Document Rj6JD2mxJLr96694eyVaRj7bB

Ea Fr AR126-0186 0E5-3 PROTOCOL PFOS: A 96-HOURTOXICITY TEST `WITH THE FRESHWATER ALGA (Anabaenaflos-aquae) ~ US.Environmental ProtectionAgency "Series 850 -Ecological Effects TestGuidelines (OPPTSNumber 850.5400 3MLab Request No. U2723 * Submitted to 3M Corporation `EnvironmentalLaboratory 935 Bush Avenue `St. Paul, Minnesota 55106 - Wildlife International, Ltd. nT 8598 Commerce Drive `Easton,Maryland 21601 (410) 822-8600 September 17, 1999 604482 WILDLIFE INTERNATIONAL, LTD -2- PFOS: A96-HOUR TOXICITY TEST "WITH THEFRESHWATERALGA (Anabaenaflos-aquae) SPONSOR: 3M Corporation `Environmental Laboratory 935 Bush Avenue St. Paul, Minnesota 55106 SPONSOR'SREPRESENTA"TIVE: RochellRe. Robidesu . TESTINGFACILITY: `W8i5l9dl8iCfeoImnmteerrncateioDnrailv,eLtd. . `Easton,Maryland 21601 STUDY DIRECTOR: Cary Sutherland, Senior Biologist LABORATORYMANAGEMENT: ~~ Heasy O. Kruoger, PhD. DireofcAtquoatric Toxicology & Non-TargetPlants FORLABORATORY USE ONLY Proposed Dates: . `Start Date: Project No.: df 4- (10 TerminationDate: ____~ Test Concentrations: _ : TestSubstanNcoe: _4675. RefercaceSubstanceNo. (fapplicable): __4526 PROTOCOLAPPROVAL te0 STUDY DIRECTOR U4 -------- LABORATORY JEMENT Em IE se DATE 2/9foo DATE B 04483 PROTOCOLNO.: 454/091799/ANA96/SUB454 3M LAB REQUEST NO. U2723 AWAILTDLITFER INNTTCERRRNLATTIVOONNAALY,LLITDD ee eeecssmmmmsseeeeemmeimme 3. INTRODUCTION `WildeInternationa,Ld.willconduct four-daytoxicity tet iththefreshwater alga,Anabaena Jflos-aguae,fo theSponsoratthe Wildlife ntemationalLid.aquatictoxicology clitinEaston, Maryland. `Thestudywillbeperformedbasedonprocedures i.the U.S.EnvironmentalProtection AgencySecs850Ecological Efects TestGuidelOiPnPeTSs Number 850.5400; Algal Toxicity, TiIaendIrldesaf) 1). RawdataforallworkperformedatWifeIntemational,Lid.and acopyofthe ialreportil befiled byprojectnumberinarchiveslocatedontheWildeInternationalLid site,orat analternativelocationto bespecinitheffinial reepodrt. "PURPOSE `Thepurposeofthi stdyis todeterminethetoxicityofPerlucrooctane SulfonicAcid, Potassium Salt(ercattereferred1 as PFOS)tothefreshwateralga,Anabaena flos-aguae. EXPERIMENTALDESIGN - Thefreshwateralga,Anabaenaflos-aguae,vilbeexposedto ageometricseriesofsixtest `concentrationsand negative culturemedium)control for 9 hours.Targetconcentrwialltnoiteoxcneesd 100mg/L orthesolubilitylimitofthe testsubstanceinwater. Generally, thenominalconcentraotficaocnh testsubstanceusedinthedefiiiv testwillbeatleast50%ofthenexthighertreatment,unlessinformation `concemingtheconceatratio-effoctcurveindicates that adiffrentdilution ctorwouldbemore appropriate. Testsolutions el be inoculatedwith 10,000celisinL. Three"biorleploicatgespierccxpaerilmea"tal groupwillbepreparedforevaluatingceldensities. Oneormore additional*malyticlreplicateswillbe includedin thetest,asneeded,toprovidetestsolution forconcentration verificationonDay 3 ofthe exposure. Anabioti replicate a thehighestconceatrationvil beincludedinthetestandwillbesampled onDay andDay4.Inordertoconirol bias,thepositon oftheflaskswillbedeterminedbyindiscriminate dra dailyduringtheexpospeurrioed. Nootherpotentialsourcesofbiasareexpectedtoaffecttheresults ofthestudy. `Theresponseofthe algaevil bemeasuredinterms ofclldensity,biomassexpressedasareaunder thegrowthcurve,andgrow rate.AnECS0 (Le,thetheoreticaltestconcentration thatproduces a 50% reductioninthemeasuredparameter) velueforclldensity willbecalculatedforeach24hourinterval. EC10,ECS0audEC90valuesforceldeasity,biomass(EC)andgrowihrae(E.C)willbe calelated,if PROTOCOL NO.: 454/091799/ANASG/SUBAS4 3MLABREQUESNOT. U2723 004484 WILDLIFE INTERNATIONAL, LTO 4 `possible, athe72and96hourintervals.Thenoobservedadverseeffetconcentration (NOAEC),which - isthehighesttest concentration that inducesno adverseinhibitoryeffectongrowdh,willbe determined `relative to each parameter at 72 and96hours baseduponevaluation of the statistical resultsand the dose- responsepattem.Atthe endofthe96-hourexposure,algistaticeffectswillbediffercatiatedfromalgicidal `effectsinthosetreatmewhnitcsharemaximally inhibited. MATEARNI DMEATHLODSS Informationon thecharacterizationoftest,controlorreferencesubstances isrequiredbyGood LabPo racr ticeaStat ndao rdsr (GLy P).The SponsorisresponsibleforprovidingWildlifeInternational,Ltd. writtenverificationthat th estsubstancebasbeencharacterizedaccordingtoGLPspri o tsusointhe `study. IfwrittenverificationofGLPtestsubstancecharacterizationisnotprovidedtoWildlifeIntemational, Ltd,itwillbenotedinthecompliancestatementof thefinalreport.TheattachedformIDENTIFICATION `OFTEST SUBSTANCEBY SPONSOR (Appendix I)istobe usedtoprovideinformationnecessaryfor GLPcompliance. TheSponsorisresponsibleforallinformationrelatedtothetestsubstanceand agreestoaceeptany unusedtestsubstanceand/ortestsubstancecontainersremainingattheendofthe stidy. Jit Seton Pega `Thedesired concentrationsofPFOSwillbe obtainedbypreparing asinglestocksolutioninmedium `anddiluting the appropriate vool fstou cksm olute ion withmedium.Thetestsolutionsmayalsobe prepared `usingsecondarydilutionsoftheprimarystock, basedupontheconcentration desired. PFOSwillbe administeredtothetestorganisminalgalmedium.Thisrouteofadministration was `selectedbecause itrepresents themostlikelyrouteofexposureto algae, which existsuspeinnadwaetder column. . TestOrganism `Thetestspecieswillbethefreshwateralga,Anabaenaflos-aguae. Thisspeciesisrepreseatativeof `animportant groupofalgac and was selected for useinthe testbased uponpast use andease of handlingin thelaboratory. Stockcultures,obtainedfromtheCultureCollectionofAlgaeattheUniversityof Texasat PROTOCOLNO.: 454/091799/ANAY6/SUBAS4 3MLABREQUESTNO. U27804485 WILDLIFE INETERRNAATIOONAAL,D LD ee ge eee Austinoranothersupplicr,willbemaintainedinculturemediumat WildlifeIntemational,Lud.for a we minimum oftwoweeksprior tousein toxicitytest.Algaeusedintoxicity estswillbeinexponential `growthphase,which sdefinedastheperiodofgrowthwhenalgal elsaredividingat aconstantrate. Justprocto begioningthetest, an inoculumofthestock culturewillbepreparedsotha eachlier ofinoculumcontainsenoughcellstoprovideennislcelldensity of spprosiaately10,000 lsh.inach replicate, CultureMedium Culturemedium preparedaccordingto Wildlife International,Ltd.StandardOperatingProcedures willbeusedasdilutionwater. Theconcentrationsofth componeatsinthemediumaepresented aTable 12). Stockmriatsolutionswillbepreparedbyaddingreagent-gradeorbette chemicalsto Wilde International, Ltd.wellwaterpurified byreverse-osmasis. Apprvoloumespofrtheistoackntutrieent solutionswillthenbedilutedwithpurifiedwel watertopreparethemedium.Themedwiillboslenr sterilized (0.22 um)orautoclavedpriortouse,Analysesvillbeperformedatleastonceanalyto determinetheconcentrationsof selectedorganicendinorganicGonstituentsofthewell ater odresultsof themostrecatGLPcompliantanalyseswillbesummarizedi thefinal report. `Specificationsfo acceptablelovlsofcontaminantshavenotbeenestablishedfo culturemedium. 3 Hower,thesare nolevelsofcontaminantsreasonably expectedtobepresentinthemediumthatare `consideredtointeferswiththepurposeorconductofthestudy. TestApparatus "Testchamberswillbe sterile250-mLpolycarbonateErlenmeyerflaskspluggedwithfoam stoppers and cont100amLiofntesitsonlutigon. Thetestchambers vilbeindiscrimpoisintiaontedeolnay `mechanicalshakestablein anenvironments!chamber ndwillbeshakencontiauouslya approximately 100 ___....Tpm. Testchamberswillbelabeledwiththeproject number,test concentration,and replicate. Emi Cnt "Testflaskswillbeheldat24 2CundercontinuouscookwhiteSuoresocatlightingatan nteasity 2200:1h0ox%. Light tensitywillbemeasatfuiverlocaetiodns surroundingthetet flaonsDkays0 PROTO0COLTOCO!NO: 454/091799I1ANASG/SUBA54 MLABREQUESTNNOO..UU27 (40 0 O W_I--LDLIFE INTERNAO TIONAL, LD OO -6- ofthetest. Temperaturewillbemeasuredtwicedailyin acontainerofwater adjacenttothetestflasksinthe environmental chamberusing aliquid-itnh-ergmolmaetsesr. The pHofeach treatmentandcontrolgroupwillbemeasuredattestinitiation andterminationusing. aFisherAccumetModel915 pHmeterorequivalent.`Samplesfor pHmeasurementattestinitiation will be. `collectedfromthe individualbatchesoftestsolution preparedforeachtreatmentand control group.Attest termination,pH willbemeasuredinpooledsamples oftetsolution collectedfrom cach ofthethreereplicates ofeach respectivetreatmentandcontrolgroup. BiologicalMeasurements Celldensitieswillbemonitoredduringthetestbyconductingcellcountsusing ahemacytometerand `microscope.Onesamplewillbecollectedfrom cachreplicate ofthetreatmentandcontrolgroups at24- `hourintervalsduringthe exposureperiod.`Thesampleswillbeheldatapproximately 4Cuntilcellcounts `canbeperformed.Eachsamplewillbedilutedusinganelectrolytesolution(Isoton),asneeded,tomaintain `countingaccuracy. Asmall amount of each samplewillbe loadedonto ahemacytometerand the total `umberofcellsin 10gridswillbecounted.`Thecelldensityofthe sample willbecalculatedbasedonthe `meannumber of cellsper grid.Using thistechnique, theminimum quantifiablecelldensityis 1000cells/mL. `Samplesoftestsolutions willbecollectedfromeachofthethreereplicatespertreatmentandcontrol groupatthe endofthetest.`Thesesamples willbepooledwithintheirrespectivetreatments,andsubsamples `willberemovedand examined microscopically foratypicalcellmorphology(e.g,changesincellshape,size. `orcolor).Growthof cellsinthe flasksalsowillbeassessedforaggregationsor flocculationsofcellsand `adherenceofthe cellsto the test chamber. Atthe eadofthetest,algistaticeffects,whichresultintheinhibitionofcellgrowth,willbe differentiated from algicidaleffects, which resultinthedeath ofcells.Aliquots(0.5mL)oftestsolutionwill `betakenfrom each replicateofthosetreatmentgroups wheregrowth is maximallyinhibited.Maximally inhibitedtreatmentsarethoseinwhichitcannotbevisually determinediflivealgalcells arepreseat (6.g., absenceofcolorto thetestsolution). Thesealiquots will be pooled byconcentrationanddilutedwith `untreated culturemediumto a concentrationofthetestsubstancethattheoreticallyshouldnotaffectgrowth. A negative contol wilbeprpurdby ding. 0.-nL alu from one egies repli 00) 1 PROTOCOLNO.: 454/091799/ANA96/SUB4S4 3MLABREQUENSOT. U2723 kp _ WILDLIFE I-- NTERNATIO-- NAL,eLTeD eese 7. mLwithculturemedium.Growthofthesesubcultureswillbemonitoredfor amaximum of 9daysto determine whetherinhibitionofgrowthobservedduringthetestisreversible. `Sampleswillbecollectedfor cellcountsatrecoveryphase initiation,terminationandatapproximately 3-dayintervalsbetweeninitiation `and termination.`Therecovery phasemay be terminated once algalgrowthis sufficienttoindicatethatthe algalcells collectedfromthetreatmentgroups havefullyrecoveredfrom effectsduetothetestsubstance, `This willbe determined through visual examinationoftherecoverytestsolutions. . "~ Samplesoftheexposuresolutions willbecollectedoaDays 0,3and 4endanalyzedfo est substance `concentrations.Day 0samples willbetakenfrom eachbatch oftestsolutionprior totheidistribution among thereplicatetestchambers.Day 3 sampleswillbecollected from the extrareplicatetestchamber(s)included inthetestto provide sampleforthat analysis and from theabiotic replicates. Ifmore thanonereplicateis includedfortheDay 3sampleanalysis,thenthesolutionsfromeachreplicatewillbecompositedtoprovide. the sampleforthattreatment. Day 4samples willbecomposited solutions from thethree replicates `rem ata theiendnofithn etegst. `Samplescollectedon Days 3 and 4maybecentrifugedor filteredtoremovealgalcelspriorto p`aonlaylypsriosp.ylSeaamcpolreposlwyieltlhbyleeannebaoltytzlee)diamnmdesdtioraetdeulnytiolrapnlaalcyezdeid.nThaenaspapmrpolpersicahteemsteoirsasgeucmonmtaairneibre(zlee.ogwd.: ce [E | wco|e bow |bG ys ooww s | Control PROPOSEDNUMBERS OF VERIFICASATMIPOLENS SolventControl (fneeded) Level1-LowConcentration Level 2 Love3t . 4 : Levels [os] Level6-HighConcentration 004488 PROTOCOLNO.: 454/091799/ANA96/SUB4S4 3MLABREQUEST NO. U2723 3 WILDLIFE INTERNNAATIOONNAL, TLDTD ei -3- ior `Theabove numbersofsamplesrepresentthosecollectedfromthetestand do notincludequality control (QC) samples suchas matrix blanks andfortificationspreparedand analyzed during the analytical chemistryphaseofthestudy. | 55 Chemical analysisofthe sampleswill be performedbyWildlife International Ltd:The analytical `methodusedwillbe based-uponmethodologyprovidedbythe Sponsorand identified in AppendixIL report. Modificationsmade tothe analytical methodwillbe documentedinthe raw data anddescribed in the final Sa `Biomassexpressedas area under the growthcurve willbecalculated forthe treatmentandcontrol groupsusing the followingformula: . A= (ONYX NNNYY rt HNrN 2NV2) rte) `Where: N A= Areaunder thegrowcutrvhe NN, =Mea nums beru ofcr ellse /mLad tt, Ny =Mena umbs erou fcer lls/e mLad tt; NN; =Mena umbs erou fcer lls/e mLad tt; 'N, =Mena umbs erou fcer lls/e mLad tt, ty =Timeoffirstmeasurementafterbeginningoftest(hours) t=Timeofsecondmeasurementafter beginningoftest(hours) ta =Timeofnthmeasurementafterbeginningoftest(hours) Growthrates (14) wil be calculatedfor the treatment and control groups using the followingformula: - INIn,No --_ mem . e . blo sree 'N, =Measured numberof cells/mLatt, t'oN,==TMiemaesoufrbedenugmbeiroonfftceneslti(lhso/numrLgas)tt, to =Timeofnth measurementafterbeginningoftest(hours) 04489 PROTOCOLNO.: 454/091799/ANA9G/SUBA454 3MLABREQUENSOT. U2723 -- WILDLIFE INTERNA_ TIONAL, LTD --r 000000 Oo -9- `andgroPwetrhcreaatteiunshiibnigtitohnewfiolllboewcianlgcfuolramteudlfao: cachtreatment group basedupon meancel deasity,biomass PercentInhibition =M_eaRnesC pono se n _-_Mt eaRner Tsrpeoo antsmel ent MeanControl Response X 100 ECvalueswillbe determined,whenpossible, usinglinear interpolation o othe suitabletechniques `ithtreatment response(i elldensities,biomassandgrowthrate)andexposureconcentrationdatafor `each 24-hour interval of the test. Amo observedadverseeffect concentration (NOAEC)willbeselectedbasedonanevaluationofthe `dose-responsepattemandtheresultsofthestatisticalanalysisusingindividualreplicatevaluesofthe ell 8roupswillbeidentifiedusing analysisofvariance (ANOVA)and atettocomparetreatment mean responsestothecontrolresponse (e.g,Dunnett's test orBonferroni'st-test).Datawillbeassessedfor `normalityandhomogeneityofvariances priorto performingthe ANOVA.`Transformationswillbe used to `correctanyconditionofnoa-normality orunequalerrorvariances,ifpossible. Ifa solveatcontrolisused inadditiontothenegativecontrol, theirresponses willbecomparedusing Student's test.Datafromthe . wocontrolgroupswillbepooledfortreatment level analysesif no statistical differencesexist. Otherwise, allcomparisonswillbe madetothesolventcontrolgroup. `Statistical comparisons fortheANOVA andthe `comparisonofmeanresponses willbecarriedoutusing TOXSTATV3.5software (4) o equivaleat. REC TOO BE MR AIND TAIS NED limitedto`Recordstobemaintainedfordata generatedat WildlifeInternational,Led. willinclude,butnotbe 1. Copyof signedprotocol. 2. Mentificationandcharacterizationof thetestsubstance,if providedbySponsor. 3. Datesof tiationandterminoaftthietoesnt. 4. Souorfalcgaee. 5. Culture conditions. 6. Growthmeasurements. 004490 PROTOCOLNO.: 454/091799/ANA96/SUB4S4 3MLABREQUENSOT. U2723 x W--I_--LDLIFE INTERNATIONAL, LTD ele OOOO 7. Calculationandpreparationoftestconcentrations. a 8. Observations. 9. Ifapplicable themethodsusedtoanalyzetestsubstanceconcentrationsandtheresultsofanalytical `measurements. - 10. Statisticalcalculations. 11. Testconditionsandphysicallchemicalmeasurements. 12. Copyoffinalreport. [FINALREPORT Afinal report oftheresults ofthestudywillbe preparedby Wildlife International, Ltd.Thereport willinclude,butnotbelimitedtoth following,whenapplicable: 1. Nameandaddressofthefacilityperforming thestdy. 2. Datesonwhichthestudywasinitiated andcompleted.Itistheresponsofithbe iSplonisotrtyo providethe finaldaethatdataae recordedforchemistry pathologyand/or supportingevaluations that maybegenerated at otherlaboratories. 3. Astatementofcompliancesignedbythe StudyDirectoraddressinganyexceptionsto Good LaboratoryPractice Standards. | 4. Objectivesandproceduresstatedintheapprovedprotocol,includinganychangesintheoriginal protocol. 5. Thotestsubstanceidentifiedbyname;chemicalabstractnumberorcodenumber,strength,purity, composition,andothercharacteristicsprovidedbythe Sponsor. 6. Stabilityandsolubilityofthetestsubstances undertheconditionsof sdmisistraton, if providedby theSponsor. 7. Adescriptionofthemethodsusedtoconductthetest. 8. Adescription ofthetestsystem,includingthesourceofthe est cganismsandtheirscieatifcname. 9. Adescriptionofthepreparationofthe estsolutions,methodsuse toallocateorganismstothetest chambersandbeginth test,numbersoforganismsandchamberspe treatment,andduration ofthe test -------------10-- Adescriftionofcircumstancesthatmayhaveaffectedthequalityorintegrityofthedata. 11. ThenameoftheStudyDirector andthenamesofothrscintiss,professionals, ndsupervisory personnelinvolvedin thestudy. PROTOCOLNO.: 454/091799/ANASG/SUB4S4 004491 - 3MLABREQUESTNO, U2723 A W ILDLIFEEINTAEe RNTATIOONAeL, TLTDs D et -u- 12. Adescriptionofthetransformations,calculations,oroperations performed on thedata, a summary `and analysisof thebiological data and analytical chemistry data, anda statementofthe conclusions drawn fromtheseanalyses. 13. Statisticalmethodsemployedforanalyzingthedata. 14. `The signed and datedreports of each of theindividualscientistsorother professionalsinvolved in the study. 15. Thelocation where raw dataand final reportaretobe stored. 16. Astatementpreparedby the Quality Assurance Unitlistingthedatesthatstudy inspections and audits `were made andthe datesof any findings reported to the StudyDirector andManagement. 17. Ifitis necessary tomakecorrections or additions to afinalreportafterithasbeen accepted, such changesshallbemade inthe formof an amendmentissued bythe StudyDirector. Theamendment shallclearlyidentify the part of the finalreport thatis being amended andthe reasons forthe alteration.Amendmentsshallbesigried anddated bytheStudyDirector. CHANGINGOFPROTOCOL . `Plannedchangestotheprotocolwillbeintheform ofwrittenamendmentssignedbythe Study Director and the Spoasor's Representative. Amendments willbe considered as partof the protocoland will `beatttoathecfinahlproetocdol. Anyotherchangeswillbeintheform ofwrittendeviationssignedbythe `StudyDirectorandfiled withtherawdata.Allchangestotheprotocolwillbeindicatedinthefinalreport. GOOD LABORATORY PRACTICES `This studywillbe conducted in accordance withGood Laboratory Practice Standardsfor EPA (40 CFRPart 160and/or Part 792); OECDPrinciples of GoodLaboratory Practice(ENV/MC/CHEM(98)17); and Japan MAFF(59, NobSan, NotificatiNoon.3850, Agricultural Production Bureau). Eachstudy `conducted byWildlifeIntemational,Ltd.is routinely examinedby theWildlifeInternational,Ltd.Quality AssuranceUnitfor compliance with Good Laboratory Practices, Standard Operating Procedures andthe specified protocol. A statement of compliancewith GoodLaboratory Practices willbeprepared for all portions of the study conducted by WildlifeInternational, Ltd. TheSponsorwillbe responsibfloer em em `compliancewithGoodLaboratory Practices forprocedures performedbyother laboratories (e.g, residue - -- anaolrpyathsoleogys). Raw dataforallworkperformedat WildlifeInternational,Ltd.and a copy ofthe final report willbefiled by project number in archives located on theWildlifeInternational, Lid.site,orat an altemativelocationtobespecified inthefinalreport. 004492 PROTOCOL NO.: 454/091799/ANA96/SUB454 3MLABREQUENSOT. U2723 WILDLIFE INTERNATIONAL, LTD 12- REFERENCES 1 US. EnvironmentalProtectionAgency. 1996. Series 850-EcologEifcfeactls Test Guidelines . (draft), OPPTS Number 850.5400: Algal Toxicity, Tiers I and IL. 2 ASTM Standard Guide 1218.90F. 1990.StandardGuideor ConduStactitc9i6-nHougr Taxiclty Testswith Microalgae. AmcricanSocictyforTestingand Materials. Philadelphia, Pennsylvania. 3 Norberg-TKJi.n19g93,. ALinearInterMetphoodfolrSuabletthaliToxoicinty:TheInhibition Concentration(IC,)Approach (Version 2.0). USS. EnviPrr oteco tionmAgee ncy,nEnvtironamenltal ResearchLaboratory, Duluth,Minnesota. 4 West, Inc. and DD. Gulley. 1996. TOXSTATRelease 3.5. Wester EcoSystems Technology, Inc. Cheyenne, Wyoming. PROTOCOL NO.: 454/091799/ANA96/SUB4S4 04493 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL, LTD 1- TABLE 1 FREAS LGH ALMEW DIUA MCOT NSTIE TUER NTS `Component `Nominal Concentration VERO 1206 mek CaCLr2H;0 440 mglL HBO, 0.1856mg/L. MaCl4H,0 0.416 mg/L ZnCl 328 gL FeCly6H,0 0.1598mg/L. CoChe6H:0 1428 gL CNuaCl;yM2oHO,#02H0 NaEDTA2H,O NaNO; MgSOLTHO KHPO, NaHCO; 0702162 ggLL 0300 mg/L, 2550 mg 1470 mg 1044 mg/L, 15.00 mg ThepHofthemedium willbe adjustead, nece07.s5+s0.a1wirngyO1,N NaOH or 10% HCL. en \ Cem PROTOCOLNO.: 454/091799/ANA96/SUB454 - 04494 3MLABREQUEST NO. U2723 W_I--LDLIFE INTERNATIOe NALe, . LTD 14. APPENDIX IDENTOFITEFSTSIUBCSTAANCETBYISPOONSNOR Tobe ComplbeySptonesadr I TestSubstance deni ReferenceStandard(if (asppalmiecatbolbe)e:uAsnealdytiitchaelrSetpaorntd)a:rdP:FOS_N(/PAerflucrooctaneSulfoicAcidPotassiumSalt TestSubstanceSampleCodeorBatch NuInmtbeerrna:l_S_tLanodta2rd1:7_1,1.2.2HHHH PerfluorooctaneSulfosicAcid TestSubstancePurity (% ActiveIngredicat): __989 ExpirationDate: _2008 IL TestSubstaeeCharacterization . Hatepa deh nysni ig pbBitEtymnbecettpoemmi poocretonstgchireiiogsietns Ys _x No IL TestSubsStotragaeConndictioens Pleasoindiatth ecumendedteragecoediionsstWd ternational,Lid. Abbeesab . 000000 oc pl fe oe Yo x Mo te ee ee sst----eeirii men ee IV. TestConcentrations: . x basedApdajnutsiettpesrtcyo(n6)pgevenntrh0oa1v0te0i%oain. } Doctoft conttion0t0%o. V. Todchy formation: Mammal: RatLDSO 251mpgMowsLDSO_NA Aquatic vercbrateToxicity (BO/LCSO) FishTaxiy(LCS0) 2 Dephoiamagna: Somgfl Fathead Minnow: 38me/l. OtherTentInfomation achingfiodingsofchronicandsubchrocic ests): fe PleaseseMsDS Sia PROTOCOL NO: 454/091799/ANASG/SUBASS 04495 SMLABREQUESTNO.U723 BEWILDLIFE INTERNATIONAL, LD Lis. APPENDIIXI AnaMel thoy dPrt ovii dedbcySa ponl sor * Samples willbeanalyzedbaseduponproceduresprovidedbythe Sponsor athefollowinganalytical ofPerfluorooct1a.neSLoilfqounidACchirdoPmoattaosgsriaupmhySMalas(sPFSOpSec)tIrnoFmeestrhya(tLeCs,MSSa)lMtewattheoradfnodrAtlhgealDeMteedrimicnation analyzAectohpyteosftstahmeplaebsovweilmlebtehdoodcwuimlelnbtemaiinntthaeirnaedwidnattahaenradwsudmamtaar.iTzheediancttuhaelfmineatlhroedpoolrto.gy usedto , AA . PROTOCOLNO.: 454/091799/ANA96/SUB454 , 004496 3MLAB REQUEST NO.U2723 . W_IL--DLIFE INTERNATIONAL Lr. PROJECT NO.: 454A-110 |Phageellodf11 STUDY TITLE: AMENDMENT TO STUDY PROTOCOL PFOS: A96-HOURTOXICITY flos-aquac) TESTWITHTHEFRESHWATERALGA (Ansbacna PROTOCOL NO: 454/091799/ANA96/SUB4S4 SPONSOR: 3M Company EFFECTIVE DATE: February 23, 2000 AMENDMENT: Page2 AMENDMENT NO.: 1 PROJECT NO.: 454A-110 3MLABREQUESTNO.: 12723 Add: ExperimentalStartDate:February25, 2000 . ExperimentalTermination Date: February 29, 2000 `TestConcentrations: NegativeControl, `Test Substance No.: 4675 39.5,61.0,92.5, 145, 225.and 345mgai/l REASON: Theabove information wesnotknown whentheprotocol was signedbytheStudyDirector. AMENDMENT:ExperimentalDesign, Page 3 . Change: `Targetconcentrationswill notexceed100mg/L o thesolubility limitofthetestsubstance inwate, To: `Targetconcentrationswillnotexosed 1000mg/L orthesolubility limitofth testsubstanceinwater REASON: Nominaltestconcentrations exceed 100.mg/L. A 7 aL2 STUDY DIRECTOR J. 4 JRATORY MANAGEMENT -- PSO-- NZE-- VARe-- s GLHiiz = hho DATE 9 [23/po DA Bj "04497 F Reviewd[7% 23300