Document ByLaxmdE19YDnMYbe3bNkBJxj

Protocol.P Freshwater .. 11::::I PROTOCOL PFOS: A 96-HOURTOMCITY TEST WITH THE FRESHWATER ALGA (Anabaenaflos-aquae) US. Ewhmmotd ftowdon Agency "@-'Scri8e5s0 -EcologicalEffectsTestGuidelines OPPTS Number 850.5400 3M Lab RequestNo. U2723 submittedto 3M Corporation EnvironmentalLaboratory .935Bush Avenue St.Pad, Mimesota 551.06 WildlifeInternational,Ltd. 9598 Comma= Ikive Easton,Mmylmd 21601, (410)822-8600 September 17, 1999 WILDLIFE INTERNATIONAL, LTD -2- PFOS: A 96-HOURT03aCrrY TEST WITH THE FRESHWATER ALGA (Anabaenaflos-aquae) SPONSO SPONSOR!S REPRESENTATIVE TESTING 3M Corporation Envkon=ntal Laboratory 935 Bush Avenue St PauL Minnesota 55106 RochelleP, Robi&AU WddUt bitemadomLLLtd. $598 Commc= Drive Easton,Maryland 21601 STUDY DIRECTO LABORATORY MANAGEUIENT: Cary SutherlandS,eniorBiologist Henry 0. Kruaga, Ph.D. Dko=r ofAquaticTo)dcology& Non-TargetPlants FOR LABORATORY USE ONLY Proposed Dates: EVak=W StartDate: ProjectNo.: TestConcw.ft6m: TestSubstam No.: 4675 t(0 Everinmientai TaminadonDatw. Rcf== Substm= No.(ifapplicable):4526 PROTOCOL APPROYAL STLj6yDIRECTOR DATE LABORA'TOKY 9ANAGEMWT DATE I s i sp@i"EP7. PROTOCOL NO.: 454/091799/ANA96/SUB454 DA-;?g 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL, LTD -3- IWRODUCRION WildliWf=eafion4 Ltdv.nuconducatfour-datyoxicitteyavnththefreshwataelrgaA,nabaena j7os-aquafeo,rtheSpmsorattheWgdhk bkmatima ld aquabctoxicolofgayciliintEyastceM,arylancl The sb* willbe performedbasedon proceduresin theU.S.EnvironmentalProtectioAngency Series850EcologicalEffectsTest GuidelinesOPPTS Number 850.5400: Algal Toxicity,YlersI and H (draft()1). Raw dataforallwork performedatWil&dc InternationaLlt,dLand a copy of thefinalreportwillbe filed by projectnumber inarchiveslocatedon theVtrildhfIcntcmtionaL LtcLsiteo,r atan altm=tlvelocatiotno be specifieidnthefinalreporl PURPOSE The purposeof thissu* istodeterminethetoxicitoyf PerfluorooctanSeulfonicAcid,Potassium Salt(h=after referretdo asPFOS) tothefirshwateralga,Anabaenaflos-aquae. EXPERIMENRAL DESIGN The freshwateralga,Anabaena flos-aquae,vaU be exposed to a geometricseriesof six test concentrationasnd a negative(culwm mc&um) controlfor.96bom. TargetconcentrationvsaU notexceed 100 mg/L or thesolubilitIyiimtofthetestsubstanceinwater.Generallyt,henomina.c1oncentratioonfewh testsubstanceusedinthedefinititvestwillbe atleast50% of thenexthigherbw=ent, unlessinformation cmcmning theconccatrauon-efficzutrvemdwates dua a chffcrcndtdutlonfactorwould be mm appropriate. Testsolutionwsillbe ino@ with10,000ceKs/mL Three"biologimrreplicatpeesr=Pmimcnw group willbe preparedforevaluatingcoU densitiesO.ne or more additional'analyticarle.plicatewsin be@ includedin the test,-asneoded@to providetestsolutionfor concentratiovnerification. Day 3 of die exposure.Am abiotircephcateatffibcighestconcentrattovnaR be mcluded m thetestand willbe sampled on Day 3 and Day 4. Inordertocontrolbias,thepositionof theflaskswillbe &t=ained by ind@@to draw dailyduringtheexposureperioclNo otbcprotentiaslourcesof biasareexpectedto affirtheresults of thestudy. The responseof thealgaewillbe measured in termsof CeU densityb,iomass expressedas areawider thegrowth curve,and growth rate.An EC50 (i.e.t,he theoreticatlestconcentratiotnhatproducesa 500/o reductioninthemeasured parameter)valueforcelldensitywillbe calculatefdoreach 24 hour mterval ECIO, ECSO and EC90 valuesforcelldensityb,iomass (EbC) and growth rate(FC) willbe calculatedi,f PROTOCOL NO.: 454/091799/ANA96/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL, LTD -4- possiblea,tthe72 and 96 hourintervalsT.he no observedadverseeffectconcentratio(nNOAEC), which isthehighestestconcentratiotnhathxhww no adverseinln'bitoerfyfecton growth,willbe &Immiined relativteoeachparameterat72 and 96 hoursbasedupon evaluatioonfthestatisticraelsultasnd thedoseresponsepatternA.t theend ofthe96-hourexposure,algistateifcfectwsillbe differentiatferdom gilgicidal effectisnthosetreatmentwshich aremaxibmuy inhibitcd MATERIALS AND METHODS TestSub Informatioonn the huactaizadon of testc,ontrolor referencseubstancesisrequiredby Good LaboratorPyracticSetandard(sGLP). The SponsorisresponsiblfeorprovidinWgild[deht=ttonal IAi writtenverificatitohnatthetestsubstancehas been dwadmizod accordingtoGLPs priortoitsuseindie sb*. IfwrittevnerificatioofnGLP testsubstancedwwtmintion isnotprovidedtoI&rildhIfnetem&onaL LtdLi,twillbe notedinthecompliancsew=int ofthefmd report-ne attachefdormIDENTMCATION OF TEST SUBSTANCE BY SPONSOR (AppendixI)istobe usedtoprovideinformationneccss@@qfor GLP compliance The Sponsorisresponsiblfeorallinformatiornelatetdothetestsubstanceand agreestoacceptany unusedtestsubstanceand/ortestsubstancecontainerrsemainingattheend ofthestiidy. TestSolution)LrM The desirecdoncmtrafionsofPFOS willbe obtainedby preparinga singlestocksolutioinnmeffiun anddilutintgheappropriatveolume ofstocksolutiownithme&unl The testsolutionmsay alsobe prepared usingsecondarydilutionosftheprimarystock basedupon theconcentratiodnesired PFOS willbe adm@@ tothetestorganisminalgalmc&um. Thisrouteofadministratiwoans selectebdecause.irtepresentshemost likelryouteofexposuretoalgaew,hichexistswpended inawater column. TestOromism The testspeciewsillbe thefirshwatearlga,Anabaenaflos-aquae.Thisspeciesisrepresenftfiovfe an importantgroup of algaeand was selectedforuse inthetestbasedupon pastuse and easeof handlingin thelaboratoryS.tockcultureso,btainedfrom theCultureCollectioonfAlgae attheUniversitoyfTexasat PROTOCOL NO.: 454/091799/ANA96/SLJB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL, L-rD -5- Austmoranothesrupplievra,n be mamftinedinculturmec&um atWildlifIentmuabonaLLtdLfora rninimilomftwoweekspriotrouseinatoxicitteyslAlgaeusedintoxicitteystwsillbeinexponential growth phase,which isdefinedas dw periodof growth when algalcellsarechvidingata constantrate. Justpnor to beginningthetcs%an inocuhraof thestockculturweillbe preparedso dW eachmilliliter of inoculum containsenough edb tc)provide an initiacleU densityof approximately 10,000 CeLs/mL in ewh replicate. Culture Moditun Culture modiun prepared according to Virildlifbetm-national Ltd. Standard Operating Procedures willbe used as dilufm water. The concmtrations of thecomponents in the medimn arepresentedin Table 1 (2). Stock mitrientsolutionswillbe prepared by adding reagent-gra& or betterchemicals to Vtrildlife InternatLonaLLtd. well water purifiedby revmw-osmosls. Appropriate volumes of the stock nutrient solutionswillthen be dilutedwith purifiedwell water to prepare the medium The medium willb@ filter stmilized(0.22 pm) or autoclavedprior to use. Analyses willbe performed at leastonce amuauy to determine the concentrationsof selectedorganic and inorganicconstituentsof the well water and resultsof the most recentGLP compliant analyses willbe swmnuized in thefinalreport- Specificationsforacceptablelevelsof contaminants have notbe= establishedforculturemc&unl However, there = no levelsof contaminants reasonably expected to be presentin the me&mn that are considered to interf= with thepurpose or conduct of die study. Test ARparatus Test chambers willbe steffle2,50-mL po@ycarbonate Erlmmeyer flasksplugged with foam stoppers and containing 100 mL of test solutiorl The test chambers will be mdisamminately positioned on a mechanicaglmkertablem amc[Lvironnentalchamber and willbe shaken continuouslyat approm=tely 100 rpnl Test chanibm willbe labeledwith the projectnumber, testconcentration,and replicatr-- Environmental Conditions Tea flaskswillbe held at 24 rc under continuous=i-wwte fiwmscmt B&fing atan intensity of 2200:kIOO/o IwL Light intensitywillbe measured at fiveImabons sunvmding the testflaskson Day 0 PROTOCOL NO.: 454/091799/ANA96/SLJB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL, LTD -6of thetest Tempm-au= vnU be measured tw= dady ina containerof wateradjacenttothete.sftlasksinthe environmentalchamber usinga liquid-in-glatshsermometer. The pH ofeachtwtinmt and cmtrolgroupwillbe =&wed attestuntiatioanndtermmtion using a FL-,haAm=et Model 915 PH meteror equivalentSamples forpH measurementattestkitiatiownillbe collccw from theindividuabla oftestsobiuonpreparedforeachft=tment and controlgroup. At test tamunhon, pH willbe measuredinpooledsamplesoftestsolutiocnollectefdrom eachof theduw replicates of each respectivbewbnent"and controlgroup. BiologicaMleasurements CeH densitiewsillbe monitoredduringthetestby conductingcellcountsusinga hemacytometer and a microscope.One samplewillbe collectefdrom each replicatoef thetreatmentand controlgroups at24hour intervaldsurmg thecqxn= penod- 'Me sampleswillbe heldatapproxmuftly4*C untilceU counts can be performed.Each smnplewillbe diluteudsingan electrolystoelutimgsaton(t)a,sneeded,toWaintilin countingaccuracy.A smallamount of each sample willbe loadedonto a hemacytometer and the total number of ceusin 10 grids.wibue counte& Ile celldensityofthe sample willbe calculatebdased on the mean number of ceuspergrU Usingthistechniquet,herniniTniTqmuantifiabclceU.densityis1000 ocUshnL Samples of testsolutionwsillbe collectefdrom each oftheffim replicatepsertreahnentand control group attheend of thetesl Thm sampleswillbe pooledwithintheirespectivtereatmentsa,nd subsamples willbe removed and maminw microscopicallfyoratypicaclellmorphology (eg.,changesincellshape,size or color).Growth of ceus intheflasksalsowillbe assessedforaggregationosr flocculationosf ceus and adherenceof theceM to thetestchamber. At theend of the testa,lgistateifcfectsw,hich resultinthe inhibitioonf cellgrowth,win be diff=nhated from algicidaelffmts,which resulitnthedeathof ceM. Aliqwts (0.5mL) of testsolutiownill be takenfrom each replicatoef thosetreatmentgroups where growth ismaximally inhibitedM.axkwUy inhibitetdreatmentsarethoseinwinch itcannotbe visualldyetm=od iflivealgalceus arepresent(e.g., absenceof colortothe = solution)T.hese aliquostwillbe pooled by concentrafioannd cwuted with untreatedculturemedium toa concentratioonf thetestsubsu= thattheoreticalslhyouldnotaffectgrowdl A negativecontrolwillbe preparedby dilutinag 0.5-mL aliquotfrom one negativecontrolreplicatteo 100 PROTOCOL NO.: 454/091799/ANA96/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL, LTD -7- mL withculbm me&um. Growth of dme subculturewsillbe monitoredfora maximum of 9 daysto detem3inewhetherinhibitionfgrowthobservedduringthetestisreverm'bl-eS.ampleswillbe collectefdor cellcountsatrecoveryphaseimtmtton,termmt:tonand atapproximatel3y-daymtenals betv@ initiation and termination.The recoveryphase may be terminatedonce algalgrowth issufficienttoindicattehatthe algalcellscollectefdi-omthetreatmentgroups have fifflryecoveredfrom effectsdue tothetestsubstance. This willbe determinedduxnighvisualexaminationof therecoverytestsolutions. Snpling forAnabgicalMeasurements Samples Of dkCeq)w= solutionwsillbe collecteodn Days 0,3 and 4 and analyzedfortestsubstance macmtrations.Day 0 sampleswillbe td= from eachbatchoftestsolutiopnriortotheir&tnbution among dke rephcatetestchambers.Day 3 sampleswillbe collectefdrom theektrareplicatea chamber(s)included in thetesttoprovidesmnple forthatanalysisand from dieabiotirceplicatesI.fmore dm one replicaties includedfortheDay 3 s=ple analysist,henthesolutionfsrom eachreplicatweillbe compositedtoprovide the sample forthatbutment Day 4 sampleswillbe compositedsolutionfsrom the0=. replicates remainingattheend of thetest. Samples collectedon Days 3 and 4 may be cmbffiged or fdteredto remove algalceus priorto analysis.Samples willbe analyzedimmediatelyor placedin an appropriatsetoragecontainer(e.g., polypropyleneor polyethylenbeottle)and storeduntilanalyzed.The sample scheme issummarized below: PROPOSED NUMBERS EVerimental Group Control SolventControlCifneed4 Level I-Low Concentration Level2 Level3 4 Level5 Lcvel 6-IfighConcentration Totals 'Includesabioticsamples. OF VEREFICANON Day 0 SAWLES Day 3 1 21 8 9 Day 4 21 9 PROTOCOL NO.: 454/091799/ANA96/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNA-NONAL, LTD The above numbers ofsamplesrepresendtme collectefdrom thetestand do notincludequality control(QC) samplessuchasmatrixblanksand fortificabopnrseparedand anah,=dduringtheanalytical chemistryphaseofthesb*. AMIY-dc-al ChemicalanalysiosfthesampleswLU be perfomiedby Wildlifkewusbonal U& The analytical method usedwillbe basedwm methodologyprovidedby theSponsorand identifiiendAppendix IL Mo&ficationsmade totheanalyticmaelthod vaU be &mm=tod inft raw dataand describeidnthefinal report. StatistiAciaallym Biomass expressedasareaunderthegrowth curvewillbe calculatefdorthehwtinentand control groupsusingthefollowinfgormula: A = ((NI-N.)/2Xtl)+((N,+N2-2N.Y2)(t2--ti)+-.-4@(Ni+N.72N.)/2Xt.-ti.1) Where: A = Area underthegrowth curve N, = Measurednumber ofceUs/mL att. Ni =Mwmtd number ofceWmL atti N2 =Mm%ued number ofceUdmL att2 N. =Mmsured number ofc"mL att. ti= Time offirsmteasurementafterbeginningoftest(houn) t2 = Time ofsecondmcas=mwt afterbc&ning oftest(hours) t.= Time ofnthmmmirement afterbeginningoftest(hours) Growth rates(u)wdl be calculatefdordieuratnent andcontrolgroupsusingthefollowinfgormula:-. InN. -lu14, to 14 Average spedfic growth rate N, = Measured number of ccUs/mL atU Nn = Measurednumber ofcelLs/mLatt, to= Time ofbeghming oftest(hours) = Time ofnthmeasurement afterbeginningoftest(hours) PROTOCOL NO.: 454/091799/ANA96/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL, LTD -9Percentinhibitiownillbe calculatefdoreachbzabnent groupbasedupon mean celldensityb,iomass and growth rateusingthefollowingformul& Mean Control- Mean Treatment Percentbihibition EMonse &MQM Mean ControlResponse x 100 EC valueswillbe determined,when possibleu,singlinearinterpolationr othersuitablWechniques withU=t3nentresponseCLeca dcmties,biomass and growthrate)and cqmm each24-hourkiwval offfitcesl concentratiodnatafor A no observedadverseeffectconcentratio(nNOAEC) willbe selectebdasedon an evaluatioonf the dose-responsepatternand theresultosf the statisticaanlalysisusingindividuarleplicatvealuesof thecell densityb,iomass and gmwth rate.Stafi@y sigdficandtiffereacebsetweenthecontroland dietreatm=t groups willbe idendfiedusinganalysisof variance(ANOVA) and a testto comparc ftutinent-mean responsesto thecontrolresponse(e.g.D,u=ctt!s testor Bonferroas t-test)D.ata willbe assessedfor normalityand homogeneityofvariancespriortoperformingthe-ANOVA. Transfonnafionwsillbe usedto correctany conditionof non-normalitoyr unequalerrorvariancesi,fpossible.Ifa solventcontrolisused inadditiontothenegativecontroltheiresponseswillbe compared usingStadent'st-testData from the two controlgroupswillbe pooled'for13reatinelnetvelanatypcsifno statisticdailfferenceesxisl Otherwise, allcomparisonsvnffbe made tothesolventcontrolgroup. StatisticcaolmparisonsfortheANOVA and the comparisonofmean responseswillbe carriedoutusingTOXSTATO V3.5 software(4)orequivalent. RECORDS TO BE MAMAINED Records tobe mamwned fordatageneratedat WildlifeInteimtionaLLtd.willincludeb,ut not be limitedto: 1. Copy ofsignedprotocol. 2. Identificatiaonnd dwactcrizationof thetestsubstance,ifprovidedby Sponsor. 3. Datesof initiatioannd tmm-iin@onof thetesl 4. Source of algae. 5. Cultureconditions. 6. Growth measurements. PROTOCOL NO.: 454/091799/ANA96/SU]3454 3M LAB REQUEST NO. U2723 WILDLIFE I.NTERNA-nONAL, L-rD -10- 7. Calculationand preparationoftestconcentrations. 8. Observations. 9. Ifapplicabled,e methods used to analyzetestsubstanceconcentrationasnd theresultsof analytical measurements. 10. Statisticcaallculations. 11. Testconditionsand physical/chemicamleasurements. 12. Copy offtd reporl ]NNAL REPORT A finarleporotftheresultosfthesh* willbe preparebdy WildliflentemtionalLtdLThe report willincludeb,ut notbe limitedto thefollowingw,hen applicable: 1. Name and addressof thefacilitpyerformingtheSh*. 2. Dateson which theshi4 was initiateadnd completed.ItistheresponsibiliotfytheSponsor to providethefinaldatethatdataarerecordedforchemistrypathologyand/orsupportingevaluations thatmay be generatedatotherlaboratories. 3. A statementof compliancesignedby the Sh* Directoraddressingany exceptionsto Good LaboratoryPracticeStandards. 4. Objectivesand proceduresstatedin the approved protocolincludingany changes in the original protocol. 5. The testsubs== idcntlficbdy chc=cal abstracntumber or code number, strengthp,urity, composition,and othercharacteristipcrsovidedby theSponsor. 6. Stabilitaynd solubilitoyf thetestsubstancesunderffieconditionosf administratioinf,providedby theSponsor. 7. A descriptioonf themethods used to conductffictest 8. A descriptioonf thetest*sten4 includingthe sourceofthetestorg=Llms and theirscientifniacme. 9. A descriptioonf thepreparationof thetestsolutionsm,ethods usedto allocatoergmiism tothetest chambers and beginthetest,numbers of organisms and chambers pertreatinen@and durationof the test. 10. A descriptionof circumstances thatmay have affocw the qualityor integrityof the dat& 11. The name of the Study Directorand fficnames of otherscientistpsr,ofessionalsa,nd supervisory personnelinvolvedin thestudy. PROTOCOL NO.: 454/091799/ANA96/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL, LTD 12. A descriptionfthehinsformationsc,alculationosr,operationpserformedon thedata,a summary and analysiosf thebiologicadlataand analyticaclhemistrydata,and a statementoftheconclusions drawn from dicseanalyses. 13. Smfisticamlethods employed forana4-zingthedata. 14. The signedand datedreportsof eachof theindividuaslcientisotrsotherprofessionalisnvolvedindie 15. The locationwhereraw dataand finalreportaretobe stored. 16. A --.t prvamd by theQualityAssuranceUnitlistintghedatesthatsu* inspectionasndaudits were made andthedatesofanyfinchngsreportedtotheSt* Directorand Managcmmt 17. Ifitisnecessartyomakr.correctionosradditiontsoa finarleportafterithas bow accepteds,uch changesshallbe made indieform of an amendment issuedby dm Sb* lhrector.Tim amcndmen t shallclearliydentiffyfipcartof thefinalreportthatisbeingamended and dw.reasonsforffic alteratioAnm.endments shallbe signedand datedby theStudyDirector CELANGRNG OF PROTOCOL PWmed changestodw protocolwillbe intheform of writtenamendments signedby the Study Directorand theSpmsoes RepresentativeA.mendments willt@tconsideredas partof theprotocolsad will be attachetdothefinaplrotocolAny otherdmges willbe intheformofwrittedneviationssignedby the Sh* Directoarndfdodwiththeraw data.Alldiangestotheprotocowlillbe indicatemd the&W report. GOOD LABORATORY FRACRICES Thissb* willbe conductedin=arda= with Good LaboratoryPracticSetandardsforEPA (4.0 CFR Part160 and/orPart792);OECD PrinciploefsGood LaboratorPyractic(eENV/MCICBEM (98).17);. and JapanMAFF (59.NohSan,NotificatiNoon. 3850,AgriculturParloductioBnureau).Ea& sb*... conductedby WildlifleawnationalLtd.isroutinelmyammed by theVFddhfcIntemtions.LLtd.Quality AssuranceUnitforcompliancewithGood LaboratoryPracticesS,tandardOperatingProceduresand the specifiepdrotocolA statemenotf compliancewith Good LaboratoryPracticewsillbe preparedforall portionosf thesb* conductedby WdcUifeInternationaLlt,d. The Sponsorwillbe responsiblfeor ...-compliancweithGood LaboratoryPracticefsorproceduresperformedby otherlaboratorie(seg.,residue analyseosrpathology)R.aw dataforallwork performedat)hrddhfe.lntematioLntadl-and a copy ofthe finalreportwillbe filebdy pmjoctnumber inarchiveslocatedon theWildlifIenternationaLlt,d.siteo,rat an alternatilvoecatiotnobe specifieidnthefinalreport. PROTOCOL NO.: 454/091799/ANA96/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL, LTD -12- REFERENCES 1 U.S.Environmental ProtectionAgency. 1996. Series850- EcologicalEffectsTest Guidelines (draft)O,PPTS Number 850.5400: AlgalToxicityT,iersIand U- 2 ASIMStanckrdCAddel2lg-90E. 1990. &wukrdCaddeforConducangStaizc96-HourToxidly TestswithMicroalgae. American SocietyforTestingand Materials.Phd*lphiaP=xsylvania. 3 Norber&)Cing,TJ.-1993. A Linear InterpolationMethodfor SublethalToxicitY.Y-heInhibidOn Concenft-adonac,dApproach (Ver@don2.0).US. Envkom=W hvtectionAgmcy, Research Laboratory,Duluth,Minnesota. 4 West, I= and D. D. Gulley. 1996. 77OMTATRelease 3.5. Western F=Systems TochnoloSy,InrChq=e, Wyoming- PROTOCOL NO.: 454/091799/ANA96/SUB454 3M IAB REQUEST NO. U2723, WILDUFE INTERNA-NONAL, LTD -13- FMHWATER TABLE I ALGAL MMIUM CONSTITUENTS Componcnt Nominal Cowentmtion Mgcl:rOH20 CaCli-2H20 H3BO3 bba2-4H20 ZnCI2 FcCI3-6H20 CoCie6H20 Na2MoOe2H20. CUC12!2H20 Na2EDTA-2H20 NaNO3 MgSOe7H20 K2BPO4 NaHCO3 The PH of tbcmc&un willbe Aust4 NAOH or l(r/oHCL 12.16 M&IL 4.40 mgtl: 0.1856mg/L 0.416 mg(L 3.28 g/L 0.1598mg/L 1.428 gIL. 7.26 g/L 0.012 g/L 0.300 mA 25.50 mg/L 14.70 mg/L 1.044 mg/L 15.QO MA as necemary@to7.5:k0.1using0.1N PROTOCOL NO.: 454/091799/ANA96/SUB454 3M LAB REQUEST NO. U2723 'WILDLIFE INTERNATIONAL, L-rD -14- APPE@DIXI IDENTMCA'NON OF TBT SUBSTANCE BY SPONSOR -TobcCmVlftdbySpmsx L TestSubstanceldcatit(yname tobe used inthereport):PFOS (PcrfluoroodmcSulfonicAcid PotassiumSalt ReferenceStandard(ifapplicable)A:nabdcal StandarcL-N/A L2tmug Standard. 1.12.21LH.FLH PcrfluorooctamSulfonic Test SubstanceSample Code or Batrh Nmbcr Lot 217 Test SubstancePtirit(y1@A/c9tivebgreffient): 98.9 ExpirationDate: 2008 R TcstSubst=Chmmdmizafim Have dr.idmfitys,mdm & x.mtym lccmposomorodxrdmradmlgbm I pw-jdyiv 9 sodrcf=mgmdardbem xxrtD itussemthissk*in aoomda=w& GLP Stmdards? HL TestSubsta= StW4F C03MM pkmi themmmnmded &W maddim atWM& Tnk"&cnal Ld x No Ambiat Has thestabiuyofdketeambs=m undrr&= stww oondidcw bom ddmmin6d inacomdmce withGL2 Stmdards? odff pa6xmt stabryifamaficm. ---LYCS x No IV. TestCanecoftwiaw. Adjustteaemomhafim toIWI. ai x basedWm ffipcuritCylo)Fm abom Do nota43SttUlCMCMhZfiMtD -&i Tcsttbemakdal AS IS. 10(r/. V.- TmiciLybftnadm- ManmiaHm- RatLD50 251 mg&g MousrLD50 N/A AWatic. kmtbmto TodciiyWAJC50) FishTcDd&Y(100) Dohma L-maena: 27 mil Da 2_h_m@amagm: 50 mg!L ' Rainbow Trout-,II M-FJL FatheadMinnow: 38 MGIL Odxr TadcityWamafim Cm&xhog ffidw ofdronicandmbchtdc tc&). Pleasesee MSDS PROTOCOL NO.: 454/091799/ANA961SUB454 3M LAB REQUEST NO. U2723, WILDLIFE INTERNANONAL, LTD APPENDIXII AnalyticalMethod Providedby Spon@" Samples willbe mmlyzw based upon procodum providedby theSponsorinthefollowinganalytical methods: Uquid Ckmatography Mass Speammetry (LCMS) Modidd for.dwDetennination OfPffffimrooetamSuMai@'idd Pdassim Salt(PFOS) InF'rc*watcrS,'dtwatearnd AlgalMedimn * A copy of dieabove method willbe mainwned infficraw datil llc actud methodologyused to analyzethetestsampleswM be domm=tod m theraw dataandsummmized.inthe&W reporl PROTOCOL NO.: 454/091799/ANA96(SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTF-RNA-noNALLTD. PROJECT NO.: 454A-1 10 Page I of I AMENDMENT TO STUDY PROTOCOL STUDY TINE: PFOS: A 96-HOUR T03aCrrY TEST Wrffl TBE FMHWATERALGA flos-aquac) (Anabma PROTOCOL NO: 454/091799/ANA96/SUB454 SPONSOP@. 3M Company EFFECrm DATE: Febnuoy23,2000 AMENDMENT NO.: I PROJECRNO.: 454A-110 3M LAB REOUEST NO.: U2723 AMENDMENT: Page2 Add: ExperimentalStartDate: Febniary25,2000 ExperimentalT=dnation Date:Febmary 29,2000 TestConcentrationsN:egativeControL39.5,61.0,92.5,145,225.an3d45 mg&i./L TestSubstanceNo.: 4675 REASON: The aboveinformatiownas notknown when theprotocolwas siped,by the Sh* Dh=wr. AMENDMENT: EncrimentaDlesim Pait3e Change: Targetconcentratiownisllnotexceed100mg/L orthesolubililtiymitofthetestsubstanceinwater. To: TargetconcentratiownisllnotexceedLOOO mg/L orthesolubililtiymitofthetestsubsw= inwater. REASON: Nominaltesctoncentrationsexceed 100 mg/L. STUNYDIR CROR LAYM RY MANAGEMEENT p ATIVE DATE . D h-?@oo DAYE o2.J.3-co