Document 82n2jBzQNzQjbZJa6mN8Bm2Yo

Protocol.PFOS: A 96-Hou Freshwater Diatom (Navicu PROTOCOL PFOS: A 96-HOUR T03UCITY TEST WITH THE FRESHWATER DIATOM (Naviculapelliculosa) U.S.Fn*omncnW ProtectioAngmcy Series850 -EcologicalEffectsTest Guiddines OPPTS Nunber 850.5400 3M Lab RecpiesNto. ln723 submittedto 3M Corporation EnvironmentalLaboratory 935 Bush Avam'..., St.Paid, Minnesotit 55 IIM WildlffeInternationalL,td.. 8599 Commera-,Drivc Easton,Maryland 21601 (410)822-8600 September 17, 1999 WILDLIFE INTF-RNATIONALT LTD PFOS: A 96-HOUR TOMCITY TEST WrrH THE FRESHWATER DIATOM (tvaviculapelliculosa) SPONSO SPONSOR!S REPRESENTA 3M Corporafion Laboratory 935 Bush Avenue St PauL Minnesota55106 RochelleP, Robideau TESTING FACELITY: W-ddHfcktmatimal Ltd. 8598 Commerce Drive Easton,Maryland 21601 STUDY DIREC 14K SeniorAquaticBiolo& LABORATORY MANAQ-EUCE-NT: Henry0. Kmgw, ]?ILD. Dkwtor ofAquaticToxicology& Non-TargetPlants Proposed Dates: Expminiental StartDate: ProjecNto.: TestConcenhifions: FOR LABORATORY USE ONLY EVaim=W TerminatioDnate: TestSubstm= No.: Referem SubstanceNo.(ifapplicable): PROTOCOL- P STUDY DMECTOR LABORJ?FO-R--Y-iMUTAGEMENT- O'e, - SP 9A9 @ - PIR STOPC*OLMNO.0:.- 454/17Y1/79N9A/VoN9l@A6V/9S6U/1S3U4B54454 riol) DATE Ila DATE . @ / 14 @(e /M DAA 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL, LTD .3- INTRODUCRIO Wd"e Inter@omdI,Adw.ilclonducatfive-dtaoyncityesvtaththefreshwadtieartnNaaiv,icula pelliculofsoart,heSponsoarttheWildlibfteanationLatld.aquatitcoxicolofgaycilintEyaston, MarylanTchLestudwyilbleperformbeadseodnprocedurientsheU.S.EnvironmenPtraoltectAigoenncy Series850 - EcologicaElffectsTestGuidelineOsPPTS Number 850.5400(1).Raw dataforallwork performedatWildlifIent=wowLL Ltd.anda copy offfifcinarleportwillbe filebdy projecntumber 'm archivelsocatedon theW"jldlfhficte=tiomd@IA& siteo,ratan alternatilvoecatiotnobe specifieidnthefinal reporl PURPOSE The purposeofthissui4 istodeterminethetoxicitoyfPeffluorooctaSnuelfoniAccid,Potassiun Salt(hereaftreerfaredtoasPFOS) tothefreshwatedriatom,Nawcula pelliculosa. EX]PERE%4ENTAL DESIGN The fi-eshwatdeiratom@Natdculapelliculosaw,illbe exposedto a geometricseriesof sixtest concentrafioannsda negative(culturmee&um) controflor96 hours.Targetconcentratiownisllnotckbeed 100mg/L or thesolubililtiymitofthetestsubstanceinwater.GenemUy, thenomitfacloncmtrationofeach testsubstanceusedinthedefinititvestwillbe atleast50% ofthenexthighcrtreatmentu,nlessinfmmfion cmcm=g theconcentration-efcfuercvtemdwates thata chffcrcnctWutLonfactorwouldbe mom appropnate. Testsolutiowngslbekowlatodwith10,000celWmL Three'biological"rcphcpaetreaspaime" groupwillbepreparedforevaluatincgelldensitiesO.ne ormore additiona'lanalyficarreplicatweisllbe mc4udad inthewst,as needed,to providetestsolutiofnorconcentratiovnerificatioonn Day 3 of the". OPOIS=. An abiotirceplicataetthehighestconcentratiwoinllbe includedinthetestandwillbe sampled.' on Day 3 andDay 4. Inordertocontroblias,thepositioonftheHa&s wiU be&tm@M byMobhM draw"ly dunng theexposurepenocl No otherpotenualsourcesofbiasareexpectedtoaffectheresults ofthesbi4. The responseofthealgaewillbe measuredintermsof ceU densityb,iomassexpressedasam under thegrowthcurve,andgrowthrate.An EC50 (i.e.t,hetheoretictaelstconcentratiotnhatproducesa 5001* reductioinnthemeasuredparameter)valueforcelldensitywillbe calculatefdoreach24 hourinterval ECIO, EC50 andEC90 valuesforcelldensityb,iomass(EbC)and growthrate(FC) willbeczlculateidf, PROTOCOL NO.: 4541091799/NAV96/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL, LTD -4- possible,atthe72 and 96 hour intervalsT.he no observed adverseeffectconcmitratio(nNOAEC), which isffielu&cst W concwtrafion&at inducesno a&cm inhlitorycffccton growth,willbe determmed relativteo each parameterat 72 and 96 hours based upon evaluationof thestatisticraelsultsand thedoscresponsepatterm At theend ofthe96-hourexposure,al&6tatiecffbcfwtillbe &ff=ntia todfrom algicidal effectsinthosetreatmentswluch = maximally inhibited. MATERIAM AND METHODS Test Sub Informationon the of U%% controlor referencesubstmm isrequu-odby Good LaboratoryPracticeStandards(GLP). no Sponsor n responsiblfeorprovidingWddffz IntemationaILA& writtenverificatiotnhatthetestsubstancehas be= chwacknzed accordingtoGLPs priortoitsuse inthe Sh*. IfwrittenverificatioofnGLP testsubstm= isnot providedtoVtrddhfelnwmabona Ltd.,itwillbe notedimthecompliancestatementof theEmal report The wadrd form IDFNMCAIION OF TEST SUBSTANCE BY SPONSOR (Appendix I)istobe used toprovideinformation:nemsmy for GLP compliance. The Sponsor isresponsibleforallinformationrelatedto the testsubstanceand agreesto acceptany unused testsubstanceand/ortestsubstance containersremaining attheend of the st@dy. Test SolutionE=aration The desiredconcentrationosf PFOS willbe obtainedby prepwing a singlestocksolutioninmedimn and dilutintgheappropnatevolume ofstocksolutionvathme&unl lU testsolutionmsay alsobe prepared using secondarydilutionosf theprimary stock,based UPM dw concentratiodneswe& ThetesstubftcewiUbe adminis@ tDdm tesotrgmisminalganlzdium Ms routoef adm4n;stratiownas selectedb=use itrepresentsthe most likelymute of exposuretoalgae,which exist suspended ina watercolium Test Organism The testspecieswillbe thefreshwaterdiatoraNia,viculapelliculosa.This speciesisreprescritative of an importantgroup of algaeand was selectedforuse inthe testbased upon pastuse and caseof handling inthelaboratory.Stockcultureso,btainedfrom theCultureCollectioonfAlgae attheUniversitoyf Texas PROTOCOL NO.: 454/091799/NAV96/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL, LTD -s- atAustinor anothesrupplierw,illbe maintaineidnculturmeedium atWildUe Int@onaL Ltd.fora oftwo weekspriortouseina toxicityest.Diatomsusedintodciiytestswillbe inacponential growthphase,wtuch isdefinedastheperiodofgrowthwhen algalceusarechvidingata constantrate. justpnortobeginningdw tes@an inoruh= ofthestockculturweillbe preparedso thateachmdhhux Ofinomd= containesnoughocustoprovidean mdid celldensitoyfapprom=tely 10,000mUshnL ineach replicate. Culture Culturemedimn preparedaccordintgoWildlifeInternationLatld,.StandardOperatingProcedures willbe usedasdilutiownater.The concentrationosfthecomponentsinUr medium arepresentedinTable 1 (2).Stocknutrienstolutionwsillbe preparedby addingreagent-gradoerbetterchemicalstoWildlife intemationaLltd.wen waterpurifiebdy reverse-osmosiAsp.propriatveolmnesof fficstocknutnent solutionwsillthenbe dilutewdithpurifiewdeuwater topreparethemedium- The medium will& filter stmifize(d0.22@m) or mwdaved priorto use.Analyseswillbe perofrmed atleastonce annuallyto determinetheconcenfimuonosfselecteodrganicandinorganiconsiatuenotfsthewellwaterand =Wts of themostrecentGLP compliantanalym willbe summulzed inthefinalreporl Spedficationfsoracceptablleevelsofcontaminanthsave notbom establishefdorculturmeediuni. However,thereareno levelosfcontaninantrseasonableyxpectedtobe presentinthemo&um thatare consideretdokterf= withft purposeorconductofthestudy. TestAMaratus Testchamberswillbesteril2e5,0,-mL polycarbonaEtrelenmcYcfrlaskpsluggedvnthfoam stoppers and conummg ioo mL oftestsoiuttorTlhe testchamberswillbe indisaiminateploysidonedon a mechanicalshakertableinan environmentaclhamberandwillbe shakencontinuouslaytapproxim@y 100 rpal Testchamberswillbelabellewdithft projecntumber,testconcentratiaopna_rep tre.. EnvironmentalConditions Testflaskwsillbe-heladt24:L.20C undercontinuouCs601-wtutfelwrm=t hghtingatan intensity of4300:h 10% IwL Lightintensiwtiyllbe measuredat"fivleocatlonssurroundintghetestflaskson Day 0 PROTOCOL NO.: 454/091799/NAV96/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL, LTD ofthetest Temperaturewillbe measured twicedailyina =teih= ofwater acentto thetestflaslcisnthe mviro=ental chamber usinga cahbrat4 hand heldm=ury thermometer. The pH ofeach tutment and controlgroup willbe measured attestinitiatiaond terminatingusing a FisherAocumet Model 915 PH meter or equivalent Samples forpH measurement attestinitiatiownillbe collectefdrom the mchvidualbaum of testsohitim preparedforeach umunmt and controlgroup. At test termmaton, pH vaU be measured m pooled samples of testsolutioncollectedfrom each of the6= replicates of eachrespectiveuratmcnt and controlgroup. BklggicalMeasurements Celldensitiewsillbe monitored duringdw testby conductingcellcountsusinga hcmacytometer and a microscope. One sample willbe collectedfrom each replicateof the treatmentand controlgroups at approximate24-hourintervaldsuringtheexposure period.The samples willbe evaluatedimmediatelyor willbe heldin thedark atapproximately40C untilcellcountscan be performed. Each sample willbe diluted using an electrolytseolution(Isotoa4D)a,s needed,tomaintaincountingaccuracy.A smallamount of each sample willbe loadedontoa hcmacytometcr and thetotaln=ber ofccus in10 gdds willbe counted.The celldensityof thesample willbe calculatedbased on the mean number of cellsper grid- Microscopicexaminationof alpe willbe made toassesschanges ingrossmorphology. Algae will . be observed toassesschangesin cellshape,cdl color,and aggregation(clunping)of thecells.Observations willbe used todeterminewhed= u=t3n=t-related effectisncludethosechanges ingrossmorphology. Simp-hLngforAngly-fic-Mamlswmcat--s Samples ofthecqmm solubm vaU be conectedon Days 0,3 and 4 and =4-md fortestsubstance Day 0 s=plewsiUbetim fim ea&batcohftatsolufipofniotrothd&rtnb.udmonong the replicatteestchambers. Day 3 samples willbe collectefdrom theextrareplicatteestch=ber(s) included inthetesttoprovidesample forthatanalysisand fi-omffiacbioticreplicatesI.fmore dm one iri.lpicateis includedforft Day 3 smnple aaabrsisd,= thesobitionsfi-omeachrepheatewiU be compositedtoprovi& the sample for thattmtment. Day 4 samples willbe compositedsolutionsfrom the threereplicates remainingattheend of the test PROTOCOL NO.: 454/091799/NAV96/SUB454 3M LAB REQUEST NO. U2723 EZLZfl Eivrviqc tSVMS/96AVN/66LI60/t,5t, lo.quopouglwtWoj4 olpjojMUMM oq W& OAM :vlnuuojSuVAollojotp Su!snsdnoig qVAog op jopun om so po=dxo ssmoie[ S Tuft laug oxpm poqpmp ptm mp jaiuotpu!M=un*T aq ipapoxpow porp@lm mp oqopum saoll=UTpoyq 11 xq=ddv u! pogpwpi pEm j=ods otp Aq popymjd Agolopotpom uodn pmq oq lptapm poqpm luonapm olql -prlpmi.4mumul OJTMPAI Aq poucuojjod oq IPIA soldms oxpjo s!s4ro po!=qo (prnsolgjoosuqdkv=qo ponapm oT Suunp p=gpm pui pomdwd sao!munlo put qnlq x@n= seqm soldms bb) lonuco @tqenbopnlou!lou OP Pug IM OID MO.IJP4wHOO osolpluo=doi sold=s p snqmu OAoqu oU 6 6 8 tz Iz I I I 1 I I I I I I I I I I I *sold=s o!3o!qusopnloul, SMO.L uOgzg=uOD RRIH-9JOA7I 5 JOAVI t JOAN P"l Z JOAN (M=jl MOTI JOAN 10.4UCO MAJOS I T t,ABU E"a o"a &wig pwmndxa S-d'ld]WSMOUVOMah 40 MEYM CCaSOcIO-dcl :mor-q pnum=s s!*=qos oldmus *U T=4= um poxns pau (omoq =IhpVqod jo =pWaxVqod '*S'O=)m Ww ogwols cqvpdaiddeuc m pomld.ID ARJPT p=4= oq TptAsold=S *=Apm cq.xo!jsdnoolaluOAOU= 01 P=RU 10 panje .4= aq &m t,puv E s,@raw popmc)o soldmms a.L-1'-lVNOIJLVNH3.LNi :3.di-la-IIM WILDLIFE INTERNANONAL, LTD A = ((NI-NXXtl)+((Ni+Nr2N@)/2)(t2-ti)+.-.4@(Nt+N.-2NX)(U-ti-i) Where: A = Area under thegrowth curve N, = Measured number ofcelLs/mLatt, N, =Mwsured number of wWmL atti N2 =Mwsmrd nmnber of celldml at t2 N. =Measured nmnber of ceUshnL atit@ t,= Time offimtmeasurement afterbcghmiag oftest(boum) t2 = Th= c@f'dwmd mcasw=cnt aft bc&ning oftest(hours) t@= Time of nth measurement afterbegkming of test(hours) Growth rates(p)willbe calculatefdorthetmtmcnt zmd controlgroupsusingthef,ollowingformuLL InN,-InNo u - to 12 Averap specifigcrowth rate N. = Measured number ofceWmL atU N. = Measured number ofcclls/mLatt@ to= Time ofbeginningoftest(hours) t@= Tin2cofnthmeasurement afterbcpming of test(hours) Percentinhibitiownillbe calmaatodforeach twuncat group based upon mean celldensity,biomass and growth rateusingthefollowingformula: Mean Control- Mean Treatment PercentInlu-bition &RMM Mean ControlResponse x 100 EC valueswiU be &ftrmined, when possibleu,singlinearintmWIation orothersuitablwechniques with treatmentresponse(Le.celldensitiesb,iomass and growth rate)and exposm-c concentratiodnatafor each 24-hour intervaolf thetesl A no observedadmw effectconcentratio(nNOAEC) willbe selectebdased on.anevaluationofthe dose-responsepatternand the resultsof the statisticaanlalysisusingindividualreplicatevaluesof the cell densityb,iomass and growth rate.StafisticasUiygnif@ differcnm bdwom thecontroland dw U=tncnt groups willbe identifieudsinganalysisof variance(ANOVA) and a testto compare treatmentmen PROTOCOL NO.: 454/091799/NAV96/SUB454 3M LAB REQUEST NO..U2723 WILDLIFE INTERNATIONAL, L-rD -9- responsestothecontrolresponse(e.g.D,unnetestestorBonferroni'ts-test)D.atawillbe assessedfor normalitayndhomogeneityofvarianm prortoperformintgheANOVA. Transformationwsillbeused.to correcatnyconditioonfnon-mrmalityorunequalerrorvariancesi,fpossible.Ifa solventcontrolisused inadditiotnothenegativecontroltheiresponseswillbe comparedusingSWdenes t-testData from the two controglroupswillbepooledforbuftnentlevelanalyseisfno statistidciaflferenceexsist Othawise, allcomparisonwsin bemade tothesolvenctontroglroup.S=sticalcompmu= fordc ANOVA andthe, comparisonofmean responsewsillbecarrieodutusingTOXSTAI* V3.5 software(4)orequivalent RECORDS TO BE MAINTAINED Recordstobe maintainefdordatagenerateadtW"ddlifIentmnatLonaLtd.vaR includeb,utnotbe limitetdo: I. Copy ofsignedprotocol 2. ldcnfificatainodnchmwtmizationofthetestsubstancei,fprovidedby Sponsor. 3. Datesofinitiatiaonndterminatioonfthetesl 4. Sourceof algae. 5. Cultureconditions. 6. Growth measurements. 7. Calculatioanndpreparatioonftestconcmbldons. 8. Observations. 9. Ifapplicablet,hemcffiodsusedtoana@ym testsubstanceconcentrationasnd theresultosf analytical measurements. 10. Sufisticaclalculations. 11. Testconditionasnd physical/chemicmaolmmm=ts. 12. Copy offinarleporl FINAL REPORT A finarleporotfd2C results ofthesb* willbepreparebdy WildlifkeWrnationaLLtcLThe report willincludeb,utnotbe limitetdothefollowin&when applicable: I. Name and addressofthefacilipteyrformingthesh*. PROTOCOL NO.: 454/091799/NAV96/SU]3454 3M LAB REQUEST NO. U2723. WILDLIFE INTERNATIONAL, LTD -10- 2. Dates on which dc su* was initiatedand completed. Itis fficresponsibilityof the Sponsor to providethefinaldatethatdatauc recordedforchemistrypathologyand/orsupportingevaluations thatmay be generatedatotherlaboratories. 3. A statement'ofcompliancesignedby the Stx* Directoraddressingany o=ptiont -to Good LaboratoryPracticeStandards. 4. Objectivesand proceduresstatedin theapproved protocol kchuhng any changes in the original protocol 5. Ile testsubstanceidenfifiebdy name chemicalabsbid munber or co& number, strengthp,uity, composition,and otherchuacteristcsprovidedby theSponsor 6. Stabilitaynd solubilitoyfthetestsubstancesunder theconditionsof on,ifprovidedby the Sponsor. 7. A descriptioonfthemethods used toconductthetesl 8. A descriptioonf the testsysten3=,Iudmg the sourceof thetestorganisms and theirscientlfincame 9. A descriptioonf thepreparationof the testsolutionsm,ethods used to allocateorganisms to thetest chambers and beginthetes@numbers of organisms and chambers per treatment,and durationof the test 10. A descriptioonf circumstancetshatmay have affectedthequalityorinteg* ofthedata. 11. The name of theSb* Directorand the names of otherscientistpsr,ofessionalsa,nd supervisory personnelinvolvedinthesh*. 12. A descriptioonf ffitcransformationsc,alculationso,roperationsperformed on thedata,a smmmy and analysisof thebiologicadlata and analyticaclhemistrydata,and a statementof the conclusions drawn from these analy=. 13. Su&dcal methods employed foranabzing the data. 14. The signed and dated reportsof each of the individualscientistsor ofl= professionalsinvolved in dw study. 15. The locationwhere raw data and finalreportare to be store& 16. A AaWment Prepared by tbcQuality Assurance Unit hsbng the ddm thatStudY MSPMUWS and audits were made and thedatesof any findingsreportedto the Study Directorand Management 17. ffitisnecessarytomake correctionosr additionsto a finalreportafterithas been accepted,such changes shallbe made inthe form of an amendment issuedby the Sbxly Director.The amendment PROTOCOL NO.: 454/091799/NAV96/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL, LTD shallclearliydentiftyhepartof thefinalreportthatisbeingamended and thereasonsforthe alteratioAnm.endments shallbe signedanddatedby theSbidyDirector CHANGING OF PROTOCOL Plannedchangestotheprotocowlillbe intheform ofwrittenamendmentssignedby theSh* Directoarnddw SponsoesRqmmtafive. Amendments willbeconsidereadspartoftheprotoooland will be attachetdothefinalprotocolAny otherchangeswillbe intheformofwrittendeviationssignedby the Sb* Directoarnd filewdiththeraw data.AJIchangestodw protocowlillbe hidicateidnthefinalrepc)it GOOD LABORATORY PRACRICES Thissb* willbe conductedinaccordancewithGood LaboratoryPracticSetandardsforEPA (40 CFR Pan 160 and/orPart792);OECD PrinciploefsGood LaboratorPyractic0e2qV/MC/CHEM (98)17);. and JapanMAFF (59 NohSan,NotificatiNoon. 3850,AgriculturParloductionBureau).Eachsu* conductedby WildlifEeat=ahonaL LtcLisroutmelyexaminedby theWddhfe figmuationd,LTCLQuality AssuranceUnitforcompliancewithGood LaboratoryPracticesS,tandardOperatingProceduresand the specifiepdrotocol.A statemenotfcompliancewithGood LaboratoryPracticewsillbe preparedforall portionosf thesh* conductedby W-ddhfeInW=tionaL Ltd. The Sponsorwillbe responsiblfeor compliancewith Good LaboratoryPracticefsorproceduresPerformedby otherlaboratorie(se.g.r,esidueanalyseosrpathology)R.aw dataforallwork performedatWildlifIent=ationaLLtd anda copy ofdo finarleporwtillbe filebdy projecntumber inmrbves ImaW on theWildlifIentcmtionaLIA& siteo,rat an altm-natilvoecatiotnobe specifieidnthefinalreport. PROTOCOL NO.: 454/091799/NAV96/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNA-NONAL, LTD -12REFERJENCES 1 US. Environmental ProtectionAgency. 1996. Series850- EcologicalEffectsTest Guidelines (draft)O,PPTS Number 850.5400: AlgalToxicityT,iersIand U. 2 ASTM Standard Guide 1218-90E. 1990. &wui6rd Giddefor Conducting &adc 96-Hour ToWdty TestsudthMicroalgae. American SocietyforTestingand Materials.Phi@a&lphin,Pemsylvani& 3 Norberg-IGngTJ. *1993.ALinearh:terpolmlonMethodforSubkthal Toxicity.Ihelnhibidon Concenowdon ac.,).4pproach (FWsion 2.0). U.S. Ft@@@ ResearchLaboratory,Duluth,Mmesoi& ProtectionAgmcy, Envia=caW 4 West, Inc.and D. D. GuIley. 1996. YIOMTATRel ease 3.5.Wwtcm EcoSystems Tecbmlogy, Inc. Cheyenne, Wyoming. PROTOCOL NO.: 454/091799/NAV96/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL, LTD -13- RZESHWANR TABLE I ALGAL MMIUM WrrH SIUCA CONSTrrUEM Component Nominal Concentration MgCI2-6H20 CaCl:e2H20 H3B% I&CI2-4H20 ZUC12 Fea3-6H20 COC12-6H20 Na2MoOe2H20 CuCI2-2H20 12.16 mg/L 4.40 mg/L 0.1856 mg/L 0.416 mg/L 3.28 -g@IL 0.1598 mg/L 1.428.gIL 7.26 g(L 0.012 g/L Na2EDTA-2H20 NaNO3 MgSOe7H20 K2BPO4 NaHCO3 Na2SiO3-9H20 Na2SeO3-5H20 0.300 mg(L 25.50 mg/L 14.70 h*/L 1.044 mg/L 15.00 mgtl 20.0 mg/L 0.010 ME& The pH ofthemedium willbe adjusted,asne=sary, to7.5 N NAOH or 10*/oHCL 0.1 us@ng 0.1 PROTOCOL NO.: 4541091799/NAV96/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL, LTD -14- iDE=cAnoN APPENDDCI OF TEST SUBSTANCE PY SPONSOR Tobe Compkbdby Spa= I. TestSubstanceidentit(yname tobe used inthereport):PFOS (PcrfluamoctaneSulfonicAcid Potassimn Salt ReferenceStandard(ifapplicable)A:nalyttealStandud. -N/A latmw Standard- 1.l2-2HJiRH PerflaomoctaneSulfonicAci Test SubstanceSample Code or Batch Number-. Lot 217 TestSubstancePm* ('/AoctiveIngredient): 98.9 ExpiratioDnate:. 2008 R TestSubstm= HavegrOcofty.@,sYa Z NdikhappWa e tmt Icomposifiomrodw dimicomistics and rcfmt= swadardbem dd=3ffi8dfrkr toks im indisswdy inaoo=Lmm vvitGhLP Sbndards? ----@yes IEL TestSdxta= StorapCaiM= x No Pkmindi de =omm=L-d staw coodw= atWWM bkmWiaW W Ambient Has Thestabkyofdc tatmbstmw uoder&= staw =dticw bernddeanit)iidnaccordmxewidiGIY Staodards7 odwmfinatswmuyinf@on Yes x No IV. TestC4nceoftafi=: Adjusttug cmcwbzdon tc)1000/&oL x basedWm de purity('/og)iN= abovr- Do nc)tadjust= conomhzfim to1000/o -&L TestthemateriaAlLI-S. V. Twi&y kbmafim- NiammaUamAquatic: RatLD50 251 mg&g -MouseLD50 kvawxzw Todcity W-A.C50) N/A FishTodcity(LOO) DaMhm@a mama: 27 mpJL Rainbow Trout: I mg)L DaRhm @a magna: 50 mgL L FatheadMinnow: 38 mpJL otbr.Trmcdy kfamatam (m&xbqg fimm ofchmm andmbcbmm tcsts): Pleasesee M5DS PROTOCOL NO.: 454/091799/NAV96/SU]3454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNA-NONAL, L-rD -15- APPENDIX ]I AnalyticalMethod Providedby Sponsor Samples willbe'analy7M based upon proceduresprovided by theSponsor inthe followinganalytical mediods: - I. Liqmd Chromatogwby Mass Spmtromtry (LCMS) Method -forthe Determinationof PerfluomoculiqSulfonicAcid PoUssium Salt,(PFOS)In Freshwater@Saltwaterand AW Mc&mn A copy of theabove method willbe maintainedin theraw data. The actualmethodology used to ana@y= thetestsampleswillbe docwnented intheraw dataand n=mafized inthefinalreport PROTOCOL NO.: 454/091799/NAV96/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNA-noNAL LTD PROJECTNO.: 454A-112 Page I of I AMENDMIENT TO STUDY PROTOCOL STUDY TITLE: PFOS: A 96-HOUR TO)UCITY TEST WITH THE FRESHWATER (NaWculapelliculosa) DIATOM PROTOCOL NO: 454/091799/NAV96/SLM454 AMYNDMEENT NO.: 1 SPONSOR: 3M Corporation PROJECrNO.:'454A-112 EFFECTIVE DATE: Febniw 23,2000 3M LAB REQUEST NO.: U2723 AMEENDMENT: Page 2 Add. ExperimentalStartDate: February25, 2000 ExperimentalTerminationDate: February29, 2000 TestConcentrationsN:egativeControL70.0,92.5,125,167,225,300 and 395 mg a.i./L Test SubstanceNo.: 4675 REASON: The above informatiownas notknown when theprotocolwas signedby the StudyDirector. AMEENDMIENT: ExRerimentaDlesi Page3 Change: Targetconcentrationwsin notexceed 100 mg/L orthesolubilitlyimitofthetestsubstanceinwater. To: Targetconcentrationwsillnotexceed 1000 mg/L orthesolubilitlyimitoft@itestsubstanceinwater. REASON: Nominal testconcentrationesxceed 100 mg/L. STUDY DiRiffcrOR 9WI ./L- LABOMTBRYUA@NAGEMEENT 4@:@@@@PRE@SE@ATIVPER@ES@E@ ATIVE /00 DATE' / 000-1 DATIE DXT-T WILDLIFE INTERNA-NONALLTD. PROJECTNO.: 454A-112 Page I of I AMENDMENT TO STUDY PROTOCOL STUDY TITLE: PFOS: A 96-HOUR TO)UCITY TEST WITH THE FRESHWATER (NaWculapelliculosa) DIATOM PROTOCOL NO: 454/091799/NAV96/SUB454 AMENDMENT NO.: 2 SPONSOR: 3M Corporation PROJECR NO.: 454A- 112 EFTECRIVEDATE: Februarv2g,2000 3M LAB REOUEST NO.: U2723 AMENDMENT: Biological-MeasurementPsage 6 Add: At theend ofthetestal&taticeffectsw,hich resulitnffiicnhibitionf cellgrowth,willbe diffenmfiated fromalgicidaelffectsw,hich resulitnthedeathof ceus.Aliquots(0.5mL) oftestsolutionwillbe taken from eachreplicatoefthosetreatmentgroupswhere growthismaximallyinhibitedM.aximallyinhibited treatmentasrethoseinwhich itcannotbe visualldyeterminediflivealgalcellsuc present(e.g.a,bsence of colortothetestsolution)T.hese aliquotwsillbe pooledby concentratioannd dilutedwithuntreated culturemedium to a concentratioonf thetestsubstancethattheoreticalslhyouldnot affectgrowth.,A negativecontrolwillbe preparedby dilutinag 0.5-mL aliquotfrom one negativecontrolreplicatteo100 ML withculturmeedium. Growth of thesesubculturewsillbe monitoredfora maximum of 9 daysto detmmmc whetherinhibitioonfgrowth observedduringthetestisreversibleS.amples willbe @oll@cted forcellcountsatrecoveryphase initiatiotre@rminatioannd atapproximately3-dayintervalbsetween initiatiaonnd terminationT.he recoveryphase may be terminatedonce algalgrowth issufficientto indicattehatthealgalcellscollectefdrom thetreatmentgroupshave fidlyrecoveredfrom effectsdue to thetestsubstanceT.hiswillbe determinedthroughvisualexaminationofthe'kecovertyestsolutions. REASON: To add a recoveryphase totheprotocol. STUDY DIRECTOR LABbP-@TZ)RVMANAGEMENT SP s RESSEE7TIVTEIV, DATE DATE DAft