Document pBOb3k2zRG17BO15waVjoYYGB

PERFLUOROOCTANESULFONATEP, OTASSIUM SALT (PFOS): A FLOW-THROUGHBIOCONCENTRATIONTEST WITH THE BLUEGILL (Lepomis mac_cMms) FINAL REPORT WILDLIFE INTERNATIONAL,LTD. PROJECTNUMBER: 454A- 134 ENVIRONMENTAL LABORATORY REQUESTNUMBER: U2723 U.S. EnvironmentalProtectionAgency Series 850 - Ecological Effects Test Guidelines OPPTS Number850.1730 and OECD Guideline305 AUTHORS : Kurt R. Drottar RaymondL. VanHoven,Ph.D. Henry O. Krueger,Ph.D.. STUDY INITIATION DATE: October 31, 2000 STUDY COMPLETION DATE: June 21, 2001 AMENDED STUDY COMPLETION DATE: June 6, 2002 SUBMITTED TO : 3M Corporation EnvironmentalLaboratory Building 2-3E-09 935 Bush Avenue SLPaul, Minnesota 55144St. Paul, Minnesota 55144 "I Wildlife International, 8598 CommerceDrive Easton, Mm3,1and2160 l (410) 822-8600 Page I of 134 Ltd. AMENDED _/rildli_ International, Ltd. Proj_tN_b_454A-134 -2" GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT SPONSOR : 3M Corporaiion TITLE: Per, fluorooctancsulfonato,Potassium Salt(PFOS): A Flow-ThroughBioconceatrationTest with the Bluegill (Lepomia machochirus) WILDLIFEINTERNATIONAL,LTD. PROJECTNUMBER : 454A-134 STUDY COMPLETION: June 21, 2001 AMENDED STUDY COMPLETION: June 6, 2002 This studywas conductedincompliancewith Good LaboratoryPracticeStandardsas publishedbythe U.S. EnvironmentalProtectionAgency in 40 CFRParts 160 and 792, 17 August 1989. STUDY DIRECTOR: Henry 0. KrUger, t6h.D, d Date Director of Aquatic Toxicology and Non-TargetPlants SPONSOR APPROVAL: Spon._r Rcpres_atativo - Date AMENDED Wildlife International, Ltd. P,oj tNb 454A-134 "3" QUALITY ASSURANCE STATEMENT ThisstudwyasexaminefdorcompliancweithGoodLaboratoPlryactiScteandaradsspublishbeydthe U.S.EnvironmentParlotectiAognencyin40CFR Parts160and792,17August1989.Thedateosfall inspections and audits and the dates that anyfindings were reported to the Study Director andLaboratory Managementwere as follows: ACTIVITY: DATE CONDUCTED: DATE REPORTED TO: STUDY DIRECTOR: MANAGEMENrf: Test Sub--lion MatrixFortification November 29, 2000 Ekccmber 8, 2000 November29. 2001 December 8, 2000 November 30, 2000 Ikeember I I, 2000 Water Chemisl_ February20, 2001 February20, 2001 February22, 2001 AnalyliealData and _rt May 14 - 17 and May 20 - 24, 2001 May 24, 2001 June2, 2001 Biological Data and Draft Report June 11- 13, 2001 June 13, 2001 June 14, 2001 FinalReport June 20, 2001 June 20, 2001 June 20, 2001 Amended FinalReport June 5, 2002 June 5, 2002 June6, 2002 T. - - // " " Quali_.Assurance ProgramSupervisor AMENDED Wildlife International, Ltd. -4. REPORT APPROVAL PromNumb4e5r4A-134 SPONSOR : 3M Corporation TITLE :Perflucxooctanesulfomtc,Potassium Salt (PFOS): A Flow-ThroughBh3conomtrati(Tmestwiththe Blu$ill (Zepom_mz acrochirus) WILDLIFE INTERNATIONAL, LTD. PROJECTNUMBER : 454A-134 STUDY DIRECTOR_ Hcmy O.Kru_, Ph.D. O Date : DirectorfAquatiTcoxicologaynd Non-TargePtlants PRINCIPAL INVESTIGATOR: 7 Directorof Re_ AMENDED ildlife International, Ltd. -5" TABLE OF CONTENTS Numb45er4A-134 Title/Cov_Pagn........................................................................................................................................... 1 Good Laborato_ Practice ComplianceStateroom...................................................................................... 2 Qnality AssuranceSt_nt ...................................................................................................................... 3 Report Approval.......................................................................................................................................... 4 Table of Contents........................................................................................................................................ 5 Summary ................................................................................................................................................. 8 Introduction................................................................................................................................................. 9 Objective ................................................................................................................................................. 9 ExperimentalDesign ................................................................................................................................... 9 Materials and Methods .............................................................................................................................. 10 Results and Discussion.............................................................................................................................. 16 Conclusions ............................................................................................................................................... 18 References ............................................................................................................................................... 19 Table 1 Table 2 Table 3 Table 4 Table 5 Table 6 TABLES Means and Ranges of WaterQuality Parameters................................................................. 20 Concentrationsof PFOS in Water Samples Duringthe Uptake Phase ................................ 21 Concentrations of PFOS in Water Samples Duringthe Depuration Phase.......................... 22 PFOS Concentrations in Edible, Nonedible andWhole Fish Tissues of Bluegill Exposed to 0.086 naga.i./L ............_......................................................23 Apparent Steady-State BCF Values for Bluegill Exposed to 0.086 mg a.i.FL..................... 26 BCFK ModelEstimatesfor Bluegill Exposed to 0.086 nag a.i./L ....................................... 27 AMENDED Wildlife International, Ltd. -6- TABLE OF CONTENTS - Continued- Numb4e5r4A-134 Table 7 Table 8 Figure 1 Figure 2 Figure 3 Figure 4 TABLES (Coat'd.) PFOS Concentrationsin Edible,Nonedible andWhole Fish Tissues of Bluegill Exposedto 0.87 mg a.i./L......................................................... 28 Day 28 BCF Values for Bluegill Exposed to 0.87 mg &i./L................................... 30 FIGURES Concentrationsof PFOS in Edible Fish Tissues of Bluegill Exposed to 0.086 mg a.i./L..................................................................................................... 31 Concentrationsof PFOSin Nonediblc FishTissues of Bluegill Exposed to 0.086 mg a.i./L..................................................................................................... 32 Concentrationsof PFOS in Whole Fish Tissues of Bluegill Exposes to 0.086 mg a.i./L ........................................................................................i............ 33 Concentrationsof PFOS in Edible.Nonedible, andWhole Fish Tissues Of Bluegill Exposed to 0.87 rag a.i./L..................................................................... 34 AMENDED rildtife International, Ltd. jectNum 4s4A-134 .7. TABLE OF CONTENTS . C_tlml_l - Appendix I Appendix 2 Appendix 3 Appendix 4 Appendix 5 Appendix 6 Appendix 7 Appendix 8 Appendix 9 Appendix 10 Appendix 11 Appendix 12 APPENDICES Specific Conductancc,Hardness,.AlkallnityandpH of WeUWater Measure_During the 4-WeekPeriodImmediatelyPrecedingthe Test ................... 35 Analyses of Pesticides, OrganicsandMetals in Wildlife Intema_onalLtd.Well Water................................................................ 36 The Analysis of Perflur_ Potassium Salt (PFOS) Concemrationsin FroshwaterandBluegill Sunfish Tissuo in Supportof W'fldlifeInternationaLl td.ProjectNo. 454A-134 ................................................... 38 TemperatureandpH of Water in the Test Chambers ..............................................89 Dissolved Oxygen of Water in the Test Chambcl_.................................................. 91 Hardness,Alkalinity, ConductivityandTOC of Water in the Negative Control....................................................................................................... 95 CumulativeMortalityandTreatment.RelatedEffects............................................. 96 . Changesto Protcol...................................................................................................105 Protocol Amendmentsand Dcviations .................................................................. 106 PersonnelInvolvedin the Study............................................................................. 130 CalculationsOf The Kinetic ConcentrationFactor(BCF_, The UptakePate Constant(ks), The DeputationRate Constant (k2), The Half-LifeFor Clearance,And The Time To Reach 90%Of SteadyState..... 131 ReportAmendment................................................................................................. 133 AMENDED Wildlife International, Ltd. Project Numb4e5r4A-134 -8- SUMMARY SPONSOR: SPONSOR'SREPRESENTATIVE: 3M _on Ms. SusanA. Beach LOCATIONOF STUDY,RAW DATAAND A COPYOFTHE HNALREPORT: W'ddlifeInternationalLtd. Easton, MD 21601 WILDLIFEIN'IERNATIONAL, LTD.PROJECTNUMBER: 454A-134 TESTSUBSTANCE: STUDY: NOMINALTEST CONCENTRATIONS: MEANMEASUREDTEST Perfl_onate, Potassimn Salt (PFOS) Perfl_-oronctanesulfcmatPe,otassium Salt (PFOS): A FlowThrougllBioconcentrationTest with the Bluegill (Lepomis macrobhirus) Negative Control,0.10 and 1.0 mg &i./L CONCENTRATIONS: Negative Control,0.086 and 0.87 mg a.i./L. Ex.Rosureto 0.87 mg a.i./L ceased aRer35 days due to fish mortality and tissue sampling TESTDATES: ExperimentalStart (OECD) - November 29, 2000 ExtxaimentalStart(EPA) - December 5, 2000 Biological Termination- April 2, 2001 ExperimentalTermination- April 11, 2001 - LENGTHOFTEST(0.086 naga.i./L): T._IGTH OFTEST(0.87 ml_a.i./L): i TESTORGANISM: SOURCEOFTESTORGANISMS: AGEOFTESTORGANISMS: 118Days (62-Day Uptake, 56-Day Dcpuration) 35 Da_ of Upta_ Bluegill (Lepomis macrochirus) Osage Caffashedes,Inc. 1170 Nichols Road Osage Beach, Missouri 65065 Juveniles MEASUREMENTOS F 10NEGATIVE CONTROLFISHCOIJ.I_.CTEDATTEST TERMINATION: WEIGHT(g): TOTALLENGTH(ram): Mean -- 2.70; Range= 2.03 to 3.32 Mean= 62; Range = 56 to 66 I[ RESULTS:(0.086mg a.i./L) KineticBioeoneentrationfactor(BCFK): I Edible Nonedible Whole Fish [ 1124 4013 279.6 : ., *Note: The BCFs were underestimateddue to fish mortalit_prior to achiovmg steady-state. AMENDED WildlifeInternationaLlt,d. 4s4A-1 -9- INTRODUCTION A bluegill sunfish,Lepomis macrochirus, bioconcentrafionstudywasamduct_ for 3MCa, pmatim at the WildlifeInternational,Ltd.aquatictoxicology facilityin Easton,Maryland. The in-life phase of the test was conductedfromDecember5, 2000 to April 2, 2001. Rawdatageneratedby Wddlife lnt__smational, Ltd. and a copy of the final reportare filed under ProjectNumber454A-134 in archives located on the Wildlife InternationalL, td. site. OBJECTIVE The objective of this study was to determine the bioconcc_tration potential of perflum'oocmnesulfonatep,otassiumsalt (PFOS)in the bluegill sunfish. EXPERIMENTAL DESIGN Thebioconcentrationtest consistedof a62-day uptakephasefollowedbya56-daydepuretionphase. Duringthe uptakephase, the test organismswere exposed inone of threegroups: 1) A negative(dilution water) control, 2) A nominal concentration of 0.10 mg active ingredient (a.i.)/L; or 3) A nominal concentrationof 1.0 rag a.i./L. At the start of the deputationphase, stock flow to the treated groups was stopped andthe bluegill were exposed to dilutionwaterwithout PFOS for the remainderof the test. Each test chambercontained90 bluegill at test initiation, and one replicatewas tested for each treatmentandthenegativecontrol.Watersamplesware collected on Day -4 (pre-test),Day-I (pre-test) on uptakeDays 0 (0 and--4 hours),1,3, 7, 14, 21, 28, 35, 42, 49, 56 and62 andon depurationDays 14, 28, 42 . and56 duringthe test andanalyzedfor PFOS using liquidchromatography-massspectrometry(LC/MS). Tissue sampleswere also collectedat selectedwatersample collection periodsduringthetest andanalyzed for PFOS by LC/MS.The resultsof these analyseswere usedto calculatetheBCF values, uptaker_t_ and depurationrates in edible tissue, nonedibletissue and whole fish. Wildlife International, Ltd. Pom Numb4e5r4A-134 - 10- MATERIALS AND METHODS The study was cmducted according to the procedures outlined in the protocol, "PerfluorooctanesulfonateP, otassiumSalt (PFOS)(Appendix9): A Flow-ThroughB_on Testwith the Bluegill (Lepomis macrochirus)'. The protocolwas based on proceduresoutlinedin U,S. Environmental Protection Agency Series 850 - Ecological Effects Test Guidelines OPPTS Nnmbm"850.1730 (l); AST/_ Standard EI022-84 Standard Practice for Conducting Bioconcentration Tests with Fishes and Saltwater Bivalve Molluscs (2); andOECD Guidelinefor Testing of Chemicals 305, Bioconcentration: Flow-Through PTshTest (3). Test and Reference Substances The test substancewas receivedfrom3M Corporationon October29, 1998 andwas assigned V(ddlife International,Ltd.identificationnumber4675. Thetest substance,a whitepowder,wasidentifiedas FC-95, Lot #217. Informationprovidedby the Sponsor indicatedapurityof 86.9% and anexpirationdateof August 31, ;. 2001. The test substance was storedunderambientconditions. Preparation of Test Solutions The nominaltest concenlxationswere 0.10 and 1.0 mg ai./L. Two stock solutions were preparedat concentrationsof 10 and 100 mg a.i./L. The appropriateamount of test substance was weighed out and dissolved in dilutionwaterfor each stock. Thestock solutions werestirredwithan electrictop-downmixer to aid in the solubilizationof the test substance. Aidermixing, thestock solutions appearedclear andcolorless. Stock solutions werepreparedat approximatelyweeldy intervalsduringthe uptakephaseof thetest. Thestock solutions wereinjected into the dilutermixing chambers(at arateof 3.5 mL/minute)wheretheyweremixedwith dilution water (at a rateof 350 mL/minute)to achieve thedesiredtest concentrations.All testsolutionsappeared clear and colorless. Test Organism Thebluegill,Lepomis macrochirus, was selected as the test species for this study. The bluegill is one of the reco_m_endedfreshwaterfish species for use in bioconcentrationtests (1, 2, 3). Bluegill usedin the test were obtained fromOsag_ Catfishmes, Inc., Osage Beach, Missouri. The fish were approximately7 months old at test imtiation. Identificationof the species was verifiedby the supplier. ]/gildlife International, Ltd. Numb4e5r4A-134 -li- The bluegill wereheadinWildlife InternationalL, td. well waterfor 103 days prioto testing. The fish were acclimatedto test conditionsfor approximately49 hourspriorto test inifiatiolz Duringthe holding and avclimatioaperiods thefish showedno signs of disease or stress. Duringthe 14-dayholdingperiodpreceding the test, water temperaturesrangedfrom 22.3 to 22.7eC. The pH of the water rangedfi-om7.8 to 8.2 and dissolved oxygen rangedfrom7.6 to 8.2 mg/L. Instrumentationusedforwaterm_ are describedin the,Environmental Conditions section of this report. At test initiation, tim bluegill were collected from the acclimationtank and indiscriminatelydistributedI to 2 at a time into the test chambersuntileach chamber contained 90 fish During holding and duringthe test, the bluegill werefed flake food supplied by ZeiglerBrothers,Inc., Oardnets,Pennsylvania. The bluegillwerefed atleast once dailyduringholdingandonce dailyduringthe test Feeding andsampling scheduleswerecoordinatedso that fish were sampled at least four hoursafterfeeding. All fish used/n the test werefromthe samesource andyearclass, andthe standardlengthof thelongest fish was no morethan twice the length of the shortest. The lengthandweight of fish in the negativecontrolwere consideredto be representative of all fish used in the test. The mean total lengthof 10 negative control fish measuredat the end of the test was 62 nunwith a range of 56 to 66 ram. The averagewet weight (blotteddry) was 2.70 grams with a range of 2.03 to 3.32 grarrt_L.oading was &freed as the total wet weight of fish per liter of test water that passed throughthe test chamberin 24 hours, andwas determinedto be 0.48 g fish/L/day.The loadingrat.was based on the averageweightof the f_h at timendoflhe test andthe initial stocking dens/ty(90 fisphertank). "Test Apparatus A continuous-fdl/ohwmaw-asusedtodeliverachconcentratoifotnhetesstubstancaendanegative controlA.peristalptuimcp(Cole-PannIenrstrumeCdonmtpany,ChicagoI,llinowiass)usedtodelivetrhetest substancsetocksolutioinnstomixingchamberasssignetdoeachPFOS treatmenTth.estocksolutiownesre mixedwithdilutiwoanterinthemixingchamberpsriotrodelivetroythetescthambersT.heflowofdilution watertothetescthamberwsascontrollbeydrotameterTsh.edeliveorfywaterfromtherotametewtasschecked priotrothetesatndatapproximatewleyeklyintervatlhsereaftAeprp.roximate6l.y3volumeadditioonfstest waterweredeliverteodthetescthamberesveTl24hoursT.hegeneraolperatioofnthedilutwearscheckeadt leasttwotimesacladyurintghetesatndonceonthelasdtayofthetest. Wildlife International, Ltd. Poj, Nt umb4e5r4A-134 - 12- Test chambers were 104-L sfainl_ssstcel aqum_ filled with approximately80 L oftcst solutiomThe depth of the test water in a rep_ve chamber was appro_im_tt4 19 cm. Test chambers were indiscriminatelypositioned in atemperature-controlledwaterbathdesigned to maintaina cc, astant tempaaturc. The water bath was enclosed in a plegiglass ventilation hood in orderto minimiTeany potential for cross- _on. Tcst chamberswac siphoacddaffyandperiodically clcmed during the test to rcmove cxcess feed andfecal matter. Test chamberswere identifiedby theproject numberandtest concentratio_ Dilution Water The water used for holdingandtestingwas freshwaterobtainedfroma well approximately45 meters deeplocatedon the WildlifeInternational,Ltd. site. ThewcIlwater is charactcriz_ as moderately-hardwater. The specific conductance,hardness, alkalinityandpH measurementsof the well waterduringthe four-week period immediatelyp.rec_,_l_tinhge test are presentedin Appendix I. Thewell waterwas passed througha sandfilterto remove particlesgreaterthanapproximately25 pro, : and pumpedinto a 37,800-L storage tankand aeratedwith spray nozzles. Priorto use, the water againwas filtered (0.45tim) to remove microorganismsand particles. The results of periodic analyses performedto measure the concentrat/onsof selected contmninantsin well water used by Wildlife International,Ltd. are presentedin Appendix 2. Environmental Conditions The targettemperaturerangeforthe test was 22 ._IC. Temperaturewas recordedcontinuouslyinthe negativecontrolwith a FulscopeER/C Recorder(1900 JSeries model no.A). Temperaturewas also measured in all test chain_herastthe bemnnin_ andendof the test and atweekly intervalsdm'ingthetest, with a liquid-inglass thermometer. Dissolved oxygen was measuredwith a Yellow SpringsInstmmmtC_xm_anyI,nc.Model5IBdissolved oxygen meter. With the exception of uptake Day 24, measurementswere made daily in each test chamber. Dissolved oxygenmeasurementswerenot madeon uptakeDay 24 dueto biologist oversight.Measure_ of pH were made in eachtest chamberatthe beginningandend ofthe test andat weeHy intervalsduringthe test using a Fisher Accumet Model 915 pH meter. Wildlife International, Ltd. Numb4e5r4A-134 - 13- Hardness,allmlim'ty,conductivityandtotal organiccarbon(TOC)wae measn_ i_ thenegativecc___Iml at the beginning and end of the test, and at wecldy intervals during the test Hardness andalkalinitywere measured by titrationbased onprocedm_ inStandard Methods for the EncanffnationoftVater and Wastewater (4). Conductivity was meama'ed using a Yellow Springs lmtnanent Company, Inc. Model 33 SalinityConductivityTemperaturemeter. Total_ganic carbonwas measuredusing aShimmy, modelTOC-5000total orphic carbonanalyzer. Ambientroom light was used to illmninatethe teatsystems. Flutmu_z_attubesthatmaitledwavelmgths similartOnaturalsunlight(Colortone 50) werecontrolledby an automatictimertoprovidea_od of 16 hoursof light and8hoursof darkness. A 30-minutetransitionperiodof low light intensitywasprovidedwhen lights went on andoff to avoidsuddenchangesin lightintensity. Light intensityat thesurfaceof thewater(over the negativecontrol)was 278 lux attest initiation. Lightintensitywas meastwednstngaSPERScientificModel 840006 light meter. Observations All fish were observed once each day to evaluate the number of mortalities and the number of individualsexhibiting signs of abnormalbehavior. Procedures for Exposure offish to PFOS The test chamberswereconditionedby deliveringPFOS to thediluter system fox al_ _ 5 days before _ad_ngthe fish. Watersamples were collected threetimes duringthe pre-test period to confirmthat equih'briumconcentrationsof test substancein the test chamberswere achievedpriorto addingthe fi_ho At the end of the pre-test period,the uptakephase of the test was initiatedon December5, 2000 by placing the f_shin thetest chambers.Bluegill wereimpartiallyremovedfromthe holdingtankin groupsof 1to 2. The groupsof bluegillweredistributedamongthe test chambersuntil each test chambercontained90 tr_h_ The durationof the uptakephasewas 62 days for thelow exposureconcentration(0. l naga.i./L, nominal).The uptake phase for the high concentration(1.0 mg a.iJL, nominal) was terminatedafter 35 days due to fish mortality. All subsequentreferencesin this reportto samplingperiodsafterDay 35 of uptakereferto the 0.1 naga.iJL (nominal)exposureconcenl_ation.At theend of the uptakephase,stock flow to thetreatmentgroups was stopped andthe blucgil!wereexposed to dilutionwaterwithout PFOSfor a periodof 56 days. VVildlife International, Ltd. projtNumb4e5r4A-134 - 14- Conection and Analysis of Water Samples Watersamples werecollectedon Days 0 (0 hoursand4 hours), 1,3,7, 14,21,28,35,47.,49,56 and62 of theuptakephase. Watersamples were also collectedon Days 14, 28, 42 and56 of the depurationphase. At each watersampling interval,two water samples werecollectedfromthenes_,e controlandthreesampleswere collected fromeach of the two PFOS treatmentgroups. Onenegativecontrol sampleandtwosamplesfromeach of thePFOS treatmentgroupswere analyzedforPFOS. Theremainingsamples were held in reserveas backup samples. All watersamples were collected from mid-depth of each test chamberusing a glass pipette. The water samples were analyzedfor PFOS by liquidchromatography-massspectrome_ (LCJMS).Proceduresfor analysis of the watersamples areprovidedinAppendix 3. Water samples were analyzedas soon as possible aftercollection without storage, t Collection and Analysis of Tissue Samples Tissue samples were collectedon Days 0 (4 hours), I, 3, 7, 14, 21, 28, 35, 42, 49, 56 and 62 of the uptakephase. Tissue samples were also collectedon Days 14, 28, 42 and56 of the deputationphase. At each :' tissue sampling interval,a sufficientnumberof fish were collected to providetwo replicatesamplesof negative controlfish andfourreplicatesamples of each PFOStreatmentgroup. Fishwereimpartiallyremovedfromthe test chambersandeuthaniz_ by severingthe spinalcordabovethe opercularregion.,The fish wereblotteddry and measuredfor total length and wet weight withinapproximately15 minutesof collection, when possible. Each fish was then rinsedwith dilutionwater, blotteddo, againanddissected into edible andnonedibletissue fractions. Dissection was accomplishedby makingan incisionfromjust posteriorto the base of thepectoralfin dorsallythroughthe spinalcord. Thehead,f_n._andviscera wereremovedfromthebodyandwe_c(msidercdto be nonedibletissue. The remainingtissue (includingskin) was consideredthe edible tissue. Tissue samples were transferredto tared scintillationvials and weighed. Prcwauturefsor extractionand analysis of the tissue samples areprovidedin Appendix. All tissue samples wereextractedimmediatelyor storedat approximately14C untilextraction. Wildlife International, Ltd. PromNnm454A-134 - 15- T'usue Lipid Content Selected fish wcvc colleO_ to determi-e lipid cov___ (Appendix3). The detmninafion of percent lipids provides thepotentialto express BCFvalues in terms of lipid contem. Fish were smnplcdon Day 0 of uptake,on Day 62 of uptakeandon Day 56 of depuratiotx All fish coll_ztcdfor lipid contentwere storedat approximately-14C untilanalysis. Inthis stady, no attempt was made tocxprcssbiocommtratiminrolatimto lipid contmt` Data Analysis Whole fish concen__tratiownesre calculatedbased on the sum of the edible and nonedibleparts. The _cady-state bioconccutrationfactor (BCF)values were determinedfromthetissue concentrationsat apparent stuady-statedividedby theaveragewaterconccntratio_ Tissue concentraticmwereconsideredtobeatapperant steady-stateif threeor moreconsecutivesets of tissue concentrationswerenot significantlydifferent_o> 0.05). Tissue conccn_ationswereevaluated fornormalityandhomogeneityof varianceusing theShapiro-Wilk's test andBartlett'stest, respectively, ff timdata did not meet ttm assumptions,the_d__wt_aswansform_ in anattempt to correctthedata. Mean tissue concentrationswere then comparedusing analysis of varianceand Dunnett's test. Inthe originalreportthetissue values wen: consideredto be at steadystate andthe computerprogram BIOFAC was used to estimate the kinetic concentration factor (BCFK), the uptake rate constant (kt), the depurationrate constant(k2), the estimatedtime to reach 90% steady state, andthe half-life for clearance. Although tissue concentrationsfi'omupt___dkeays 49, 56 and62 werenot siLmificanflydifferent (p >0.05), the mtrations of PFOS in tissues appearedto still be increasing. Therefore,itcanbe arguedthatthe fish did not reach an apparentsteady state andthat using the BIOFAC programmay not havebeen the best way to estimate these parameters.Therefore,the data were reanalyzedusing the equationsand graphicalmethods outlinedin the draftOPPTS850.1730 GuidanceDocument(Appendix 11). The recalculatedrateconstants were used to calculatea BCFK (BCFK ffikl/k2). AMENDED Wildlife International, Ltd. 454A-134 -16- RESULTS AND DISCUSSION Water Chemistry Means and ranges of temperature,dissolved oxygen and pH of the water in the test chambersare presentedin Table I. Thcindividualmcasmcmmts arcgiven inAppendicim 4 and5. Water_ inthe test chamberswere withinthe temperaturerangeof 22 :_ICestablishedfor thetestwithcue exception.OnDay 19 of depuration,the continuous temperaturerecordermeasured20 C. The durationof this temperature deviation was approximately2 hours. Dissolved oxygen concentrationsremained>6.4 mg/L (74 percentof saturation) throughoutthe test. Measm'emcntsof pH ranged from 7.9 to 8.2. Weekly memmcments of hardness,alkalinity, Appendix 6). conductivity and totalorganiccarbonremainedconsistentthroughoutthe test (Table I and Observations of Mortality and Clinical Signs Observations of mortality andclinical signs arc presentedin Appendix 7. Bluegill in the negative control appeared normal andhealthy throughoutthe test. Two bluegill died in the 0.1 mg a.i./L (nominal) trcatmentgroup;all other fish appearednormalandhealthy. Bluegill in the 1.0 mg a.i./L (nominal)treatment group started to die on Day 9 of the uptakephase. By Day 35 of the uptakephase, all fish in the 1.0 mg/L treatmentgroup hadeither diedor beensampled for tissue analyses. Any fish founddead in the aquariawere stored frozc_ Concentrations of PFOS in Water Concentrationsof PFOS in the negative controlwere <LOQ (0.0500 rag a.i./L) (Tables 2 and 3), Measured concentrationsof PFOSduringthe uptakephase inthe 0.10 mg aJJL _ grouprangedtics, 68 to 113% of the nominal test concentration(Appendix 3). When concentrationsmeasuredduringthe uptakephasewere averaged,themeanmeasuredconcentrationwas 0.086 m8 a.i./Lwhich represented86% of the nominal tcst concentration. Measuredconcentrationsof PFOS duringthe uptakephase in the 1.0 mg a.i./L treatmentgroup rangedfrom 73 to 101%of the nominaltest concentration. When concentrations measuredduring the uptake phase were averaged,the mean measured concentration was 0.87 mg a.i./L, which represented87%of the nominaltest concentration. Concentrationsof PFOS duringthe deputation phase were all <LOQ Wildlife International, Ltd. jtN b= 454A-134 - 17- Concentrations of PFOS in F'mhTissues Concentrationsof PFOS in thenegative controltissue _mples containedno quantifiablePFOS concen_ations (Appendix3) The concentraticqaosf PFOS in tissues of fish exposed to 0.086 mg a.i./L arepresentedin Table 4. Althoughtissue concentrationsfrom uptakedays 49, 56 and62 werenot significantlydifferent (p >0.05), the _ntrations of PFOS in tissues appearedto still be increasing The meanmeasuredtissue concentrationsfromthese last threeuptakesamples were41.6, 96.7 and73.6 mg a.i./Kg foredible, nonedibleand whole fl.qb_respectively. Steady-stateBCFvalues calculatedfrom thelast threedays of uptakeranged from484 in edible tissue to 1124 in nonedibletissue (Table 5), however, these values were believedto be unreliablesince tissue concentrationswere still increasingwith time. Itwas believed thatbetterestimates of the bioconcentrationfactorcould be obtainedby determiningthe kinetic concentrationfactor(BCFK). Inthe originalreportcomputerprogramBIOFAC was used to estimatethekinetic concentration factor (BCFK), the uptakerateconstant(k0, thedepurationrateconstant(k2), the estimatedtime to reach 90% steady state, andthe half-life for clearance.However, afterthe report hadbeenfinalized it was determinedthatusing the BIOFACprogrammay not havebeen the best way to estimatethese parameters.Therefore,the datawere reanalyzedusing the graphicalmethods outlinedin the draf_ OPPTS 850.1730 GuidanceDocument(1). These new calculationsare describedin Appendix 11. Plots of the tissue concentrationsversus time were constructedto evaluatethedata, The raw data wereplotted as well as acurve connectingthe meanvalues, anda curve throughthe predictedvalues fromBIOFAC. This graphical approachwas a simple way to evaluate how well the curves fit the data. The end of the uptakephase (Day 62) was akey date to evaluate,since data fromthis day has a pronouncedinfluenceon the estimatesof the uptakeand depurationrateconstants (kt andt2). The plottedresults in Figures 1-3 show that the BIOFAC modelpredictedvalues for Day 62 are lower than the Day 62 means for all threetissue types (whole, edible,and non-ediblefish tissue). Thus, the BIOFAC model slightlyunderestimatedthe uptakeanddepurationrateconstants, andoverestimatedthe kinetic cortcentratioufactor,theestimatedtime to reach90% steadystate, and thehalf-life for clearance in the originalreport. The recalculatedBCFKvalues for edible,nonedible, andwhole fish were 1124, 4013, and2796, respectively(Table 6). AMENDED Wildlife International, Ltd. Poject 4S4A-134 -18- Recalculatedestimatesof thetime toreach90% of steadystate inedible tissue,nmedibletissue end whole fish were 287, 383, and371 days, respectively (Table 6). Duringthe deputationphasc of the test, eslimates of thetime to reach50%clearanceof PFOSwere 86, 116, and112daysfor edibletissue,nonech'ble tissue andwhole fish, respectively(Table 6). The concentrationsof PFOS in tissues of fish exposed to 0.87 mg a.i./L arepresentedin Table 7. PFOS concentrationsin edible,nonedibleandwholefish tissues did not appearto reachsteady-stateby Day 28 (Figure 4). By Day 35, all fish in the0.87 mg a.i./L treatmentgrouphad been sampledor were dead. Consequently, the only informaficmon bio_ncentration that could be detennin_ from the 0.87 mg e.iJLtreatment groupwas the Day 28 BCF. Day 28 BCF valuesranged from 136 in edible tissue to 386 in nonedibletissue (Table 8). Based on the lengthof time requiredto reach90%of steady-statein the 0.086 mg/L treatmentgroup,these values underestimatethe bioconccntrafionpotentialof PFOS. CONCLUSIONS "' Perfluorooctanesulfonate,potassium salt (PFOS) bioconcentratedin the tissues of bluegill sunfish (Lepomis macrochirus). Bluegill exposed to 0.87 mg a.i_ wereeitherdeadorhadbeensampledby Day35 of the uptakephase. Consequently,no reliableinformationwas gainedfromthis treatmentgroup.Apparent steady-state c_c_ntrations werenot reachedin the0.086 rag a.i./Ltreatmentgroup. PFOS concentrations appearedto be increasingup to Day 62, the last day of exposure. BCFK values for edible,nonedible,and whole fish tissues were 1124, 4013, and2796, respectively. Estimatesfor timeto reach50%clearance for edible, nonedibleand whole fish tissues were 86, 116, and 112 days, respectively. AMENDED Wildlife International, Ltd. - 19REFERENCES ProjectNumbex454A-134 1 U._ Environmental Protection Agency. 1996. Series 850 - Ecological Effects Test Guidelines (draft), OPPTS Number850.1730: Fish BCF 2 ASTM Standard E1022-84. [988. Standard Practlce for Conducting Bioconcentration Tests withFishes and Saltwater Bivalve Molluscs. AmericanSociety for Testing andMaterials. 3 OECD Guideline for Testing of Chemicals 305. 1996. Bioconcentration: Flow-Through Fish Test. 4 API_,AWWA, WPCF. 1985. StandardMethodsfortheExaminatLonofWaterand Wastewater. 16th Edition,AmericanPublic HealthAssociatioL AmericanWaterWorks Associatio_ WaterPollutionControlFederation,New York. 5 BIOFAC. 1991. September19, 1991 versio_ The Dow ChemicalCompany, Midland, MichigAn Wildlife International, Ltd. Numb4e5r4A-134 - 20 Table I Meansof Rangesof WaterQualityPmmneters Sponsor:. Test Sustaucc: Tcst _: Dilution Water':. Uptakc Phase Nomad Concentration {ragzi_) 3M Corporation PFOS Blue_ll, Lepom/A maciwochrua Well Water Temperature1 DO2 (_) (me/L) PH Conductivity 0m_n) 0nffL as CaCO_) I-hudncu (mgn_u_ _V(_ Control 2|.9 21.8 - 22.0 7.8 i 8.1 6.8 - 8.6 7.9 - 8.2 324' 310 - 330 i_ I 174 - 185 127 104 - 138 T(_ (mS II _I <l - <I 21.8 7.8 8.1 0.I0 21.7-22.0 6.8-8.6 7.9-8.2 21.8 7.5 8.1 1.0 21.7 - 21.9 6.4 - 8.2 7,9 - 8.2 i I lllll :" mTemperatu_n_ continuousilnythenegativceontrorlangcdfrom20.0to22.0C. 2 At a tcmly_turc of 22C, thc dissolved o_en saturaSonconcentration is 8.7 mf/L and 60% saturationis 5.2 m_/L. -21 - Project Number 454A-134 Table 2 Concentrations of PFOS in Water Samples During the Uptake Phase Sponsor: Test Substance: TestOrgani_n: DilutioWnater: 3M Corporation PFOS BluegilLle,pomismacrochirus WellWater Nominal Concontration 0 (mga.i./L) Hour Negative Control <LOQ t 4 Hours I <LOQ <LOQ 3 _'LOQ 7 <LOQ Day ofUptake 14 21 28 <LOQ <LOQ <LOQ 35 <LOQ 42 <LOQ 49 <LOQ 56 <LOQ Mean Measured Percent Conc_mtration of 62 (raga.i./L) Nominal <LOQ <LOQ -- 0.I0 0.0717 0.0791 0.0702 0.0751 0.0826 0.0680 0.II0 0.0822 0.0915 0.0983 0.103 0.0853 0.0887 0.086 86 0.0692 0.0741 0.0734 0.0717 0.0781 0.0709 O.ll3 0.0843 0.0914 0.II0 0.I03 0.0948 0.0914 1.0 0.797 0.891 0.867 0.813 0.845 0.900 0.988 0.913 0.838 ..z -- -- -- 0.87 87 0.802 0.930 0.820 0.734 0.818 0.875 1.01 0.925 0.871 _2 ..... '"l The Limitof Quantitation(LOQ)was 0.0500 mg a.i./L. '" 2Samplesnotcollcotdeudeto1006mortality. Note: Values presented foreach samplin_intervalare two replicate samples collected from each test chamber. Wildlife International, Ltd. ProjectNumb4e5r4A-134 - 22 Table 3 Concenu_ons of PFOS in Wate_Samples Duringthe Depurafion Phase j Sponsor:. TestSubstance: 3M _on PFOS TestOrgamsm: Bl_ll,Lel_m_m_c_J DilutiWonatt. WellWater i i| UptakePhase MeanMe.asurcd Concentration DayofDeputation (ms a.i./L) (mga.i./L) 14 28 42 56 Nc_galivCc ontrol <LOQI <LOQ <LOQ <LOQ <LOQ <LOQ o.os6 <Loq <LOq ] ,'_ Limitof quantitation_LOQw) aso.osoo m_Li./L. , _,OQ <LOq <LOQ <LOq 2" - 23 - Project Numbm- 454A- 134 Table 4 i Sponsor: Test Substance: Test Organism: Dilution Water: l|ll PFOS ConcenlzatioinnsEd_lerNonedibleandWhole FishTissuesofBlue_ilElxposedto0.086m[_a.i./L 3M Corporafon PFOS Bluegill, Lepomls macrochirus Well Water , SampleID F_,4/N4 E5/N5 E6/N6 E7/N7 Uptake I_y 0 (4 hours) 0 (4hours) 0 (4hours) 0 (4hours) EdiblcTissue Conccntration (ms a-i./KS) 0.167 0.155 0.144 0.182 EdibleTissue Wcight (g) 2.0545 1.8960 2.5219 1.3561 NoncdibicTissue Concentration (ms a.i./Kg) 0.415 0.519 0,417 0.497 NonedibleTissue Wcight (g) 2.I153 2.2103 2.7449 1.83T7 " WholeF,sh Concentratiotn _m8 a.i./K_ 0.293 0.351 0.286 0.363 EI7/NI7 I E18/NI8 I El 9/N19 I E20/N20 I 0.734 0.726 0.631 0.806 1.6517 1.9269 1.2074 1.0777 1.68 1.85 1.72 2.07 2.0826 2.2925 1.8419 1.4272 1.26 1.34 1.29 1.53 E30/N30 3 E3 I/N31 3 E32/N32 3 E33/N33 3 E43/N43 7 E44/N44 7 E45/N45 7 E46/N46 7 1.73 2.07 2.03 2.11 3.73 4.25 4,73 6.25 1.7643 1.3794 1.2621 1,1883 2.2291 1.6419 1.2994 1.2686 4.59 5.50 5.47 5.97 10.2 10.6 11.9 15.2 1.8836 1.8505 2.0994 1.7014 2.7943 1.8984 1.6429 1.7110 3.21 4.04 4.18 4.38 7.33 7.66 8,73 11.4 E56,rN56 14 E57/N57 14 E58/N58 14 E59/N59 14 I1.4 9.07 13.7 12.6 2.0423 2.1548 1.0709 1.1804 27.3 23.2 35.3 32.6 2.5002 2.6582 1.4046 1.7455 20.2 16.9 26.0 24.6 E69/N60 21 E70/H70 2! E71/N71 21 E72/N72 21 11.7 2.1161 33.3 2.47]2 23.3 12.0 1,2984 22,7 1,9396 18.4 12.9 1.4092 24.6 1.99 I0 19.8 10.6 1.6268 24.4 2.1872 18.5 l Whole Fish Concentration= (edible wt, X edible cone.) + (nonedible wt. X nt'_._dih!ec,_er) .......... _diblwet. + nonedible wt.) Wildlife International, Ltd. ' .24 - Project Number 454A.134 Table 4 (Continued) , PFOSConcentrationinsEdiblerNonedibleandWholeFishTissuesof Blue_illExposedto 0.086 m8 a.i./L Sponsor: 3MCorporation Tesl Substance:PFOS TcslOrganism: Bluegill,Lepomlamaooehirua DilutionWater. WellWater IIIII II ii SamplIeD E82/N82 E83/N83 E84/N84 E85/N85 Uptake .Day 28 28 28 28 EdibleTissue Coneentralion (msa.i/Ke) 18.3 13.7 23.9 23.1 EdibleTissue Weight ._) 1.4636 1.4113 1.2081 0.8830 NonedibleTissue Concentration (msa.iYKs) 49.4 40.7 65.3 57.9 NonedibleTissue Weight ,, (_) 1.7564 !,8903 ! .3062 !.3512 E95/N95 35 E96/N96 35 E97/N97 35 E98/N98 35 22.6 27.7 23.8 20.6 1.7888 1.5897 1.1136 1.2226 67.1 73.3 62.0 59.1 2.0434 2.1281 1.6454 1.3627 E IO3/NI03 42 E 104/N 104 42 ElOS/N105 42 ElO6/Nl06 42 27.6 25.3 21.2 27.6 1.2037 1.4942 1.4367 1.3033 64.0 68.1 54.4 79.6 1.9474 1.9266 2.0920 1.6100 E11 l/N111 49 E 112/N 112 49 El 13/NI 13 49 E 114/NI 14 49 33.3 !.7249 85.0 2.2873 36.2 1.2185 95.1 I. 5993 39.0 1.5506 93.1 2.2184 30.6 1.4650 77.7 1.9337 E 119/N 119 56 E120/NI20 56 E 121/N121 56 El22/N122 56 48.3 38.9 44.I 38.3 1.6058 1,0946 1.0521 1.8592 122 94.2 73.2 106 2.1688 1.5831 2.0532 2.1739 EI27/NI27 E 128/N2I8 E 129/N129 E 130/N130 I Whole Fish _U'afion 62 42.4 1.2209 62 66.2 1.3902 62 42.2 1.0832 62 39.2 1.6697 = (edible wt X edible ooac.) + (nonediblewt. X nonedible co;lc,) _ediblwe t +_'ble wt.) 101 I12 105 96.4 1.7596 1.9170 I.5965 2.4378 WholeFish Concentratiotn (roba.i_,_) 35.3 29.2 45.4 44.1 46.3 53.8 46.6 40.9 50.I 49.4 40.9 56.3 62.8 69.6 70.8 57.4 90.6 71.6 63.3 74.8 77.0 92.7 79.6 73.I - 25 - Project Numb_ 454A- 134 Table 4 (Continued) PFOS Concentrations in Edible, Nonedibk and Whole Fish Tissues of Bluegill Exposed to 0.086 mg a.i./L Sponsor: ..... 3M Corporation Test Suhstan_: PFO8 ................... Test Organism: Bluegill, Lepoml$ maerochirus Dilution Water: Well Water ii Edil_leTissue Uptake Concentration SampleID Day (ms a.i./Kg) E135fN135 14 48.5 E136/N136 14 31.8 EI37/NI37 14 31.6 E138/N138 14 42.0 Edible Tissue Weight (g) 1.3634 1.4503 1.2626 0.9340 Nonedible Tissue Concentration (rag a.i./Kg) 124 79.4 81.8 113 E143/N 143 28 E144/N144 28 E 145/N145 28 E146/NI46 28 26.0 !.7691 85.7 33.3 1.1358 95.1 38.7 0.8845 85.7 55.8 0.6909 94.8 E 150/N 150 42 EI51/NI51 42 El52/NI52 42 El53/NI53 42 24.1 31.2 30.0 33.0 1.7590 1.1220 0.8440 0.8672 71.7 80.6 78.3 82.I E 157/NI57 56 21.1 1.5471 57.7 E158/N158 56 37.6 0.5892 80.3 E 159/N 159 56 EI60/N160 56 ii 32.9 0.6244 85.4 31.2 0.7223 u 84.4 Whole FishC_centration = (edible wt. X edible cone.) + (noncdible wt. X nonedible c_1,) ....... _xtibl,w,et,,.+none&_le wt.) i iiii Nonedil_teTis,_e Weight (g) 1,6928 2.1909 1.8708 1,4560 i ii [ WholeFish Concentration (rag a.i./Kg_ 90.3 60.4 61.6 85.3 2.0744 58.2 1.6772 70.1 1.4801 68.1 1.2803 81.1 2.3578 1.7635 1.5181 ].2687 51.4 61.4 61.0 62.2 1.9735 1.2353 1.0439 0,9975 ill 41,6 66.5 65.8 62.1 ...... IIII II /rildlife International, Ltd. 454A-I34 - 26 - Table 5 ApparentSteady-StateBCFValues for Bluegill Exposedto 0.086 mg &i./L Sponsor: Test Substance: Test Organism: Dilution Water: , ,, Tissue T_e Edible .un 3M Corporation PFOS Bluegill,Leporms macrochirus Well Water Mean _ Test Ccmcentration ,, _mga.L/L) 0.086 Uptake Days at Apparent Steady-State 49, 56 and62 Mean M_red AppareatSteadyState Tissue Concentration ,, (mg a.i_/Kg) 41.6 " Apparent Stead},-StateBCF 484 Noneth'ble 0.086 49, 56 and62 96.7 1124 i Whole Fish 0.086 49, 56 and62 73.6 . i | 856 Wildlife International, Ltd. - 27 - ProjeNcmt nb4e5r4A-134 Table 6 BCFK Estimntmfor Bluegill Exposed to 0.086 mg _i./L Sponsor: 3M Corporation Test Substance: PFOS Test Organism: Bluegill, Lepomts macrochlrus DilutionWater: Well Watt" Kinetic Biocc_cc_tration UptakeRate Factor Constant Tissue Type (BCFIC) (kt) Depur_on Rato Constant (k2) EstimateTime to Reach 9(PA of Steady State (Days) Estimated Time to Reach 50% Clearanc_ Edible 1124 9.022 0.0080 287 86 Nonedible 4013 24.08 0.0060 383 116 Whole Fish 2796 17.35 0.0062 371 lI1 :. AMENDED Wildlife International, Ltd. .2S- promNuml4r54A-134 Sponsor: Test Substance: Test Organism: Dilution Water: imlm Sample ID E9/N9 E I0/NI0 E11/NI I E12/N12 Table 7 " PFOS ConcentratioinnsEdibloN,oneMibloandWhole FishTissuesofBlue_ilElxposedto0.87m_ a.i./L 3M Corporation PFOS Bluegill, Lepomi_ macrochirua Well Water Edible Tissue Edible Tissue Nonedible Tissue Nonedible Tissue Concentration Weight Concentration Weight Uptake Da_, 0 (4hours) 0 (4hours) 0 (4 hours) 0 (4 hours) _m_ a.i2K_) 1.46 1.48 1.19 1.39 . (g) 0.9795 1.3226 1.6284 1.5205 (m_ a.i./K_ 3,52 4.37 4.22 4,06 (g) 1.5183 1.6397 1.9391 1.9489 Whole Fish Concmtratiton (r_ a.i./Ks) 2.71 3.08 2.84 2.89 E22/N22 I E23/N23 1 E24/N24 1 E25/N25 I 4.68 1.7060 11.1 6.59 1.3724 14.2 5.56 1.1272 13.3 5.64 1.0507 12.I 1.8323 1.7515 1.7081 I.5273 8.00 10.9 10.2 9.47 E35/N35 3 E36/N'36 3 E37/N37 3 E38/N38 3 17.3 15.8 19.0 20.8 0.9889 1.2666 0.8348 1.1215 39.3 42.0 43.8 51.8 1.4781 1.6727 1.4005 1.6070 30.5 30.7 34.5 39.1 E48/N48 7 FA9/N49 7 ES0/NS0 7 E51/N51 7 42.0 1.7575 44.0 1.5527 57.7 1.3391 46.8 0.9532 100 ' 2.3064 74.9 102 2.0505 77.0 102 2.2079 85.3 120 1.3562 89.8 E61/N61 14 87.1 1.9813 177 E62/N62 . 14 81.6 1,2152 207 E63/N63 14 90.7 1.1290 245 E64/_64 ]4 73.3 1.0811 214 t Whole Fish Cgncentration= (edible wt. X edible cone.'}+ (nonedible wt. X nonedibl conc.) ' _edi'blewt. +.nonedible wt.) 2.9193 141 1.8260 157 1.5376 180 _[,619_ 158 WiMlif International, Ltd. - 29 - Project Numbm'454A-134 Table 7 (Continued) Sponsor: TestSubstance: TestOrganism: DilutionWater: SampleID E74//N74 E75/N75 E76/N'/6 E77/N'77 PFOS Concenlrations in Edible, Nonedible and Whole Fish Tissues of Bluegill Exposed to 0.87 mg a.iJL ii 3M Corporation PFOS BluegillL, epomismacrochtrus WellWater EdibleTissue UptakeDay ,,, 21 21 _tration (mga.iJl_) 79.4 117 21 104 21 102 EdibleTissue Weight (g) 1.8300 . 1.4526 1,6824 1.4076 NonodiblvTissue Coacentrafion (mflLi./Ks), 201 278 246 229 Nonedibl8Tissue Weight (g) 2.1803 1.9786 2_130 1.755g E87/N87 28 E88M88 28 102 1.6679 289 131 1.0923 372 E89/N89 28 107 1.167S 320 E9i 0i i/NlilJ 90 i 28 133 1.1466 361 WholeFishConcentrat=io(nedibwlte.X edible co,nc.)+(nonedibwlte.X nonedibleonc.) (ediblwet.+nonediblweL) 2.0549 1.4045 1,6595 1.5186 Note: Samplingended afterDay28, By Day 35 allthe fish in this treatmentBrouphad died or werepreviouslysampledfortissue analysis. WholeFish Concentratioln (mg a.iJKg) 146 210 185 172 205 267 232 263 Wildlife International, Ltd. -30- PmjectNumber454A-134 Table8 Day28BCF ValuesforBluegiElxlposedto0.87mg a.ifL i Sponsor:. 3M Corporation TestSubstance:PFOS Test Organism: Bluegill, LeporaJs macrochtrus Dilution Water: Well Water MeanMeasured TestConcentration Mean Day28 Tissue Concentration Tissue Type .... Edible (rag a.i./L) 0.87 (rag a.i./Kg) I18 Day 28 BCF 136 Non Edible 0.87 336 386 Whole Fish 0.87 ii i 242 278 Note: BCF Values presentedhere _ bia_ low due to fish mortality. Wildlife International, Ltd. -31 - P_ N._ 454A.134 Figuro 1. Concentrationsof PFOS in Edible Fish Tissues of Bluegill Exposedto 0.086 raga.i./L. 70 X 60 x x [] MeanTissue_cms 10_ [] 0 i ! t w I 0 20 40 60 80 100 120 140 Days AMENDED 'z Wildlife International, Ltd. -32 - ProjcctNmnlm'454A-134 Figure2. Concentrationsof PFOS inNonexliblFish Tissues of Bluegill Expos_ to 0.086 mg a.i./L. 140 120 X g X X _1oo x _x 2o] f rt i i 0 2O 40 ...a. I_n'ri_sue_ons i i 60 80 Days i _ 100 120 140 AMENDED Wildlife International, Ltd. -33 - ProjeetNumber454A-134 Figure3. Concentrationsof PFOS in Whole Fish Tissues of BluegiUExposedto 0.086 mg a.i./L. 100 90 XX X __ _4o o x j'x x -_.._ x _ x Observed'tissueConcentrations , ,.., , , , , , , , , , , , , , 0 10 20 30 40 50 60 70 80 90 100 110 120 130 Days AMENDED ': Wildlife International, Ltd. _oj_Numb4c5r4A-134 -34 Figure 4. Concentrations of PFOS in Edibl,Noneible and Whole Fish Tissues of Bluegill Exposed to 0.87 mg a.iJL. 43O500 i 300 ""_ 250 _, == 200 e_ 1 150 = 100 5O 0 A A " x x " Non-Edible = Whole Fish Edible ! I 10 20 30 Day Wildlife International, Ltd. - 35 - ProjecNtumb4e5r4A-134 Appendix 1 Specific Conductance,Hardness,Alkalinity andpH of Wen Water Measured Duringthe 4-Week PeriodImmediatelyPrecedingthe Test i Sponsor: 3M Corporatien TestSubstance:PFOS Test Organ/sm: Dilution Water: I Bluogfll,Lepomls macroc_#rus Well Water Mean Range Specific Conductance (ttmhosl_n) Hardness (mg/LasCaC03) 313 (N = 4) 130 (N = 4) 310 - 315 128- 132 Alkalinity (mg/LasCaC03) 178 (N = 4) 176- 178 pH i 8.1 (N = 4) 8.0 - 8.2 I II I II III Wildlife International, Ltd. -36- Appendix2 AnalysesofPesticidesO, rsanicsandMetalsinW'ddlifeIntmmim_ Ltd.WellWate_r Pesiiaidemand Orgmies Componcnt Aionifm Akchlor Ame_ A_ Azinphos..cth_ Azinpho_methyl Azoxysu_in Bifenthrin Bimlkthfin Bitmtanca Bronmcil Bromophm Bmmophcm-cthyl B_pylat Bupirimate Carbav/l Carbofuran Chiorfenvinphes Chknidazon Chlorpropham Chlorpy_fos Chlorpyrifos-methyl Chlorothalo_ Coumaphoa Cyanazine Cyfhth_ Cypcrmtluin Cyproemmzole Ikltam_in Dmeton l_m_oa-O DesmhytaUazine r_x'o_.aUazin Diazinon Dichlobenil Dichlm'an Dichlorbenzamide Dichlm'fenthion Dichlodlmmid Mmsumd Conom_ion <0.03 ,g/L <0.0t pg/L ._.01ps/L <0.01 pg/L <0.04 pg/L <0.08 pg/L _.25 ps/L <0.05 pg/L <0.05 .g/L ,0.05 _g/L <0.05 ps/L <0.02 pg/L <0.02 pg/L ._.02 pg/L ,:0.05 lag/I, <0.05 pg/L <0.03 _g/L <0.132_g/l, <0.02 pg/L <0.05 pg/L <0.02 pg/L <9.01 pg/L <0.01 pg/L <0.04.8/L ,c0.02 pg/L <0.05 pg/L <0.05 pg/L <00.5 Pg/L <0.05 pg/I., <O.0t2tg/L <o.o2_/L <o.o2 pg,5. <o.olp_JL <o.o2.NL <0.o]_g/L <o.ot_g_ <0.01 _g/L .<0.03 ttg/L <0.02 pg/L <0.01 pg/I. <0.03 I_g/L Compon_t Mmmm:d Conccatmtion Dichlorvm Dieofol DiethyUohmmm Difenoconazolc Dimefl_te Dimetlummrph Disutfoum DMST Do&nuocph F.axdoml_-ot Endmulfan._ Endom_amsulfate Epoz_0_.z_ Epmn Esfenvalerate Ethion Ethofunu_ate Ethoprophm Etridia2ole Etrimfm Fe,nafimol Fenchlmphos Fenitrothion Fenoxyearb Fenpielonii Fenpropat_n Fcnlxopimorph Fenthion Fenvmlerate Fluazifop-bu_ Fluomslyoofcn-thyl Hw_yp_-meptyl _ Fo.ophm FumU_ mp_nop_s Imazalil lpmdion Kt'__ _ Lindane <13.01Ps/L .,:0.25 pg/L _.02 ps/L <0.03 pg/L .<0.02 pg/L <0.05 pS/I., <0.02_8/L <0.051_8/L <0.01 pK/L <0.01 _g/L <0.01 _ts/L <0.02 pg/L <0.05 p.g/L .<0.02 ps/L <0.02 lag/I, <0.05 pg/L <0.02 pg/L <0.01 pg/L <0.02 pg/L <0.05 pg/L <0.05 pg/L <0.01 pg/L <0.03 pg/L <0.03 pg/L <0.05 pg/L .<0.25 pg/L <0.01 tt8/I., <0.0I ps/L ,_0.02 Ps/L <O.O.s2/L <o.o2t_g/L ,:0.05 ps/L <o.o2pg/L <o.ol.s/L _.o2 tts/L <0.o2pg/L <0.01 pS/L <0.05 pg/L <0.02 pg/I., <0.05 pg/L <0.02 pg,_., _Analysmperformed by TNO Nutrition and Food Institute on samples collected on November 15, 2000. Continued I Ill I Wildlife International, Ltd. -37- Analyses of Pesticides, Appendix 2 (Continued) Organics and Metals in Wilali_ Intmmtk_ Pcstiaks _ org_cs (P_ 2) Ltd. Well Water t Component Meamm:dCota=mmtlen Compmamt Meastm:d Coneentratitm M_lathion <0.02 _8/L Meth_ot" <0.01 pg/L McUdax_ <0.05 _/L l_olachlor <0.0t _StL Metamitron <0.05 p_rL Metdbutzin <0.02 p_ 1_. or <O.02Fg/L Mm_nphos " <0.01 _8/L _c_hidau_on <0.02 _gtL _Uroth_-_ropyt <0.05 p_, Paeaobutazolc <0.05 Ixs/L Pyfifenox-1 <0.01 pEeL Parathion <0.01 pg/L Pydf_ox-2 <0.01 pg/L Parathion-methyl <0.01 pWL _il <0.01 pS/L Pmconazo_ <0.05 ps/L Qu_ofop_h# <0.02 ps/L Pmdim_thalin <0.03 pg/L Simzinc <0.0l pgtL PmaeOuin-_ -'0.01 pg/L Sulfotep <0.02 pStL Perrn_tmns <0.01 p_, T_conazal <0.05 ps/L Phoealotte <0.05 Ps/L Tebufm_pyrad <0,05 pg/L Phosmct <0.02 _gtL Terbut_ <0.01 pg/L Phcmphamidon-cis <0.05 _8/L Terbuthytazin <0.01 pg/L Ptfmicarb <0.01 _g/L Tetra_hlorvinphos <0.01 pg/I, Pirimiphos-cthyl <0.01 pg/L Tetrahydroi_alimide <0.05 ps/L :" Pirimiphos-mcthyl <0.01 pg/L Tctnmg't_n <0.01 pg/L ProcMoraz <0.02 pg/L Thisl_lazok <0.05 p_/L Procyrnidon <0.01 p_L Thiomcton <0.04 pg/L Promct_'n <0.01 pg/L Toklofos-methyt <0.0l pg/L Propachlor <0.01 pg/L Tolylfluanid <0.04 pg/L Propazinc <0.01 pg/L TriKlim_on <0.05 pg/L Propham <0.02 ps/L TrisKIimcnol <0.05 ps/L Propiconazolc <0.05 p,8/L Triallate <0.02 pSIL propoxus <0,03 m/L Triazophos <o.o2_s/L Pmpyzamide <O.O2Ps/L Trmun_in <O.O2pen. Prmuifocarb Pymzophos <0.02 pg/L <0.03 pg/L Vamidothion 'V'mclozolin <0.01 ttf/L <0.01 pg/I., I_um Sodium Calcium Iron Potassium Aluminum Manganese Beryllium Chromium Cobalt 13,2 ms/L 21 tns_ 35 m$/L <0.02 mg/L 6=2mg/L <0,09 m$/L 0.72 _g/L <0,3 pS/I, <0.6 pg/L <0A pgtL Metals Nickel Copper Zinc Molybdenum Silver Cadmium Arsenic Mercury Selenium <1.4 pg/L <1.0 pg/L <:2.3pg/L <0.7I*S/L <0.3 pg/L <0.3 pg/L <0.25 ME/L <0.025 p.8/L <1 pg/L tAnalyscs pcfformcd by TNO Nutrition andFood Instituteon samplcs collated on November 15, 2000. Wildlife International, Ltd. -38 Appendix 3 THE ANALYSIS OF PERFLUOROOCTANESULFONATEP, OTASSIUMSALT (PFOS) CONCENTRATIONS IN FRESHWATER AND BLUEGILL SUNFISH TISSUE IN SUPPORT OF WILDLIFE INTERNATIONAL, LTD. PROJECTNO.: 454A-134 _. II I -Wildlife International, Ltd. -39- SPONSOR: REPORT APPROVAL 3M Corporation _j_tNum4_54A-134 TITLE: Perfluorooctan_ulfonate,PotassiumSalt (PFOS): A Flow-Through BioccmcentrationTest with theBluegill (Lepomis macrockirus) WILDLIFEINTERNATIONAL,LTD.PROJECTNUMBER: 454A-134 PRINCIP&LINVESTIGATOR: Scientist Date 2' WILDLIFE INTERNATIONAL, LTD. MANAGEMENT WillardB. Nixo_ Ph_D/ Director,AnalyticalChemistry Date" _' AMENDED Wildlife International, Ltd. -40- Introduction Freshwater samplesand tissue samples were collected from a flow-through aquatic test to determinethe bioconcentrationpotential of perfluorooctanesulfonate,potassiumsalt (PFOS) in the bluegill (Lepomis macrochirus). The study was conducted by Wildlife International,Ltd. and identified as Project Number 454A-134. The analyses of freshwater and tissue samples were performedat Wildlife International,Ltd. by high performanceliquid chromatography(HPLC) with mass spectrometric detection. Water samples were diluted and analyzed by HPLC with single quadrupolemass spectrometricdetection (LC/MS). Tissue sampleswere homogenized, extracted, diluted, and analyzedby HPLC with triple quadrupolemass spectrometricdetection (LC/MS/MS). Freshwatersampleswere collected and analyzedfrom December 1, 2000 to April2, 2001. Tissue samples were collected and analyzedfrom December 5, 2000 to April2, 2001. Additional tissue sampleswere collected and analyzedgravimetricallyfor lipidcontent. Samples forlipidcontent were collected on December 5, 2000, February5, 2001 and April2, 2001 and analyzed between April4 and April 11, 2001. Test SubstanceandInternal Standard The test substance, PFOS, was used to preparecalibrationstandardsandmatrixfortification samples and was identifiedas Wildlife International,Ltd. identificationnumber4675. The internalstandardwas received _om 3M Corporationon Iuly 2, 1998 and was assigned WzldlifeInternationalLtd. identificationnumber4526 upon receipt. The internalstandard,a granular material, was identified as: IH, lI-l, 2H, 2H Perfluorooctane Sulfonic Acid, Chemical Abstract Number: 27619-97-2. The internal standard, referredto hereafter as 4HPFOS, was stored under ambient conditions. Analytical Method Water and tissue samples wm'e analyzed for PFOS using high performance liquid chromatography (HPLC) with mass spectrometric detection. Water sampleswere analyzedaccording to the method entitled "'Analytical Method Validation for the Determination of Perfluorooctane Sulfonic Acid, Potassium Salt (PFOS) in Freshwater, Saltwater and Algal Media" (Wildlife International, Ltd. ProjectNo. 454C-I09). Tissue samples were analyzed according to the method entitled "'Analytical Method Validation for the Determination of Perfluorooctane Sulfonic Acid, Wildlife International, Ltd. -41 - PotassiumSalt (PFOS) in FishTissues" (Wildlife Intemation_ Ltd. ProjectNo. 454C- 119). Forwater_yses, oneminormodificationfromthevalidation was incorporatedinto the present study. This changewas use of 10 ppb in place of 1O0ppb 4I-IPFOSinternalstandardconcentrationin all calibrationstandardsandstudysample dilutions, Thelower internalstandardnominalconcentrationwas closer to therange of PFOS calibrationusodin thepresentwork. The analyticalmethodologyimplementedfor tic determinationof lipid contoatin fi_ tissue is presentoendpage42. Freshwater Samples A method flow chartfor the analysis of PFOS in freshwateris presented in Figure 1. Samples were dilutedin a 50% methanol(HPLCgrade, 99.9+%): 50% NANOpure. water solution containing 10.0 Itg 4H PFOS/Land 0.05% formic acid (v/v) so thatthey fell within the calibrationrangeof the LCMS methodology. Aliquots of the dilutions we_ transfemredto autosamplexvials andsubmitted for analysis by direct injection. Concentrations of PFOS in freshwat_ samples were detetmimxi by reverse-phasehigh pexformance l/quid chromatographyusing a Hewlett-PackardModel 1100 High PerformanceLiquid Chromatograph(HPLC) :" interfaced with a Perkin-ElmerAPI 3000LC m_s spectrometeroperated in selective ion monitoring (SIM) detection mode. The mass spectrometerwas equipped with a Peddn-Elmer TurbolonSpray ion source. Chromatographicseparationswere achievedusing aKeystone Betasil Cls analytical column(50 mmx 2 mm I.D., 3 pm particle size) fitted with a KeystoneJavelin Ct, Guard Cartridge(20 nun x 2 nan). The instrtanent parametersare summarizedin Table 1. Freshwater quality control (QC) samples (matrix blanks and fortitlcations) were processed in the same manner as the test samples. Freshwater was fortified with the appropriate PFOS in methanol stock solution using a gas-tight syringe. Matrix blank samples were not fortified with the test substance. Tissue QC Samples Bluegill sunfish (Lepomis macrochirus) tissues were obtained from a breeding stock maintained at Wildlife International, Ltd. Approximately 200 individual fish (ages 3 to 5 months) were removed from a breeding tank and euthanized by severing the spinal cord above the opercular region. Heads, fins and viscera were removed from the body and were considered to be nonedible tissue. The balance of the tissue was considered to be edible tissue. The edible and nonedible tissues were separately inn I 1 Wildlife International, Ltd. - 42 - homogenized with a Waxing stainless steel blender. An appropriate number of l-gram aliquots of edible and nonedible homogenate were weighed into separate, 20-mL glass scintillation vials. Each vial was uniquely identified and labeled with a facility log identification number. The tissue QC samples were stored frozen until they were used in the preparation of matrix blank and matrix fortification samples, or storage stability samples. Tissue Samples A method flow chart for the analysis of PFOS in fish tissues is presented in Figure 2. Edible and nonedible fish tissue samples were collected from the test in 20-mL glass scintillation vials and stored frozen, if necessary, until analysis. Upon analysis, tissue samples were batched by sampling interval and tissue type. At that time, three of the appropriate tissue QC samples also were removed from the freezer. Any frozen samples were allowed to thaw. One tissue QC sample was designated as the matrix blank sample. The other tissue QC samples were designated as matrix fortification samples and were fortified with the appropriate PFOS stock solution(s) in methanol using gas-fight syringes. Test and QC tissue homogenates were extracted as follows. Ten milliliters of methanol were added to each vial. The samples were homogenized with a tissue shredder for approximately one minute. The samples were then sonicated with a sonic dismembrator for approximately five minutes. The samples were capped, shaken, and centrifuged at approximately 2000 rpm for approximately 5 minutes. Aliquots of the extract were then volumetrically diluted into the calibration range of the LCMS methodology with 50:50 methanol: NANOpure .water dilution solvent. Aliquots of the diluted extracts were transferred to autosampler vials and submitted for analysis. Back-up tissue samples were collected and stored frozen. If necessary, the back-up tissue samples were removed from the freezer, allowed to thaw and processed using the same procedures described above. Tissue Storage Stability Samples Stability samples were prepared at test initiation to establish test substance stability in fish tissues stored frozen during the study. Two tissue QC samples of each fish tissue type (edible and nonedible) were removed from the freezer and allowed to thaw, The edible and nonedible fish tissues Wildlife International, Ltd. - 43 - were each fortifiedat 0.100 and 10.0 mg a.i./Kg usingthe appropriatePFOS stock solution anda gastight syringe. The stability samples were returnedto the freezer. Following 119 days of frozen storage, the stabilitysamples were removed from the freezer and amdyzed. Fresh fortificationand matrixblanksamples also were preparedandanalyzedat this time. The samples(new and old) were processed using the same proc_ures describedfor tissue sample analyses. Concentrationsof PFOS in fish tissue were determinedby reverse-phase high performance liquid chromatographyusing a Hewlett-PackardModel 1I00 HPLC interfacedwith a Perkin-Elmer AP130OOLCMass Spectrometeroperatedin multipleion reactionmonitoring(MRM)detectionmode. The mass spectrometer was equipped with a Perkin-Elmer TurbolonSpray ion source. Chromatographicseparationswere achievedusing a KeystoneBetasil C18analyticaclolumn(50 mmx 2 mm I.D., 3 pm particlesize) fitted with.a Keystone :lavelinC_sGuardCartridge(20 mmx 2 ram). The instrumentparametersare summafiz_ in Table 2. :, Tissue Samples for Lipid Content A method flow-chart for the analysisof lipid content in fish tissue is presented in Figure 3. Edible and nonedible fishtissue samplesto be analyzedfor lipidcontent were collected fromthe test at initiation,on Day 62 of the uptakephase (Day 0 depuration),and at termination(Day 56 depuration). Only fish from the Negative Control and 0.10 rng a.i./L nominal test level were sampled for lipid content. Samples were collected in 20-mLglasscintillavtiailsonand stored fi'ozuentialnalysis. Sample weights were recordedatthe timeof collection. Upon analysis,tissue samplesdesignated for lipidcontent determinationwere removed from the freezerand allowed to thaw. Foreach sample, l0 mL of NANOpure@water was addedto the fishtissue inthe vial andthe samplewas homogenizedfor approximately one minute using a tissue shredder. Each homogenate was transferredto a 250-mL separatory funnelthat contained25 mL or'chloroformand 50 mL of methanol. Each sample vial was rinsed with an additional 10 mL of NANOpure_. water and the rinsewas poured into the respective separatory funnel. The separatoryfunnelswere shakenwith venting for approximatelyone minute. Fifty millilitersoFchloroformfollowed by 50 mL of saturatedsodium chloride were added to each separatoryfunnel. The separatoryfunnelswere brieflyswirledwith venting. Thephaseswereallowed to separate. For each sample,the chloroformlayer was drainedthrough a powderfunnel packed with Vildl International, Ltd. -44 - glass wool and anhydroussodiumsulfateinto a 250-mL round-bottom flask. An additional50-mL aliquotof chloroformwas addedto each separatoryfunnelandthe extractionand drainin8 procedures were repeated. The extractswere rotaryevaporatedina waterbathmaintainedatapproximately40 C to neardryness. Eachextractwas tranfferredto a pre-weighedlabeledscintillationvial. Each250-mL round-bottomflaskwas rinsedwith a smallvolume of chloroformandthe rinsewas transferredto the respective scintillationvial. The remainingsolvent in each vial was evaporatedunder a gentle stream of nitrogenor clean dryair. Thevials were reweighed and the weights were recorded. Lipid content was calculated for each sampleas the ratioof lipidto fish tissue weights (mg,/Kg). Calibration Standards For water analyses, calibration standards of PFOS prepared in a 50% methanol : 50% NANOpuree.water solution containing 10.0 jtg 4H PFOS/L and 0.05%formicacid (v/v), ranging in concentration from0.500 to 5.00 g a.i./L, were analyzed with the each sample set. PFOS was fortified into the standardsby appropriate dilutionsof a 1.00 mg a.i./L stock solution of PFOS in methanol. Five calibration standards (different concentrations) were analyzed with each sample set. The calibration standard serieswas injectedat the beginning and end of each run, and one standard was injected, at a minimum,aiderevery five samples. Linear regressionequations were generated using peak area response ratios (PFOS : internal standard)versusthe respective concentrationratios (PFOS" internal standard) of the calibrationstandards. A typical calibration curve from water analyses is presented in Figure 4. The concentration of PFOS in the fi'eshwater samples was determinedby substituting the peak area response ratios into the applicablelinearregressionequation. Representative ion chromatograms oflow and high calibrationstandardsused forfreshwater analyses are presentedin Figures 5 and 6, respectively. Fortissue analyses,calibrationstandardswere prepared in 50:50methanol:NANOpure.water by appropriate dilutions of either a 1.00 nag a.i./L or a 10.0 rag a.i./L stock solution of PFOS in methanol. Two separate PFOS cah'brationstandard sets were employed in the study. For Days 0 through 7 of the uptake phase, andfor stabilitysample analyses, PFOS calibrationstandardsrangingin concentration from0.500 to 5.00 _g a.i./L, were analyzed with each sample set. For all subsequent samplingintervals,a higher(5.00 to 50.0 PFOS _g a.i./L) calibration rangewas requiredto minimize dilution of the sample extracts. For each sample set, five calibration standards (different Wildlife International, Ltd. - 45 - concentrations) were analyzed. The appropriate calibration standard series was injected at the beginning and end of each run, and one standard was injected, at a minimum, ailer every five samples. Linear regression equations were generated using the peak area responses versus the respective concentrations of the calibration standards. A typical calibration curve from tissue analyses is presented in Figure 7. Concentration of PFOS in fish tissue samples was determined by substituting the peak area response into the applicable linear regression equation. Representative ion chromatograms of low and high calibration standards used for tissue analyses are presented in Figures 8 and 9, respectively. For both water and tissue analyses for PFOS, the same and most prominent peak response for PFOS was u)iliT_edto monitor PFOS in all calibration, quality control, and study samples. No attempt was made to quantify PFOS on the basis of individual isomeric components. .-, Fortification Stocks Freshwater and fish tissue homogenates were fortified with the appropriate stock solution of PFOS prepared in methanol. Each stock solution was assigned a unique identification code that was recorded on a stock preparation log sheet. Limits of Quantitation The method limit ofquantitation (LOQ) for PFOS in freshwater samples was 0.0500 mg &i./L, calculated as the product of the lowest PFOS calibration standard (0.0005 mg a.i./L) and the dilution factor of the matrix blank sample (100) analyzed concurrently with the test samples. The LOQ was calculated on an individual basis for each tissue sample since each entire submitted sample (of differing weight) was extracted without an adjustment to a constant weight. The LOQ (rag a.i./Kg) for a given tissue analysis was calculated as the product of the lowest PFOS calibration standard (0.0005 or 0.005 mg a.i./L) and the overall dilution factor (l_/Kg) of the tissue sample. To illustrate for a 1.207 gram sample analyzed with the low-level calibration standard set, extraction with l0 mLs of methanol followed by a 10x volumetric dilution (overall dilution factor = 82.85 L/Kg), gives an LOQ = 0.0414 mg a.i./Kg. Wildlife International, Ltd. -46- Freshwater Matrix Blank and Fortification Samples Along with the actual freshwater sample analyses, 20 freshwater matrix blank samples were analyzed at periods throughout the study to determine possible interferences (Table 3). No matrix interferences were observed at or above the limit ofquanfitation (0.0500 rag a.i./L). A representative ion chromatogram of a freshwater matrix blank sample is presented in Figure 10. Freshwater samples were fortified at two levels (0.0750 and 0.500 mg a.i./L) or three levels (0.0750, 0.500 and 2.00 mg a.i./L) at each sampling interval using appropriate stock solutions of PFOS prepared in methanol. Freshwater matrix fortifications were analyzed concurrently with each sample set and the analytical results were not corrected for mean procedural recovery. Recoveries ranged from 84.9 to 120% of nominal concentration (Table 3). The mean and standard deviation of fortification recoveries at the 0.0750, 0.500 and 2.00 mg a.i./L fortification levels were 94.7% + 5.12% (n = 20), 98.7% :t:6.60% (n = 20), and 96.9% :t:2.95% (n = 12), respectively. A representative ion chromatogram of a freshwater matrix fortification is presented in Figure 11. Tissue Matrix Blank and Fortification Samples Along with the actual tissue sample analyses, 16 edible and 16 nonedible fish tissue matrix blank samples were analyzed to determine possible interferences (Tables 4 and 5, respectively). No interferences were observed at or above the applicable litnits of quantitationduringthe test. Representative ion chromatograrns of edible and nonedible fish tissue matrix blank samples Are presented in Figures 12 and 13, respectively. Edible and nonedible fish tissue homogenates were fortified with the appropriate stock solution(s) of PFOS prepared in methanol. Tissue matrix fortifications were prepared and analyzed concurrently with each sample set and the analytical results wexe not corrected for mean procedural recovery. The levels of fortification were chosen to bracket the expected concentration of test samples. Because measured concentrations increased with time for the duration 0fthe uptake phase, the fortification levels were frequently revised. Low-level fortification levels ranged from 0.100 to 5.00 mg a.i./L and high-level fortification levels ranged from 10.0 to 500 mg a.i./U Edible fish tissue recoveries ranged from 948 to 111% of nominal concentration for the low-level fortifications, and Wildlife International, Ltd. - 47 - from 92.0 to 10"PA of nominal concentration for the high-level fortifications (Table 4). The mean and standard deviation of fortification recoveries at the low- and high-level fortifications (n = 16) for the edible fish tissue were 102% 4.61% and 100% 4.40%, respectively. Nonediblc fish tissue recoveries ranged from 93.9 to 116% and from 90.1 to 122% of nominal concentration for low- and high-level fortifications, respectively (Table 5). The mean and standard deviation of fortification recoveries at the low- and high-level fortifications (n = 16) for the nonedible fish tissue were 106% =_5.55% and 103% + 8.49%, respectively. Representative ion chromatograms of edible and nonedible fish tissue matrix fortification samples are presented in Figures 14 and 15, respectively. Example Calculations The analytical result and percent recovery for freshwater sample 454A-134-68, from the 0.10 mg a.i./L nominal PFOS treatment group, was calculated using the following equations: Peak Area Ratio = AnalytePeakAreaflntemal StandardPeakArea PFOS Area = 593240 : InternalStandardArea= 1295042 Peek area ratio= 0.4581 InternalStandardConccnwation:I0.0 _g a.i./L Y-intercept= -0.0187 Slope = 5.2182 Initialvolume (Vi) = 0.100 mL Finalvolume (Vf) = 10.0 mL Dilution factor(Vf/Vi) = 100 PFOSOzga.i./L)atlnstmment = Peak area ratio-Or-intercep0 xlntemalStandardConcentration Slope = 0.4581-(-0.0187) x 10,0 5.2182 PFOS (rag a.i./L) in sample = PFOS(mg a.i./L) at Instnnnentx DilutionFactor = 0.0009137 x 100 -- 0.09137 Percentof Nominal Concentration= PFOS (raga.i./L)in sample x 100 PFOS(mg a.i./L) nominal = 0.09137 m8 a.i./L xlO0 0.10rag a.i./L - 91.4% Wildlife International, Ltd. -48- The analyticalresultfor ech_olfeishtissue sample454A-134-E-43, from the 0.10 mg &i./Lnominal PFOS treatmentgroup, was calculatedusingthe following equations: Peak Area = 11192 Y-intercept = 713.81 Slope ffi6287.33 PrimaryDilution: InitialWeight (Vii) = 2.2291 g ExtractionVolume (Ve) = 10.0 mL SecondaryDilution: InitialVolume (Vi) ffi0.0500 mL FinalVolume (VO = 25.0 mL Overall Dilution Factor (Ve/Wi x VOVi) = 2240 mL/g = 2240 L/Kg PFOSatinstnnmen(t]_ga.iJL)= (peakarea- y - intercept) slope (11192-713.81) 6287.33 = 1667 ttg a.i./L PFOS in sample(rag a.i./Kg) = PFOS at instrument(_tga.i./L) x overalldilution factorx unit conversion factor PFOSinsample(raga.i./Kg)= 1.667gga.i.x 2440L x lmg L Kg 1000pg ffi3.73 mg a.i./Kg RESULTS Freshwater Sample Analysis Freshwatersampleswere collected andanalyzedfor PFOS concentrationson Days -4 and -1 (twice) duringthe pre-uptakephaseofthe test and Days 0 (0 and 4 hours), 1, 3, 7, 14, 21, 28, 35, 42, 49, 56, and 62 duringthe uptakephase of the test. Uptake was suspendedon Day 62 of the test and deputation began. During the depurafion phase of the test, freshwatersamples were collected and analyzed for PFOS concentrationson Days 14, 28, 42, and 56. Measured concentrationsof PFOS in the pre-test diluter verification (pre-uptake phase) samples were <LOQ in the negative controls. Measuredconcentrationsinpre-test samplesranged from 64.6 to 95.4% of thenominalconcentration Wildlife International, Ltd. - 49 - in the 0.10 mg a.i./L treatment group and from 71.8 to 111% of the nominal concentrationin the 1.0 mg a.i./L treatmentgroup(Table6). Duringthe uptake phase of the test, measuredconc_-aations of PFOSin the negative controlfreshwatersampleswere <LOQ. FreshwatersamplescoUectedfrom the 0.10 and 1.0 mg a.i./L treatment groups rangedfrom 68.0 to 113% and from 73.4 to 101%ofthe nominal concentration, respectively (Table 7). During the deputation phase of the test, measured concentrationsof PFOS in all freshwatersampleswere <LOQ. A representativeion chromatogramof a freshwater sample is presentedin Figure 16. Tissue Sample Analysis Tissue samples were collected on uptakeDays 0 (4 hours), I, 3, 7, 14, 21, 28, 35, 42, 49, 56, and 62 and deputation Days 14, 28, 42, and 56 of the test. Measured concentrations of PFOS in all negativecontrol edible andnonediblefishtissue sampleswere <LOQ (Tables 8 and9). The resultsof analyses of edible and nonedible fish tissue samples collected from the 0.100 and 1.00 mg a.i./L treatmentgroups are presentedin Tables8 and9, respectively. Representativeion chromatogtams of :" edible and nonedible tissue samples from the same study fish are presented in Figures 17 and 18, respectively. Stability Sample Analysis Stabilitysamples were prepared at test initiation(uptakeDay 0) and storedfrozen. The results of stability sampleanalysesarepresentedinTable 10. These dataindicate that thetest substancewas stable during relatively long term (119 days) frozen storage at the 0.100 and 10.0 mg a.i./Kg concentrationlevels. Definitivetissue samplestorage did not exceed 7 daysduringthe conductof the study. Tissue Sample Analysis for Lipid Content Tissue samples were collected on uptake Days 0 (0 hour) and 62 and deputation Day 56 to determinelipidcontent in fishtissues on awet-weight basis. The resultsof lipidanalyses inedible and noned_le fish tissues are presentedin Tables 11 and 12, respectively. Wildlife International, Ltd. - 50 TableI TypicalHPLC/MSOperationalParametersfor Analysisof Aqueom Samples i mira ,ill i i I Im I _ I . INSTRUMENT: Hewlett-PackardModel 1100 High PerformanceLiquid Chromatograph (HPLC) with a Perkin-ElmerAP13000 Mass Spectrometerequipped with a Perkin-ElmerTurbolonSprayionsource. Operatedin selective ion monitorimngode(StM). ANALYTICALCOLUMN: KeystoneBetasil Cn cohen (50 mmx 2 mm I.D., 2-lt3m part_le size) GUARD COLUMN: KeystoneJavelinCn column(20 mmx 2 mm I.D.) OVEN TEMPERATURE: 40 C STOP TIME: 5.00 minutes FLOW RATE: MOBILEPHASE: INJECTIONVOLUME: PFOS RETENTIONTIME: 250 td..tminute 70.0% Methanol: 30.0%NANOp_e Watercontaining0.1% FormicAcid I0IlL Approximately3.7 minutes INTERNALSTANDARD RETENTIONTME: PFOS MONITORED MASS: Approximately2.5 minutes 499amtm INTERNALSTANDARD MONITORED MASS: i i 427 ainu ii i i Wildlife International, Ltd. -51 - TRhlo2 TypicalHPLC/MS/MS Ol_rationa] Porametersfor Analysis of Tissue Samples INSTRUMENT: Hcwlctt-PackardModel 1I00 High Pcrfonnm_ Liquid C_mato-graph (HPLC) with aPerkin-ElmcrAP13000 Mass Spectrometerequippedwitha Perkin-ElmerTurbolonSprayionsource. Operatedin multipleionreaction monitoring(MRM) mode. ANALYTICAL COLUMN: KeystoneBetasil Cts column (50 nun x 2 mm I.D., 2-tt3m particlesize) GUARD COLUMN: KeystoneJavelinC_.column (20 _ X:2mm I.D.) OVEN TEMPERATURE: 40 C :, STOP TIME: 5.00 minutes FLOW RATE: MOBILE PHASE: INJECTIONVOLUME: 250 _d_Jminute 70.0% Methanol: 30.0% NANOpure Watercontaining0.1% For'talcAcid lO.0 ttL PFOS RETENTION TIME: PFOS MONITORED MASS: Approximately4.1 minutes 499.0 AMU PFOS MONITOREDMASS: 499.0 _IU --4'99.1_lU it Ill Wildlife International, Ltd. - 52 - Table 3 MatrixBlanks andFortificationsAnalyzed_y with FreshwaterSamples I i I II I lil I I II Illil I Concentrationof Periluomoctaneaulfonate_ Sample l_mnber Sampling Interval Puttmum Salt ('PI:OS)(ms e.i./L) Perumt I II I I t p-_ -4 _r-M_ .4 _r-_2 -4 PT-M_-3 -4 _-uAn-2 -_ VT-_t_-4 -_ PT-MAS,.5 -1 PT-MAS-6 -1 PT-MAB-2 -1 PT-MAS-7 -1 I:rI'-MAS-8 -1 PT-MAS-9 -1 MAB-I 0 MAS-I 0 MAS-2 0 MAS-3 0 _ . It II III.I _ _ Hour0 - Up_ko 0.00 0.0750 o_00 2.00 0.00 o..o 0.500 2.00 0.00 0.0750 0.500 2.00 0.00 0.0750 0.500 2.00 I t _oQ 0.0707 "0.493 _.98 _ 0.0779 0.464 1.91 <LOQ 0.0724 0.483 1.86 <LOQ 0.0712 0.488 2.00 I I .n - 94.3 98.6 99.0 lo4 92.9 95.3 - 96.5 96.5 93.2 - 95.0 97.5 99.9 MAB-2 MAS-4 MAS-5 MAS-6 0 Hour 4 - Uptake 0 0 o 0.00 0.0750 0.500 2.00 <LOQ 0.0728 0.490 1.97 97.1 98.0 98.6 MAB-3 1 MAS-7 l MAS.8 1 MAS-9 1 MAB-4 3 MRS-t0 3 MAS-1 ! 3 MAS-12 3 MAB-5 7 MAS-13 7 MA8-14 7 MAS-I 5 7 Uptake Uptake Uptake 0.00 0.0750 0.500 2.00 0.00 0.0750 0.500 2.00 0.00 0.0750 0.500 2.00 <IJ3Q 0.0745 0.498 1.95 <LOQ 0.0666 0.509 1.86 <LOQ 0.0753 0.508 1.96 99.3 99.7 97.6 88.9 102 92.8 - 100 102 98.2 MAB-6 14 MAS-16 14 Uptmkc 0.00 0.0750 MAS..17 14 0.500 M_18 14,, 2.00 , _The limit of quantitation(LOQ) was 0.0500 mg a.iJL, calculated as the product of (0.0005 mg a.i./L) and the dilution factor of the matrix blank sample (1(30), <LOQ 0.0671 0.450 r85 the lowest calibration 89.5 90.0 92.7 standard ,2R_ultswere _erat_l usingMacQmm, version 1.6soflw__. _ c_culations may differ slightly. Wildlife International, Ltd. - 53 - MatrixBlanks andFc_fications Analyzed_ with Freshwater Samples i i i Coneentnttim of Pedluorooetanetulfmate, Sample Nmnl_ SamplingInterval Potmium Salt(PFOS) (rag aJJL) Pereeat (454A-134-) (Day) ' Fotlili_l ]_mxl' [i R_ MAB-7 21 MAS-19 21 Upcld_ 0.00 0.0750 <LOQ - 0.0710 94.6 MAS-20 21 0.500 0.600 120 MAS-21 21 2.00 1.99 99.6 MA_ 2S MAS-22 28 MAS-23 28 MAS-24 28 Upt_ 0.00 0.750 0.500 2.00 _3Q - 0.0659 87.6 0.517 103 1.91 95.4 MAB.9 35 MAS-25 35 MAS-26 35 MAS-27 35 Uptate o.oo 0.0750 0.500 2.00 <LOQ - 0.0727 96.9 0.470 94.0 2.01 10 l MAB-10 42 MAS-28 42 MAS-29 42 Uptake 0.00 0.0750 0.500 <LOQ - 0.0734 97.9 0.517 104 MAB- 11 49 MAS-30 49 MAS-3I 49 Uptake 0.00 0.0750 <LOQ - 0.0712 95.0 0.500 ' 0.463 92.7 MAB-12 56 MAS-32 56 MAS-33 56 Uptake 0.00 0.0750 . 0.500 <LOQ -" 0.0701 93.4 0.477 95.3 MAB- 13 62 MAS-34 62 MAS-35 62 Uptake 0.00 0.0750 0.500 <LOQ - 0.0745 99.3 0.481 96. I IVIAB-14 14 MAS-36 14 MAS-37 14 Depuration 0.00 0.0750 0.500 .cLOQ -- 0.0751 I00 0.494 98.7 MAB-I5 28 MAS-38 28 Dep_ 0.00 0.0750 <LOQ 0.0644 85.8 MAS-39 28 0.500 , 0.525 ....1.,.0.5. 'iThe limit of quantitatk_OQ) was 0.0500raga.i./L, cal_a_d as the product of the lowesl calibration standard __esul.0005mg a.i.fL) and the dilution factor of the malrix b!ank sample (100). Iswere _eneratcd,nsin_ MacQuan, ve_,,i,o, n 1.6 software. Manual calculations ma_ ,differ slightly. Wildlife International, Ltd. - 54 - Table3 (_ MatrixBlank_andFortificationsAnalyzed_y with FreshwaterSamples ii I Cone,eatrafionof Perfluoroootaneaulfonate, .sam_Num_ s_,,._-sI_t_d roUi."m Slit(FPO0(ms_a.) (454A.134-) (Day) i Fortified Moasurcd' ' '/b_-_6 4:2 _ _u_.4o 4:2 o.oo o.0750 <o_ o.0r_ MAS41 42 0.500 0.452 pen=nt Rnoova_s u.9 90.4 _,B.17 56 MAS-42 56 _tion 0.0o 0.75o <LOQ - 0.0712 94.9 _3 56 0.500 i I 0.484 96.9 LThelimitof quantitation(IX)wOa3s0.0500mg&iJL,cadmdataasctlheproduootfthelowesct adibratiosetandard (0.0005mga.L/L)andthedilutionfactorofthematrixblanksample(100). 2Resultwsere8e_r._t_ _ MacQuanv,ersion1.6soitwareM. anuael adculatiomnsaydietesrliehtly. IIIIII Wildlife International, Ltd. -55 - Table 4 Matrix Blanks and Fortifications .Annly_ Cxmaa'renfly with Edible Fish Tissue S_les SampleNmnber (454A-134-) E-MAB-I 0 B-MAS-1 0 E-MAS-2 0 SmnplingInterval (Day) i i Hour 4 - Uptake Can_ion of Pafloc_oc_z_s_, PotassiumSalt (PFOS) (rag a.i.gtQ,) Fortified _t 0.00 0.100 I0.0 _.0500 0.0970 9.65 Pe_rvamt Reoovefy2 97.0 96.5 E-MAB-2 1 E-MAS-3 1 E-MAS-4 1 Uptake 0.00 0.100 10.0 <0.0500 - 0.0993 99.3 9.77 97.7 E-MAB-3 3 F.,-MAS-5 3 E-MAS-6 3 Uptake 0.00 0.500 50.0 <0.100 - 0.494 98.8 48.6 97.2 E-MAB-4 7 E-MAS-7 7 E-MAS-8 7 Uptake 0.00 <0.200 - 1.00 0.948 94.8 100 95.0 95.0 E-MAB-5 14 E-MAS-9 14 E-MAS-10 14 Uptake 0.00 <I.00 - 5.00 5.31 106 250 268 107 E-MAB-6 21 E-MAS-11 21 E-MAS-12 21 Uptake 0.00 <1.00 - 5.00 5.21 104 250 251 101 E-MAB-7 28 E-MAS-13 28 E-MAS-14 28 Uptake 0.00 <I .00 - 5.00 5.27 105 250 249 99.5 E-MAB-8 35 E-MAgI5 35 E-MAS-16 35 Uptake 0,00 5.0O I00 <1.00 - 4.92 98.3 104 104 E-MAB-10 42 E-MAS.19 42 E-MAS-20 42 Uplake 0,00 5.00 100 <I.00 4.97 105 -99.4 I05 E-MAB-I 1 49 E-MAS-21 49 E-MAS-22 49 Uptake 0.00 <l.00 - 5.00 5.27 106 I00 106 106 E-MAB-12 56 E-MAS-23 56 Uptake 0.00 5.00 <I.00 - 5.31 106 E-MAS-24 56 200 208 104 ' 'Thetincot rquantitat(/LonOQw) as0.05OmOSa.i./L_,late, t asmeproducotfthelowest calibration's_mdard (0.0005 m 8 a.i./L) and the dilution factor of the matrix blank sample (1(30). 2Results were _enerated using Mac(_an, version 1.6software. Manual calculationsmay differ slightly.. ..... - II Wildlife International, Ltd. -56- Table 4 (Continued) Matrix BIm_ks and Fostific,ations Analyzed Conmrrmdy with Edible Fish Tissue ili i ii i i i,w i Conccatr_on of Pcrfluoroocmmsulfonatc, s_ ramher (454A-134-) Samplirn-g_v_ (Day) :l ii roemi.sm_t (eros()met_ Fo[tificd i Mea, sm_ _ J E-MAB- 13 62 F,-MAS-2$ 62 Uptake 0.00 <I .00 5.00 5.54 E-MAS-26 62 200 192 Samples Percent Reoova'y= i Il1 96.0 E-MAB-14 14 F_,-MA_-27 14 E-MAS-28 14 Delmration 0.00 5.00 200 <I.00 -- 5.37 107 205 102 E-MAB-I5 28 E-MAS-29 28 E-MAS-30 28 Del:m_ioa 0.00 5.00 100 <I.00 - 5.15 103 104 104 E-MAB-16 42 Depuration 0.00 E-MAS-31 42 5.00 <!.00 - 4.82 96.5 E-MAS-32 42 100 92.0 92.0 2 E-MAB-17 56 Deputation 0.00 E-MAS-33 56 5.00 E-MAS-34 56 100 <1.00 - 4.99 99.7 IOI 101 _[ i i i BieR U il _ i i [I I lili i Lessthanvaluecsone.spotn_dlimit ofquanlitat(LiOonQ). Foreachanalysitsh,eLOQ wascalculataesdthe productof the lowest oalibrationstandardand the overall dilutionfaotorofthematrixblanksample. All sample weights = 1.00gram. 2Resultswere _ene_ated,Li,n_,,MacQuanv_ersio1n.6 soltwareM.anualcaflculatmiaoyndsiffesrlightly. Wildlife International, Ltd. - 57 - Table 5 Matrix Blanks and Fortifications Analyzed Concmxmtly with Ncmedible Fish Tissue Samples Snmple Number (454A-134-) N-MAB-I 0 N-MAS-I 0 N-MAS-2 0 ' SamplinglnUmnd (Day) Hour 4 - Uptake Cc_c_ou ofP_fluo_om_ Pctanium Salt (PFOS) (rag Li./KS) ' Fortified Measured] 0.00 0.100 10.0 <0.0500 0.109 9.26 ' Pen_t Recove_ 109 99.6 N-MAB-2 1 N-MAS-3 1 N-MAS-4 1 Uptake 0.00 0.100 10.0 <0.0500 - 0.106 106 9.36 93.6 N-MAB-3 3 N-MAS-5 3 N-MAS-6 3 Uptake 0.00 0.500 50.0 <0.100 - 0.470 93.9 45.8 91.6 N-MAB4 7 N-MAS-7 7 N-MAS-8 7 Uptake 0.00 <0.200 - 1.00 0.975 97.5 100 90.1 90. I N-MAB-5 14 N-MAS-9 14 N-MAS-10 14 Uptake 0.00 <! .00 -. 5.00 5.25 105 250 255 102 N-MAB-6 21 N-MAS-11 21 N-MAS-12 21 Uptake 0.00 <l.00 -. 5.00 4.98 99.7 500 505 101 N-MAB-7 28 Uptake 0.00 <1.00 - N-MAS-13 28 5.00 5.25 105 N-MAS-14 28 500 5.16 103 N-MAB-8 35 N-MAS-15 35 N-MAS-16 35 Uptake 0.00 <I,00 - 5.00 5.15 103 100 108 108 N-MAB- 10 42 N-MAS-19 42 N-MAS-20 42 Uptake 0.00 <I .00 -- 5.00 5.26 105 100 104 104 N-MAB-11 49 N-MAS-21 49 N-MAS-21 49 Uptake 0.00 <l.00 -. 5.00 5.40 108 100 108 108 N-MAB-12 56 Uptake 0.00 <1.00 -- N-MAS-23 56 5.00 5.41 108 N-MAS-24 56 200 245 122 1Lessthanvalues correspondto limit ofq_tation (LOQ). For each analysis, the LOQ was calculatedas the product of the lowest calibrationstandardandthe overall dilution factor of the matrixblanksample. All sample weights ffi1.00 gram. _Resultswere _eneratcdusin_ Mac(_uan,version !.6 software. Manualcalculations ma_ differ sli[_htly. Wildlife International, Ltd. -58 - Tabl5e (Co.enued) Matrix Blanks and Sample Number (454A-134-) N-MAB-13 62 N-MAS-25 62 N-MAS-26 62 Fortifications Analyzed Concurrently with Nonecb'bl Fish Tissue i t ConccuU'ationof Perfluorooctmesulfonate, SmnplfingInterval (Day) Potassimn Salt (PFOS) (rag LL/Kg) Fot_iod t Measure_d Uptake 0.00 <1.00 5.00 5.68 200 212 Samples iJ i i Pcmmt Recow:r2Y 114 106 N-MAB-14 14 N-MAS-27 14 N-MAS-28 14 N-MAB-15 28 N-MAS-29 28 N-MAS-30 28 Depuratio_ Depuralion 0.00 5.00 200 0.00 5.00 100 <! .00 5.29 207 <i.00 5.45 Ill 106 .. 104 -i 09 Ill N-MAB-16 42 N-MAS-31 42 N-MAS-32 42 Deputation 0.00 5.00 100 <I.00 - 5.19 104 98.9 98,9 N-MAB-17 56 Deputation 0.00 N-MAS-33 56 5.00 <l.00 - 5.80 116 N-MAS-34 56 i i 100 i 112 112 tLess thanvalues c,orresl_nd to limit of qtmatitation(LOQ). For each analysis,the LOQ was calculated as the pttxiuct of the lowest calibrationstamiardand the overall dilutionfactor of the matrixblank sample. All sample weights = 1.00 gram. _ult_ _e_teud.e_bn_,_q,_m,,,_!6 soJ_.Mm_ _cula_moa_ys"di__teir_, Wildlife International, Ltd. -59Table 6 Measured Concentmfiom of Perfl_onate, Potassium Salt (PFOS) in Pre-Test Diluter Verification Semples i Nnm_,! Test Cone_m_a6on (mR a.i./L_ 0.00 (Negative C_trol) Sample N_nbe_ _454A-134-_ PT-1 PT-9 Phase Prc-Uptak S_pling Time (Day_ -4 -1 PT-17 -I ktumuzfl Comentmtion of pe_t_ootn_fon_, Pot_miumSalt (PFOS)_ (m_:Li./L_ <LOQ <LOQ <LOQ ii i| Peseta of Nomi-,12 --- -. 0.10 PT-3 PT-4 PT-I 1 PT-12 PT-19 PT-20 -4 0.0850 85.0 -4 0.0844 84.4 -1 0.0695 69.5 -1 0.0646 64.6 -1 0.0954 95.4 -1 0.0937 93.7 1.0 PT-6 PT-7 PT-14 PT-15 PT-22 PT-23 -4 0.917 91.7 -4 0.949 94.9 -I 0.718 71.8 -1 0.738 73.8 -1 1.11 11! -1 !.01 101 IThe limit of quantitation(LOQ) wu 0'.0500 m8 a.i./L, calculated as the productof the lowest calibration standard(0.0005 mg a.iJL) andthe dilutionfact_"of the matrixbleak sample (100). 2Resultwsere_nerateudsing M_Quan r vm'sio1n.6software. Manualoelculatiommay differ slightl]t. , International, Ltd. .60. Table 7 Concentrations of Petfluoroo__a _n_nlfonate, Potassium Salt (PFOS) in Freshwater Samples f_om a Blue_ill Sunfish Bioc,oncentrafion Test M_ Concm_trationof Sample Perfl_onate, Number Sampling Pota_tan Salt (PFOS)l Percent of (L454A-134-) l 9 17 25 33 41 49 57 65 73 78 83 88 Phase Uptake Time _D_) 0, 0 hou_ 0, 4 hours 1 3 7 14 21 28 35 42 49 56 62 (m8 a.iJL_ LOQ <LOQ <LOQ <LOQ <LOQ <LOQ _OQ <LOQ <LOQ .LOQ <LOQ "LOQ <LOQ Nominal2 - -- --- -- - - -- - - 93 Deputation 14 98 28 103 42 108 56 _LOQ <LOQ <LOQ <LOQ - - : - - 3 Uptake 0, 0 hours 4 0, 0 hours 11 0, 4 hours 12 0, 4 hours 19 1 20 1 27 3 28 3 35 7 36 7 43 14 44 14 51 21 52 21 59 28 60 28 67 35 68 35 75 42 76 42 80 49 81 49 85 56 86 56 90 62 0.0717 0.0692 0.0791 0.0741 0.0702 0.0734 0.075 ! 0.0717 0.0826 0.0781 0.0680 0.0709 0.110 0.113 0.0822 0.0843 0.0915 0.0914 0.0983 0.110 O.!03 0.103 0.0853 0.0948 0.0887 9] 62 0.9914 (LOQ) was 0.0500 mg a.i./L,calculated as the productof the lowest calibration a.i./L) andthe dilutionfactor of the matrix blank sample (100). usin_ MacQuan,version 1.6 sotIware. Manual calculationsmay differ slightly. 71.7 69.2 79.1 74.1 70.2 73.4 75. l 71.7 82.6 78.1 68.0 70.9 110 113 82.2 84.3 91.5 91.4 98.3 !10 103 103 85.3 94.8 88.7 91.4 International, Ltd. -61 - Table 7 (Continued) Concentrations of Peffl_tdfonate, Potassium Salt (PFOS) in Ft_hw____t_r_ .... Samples from a Blue_ill Sunfish Bioconumtrafion Test IIII I Memm_ _on of Sample Number Sampli8n Pe_fluoroootanm_onate, Potas_umSalt(PFOS)_ Peroent of _454A-134-) 95 96 100 101 105 I06 110 111 Phase Deputation Time(Day) 14 14 28 28 42 42 56 56 (m_ &iJL) <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ Nomina2l - - -- 6 Uptake 0, 0 hours 0.797 79.7 7 O,0 hours 0.802 80.2 14 O,4 hours 0.891 89.1 15 O,4 hours 0.930 93.0 22 I O.867 86.7 23 l 0.820 82.0 30 3 0.813 81.3 31 3 0.734 73.4 38 7 0.845 84.5 39 7 0.818 81.8 46 14 0.900 90.0 47 14 0.875 87.5 54 21 0.988 98.8 55 21 1.Ol lOl 62 28 0.913 91.3 63 28 0.925 92.5 70 353 0.838 83.8 7| ,,, 35' 0.871 87.1 (LOQ) was 0.0500 mg a.i./L, cadoulatedas the produrt, of the lowca_oah_orati_m a_k/L)andthe dilutionfactor of the matrixblank sample (1OO). using _ versioa 1.6 soaware. Manual calculaticm may differslightly. aRerDa_ 35 Uptake clueto 100% mortalit_at the 1.0m_ _LL/Lnominal test level. Wildlife International, Ltd. - 62 - Table 8 Measm_ Ccmeea_tions of Perfluorooetauesadfonate, Potassium Salt (PFOS) inedible Fish Tissue Samples from a Bluegill Sunfish Bioeoneentration Test ......... M= =i Nominal Test Ccmumtralion SampleNumber Sampling Time P4l_tl_talaeSuIfolla_ Pota"_m_m Salt (PFOS)I (nag s.i./L_ _454A-134-_ Phase ,, _13ay_ (mg s.i./L1 0.0 E-1 Uptake 0, 4 hours <0.0510 (Negative E-2 E-14 0, 4 hours 1 <0.0665 <0.0960 E-15 I <0.0835 E-27 3 <0.114 E,-28 3 <0.0910 E-40 7 <0.0965 E-41 7 <0.162 E-53 14 <0.468 E-54 14 <0,560 E-66 21 <0.730 E-67 21 <0,710 E-79 28 <0.740 E-80 28 <0.800 E-92 35 <0.970 : E-93 E-lO0 35 <0.575 42 ":0.399 E-101 E-108 E-109 42 <0.590 49 <0.575 49 <0.520 E-116 56 '<0.775 E-117 E-124 56 <0.820 62 <0.660 E-125 62 <0.615 E-132 E-133 E-140 E-141 E-147 E-148 E-154 E-155 Deptwation 14 14 28 28 42 42 56 56 <0.685 <0.710 <0.585 <0.590 <0.830 <0.825 <0.755 <1.07 0.10 E-4 Uptake 0,4 hours E-5 0,4 horus 0.167 0.155 E-6 E-7 E-17 E-18 0, 4 hours 0, 4 hours ! 1 0.144 0.182 0.734 0.726 E-19 1 0.631 E-20 1 0.806 E-30 3 1.73 E-31 3 2.07 E-32 3 2.03 ,. E-3_ 3 2.11 1Lessthan values correspondto limit ofquantitation (LOQ). For each analysis, the LOQ was calculatedas the product of the lowest calibration standardand the overall dilution factor of the sample (L/Kg). 2Resultswere _nerated usin_MaoQuan,version 1.6 soRware. Mammlcalculations may_ sli_tl},. i II I Wildlife International, Ltd. - 63 - Table 8 (Continued) Measured Conccntr_iom of Pcrfluorooc_on_, Potassium Salt (PFOS) in Edible Fish Tissue Samples from a Bluo_ll Sunfi?h Bioconc_rafion Test MeasuredCommatratim of Conc_mralion (n_ a.i:/L), , 0 Sample Number (454A-134-) E-.43 E-44 E-45 E-46 E-56 E-57 E-58 E-59 E--69 E-70 F__-71 E-72 E-82 E-83 E-84 E-85 E-95 E-96 E-97 E-98 E-103 E-104 E-105 E-106 E-111 E-112 E-113 E-114 E-119 E-t20 E-121 E-122 E- 127 E-128 E-129 E-130 Phase Uptake "r'-m_ (D_,) ,, 7 7 7 7 14 14 14 14 21 21 21 21 28 28 28 28 35 35 35 35 42 42 42 42 49 49 49 49 56 56 56 56 62 62 62 62 Potassium Salt (PFOS)' (rag a.i./L) ....... 3.73 4.25 4.73 6.25 11.4 9.07 13.7 12.6 11.7 12.0 12.9 10.6 18.3 13.7 23.9 23.1 22.6 27.7 23.8 20.6 27.6 25.3 21.2 27.6 33.3 36.2 39.0 30.6 48.3 38.9 44.1 38.3 42.4 66.2 42.2 39.2 E-135 Depurafion 14 48.5 E-136 14 31.8 E-137 E-138 E-143 E-144 E-145 E-146 E-150 14 31.6 14 42.0 28 26.0 28 33.3 28 38.7 28 55.8 42 24.1 E-151 E- 152 E- 153 42 31.2 42 30.0 42 33.0 'I]_ss'ihanValuescorrespondto limit of qumtitation (LOQ). For each analysis, the LOQ was calc_ated as'_e productof the lowest calibrationstandardand the ovea_alldilutionfactor of the sample (L/K$_: "Results.w. ere [_eneratedusinBMacQuan_version 1.6 software. Manual calculadonsmay._. ffer slightly. ]/Vildlife International, Ltd. -64- Table 8 (Cant/mu,A) Measured Conceatrations of Peffluorooctanem_onate, Potassium Salt (PFOS) in Edible Fish Tissue Samples from a Bluegill Sunfish Bioconcenlzation Test ...... Nominal Test Conoentratkm (m_ a.i./L) 0.10 Sample Number (454A-134-) E-157 E-158 E-159 E-160 _ Xk.ptio Samplin8 Time _I3ay) 56 56 56 56 Perfluorooctaneadfonate, Pota_ium Salt (PFOS) ! _m_a.i2L_ , ,, 21.1 37.6 32.9 31.2 1.0 E-9 Uptake 0, 4 hours 1.46 E-10 0, 4 houri 1.48 E-ll 0, 4 hotma 1.19 E-12 0, 4 hours 1.39 E-22 1 4.68 E-23 l 6.59 E-24 ! 5.56 E-25 1 5.64 E-35 3 17.3 E-36 3 15.8 -_ E-37 3 19.0 E-38 3 20.8 E-48 7 42.0 E-49 7 44.0 E-50 7 57.7 E-St 7 46.8 E--61 14 87.1 E--62 14 81.6 E-63 14 90.7 E-64 14 73.3 E-74 21 79.4 E-75 21 117 E-76 21 104 E-77 21 102 E-87 28 102 E-88 28 131 E-89 28 107 E-90 28 133 tLess thanvalues correspondto limit of quantitafion(LOQ). For each analysist,he LOQ was calculatedas the productof the lowest calibrationstandardmd the overall dilutioa fact_ of the sample 0-./Kg). Re_21tswea'e_enerated _qin_Magnum, version 1.6 sottware. Manual calculatic_ m_ differslisl_. Wildlife International, Ltd. - 65 - Table 9 Measured Conce_ations of Perfluor_ulfonate, Potassium Salt (PFOS) in Nonedible Fish Tissue Samples from a Bluegill Sunfish Bioeamomtration Test Nominal Test Conomtration (mg a.i3L_ 0.0 (Negative 0.I0 Sample Number (454A-134-_ N-! N-2 N-14 N-15 N-27 N-28 N-40 N-41 N-53 N-54 N-66 N-67 N-79 N-80 N-92 N-93 N-100 N-10I N-108 N-109 N-116 N-117 N-124 N-125 N-132 N-133 N-140 N-141 N-147 N-148 N-154 N-155 N-4 N-5 N-6 N-7 N-17 N-18 N-19 N-20 N-30 N-31 N-32 Phase Uptake Sampling Time _Day) 0, 4 hours O,4 hours 1 1 3 3 7 7 14 14 21 21 28 28 35 35 42 42 49 49 56 56 62 62 Deputation 14 14 28 28 42 42 56 56 Uptake 0,4 hours 0, 4 hours 0,4 hours 0, 4 hours 1 1 I l 3 3 3 Meam_ Concentrationof Perfluofoob'tmu_ffo_me, Potassium Salt (PFOS) n _m8 a.UL) <0.0458 <0.0575 <0.0820 <0.0645, <0.0820 <0.0795 <0.0805 <0.0965 <0.434 <0.457 <0.560 <0.560 <0.580 <0.620 <0.590 <0.417 <0.363 <0.480 <0.555 <0.375 <0.540 <0.520 <0.505 <0.505 <0.470 _0472 <0.449 <0.505 <0,590 <0.570 <0.630 <0.780 0.415 0.519 0.417 0.497 !.68 1,85 1.72 2.07 4.59 5.50 5.47 ILess than values correspondto limit of qumtitation(LOQ). For each analysis, the LOQ was calculated as the p21roductof the lowest calibrationstandardand the overall dilution factorof the sample tTIKg). Results were [_eneratedusin6 MacQuan, version !.6 software. Manual calculations maydiffersli_tl},. Wildlife International, Ltd. - 66 - 9(Coatinu Measta'ed Cone_arafiom of Perlluc_oo_,mesutfom_, Potassium Salt (PFO$) in Nonedible Fish _NominaTl est _u'afion (me a.i./L) O.lO Tissue Samples from a Blue_ill Sunfish B_Wation Sample Number (454A- 134-) N-43 N-44 N-45 N-46 N-56 N-57 N-58 N-59 N-69 N-70 N-71 N-72 N-82 N-83 N-84 N-85 N-95 N-96 N-97 N-98 N-103 N-104 N-105 N-106 N-I 11 N-112 N-113 N-114 N-I19 N-120 N-121 N-122 N-127 N-128 N-129 N-130 Phase Uptake S_ling Tkne _I3ay_ 7 7 7 7 14 14 14 14 21 21 21 21 28 28 28 28 35 35 35 35 42 42 42 42 49 49 49 49 56 56 56 56 62 62 62 62 Test MeasuredConcentrationof Ped_omoetanesulfomte, Potassium Salt (PFOS)t (m_ Li./L) 10.2 10.6 11.9 15.2 27.3 23.2 35.3 32.6 33.3 22.7 24.6 24.4 49.4 40.7 65.3 57.9 67.1 73.3 62.0 59.1 64.0 68.1 54.4 79.6 85.0 95.1 93.1 77.7 122 94.2 73.2 106 101 112 105 96.4 o : N-135 Deptnfion 14 124 N-136 14 79.4 N-137 14 81.8 N-138 14 !13 N-143 28 85.7 N-144 28 95. ! N-145 28 85.7 N-146 28 94.8 N-150 42 71.7 N-151 42 80.6 N-152 42 78.3 IN-I_3 42 8_.1 |[.,e_ thanvaluescorr_pond to limitof quanflta(ion(LOQ). For eachanalysist,he LOQwascalculatedas the producot f the Iowcstcalibrationstandardandtheoveralldilutionfactorofthe sample(I_8). 2Resultswere_nerateudsine Mae(_ ver_n 1..6software.Manuacl alculationms a_d' iffcrslightS. Wildlife International, Ltd. - 67 - Tabl9e(Continued) Measured Concenlrafions of Perllucsooctanesu_onate, Potassium Salt (PFOS) in Nonedible Fish Tissue Samples from a Bluegill Sunfi_ Bioconcentration Test Nnmina! Test Concentration (mg a.i./L) o.10 i Sample Number _454A-134-_ N-157 N-! 58 N-159 N-160 Phase rX mo Sampling Time (Day) 56 56 56 56 Me._mu'_Clonocntrslioofn Peffluorooetanesulfcmte, Potassium Salt (PFOS)1 (me a.i./L), 57. 80.3 85.4 84.4 1.0 N-9 Uptake 0,4 hours 3.52 N-10 0,4 hours 4.37 N-II 0,4hours 4.22 N- 12 N-22 N-23 0,4 hours 4.06 l 1i. 1 i 14.2 N-24 1 13.3 N-25 N-35 1 12.1 3 39.3 N-36 N-37 3 42,0 3 43.8 N-38 N-48 3 51.8 7 I00 N-49 N-50 7 102 7 102 N-51 N-61 7 120 14 177 N-62 14 207 N-63 14 245 N.64 N-74 14 214 21 201 N-75 N-76 N-77 N-87 21 278 21 246 21 229 28 289 N-88 N-89 28 372 28 320 N-90 2_ , 361 _Lessthanvalues correspondto limit of quantitad_nCLOQ). For each analysis, the LOQ was calculatedas the _roduct of the lowest calibrationstandardatld the overall dilutionfactor of the sample (L/Kg). Results were _esato.t usin_ Macquan, version 1.6 software. Manual calculations may differ sli_,lb/. i II Wildlife International, Ltd. .68. Table 10 MeasuredConcentrationsof Pe_fluc_oc_fonat, PotassimnSalt (PFOS) inTissue Storage StabilitySamplesfroma Bluegill SunfishBioconcentrationTest ml II Nominal Concentration _m_a.i./Kg) Negative Conlrol SampleNumber .... (454A-134-_ E-MAB-18" N-MAB- 184 Tis,sueType Edible Nonedible Im MeasuredConcentrationof PeriluorooctanesulfonatPec,xccnt PotassiumSalt (PFOS) (mg of a.i./Kg) <LOQl <LOQ1 Nomin_ _ - -- E-STMAS-1_ Edible 0.109 109 O.100 E-MAS-354 Fd, ible 0.111 111 N-STMAS-I 3 Nonedible 0.114 114 N-MAS-35" Nonech_ole 0.112 112 E-STMAS-23 Edible 8.43 84.3 10.0 E-MAS-364 Edible 11.3 113 N-STMAS-23 Nonedible 9.30 93.0 ...... N-,MAS-364 Noncdible , 11:3.,..... 113 ] Lessthanvaluescorrespontdo limitof quantitatio(nLOQ).TheLOQwas0.0500mga.i./Kg,calculatedas the productofthelowestcaI_rationstandard(0.0005mga.iJ:))endtheoveralldilutionfactorofthe matrixblank sample(100 L/Kg). All sampleweights= 1.00gram. 2ResultsweregeneratedusingMacQuanv,ersion1.6sottware.Manuaclaloulationms aydifferslightly. 3The stabilitysampleswea'efxxtifiedon Decembex5, 2000andstoredinthefi-eezer.Thesampleswereremoved fromthefreeze,onApril3, 2001(after119daysoffrozenstorage)emdanalyzed. 4Thesesampleswere[a'cparcodnApril3, 2001andtheresultswerecomparedtothestabilitysamplereacts. s ThecritcrionforstoragewasPcromtof Nominabl etwcc_80 and120%. Wildlife International, Ltd. - 69 Table 11 LipidContent in F_A_leFish Tissue Nominal Test Concentration (rag _iJL) 0.0 (NegativeControl) Sample Number (454A-134-) EL1 EI.2 EL3 EL4 Phase Uptake Sampling Time (Day) 0, 0 hour 0, 0 hour 0, 0 hour 0, 0 hour EL6 Uptake 62 EL7 62 EL8 62 EL9 62 ELI6 ELI7 ELI8 ELI9 Depuration 56 56 56 56 0.10 ELI 1 Uptake 62 ELI2 62 ELI3 62 ELI4 62 Lipid Weight 0g) 0.0314 0.0453 0.0453 0.0463 0.0231 0.0135 0.0134 0.0162 0.0254 0.0163 0.0158 0.0256 0.0162 0.0327 0.0329 0.0188 Fish Tissue Weight (g) 1.7499 2.1445 1.8577 1.8335 1.5122 1.0670 0.9726 1.0650 1.3549 1.0890 1.1234 1.3777 1.0411 1.3551 1.4893 0.9054 EL21 Deputation 56 EL22 56 EL23 56 0.0172 0.0190 0.0250 1.2362 1.3483 1.3303 EL24 56 0.0252 1.1040 _Ratioalgulatedas [lipidweif_ht(_9'+ fish tissue weight (g)] x 1000 mg/gx 1000 _/Kg. , Lipid/Fish' Tissue weigh t_ (n_]Kg) 17,900 21,100 24,400 25,300 15,300 12,700 13,800 15,200 18,700 15,000 14,100 18,600 15,600 24,100 22,100 20,800 13,900 14,100 18,800 221800 Wildlife International, Ltd. -70- Nomin_ Test _rmtion (m_ a.i./L) 0.0 (Negative Control) Sample Numba" (454A-134-) NLI NL2 NL3 NL4 Table 12 Lipid Con_t in Nonc&'blc F/sh Tissue Phase Uptake SJm2plin8 Time (DJy) .... Lipid Weight _ 0,0 hour 0, 0 hour 0, 0 hour 0, 0 hour 0.1363 0.2383 0.2258 0.1992 Fish Tissue Weight , (8) 2.0649 2.6355 1.9568 2.0812 Lipid/FiSh Tissue Weight_ (ms/Kg) 66,000 90,400 115,400 95,700 Uptake 62 0.0506 1.8821 26,900 NL7 62 0.0274 1.5901 17.200 NL8 62 0.0298 1.7596 16,900 NL9 62 0.0518 1.7302 29,900 NLI6 Depuration 56 0.06]6 1.7263 35,700 NL17 56 0.0318 1.6746 19,000 NLI8 56 0.0360 1.5847 22,700 NL19 56 0.0966 1.8165 53,200 ? 0.10 NLI 1 Uptake 62 0.0503 1.5484 32,500 HLI2 62 0.1370 1.7642 77,700 NL13 62 0.1276 2.0113 63,400 NL14 62 0.1221 1.2898 94,700 NL21 NL22 NL23 NL24 Deputation 56 56 56 56 0.0343 0.0404 0.0710 0.0714 _Ratio calculated as [lipid weight (g) + fish tissue weight (g)] x 1000 mg/g x 1000 g/Kg. 1.7085 1.8394 1.7652 1.5682 20,100 22,000 40,200 45,500 II Wildlife International, Ltd. -71 - METHOD OUTLINE FOR THE ANALYSIS OF PERFLUOROOCTANESULONATE, POTASSIUM SALT _'OS) IN FRESHWATER Preparematrixfortificationsamplesin freshwatermaldx by spiking_herequhite volume of PFOS stock solut/ons direc_ lmo freshwat_'.Performfortificationsw_ gas_ght syringesand Class A volumeffic flasks. Prepareappropriatedilutionsofstudy snd QC s_q/es to wlthk the csllb_lon range office PFOS LCMS u,_blogy:. Par_lly fill Cl_s A volumetrlcflaskswith 50% medm_S0% water dIIu_ solvent _ 0.0100 mg/L41-[PPOS/men_ s/m_landand0.05% v/v formicadd. Add_prolrlate volume o/'sa_ole ml bd_ to vol,me w_ dilution solvent. Processn_ddx blanksamplesf_ a glven matrixusin8 the nine dllutloe sad aliquotvolmnesas fo the lowest fortification level In thatmah.ix. Mix well by sevend repeat/nversions. Aml_datesamplesandsubm/tforLCMSanalys/s. Figure 1. Method flow chart for the analysis of Pcrfluorooctanesulfonatv, Potassium Salt (PFO$) in freshwater. Wildlife International, Ltd. .72- METHOD OUTIAN]g FOR Tlig ANALYSIS OF PERFLUOROOCTANgSULFONATF., POTA,qSlUM SALT 0PFOS) LN FiSH TISSUES Qualitcyon_tsamplems Fepred_ _ (_pr0zLmtOtJg}ot'l_kcontrol_l tissue b,,,_$emZ Rema__ (edib_lernm-d_) Jquo_ fromfrm__n andglow to thaw. Preparef_tLflcaflmt samples with the apjnprlate PFOS stock solutim using gu-clght ,_ya_e(O". Fhte_r_ bl_lt:am#ewmt_tt..ff_ flattl._e _te. Add I0.0 mL of metlmml to ach ttm#e wtflt a glm Cl_ A vo_ lglm_ Homogetg_ e_h te_mple for apprmdmaely I mtrmtemingatimtmtdm_l___d__R. intmthehomgenlterwlthtlm _pmp_tesoh_M mbztwe_samples. ,L Sonkate each sample fur aplmml_tdy $ minutes wlOsa s0_c disnmnbmtor L Cap the vials and shake welL Cen_duge the vials at agrozlmately 2000 rpmfor approximately 5 mimSeL l, Prepare appropmte dilutions of study and QC samples to within the calibration range oflhe PFOS LGMS metlmdolog_ Partially fir Class A volmuetric flasks with 50%: 50% methanol/NANOpuree w-_r dilution solvent. Add appropriatevolame of sample and bring to valume with dilution solvent. Frocess mamx blank uunples for a given mauix ustng the same dilution and aliquet velumes as for the lowest fortification level _nthat ma_tx. Mix well by several repeat Inversions. Tnmsfm"an a,]lqttotof eadl sample to amautoumpler vial and submit for L_ analysis. Figure 2. Method flow chart for the analysis of Periluorooctanemdfonate, tissues. Potassium Salt (PFOS) in fish Wildlife International, Ltd. - 73 - METHODOUTIANEFO2 THEANALYSIOS FIAPIDSINYLSHTISSUF..S l_zmovvelabtobcmmly'amfrtomtf_freezer.AHowsamglestothaw. ,t Foreachsamplea, dd10mL_ NAN0puArwesetertofishtissueInvkl andbomogemuf_m approztmtely1___ uslug lumcl-i/gtidssueshredderR. insethelmumgenlzwextththe appmptmesolvemt(i_n betweensamples. Tnmsfeerachhonmgmatteea _0 mL__fm_ timcontain2s ,5mLof chloroformand _ AUJI.e _ MIl_ltlala4mtU-- t P_usinechviawl Jua_nadd.iomml __" .er.nd pou_r _m_ SimZeaclzSelmmmzfuynnelfora_ely oaematurwe _ venax_g. Add50mLofchloroformfellowedby50mLof satm'atesdodiumcklorldetoeachsepmlmyfurmel. J, Brieflyswirleachselpm__ofunnewl ithventing. Allowthep. hasestoseparateF. oreachsampled, rainthechloroformlayerthnmghapowderfummei packedwithglasswoolandanhydrousosdiumsulfateintoa250-mLround-bottofmlask. J, Addanadditiona5l 0-mLaliquootfchloroformtoeachseparaterfyu_el andrepeatheextraction anddm_procedures. Rotaryevaporatteheextractisnawaterbathmaintaineadtapproximate4l0y0Ctoneardryness Transfeerachextracttos pm-w_J_l. _ _,_Jmln,,gv_inal. ]TJnseeach2$0-mLmmzd4mttaflmaskwithssmallvolummofechloroformsadtrm_errtnmtoe sct,ellatt0mvUd. J, Evapo_ethe_-mainlnrg,_ventmeachvialu4n,dear 8entleslreamof nllroseam r4_ _ _r. Rewe_ eachvialandrecontweigl_. Figure 3. Methodflow chartfor the analysisof lipids in fish tissues. Wildlife International, ;Ltd. - 74 - _F_o) 0 o.0 0.06 0.12 0.18 0.24 0.30 0.36 0.42 0.48 0o_ (R_o) Figure 4. A typical calibration curvefor Peflluc_ooctanesulfonateP, otassimnSalt 0PFOS)in freshwater. Slope = 4.64, _ = -0.01; r = 0.99932. Curveis weighted( 1Ix). (monitoredmasses = 499 ainu (PFOS) and427 ainu (4HPFOSinternalstandard)). Wildlife International, Ltd. - 75 - PFOLI 8'10 0..S00q -.L/t. 487'SA.OIID ..11 4.m. _nI pedod FllVRe:)nGvd81andald_: Uw Aras Abeokm _ Time I: 4,97 Ol MI 296 m 499.0 14olu11ml4, 20.0 Quant Thn_ Mm.Wldlh Mull. Wldm Due. Wl_h RT Win. (uai) 16.0 5 6 40 aO 8_mnoToUi 31 Jo Ama 2SlO66 Height 236"/3 Time 3.61 EndlTml 4,28 kVeorst_ Width FUna_n'nnm IntegrationType 0.67 3.13 A. B8 "rue.oeo & 200o 10:40 10o 0@ 80 7O 60 In,..ens,y=25OOO0cps 60 4o 30 20 t0 2t8 3_! eD .tOS. . 167 108 _ C " 4.'I " 8"I t h " 161 ""_'*'_ _1 228i1I 8ran 0,119 1,36 _D_ 2.69 3.38 4.03 4.70 Tklne PfOS_I 8TD 0,500 lag I.L/L 4675A-0110 -1 ! 4.Zin _ pe_od tmoas kdomal8tanclard 1:4.g7 01.296-427.0 Hobe Thras. 2.0 Quwd _ 1.0 Idln.Wk_ g Wide1 30 MUlLWldm 6 Height 141549 8tan TmJ EndTJme w_'ononfms, w_ IntaOmsk__ 2.44 2.9S o_.so A- 68 Tue, 0o(:6. 2000 10:40 1;_--- :1 ._ intensity':._SOGOcDps llZ o"! " 4s"o1," s'J _"o'tTdla.'2l"J_a_i'0" 12.6z_2_" 2,zist o" 2d_scan 0.60 1,_0 2,0e l.OO 3.,10 403 4.7OTime Figure S. A rcpresentativc ion chromatogram of a low-level (0.500 pg a.i./L) Pcrfluorooctanes_0nate, Potassium Salt (PFOS) standardfor frcshwatcranalyses. (monitoredmasses ---499 amu (PFOS - top) and 427 amu (4HPFOS internalstandard- bottom)). Wildlife International, Ltd. - 76 - PI=OS_S 811)500 uo 8JJl. 467A-011D -15 4.Mm1ped_l b_nvd StandUr_4Hi=F_ Lira Mini Absoh_eRetention_ 1: 4.I? Q1 Mi, 2118scans 499.0 No_e "llwm;. 2O.0 Qk4ukintW.t T_hthins. JAdLWdh Btse. ',Mclm m"w_. (:euJ) Smooth 315.0 a 4O 8O 1 Area ,_S05786 Height 227264 Grad 11mo 3.81 End'tlme 4.28 RIInIkIlIg_r_ TIi_rol a0.I6:7I l"u_ Dec5. _ I1:10 100. IO 8g 7G I0 SO _ :ZSO000ps 229 j _ Ill 1111Zl _51_1 201 241 217_4 PFOS_| STD i.00 -11 Id./t. 4675A-01!D -15 4.m in : ped0d use "-' Inlemd _8ndttd Tta, DecS. 2000 1t:10 :" intense. 2S0000 ciss 1:4.9701 Mt, 298m 427.0 14oi_Yhre_ 2.0 Ouant"llvu. 1.0 Mn, Wld_ 3 MulL IMcN1 6 BIIm. V_II_ gO 'i1 152 _ IQ511782 He/0ht lS$897 Staff Time 2.44 End Time 2.95 IntegmtionW'_tb Retent_nl_m_ kv_0,_on _ 0.50 _ A-Be 01 S0_ 81 1081ILL 207 236 267 u " 4"1 " I'_ " ti5 " s61 " 161 " I45 " :t61 I.II t.3e _ slug a.as 4.os 4.70 Tlme Figure 6. A representativeion chromatogramof a high-level (5.00 ttg a.i./L) Pcrfluorooctanesulfonate, Potassium Salt (PFOS) standardfor freshwateranalyses. (monitoredmasses .- 499 ainu (PFOS - top) and427 ainu (4HPFOSinternalstandard- bottom)). Wildlife International, Ltd. - 77 - Anm ._040 !1000' r|OOO _SOOO 12000, DO00 8000 _O0 _.o' 'o._o' _1._o"' 1_" "_o' "sJc_"'s.bo""i"._o'"4._'"' cmle. Ul;ida. Figure 7. A typical calibrationcurve for PerfluorooctanvsulfonatcP, otassium Salt (PFOS) in fish tissue. Slope = 4552.41; Inte_:pt ffi69.58; r = 0.99940. Curveis wvighted(l/x). (monitoredmass = 499 ainu--*99.1 ainu). Wildlife International, Ltd. - 78 - 100 9(Y 8& intensity:,25Q0 cps 6_ 5& 4(_ 2(> I0 220 iI | v' i i 41 81 121 1_1 201 2,tl 281 0.6g 1.36 2.03 2.70 3.37 4.04 4.7t Time :.- Figure 8. A representative ion chromatogram of a low-level (0.500 _g a.i./L) Perfluorooctanesulfonate, Potassium Salt (PFOS) standard for fish tissue analyses. From low-range (0.500 to 5.00 Ixg a.i./L) calibration s_t. (monitored mass: 499 ainu --*99.1 ainu). I Wildlife International, Ltd. -79. 100 9O 8O 7O6050 intensity: 30000 ops 209 2 183 !o s.o g3 _22142 287 "" ' 4o"..1" e1'.l_" _21.o3'1621.7"0 23_.137' _41.04"24_.17___,._ Figure 9. A r_)rcscntativcion chromato#am ofa ld_-l_l (50.0 _$ a.iJL) Pcriiuorooctaaos_onate. Pot___,'iumSalt {PFOS) smadardfor fish tissue analyses. From high-rang, (5.00 to 50.0 tag a.i./L) calibrationset. (monitoredmass - 499 amu -.99.1 ainu). . i II I Wildlife International, Ltd. - 80 - PFOS_7 454A-I 34" MAB.1 Tue, Oec 6, 21100It_2 4.min1petad InW_l Glmclm_,.U.ml:OS UmAma Alarum i:lmmUonTime 1:4.87Q11dl, ;U)8m 4119,0 Noke "thces. 20.0 QuantThins. MIn.W_h MulL YAdlh Base. wkm RT Wtn.(m_s) 15.0 3 6 4o SO 8Fm..oxopUte_cr_ Anm 0 14_OIV 0 StUnTime a1 .w 0O EnUTime iRneletegrn4doi.__Wldth Int,gr_ Type 0,00 O0.J0O0 0 lOCI. 8O` 80. TO' 60` SO' 4O" 30, 2O 10. SO 12 _ , 4o1.m " .....1..,1 e1'.!._ 12tl.o2 _ ,59. ,91 224248 1_6..ta z_61 2_4_ 2r_oOo 271 ' 2641.7o TI__n PFO8_7 dS4A-|34- MAB-1 4.98in 1 I)e_od useas h_rnal 8W_latcl Tuo, Dee 6. 2000 11:22 1: 4.fr Q11dL29Bin N_ 11WU. 2.O Quant_ 1.0 4ki2n7..0_AUth $ l;_/:_n,.,. _ RTMAn(.eKS) 20 smoom t I s0726 81anT/me ?..44 EndTime _M I 2.66 tn_ns/Sy:;_0(mo cps Figure 10. A representative icmcl_omato_ramof a fxeshwatexmatrixblanksample (454A - 134- MAB - 1, dilution= 100x). The arrowindicatestheretentiontime of Pertluorooctancsulfcmte, PotassiumSalt (PFOS). (monitoredmasses ffi499 ainu (PFO$- top)and 427 ainu (4HPFOS internalstandard- bottom)). Wildlife International, Ltd. -81 - PFOS.II 414A-134- ll,kS-2 Tue, 10eS, 2000 11:34 4,1m[nI J FFOS Inwnd Slandald4: H:'FOS UXbNe__ R_ T_ 1:4.B7Q1 MI,_NIm 489.0 "llvU, 20.0 OMu_anWlTkhllrle_ Mull _ BlU. _ RT_n. (secs) laS.O 4 40 30 Smoollt 1 100' 90 80' 7_ 60" 5_ 4 _u- IntledlF. :S0000 =p, 229 92SI7 $llJt Tlmll 3.61 End'llm ,I.28 Rmr_on 9_95049 _ Jr,_mtim'_jpe A._s 2 ,o A 11! .... 3S 4o1.u lg? 'Qs '" '" i e"i " '_iI " I11 " _'61 2_1 " 22i,i, Scan 1.s _ e.e_ =.,Is 4.o; 4.'to PFOS II 454A-134. MAS-:_ 4.g8 k'l I pedod 4.1m0S usoaslnlenudStar,an/ Tue, Dec 5, 2000 '11:34 :. r_wwny: _ o_ 427.0 Nob* lhml. 2.0 OuantTlv_ !.0 k_. WkSh S Mul. Wklh | Ease.W_ $0 _l 1109335 l_t 1_g3P_ Slid Time EndTkm Inl, waUonWkZh m_.ln_ 2.43 2.93 O.SO 2_s :1eO] I_3 1 c! s4so . I_ _ 20s =40 =17 Figure ll. A rcprcsontativion chromatogramof a freshwater matrixfortification samplo(454A-I34MAS-2, 0.500 mg a.iJL nominalconccnitaiion,dilution= 250x). (monitoredmasscs ffi499 ainu (PFOS - top) and427 ainu (4HPFOSinternal standard- bottom)). I II I III Wildlife International, Ltd. - 82 - 10090 80 70 80 intensi_y:2. 500 c;)$ 3(_ 111. 10 3_ s5 e,o., 183 19_21__9 ' " _1 " e'_ 1_ "lil " 261 " 2_ " 26i s_. 0.69 1.38 2.03 2.70 3.37 4.04 4.71 Time Figure 12. A representativeion chromatogramof an edible fish tissue matrixb!Anksample (454A-134- E-MAB-1, overalldilutionfactor= 100 L/Kg).The arrowindicatestheretentiontime of Perfluor_ulfonate, PotassiumSalt (PFOS). (monitoredmass= 499 ainu _ 99.1 ainu). Wildlife International, Ltd. - 83 - Intensity:.2500 ops 100 9_ 8O- 7e 6e 50 413. 211- O- 16 _0., 1._100 _1028_ 218.7__0818_732z1,,_842.1_o42_47..471r_m Figure 13. A rqx_seatative ionchromatogramof a nonvdiblv fish tissue matrixblank sample (454A134- N-MAB-I, overall dilutionfactor = 100 L/Kg). The arrowindicatestheretentiontime of PcrfluorooctanvsulfonateP, otassiumSalt (PFOS). (monitoredmass = 499 ainu --*99.1 am,). Wildlife International, Ltd. -84- 1009O80, 7O 6@ 50 40 30 intensity: 3500 ps 212 10 188 0 29 54 73 135 161 41 81 t21 101 201 241 281 Scan 0.6,9 1.36 2.03 2.70 3.37 4.04 4.71 Time Figure 14. A rcpresmtativc ion chromatogram of an _liblc fish tissuc matrix fortification sample (454A-134-E-MAS-6, 50.0 mg a.i]Kg nominal concentration, overall dilution factor = 20OOOL/Kg). (monitored mass = 499 ainu --,99.1 ainu). II Wildlife International, Ltd. -85- 1009O- 7O lntenslty: 3000 cps 4_ 210 I 183 olO 28 .s, _85_2_a41s7 51 284 41 81 121 161 201 2 281 _ " 0.69 1,36 2,03 2.70 3,37 4.04 4.71 "time Figure 15. A representativeion chromatogramof anonediblefish tissue malrix fortificationsample (454A-134-NMAS-8, 100 mg a.i./Kgnominalconcmlration, overalldilutionfactor = 50000 L/Kg). (monitor_ mass = 499 mnu---,99.1ainu). I III Wildlife International, Ltd. - 86 - p_'ls s 454A-134- Tuo,Dec5. _00 t!:56 4.eeIn 1 peded Inienm_landa_ 4HPJ:QS Llm_ Nmo_ ReWt_n"llme I: 4.1;7Q! MI.2M,m 4N9O9i.s0"erives. 20.o QuamTlvm. M_.Wkm MulLWidlh Baset.Mmh 15.o 3 6 4O RTV_. (sees) 30 8_nwvrsMh qI .eo Area 3551"F'3 H4(gN 33188 SlattTime 3.63 EndTime 4.30 k_Om_n1W1m_he __eL'8/ 3 100. 8_ 70" 6(_ S_ 4G SO" 2010 ss 0 ! 8 .A_ . Imnsi_. _ _s . 124 . I es . _ 2229 .: PFOS_13 3 454A-! S4- Tue, Die S, 2000 11".S8 4.U i. 1 pedod 4-F_S tamu I_emai8W_lan_ _,may:. 2so000c0e I: 4.97Q! U,.298scans 427.0 Tlvm, Z.O GuanTt hr_ 1.0 k_ Wknh 3 Mull _ 6 RTWin.(,sacs) ZO 8,moam I sq.x,_ wr ,,s: Are4z 1084547 He_ht _mS4Z 81W1Time EInlnedgTrM_Imon Wldlh neu.vion_mu _0.-5,0S _ 19_:_rJ, 5 183 11 ,f4,_.._.. Q_, lS0 L 208 242 276 "' " 4't " s'_ " 1_1 " 161 " =6_ " =4_ " =i_ Sem Figure 16. A representativeion chromatogramof a freshwatersample (454A-134-3, dilution,, 100x) from the 0.10 mg a.iJL Izeatmmtgroup.(monitozedmasses ffi499 amu(PFOS - top) and 427 amu(4FIPFOSinternalstandard-bottom)). Wildlife International, Ltd. - 87 - In_nsi_. 3500 cps 100 gG 80 7ff 6_ 50 4O 30 2fl 4e lse _ - 274 iI s e - .... .... e 41 61 121 161 201 241 281 Scan 0.69 1.36 2.03 2.70 3.37 4.04 4.71 Time ._ Figure 17. A representativeion chromatogramof an edible fish tissue sample (454A-134-E-30, overall dilutionfactor = 1420) fromthe 0.10 mg Li./L treatmentgroup). (monitoredmass = 499 ainu --,99.1 ainu). Wildlife International, Ltd. - 88 - 100" 8O70 60 50 4.0' . intensny: 3000 ops 210 , 45 131 _ _ 262. 285 41 81 121 161 20! 241 201 Scan 0.69 1.36 2.03 2.70 3.37 4.04 4.71 Time Plsure 18. A representativeion da'omato_am of anonedible fish tissue sample (454A- 134-N-30, overall dilutionfactor- 2650) fromthe 0.10 m8 a.i./Ltreatmentgroup).(monitcgedmass 499 ainu -,99.1 amu). .89 - ProjectNumber 454A- 134 Appendix 4 Sponsor: Test Sustance: Test Organism: Dilution Water: Temperatta_of pH of Waterin the Test Chambers Uptake Phase 3M Corporation PFOS Bluegill,Lepomis raachrochirus Well Water Uptake Phase Mean M_ut_ Day0 ,,. Day7 Day 14 Day21 ,, Concentxation Tempt (rag a.i./L) _'C) pH Negative Control 22.0 8.2 TempI _'C) pH 21.9 8.0 Temps _'C) pH 21.8 8.0 Temps CC_ pH 21.8 8.1 D_28 Temps CC) pH 21.9 7.9 0.086 21.9 8.2 21.8 8.0 21.8 8.0 21.8 8.1 21.9 7.9 0.87 21.9 8.2 21.8 8.0 21.8 7.9 i 21.8 8.1 21.8 8.0 _ _Continuousmeasurementsof temperaturrean_edfrom20.0 to 22.(Y'C. Uptake Phase M_m Measur_ Concentration (raga.i./L) Negative Control Day 35 Temps CC), pH 21.8 8.1 ,, Day 42 Temp' _C1 pH 21.8 8.0 Day 49 Temps _C_ pH 21.9 8.0 0.086 21.7 8.1 21.8 7.9 21.8 8.0 0.87 21.7 8.2 ..2 _2 ...... i s Continuousmeasurementsof temperaturerangedfrom 20.0 to 22.0C. Measure,,mentdsiscontinueddueto 100%mortality. Day 56 Temps _C_ pH 21.9 8.1 21.9 8.1 i ii ProjectNumber454A-134 - 90- Appendix 4 (Continued) Sponsor. Test Sustan_: Test Organimm: DilutioWnatca: TemperatureofpH of Watu"in the Test Chambers DefuratJoPnhase , 3M Corporation PFOS Bluegill, Ltpomis machrochi, us WellWater .................. Uptake Phase Mean Measured Da],0 IIIIII I DII ayo I I II IIII Dayl4 I Da), 21 II Conc_mtration TempI Ternp1 TempI Temp1 (raga.i./L) {_C) pH Negative Control 21.9 8.1 , (C) pH 21.8 8.2 (C) pH 21.9 8.0 {_C) pH 21.9 8.1 I I Day28 TempI _'C), pH 21.8 8.1 0.086 21.8 8.1 21.8 8.2 21.9 8.1 "_Continuous ramsi,,lul _mnentosf temperature'mn_edfrom20.0ito22.0C........... i i 21.8 8.1 21.7 8.1 mm ii ,i ii Hi mH i ill Uptake Phase Mean Measured D'_ 35...... __ ,, Da_',42 , Da_ 49' , ..... Des/56 Concentration Tempi (mga.i./L) _C) .pH NegativCeontrol 21.9 8.1 TempI _C) pH 21.9 8.1 TempI (C) pH 22.0 8.1 Tempj _C_) 21.9 pH , 8.1 0.086 21.8 8.1 21.9 8.1 22.0 8.1 _Continuoumseasu_mts oftempei rmlaJ turi raen_cdfrom20.0toi '22.|.ll.0..C.. i 21.9 ni _I i : PVildlife Infernational, Ltd. -91 - P oj tU.m 454A-134 Smtmme: Organism: Water: AppendSix Dissolved Oxygen (rap'L) of Water inthe Test Chamba_ t 3M Cotp(a'ation PFOS Bluegill Z_pom/a machroch/rua Weft Water Day NeBa//v_ C.mtrol 0.086 m_ a.i./L 0.87 m_ a.i./L 0 8.2 8.2 8.2 1 6.8 6.8 6.8 2 7.4 7.2 6.9 3 7.3 7.1 6.5 4 7.3 7.2 7.3 5 7.3 7.3 7,1 6 7.3 7.3 6.8 7 7.0 7.0 7.0 8 7.6 7.6 7.4 9 7.6 7.4 7.4 10 7.8 7.6 7.2 11 7.7 7.6 7,5 12 7.5 7.3 7.0 13 7.8 7.6 7.1 14 7.3 7.1 6.4 15 7.5 7.2 6.7 16 7.5 7.3 7.4 17 7.9 7.8 8.0 18 8.0 8.0 8.0 19 7.4 7.5 7.5 20 7.8 7.8 7.7 21 7.8 7.8 7.8 22 7.8 7.6 7.7 23 7.8 7.8 7.9 24 NC 2 NC NC 25 7.5 7.4 7.4 26 7.5 7.4 7.6 27 7.6 7.6 7.8 28 7.3 7.2 7.4 29 8.0 7.8 8.0 30 7.6 7.4 7.9 31 7.8 7.6 8.0 32 7.5 7.4 7.7 33 7.8 7.9 7.8 34 7.8 7.8 8.2 35 iw 7.5 7.4 8.1 dissolvedoxygenoonoentratioonf 52.mg/L represents60% saturationin freshwaterat22C. inadvettentl_tnot oallected. I i I II Wildlife International, Ltd. - 92 - t_oj_Nt _mb4_S4A-134 Sustancv: Organism: Water: &opendix5 (Continued) 3M_o_ Dissolved Oxygen (m_.,) of Water in the Te,st Cbamlx:rs' PFOS Bluegill,/.,epomLt macAroch./rus Well Water up'_P_, ......... D3a6y N_ativ7c.8Control 0.086 7m.6S a.i./L 0.87 n-_ a.i./L .... 37 7.7 7.8 -- 38 8.2 7.8 - 39 7.8 7.6 - 40 7.8 7.8 - 41 7.8 7.7 - 42 7.4 7.2 - 43 7.7 7.4 - 44 7.3 7.0 -- 45 7.8 7.4 - 46 7.8 7.2 -- 47 7.6 7.3 - 48 7.9 7.6 - 49 8.0 8.0 - 50 7.5 7.2 - 51 7.6 7.5 - 52 7.5 7.1 - 53 8.0 7.9 - 54 7.4 7.1 -- 55 7.g 7.2 -- 56 7.8 7.4 - 57 7.7 7.4 - 58 7.4 7.4 -- 59 7.6 7.4 - 6O 7.6 7.6 -- 61 7.6 7.6 -- 62 7.6 7.6 - dissolved oxygen concenb-ationof 52. mg/L repres_ts 60% _m_tion in freshwaterat 22_. inadv_, notcollcc_..:..,.... International, Ltd. - 93 - Pom Numb4e5r4A-134 AppendixS (Coutinm3 Dissolved Oxyl_m (mg/L) of W_r SIxmsor." 3M C.apta_oa Substance: PFOS in the Test Chambers: Or_ Bluely, Lepom_ macrochirus Dilufica Watch. Well Water i i Day I?_t_afi_ Pl_e, , Negative Control 1 %5 2 7.6 3 8.3 4 8.0 5 8.2 6 8.0 0.086 m_ a_iJL 7.6 7.7 8.4 8.0 8.2 8.0 7 8.4 8 8.3 9 7.9 10 8.0 11 8.0 12 7.9 13 8.0 14 7.9 15 7.6 8.4 8.4 8.0 8.0 8.0 8.0 8.0 2" 7.9 8.0 16 8.1 8.2 17 8.0 8.0 18 7.9 8.0 19 7.8 7.8 20 8.2 8.2 21 8.0 8.1 22 8.1 8.2 23 8.4 8.4 24 8.4 8.4 25 8.1 8.2 26 7.5 7.6 27 "/.9 7.9 28 7.5 7.6 29 8.2 8.2 30 8.0 8.1 31 8.3 8.3 32 8.4 8.4 33 8.0 8.1 34 8.3 8.4 35 8.5 8.5 dissolved ox_[:en concentrationof 5.2 m[/L represents6% saturationin freshwaterat22C, ,s I lI [ International, Ltd. .94. v_omNu=_ 4S4A.t34 Appendix 5 (Continued) . Dissolved Oxygtm (.re#L) of Water in the Test Clmmbcrs' Sub_,,,_" 3M Corporation PFOS Org_ Bluegill,/,_/_m_ macroc_rua Water:. Well Water Day ' D_umo_Ph_ '" _,,o c,_-ol ...... o.o86'2m_g'_ 36 8.4 8.5 37 8.2 8.1 38 8.0 8.0 39 8.0 8.0 40 8.1 8.2 41 7.7 7.7 42 7.8 7.8 43 8.2 8.3 44 7.8 7.8 45 8.2 8.3 46 8.1 8.1 47 8.6 8.6 48 8.4 8.4 49 8.5 8.6 50 8.1 8.1 51 8.6 8.6 52 8.1 8.1 53 8.2 8.2 54 8.4 8.4 55 8.2 8.3 56 8.2 8.3 dissolvoex_T,_,ecnono_trafioofn5.2mg/L_'pre.um6_% __m__ationfni'eshwaatte2r2C. /Vildl e International, Ltd. - 95 - momN ,, 454A-134 Appendix 6 HardmwaA, lkalinity,Conductivity andTOC of Water in the Negative Control Sp(msor:. Test Substance: 3M Corporation PFOS upt Test Organis_ Bluegill Lepomia marrochirua __ Dil,utima,Watcr: WcllWatc* Parameter 0 7 14 21 t-I_:TJ_css 132 112 126 130 (mf/LasCaCO3) , Day 28 136 SH 'l ,, 35 42 49 132 10]l 134 ,, 56 "130 Alkalinity 183 174 179 183 181 183 183 184 181 (mg/LasCaCO3) Condunfivity 310 325 330 330 325 325 310 320 330 0mlhos/cm) TOC <I <I <I <I <I <I <I <I <I : CtL) .... Sponsor: 3M Coqx_ation Test Substance: PFOS DepuratioPnhase .... Test Organism: Blu_gillL,epom_ macrochirus DilutioWnater:. WellWater J Pmameter Day , ! 7 14 21 28 35 42 49 56 I-I_md_'ss 134 110 126 124 130 132 132 128 126 (mgtLas CaCO3) Alkalinity 179 178 178 180 183 180 175 179 178 (m_/L asCaCO 3) Conductivity 330 330 325 320 325 325 330 320 325 (Umhos/cm) TOC Ill (rob C/l.,) <1 <1 <1 <1 <1 <1 <1 <! <1 I II I Wildlife International, Ltd. - 96 - Numb4e5r4A-134 Appendix 7 Cumulative Mortality and T_-Rolat_ F__octs_ Negative Control- U_.,ta_ Phase .., Sponsor:. '3MCorporaficm Substance:PFOS Organk_: Bluegill Lepomi8 macrochi_s Water: Well Water Cumulative Numlm" Nmnbcr Da_ ,, 0 1 2 3 4 5 6 7 8 9 10 1! 12 13 14 15 16 17 18 19 20 2t 22 23 24 25 26 27 28 29 30 31 32 33 34 35 ObscrvcEdffects: AN = Obs_'vafion..s..i AN AN AN AN AN AN AN AN AN AN AN AN AN AN AN AN AN AN AN AN AN AN AN AN AN AN AN AN AN AN AN AN AN AN AN AN al iDead () 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Remaini._ 90 85 80 80 75 75 75 75 70 70 70 70 70 70 70 65 65 65 65 65 65 65 60 60 60 60 60 60 60 55 55 55 55 55 55 55 ........ Nnmber Sangled 5 5 0 5 0 0 0 5 0 0 0 0 0 0 5 0 0 0 0 0 o 5 0 0 0 0 0 0 5 0 0 0 0 0 0 5 Wildlife International, Ltd. - 97 - Numb454A-134 Appendi7x (Continued) CumulativMe ortalitayndTreatmmt-RelaEtefdfect_s NegativeControl-UptakPe hase Spons_'. 3MCorporation Substan_: PFOS Organi,m'_: BluegilLlep,omia macrochirua Watt: Well Watot Cumulativ Number Numbe= D_ Obviations Dead m_.,._._ 36 AN 0 50 37 AN 0 50 38 AN 0 50 39 AN 0 50 40 AN 0 50 41 AN 0 50 42 AN 0 50 43 AN 0 45 44 AN 0 45 45 AN 0 45 46 AN 0 45 47 AN 0 45 48 AN 0 45 49 AN 0 45 50 AN 0 40 51 AN 0 40 52 AN 0 40 53 AN 0 40 54 AN 0 40 55 AN 0 40 56 AN 0 40 57 AN 0 35 58 AN 0 35 59 AN 0 35 6O AN 0 35 61 AN 0 35 62 AN 0 35 Obsorvod Effects: AN = A_ Normal Numb_ sampled 0 0 0 0 0 0 5 0 0 0 0 0 0 5 0 0 0 0 0 0 5 0 0 0 0 0 i 10 ,, I I WildlifeInternational, Ltd. - 98 - Numb4e5r4A-134 Appendix 7 (Continued) Cumulative Mo_lity n.d Trcatmmt-Rolated Effects I 3M C_on Substance:PFOS N.e_ativc Control- Uptake Phase Org_mlm1: BluegdlL,epomJamacnx:hirua Water: Well Water II I Cumulative Number Number D._ Observations , 1 AN 2 AN 3 AN 4 AN 5 AN 6 AN 7 AN 8 AN 9 AN 10 AN 11 AN 12 -* 13 AN 14 AN 15 AN 16 AN 17 AN 18 AN 19 AN 20 AN 21 AN' 22 AN 23 AN 24 AN 25 AN 26 AN 27 AN 28 AN 29 AN 30 AN 31 AN 32 AN 33 AN 34 AN 35 AN O_'rvexi'Effects: AN = Appea=rNsormal *Biolo_aileo,bservatinoontsrccamieodnth/dsa_. Dead 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 o 0 0 0 0 0 0 0 0 0 0 0 0 Renminin_ 25 25 25 25 25 25 25 25 25 25 25 25 25 25 20 20 20 20 20 20 20 20 20 20 20 20 20 20 16 16 16 16 16 16 16 ........ ........... I II III Number .,, Sm.,pl_l = 0 0 0 0 0 0 0 0 0 0 0 0 0 5 0 0 0 0 0 0 0 0 0 0 0 0 0 4 0 0 0 0 0 0 0 , , , Wildlife International, Ltd. -99- VroimNum4b5e4rA-134 Appendix7 (Continued) CumulativMeortalityandTre_,m,mt-RchEt_ffects NegativCe ontro-lUptakPe hase $_ 3M Cotp_on Substance: PFOS Ors_i._" Bluegill, LepomLvmaovchwua Water: Well Water Cumulative Nmnl_'r Number i Numb_ Day Observations Dead 36 AN 0 37 AN 0 Remain_ 16 16 Sampled 0 0 38 AN 0 39 AN 0 40 AN 0 41 AN 0 16 _,. 0 16 0 16 0 16 0 42 AN 0 43 AN 0 44 AN 0 45 AN 0 46 AN 0 47 AN 0 48 AN 0 49 AN 0 50 AN 0 16 4 12 0 12 0 12 0 12 0 2' 12 0 12 0 12 0 12 0 51 AN 0 12 0 52 AN 0 12 0 53 AN 0 12 0 54 AN 0 12 0 55 AN 0 12 0 56 AN 0 12 12 Obm'v_EffectsA: N=App_Normal I Vildlife International, Ltd. - lO0- Proj Numb4e5r4A-134 Appeadix7 (Cendneed) Cumulative Morality end Treatmmt-Related Effects ! Spoeaor: _: 3M Corporation PFOS 0.086 mg a.i./L - Uptake Phase Orsmi_n: Bluegill,/_om_ _crodarua iWater: Well Water i Cumulative Number Numbcr Day O_ervatiom Dead ,Remainin_ 0 AN 0 90 1 AN 0 85 2 AN 0 80 3 AN 0 80 4 AN 0 75 5 AN 0 75 6 AN 0 75 7 AN 0 75 8 AN 0 70 9 AN 0 70 10 AN 0 70 11 AN 0 70 12 AN 0 70 13 AN 0 70 14 AN' 0 70 15 AN 0 65 16 AN 0 65 17 AN 0 65 18 AN 0 65 19 AN 0 65 20 AN 0 65 21 AN 0 65 22 AN 0 60 23 AN 0 60 24 AN 0 6O 25 AN 0 6O 26 AN 0 60 27 AN 0 60 28 AN 0 60 29 AN 0 55 30 AN 0 55 31 AN 0 55 32 AN 0 55 33 AN" 0 55 34 AN 0 55 35 AN 0 55 i ii ]i i Observed EffIectsII: AN = Appears NormalI I ,,, Number Sampled 5 5 0 5 0 0 0 $ 0 0 0 0 0 0 5 0 0 0 0 0 0 5 0 0 0 0 0 0 5 0 0 0 0 0 0 i5 II Wildlife International, Ltd. - 101 - Project Numb4e5r4A-134 Appendix 7 (Continued) Cmnulativc Mortality and Trcalnu_-Rdated Effects _ S_. 3M Corpor_oD Substance: PFOS 0.086 m_ a.i./L- Uptake Phase., Orgmi._n: Bluegill,/_pomis maeroe_rus Water: Well Water Ctmmlative Numb_ Nmnber Number Day Obs_=vations Dead 36 AN 0 37 AN 0 38 AN 0 39 AN 0 Rcmainin_ 50 50 50 5O Sampled 0 0 0 0 40 AN 0 50 0 41 AN 0 50 0 42 AN 0 5O 5 43 AN 0 45 0 44 AN 0 45 0 45 AN 0 45 0 46 AN 0 45 0 :. 47 AN 0 45 0 48 AN 0 45 0 49 AN, IX I 45 5 50 AN 1 39 0 51 AN 1 39 0 52 AN 1 39 0 53 AN 1 39 0 54 AN 1 39 0 55 AN 1 39 0 56 AN 1 39 5 57 AN ! 34 0 58 AN 1 34 0 59 AN, IX 2 60 AN 2 61 AN 2 62 AN 2 34 0 33 0 33 0 33 10 ObservedEffectsA:N = AppearsNormal.X = Dead. ..... Ill Wildlife International, Ltd. - 102- Project Numb4e5r 4A-134 Appendix7 (Continued) Ct.nulativMortalityandTreaUnmt-RelateEdffect_s , 0.086 m_,a.i./L-Dc_tion Phase ..... Sponsa': 3M _ation Substance: PFOS Organism:Bluegill, Lepomia macroehlrus Water. Well Water Cmn_ative Number . Number Day Observations I AN 2 AN 3 AN 4 AN 5 AN 6 AN 7 AN 8 AN 9 AN 10 AN 11 AN 12 -* 13 AN 14 AN 15 AN 16 AN 17 AN 18 AN 19 AN 20 AN 21 AN 22 AN 23 AN 24 AN 25 AN 26 AN 27 AN 28 AN 29 AN 30 AN 31 AN 32 AN 33 AN 34 AN 35 AN / Observed Effects: AN = AppearsNormal *Biological observi ationsnoti recordedon this da_. Dead 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 Remainin_ 23 23 23 23 23 23 23 23 23 23 23 23 23 23 18 18 18 18 18 18 18 18 18 18 18 18 18 18 14 14 14 14 14 14 i 14 mR ii i i Number Sampled 0 0 0 0 0 0 0 0 0 0 0 0 0 5 0 0 0 0 0 0 o 0 0 0 0 0 4 0 0 0 0 0 0 0 allll Wildlife International, Ltd. - 103 - ProjNumb45er4A-134 Spot. Substan_: Org_,_sm: Water:. Appmdix7 (Conttued) Cumulative Mortality and Treatment-Related 3M _on PFOS 0.086 m_:a.i./L- D_uration Phase Bluegill, I.,epomi_ macrochirus Well Water _.lA_ve Effects I Day Observations 36 AN 37 AN 38 AN 39 AN 40 AN 41 AN 42 AN 43 AN 44 AN 45 AN 46 AN 47 AN 48 AN 49 AN 50 AN 51 AN 52 AN 53 AN 54 AN 55 AN 56 AN ObservedEffects: AN = AppearsNormal Dead 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 Rem.inln_ 14 14 14 14 14 14 14 14 10 I0 10 I0 10 10 10 l0 I0 10 10 I0 lo Sampled 0 0 0 0 0 0 0 4 0 0 0 0 0 0 0 0 0 0 0 0 ,19, Wildlife International, Ltd. - 104 - ProjectNumber454A-134 Substance: Organimn: Wa_. Day 0 ! 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 Appeadix 7 (Contiaued) Cumulative Mortality and Treatment-Related Effects i 3M Corporation PFOS 0.087m_ ai./L - Uptake Phase Bluesi]l , Lepomis macrochtr_ Well Water Cun_lative .... Number Numb_ Observsti_ AN AN AN AN AN AN AN AN AN AN, IX AN AN AN, 1R AN, IX AN, 6R, 14X AN, 8X AN, 6X AN AN AN A_hl AN, 3R, 5X AN, IR, 4X AN, IR, 2X AN, 1X AN, 5R, IX AN, 6X AN, IR AN, 3X AN. IC,IX IC,IX IC IC IC IC IX Dead 0 0 0 0 0 0 0 0 0 1 1 1 1 2 16 24 30 30 30 30 30 35 39 41 42 43 49 49 52 53 54 54 54 54 54 55 R.emaininwa 90 85 80 80 75 75 75 75 70 70 69 69 69 69 68 49 41 35 35 35 35 35 25 21 19 - 18 17 11 11 3 2 1 1 1 1 ! ObservegElffectAsN: = AppearsNormal,C = Lathar_iRc=,Lyin_onBottomX, = Dead. Nm_b_ Sampled 5 5 0 5 0 0 0 5 0 0 0 0 0 0 5 0 0 0 0 0 0 5 0 0 0 0 0 0 5 0 0 0 0 0 0 0 " I Wildlife International, Ltd. -I05- Numb4e5r 4A-134 Appendix S Changesto Protocol This studywas conductedin accontanccwith the approvedProtocolwith the following changes: I.Amendment:Theproposeedxlm'/nmmlstaratndterm/nat/doantewse_'aeddedtotheprotocol. 2. Amendment: The frezlumcyof light intensitymeasurementswas addedto the protocol. 3. Amcadmc_ The frequencyof TOC _ was addedto the protocol. 4. Amendment: The methodologyfor TOC mea, s_ was added to the protocol. 5. Amendment: Storagestability QC samples vccrcadded to the protocol. 6. Amendment: The protocol was clarifiedto indicatewhen fi_5would be sampled for lipid ,-olysis on Day 0. 7. Amendment: The internal_fcrmce standardwas _ded to the protocol. 8. Deviation: Dissolved oxygen was not measured on Day 24 of the uptakephase. 9. Deviation: Test tomporaturowas out of rangefor approximately2 hours. :" I0.DeviatioTnh:e0.87m s e_iJtLesctoncentrataidovncasealfyfecttehdetestorganisms. II.DeviatioBni:ologicoablservatiownes_ notrecordeodnDay 12ofthedeputatipohnase. 12. Doviation: Fourfi_h were collected fromeachtreatme_ on Days 28 and 42 of the dcpurationphase. 13. Deviation: The dcpuration phase was terminatedo_ Day 56. 14 Doviation: Fish in the 0.87 mg oA./L lzea, tmmt groupwere not collocted for lipid analysis at the end of the uptakeor deputationphases. Wildlife International, Ltd. -I06- ProjoctNuml_454A-134 Appendi9x Protocol_, ts andDeviatiom , i II Illll I Wildlife International, Ltd. - 107- Project Numb4e5r4A-134 PKOTOCOL Pm,_,FI.UOROOCT_NATI_ POTASSIUSMALT(PlrOS):. A FLOW-'rHP.OUBO0[HOCONCI_TRATTIOESNTWITH BLUBOIL(LLepom_mm:muc_nu) U.S._t PmedimAp, mcy OPPT_ 850.1V30 sad O_Outldm 305 envirmmmtt_adxxmmSya_a Nms_ uzn3 Submiuatoi 3MCoqmutim l_vilmmm__ry 935BushAveaee .' St Pm,dt_. sma 5S144 Wildtife International, tSN Cmmm_Drive Sut_va,yla_ 2_6ol (41os)22-s6o1 Ltd. Octobe1r8,2000 I Illll Wildlife International, Ltd. - 108 - Project Numb4e5r 4A-134 Wildlife InternatioLntda.l, -2- ?_FLUOP.OOCT_NA.TK POTASSIUSMALTtYF0S): A FLOW-TI'IZOUG_ mOCONCI_t'flIA'noN TEST THSBL_ILL (_po_s mam.o_vm) s_o_on= 3M le.O.BoR_J'J_I SPONSOR.'SRlgPR]_I_'_TATI_: Ms. StomaA..Beach 'W'ddlr_azmmimLalUJ. _91l Commecxe, IX'ire t_u-yUm2d1_t x_x. Dmusr LAJ_RATOR.Y MANAGm_I_qT: Hazy O. r,xucp., Ph.D. Dh_torofAquafiTco_ & Noa-Tat_tPlants Proeo_ Dam: lrOR t,_OIO.TORY USE ONLY ...... Stsa Din: _o.: Tcstt'_ qSq-A -/_q- Ne_iwrnm_lLo.! r_ S_mmzHo.: 4675 _ T_ Dsm zml 1.0maLL/L _ 14o0. i'_Ii_ s'rt_ D_I DA/T_B,p//oo . PKOTOCOLNO,:4.54/10t|00/BLU.810_'UB4_ Eavirmmm_ LmboratmRycqucstNumbcr1,12"/23 I Vildlie International, Ltd. - 109- IIII Numb454A-134 wn_- _ IHTI_D_C'TION IzdLwiBemd_ a I_mememem__t _b _heId_giD_nt_h _ mq_,_. "he m_ wm tz paeem_ lined m lt-,-.,_ _ US. V.m_mmud Pro_ Asaw Sam 85O- _ Sffa=sTea GaJddimoF]rrs Numt_ 8.50.1_0(1); _STM3m.dl_ Rl02_r_.$mda.d/,mce_J(bTrmmbaq_/Ik_m:ca_:.r'_ _,_ FLs/aa,rnd _W_ _ _ (2); tad 0GCl) 0middimfor T_ of _ 305, _m_.: ,_-_b,,qk_ Ta*0). hwdmtcrsllm_pa/imcdmW',Jdl_tm_a- _d,I_ md amp_dk _al_ wilb_u_ by_ _ _aazcb__ m b w_ie rlmtim_ Ltds. ire._ _tm Jdmm_ tzcet_ tohespecif_ _.theflintrep_ rboOt_ _ dds:u_ ot t_.r_um_tm_ mnc_oddr_. OBJECIWE is to ob_ tdmmmydam_ tbcblocm_tmlm 0znna_ tda_ to u PPOSi)n thebl,,qsillr,-pomps _ v_l_ _ c,cmlsdt me sat _ I_q'I_IME,KrALDlr-qIGN _ _ t_ embryos ae_a o_sa_o_ze_ Ba_Ueup_ w_bup sosRpuci:m_y4S0ipznud'Ask t_msss in(_cbr,l:am_. tn_mmtgroupwsUlI_zqxmztlombleth_Mm_mtnsfi_oIm_OS,wh_ fisihsthea_ml _ w_gbccssxmtdo,m_,-rotor".l=dmmCmao_tba._o_s_u=0 _,_.So_8dm_)md _ _os _.w._ Tbm wdmswm_ min_d dmm:.__ ,qmlmrm cmmat0_).the4q,usttm ,_ am_ 0_. _ bi,,_ biocmamSa_tbcm(l_ m:dtb_stmty-stmt/oamm,t,',_ MATERIALASNDM]CI_ODS Tut Salm_ "rigrestsubstm_willIx:_N_zaneJdg_sm. PotudmaFairb. aesftcrn_tb_dto as I_FOS_, onthodmmctmbat_t.az:tm4 ocr_..._subJtmmesis mquindhyGood PROTOCONLO.:4_WIOISOO/BI..U-BIO/SUE]m_$d4mmnm_fM _ Nt,qnbU_ 2723 l' rildlIinfteernationaLlt,d. -II0- ProjecNtumb4e5r4A-134 WildlifeIn l, Ztd. -4" _ _s_ sub,_ C(7,_."neSpmuois,,cqxxwg_, _-_ _ Immat_ml. Ltdv._m_nv_,,_,,a,,__ teasubu_mhasbmadmw_ aeee_ _ Qt_s_ac w _,a_t_,.ef lbestndy,lf ve_m verikaemofOLPt_t mbemmdwaetexiza_ is netpmvidetdo W'd_ lm_u_ml, Ltd.,ttwalbem_d tathee_aanee mUawntefthefml rqx_ _ Spm_ is_d_ _ _ _ _ m b mt mlm_ce,inck_8 the retmfieoaf t tam'renmpbd gholotw batchd h trot_ usedi_g_isstudy.TimSpeansor admuwe=toaazptmy unnusorudtsdmanNud_ teataulbatm_;.,_u mmbsle8ntthecad etenmd_,. PreparathomfTest(3memtrafiom TimtoJtndmmmwia _ odmbistautlo ehetm _pa_uJ ia _ter. Tt_ muted admiaistraxvmm ndec_ _ it rept'mclsthe mintlikly routeef ezpea_ to aq_ti_ oqp_. A pehna_atocitselu6onof PFOSwillbe pzeptarebdy tnmfert'_8the PFOSto nt vohmmm_dmcsmk l di_ ton mpp_ _dmnw_ itkdilm_nws_. Tk ta__nhm usmiml thism_ winI_ lhob1_g31l_,x,edama'md_,m.Bl_m mo ofthe,--.-._..".-.-f:isshpeci_ersuu _,bloeanam_ tats (1.2,.3).Dluq_ willbe esqn'aanu that_f_t:rfrm ab:_t t_.,,_,, th_ m'abotm_ tokst tanpaatmat t _ meam,s_ _'C ta,rr2_ _ t_ts_, xemmu_ .___ S%,_uue_b_4a'anwn_ed mme_te__ dmtin.ememlmbatceht"fishwigbee____e_rkid _ _ _ 14- dayimi_!tom I]_ tl_yamt,em-. ,__. FahwillI_ imxll_l-. litde-- ima'bleb, ,t whmhaedlinisitm_'. it will1_do_ aue_. pe_. ud q,t_. To mind bla,.ashwla_ mmvedem bd_m_tma_withnm and impattt_ydlaaibe_ _ en _t ctumbeml,_teedterfomuet blahasneexpectetdo tebettheresulU PROTOCOLNO4.5:4810180QOBIJJ-B_ Ea_mamentstLabe_teayltequestlqumberU2T'7.3 Wildlife, International, Ltd. -111- ProjNt umb4e5r4A-134 Wz'zgc x z, r_ ___ _-e,_v_._ slze _i_d tm oqt_ vn'aso _i_ bT_ qmaity ef timm __ ",udrm _m._ ef aatduet t,mdtg; _ln fetal wet weiOt ef fish 1_ etm"ef tm __,_,,_)'Inet_oeulI_ Ip-aomfrisikNrlitoefrmlntim_satl_nsmtl_ a(mr_ m 24 hours. lJlu_i_nIIbe fmlIWm feederaaee_ _ fml,__-'d_a_i_|y_. B_IwelI_ _-'.-a-f-__m_tlnmh ,vv,,.._..3k0bm_utcsatkrfmb,. _llqs ml smpli_ _ wl] be_ m ht fahwin_ mmekd attrotfe__em _ feedt___ ----:J_-__le_ el umamlmm in fah _rm' st,,,vuact bern _u_a.,a ELm. themam m tm_ levd.ud emtmim_ mumtbb, e_:_l _ b inmm ia tho _a dat am umside_ m interneewi_hh lmpme m eemlua ef'dte_. Watt*" Wmt_uml fro.k heldiq ml _ efbl_Ol wal be _ f_m t,_U 40 mmu__le_ h_a_l a _ W'ddl_ lammn_ml,L,_Lsi_ 'r'_ wa_aw' ill bui_ _bm_ha sandf_t_md l_umpeldetae 37.gOO-mUem_ Umk _em the_aterw_nbe_ __ _.,y--_P_r.iorto_edmwaterv_llbet_mite0.45mimonketormmvuempaaideWsa.ter ami _r holU,_ aedtmh_ _-darmeri_ as_ ka_L T_i_ va_s r_ _. _y _ 17o ' $3 _'_v. pH .,,,a _ _.,a.,.,.,... will be mea.um:d_ to azalter mmhtm_ _"_ wa mte_. Mma md raal_ e_ em msmm tma_ _ e_e _ee_ S_ gumt_ _ tm wia be ptw, ,_ inew emt n_m. Amty_ wia _. t_r_a_ ,.*lint eeee amm_ to dJn_m a-. _ _ sdeutedorgaak mndi_ mmemm ef _e wU v,_r ,,,.a rmOaat'thea_t m OLPmalysm willbe mmmm'im_in tt_rmsl _upea. $918mnamd'ramCond_t'L_a A _..e.,o--,et..l_awdilul_will I_ uml._olmovidtewow,_m _ IL_OSanda _m_a_L 11m_ efdilmm v_m ta matmi _4 t:mmm_tgmt_ _ltl be _lpdued by mtM_teaL A W,eOTOCONLO.: ,LT,,OI0_S0e_L.U-BXO_t_4_I_n,kmmema_i _ Nu_be.rU2"J23 Vi/ildliIfneternationalL,td. - I12- Numbe4a54A-134 WildliIfneternatioLntda.l, _ pz_ _Y k v_edm hr_x _,,,,_ mbmam ,mdd_ fm:k _ dkmO_rlbm_ _ mr_ _ _ oocm. tam t _ _ _ _ _. i_ay _ c,_,m- _ m_ _rk_4B mJdkm_m v_l lmirJtlm__ k_s_ U km_ _r _ r_ _m_.l_p_m wOmmmpl_ _,. ,,,,_ mt d_d_r wiNImm_ml m kU tv_c_c _dfy_ _ d -?._@Jm_ otM_ is,_ _ dm_m b_$ate mm_d_y_m_ Stskdmsmd aqmm_mt,_withappmzhnm_80 L _"t_. w'd_ wflll!_ usodm test Tat dinl_m willImplal:ailn &wltmb- alhtomabtaJanlcstInupa'also_f_1t ! "C. "I_ m_ dmmlm__mi_ bi wia_me_lo_d hs8pim_hm ht_d m tmlw_ _ _ ". Ambi_mi_wm_pmvi_l__d_,_,,at w_.kmg_ nhm'lm-.m-,_ m_i_ {q. C_mm_ 50_ cq_ah_0. A pimmi_kxlof I_ houm offi_ah_tdIkxm oi'daud_mImI5omo_ndle_dntah_a_xmS_t_. A 30-m_ _._ p_ni,oodll"owI_g imtam__ Iz pmvid_1xdl un_ IlOm o_c_tto' r,'okl_ c,_-- Tm mt _a _e_v_ml_ m ulm_md a_ pht_ o_ _ _m_ismup TI_ t_t _ ndmal will mZram, init_, a"otbawmalvmdy _cS tbo ._r,_,L,__T- Iz tesat mx_nlimswinbesdectakl _ wt tl_Spm_ ml s_llbslined m m_ ordmsm tmicit_din. Thotwoe_am _ dsxdd_Jf_ebr3,mk,tcr _ I0. PROTOCONLO.:4.54/i@iSO(OLU-DfO/SU]BP4..5m4dronmmtudL,zbmR_"alutmC_ I.,127"_ i i I Wildlife International, Ltd. - 113 - 4 A.134 Wjllife lntemaonal, Ltd. f _ui_aF_n_:_(Igools_im_ _0,_ z._ miq a _ thmmm_. 1"mpm,tm_ _I _ mmwod in_t_ _ _ at_ _mS m/-._ d _ tm m/ .t _ _mb _mS _ tm urns _-i_0m Dimdv_ aa.jBmvn'lbl ommmndd_lym_vco,trotdgmbgrdm_Sfl_ t_t miq a Y_ Sp_ _mtm_s Modd $IB dirtied mAgm m_er, zr _h_t,_ k tl_ _ _hat d/_l aa,/gmk._h _ Wiow60"Asmm_im._ s,_m _ll be.-t-- a/h_ommlta_a _ h _._. _ di_l _ii bo_do i_ ,mr/t_t _ attimbq_,_ ml ml oftl_ _ .. and8t_fldy _:_rab duringd_ l_t _/g_ a Irul_rPo/_.,_tModd915pHn_'. or equivalmt. _ o_._._/, ml _ _iI b_mm_l ia b omt_2 _,em_t 8t_z bcsim_ m/ Wa_wad Wm.'mt_r (4). '_.9___.q_/yMll be mmmmlu_8 aYdow Spwp _Madd Gl_'va6mm_ behaviomr ad_. willbemadedm'_.F_ts _cst_ _ _d va/_ motUdita_ndalr_0mabldmvimd"_oddn0tbesppu_t blmm't_han10%_d_ ff.dlb.d_et_m_'umt or _omtzol iF,,,,.,,,qm. _ntum dUptm_ _qume "_ _ q_mm d_ _tt_ _,_ I_,_ (u)i, nm u= n._k2b, utnot_tu 3.0&2, width it equiv.dmt _o 95 perumt _ stm_ am_ "I_ _ flaw will ce"_-- m_il d_ cmmmm_ _t I,FOS _..-- _mhod,/timum _ stcmdy..esmeis thnt _br_ _mam_ti_ PFOS nmldu__ from ea_ _hm. mmdy.mm_ 3"_ cx_e fro"8mtam_ o_ in Fmh,mu nuxstatis6m_ dil_mu_ PROTOCOLNO.: 454/10 I800/BLU.81C)/SUB454 EJwtroemem_Lmbo_tm'7Request Sundm" LI2TJ3 , I II I - Wildlife International, Ltd. - 114- Proj ,,m 454A-134 W'i!d!_fe International, Ltd. Tw tlmze h mml_ a _ 111 fm _mut95pm_t mmvd dtlffi _ _ (_- 0._. ho_'_. _ _p_ Ome _! _ milltin _mmti_ d PFO$midueimk o_ffii_m _ _ am dfln fi_i_ dffimmulitim_ L_ rink ima-- l_dm_'y_ _ 2, F_dm ill I_owb LO_w 3. s_C6O)_d_hM_. _ Ittern_ forWmr tadlrmlCt ated_ FithmSwing_ t mmp_ Jo tm tim ftw,_,_ dm-kldtoqmtg l_am md_ _ duri_tboq_a_m kuod_ tt_ a _ m_ _. k _,_-_ -,,a tnatnt_r will k mq_d m km dm t_ffi p_ _ _iJion of t_ W _ dm_m m _ h zcmcmtmims4mimd.ia du 8bsmmog kgomatimonthebiosccumdatmpcemtiadl thetrot ndstauceandhxndiabkph)5_bemicadl aisfromwhichbioe_m_-._- _ maybo ,atinud,_thde.ngomm=demd unqp_.,n:bSodluilsan fz&lo_,s" -_ "Sm_pt_o'" wm _ _e ,, , , x o X - ~4bom l_ay X X X X 3_ay 7 x x X X : 14 X X 21 X X 28 =, x x tawmii= t x x $ x x ? x x 10 X X Addfud_ Tmmm_ , , tO x xm _05. Additiamal mpk8willbedmuuu noaemyk_ the_ ofpH.In/dnm. "nepm_ _ _ Wm_me uSS_ _eceen_ _OS ._J.o" ._ _pt_e euuuu_ymeddm;--x_.-.,_--a. b 5bedmstim_ dopuratpimintoasp_ u. PROTOCONLO.:4.54/101JOOAILI_-BIO_IEJa9t4v5i4rmmuLmstbaol eutI4D_._luaHtvmb_1"ff2'l_l rildlife International, Ltd. - 115 - Numb4e5r4A-134 DrildlifiIn ionaLlt,d. T_ W mS Yxu_le_ of I pmpe_ ot d)o_8 ndmtanoferom_ intovds my te emit _ d_ _sed m _t_ rm 8a t_ 4c_e4 mdTor _ esd_ nmplb8 kmn81otw wlr amplm uUl tffimUn:mifkumabocmtml md d_e _ 8mplu v_i !_ _ fnuudu WOS gnmunt 8mt_ Tin PFO$emmmtim _in be _ Ihra aialmm t om a_di_m eta _amd amlmu al_n_s x ea_ t'maa_ _:I k _r_.m-,_d_ sam_sdram hr PFOSmstT_ wii I_ bdd in r_s_ mt u_, !_ am,',_edm h _ of k Study_ or(:_m_q, l_d In_dl;mr. T_ mn_mot P_OSin mo _ m a_ll__ _:_t _1 1_ t_id u d_ avmSod d_ nai_ _, as _ -n.-_,,, _ samp_ u_dl te drawnas nu:msaryfor lbe dt_mJnationd_pit, tmdness. amdumn_restoU,d_8n_uubm(TOe). ]_shSmprms To mm b FFO$mmmci ca. tmm_. _ka_ r_ _IL 1_ _x_ w _ I_ oamul group. Oas additlmalsm_ fa_ uu:hStouproll _ _.____h,_ md I_ ia maem md _ uua_wa_ ort'l_OSInIsh at m_ mq_ lam'val_ ta lxmm_ as th_a_ _ _ F'u_mnple4Gx"ck_ d ]flK)So_m_ vn'UbebnlPeni_lnymov_ _mmtbo_ea dry)mdt_a__e_tb_.-,_, Sd_wiU_ _*-,,,-_ wi_n _ ts m_sesadcmliSecti_fn, Fm_k_ SadstL_km_gbo_ im oditdmo d mmdZldd, amo-,-a a5ow_ _ _ mc_ dssuioSactio(ni.o.edk'bukds ,,._._rdc)wslI_ nxa_d. Fishvfll boso_ i_o_mif uc naJysod iuixlia_y. PIt.OTO_OLNO: 4_,IIOISOCCBLU-BIO/SUB4ESu4vitmmaDlLabmatoryRapx:uNt umberU2'F23 Wildlife International, Ltd. - 116- Numb4e5r4A-134 _rildlife International, Ltd. - 10_f_ad mgo_ _n b__m _ mcemmy_c _ _lysb ot Y_ _m',_. And_ MlCk_ w_ _ _ _ _ __ _ _-os _ _v_ _ sl_:aua_. Wm _ re'Itk mmtTgd=m'e_tot_ mm_t ,'.._u'd"AattyumMl mlmd Vatid=i= for t_=]_=mimtim =r l'm'_sm=am=Stafmi=Acid.t_m.ummSatt (PI=OS) l_mnnr_=s==.,nm,=-,,a._uo=na_aar=(w- =_ k=am=i===L_==J'=#= Z,b.4.5_-.-L09_ mq_ _'_ I= ==d,p=d=,,_== ,= b__ "Anht,_ _ vslidmi= fro.b 4.q4C-119)_. _ dcsar_ _stboJzd s_z_ tothcaba_ nzdmddol_ wJl bc _ ink nnvdm _1 T'nmeL_t Cmt_ Ata "_'_"-. r_s walbcsmnpkdr_m tb_co,m_JqJ'_m g DaY0 (uPoausm_ d/_ m=_,ia)a. t==i=,=r_-,=mtl,=PIFO=S=mat_i=(=him n=sd==,lml_ =a=(o=rf=aowi_ 2t _ _ mq_.me.)m, a at t_ md_rt_ dCpur_,_tpme_eredr,mht_t t= nmpt=tm==mm=gmmr mmplm.".--_ti=mtmltad t=,=tnat=l,quari=atmghoftimimmv,=l=treed. TimKpldmmtmo=f _ _,,,,-_mmpmn wint_ _ b,/_ =am=ira. UVta,_t_ (s}. RI_O]IU_I"OBEMMNI"_ IP.=o_mbe_ b=rm_=d_ _=ra.,_gmmt_ atW'd411l=fe===_o=_Ltd._ i. Amlpy_'tl==_S,=pd,r.,_,.._ Z id==tifi=,tim-,.a_,motth=t=t=utnm_.if_twa,,_Spomo= 3. Dat_oF_ md_ofthe test. 4. "r_ ,=,_=_mb=_U=,.=-, _ _. includbeut_t ". PROTOCOLNO4.:54_IOI|00/BLU-BIO/SUBF45.w4dromna_md Rcqucstl_mb_U2723 Wildlife International, Ltd. - 117- Numb4e5r4A-134 4rildlife International, Ltd. - II- 6. _ oVmm_km. 7. Wmr _ _ (_., lumUm _h_nity). S. OCpaUmu_Ut am Im_UmummmU. 9. The umb_ u._eclm md_ ummmmB. _ut mlumee ememmim 10. Stat_i_ c_udaJm. II. A col[_db rmal mpull. _l _e _n_ _" _ gIIqALRgJPORT A _xxt d mn ,_ _ d_u_ m_ _'U be _pmRl1_ W___'__mmlm_ laxL "nwnpo, _vilL_-_,a- Im uQtbc runi_ _ du idkm_ t. _ u_ _u_u _ _u:a_pc_mq _ mu_,. 2. D_esouwh_tbestndywssini_micoaq_ kistlz_dthoSponsorto pro_de dssfinsJdsmth_ detsm maxdedr_ chemiC, Imi_4oSysuccormppoma8 ev_ tbZ m_ t_ 8az_ ntodsal_---:-- _Pm_mSmdm_ s. s_ n_od_mp_,_ _ d_ 6. "The_, _n_rd ml n_race mbmuc_ u_,,,_ bymine, _u_h_l d_uu_ uumbee_r :. mk _. mmO_._, m_ _ oro_r _ _um_, ifprovi_ _. s_y m_ yamw.k.vm_ _ eondm_'_e m_, _ _luUi_ _xhe_st.mindand _mm mamamm a_ _ mudi_m _ a_u_ma_m, i_ pmvk_ _y _ ._cmsor zr enmu_ _ WlklU_inUmmim_ t,Ul. s. A _ _the,nuduxh,a,uL 9. Ad_e_d_so_s_--a WS,_al_5_d_mt_p@,_,i,dl_Jodome to. a __em _ _ _m, _,_m_ 11. A dea_p_ _'ad_lumstancet8hamty havead_ _s_ thequalio__i,_ _h_ dst_ PROTOCOLNO.4:..q4/101_0/_UB4_4 Ruvimmum_ _i_iu_Numb_ Wildlife International, Ltd. - 118- Numb4e5r4A-134 Wtla/ e IntarnaaonalL, td. - 12- 12. The t_e of !_ Sludy13i_ter, the m ofOl_' seimlittge_, a_l dieI_1 d 13. A deNt,il:dim_L"k _ aleulslio_ _ opmlie_ perfonmd oa t_ dala, t mma_ mim_sh era..-din.mls _ of__ _ fiua_ m_b. 14. "tim silnnd_d da_ _Nwts ut cad__"_ _ _. _ elbar_ invol_d h ea _'y. e',v_:a,tL 15. "I'aeu,_._ _t aa q_ktem, rim,da_ mtem fiml mlma m to be anml. i_ am,mm 1_pm_bymsqmn_Ammmm UM*m_q__hodin=m_ _m_fm_m_mml _ weremacml e,_a._mor'mrfmU_ _ ma_erandyDim_ i'7. ILlit k neceuasyto m*t_'a_xsecfi0mm"alddit_mto t RindmJ_ addsk" lau hemscce_ mobSsq_sd_Ltt_ in tbo5_nmd.---_-,,-v 5y b StudyD"_u,____"1. _ -_-___,___,_ ckedy_,,v_rd_pm_d_ rml _po_d_ is5nnipdd_Stoa _m_i_dud_ ms_m_ theconeclimor_l_tia=. AmmJkm_ ,_,. bes_m_ndd dztedby1heStud_Oi__py_,,__. CIIABGaNGOF PROTOCOL fknmdduaSom,_-pnanmivIn_nind_ tanaofwrlttm,-,,._,,_- aillmi1_tt, 8lady Di,m_ md theSi._j_tP.a_pmm_ve_.e._,,,_ _il becemidem_l lint_"theimaecoi n,l wgl_ _ _ e,_r...azzretoo,*_,.yetbacrS_mws m1_izeze_.,,,.otwdu,=dev_lms ;_-,,,b,,yatl_ StudDyk'a:z_ml el_x,wl ithamrawam. j_jz_ _o_ pnxo_wiaI_ ;,,,..,-,,_*;_,d, zfeiezZl lXXt. GOOD1,ABORATORPYRaCTICeS _ mn_wm _ om_mm_m Mam_mmv_h__c_L_s_bomu_Pm_m Sumdm__o_rA j_ M.t._ O9ttaS._ Wmm=t__. st_o.._-,,u,,nd _ ltme_. Eachaudy madm=lbyWddlifleamatim_ lad.i_ mai_ _ bye_ W_w- lamaam_ lad, q_, _mmam Uat _ ma_tm_ _ Oeeataemwy X'_mk_ _ Ot_e_ Pm_m_ mt _ _ pmeoa_Amtm_t ofeoaW_amv_thOo_ _ _e_ .watl_ pa:pm__r d pma,,,-_d_ su_ madua_b_Wa_r-IsaanaSimL_td._1_SW=_ b_abxim (e.8.,midmsml,_esm"psdsoio_).Patwdm fro.,n workpes_med8tW'_m_, _,I._ mdaooio_f_h__mam]pmwt ifbl o_tla11i5nyject_ inasddwtaooa,tm_ d_'Krskgi_bammimd. Ltd.sisr.,m _t ss Jdtamtiv__ to bc sipa:_zJ in dmfinsl rePmt. PR,o'rq:_OLNO.: 45#/101800_LU-BIO/SUB454 _ Labontory P.a:tucsINumb__,F_725 , , Wildlife International, Ltd. - 119- I II mjmNumb4e5r4A-134 i. uS. ]gevlmmmmd hotectka Aipzcy. 1996. Scai_ 850 - F_ Guidegmc(s/_b_D_ oPgrgNtnb_U0.l_0: I_:kBCT. 2. ASTMtStmdzrdEt022.4Ml._g|..._tandard_for_B/ooom:emm_on _ Tat Tern 3. OECD Guiddlu for Testingof CImnkab305. 1996. __on: F/o_-Th_ 4. AXqL4,aww_ WFCF. I_LS. Sta_nl Maho_ for th# _ of Fahw awl w_r. Ifgh_AmmlmnlPM_Hgal_A._glatloL Ame_i_W_Worll_ b____"-_,__W_ Paktim Cczm_Fedmti_ NowYodc. 5. G.E. Bln sndG. L. Af0n.197|. BIOFACT. hoDow_. MidbmdM, _. PROTOCOLNO.:454/101|00/BLU-BgO/SUB45E4 n_anumr_dL_ Requ_Numh_ Yo_ Wildlife International, Ltd. - 120 - Po, j=Nt umb4e5r4A-134 WILDLIFE: I_TIONAL L'm. PRO,I_CNTO.:4.NA-134 _ I _2 AM_IlXMm'_TO_rUDYlqtOTOCOl, STUDYl"rrl_ PF..q.FI,UOR.OO_ANi_UL.u'ONATF.,.SI*,_oT'r_AssIUMAFt.OW-THROUGH BIOCONCI_r_TtO_N WITHTl-mBU.,'F.GI_ mcr_Itm=r) I=ROTOOOLNO- 4._I/10|I00/BIJJ-BIO/51JB4$4 AMEHDMEHI' NO.: I $1=ONSOE:3M _ Plt_ NO.: 454A-134 ' m_'gCtlVl DATi_14om=_ 7.2000 ICNVIROHMERLrAALBORATORYIUIQLI__SOr.:U_TZ$ _: 1'=1=2 Add:r=xp"___,,,,_-__StmD_. 12/5/00 _ Tcmimlfio= ____-_!/1(_1 REASON1=1_#0o_ i_m.udlo,v_ =orI=o_ wl_ d" Stsly_ =ilmdd_protocol. AMENDMENT_; $.Vxte-m=,dTe=_t , P_o6 :" T= Hmdmm=_nk=lim_i._e,--.a._ =md*om*d'm_ mdmcroc) wlmb,mmm=_i=me=mm_tmmnmm b _#=i_ ,.,_==!_tl= trotJ =tmtly i=m_ dmi_d=t=t. R]_.ASOR:Tolqm=_l_ _qmmq, olt'TO_mmm_mm_. AMm'VX_Vam__: Mmmrm==__"q_7 Add: TOC _ll Ix:mmmml mime SIMmml,mMmldTOC-5000TOC_tly=='. RRASON* To _Id I_ m_ fm'TO_ _ (_/l.lc- t,., Il-"l -'oo Wildlife International, Ltd. - 121 - _j_ Numb4e5r4A-134 WILDLIF'E INTERNATIONALL_. PROJECT NO.: 4_4A-134 P=_ 2 or2 AMENIM_NT:AnalyticaMl cdzedP._,otO Add: T'mmeztabili_ samplm,_ll be prepmedatI_ initiationto _blish test substm_e stabilityin M stemtfrmzadu_ study. REASONT: oaddr,_smhitity QCnmpl_. AMIgmMeh,'r: Thsuel,lpldC.smmrlh, _ 10 CZanSA_ ta mbkmmf,ishz_llbusampled_mthe_ntraoql umaaDt ay0... To:Atami_zm, Ish m'llbu_ m thy 0... REASONF."khmepkdm Da,/0farlipid_ _ beeollee_diz_m d_n1_on md_e__. _ m . SPONSOR_'SATIVZ DATZ . Wildlife International, Ltd. - 122- _oj_Numb45er4A-134 WILl)LIFE INTERNATIONALLTo. PROJ_"TNO.: a.._lA-I)l IPqle| _! .OOflqDMgNTTOri'l,_y MIOTOCOL STUDY -[-_-ulg_PEIEOUOROOCT._I20_T_ POTA._IL_SAlT CI'FOS^):F. t,OW-THRIX.q_ m_TIO_ TI_TWITHTI'_ _ _._nows m_toc#_) PRo'rOO_L[_.: 4$4/1011_0/BLIJ-B[O/$U_ai AMENDME_rrNO.:2 _OI_BOI_ SM_ _RDJI_"TNOa 4_tA-I_ _l-l_B DATP.:_ 212_0 I_VIROI_qTAL I,ABORATORYll_Q_ NO.: Lr_7_ ZZ_NT. Pqp2 RRA$ON:To_ldtis:_lcmsln:_tazc stmdad. STUDYDIRECTOR ///so/c_ DATE . SIPONSORI'SU_J'RKSENTAI'IVK _ Wildlife International, Ltd. - 123- Hum4b5e4rA-134 WILDLIFE _TIO_ Llo. PI_OJF.,CNTO.: 454A-134 ]PI_ JO[ i I_VIATION TO b'TI_Y PROTOCOL STUDY TITLE: PERFLUOR.OOCI'AI_ES_AT_POTASS[I.IMSALT(PFO_. AIq.OW-TI_OUGH BIOCOH_TI[OI'I "I1_T WITH _ BIAJEGiI_ (pom/s macroch/rw) PROTOCOL NO: 4541101800_I.XI..BIO/SUB4._4 DKVI&TION NO- 1 SPONSOR: 3M _ PROJECTNO.: 454A-134 DATE OIcDEVIATION:. _ DgVlATION: _ _ _ _vel _ 29, 2000 tl_ db_val _ not _l en _ _11Im_ _ k_ 24 are_eq_lz i_e _ee _lt _t dmnl_. RF_A_OIt: Bidil_t_r_hL AIl_a_enme_Jn_ms,ll_tltr,_t_01C, of_. _, it is tbo best jud_m_ of tl Study Dbector Ihatthis doviat/on did not advmely alrcct _ malts oftl_ study. STUDY DIP.ECTOR DATE II Wildlife International, Ltd. - 124 - _j_ Numb4e5r4A-134 W|I,.DI_IF'_ III'Ea:_IqA'rIONAL.I.-m. PPd3JE-CTNO4.:.54A1. 34 Ptlle I ell I I II DEVIATIONTO STUDYPROTOCOL STUDYTITLE:PERIq.DORDOCT_OHA'[_ POTASSIUMSALT(PFOS)A: PLOWol'HROUOH BIOC_t_EIqTRATION'IESwTrrll _ B_ _f._,_o_ _tn_r) PROTOCOLNO:.454/1011100/BIJJ.BI0/SUB4.54 DEVIATIONIqO.:2 SPOIqSOR:3MCeqmntim EtiO,,I'ECNTO.: 454A.134 DATEOll'DE'_k'i"ION: Febm..r2v4.2001 , D_ILATIO_ TIwlxala_ su_s tel _buu_:sl_ _1 _ pistol in a _Rr b_ mmsimaiaa trot tmnpemtureef224,l_. QnDq 19afdelR'atim,h___read_ 2O'C.Theumpemumma mt dungs tr _ 2 ___m__ REASON:_ Bmxlonthc_x_du-_of'the __.tn, Se,,_/_ thatthi_dev_.___didnetm'verutyte_:t the_ itistlnbeEt_lga,_ of theUu_. STUDYDIRECTOR Mrm, " ._'/a 3/d t DATE DArK " , 11 I I Wildlife International, Ltd. - 125- Numb4e5r4A-134 WILDLIFE INTERNATIONAL LID. PRO,ff_CTNO.: 4.S4A-134 P'_ ! oti DEVIATION TO STUDY PIuYrOCOL STUDY _ PEP.r"iJ,_iR.OOCT_ONAT_ POT_ SALT (PFOS):AFLOW-TH]tOUQH BIOCONO_n'KATIOTN_STWITHTI_BLUP_ILL(_.._,mcm_i,_) PROTOCOL NO: 4_I/101g00_LU.BIO/SUB454 DI_'IATION NO.: 3 SPONSOR: 3MC.mp, madm PROJBCr NO.: 454A-134 DATE OF DEVIA'I['ION: _ 14,200Q DEVIATION: ThoWotaz_simsflu_tbIccsat_ fin mllmima. 11im0.17 m8 LiJL _ scloctwcidlmlotstn_,_".or _ *m',J Snmpwm tmdly d_ _ m bl_ I_ZASON: Ib_mmalloa_, II_ dlm:m_w_cky _PFOS m bluqlll wss _mzlmovmwlam_ _ _ ,_ _I_I. "n,bdm.bdou_ dr_ ,c ,,_ ot tl_ may ____,,- ,d,_,,_ b_m,tim, dlalavmsnot ob,,_,'_ fxmnelm0.ST m8 a.i.A, lacmam B'oup. DA.TlE,,qlot Wildlife International, Ltd. - 126- P_ojmNuml4x5_4rA-134 WILDLIFE INTrr.R_'no_m.. L'_. PROTSC'rNO.: 454A-134 V"_ 1ott DKVIATIONTO STUDY PROTOCOL STUDY TITLE= PI-_.FLUOPD,OCTANESULFONATF_POTASS[UMSALT(PFOSA):FLOW-THROUOH BIOCONCENTRATION TEST WITH THE BLUEGI/,L(Lepom_ mac_.Alruz) PROTOCOL NO: 454/IOI$00/BIA_BIO/$UB454 DEVIATION NO.: 4 SONSOR; 3M _ PROJECT NO.: 454A-134 DATE OF DEVIATIOIq=Fr__ruL1w7.204)1 . ,, DKVIATION: "n_irctocoi stat_s lbal,_,lia_ o/_md motq_'t'y'vdnI_ nutdodaily. _gtead obm,mtiouws_ notre_'ded_l_ 1:2ofd_3umi_ REASON:Biologis_t Biologicaolbm'vafia_msDay1t E! 13ofdcputst_ w_ t]_ mun_ _, irist_ i_tj_m ot't_m@ _a_:tott_ t_ _.oaim cr_no_t _ DATE ii II III I Wildlife International, Ltd. - 127 - 454A-134 WILDUFE INTE3RI_'IION_ LTo. J PROJHCTNO.:454,%-134 _ 1ot] DitVIATIONTO STUDYPROTOCOL STUDYTIT_: PERFIMOROOCTANESULk'ONA3T+POTSAASSLiTI(_FIfO_ AFLOWoTHR(XIGFI mOCON_llON TF.SIw"rrHTi_ _ (/a,_ mcr.a_Ir_r) PROTOCOLNO: 454/I01_IOASUB454 DXVlATiONNO..: SPONSOR3:M_ l_OJ_ NO.: 454A-134 ]_TES Or DEVL_TIOr/:Mm:h5ud 19,2_! DEYLATIOK:Thop._d M _. mE_ad fisuh_l_ cok_ _ dbw __ .tFo.r_i_. smnphs_m each_.b_--l__upmd d.plk_u,m_s kxn b anm_ pu_ O_ xid_kn.d._b hm escpkrop_ k coSa=dmdhdd_ msa_ J.dnq boauly=d_ RFASO_. Rescrvesmptes'u:reaetcdleaJedtoccmu'vethe_off_r,_,_,,,,inlhestudy. bestjudp:mestorthestudy:_a____dut thisdeviatimdidnotIdvmety sffect_ _ Jt_,. ltisthc __ .STUDYDIRECTOR DATE DATE ' " I I I Wildlife International, Ltd. - 128 - Numb4e5r4A-134 WILDLIFE I_TIONAL LtD. PIE)iFC, T NO.: 454A.134 Pase! ofl DEVIATIONTO STUDYPROTOCOL STUDYTITL_ PF.2ICUOP..0OCT/d4F.SULIFONKKnt'_.t.SSKJMSALT0FR)S_,t.FLOW._ Br_CONCENTKATIOTNBST_JPrll TI_ BLUFG, IM,(/.Rmm_rmacro_/,.'u:) PROTOCOLNO: 454/101800/BI.U.BIO/SUI34.54 DEVIATIONNO.:6 SPONSOR:3M r_q3efttim PROJECTNO- 454A-134 DATEOFDEVIATIOINAr:p_ 212001 ,, DgVIATION: Thepeteml mtm thatIw &:pumtiopolanewill oetimmuntiltheoscmUUioaof PPOS sesidmitho m'_dsms attainsayooc _k foiiowiastlzw _-'x-_'__'-"_t_)P,midumseaht._ thin 10%ofmeady.etm_e2r)Reddnm_dlbel_the LOQo. t 3] Stxty(60)d_c_depml_ In ehpe_ Thensideesdidnotmeet1or2jdboyeml_ ef_ wmmminm_onI_ 56 REASON: TheSpem_mqumtedthatnmpl_gec_evcrytwowa:ksdudnsdepumim. ltisehebeu .b_mmt ofthe seudydim_ tha thisdeviatioudidua advu_ly afire _ gsula oftl_ study. :" STUDYDIRECrOlt / DATE ;,//Of ,I i II i i ii i i I| _Vildlif e International, Ltd. - 129- P_j_N_m_454A-134 W! t..DW FE l__ Lm. PRO_ NO.: 454A.D4 I'_,_ I efi DEVIATIONTO STUDY PROTOCOL STUDYTITLK: PERFIJJOKOOCTANESU_ONATEP, OTASS_VlSALT(PF.O$):An_/-TblROUGI-[ BIOCONCI_qTg_TIONTESTWITHTHE BL_.,O[LL (y_p0_ au_o-oo_r_) I'ROTOCOLNO: 4.q4/I0|$00_I.,U.B_54 DEVIATION IqO.: 7 SPONSOR: 3M_ PRGJEcr NO- 454&.134 DATES OF DEVIATION: Fdmmmr$ axl Am']2, 2001 DEVIATIOIV=TimFrctomrlamst_tfl_wkUlbesznplcRsr _nidmalyakatSmKly.m_ _d atlbemd_ STUDY DIRECTOR a,/,_,/o/ DATE Wildlife International, Ltd. - 130 Appendix 10 Project Number 454A-134 Perscenel Involvedin the Study The following key WildlifeInternationalLtd.personnelwexe involvedin theconduct or managemmt of this study: 1. Mark Jaber,Directorof Research 2. HenryO. Krueger,Ph.D., Directc,Aqua/k Toxicology andNon-Target Plants 3. WillardB. Nixon, Ph.D., Din:trot,AnalyticalChemistry 4. Kurt R. lkrottar,SenioxAquatic Biologist 5. Ca_yAS. utherlanLda, bont_ Sepervi_ 6. RaymondL. Van Hoven,Ph.D., Scientist 7. Susan T. Plantania,Biologist AMENDED Wildlife International, Ltd. - 131 - ProjectNumb4e5r 4A-134 Appendix 11 CalculationsOf The KineticConce,_ration Factor(BCFK), The UptakeRate Constant(t0, The DeputationRate Constant(k2), The Half-Life ForClearance,And The Time To Reach 90% Of Steady State. 1. The deputationrate constant(k 2) was determinedfromdata collected duringthe deputation phase andwas definedas the slope of the regressionline where x equals days of depurationand y equals the natural log of the tissue concentration. Resultsof all calculations were generated using Excel 2000 in full precisionmode. Manualcalculations may differslightly. deputationrateconstant (k 2) values 2. The half-life for clearancein tissue duringthe depurationphasewas detamined by the following equation: t,,_=0/t_)ln 2 Half-lives for Clearance(days) [ Edib1le8F6i.s4h Tissue Nn'EdIi1b1l5e.F5ish Tissue WholTe 1isF1sui.s7ehl 3. Theuptakreat_mnstant(kl)wasdetmninefdromthefollowienqguetion: C/k2 k,= C.(]-e-") whereC/istheco_ation offisthissuferomtheuptake/deputactuirovneneartheuptake midpoin(tmeanumc,entrafiofnrsomday35wereusedincalculations); whereC_,isthemeanmeasurecdoncentratiinownater(0.08m6g a.i./L); and wheret-- 35 days. AMENDED Wildlife International, Ltd. - 132 - ProjNumb45=4A- 34 Appmdix 11 (coued) uptake rate constant (k_) values F-_I/1Fb9il.se0hT2i2ssue I Nn'Edi2b4l.Fe0i8shTissue I Whle1F7/.s3hT5issue 4. Itisassumcdthathotwocompamncntt,wo-p_ modelappliesT.hismeansthathetin_ to reach90% steadsytatientheuptakp_haseisaboutequaltothetimeittaketsoreach90% of deputatiionnthodepuratipohnase.The_forck,2,th_deputatiroanteconstanwta,susedinthe followinegquatiotnodetermintehetimetoreach90% steadsytatfeoruptake: 0.9 = 1- e -t2t or t_ = 2.31k 2 Time to reach 90%of steady-state(days) 5. BCFK values (k 1/ k 2) areprovidedbelow. BCFK values I Edib1le1F24ish Tissue Non-Ed4i0b1l3eFish Tissue Who2l7e9F6ish Tissue AMENDED Wildlife International, Ltd. - 133- ProjN=ut m4b5e4rA-134 Appendix 12 ReportAmendment 1. Original Report: Pages 1-8 Amendment: The pages were changed to include the amended report date, revised page numbers, and new signatures and dates due to the addition of the report amendmentas Appendix 12. Reason: To reflectthe issuingof an amendedrcport. 2. OriginalReport: Page 2 and 4 Amendment: The StudyDirectorwas changedto HenryKruegerandManagementwaschanged to MarkJaber. Reason: Reassignment of responsibilities due to the departureof the original Study Dircctc. 3. OriginalReport: Data Analysis Section, Page 15 Amendment This section wasmodifiedto clarifythatthetests for normalityandhomogeneity met the assumptionsof the tests. Additionally,the section was modified to explainwhy thecomputerprogramBIOFAC was not usedto estimatethe kinetic concea_uationfactor (BCFK), the uptake rateconstant (k0, the deputationrate constant0c2),thehalf-life for clearance,andthetime toreach90%of steadystate. However,afterfurtherreview,the BIOFAC programmay not be thebest wayto est_naatt=hese parameters. Reason: Afl=r consultation with the Sponsor it was befieved that the BIOFAC model underestimatesthe uptakeanddeputationrateconstants, andoverestimatesthe kineticconcentrationfactor,theestimatedtimeto reach90%steadystate,andthe half-life for clearance. Therefore, the data have been reanalyzed using the equationsandgraphicalmethodsoutlinedinthedraftOPPTS850.1730 Guidance Docum_t. 4. OriginalRel_rt: Pages17-18 and31-33 Amendment: The resultsandconclusionsections, as well as theTable 6 andFigures 1-3have beenchanged to reflecttherecalculatednumbers. Reason: To reporttheresults of the test based on recalculatednumbers. AMENDED Wildlife International, Ltd. - 134- p,oj_Nt umb4e5r4A-134 Appendix 12 -Continued- Rq:mrtAmendm_ 5. OriginalReport: Addition of Appendix 11. Amendment: Reason: Appendix 11 was added to detail how calculations in the amended report were performed, To provide adequate explanation of how the new calculations were performed. AMENDMENT SIGNATURES: Henry O. Kr_er, Ph/.D_ ." _._'e_Directorof Aquatic Toxicology andNon-TargetPlants j. Mark_l_ -./ '_-- - . Direct6rof Re,_._ REVIEWEDBY: DA_T"E _J_t._-'-_-C__,.__ : DATE/ [ AMENDED