuattro11 triplequadrupolesystems(post1998) utilizae"Z-spray"conformation.The sprayemitted from a probe isorthogonalto the cone aperture.In the conventionalconformationitisaimed directlaytthe cone aperture,afterpassingttitougha tortuouspathway inthe counter electrode.Though the configuratioinsdfferent,themethods of operationc,leaning,and maintenance arethe same. However, Z-spray components and conventionalcomponents are not compatiblewith one another,but only with similarsystems(i.e.Z,-spraycomponents are compatiblewith some otherZ-spraysystems,etc.) 3.5 Mass Lynx Software: System softwaredesignedforthe specifiocperationof these Quattro 11triplequadrupole systems. CurrentlyMassLynx has Windows 95 and WindowsNT 4.0 versions.Allversionsare similar.For more detailsee the manual specifitco the instrument(NEcromass Quattro IItriplqeuadrupole MassLynx or MassLynx NT User's Guide). 4.0 WARNINGS AND CAUTIONS 4.1 Health and Safety Warnings: 4.1.1 Use cautionwith the voltagecablesforthe probe. When engaged, the probe employs a voltageof approximately5000 Volts. ETS-8-5.1 AnalysisofSenan ExtractUsingES/MS Page 2 of 9 4.1.2 When handlingsamplesor solventswear appropriatperotectivgeloves,eyewear, and clothing. 4.2 Cautions: 4.2.1 Do notoperatesolventpumps above capacityof400 bar(5800 psi)back pressure. Iftheback pressureexceeds400 bar,theBPI 100 willinitiataeutomaticshutdown. 4.2.2 Do not run solventpumps to dryness. 5.0 INTERFERENCES 5.1 To minimizeinterferencwehsen analyzingsamples,teflonshouldnot be used forsample storageor any partof instrumentatiotnhatcomes incontactwiththesample or extract. 6.0 EOUEPMENT 6.1 Equipment listebdelow maybe modifiedinordertooptimizethesystem.Documentany modificationisntheraw dataas method deviations. 6.1.1 Mcromass QuattroR triplqeuadrupoleMass Spectrometerequippedwithan electrospraiyonizatiosnource 6.1.2 BPI 100 low pulsesolventp,u.mping system,solventdegasser,column compartment, and autosampler 7.0 SUPPLIES AND MATERIALS 7.1 Supplies 7.1.1 High puritygradenitrogengasregulatedto approximately100 psi(House air system) 7.1.2 BPLC analyticaclolumn, specifictso be determinedby theanalystand documented intheraw data. 7.1.3 Capped autovialosr capped 15 ffilcentrifugteubes 8.0 REAGENTS AND STANDMWS 8.1 Reagents 8.1.1 Methanol,BPLC gradeor equivalent 8.1.2 Nfilli-Qlwmater,allwaterused inthismethod shouldbe MIR-QTM water or equivalenta,nd may be providedby a Nffi-Q TOC Plussystemor othervendor 8.1.3 Ammonium acetater,eagentgradeor equivalent 8.2 Standards 8.2.1 Typicalltywo method blanks,two matrixblanks,and eighteenmatrixstandardsare preparedduringtheextractiopnrocedure.See ETS-8-4.1. 9.0 SAWLE HANDLING ETS-8-5.1 AnalysisofSenun ExtractUsingES/MS Page 3 of9 9.1 Fresh matrixstandardsarepreparedwith each analysisE.xtractedstandardsand samples arestoredincapped autovialosr capped 15 mL centrifugteubesuntilanalysis. 9.2 Ifanalysiswillbe delayed,extractedstandardsand samplescan be refrigerateadt approximately4' C, or atroom temperature,untilanalysicsan be performed. 10.0 ()UALITY CONTROL 10.1 Solvent Blanks, Method Blanks and Matrix Blanks 10.1.1 Solventblanks,method blanksand matrixblanksareprepared and analyzed with eachbatch to determinecontaminationor carryover. 10.1.2 Analyze a method blankand a matrixblankpriorto each calibratiocnurve. 10.2 Matrix Spikes 10.2.1 Matrix spikesarepreparedand analyzedto determinethematrixeffecton the recoveryefficiency. 10.2.2 Matrix spikeduplicateasrepreparedand analyzedto measure theprecisionand the recoveryforeach analyte. 10.2.3 Analyze a matrixspikeand matrixspikeduplicatpeerfortysamples,with a minimum of 2 spikesperbatch. 10.2.4 Matrix spikeand matrixspikeduplicatceoncentrationwsillfalilnthemid-rangeof theinitiaclalibratiocnurve.Additionalspikeconcentrationmsay faUinthelowrangeof the initicaallibratiocnurve. 10.3 Continuing CalibrationVerifications 10.3.1 Continuingcalibratiovnerificatioanrseanalyzedtoverifythecontinuedaccuracyof thecalibratiocnurve. 10.3.2 Analyze a mid-rangecalibratiosntandardaftereverytenthsample,with a minimum of one per batch. 11.0 CALIBRATION AND STANDARDIZATION 11.1 Analyzethe extractedmatrixstandardspriorto and followingeach setof extractsT.he averageof two standardcurveswdl be plottedby linearegressio(ny= my + b),weighted I/x,not forcedthrough zero,usingMassLynx or othersuitablseoftware. 11.2 Ifthe curve does notmeet requirementsp,erformroutinemaintenanceor reextracthe standardcurve (ifnecessary)and reanalyze. 11.3 For purposesof accuracywhen quantitatinlgow levelsof analytei,tmay be necessaryto use the low end of thecalibratiocnurveratherthanthefullrangeofthe standardcurve. Example: when attemptingto quantitataepproximately10 ppb of analyteg,eneratea calibratiocnurveconsistinogfthe standardsfrom 5 ppb to 100 ppb ratherthantheflill rangeofthecurve (5ppb to 1000 ppb). Thiswillreduceinaccuracyattributetdolinear regressionweightingof highconcentratiosntandards. ETS-8-5.1 Analysisof Senim ExtractUsing ES/MS Page 4 of 9 12.0PROCEDURES 12.1AcquisitiSoentup 12.1.C1lickon startbuttonintheAcquisitioCnontrolPanel.Setup a samplelist.Assigna filenameusingMO-DAY-last digitofyear-samplenumber, assigna method (MS) foracquiringa,nd typeinsample descriptions. 12.1.2 To createa method clickon scanbuttonintheAcquisitioncontrolpaneland select SIR (SingleIon Recording)orN4RM. SetIonizatioMnode asappropriataehd mass to 499 or otherappropriatmeasses. A fullscanisusuallycollecteadlongwiththe SIRS. Save acquisitiomnethod. IfMS/MS instrumentasreemployed,additional productionfragmentationinformatiomnay be collectedS.ee NEcromass MassLynx GUIDE TO DATA ACQUISITION for.additionianlformatioannd NIRM (Multiple ReactionMofiitoring). 12.1.3Typicalltyheanalyticbaaltchrun sequencebeginswitha setofextractemdatrix standardsand ends with a setof extractedmatrixstandards. 1.2.1.4Samples are analyzedwith a continuingcalibratiocnheck injectedaftereverytenth sample. Solventblanksshouldbe analyzedperiodicaltloy monitorpossibleanalyte carryoverand arenot consideredsamplesbutmay be includedas such. 12.2 Using the Autosampler 12.2.1 Setup sample trayaccordingto the samplelisptreparedinSection12.1.1. 12.2.2 Set-uptheBPI 100/autosampleratthefollowingconditionsor atconditionsthe analystconsidersappropriateforoptimalresponse.Record actualconditionsinthe instrumentlogbook: 12.2.2.1 Sample size= 10 pL injection 12.2.2.2Inject/swnpl=e 1 12.2.2.3Cycletime= 13.5minutes 12.2.2.4 Solventramp = Time 0.00 min. 8.50 rnin. 11.0 min. 12.0 n. MEOH 40% 90% 90% 40% 2.0 mM Ammonium acetate 60% 100/0 10% 60% 12.2.2.5 Pressthe"Start"button. 12.3 Instrument Set-up 12.3.1 Referto ETS-9-24.0 formore details. 12.3.2Check the solventlevelinreservoirasnd refililfnecessary. ETS-8-5.1 Analysisof Senun ExtractUsing ES/MS Page 5 of 9 12.3.C3heckthestainlsetseseclapillatrtyheendoftheprobe.Useaneyepiecteocheck thetip.The tipshouldbe flatwithno jaggededges. Ifthetipisfound tobe unsatisfactordyi,sassembletheprobe and replacethestainlesssteelcapillary. 12.3.4 Set HPLC pump to "On". Settheflowto 10 -500 uL/min or asappropriate. Observe dropletscoming out ofthetipoftheprobe. Allow to equilibratfeor approximately10 minutes. 12.3.5 Turn on thenitrogen.A finemistshouldbe expelledwithno nitrogenleaking aroundthetipof theprobe.Readjustthetipoftheprobe ifno mistisobserved. 12.3.6 The instrumentusestheseparametersatthefollowingsettingsT.hese settingmsay change inorderto optimizethe response: 12.3.6.1Drying gas 250-400 liters/hour 12.3.6.2ESI nebulizinggas 10-15 liters/hour 12.3.6.3BPLC constantflow mode, flow rate10 - 500 gL/min 12.3.6.4Pressure<400 bar (Thisparameterisnot set,itisa guide to ensurethe IHPLC isoperatingcorrectly.) 12.3.7Carefullgyuidetheprobe intotheopening.Insertprobe untilitwillnot go any furtherC.onnect thevoltagecablesto theprobe. 12.3.8 Printthetune page,with itsparametersa,nd storeitinthestudybinderwith a copy tapedintotheinstnunentlog. 12.3.9Using thecross-flowcounterelectrodeintheES/MS sourceisrecommended forthe analysiosf biologicamlatrices. 12.3.1OClickon startbuttonintheAcquisitionControlPanel (thismay vary among MassLynx versionss,eeappropriatMeassLynx USER'S GUIDE). Pressthe start button.Ensure startand end samplenumber includesallsamplesto be analyzed. 13.0 DATA ANALYSIS AM 13.1 Calculations: CALCULATIONS 13.1.4 Calculatematrixspikepercentrecoveriesusingthefollowingequation: % Recovery ObservedResult-Background Result x 100 Expected Result 13.1.5 Calculatepercentdifferencuesingthefollowingequation: % Difference Enected Conc.-CalculateCdonc. x 100 Expected Conc. 13.1.6CalculateactualconcentratioonfPFOS, or otherfluorochemicali,nmatrix(@Lg/mL): (ngofPFOS calcf.rom std.Curvex DilutioFnactorj x I gp, (InitiVaollume ofmatrix(mL) + mL ofSurroizatSetandard) 1000 ng FinalVolume (mL) ETS-8-5.1 AnalysisofSerum ExtractUsingES/MS Page 6 of9 14.0METHOD PERFORMANCE 14.1 Method DetectionLimit(MDL) and LimitofQuantitatio(nLOQ) aremethod,analytea,nd matrixspecificP.leaseseeETS-8-4.1,Attachment B, fora listinogfcurrentvalidated MDL and LOQ values. 14.2 SolventBlanks,Method Blanks,and Matrix Blanks 14.2.1Solventblanksm,ethod blanks,and matrixblanksvaluesaremust be belowthe loweststandardinthe calibratiocnurve 14.3 CalibrationCurves 14.3.1 The r2valueforthecalibraticounrvemust be 0.980orbetter. 14.4 Matrix Spikes 14.4.1 Matrixspikepercentrecoverieasremust be within 30% ofthespiked concentration. 14.5 Continuing Calibration Verirications 14.5.1 Continuingcalibratiovnerificatiopnercentrecoveriesmust be 30% of the spiked concentration. 14.6 Ifcriterilaisteidnthismethod performancesectionisn'mtet, maintenancemay be performedon the system and samplesreanalyzedor otheractionsas determinedby the analyst.Document allactionsintheappropriatleogbook. 14.7 Ifdataareto be reportedwhen performancecriterihaave notbeen met,the datariiusbte footnotedon tablesand discussedinthetextof thereport. 15.0 POLLUTION PREVENTION AND WASTIE MANAGEMEENT 15.1 Sample extractwaste and flammablesolventisdisposedinhighBTU containersa,nd glass pipettewaste isdisposedinbroken glasscontainerslocatedinthelaboratory. 16.0 REcoRDs 16.1 Each page generatedfora studymust have thefollowinginformatioinncludedeitherinthe headeror hand writtenon thepage: studyor projectnumber,acquisitiomnethod, integratiomnethod,sample name, extractiodnate,dilutiofnactor(ifapplicablea)n,d analyst. 16.2 Printthetunepage,samplelista,nd acquisitiomnethod from MassLynx to includeinthe appropriatestudyfolder.Copy thesepages and tapeintotheinstrumentrunlog. 16.3 Plotthe calibratiocnurveby linearegressionw,eighted I/x,thenprintthesegraphsand storeinthestudyfolder. 16.4 Printdataintegratiosnummary, integratiomnethod,and chromatograms,from MassLynx, and storeinthe studyfolder. 16.5 Summarize datausingsuitablseoftware(Excel5.0)and storeinthestudyfolder,see Attachment A foran example of a summary spreadsheet. ETS-8-5.1 AnalysisofSerum ExtractUsingES/MS Page 7 of9 16.6 Back up electronidcatato appropriatmeediuni.Record instudynotebookthefilename and locationofbackup electronidcata. 17.0 TABLES, DiAGRAms, FLOWCHARTS, AND VALIDATION DATA 17.1 AttachmentA: ETS-8-5.1 Data summary spreadsheet. 18.0 REFERENCES 18.1 FACT-M-4. 1,"ExtractionofPotassiumPerfluorooctanesulfonaotreOtherFluorochen@cal compounds from Serum forAnalysisUsing BPLC-Electrospray/MassSpectrometry 18.2 ETS-9-24.0,"Operationand Maintenance of theNEcromass AtmosphericPressure Ionization/MasSspectrometerQuattroH triplqeuadrupoleSystems" 18.3 The validatiorneport-associatewdith thismethod isETS-8-4.0 & 5.0-V-1. 19.0 AFFECTED DocumiENTs 19.1 ETS-8-4.1,"Extractionof PotassiumPerfluorooctanesulfonaotreOtherFluorochemical Cqmpounds from Serum forAnalysisUsing BPLC-Electrospray/MassSpectrometry" 20.0 REvisioNs - Revision Number. I Reason For Revision Section6.1.2 ClarificatioofnBPI 100 systemcomponents. Section11.1 Average oftwo curves,not standardvalues,areused for plottinlginearegressionand added the 1/xweightingof the curve. Section12.2.2.4Clarificatiofn solventramp. Section17.1Changed from attachment8 to A. Revision Date 04/02/99 ETS-8-5.1 AnalysisofSenun ExtractUsingES/MS Page 8 of9 Laboratory Study Study: TestMaterial: Matrix/FinalSolvent: Method/Revision: AnalyticalEquipment System Number: InstrumentSoftwareNersion: Filename: R-Squared Value: Slope: Y Intercept: Date of Extraction/Analyst: Date of AnalysistAnalyst: Group Dose Sample# Concentration UZLML InitiaVloL mL Dilution FinalCone. ugtml Slope: Taken from linearregressioenquation. Group/Dose: Taken from thestudyfolder. Sample#: Taken from the studyfolder. Concentration (ugtn3iL)T:aken from theMassLynx integratiosnummary. InitiaVlolume (mL): Taken from thestudyfolder. DilutionFactor: Taken from thestudyfolder. Final Conc. (ugImL): Calculatedby dividingtheinitiavlolume from theconcentration Attachment A: Summary Spreadsheet ETS-8-5.1 Anal;sisof Senim ExtractUsing ES/MS Page 9 of 9 3M ENVIRONWNTAL LABORATORY METHOD ANALYSIS OF POTASSIUM PERFLUOROOCTANESULFONATE OR OTHER FLUOROCHEMICALS IN LIVER EXTRACTS USING UPLC-ELECTROSPRAY/MASS SPECTROMETRY Method Number: ETS-8-7.0 Author: Lisa Clemen, Glenn Langenburg ,W. Approved By: Adoption Date: RevisionDate: LaboratoryManager Date Group Leader Date TechnicalReviewer Date 1.0 SCOPE AND APPLICATION 1.1 Scope: This method isforthe analysisofliverextractsforfluorochemicalsurfactantussing BPLC-electrospray/mass spectrometry. 1.2Applicable Compounds: Fluorochemicalsurfactantosr otherfluorinatecdompounds, or otherionizablecompounds. 1.3 Matrices: Rabbit,rat,bovine,monkey livero,r othertissuesas designatedinthevalidation report. Word 6/95 ETS-8-7.0 Anal;sisofLiverExtractUsing ES/MS Page I of 10 2.0 SUMMARY OF METHOD 2.1 Thismethod describestheanalysiosf fluorochemicaslurfactantesxtractedfrom liverusing HPLC-electrospray/massspectrometryo,r similarsystemas appropriate.The analysisis performedby monitoringa singleioncharacteristoifca particulafrluorochemicals,uch as the perfluorooctanesulfon(aPtFeOS) anion,m/z = 499. Additionallys,amplesmay be analyzedusinga tandem mass spectrometerto furtherverifytheidentitoyf a compound by detectingdaughterionsof theselectedparention. 3.0 DEFLNITIONS 3.1 Atmospheric PressureIonization(API): The Mcromass QuattroIItriplequadrupole systemsallowforvariousmethods of ionizatiobny utilizinvgarioussources,probes,and interfacesT.hese includebut arenotlimitedto:ElectrospraIyonizatio(nESI),Atmospheric PressurechemicalIonizatio(nAPcl),Thermospray,etc.The ionizatiopnrocessinthese techniquesoccursatatmosphericpressure(i.en.ot undera vacuum). 3.2 ElectrosprayIonization(ES,ESI): a method of ionizatiopnerformedat atmospheric pressurew,hereby ionsinsolutionaretransferretdo thegas phase viatinycharged droplets. These.chargeddropletsareproduced by theapplicatioonf a strongelectricfaileld. 3.3 Mass Spectrometry,Mass Spectrometer (MS), Tandem Mass Spectrometer (MS/MS): The API QuattroIItriplqeuadrupolemass spectrometerisequippedwith two quadrupolemass selectivdeetectorsand a collisiocnell.Ionsareselectiveldyiscriminatebdy mass to chargeratio(m/z)and subsequentldyetected.A singleMS may be employed forion detectionor an ion may be selectedinthefirsqtuadrupole,fragmentedinthecollisiocnell, and thesefragmentsmay be analyzedinthesecond quadrupole. 3.4 Conventional vs.Z-spray probe interface:The latesmtodels ofNficromassQuattroI[[ triplqeuadrupole(post1998)utilizae"Z-spray"conformation.The sprayemittedfrom a probe isorthogonalto thecone aperture.In theconventionaclonformationitisaimed directlaytthecone aperturea,fterpassingthrougha tortuouspathway inthe counter electrode.Though theconfiguratioinsdifferenth,emethods of operationc,leaninga,nd maintenancearethesame. However, Z-spraycomponents and conventionalcomponents are not compatiblewith one another,but onlywith similarsystems(i.eZ.-spraycomponents are compatiblewithotherZ-spraysystems,etc.) 3.5 Mass Lynx Software: System softwaredesignedforthe specifiocperationofthese QuattroH triplqeuadrupolesystems.CurrentlyMassLynx hasWindows 95 and WindowsNT 4.0versions.AllversionsaresimilarF.or more detailrseferto themanual specifitco the instrument(Mcromass QuattroIItriplqeuadrupoleMassLynx orMass]LynxNT User's Guide). 4.0 WARNIINGS AM CAUTIONS 4.1 Health and SafetyWarnings: 4.1.1 Use cautionwiththevoltagecablesfortheprobe.When engaged,theprobe employs a voltageof approximately5000 Volts. ETS-8-7.0 AnalysisofLiverExtractUsing ES/MS Page 2 of 10 4.1.2 When handlingsamplesor solventswear appropriatperotectivgeloves,eyewear, and clothing. 4.2 Cautions: 4.2.1 Operatethe solventpumps below a back pressureof400 bar (5800 psi).Iftheback pressureexceeds400 bar,theBPI 100 willinitiataeutomaticshutdown. 4.2.2 Do not run solventpumps todryness. 5.0 L'QTERFERFNCIES 5.1 To minimizeinterferencwehsen analyzingsamples,Teflonshallnotbe used forsample storageor any partof instrumentatiotnhatcomes incontactwith thesampleor extract. 6.0 EouipmmNT 6.1 Equipment listebdelow may be modifiedinorderto optimizethe system.Document any modificationisnthe raw dataas method deviations. NficromassQuattroIItriplqeuadrupoleMass Spectrometerequippedwith an electrospraiyonizatiosnource. 6.1.2 BPI 100 low pulsesolventpumping system,solventdegasser,column compartment, and autosampler 7.0 SUPPLIES AND MATERL4,LS 7.1 Supplies 7.1.1 legh puritygrade airregulatedto approximately100 psi(house airsystem) 7.1.2 BPLC analyticaclolumn,specifictso be determinedby theanalystand documented intheraw data 7.1.3 Capped autovialosr capped 15 ml centrifugteubes 8.0 REAGENTS AND STANDARDS 8.1 Reagents 8.1.1 Methanol,BPLC gradeor equivalent 8.1.2 water(ASTM typeI),allwaterusedinthismethod shouldbe ATSM type I,or equivalenta,nd be providedby a Milli-QTOC Plussystemor othervendor 8.1.3 Ammonium acetater,eagentgrade or equivalent 8.1.3.1 When preparingdifferenatmounts thanthoselisteda,djustaccordingly. 8.1.3.2 2.0mM ammonium acetatesolutionW:eigh approximately0.300 g ammonium acetateP.our intoa 2000 mL volumetriccontainercontaining 2000 mL Mili-Q"m water,mix untilallsolidsaredissolved.Storeatroom temperature. ETS-8-7.0 Anal;siosfLiverExtractUsingES/MS Page 3 of 10 8.2 Standards 8.2.1Typicaltlwyomethodblankst,womatribxlanksa,ndeighteemnatrisxtandaradrse preparedduringtheextractiopnrocedure,Referto ETS-8-6.0. 9.0 SAMEPLE HANDLING 9.1 Freshmatrixstandardsarepreparedwith each analysisE.xtractedstandardsand samples arestoredincapped autovialsor capped 15 ml centrifugteubesuntilanalysis. 9.2 Ifanalysiswillbe delayed,extractedstandardsand samplesmay be storedatroom temperature,or refiigerateadt approximately4' C, untilanalysiscan be performed. 10.0 OUALITY CONTROL 10.1 Method Blanks and-Matrix Blanks 10.1.1 Solventblanks,method blanks,and matrixblanksare prepared and analyzedwith each batch to determinecontaminationor carryover. 10.1.2Analyzea method blankand a matrixblankpriorto each calibratiocnurve. 10.2 Matrix Spikes 10.2.1 Matrix spikesarepreparedand analyzedto determinethematrixeffecton the recoveryefficiency., 10.2.2 Matrix spikeduplicatesarepreparedand analyzedto measure theprecisionand the recoveryforeach analyte. 10.2.3 Analyzea matrixspikeand matrixspikeduplicateperfortysamples.With a minimum of 2 spikesper batch. 10.2.4Matrixspikeand matrixspikeduplicatceoncentrationwsillfallintheniid-rangoef theinitiaclalibratiocnurve. Additionaslpikeconcentrationmsay fallinthelowrangeof the initiaclalibratiocnurve. 10.3 Continuing CalibrationChecks 10.3.1Continuingcalibratiovnerificatioanrseanalyzedto verifythecontinuedaccuracyof thecalibratiocnurve. 10.3.2Analyzea tnid-rangcealibratiosntandardeverytenthsample,with a minimum of one per batch. 11.0 CALIBRATION AND STANDARDIZATION 11.1 Analyzetheextractedmatrixstandardspriorto and followingeach setof sampleextracts. The averageof two standardcurveswillbe plottedby linearegressio(ny = nLx+ b), weighted I/x,not forcedthroughthe originu,singMassLynx or othersuitablseoftware. 11.2 Ifthe curvedoes not meet requirementsperformroutinemaintenanceor reextractthe standardcurve(ifnecessary)and reanalyze. ETS-8-7.0 Ana@sis ofLiver ExtractUsing ES/MS Page4 of 10 11.3 For purposesof accuracywhen quantitatinlgow levelsof analyte,itmay be necessaryto use thelow end of thecalibratiocnurveratherthanthefullrangeof thestandardcurve. Example: when attemptingto quantitataepproximately10 ppb of analyteg,eneratea calibratiocnurveconsistinogf thestandardsfrom 5 ppb to 100 ppb ratherthanthefull rangeofthecurve(5ppb to 1000 ppb). Thiswillreduceinaccuracyattributetdolinear regressionweightingof highconcentrationstandards. 12.0 PROCEDURES 12.1 AcquisitionSetup 12.1.1 Setup the samplelist. 12.1.1.1Assigna samplelistfilenameusingMO-DAY-last digitof year-increasing letteorf the alphabetstartinwgith a 12.1.1.2Assigna method (MS filef)oracquiring 12.1.1.3Assignan BPLC program (Inleftile) 12.1.1.4Type insample descriptionasnd vialpositionnumbers 12.1.2 To createa method clickon method intheAcquisitioncontrolpanelthenmass spectrometerheadingsand selectSIR (SingleIonRecording)orNIRM (Multiple ReactionMonitoring).Set IonizatioMnode asappropriataend mass to 499 or otherappropriatemasses.'A. fullscanisusuallycollecteadlongwith the SIRS. Save acquisitiomnethod. IfMS/MS instrumentasreemployed,additionaplroduction fragmentationinformatiomnay be collectedR.eferto NEcromass Mass]Lynx GUIDE TO DATA ACQUISITION foradditionailnformatioannd MRM. 12.1.3 Typicallythe analyticablatchrun sequencebeginsand ends witha setof extracted matrixstandards. 12.1.4 Samples areanalyzedwitha continuingcalibratiovnerificatiionjectedstandard aftereverytenthsample. Solventblanksshouldbe analyzedperiodicalltyo monitor possibleanalytecarryoverand arenot consideredsamplesbut may be includedas such. 12.2 Using the Autosampler 12.2.1 Set up swnple tray according to the sample Estprepared in Section 12.1.1. 12.2.2 Set-up the BPI I00/autosampler at the followingconditionsor at conditionsthe analystconsidersappropriateforoptimalresponse.Record actualconditionsinthe instrumentlogbook: 12.2.2.1Sample size= 10 pL injection 12.2.2.2Inject/sampl=e 1 12.2.2.3Cycle time= 9 minutes ETS-8-7.0 Anal;siosfLiverExtracUtsingES/MS Page5 of10 12.2.2.4Solventramp conditions Time MEOH 0.00 min. 40% 1.0 min. 40% 4.5 min. 95% 6.5 min. 95% 7.0 min. 40% 9.0 rni. 40% 2.0 mM Ammonium acetate 60% 60% 5% 5% 60% 60% 12.2.2.5Pressthe "Start"button. 12.3 Instrument Set-up 12.3.1 Refer to ETS-9-24.0, "Operafionand Maintenance ofthe Mcromass Quattro]I TripleQuadrupole Mass SpectrometerFittedwith an Atmospheric Pressure IonizationSource,"formore details. 1.2.3.2Check the solventlevelinreservoirasnd refililfnecessary. 12.3.3 Check the stainlesssteelcapillaraytthe end of the probe. Use an eyepieceto check thetip.The tipshouldbe flatwith no jagged edges.Ifthetipisfound to be unsatisfactoryd,isa@semblethe probe and replacethe stainlesssteelcapillary. 12.3.4 Turn on the nitrogen. 12.3.5 Open the tune page. Clickson operateto initiatseource block and desolvation heaters. 12.3.6 Open the InletEditor. 12.3.6.1Set BPLC pump to "Ot@' 12.3.6.2Set the flow to 10 - 500 uL/min or asappropriate 12.3.6.3Observe dropletscoming out of thetipofthe probe. A finemistshouldbe expelledwith no nitrogenleakingaround thefipofthe probe. Readjustthe tipof the probe ifno mistisobserved 12.3.6.4Allow to equilibratfeorapproximately10 minutes. 12.3.7 The instrumentuses theseparametersatthe followingsettings.These settingsmay change inorder to optimizethe response: 12.3.7.1Drying gas 250-400 liters/hour 12.3.7.2ESI nebulizinggas 10-15 liters/hour 12.3.7.3BPLC constantflow mode flow rate10 - 500 liL/min 12.3.7.4Pressure<400 bar (Thisparameterisnot set,itisa guide to ensurethe BPLC isoperatingcorrectly.) 12.3.7.5Source blocktemperature150* 12.3.7.6Desolvationtemperature250' ETS-8-7.0 AnalisisofLiverExtractUsing ES/MS Page 6 of 10 12.3.8Printthetune page,withitsparameters,and storeitinthestudybinderwitha copy tapedintotheinstrumentlog. 12.3.9Clickon starbtuttonintheAcquisitioCnontrolPanel(thismay vaiyamong MassLynx versionsr,eferto appropriatMeassLynx User'sGuide).Ensure startand end sample number includesallsamplesto be analyzed. ,13.0DATA ANALYSIS AND CALCULATIONS 13.1 Calculations: 13.1.4 Calculatematrixspikepercentrecoveriesusingthefollowingequation: % Recovery Observed Result-Background Resultx 100 Expected Result 13.1.5 Calculatepercentdifferencuesingthefollowingequation: % Difference Expected Conc. - CalculateCdonc. x 100 Expected Conc. 13.1.6 Calculateactualconcentrationisnmatrix(gg/g): (ngofPFOS calcf.romstd.Curvex DilutioFnactorj x I gg (InitiWaelightofLiver(g)- 1000 ng FinalVolume (niL) 14.0 MizTHoD PiERFoRmANcF, 14.1 Method DetectionLimit(MDL) andLimitofQuantitatio(nLOQ) aremethod,analytea,nd matrixspecificR.efertoETS-8-6.0,Attachment B fora listinogfcurrenvtalidateMdDL and LOQ values. 14.2 SolventBlanks, Method Blanks and Matrix Blanks 14.2.1 Solventblanks,mdthod blanks,and matrixblanksmust be below theloweststandard inthecalibratiocnurve. 14.3 CalibrationCurves 14.3.1 The r2 valueforthecalibratiomnust be 0.980 or better. 14.4 Matrix Spikes 14.4.1Matrix spikepercentrecoveriesmust be within 30% ofthespikedconcentration. 14.5 Continuing CalibrationVerification 14.5.1 Continuingcalibratiovnerificatiopnercentrecoveriesmust be within 30% of the spikedconcentration. 14.6 Ifcriterilaisteidnthemethod performancesectionarenot met,maintenancemay be performed on the systemand samplesreanalyzedor otheractionsas determinedby the analyst.Document allactionsintheappropriatleogbook. ETS-8-7.0 AnalysisofLiverExtractUsingES/MS Page 7 of 10 14.7 Ifdata are to be reportedwhen performance criterihaave not been met, the datamust be footnoted on tablesand discussedinthetextofthe report. 15.0 POLLUTION PREVENTION AND WASTE MANAGEMIENT 15.1 Sample extractwaste and flammable solventisdisposedinhighBTU containers,and glass pipettewaste isdisposedinbroken glasscontainerslocatedinthe laboratory. 16.0 RECORDS 16.1 Each page generatedfora studymust have the followinginformationincludedeitherinthe header or hand writtenon the page: studyor projectnumber, acquisitiomnethod, integratiomnethod, samplename, extractiodnate,dilutiofnactor(ifapplicable)a,nd analyst. 16.2 Printthe tune page, sample lista,nd acquisitio-nmethod from MassLynx to includeinthe appropriatestudyfolder.Copy thesepages and tapeintothe instrumentrunlog. 16.3 Plotthe calibratiocnurveby linearegressionw,eighted I/x,then printthesegraphs and storeinthe studyfolder. 16.4 Printdataintegratiosnummary, integratiomnethod, and chromatograms from MassLynx ana toreinthe studyfolder. 16.5 Summarize datausingsuitablseoftware(Excel5.0+)and storeinthe studyfolder,referto Attachment A foran example of a summary spreadsheet. 16.6 Back up electronidcatato appropriatemedium. Record instudynotebook the filename and locationofbackup electronicdata. 17.0 TABLES. DiAGRAms, FLowcHARTs. MM VALIDATION DATA 17.1 Attachment A: ETS-8-7.0 Data summary spreadsheet 18.0 REFERENCES 18.1 FACT-M-2. Compounds 1,"ExtractionofPotassium Perfluorooctanesulfonaotre Other Fluorochemical from LiverforAnalysisUsing BPLC-Electrospray/N4assSpectrometry" 18.2 ETS-9-24.0, "Operationand Maintenance ofthe NficromassAtmospheric Pressure Ionization/MassSpectrometerQuattro IItriplqeuadrupole Systems" 18.3 The validationreportassociatedwith thismethod isETS-8-6.0 & 7.0-V-1 19.0 AFFECTED DocLTmNTs 19.1 ETS-8-6.0, "Extractionof Potassium Perfluorooetanesulfonaotre Other Fluorochemical Compounds from Liveror FluidforAnalysisUsing BPLC-Electrospray/Mass Spectrometry" ETS-8-7.0 Anal;sisofLiverExtractUsingES/MS Page 8 of10 20.0REVISIONS Revision Numbe Reason For Revisio Revision Date ETS-9-7.0 Analy;isof LiverExtractUsing ES/MS Page 9 of 10 Laboratory Study Study: TestMaterial: Matrix/FinaISolvent-. Method/Revision: AnalyticalEquipment System Number: InstniinenStoftwareNersion: Filename: R-Squared Value: Slope: Y Intercept: Date of Extraction/Analyst: Date of Analysis/Analyst: Group Dose Sample# Concentration InitiaWliL Dilution Factor Final Conc. up./a Slope: Taken from linearregressionequation. Group/Dose: Taken from thestudyfolder. Sample#: Taken from thestudyfolder. Concentration (ng/g):Taken from theMassLynx integratiosnummary. InitiaWlt. (g):Taken from thestudyfolder. DilutionFactor: Taken from thestudyfolder. FinalConc. (ug/g):Calculatedby dividintgheinitiavlolume from theconcentration AttachmentA: Summary Spreadsheet ETS-8-7.0 AnalysisofLiverExtractUsing ESIMS Page 10 of 10