Document Ozg6evz9Zk3KRaGNxxq33J5d1

Protocol.PFOS: A 96 Marine Diatom (Skele -0--- PROTOCOL PFOS: A 96-HOURT03aCITY TEST WITH THE MARINE DIATOM (S7celetonecmoastatum) US. EnvironmentaPlrotectioAngency Series850 -EcologicalEffectsTestGuidelines OPPTS Number 850.5400 3M Lab RequestNo. U2723 Submittedto 3M Coq)oration EnvirotuncntaLlaboratory 935 Bush Avenue St PauL Minnesota 55106 Wildl@feInternationalL,td. 8598 Commerce Drive EastoriM,aryland 21601 (410)822-8600 September 17,1999 WILDLIFE INTERNATIONAL, LTD -2- PFOS: A96-HOUR T03aCITY TEST WITH THE MARINE DIATOM (Skeletonemcaostatum) SPONSO 3M Corporation EnvkomnentalLaboratory 935 Bush Avenue St PauL Minnesota55106 SPONSOR!S REPRESENTATRVE TESTING FACILITY Rochelle1;LRobideau W"dMc L2ternationLatLd. $599 Comma= Drive Easton,Maryland21601 STUDY DRRECTO K-wtgretW Cgv SeniorAW,36c i@io?o@; LABORATORY MANAGEMENT: Henry 0. Kmega, Ph.D. Dzect" ofAquaticToxirolog&y Non-TargetPlants FOR LABORATORY USE ONLY Proposed Dates: Expaime@ StartDate: PmjectNo.: 4gS4,4. EVerimental Tamibmtion Date: Test CDncentradons: TestSubstanceNo.: Rc&m= Subsu= No.Cifapplicable,): PROTOCOL APPROVAL STUDYDIRECTOR UA-T E- 7 A Lk]3@OMTCfRI IRWACYEMRIT B@ATE R@EP i@-E-PrENTAATTrRVWE PROTOCOL NO.: 454/091799/SKE96/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL, LTD -3- MROD WildliIfnetemadonAU& wilclonducat96-hottwoxicitteyswtiththemarindeiatonS27,celetonema costatumf,ortheSpamuratthelWilcUIinfte=;itionI4AiaquatitcoxicolofgiymU4inEastonM.myWA Thestudwyilbleperformebdasedonprocedur*eisdnieUS. FmvimmentalProtectiAognencySerie8s50EcologicEaflfectTsestGuidelinOePsPTS Number850.540(01).Raw dataforallworkperformeadt WildlifeIntmmationaLlAi and a copy ofthefinalreportwin be filebdy projectnumber inarchiveslocated on theWildlifeIntemationa%Ltd siteo,r atan altmma@velocationtobe specifiedinthefinalreport. PURPOSE The purpose of thissb* isto determinethetoxicitoyf Peffluorooct= SulfonicAcid@Potassium Salt(h=after refwcd toas PFOS) to themarine diatom,Skeletonema costalum. EXPERIMENTAL DESIGN The marine diatom,S7xletonema costatum,willbe =posed to a geometricseriesof.sixtest concentrationasnd a negative(culturmeeffium)controlfor96 bours.Targetconcentrationwsillnotexceed 100 mg/L or thesolubilitlyimitof thetestsubstanceinwater.Generallyt,hewminal concentratioonf ewh testsubstanceused inthedefinitivteestwillbe atleast50*/oof thenexthi&a treatinenutn@lessinformation concerningtheodnccaftabca-effecaurvem&mtes thata differencthlutimfactorwould be more appropriate. Test solutionswillbe inoculatedwith approximately77,000 ecUs/mL Test solutionswillbe inoculatewdith 10,000cefls/mT. llu= wbiologicarleoplicatepserexperimentaglroupwillbe preparedfor evaluatingcelldensitiesO.ne ormore additiona"lanalyficarle'plicatewsillbe includedinthetcs4asneeded, toprovidetestsolutionforconcatrationverificatioonn Day 3 of theexposure.An abioticreplicataetthe higbestconcentratiownillbe includedinthetestand willbe sampled on Day 3 and Day 4. Inordertocontrol bias,thepositionof theflaskswillbe determinedby indiscriminatderaw dailyduringtheexposureperiod. No oftr potential sources of bias are expected to affect the results of the sh*. The resp'onsoef thealgaewillbe =asured into= of celldensityb,iomassexpressedas areaunder thegrowth curve,and growth rate.An ECSO (i.e.t,hetheoreticatlestconcentratiotnhatproducesa 50019 reductionin themeasured parameter)valueforcelldensitywillbe calculatefdoreach 24 hour intmal EC 10,EC50 and EC90 valuesforcelldensityb,iomass (EtC)and growth rate(F4C)willbe calculatedi,f possible,atthe72 and 96 hour -intervalTsh.e no observedadverseeffectconcentratio(nNOAEC), which PROTOCOL NO.: 454/091799/SKE96/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNA-NONAL, LTD -4- isthehighesttestconc=trationthatinducesno adversemiubitoryeffecton growth,willbe determined relativteoca& pameter at72 and 96 hoursbased upon evaluatioonf thestatisticraelsultsand thedoseresponsepanern At dw end of the96-hourexposure,al&tafleeffectswillbe diffawtiatedfi*omalgicidal effectsinthosetreatmentswhich am mmdmauy inlubite& MATERIAM AM METHODS Test Sub informationon thecharacterizatiofn test,controlor referencesubstancesisr"Xired by CTood laboratoryPracticSetandards(GI.P).The SponsorisresponsiblfeorprovidingWildlffIent=nada3A It& wnttm venficatiotnhatthetestsubstancehas be= characterizeadccordingto GLPs priorto itsuse inthe sb*. Ifwittm verificatioofnGLP testsubsm= charactrrintiiosnnotprovidedtoWddlifeIntmnatiomd, Ltd.,itwillbe notedinthecompliancestatmentof thefinalreport.The attachedform IDENTMCATION OF TEST SUBSTANCE BY SPONSOR (Appendix 1)istobe used toprovideinformationeces@sarfyor GLP compliance. lle Sponsor isresponsiblfeorallinformatiornelatedtothetestsubstanceand a'p= to acceptany unused tea substanceand/ortestsubstancecontainersremainingattheend of thesb*. Test SolutionP The desiredcon=tmtions of PFOS willbe obtainedby preparinga singlestocksoludoninmedium and dtlutmgtheappropriatveohime of stocksolutlonwithmechum. Ilm ttstsolutlonmsay alsobe prepared usingsecondarydilutionosf theprunarysw* basedupon dw concenbiton desired. PFOS wdl be adnnmsteredto thetestorgamsm m algalmechunl This routeof adnunlstratiownas selectebdecauseitrepresentsthemost likelyrouteof exposure to algae,which existsuspended ina water column. PROTOCOL NO.: 454/091799/SKE96/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL, LTD -5- Test ne tesstpeciewsilblethemu= algaS,7celetonecmoastatumT*h.isspecieissreprcsentaotfiamn iinportangtmW of algaeand was selectedforuse inthetestbasedupon pastuse and easeof handlinginthe laboratoryStock mdhm, obtmned from theCultmr.CollectioonfAlgae attheUniversitoyf Tom at Austinor anothersupplierw,illbe mamtined inculturemedium undertestcoaftons atVvrildliIfnetm-na- tiomd,IA& fora minirninnof two weeks priortouse in a toxicittyest.Algae used intoxicitytestswillbe incxpon=tialgrowth phwe, which isdefinedastheperiodof growthwhen algalcellsarcdmding ata constantrate. Justpriortobeginningdc tes@an komdum ofthestockculturewillbe preparedso thateachMmiliter of mocalum containsenough cellstoprovidean mitmlcelldensityofapproximatc@y77,000 cdWmL ineach replicate. CultureMedium Culbze medtmn preparedfrom nawral or mtificiasleawateraccordingtoVtrddhfeIntmmatonaL U& StandardOperatingProcedureswillbe used as dilutiownater.*Ibeconcentrationosfthecomponents inthe mcdium am prw=W in Table 1 (2). Stock mrtrienstolutidnswillbe preparedby adding reagent-grade chcnucalstoVrildhfehd=auonal Ltd.wellwaterpunfiedby revcrw-osmos.is-.Appropriatveolumes of thestockmtdcnt solutionwsfllthenbe dilutewdithartlficouramhn-alwzwater tD preparetheme&= Ilm mcdiun willbe filtesrtmihzed(0.22pm) or autoclavedpriortouse. AnalysesYaH be pcrfomlcdatleast oncemmuauy to ddamine thecmomtrations of selecteodrganicand inorganiconstiumts ofLbewellwaW. and resultosf themost recentGLP complumt mmlym vaH be summarized inthe finalreporl Specificationfsoracceptablelevelsof contaminantshave not been establishedforculturenlc&unx However. therearc no levelsof contaminantsreasonablyexpectedto be presentin the mcchmn thatare consideredtokaaf= withthepurposeor conductof thesuidy. Test ARparatus Test chambers willbe steril2e50-mL poly=wnat@-, Erlenm.qu flaskspluaw withfo= stoppas and conmining 100 mL of testsolution.The testchambers willbe indisaiminatelypositionedon a PROTOCOL NO.: 454/091799/SKE96/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL, LTD -6- mechanicalMaker tableinan environmentalchamber and willbe diakm continuouslyatapproximately100 rpnl Test chambers willbe labeledwith theprojectnunber, testconcentratioannd replicate. EnvironmentalConditions Test flaskswillbe heldat20 :k2cC under 14 hoursof cool-whiteflua=ccnt lightinagtan intensity of 4300:h 430 lux and 10 hoursof &rkom. Lightintensitwyillbe measured atfivelocafionsurrounding thetestflasksan Day 0 ofthetest Temperaturewillbe =corded twicedailyina containerof wateradjacent to thetestflasksusingk4quid-in-glassthermometer. The pH of each teatinentand controlgroup willbe measured attestuntiatioannd terminatiounsing a FisherAcmmet Model 915 pH meteror evivalcat Samples forpH measurement attestinitiatiwoinllb e couectedfrom themchvubw batcbesof testsolutionpreparedforeach U=tinent and controlgroup.At test tammatton, pH willbe measured m pooled samples of testsolutioncollectefdrom each ofthe replicates of each respectivbewttnent and controlgroup. BiologicalMeasurements Celldensitiewsillbe monitored duringthetestby conductmg cellcountsusinga hemacytometerand a microscope. One sample willbe collectedfrom each replicatoef the treatmenta,nd controlgroups at approximate24-hourintervalAsning theexposureperiod.Ile sampleswillbe evaluatedir=ediatelyor willbe heldinthedark atappm)C@ 4:-Cuntiloellcountscan be performed.Each samplewillbe diluted usinganclectrobftsolution(Lwtm(g),asneeded,tomaintaincountingaccuracy.AsmaU amountof each sample willbe loadedonto a hemacytometer and thetotalmunber of cers in 10 gridswillbe counted.110 ceU densityof the sample willbe calculatedbased on.themean number of ceus pergnd.. ]N&croscopicexaminationof algaewillbe made toassesschanges ingrossmorphology. Algae will be observedto&csm changesm cdl shapi;ceU color,and aggregation(cltunpingo)f thecrus.Observations will be used to,determine whether growth treatmcnt-relateedffectsincludethose changes in &Toss morphology. PROTOCOL NO.: 454/091799/SKE96/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNANONAL, L-M -7- SnpliniftorAnbaicia-M-lewurements Samplesoftheaq)os= sohtionswinbe collectoendDays0,3 and4 and =&yzed fortestsubstance emccauldons. Day 0 sampleswillbe W= fromeachbatchoftestsolutiopnriortodi& distributiaomnong thereplicatestchambem Day 3 sampleswillbe co&ctedfromtheextrareplicatestchamber(s)included inthetesttoprovidesmnpleforthatanalysiasnd fromtheabiotirceplicateIsf.more thanone replicaties includefdortheDay 3 smnpleanabsis,d= ffiscohffiowfiomeachreplicawtiellbe compositedtoprovide diesamplef,orthattw=mt Day 4 s=plcs vnu be compositosdolutonsfi-omthe&= replicates remainingatthecad offfitcest. Samplescollecteodn Days 3 and4 may be cmtriffigoerdfilkretdo remove algalceuspriorto analysis.Sampleswillbe analyzedinunodiateolryplacedinan appropriatsetoragecontainer.(eg., polypropylenoerpolyethylebnoettlea)ndstoreudntialnabrm& no sampleschemeisswnmadzed belovr PROPOSED NUIVBERS OF VERMCATION ExperimentaGlroup Day 0 Control I SolventControl(ifneeded) 1 Level1-Low Concentration I Level2 1 Level3 1 Level4 1 I.eve5l 1 I&vcl6-Mgh Concentration 1 Totals 8 ll'Ioenrnlcluuddeaesbsio@taiscbamip@leos. SAWLES Day 3 1 1 1 1 1 1 1 21 .9 Day 4 1 1 1 21 9 ,Iheabovenumbersof S=plcs representthosecollectefdromdz testand do notincludequality contro(lQC)sampleswh asmatrixblanksandfortificafpiroenpsuedmd ma@ga duringthe"Yfic4l chemistryphase'ofthesh*. And3lLcal Chemicalanalysiosffficsampleswillbe performedby WildlifIenternationLatld. The analytical method usedwillbe basedupon methodologyprovidedby*theSponsorand identifiiendAppendix IL PROTOCOL NO.: 454/091799/SKE96/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNA-NONAL, LTD -8- Mochficationmsade totheanalyticmaelthodwillbe d=m=tod intheraw dataanddescribeidnthefinal rq)ort StatistiAcgaWlm Biomass expressedas areaunderthegrowtharve willbe calculatefdorthetreatmenatnd control groupsusingthefollowinfgormulaA= ((NI-N.)/2Xtt)+((N,+Nr2N.)t2)(trtl)+...-@Oi+Nr2No)t2Xts-t..i) Where: A Area underthegrowthcurve N, Measurednumber ofcelWmL att, N, =Mmurod number ofceUshnL atti N2=Mmsured number ofcelWmL att2 N,,=Mmsured number ofcclWmL att@ t,= Tm2c offirsmtewwm=t aftebreginninogftest(hours) t2 =Tune ofsecondnwam=cnt aftebreginninogftest(hours) k = Time ofnthmeasurementaftebreginningoftest(hours) Growthrates(,uw)illbe calculatefdorthetwuncat andcoigroglroupsusingthefollowinfgomua&InN,. InNo tn tO Az Avwap specifigcrowthrate N. = Measurednumber ofcoUsImL atU N. = Measurednumber ofceUs/mL att. to= Time ofbeginninogftest(hours) tn= Tum ofnthmeasurementafkxbeginninogf tes(thours) Percentinlu'bitwiiolnlbe calculatefdoreachtraftnengtroupbasedupon mean ecUdensityb,iomass and growthraw usmg thefollowmgformula. Mean Control-Mean Treatment PercentInlubition Res2q= BWonse x 100 Mean ControlResponse PROTOCOL NO.: 454/091799/SKE96/@UB454 3M LAB REQUEST NO: U2723 WILDLIFE INTERNATIONAL, LTD -9- EC valueswillbe detmm2inectwhen possible,usinglinearinterpolationr othersuitabletechniques with bwtinentresponseCLe.celldensitiesb,iomass and growth rate)and exposureconcentratiodnatafor each 24-hourbgaval of ffitcest. A no observedadverseeffectconcentratio(nNOAEC) willbe selectedbased on an evahiationof the dose-responsepatternand theresultsof thestatisticaanlalysiussingindividuarleplicatvealuesof thecell densityb,iomassand gm& rate.SudsticaUy significandtffamm bdvxm thecontroland dw treatmmt groupswillbe xkntlfiedusinganalysiosf vana= (ANOVA) and a testto compare treatmentmean responsesto thecontrolresponse(eg.,DunneWs testor Bonferroni'ts-test)D.ata willbe assessedfor. normalityand bomgeneity of varianm priortoperformingtheANOVA- Transfonnationswillbe used to correctany conditionof non-normalityor unequal mTm variancesi,fpossible.Ifa solventcontrolisused in additiontothenegativecontroltheirresponseswillbe compared usingStudmlVst-teslData from the two controglroupswillbe pooledfortreat3nenltevelanalysesifnostatisticdailffa== exisl Odmrwise, allcomparisonswillbe ma& tothe solventcontrolgroup.StatisticcaolmparisonsfortheANOVA and the comparisonofmean responseswillbe carriedoutusingTOXSTATO V3.5 software(4)or equivalent RECORDS TO BE MAINTAINED Records tobe maintainedfordatageneratedatWildlifeIntmmtionaL Ltiiwfilinclude,but not be limitedto: 1. Copy of signedprotocol.. 2. Idenfificatiaonnd characterizatiofn thetestsubstance,ifprovidedby Sponsor. 3. Dates ofinitiatioand terminationof thetosl 4. Source of algae. 5. Culturecmditions. 6. Growth measurements. 7. Calculationand preparationof testconcentrations. 8. Observatipm. 9. Ifapplicablet,hemethods used toanalyzetestsubstanceconcentrationasnd theresultsof analytical measurements. 10. Statisticcaallculations. 11. Testconditionsand physicaychemicalmeasurements. PROTOCOL NO.: 454/091799/SKE96/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL, LTD 12. Copy offinalreport. -10- MAL REPORT A finalreportofffircesultsofthesb* willbe preparedby W"ddlifehdmmafionalLtd. The report willincludeb,utnotbe limitedtothefollowingw,hen applicable: 1. Naiiiiand addressofthefacilipteyrformingthesu*. 2. Datesan which dm studywas initiateadnd completed.Itisdieresponsibiliotfythe Sponsorto providethefinaldated2atdataarerecordedfor'chemistrpyathologyand/orsupportingevaluations thatmay be gm=zw atotherlaboratories. 3. A statementof compliancesignedby theStudy Directoraddressingany meptions to Good LabmVmy PracticSetandards. 4. Objectivesand proceduresstatedintheapprovedprotocolincludingany changesin theoriginal protocol 5. The testsubstanciedentifibeydname,chemicalabstracntumber orcodemunbcr,strengtkpurity, composition,and othercharacteristipcrsovidedby theSponsor. 6. Stabilitaynd solubiliotfy thetestsubstancesundertheconditionosf atiminiztatioinf,providedby theSponsor. 7. A descriptioonfthemethods usedtoconductthetest. 8. A descriptioonfthetestsystem,includindgw sourceofthetestorganismsand theirscientifniacme 9. A descriptionfffipcreparatioOnfthetestsolutionsm,ethodsused toallm#z orgmsins tothetest chambersandbeginthetestn,umbers oforgm= and chamberspertreatmenta,nddurationOfffic tesl 10. A descnption of cunnnstances thatmay have affectedthequalityor integritoyf thedata. 11. The n of theStudy DirccWr and ft names of otherscimflstsp,rofessionalsa,nd supervisory personnelinvolvedin thesu*. 12. A descriptioonf thetransformationsc,alculationosr,operationpserformedon thedata,a summary and analoisofthebiologlcadlataand arwyticalchenustrydata,and a statementoftheconclus* drawn from theseanabmm 13. Statisticmaelthodsemployed foranalyzingthedat& 14. The sigoodanddatediepa ofeachofthein@@ sricnfisotrsodw professionailnsvolvedinffic study. PROTOCOL NO.: 454/091799/SKE96/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL, LTD 15. 'Melocatiwohnererawdataandfinarleporatretobestore& 16. A staementpreparedby theQualityMsm-mm UnitlistintghedatesffigM* in@-ons and audits were ma& and fficdatesof any findingsreportedto theSu* Directorand Management. 17. Ifitisnecessaryto make conwfions or additionsto a finalreportafterithas be= accepted,such changes shallbe made in theform of an amendment issuedby theStudyDirector Ilm amendment shallclearlyidentifydiepartof the finalreportthatisbeingamended -andfficreasonsforffic alteratiomAmendments shallbe signedand datedby theSu* Dn=W CEL4,NGING OF PROTOCOL Plannedchangestotheprotocolwillbe intheform of writtenamendments sigmedby theSb* Directorand theSponsoes RepresentativeA.mendments willbe consideredaspartoftheprotocoland win be attachedtothefinalprotocol Any otherchanges willbe intheform ofwrittm deviationssignedby the Sh* Directorand filedwiththeraw dat& AU changestotheprotocolwillbe mchcatedinthefinalreporl GOOD LABORATORY PRACRICES This sU* vnU be conductedin accordancewith Good LaboratoryPracticSetandardsforEPA (40 CFR Part160 and/orPart792);OECD Principleosf Good LaboratoryPracticQeWV/MC/CHEM (98)17); and Japan MAFF (59 NohSan, NotificatioNno. 3850, Agriculan-aPlroductionBureau). Each sh* conductedby Wildhfe InWnationaL LtcLis-routinelyexanuned by theWildlifehitcmtiomd,Lt(LQualityAssurance Unit forCC)MPH=CC with Good LaboratoryPracticesS,tandardOperatingProceduresand the specifiepdrotocol A statementof compliancewith Good LaboratoryPracticewsillbe preparedforall portlonsof the sb* conductedby WildlifeIntc=tLonaL Ltd- The Sponsorwillbe responsiblfeo@ compliancewith Good LaboratoryPracticesforproceduresperformodby otherlabmvmes (0.&,residcu analysesorpathology).Raw dataforallwork performedatWildliffeirter@onalLtd.and a copy ofthe finalreportwillbe filedby prcjectnumber inarchiveslocatedon theWildlifhea=ationa Ltd.siteo,rat an alternativleocationtobe specifiedinthefinalrepoit PROTOCOL NO.: 454/091799/SKE96/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNA-noNAL, LTD 12'- REFERENCES I US. Eavirommental ProtectionAgency. 1996. Series850-EcologicaElffectsTest Guidelines (draft)O,PPTS Number 850.5400: AlgalToxicityT,iersIand H. 2 ASTM Standard Guide 12.18-90L 1990. S@z@d Giadefor Conduchng Static96-Hour Tauc7ty TeStS*7thMicroalgae. Amawan SocietyfirTestingand Matmials. PU[acUphu4P=nsylvama. 3 Norberg-lCmgTJ. 1993. A Linearlnterpoladon Mediodfor Sublethal Toxidly. 27seInhibidon ConcmoWona(@,) Approach (FWWon 2.0).US. Bwkomnental ProtectionAgency, EwAmmental ResearchLabomtofy, Duluth,Wmnesota. 4 West, Inc.and D. D. Gulley. 1996. YOMTATRekase 3.5.Western EcoSystmm Twhwlosy, brchrimile@Wyoming PROTOCOL NO.: 454/091799/SKE96/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL, LTD -13- SALTWAMR TABLE I ALGAL KIEDIUM CONsTnuENTS Component Nominal Concentration FeCI3.6H20 NiuCl2.4H20 ZdgO4.7H20 CuSO4.5H20 CoC66H20 H3BO3 Na2EDTA.2H20 K3PO4 NaNO3 Na2SiO3,9H20 Thiamine Hydrochloride Biotin B12 0.72 mA 2.16 mg/L 0.675mgtL 2.36 gg/L 6.06 lig/L 17.1 mg/L 15.0 mg/L 3.0 mg/L 50.0 mgtl 20.0 mg/L 0.25 mg/L 0.05 itg/L 0.5 lig/L lie abovecomponent win be afdod toinstansaltwatearta salinitoyf approximately30 partsper thousand. The pH of themodiun willbe adjusted,as necessaryt;o between 7.8 iuid8.1 using0.1 N NaOIi or 10'/oHCL PROTOCOL NO.: 454/091799/SKE96/SUB454 3M LAB REQUEST NO. U2723. WILDLIFE INTERNATIONAL, LTD -14- IDENMCATION APPENDIXI OF TM SUBSTANCE BY SPONSOR To beCcnvlded by Spcn" 1. TestSubstanceIdentit(yname tobe used intilereport):PFOS (PafluorooeteneSWfonicAcid_PotassiumSal ReferenceStandard(ifapplicable)A:nalyticalStandard: N/A InternalStandard: 1.12.2HILILH PufluorooctaneuifonicAcid TestSubstanorS,ample Code or Batch Number Lot 217 TestSubsu= ActiveIngredient): 98.9 R TestSubstm= Chmxtmfinfim Fxpirad.onDate. 2.008 Havr.de Wmt4y.. =and compostm orcdxrdumcwdstics wtkh.FF.=7'r abstsom andnfirmw dmxkd bom . deteadfi8d ftior tD its use in this sb* in acomlmce mi& GLP Swndards? --L-YCS No in. TestSubstm= Stonp Ca2ffifi= Pk= ffi=c==n&)d Amp c=Wm Anibiat atW&H& bwmfic* IAi Hm ffidcabltyofffitemtmbsta= uodw &= stmw =ddim b= &*nnki@dinac=&= vi& GLP Sftxlm&? Odxr pmfinentstabilkkykm3atim- _Yes x No IV. Ted C03ceabudms. A4ustW tmfm to 100'0a/i0 x -basedwm ffiperiy Clo)givm abom V. TcDdcithy&nna&m- Do nota&justttd OMCCUUUfiM tD 1000/0 ai TestffinciatmiaAlS IS. hfammaem- Aquatic,. RatU)50 251 mg& -Maw hmzWmftTodcit@Q3C4-C50) ID50 N/A FishTTd&y WO) -Piabm:a magna: 27 mF JL Dakhm @a ma=: 50 mg! Rainbow Tmut II mg& FaffieadMinnow: 38 mvJL Odw Tod(iy Wmiafim Cm&idipg6--iinFo.fSdroicandwb@b@ te&): Please see MSDS PROTOCOL NO.: 454/091799/SKE96/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL, LTD -15APPENDIX ]I AnalyticaMlethod Pwvided by Sponsor Samples willbe ana4-zedbased upon proceduresprovidedby theSponsor inthefollowingam@ydcal methods: 1. Liquida@@gWby Mass Specftmetry(LCMS) Method fortheDd=aination of Pedhmrooctme SulfonicAcid Potass'nimSalt(PFOS) In Freshwaw, Saltwaterand AlgalMedim A copy of theabove method willbe 112@@@ intheraw data. The acuw methodologyusedto analy= thetestsampleswillbe dommented intheraw dataand summarized inthefinalreport. PROTOCOL NO.: 454/091799/SKE96/SUB454 3M LAB REQUEST NO. U2723 )o rAA 4.Lu.044 0632 WILDLIFE INTERNATIONAL WILDLIFE INTF-RNA-noNAL-LTD. Q008 PROJECT 140.:454A.1 13 Page I of2 ANCENDACENT TO STUDY PROTOCOL STUDY ITRLE: PFOS: A 96-HOUR T03aCITY TEST WrM TliEMAPJM DLATOM (Shelcionema costatum) PROTOCOL NO: 454/091799/SKE96/SU]3454 SPO NSOR. . EFFECNVE 3M Corporation DATE: Fcbnmry 28,2000 DRAFT AMENDMENT NO.: I PROJEC]R NO.: 454A-1 13 3M LAB REQUEST NO.: TJ2723 AMNDNW.NT: Page 2 Add- ExperimentalStartDate- March 3.2000 ExperimentalTerminationDate: March 7,2000 TestConcentrationsN:egativeControland 4.0mg ai./L TestSubstanceNo.: 4675 REASON: Ile above informatiownas notknown when theprotocolwas signedby theStudyDirector. AMIENDMENT: Emeria=W Desi&LPagc 3 ChanSe. The numinc diatom,Skclcton=a costabn33w,illbc exposed to a goomctricseriesof six test concentrationasnd a negative(culturmeedium) control-fo9r6 hours. To: The marinediatom,Skeletonamacostawni,willbe exposedto a testconcentratioantthelimitof solubilitaynd anegative(culturmeedium) controlfo9r6 hours. REASON-. To changewqxuii=LAI designtoa ffirltittsl AMENDMENT: E2MgdmeaW Desi Page3 Change: Test solutionwsiU be inoculatedwith appro)dmately77,000 ccUs/mL Test solutionwsillbe ijioculatwoidth 10,000cells/mL. To: Testsolutionwsillbe inoculatowditlaipproximately77,000cells/mL. REASON: Typogriaphicaclrror. AMENDMENT: BUcrimentalDesi= Page3 Chanse: Three"biologicalr"eplicatepsercxpcrirnentgarloupwillbe prcpamd forevaluatincg4l de=ities. To: REASON: Six"biologicalm"vlicatesPerexperimentaglroup willbe preparedf6revaluatincgelldmsil'ies. To change=perimentaldesigntoa limittcst. 4009 WILDLIFE INTF-RNA-noNALL-m. PROJECT NO.: 454A- 113 Page 2 of2 R A FT ,khEMMENT: S=plinizforAnalyticaMlcaweMents.Page7 reMW=g Change: Day 4 samplesvallbe compogitedsolutionfsmm the&w repheates attheend ofthetest To: Day 4 sampleswillbe compositedsolutionfsromthesixrepEcaWsremainingattheendofthetest REASON; To changemwyticalsampljn$basedon amitionalreplicatpersovidedinthelhnitLmt STUDV DIMCROR LABORATORY MANAGEMNT DATE DATE hllm DATE7-7