Document YVBvmBy0awDm1DLnedVvYR6E

AR226-00%3 pros `A 96-HOUR STATIC ACUTE TOXICITY TEST `WITH THE FATHEAD MINNOW(Pimephales promelas) FINAL REPORT `WILDLIFE INTERNATIONAL LTD. PROJECTNUMBER: 454A-102 3M LAB REQUEST NO. U2723 SE CEPTS Number 350.1075 U. Environmental Protection Agency `Series 850 ~ Ecological Effects Test Guidelines and `OECD Guideline 203 AUTHORS: Kurt R. Drottar Henry O. Krueger, Ph.D. STUDY INITIATION DATE: December 4, 1998 STUDY COMPLETION DATE: July 14,1999 AMENDED REPORT DATE: April 26, 2000 `Submitted to Envi3rMomCmorepnorLataibonora 935 Bush Avenue St. Paul, Minnesota 55144 Wildlife I85n95tCeomrmenrac Dtriiveonal Ltd. Easton, Maryland 21601 (410) 822-8600 Page 10143 000543 AMENDED WILDLIFE INTERNATIONAL Lm. 2. PROJECT NO.: 454A-102 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT SPONSOR: 3M Corporation TITLE: PROS: A 96-Hour Static Acute Toxicity Test with the Fathead Minnow (Pimephalespromelas) WILDLIFE INTERNATIONAL LTD. PROJECT NUMBER: 454-102 STUDY COMPLETION: July14, 1999 AMENDED REPORT: April 26,2000 Envir"oTnhmiesnsttauldPyrwotaesctcioonndAugcteendciynicnom4p0liCaFnRcePvairtthsG1o6o0daLnadbo7r9a2t,or17yAPuragcuisote 1S9t8an9d;arOdEsCasDPpurbliisnhecdbioypfthlGoUeSos.d `LNaobhoSraant,orNoytPirfaicctaitcieo,nONCo.DE38/5G0,DA(g9r2i)cu3l2t,urEavliPrroodnumcetinotnMBounroegaru,ap1h0NAou.gu4s5t, 1P9a8r4isw1i9t9h2t;heafnodllJoawpianngeMxcAeFptFi,on5s9: chaThretaesctwastusbpseetrafrnocreimweadzascnacoorttdcihniagrtaooct3enrMiSzteadnidnaarcdcOopredraantcinegwPirtohcfeudulrleGs LanPdcoMmeptlhioadnsc,ea;nhdoawlelvrearw,dtahtea `arebeingmainitntahe3iMnarcheiveds. Thetestsubstanceisbeingrecharacterizedinaccordance with GLP. accord"aTnhceeswitthaGboooifdltLhaiebtotersayttosruybsPtraacntciecuenSdtearndacrodnsd.itionsofstorageatthtestsit wasnotdetermined in STUDYDIRECTOR: K`SuerntioRr BDiorlootgairst 2A) faeko DATE SPONSORAPPROVAL: AlBosch `Sponsor 4DATEazf2o0r 000544 AMENDED WILDLIFE INTERNATIONAL Lo. 3. PROJECT NO.: 454A-102 QUALITY ASSURANCE STATEMENT Management were s follows: `Thisstudywasexaminedfor compliancewithGoodLaboratory PracticeStandardsaspublishedby the US. Environmental Protection Agency, 40 CFR Parts 160and 792, 17 August 1989; OECD Princiopf lGoeosd `Laboratory Practice, OCDE/GD (92) 32, Environment Monograph No. 45, Paris 1992; and JapanMAFF, 59 NohSan, Notification No. 3850, Agricultural Production Bureau, 10 August 1984. The dates of all inspectionsand auditsandthedatesthatanyfindingswerereportedtotheStudyDirector and Laboratory Acti: DATE CONDUCTED: TestSubstancePrepuntion. ~~ Febrary 11,1999 Tes nition February15,1999 MPraetpairatFioorntiScaion February 15,1999 MeWastseareCnheeamtissty Febrary 17,1999 BDiotkgRieaplorDtut ed Mach 17,1999 DsaafyReapDtatand FiatReport AmendedReport March 2-24,1999 ly 14,1999 Api 19.85420,2000 DATE REPORTED TO, STUDYDIRECTOR: ~~ MANAGEMENT: Feary 11,1999 Febuary 11,1999 Fenary15,1999 Februay 16,1999 Feary 15,1999 Febuary 18,1999 Feniy17,1999 March17,1999 February 19,1999 N3een 26,1999 March24,1999" Jy 14,1999 April 20,2000 Mach24, 1999 iy14,1999 Apel 24,2000 A Lo -- Timothy A. Springer, Ph.D: Manager, Regulatory and Technical Support A sa%/20 DATE 000545 AMENDED WILDLIFE INTERNATIONAL Lr. "4. REPORT APPROVAL PROJECT NO: 454A-102 SPONSOR: TITLE: 3M Corporation PFOS: A 96-Hour Static Acute Toxicity Test with the Fathead Minnow (Pimephales promelas) ~ `WILDLIFE INTERNATIONAL LTD. PROJECT NUMBER: 454A-102 STUDYDIRECTOR: AN Kurt R_ Drottar `Senior Biologist MANAGEMENT: 4L Fear0y. Kheger, PhD. Director, Aquatic Toxicologyand 'Non-Target Plants 4/36/00 DATE 4 a Joo DAH 000546 AMENDED WILDLIFE INTERNATIONAL Lm. 5. `TOAFCBONLTENETS PROJECTNO: 454A-102 TBbIEOFCOMERS. OBST rrr SR | TABLES Table 1 -Summary ofAnalyticalChAT Dat. `Table 2 - Temperature,DissolvedOxygenandpHofWaterinthe TestChambers Table 3 -CumlativePercentMortalityandTreatmentRelatedEF... 15 rn 16 11 000547 WILDLIFE INTERNATIONAL Lm. -6- `TABLE OF CONTENTS PROJECT NO: 454A-102 FIGURE Figur1e. ConoentratiCuornve-(9R6-eHOsUEpDaotan).s..e - 19 i APPENDICES Appendix 1- SpecificConductance,Hardness, Alkalinity,and pH ofWellWater Measured During the 4-Week Period Immediately Preceding the Test... 20 Appendix TI iAnnaWliylsdeisiofefIPnetsteimcaitdieosn,alOLrtgdan.iWcesl,lMWeattarl.s.a.n.d..O.t.h..erInorganics-- 2 AppendiIlxl- TheAnaloyfsPFiOsS in FresinShupwporatoftWieldirife InternationalLtd.ProjectNO: 4S4A-102 vss 22 "Appendix IV = Changes (0 BOOCOL..cvrrnso smn) Appendix V - Personnel Involved in the Study..... sense snmmmonmmonn tl 000548 AMENDED WILDLIFE INTERNATIONAL Lm. 7. PROJECT NO.: 454A-102 SPONSOR SPONSOR'S REPRESENTATIVE: 'LDOATCAAATNIDOANFCOSPTUYDOYF,TRHAEW FINALREPORT: SUMMARY 3M Corporation Ms. Susan A. Beach `EWialsdtloinf,eMInatreyrlnaantidon2a1l6L0td1. LDL NTERATION. LTD. PROJECT NUMBER: TESTSUBSTANCE: stub: MEAN MEASURED TEST CONCENTRATIONS: TESTDATES: LENGTHOFTEST: 454A-102 PFOS (Perfluorooctane Sulfonic Acid Potassium Salt) PFOS: A 96-Hour StaticAcuteToxicity Testwiththe Fathead Minnow (Pimephalespromelas) NegativeControl, 33,5.6,9.5,17and 28 mg a.i/L. ExperiStmarte-nFetbruaarly15, 1999 Biological Termination~ February 19, 1999 Experimental Termin--aFtebiruoarny 19, 1999 96 Hours "TESTORGANISM: Fathead Minnow (Pimephales promelas) : SOURCEOFTESTORGANISMS: `EWailsdtloinf,eIMnatreyrlnaatnidon2al1L6i0d1. cultures AGEOFTEST ORGANISMS: NMEGEAATSIUVREECMOENTNRTOOFSLF1S0 H: WTEOITGAHLTLE@)N:GTH (mm): Juveniles MMeema==n03356;; RRaanngg=ee=3002011308049 SEHOURLCS0: 95% CONFIDENCE LIMITS: NOMORTALITY CONCENTRATION: C'ONNOC-EONBSTERRAVTEDI-OENF:FECT- T3mgaill 8011n mgad i/L 33 mgai/L 33mgail 000549 AMENDED I WILDLIFE ITNETERRNNAATTIIOONNAALL Lm. eee "8. PPRROOJWECCTTNNGO:: 540540A-010020 INTRODUCTION `Thisstudywasconductedby WildlifeInternationalLtd.for3MCorporationatthe WildlifeInternational Ltd aquatictoxicologyfacilityinEaston, Maryland. Thein-life phaseofthetestwasconductedfrom February 15to February 19, 1999. RawdatageneratedbyWildlifeInternationalLtd.and acopyof thefinal `reportarefiledunderProject Number 454A-102inarchives locatedonthe WildlifeInternationalLtd.site. OBJECTIVE `Theobjectiveofthisstudywastoevaluattheeacutetoxicityof PFOS (Perfluorooctane Sulfonic Acid conditions. PotassiumSalt)tothefatheadminnow,Pimephales promelas,during a96-hourexposureperiodunderstatictest EXPERIMENTAL DESIGN Fatheadminnowswereexposedto ageometricseriesof fivetestconcentrations and anegative(dilution `water)control. Tworeplicatetestchambersweremaintainedineachtreatmentandcontrolgroup,with 10fathead `minnows ineach testchamberfof atotalof 20 fatheadminnowspertestconcentration. Nominaltest `concentrationswereselectedinconsultationwiththe Sponsor,andwere basedupontheresultsofanexploratory `rangefindingtoxicitytest. Nominaltestconcesneletcterdwaeret3.i6,o5.9n,9.s9, 16 and 27mgactive ingredient (.i./L. Meanmeasuredtestconcentrationsweredeterminedfromsamplesoftestwatercollectedfrom cachtreatmentandthecontrolgroupat thebeginningofthetest,atapproximately48hours, andattest termination. Fatheadminnowswereindiscriminatelyassignedtoexposurechambersattestinitiation. Observatioonfs `mortalityandotherclinicalsigns of toxicityweremadeatapproximately 2.5,24, 48,72 and 96hoursaftertest initiation. Cumulativepercentmortalityobservedinthetreatmentgroupswas usedtoestimateorcalculateLCSO `vaatl24u,48e, s 72 and96hours.Thenomortality concentration andtheno-observed-effect-concentration (NOEC)weredeterminedby visualinterpretationofthemortalityandclinicalobservationdata. 000550 AMENDED WILDLIFE INTERNATIONAL Lo. 5. PROJECTNO.: 454-102 MATEARNI DMA ETL HOS DS `Thestudywasconductedbasedonthe proceduresoutlinedintheprotocol, "PFOS: A 96-HourStatic AcuteToxicityTestwiththeFatheadMinnow (Pimephales promelas)"". Theprotocolwasbasedon procedures outliinnUe.Sd. EnvironmentalProtection AgencySeries850 --EcologicalEffectsTestGuidelines, OPPTS `Number 850.1075 (1): OECD GuidelineforTestingofChemicals 203: Fish, Acute Toxicity Test (2); and ASTM Standard E729-880," Standard Guide for Conducting Acute Toxicity Tests with Fishes, `Macroinvertebrates andAmphibians (3). TestSubstance `Thetest substancewasreceivedfrom 3MCorporationon October29, 1998 andwasassignedWildlife InteLtr d.in dena tifit cati ionnoumn bera 467l5.Thetest substancewasdescribedas awhitepowder. It was identifiedas FC-95fromlotnumber217(T-6295). Informationprovidedbythe Sponsorindicated apurity of 98.9%,andanexpirdaatatoif2o00n8.Thetestsubstance was reanalyzedbythe SponsorandtheCertificateof `Analysis dated March 9, 2000 indicated apurityof 90.49%. Thetest substwaassntocreedatambient room temperature. PrepofaTesrtCaonctentiratoionns Nominaltestconcentwerrea3.t6,i5.o9,n9.s9,16and 27mga.i/L,basedonatest substancepurity of 90.49%. Allmaterials whichcameintocontactwiththetestsubstanceduringpreparationoftestconcentrations `wereconsoft plar sticu orsc tait nlese sstd eel. Aprimarystocksolutionwaspreparedin dilutionwaterat a `concenotf2r7amgti8.oi./nL. Theprimarystocksolution wasmixedwithan electricmixerforapproximately22. hourstoaidinthesolubilizationofthetestsubstance.Aftermixing,theprimarystocksolution was `proportionallydilutedwithdilutionwaterto preparethefouradditionaltestconcentrations.Alltestsolutions `apcpleaer anadcroloerledss. TestOrganism "Thefatheadminnow,Pimephales promelas, was selectedasthetestspeciesforthisstudy. Thefathead `minnowisrepresentativeof animportantgroupofaquaticvertebratesand wasselectedforuseinthetestbased uponpasthistoryofuseinthelaboratory. Fatheadminnowsusedinthetestwereobtainedfromcultures at `WildlifeInterLtnd.,aEatstion,oManryalanld. 000551 AMENDED WILDLIFE INTERNATIONAL Lm. "10- PROJECT NO.: 454A-102 Fatheadminnowswereheldatapproximatelythe sametemperatureasusedduringthetest. Thefish `wereheldforapproximately 126dayspriortotesting. The fishwereacclimatedtotestconditionsfor appro48hxourisprmioratottesteinitliatiyon. Duringthe holdingand acclimationperiods,thefishshowedno. signsofdiseorastrseses. Duringthe 14-dayholdingperiodprecedingthetest,watertemperaturesrangedfrom 22.01022.8C. ThepHof thewaterrangedfrom8.2to 8.5anddissolvedoxygenrangedfrom7.7 10.8.4mg/L. Instrumanedmnetthoadstusiedofonr watermeasurementsaredescribedintheEnvironmental Conditions secoftthiisreopornt. Attestinitiation,thefatheadminnowswerecollected fromtheacclimationtankand trants othfe teestrchrambeerds. Duringtheholdingperiod, fatheadminnowswerefed a commercially-prepadrietd.Thefishwerenot fed duringthe acclimationperiod(atleast48 hourspriortothetest)orduring thetest. Allfishusedinthetestwerefromthesamesourceandyearclass,andthetotal lengthofthelongestfish `wasnomorethantwicethelengthoftheshortest.The averagetotallengthof 10negativecontrolfishmeasured "attheendofthetestwas 35mmwith arangeof30to 38mm.Theaveragewetweight(blotteddry) of 10negativecontrolfishattheendofthetestwas 0.36grams with arange of0.21 to0.49 grams.Loading was 0.24 gfish/Loftest waterpresentinthetestchambersatanygiven time. TestApparatus `Testchamberswere25-L polyethyleneaquariacontaining approximately15 Lof testsolution. The depthof waterin arepresentativetestchamberwasapproximately 17.6 cm. Testchawmerb eime parr tias lly positionedinanenvironmentalchambersettomaintain atemperatureof2242C. Thetestchambewrerse labeledwiththeproject number,testconcentrationandreplicate. DilutionWater `Thewater usedforculturingandtestingwasfreshwaterobtainedfrom awellapproximately 40meters deeplocatedontheWildlifeInternatLitod.nsiatle. Thewellwaterischaracterizedasmoderately-whataerrd. Thespecificconductance, hardness, alkalinity, and pHmeasurementsof thewellwaterduringthefour-week `periodimmediatelyprecedingthetestarepresentedin Appendix L 000552 A W-- ILDLIFE IN-- TEO RNATIONR A-- L Lm. --E PROJECTNNT O0:3A45S4AA-102 BT "Thewellwaterwas passed through a sandfilertoemove particlesgreaterthan spproximately25wm, and pumpedinto a 37,800-Lstoragetankandaeratedwithspraynozzles.Priortouse,the wateragainwas. filtered (0.45 jum)to removemicroorgaandnpairtsicmless. The resultsofperiodic analysesperformedto `measuretheconcenoftselrecatedtcointaominnanstsinwell waterusedby WildlifeIntematiLodn.aarle presineApnpetndeixIdL Pk Conditions Lightingusedtoilluminatetheculturesandtestchambersduringholding, aclimton andtesting was providedby fluorescenttubesthatemitted wavelenghssimilartonaturalsunlight(Colortonc 50), A photoperiodof 16hoursoflightand 8hours ofdarknesswascontrolledwithanautomatictimer. A 30-minute transitionperiodoflow lightintensity wasprovidedwhealightsweatonandofftoavoidsuddenchangesin lighting. Lightintensityattest initiationwasapproximate3l91y uxatthesurofftahewcateer.Lightintensity `wasmeasuredusing aSPERScientificLid.lightmeter. `Temperaturewasmeasuredineachtestchamberatthebeginningofthetestandatapproximately 24-hourintervalsthereaferusingaliquidin-glassthermometer, Tempalsoewasrmeasauretdconutinruouselyin `onenegativecontrolreplicateusing aFulscopeER/CRecorder.Thetargettesttempedurrinagthtestuudryweas 2242C.Dissolvedoxygenand pH measurementsweremadeonwatersamplesfromalreplicatetestchambers ofeachtreatmentandcontrolattestinitiationandatapproximately 24-hourintervalsthereafer. Hardness, alkalinityand specificconductanceweremeasuredinthedilution wate a testinitiation. MeasurementsofpHweremadeusing aFisherAccumetModel915pHmete,anddissolvedcvygenwas measuredusing aYellow SpringsInstrumentModel S1Bdissolvedoxygenmeter. Specificconductancewas measuredusingaYellowSpringsInstrument Model 33 Salinity-Conductiviy- Temperaturemeter.Hardoessand alkalinitymeasurementsweremadebytitration basedonproceduresinStandardMethodsfortheExamination of Water and Wastewater (4) Observations Observationsweremadetodeterminethenumber ofmortalities. Thenumofibndiveidurals exhibiting clinicalsignsoftoxicityorabnormalbehavioralsowereevaluated. Obsewerrevmadaestpproiximoatenly2s.5, 24,43,72and 96 hoursafte estinitiation. 000553 WILDLIFE INTERNATIONAL Lm. S12. PROJECTNO: 454A-102 `StatisticalAnalyses `The 24,48,72and 96-hourLCSOvaluesandthe95%confidenceintervalswerecalculatedwheapossible: byprobitanalysis,the movingaverage methodorbinomialprobabilitywithnon-linearinterpolation(5,6, 7) usingthecomputersoftwareofCE.Stephan (8). Inthisstudy,LCS0valuescouldnotbecalculated at24and48 oursduetothelackofanadequateconcentration-responsepattem, however,the probitmethodwasusedto evaluatemortalityat 72hoursandthemovingaveragemethodwasusedtoevaluatemorality at 96 hows. Theo `mortalityconcentration and NOECweredeterminedbyvisualinterpretation ofthemortality andclinical observationdata. AnalyticalChemistry `Watersampleswerecollectedat mid-depthfromeachreplicatetestchamberofeachtreatmentand controlgroupa thebeginningofthetes, at 48 hoursandattestterminationtomeasureconcentrationsofhotest `substance. Thesampleswere collected in plastic (Nalgene) vials andanalyzedas5000aspossiblewithout storage. Analyticalproceduresusedintheanalysisofthesamplesareprovidedin AppendixIIL RESANU DDILSCUTSSISON `MeasofuTerstCeoncmenetrantiotns ResultsofanalysestomeasureconcentrationsofPFOS inwatersamplescollectedduringth testare: preseatedin Table |andintheanalyticalchemistryreport(AppendixI) Nominalconcentrsaelteciteodfnosr useinthisstudywere3.6, 5.9,9.9, 16and27mg aL. Samplescollectedattestinitiationhadmeasuredvalues thatrangedfrom85to 117%ofnominal values. Measuredvaluesforsamplestakeaat 48hoursranged from 86t010of1 nomi% nal.Measuredvaluesforsamplestakeaat 96hoursrangedfrom 88to 98%ofnominal. `Whenmeasuredconcentrationsofthesamplesanalyzedattestinitiation,approximately 48hoursandattest terminationwereaveraged,themeanmeasuredconcentrationsforthisstudywere 3.3,5.6, 9.5,17an28dmg iL. Meanmeasuredconcentrationswereusedintheestimationorcalculation ofLCS0values. ObseravndaMetasiuroemnentss Measurementsoftemperature,dissolvedoxygenandpHarepreseatedin Table2. Temperatureswere. withinthe22:2Crange establishedfo th test.Dissolvedoxygenconcentrationsremained 27.7mg/L(88%of saturatiion) on) throughoutthroughoutthetest. MeasuremenotfspHHrraannggeedfrom8.3to8.6duriduringngthetest. 000554 AMENDED WILDLIFE INTERNATIONAL Lo. BER PROJECTNO.: 454A-102 "Dailyobservationsofmortalityandothe clinicalsignsoftoxicityobservedduringtheestareshownin Tabl3e.Fatheadminnowsinthenegativecontrolandthe 3.3mga.i/L treatmentgroupappearednormaland healthy during the test. Afler 96-hourofexposure, mortality in the 5.6, 9.5, 17 and 28 mg a.L/L treatment groupswas20, 50, 80 and 100%,respectively.LCS0valuesand 95%confidencelimitsat24,48, 72and96 hourswereestimatedorcalculatedfromthemortalitydata, andareshowninTable4. A graofpthhe concentratciuroveinp-rerseeatsedipnoFingusree1. CONCLUSIONS "The96-hourLCS0 valueforfatheadminnowsexposedto PFOSwas9.5mga.i/Lwith 95%confidence. mitsof 8.0and 11mg ../L.The 96-hournomortalityconcentrationandtheNOECwere 3.3mg aL. 000555 AMENDED WILDLIFE INTERNATIONAL Lm. 14- PROJECT NO.: 454A-102 REFERENCES 1 US. Environmental Protection Agency. 1996. Series 850 -- Ecological Effects Test Guidelines (drafh, OPPTS Number 850.1075: Fish Acute Toxicity Test, Freshwater andMarine. 2 OChregmainciaslast.ioGnuifodrelEicnoen2o0m3i:cFCiosoh,peArcauttieoTnoxaincditDyeTveestl.opAmdeonptt.ed19b9y3.thOeECoCuDncGiulidaenli1n2esJfuloy T19e9s2t.ionfg 3 MAaScrToiMnvSetratnedbarartdeEs7,2a9n-d88Aam.phi1b9i9a4n.sS.tanAdmaerrdicGaunidSeofcioertCyofnodruTcetstiinnggAacnutdeMaTteroialx.TeistscwitihFtishyes, 4 APHA, AWWA, WPCF. 1985. StandardMethodsfortheExamination ofWaterand Wastewater. 16th Edition. American Public Health Association. American WaterWorks Association. Water Pollution Control Federation, New York. 5 Stephan, CE. 1978. U.S. EPA, Environmental Research Laboratory,Duluth,Minnesota. Personal communication. 6 Finney,DJ. 1971.StatisticalMethods inBiologicalAssay. Second edition. Griffin Press, London. 7 Thompson, W.R. 1947. BacteriologicalReviews. Vol.TL, No. 2. Pp. 115-145. 8 Stephan, CE. 1977. "Methods for Calculating an LCS0," Aquatic Toiicology and Hazard `Evaluations, American Society for Testing and Materials.Publication Number STP 634, pp 65-84. 000556 WILDLIFE INTERNATIONAL Lr. -15- PROJECTNO.: 454A-102 Table 1 `Summaryof Analytical Chemistry Data BR ``TTeesstt SOurbgsatnainscme:: PFaFtOhSeadMinnow,Pimephales promelas Dilution Water: Well Water "No`mComnacelatTraetsiotn -- SaTpilmeog MCoenwcewnteradtion ~~MeCmonMceenatrsatwioend Paofm iL) y 2/0) RNa A ai/0) aNom~ianal) BA 408 BA a%8 <<u B % Lo 36 BA 00 AB 4a88 AB 9%% 335136 33 2 330282 33436 59 AB 00 BA 4a33 AB %% 650057 56 95 558498 555750 99 AB 00 AB 448 BA 99%% 894979 95 % 99.8383 995720 16 AB 0Q 118923 " 106 AB 4a88 115560 AB 9%% 114682 2 AB o0 228855 AB aa88 72708 AB %% 2266368 Thelimitofquaniitation(LOQ)wes 0.458 mg a:i/L. 2 104 000557 AMENDED FRoECTNO, 45402 Se ea Tempers,Dio Oxyd ofWace TetChanter [T= sooorm ebmaartm,mtrei CT ete T oe or w (8 SD ow O= S eoemow T =0)eaboe m =0 e amo ee S Tom UEe momo Smoenoi uomeohnne omod nu BaPomms ownoEm oOmM mOoBwR oOm BMEoRw oome MtRouuomMoRw uow apommsomoowoum oMmeonmowoommoowunow oms MwmoRw uomomwauow ssaPomms owro Ems moam mooBwEoOomme EmoLw oOm MmRoLw oOm MwoIm WaPommaMo on oowys mosmomnoomw omm ooum nom mme ewodm m moosI Tp E waToedEoemoTmeBiooe emmooamwi oomwe eymoBonEemyLrO Hwm T woooAm Semne owmR oiw e ms aon wou mnrShp AS i: sg3 aa mom PROJECTNO. 4814-104 -- r T Boro oie F tavi a promis ou. Tobie CmltivePercentMorality ndTrestmentRelatedEc CTRAtatH eoe Doelpemm ebelmeas Du meeemDou mo ee m wnefe mleA Me Pook oL EA 0 RN Who ek Hd Ako Gm Ww co wh En Com 0 tNe am reg Lom spin 282 aa HE AMENDED WILDLIFE INTERNATIONAL Lo. -18- PROJECT NO.: 454A-102 Table 4 LCS0 Values `Sponsor: 3M Corporation --_-- `Test Substance: PFOS. Test Organism: Fathead Minnow, Pimephales promelas -- ~ Dilution Water: _ Well Water - LowesUpper Confidence Confidence LCSo Limits Limits Time (mgai/l) (mg a.i/L) (mg 2.i/L) 24 Hours >28 = Pe, 48 Hours >28 El - `Statistical Method Visual Interpretation `Visual Interpretation 72 Hours" 27 2 41 Probit 96 Hours 9.5 80 11 MovingAversge `Confidencelimitscouldnot be calculatedwith themortality data obtained. TheusefulnessofthisLC50isquestionable because aconcentration-effectrelationshipwasnotdemonstrated over areasonablerange (e.g, <37to >63) of percentdead. 000560 AMENDED WILDLIFE INTERNATIONAL Lo. "194 Figur1e. ConcentratioCunr-veR(e96s-HpouornDastae) PROJECTNO.: 454A-102 8 7 2 86 Zz 85 E E 4 g =3 2 1 1 > . 3 10 100 Mean Measured Concentration (mg a.i./L) 000561 AMENDED WILDLIFE INTERNATIONAL Lm. 20. PROJECTNO.: 454A-102 APPENDIX `Specific CDounrdiuncgttahnee4,-WHeaerkdacPsesr,ioAdlkIamlmienidtiyataenldypPHroefcWeedliltnWhgaetTeerstMeasured S--p_o--nsor. SM Corporation TTeesstt OSurbgsatnainscme::~~ FPaFtOhSead Minnow, Pimephalespromelas Dilution Water: __WellWater Spe(ciufmibcoCso/nedmu)ctance Mean N-4) Range 310-315 Hardoss (mg/L as CaCO) BIN=4 128-136 Alkalinity (mg/L as CaCOs) ma=9 176-178 s pH o 330-4) e 83 000562 a. WILDLIFE INTERNATIONAL Lm. PROJECT NO.: 454A-102 APPENDIX II Analyses ofPesticides,Organics, Metals andOther Inorganics -- e ANALYSp2 ISe T MEASUREDFLECONoCENmTRATION NwrE in Wildlife International Ltd. WellWater OrganocftlNerrpuespacndPCBs DGBomEa Gino <T Tm m0005 o==l T :Te E i=F] Shir BBEE, TI O B ==z EnE 2dosuplEfe--n, B-re <Soo0m.005 FE =gl = iE Bite CEEom a= Ra eoEmm <sFsE uuee33 4a=ar Reais R < 85 i < 0720 ual PCB-1260 Re `Metals and Other Iyorganics. BlBi=nd -: BZ p0% = Sr Gore BowF= Moaemmen =< 11039 aFa Vien Ee <8a SE Ea <Lm19 =rg FB Br Renin 1. 197. 000563 WILDLIFE INTERNATIONAL Lo. -2- APPENDIX PROJECT NO.: 454A-102 `THE ANALYSIS OF PFOS IN FRESHWATER o IN SUPPORT OF WILDLIFE INTERNATIONAL LTD, PROJECT NO.: 454A-102 000564 WILDLIFE INTERNATIONAL Lm. 3. PROJECT NO: 454A-102 REPORT APPROVAL SPONSOR: 3M Corporation : TITLE: PFOS: A96-HourStaticAcute ToxicityTestwiththe FatheadMinnow (Pimephales progelas) `WILDLIFE INTERNATIONAL LTD. PROJECT NO.: 454A-102 PRINCIPAL INVESTIGATOR: AlJr L. Van Hoven, Ph.D. Scientist MANAGEMENT: Mat Gh `Willard B. Nixon, Ph.D. Manager, Analytical Chemistry porn DATE V7.4 DA' 000565 AMENDED WA ILDLIFE INTERNATIONAL Lr. PPRROOJKECCTTNNOO.::4d54sAi-1A02z PY Introduction `Freshwatersamples werecollectedfrom astaticacuteaquatictoxicitystudy designedtodeterminethe effectsof PFOS (Perfluorooctane Sulfonic Acid Potassium Salt) to the fathead minnow(Pimephales promelas). `This studywasconducted by Wildlife InternationalLtd.and identified asProject No.: 454A102. Theanalysesofthesewater samples were performedat WildlifeInternationalLtd.usinghigh performance liquid chromatography with mass spectrometric detection (HPLC/MS). Samples were received foranalysisonFebruary15,17 and 19,1999 and were analyzedon eachsamplereceiptday. TSestubsantdInatemnalStcandeard Thetestsubstanceused fortheanalytical portion ofthis study was WildlifeInternational Ltd. identification number4675. The test substancewas used to prepare calibration andmatrix fortification `samples. - Theintemalstandard wasreceivedfrom 3MCorporationon July2, 1998 and wasassignedWildlife International Ltd. identniumbferi45c26auptonirecoeipnt. The internalstandard, agranularmaterial,was identified as: 1H,1H,2H,2HPerfluorSoulofconticaAcnied,Chemical AbstractNumber: 27619-97-2. `The standard was storedunderambient conditions. AnalyticalMethod `Themethodusedfor the analysisof thefreshwatersampleswasdevelopedatWildlife International Led. andentitled "Analytical Methodforthe Determination of PFOSin Freshwater,Saltwater, andAlgal Medium". ThismethodologywasincludedasAppendixIIofWildlifeInternationalLtd.protocolnumber 454/011299/MVAL/SUB454. It was baseduponmethodology provided by 3M Corporation. Samples were diluted in a 50% methanol : 50% NANOpure water solution containing 0.100mg 4H PFOS (internal standard)/L and 0.05% formic acid (v/v) so that they fell within the calibrationrangeofthe PFOSmethodology. Concentrationsof the PFOS in thestandardsandsamplesweredetermined byreverse-phasehigh performanceliquid chromatography using a Hewlett-Packard Model 1100High PerformaLinqcuied Chromatograph (HPLC) with a Perkin-Elmer API 100LC Mass Spectrometer equipped with a Perkin 000566 W--I_L-- DLIFE CINe TERNATIONAL Lr. 25. PPRROOEJECCTTNNOO.::d454sA-1l 02 Elmer TurbolonSpray ion source. HPLC separations were achiovod using a Keystone Betasil Cig analyticalcolumn (100mm x 2mm LD, 3 unparticle size). Theinstrumentparametersaresummarized inTabl1e. Amethod flowchartis provided in Figure 1. : Calfrtien _-- Calibration standardsofPFOS prepared in a 50% methanol : 50% NANOpure water solution containing0.100mg 4H PFOS(internalstandard) and 0.05%formicacid (v/v), ranginginconcentration from 0.00915to0.0457mga../Lwereanalyzedwiththesamples.Linearregressionequationswere generatedusingpeakarearesponseratios(PFOS : internalstandard)versustherespectiveconcentrations ofthecalibrationstandards. AtypicalcalibrationcurveispresentedinFigure 2. Theconcentratioofn PFOSinthesampleswasdeterminedby substitutingthepeakarea response ratios intotheapplicablelinear regressionequation.Representativeion chromatogramsof lowandhighcalibration standardsarepreseated inFi3gandu4,rrespeectivsely. `Themethodlimitofquantitation (LOQ)fortheseanalyseswassetat0.458mg a../Lcalculatedasthe productofthelowestcalibrationstandardanalyzed (0.00915 mga./L)andthe dilution facotfothre matrix blanksamples (50.0). `MFatroixrBlatnkianfd icatSiamoplnes `Threematrixblanksampleswereanalyzedtodeterminepossible interference, Nointerferenceswere Observedat orabovetheLOQduringsamplesanalyses(Table 2). Arepresentativechromatogramof a `matrixblankispresiennFitguere5d. Freshwaterwasfortifiedat 0.915,9.15and45.7mga.i/Landanalyzedconcurrentlywiththesamples odeterminethemeanproceduralrecovery(Table3). Sampleconcewnertenrotacortreicteodfonrtshe meanprocreecodveruyorf97a.9l%. A represeatativechromatogramof amatrixfortification spresented inFigur6e. 000567 AMENDED JLWIDLLDILITFEE IINNTTEERRNNAATTIIOONNAALLtLero,. 26. TPRROOJEECCTTNNOO.:: 4m54AA-102 Example Calculations Sample number 454A-102-27, nominal concentrationof 3.6 mg a.i./L in freshwater. Initial Volume: 0.250 mL Final Volume: 25.0 mL Dilution Factor: 100 PFOS Peak Area: 359685 Internal StandardPeakArea: 415648 Peak Area Ratio: 0.865 Calibration curve equation. Slope: 0.023 Intercept: 0.062 Curves linear - PFOS(ug2./L)at instrument =Peakarearatio ~(Y-intercept) . Slope PFOS (mg 2.i/L) in sample =PFOS(ug2/1)atinstrumentxDilutionFactor 1000 Calculated concentration: 3.46 mg a.i/L Note: manual calculations may differ. Percentof Nominal Conc. =PFOS(mg&.i/L)insample x 100 PFOS (mg a.i./L) nominal Calculated recovery: 96.9% Note: manual calculation may differ. 000568 AMENDED -- WILDLIFE INTO ERNATe IONA, L Lm. 27. PPRROOEJECCTTNNOO.::4454AA-102 RESULTS SampleAnalysis Freshwatersamples werecollectedfromtheacutetoxicitystudy withthefatheadminnow(Pimephales promelas) at test initiation, February 15, 1999 (Hour 0), on February 17, 1999 (Hour 48), and at test termination, February 19, 1999(Hour 96). Themeasuredconcentrations of PFOSinthesamples collected atinitiation ofexposureofthetestorganisms (Hour 0) rangedfrom85.3 to 117% of thenominal concentrations. SamplescollectedatHour48had ameasured concentrarnagetofi8o6.n3 to 101%of `nominalvalues. `Samples collectedat testtermination(Hour 96) had a measuredconcentrationrange of Figure 7. 87.6%to 98.3%ofnominalvalues(Table4). Arepresentativechromatogramof atestsampleisshown in 000569 AMENDED -- WILDLIFE INTERNe ATIONe AL Lm. -28- PRPOREOJCETCTNNOO.:: S454AA-I10?2 INSTRUMENT: ANALYTICAL COLUMN: OVEN TEMPERATURE: Table 1 `Typical HPLC Operational Parameters C`Hewhletrt-PoackmarwdaiMtothdaeolPer1gk10i0nr-HEialgmheprPAehrPfIor1m0a0nLceCMLaiqsusidSpectrometer `seeqleucitpipveediwointmhoaniPteorrkiinng-Emlomdeer (TSuIrMbDo.lonSpray ion source. Operated in KeystoneBetasilCyscolumn size) (100mm x 2rm LD,, 3pmparticle Ed STOP TIME: FLOW RATE: 100 minutes 0.220 mLminute. . "MOBILE PHASE: 072..10%%FoMremtihcanAoclid: 28.0% NANOpure Watercontaining. INJECTION VOLUME: 500UL PFOSRETENTIONTIME:~~ Approximately 7.1 minutes RIENTTEENRTNIAOLNSTTIAMNE:DARD Approximately 4.9 minutes PFOS MONITORED MASS: _MINO--TNEIRTNOARLESDTMAANSDS:ARD 498.6 amu 4267 amu 000570 W_I--LDLIFE INTERNATIOe NALe Lm. 29. PRPROOEJCECTTNNOO.::4454AA-102 Table 2 Matrix Blanks Analyzed Concurrently During Sample Analysis ("4N5u4mAb-e10r2) MAB-1 Sample Type > `Miatrix Blank Measured Concentration of (mPgFaOiSl'l) <I0Q MAB2 Matrix Blank <LoQ "TheliMmiAtoBfq3uant(iLtOQa)wtasi0M.oa4tn5ri8xmBgla.nik./Lbased upontheproductof<th1e0lQowest calibration standard analyzed (0.00915 mg aJL) and the dilution facto ofthe matrix blank samples (50.0). 000571 AMENDED TWIILLDDLLIIFFEE NITNETERRNNAATTIIOONNAALLLLom.. ~ "30- FPPRROOJEECCTTNNOO:: 445s44a-1l022 Table 3 -- ee Matrix Fortifications Analyzed Concurrently During Sample Analysis Sample Number Concentrations of PFOS (mga. i/L) Percent (454A-102-) Fortified Measured Recovered MAS-1 ~ 0915 1.03 13 MMAASS4T 00991155 00.587402 9120.60 MAS-2 9.15 8.65 94.5 MMAASS-35 99.1155 88.7745 9955.56 `Note: MAS3 457 483 106 MMAASS-)6 4455.77 434706 896222 - Mean =97.9 `Standard Deviation = 9.12 CV N==9932 Res anu dcol rret ctiosnsfor new testsubstancepurityweregeneratedusing MacQuanversion1.5 sof and t manuw al a calcur latie ons. Valueshave been rounded for reportingpurposes. 000572 AMENDED ZT N WILDE T LIFEE INTER RNATN IONAA L Lr.TIONAPRPL ROOEJCECTL TNNOO..: TM 445544AA-11. 0022 La Table 4 Measured Concentrations of PFOS in Freshwater Samples from a Fathead Minnow Static Acute Toxicity Test Nominal Test Concentration (mga.i/L) 00 Sample `Number (454A-102-) 2T 13 1 22%5 Sampling `Time (Hours) 00 8 a8 9%96 PFOS Measured Concentration' (mga.i/L) <<rLo0QQ <L0Q. <LoQ? <<LLoOQQ Percent. of Nominal -= - - -- 36 43 00 33.5136" 9888..96 15 48 3.08 86.3 716 49%8 - 3362 990%.3 28 96 313 876 59 5 0 6 0 17 48 18 48 2390 99%6 605 102 5.07 853 5.48 922 5.89 99.0 55.5750 99558 99 7 0 18 80 3210 49%8 32 96 8.99% 893 994487 0947.80 9.7303 %9238 952 943 Note:Rseosfutlwtasraenadncdormraencutailoncsaflcourlnateiowntse.stVasluubesstahnacveepbuereintyrwoeurnedgeednfeorrarteepdourtsiinnggpuMrapocsQeus.anversion 1.5 "The limitof quantitation(LOQ)was 0.458mga.i./L based upontheproductofthelowestcalibration standard analyzed (0.00915mga.i./L) andthe dilution facotfothrematrix blank samples (50.0). Mean resultofduplicateredilutionsofthe original sample. Duplicate results (in parentheses) for the following 454A-102 sample numbers are: 3 (3.07 and 3.25), 5(6.11 and 5.99), 7 (9.01 and 8.96), 8 (9.70 and 9.24) and 14 (<0.458 and <0.458). The original results for these samples (4.44, 7.98, 21.2, 2.21, and 0.74, respectively) are not included in the statisticalanalysisofthe data due to suspectdilutions (e.g. contamination). 00057 AMENDED JWI IILLDDLLN IIFFEE T INTERE NATIR ONAL N Lo. ATIOPN RPROOEJCEA CTTNNOO.::L 445564AA-d1. 0022 -32- "Table 4 (Continued) Measured Concentrationsof PFOS in Freshwater Samples from a Fathead Minnow Static Acute Toxicity Test Nominal Test Concentration (mg a.i./L) 16 Sample `Number (454A-102-) 190 21 2 333 Sampling Time. (Hours) 00 48 48 9%%6 PFOS Measured Concentration' (mgai/L) 119832 15.0 15.6 114368 2 Percent of Nominal 1710 90.9 94.5 9908.32 27 n2 00 228555 110044 223. 488 35 96 36, 96 2277.80 9180.14 268 97.6 26.6 91.0 Note: Results and corrections for new test substance purity were generated using MacQuan version 1.5 softwareandmanual calculations. Valueshavebeen rounded for reporting purposes. "The limitofquantitation (LOQ) was 0.458 mg .i/L based upon the productofthe lowest calibration standardanalyzed (0.00915mga.i./L)andthedilutionfactorofthematrixblanksamples (50.0). Meanresultofduplicateredilutionsof theoriginal sample. Duplicate results (in parentheses)for the following 454A-102 sample numbers are: 3 (3.07 and 3.25), 5(6.11 and 5.99), 7 (9.01 and 8.96), 8 (9.70 and 9.24) and 14 (<0.458and <0.458). The original results for these samples (4.44, 7.98, 21.2, 2.21, and 0.74, respectively) are not included in the statistical analysisofthe datadue tosuspect dilutions (e.g. contamination) 000574 AMENDED We --I_L--DLIFE INTERNATIONAL Lo. r PPRROOJCETCTNNOO.::44S54A-102 3. METHOD OUTLIINNEFRFEOSRHTWHAETAENRALYSIS OF PFOS Preparematrixifnotrtoiffriceasthiwoantsearmupsliensgbgays-stpiigkhitnsgytrhienrgeeqsuaisnidtCevloalsusmAeovofluPmFeOtSrisctfolcaskkss,olutionsdirectly + DiluteCmlaatsrsiAxfvortioficaltionufalnasdmktsewsiettsha5mtp0l%ermsientithoatnhcoelr:an5g0e%ofNthecAalibNratiwOonastteparnsdoau lrudtsibr onycpoanerttaiian lilnygfilling s0a.m10p0lmeagn4dHbriPnFgOtSh(eifnltaesrkntaolsvtoalnudmaerwdi)thatnhded0i.l0ut5i%ofnosromlivecaatc.iPdro(vc/ev)s.stAhdedmtathreiaxppbrloapnrkisaatmevpolleuusmienogf thesamedilutionandaliquotvolumeasforthveelroswieosntsf.ortification level.Mixwellbyseveralrepeat + Ampulatesamplesandsubmitfor LCMS analysis Figur1e. Analyticalmethod flowchartfortheanalysisof PFOSinfreshwater. 000575 WILDLIFE INTERNATIONAL Lo. 3. PROJECTNO.: 454A-102 120 100 5 0% do 040 on] 000 000 o o 0010 0m0 000 000 0050 Concentr(amgtai.io/Ln) Figure. Apicalcalation curvfor POS.Slape = 0.03;Inrept = 0062, =0996 00 AMENDED TWLIDLLDLIIPFEE NITNETRERNNAATTIIOONNAALLLLem.. ~~ Z35. PPRROOEJECCTTNNOO..:445544AA-110022 ~ To 169 inen83s51ycp:s o o 7 5. . " 3 . 148 20 ' SoTo e2th 931m iWEoo aWe 4781x zaism | TSiemne i| Figure3. A representativeionchromatogramof alow-level (0.00915mga.i./L)PFOS standard. 000577 AMENDED A m WILDLIFE INTERNAi TIONAL Lm. rTPRRe OJOECLTNNOO.,:445540A0-10002 -36- To 109 intnsiy:29671cps o 8 7 5 - . 4 17 2 1 124 i 31172 e2l5 eat8 152018 A6st% 17815 62181 |TiSmeen Figured. Arepresentativeionchromatogram ofa high-level (0.0457mg a.i/L) PFOS standard. 000578 AMENDED LWN LIOLLDLT IIFFEE E INTER RNATN IONAA L Lo.T ~ I~ ONAPPRL ROOFJEECCL TTNNOO.::dm 4s5e4A4-d1e. 0r2 -37- 100 a 90 | 80 70 60 so 10 a0 nten4i26ycp:e 0 - 4 . #2 a1 174 1a0 sanTeh, Mal v WAAR | TiwUG25 e3it aSe AeBTm a7s asii's ||TiSmane Figure 5. Arepresentativechromatogramof amatrixblank sample (454A-102-MAB-3). `Thearrow indicatestheretention timeofPFOS. 000579 We ILDLIFE INTEe RNATIONAL Lm.st PRODJESCi LTNRO.: 445554A8-1100022 3. To 170 intnsiy: 13084cps o o 7 o 5 . "3 148 2 F, ' 12 SiTW e2t5 eSTm ASIe AeSm 178La eslm | STiamme Figure 6. A representative chromatogramof amatrix fortification sample (454A-102-MAS-7). 000580 SWT Io LDLIE TF=E INR TERNAN TIONAA L Lo. TIONPPRROA OJJEECCTTNNOL O.:: 445s4AA-. 1l02z 39 " 170 Intonsty:23428cps 9 8 7 s 5 . " 5 . 148 2 1 26 125 3117% e2t85 eans 15301s 1631% e7r&'7 aaiis | STiamme Figure. Arepreseatativechromatogramofatestsample (434A-102:27). 000581 WILDLIFE INTERA NATIONAL Lm. 40 PPRROOJJECCTTNNOO.:: d454sA-1l02 APPENDIIXV ChatnoPgroteocsol "Thisstudywasconductedinaccordancewiththeapproved Protocolwiththe followingchanges: 1. Tsuhbesptraontcoeicolwnasfamoenadrpedmttoeaatdcodnttcheeniptrraootpioonsnse.dexperimentaltartandterminationdates, studyroom,test: 2. Theprotocolwasamendedtoremove annualfeedanalysis. 3. Theprotocol wasamendedtoremovethecalculationofanincipientLCS0value. 4. Theprotocolwasamendedtospecifytheanalyticalmethod. 000582 _WI--LDLIFE INTERNATIONAL Lo. 41 PO ROJE ECTR NO.: 45a4Ai-10l2 APPENDIX V Personnel Involved in the Study `The study: following key Wildlife IntemationalLtd.personnel wereinvolvedinthe conductor managementof this 1. Heary O. Krueger,Ph.D,Director, Aquatic Toxicology and Non-Target Plants 2. WillardB.Nixon,Ph.D., Manager,AnalyticalChemistry 3. Mark A. Menk, Laboratory Supervisor 4. Timothy Z. Kendall, Laboratory Supervisor 5. Kurt R. Drottar,SeniorBiologist 6. Raymond L. VanHoven, Ph.D, Scientist 000583 WILDLIFE INTERNATIONAL Lm. a. PROJECTNO.: 454A-102 APPENDIVXI Report Amendment 1. Original Report: Pages 14,6and23 Amendment: nTehewspiaggaeastwuererseacnhdadnagteedsdtouiencolutdheetahddeiatmioennodfetdhreeproerptodratatm,ernevdimseendptaagseAnpupmebnedrisx,Va1n.d Reason: Toreflecttheissuingofanamended report. 2. OriginalReport: Page2 Amendment: Thecompliancestatementwasrevised. Reason: "Toclarifyhowthetestsubstancewascharacterized. 3. OriginalReport: Paged Amendment: iInncflourdmiatnigotnheprreoavniadleydsbisydtatheeanSpdotnhseoprurreiftlye,cwtiansgtahdedreedatnoatlyhseiTseosftStuhebtsetsatnscuebssetcatnicoen, Reason: "Toreflectthecurrent est substanceinformationprovidedbytheSponsor 4. OriginalReport: Entirereport Amendment: `AalsldteetsetrsmuibnsetdabncyetchonecSepnotnrsatoiroinnswaereraencahlaynsgiseodfttohretfleescttstuhbsptuarnitcye o(fFtCh-e9e5s,tLsoubtst2a1n7c)e.: cTeerstitfcicoantceeonftraantailoynsisosdragtineadlMlayrwcehr9e, b2a0s0e0idnodnictahteedraeppuorritteydopfur0i.t4yo9f%.9T8h.e9%refoTrahele, cloensctentsruabtsitoanns,ceandcoLncCeSn0trvaatliuoenss,,weriencrleucdailncgulantoemdainnadlrepcoorntceendt2rsamtigonas,/Lmbeaassuerdeodn. the90.49%purity. Reason: rToreepoqortftuthheeeSrpesosunlstsotor.fthetestbasedonthetestsubstance purityof90.49%atthe 000584 AMENDED WILDLIFE INTERNATIONAL Lo. a3 APPENDIX VI ~Continued- Report Amendment PROJECT NO: 454A-102 = AMENDMENT SIGNATURES: Kurt R. Drottar `Study Director Y4 Henry' Director, Aquatic 3. Toxicology andNon-TargetPlants < REVIEWED BY: - mothy A. Springer, PRD. Quality Assurance DATE Yh Joo DATE Cf fsko DATE - 000585 AMENDED om WILDLIFE INTERNATIONAL LTD. PROJECT NO.: 454A-102 Page 1 of2 um AMENDMENT TO STUDY PROTOCOL i] STUDY TITLE: PFOS: A 96-HOUR STATIC ACUTE TOXICITY TEST WITH THE FATHEAD MINNOW (Pimephalespromelas) HH PROTOCOL NO.: 454/110998/FAT-96H1/SUB454 AMENDMENT NO.: 1 m SPONSOR: 3M Corporation PROJECT NO.: 454A-102 EFFECTIVE DATE: December 21, 1998 AMENDMENT: Page2 Add: EExxppeerriimmeennttaall TStearrmtinDaattei:on2D/a1t5e/:992/19/99 Test Concentrations: Negative Control, 3.9, 6.5, 11, 18 and 30 mg a.i/L Test Substance No.: 4675 REASON: The above information was not known when the protocol was signed by the Study Director. AMENDMENT:TestOrganism, Page 5 - Delete: aFreeednoprcoovnitdaemdintaonttsheatfilsehvewlisllkbneowannatloyzbeedcaatplaebalsetoofnicnetearnfneurailnlgywtiothentshuersetutdhya.t there, REASON: HciosnttoarmiicnalanatnsalayrseesproefseWnitldaltilfeeveIlnstekrnnoatwinontaol bLted.caapqaubalteiocffienetderhfaevreinsghwoiwtnh tthhaetsntuody. AMENDMENT:DataAnalysis, Page 8 Change: T4h8e,7L2CaSn0dv9al6uheowuirlsl,baescwaellcullaastetdh,ewihnecnipipeonstsiLbClSe0,.using mortality data collected at 24, To: The LCSOvaluewil be calculated, when possible, using mortality data collected at 24, 48, 72 and 96 hours. REASON: The incipient LCS0 cannot be determined in a 96-hour static acute test. 000586 Po 6he J WiLoLiFe INTERNATIONAL uD. PROJECT NO.: 454A-102 Page of2 1 AMENDMENT: APPENDIX IT, Page 13 Add: Liquid Chromatography Mass Spectrometry (LCMS) Method for the Determination of 1 Perfluorooctane Sulfonic Acid, Potassium Salt (PFOS)in Freshwater, Filtered Saltwater and `Algal Medium 1 REASON: To add the analytical method to be used in the study. part\ 1 STUDY DIRECTOR Vda 1 LABORATORY MANAGEMENT 219 [97 DATE HY Da; -REPRESENTATIVE zl 1 pr-- 000587 PROTOCOL o WPIFTOSH:TAHE96F-AHTOHUERASDTMAITINCNAOCWU(TPEimTepOhXaIlCesIpTrYomTeElSasT) U.S. Environmental Protection Agency on `Series 850 - Ecological EffectsTestGuidelines 'OPPTS Number 850.1075 Ho wt a (OECD Guideline 203 H 3M Lab Request No. U2723 i Submitted to 3M Corporation Environmental Laboratory P.0.Box 33331 St. Paul, Minnesota 55133 WILDLIFE INTERNATIONAL LTD. 8598 Commerce Drive `Easton, Maryland 21601 (410) 822-8600 November 9, 1998 000588 PROTOCOL NO.: 454/110998/FAT-96H1/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL LTD. 2. WPIFOTSH: TAHE96F-AHTOHUERASDTMATIINCNAOCWU(TPEimTepOhXaIlCesIpTrYomTeElSasT) SPONSOR `SPONSOR'SREPRESENTATIVE: TESTINGFACILITY: 3EnMviCroornpmoernattailonLaboratory PSOt,PaBuo,xM3i3n3n3e1sota 55133 Ms. Susan A. Beach W85i9l8dCeomImnteermcaetiDonrailveLid Eason, Maryland 21601 STUDYDIRECTOR: KSuenritoDrrAoqtutaartic Biologist LABORATORYMANAGEMENT: DHiernercytOo.ofKAruqeugaetri,cPTho.xDi.cology & Non-Target Plants Tm---- OACATORYUSEON er Proposed Dates: : I Start Date: Termination Date l ProjectNo: _ SHA - [02 | "Test Concentrations: Test Substance No: ReferenceSubstance No.(ifapplicable): PROTOCOLAPPROVAL STUDY OR DATE depellnch oTAlBORAlTORY MANAGEMENT SPONSOR'S REPRESENTATIVE BlR le Ys yls0)i2 000589 PROTOCOL NO: 454/110998/FAT-96HISUB4S 3MLAB REQUEST NO. U2723 VILDLIFE INTERNATIONAL LTD. 3. INTRODUCTION Wildife International Ltd. will conduct a static acute toxicity test with the fathead minnow (Pimephaespromelas)forthe SpoatnthesWioldlirfe Intemational Ltd. aquatic toxicology facility in Easton, Maryland. The study will be performed based on procedures in the US. Environmental Protection Agency Serics 850 - Ecological Effects Test Guidelines OPPTS Number 850.1075 (1); OECD Guideline for Testing of Chemicals 203: Fsh, Acute Tosicity Test (2); and ASTM Standard 729-884StandardGuideforConductingAcute Toxicity Tests with Fishes, Macroinvertebrates and Amphibian3)s. RawdataforallworkperformedatWildifeIntemationalLid.and a copyofthefinal reportvillbefiledbyprojectnumberinarchives locatedontheWildeIntemational Lid. siotraeta,n alternative locationtobespecifiedi thefinalreport. PURPOSE "Thepurposeofthisstudy i todeterminetheacuteeffects ofatestsubstanceonthefathead `minnows (Pimephalespromelas) ding 96-hour exposure period under stati test conditions. EXPERIMENTALDESIGN Fatbeadminnowswillbeexposedto geometricsri of at leastfivetestconcentrationsand a negative(dilutionwater)contol or96hours.Tworeplicatetestchamberswillbemaintainedincach tretmeatandcoutrolgroup,with 10ftheadmitmowsincachchamber for atote of 20fatheadminnows pertestconcentration. `Nominaltestconcentrationswil beselectedinconsultationwiththe Sponsor,and wilbebased `uponinformationsuchasthe resultsofexploratoryrange-findingtoxicitydata, known toxicitydata, `physiallchemical propertiesofth testsubstanceorotherrelevantinformation.Targetconcentrations `need not exceed 1000mg/Lorthe solubilitylimitofthetestsubstanceinwater (whicheverislower). Generally, eachtestsubstanceconcentrationusedinthedefinitivetestwillbe ee higher concentration unless information concerning the concentration-cffe different dilutionfactorwouldbemore appropriate, Watersamplesfrom collectedatspecifiedintervalsfor analysisofthetestsubstance. Results calculate meanmeasuredtestconcentrations. To control bias, fathead minnows will be impartially assigned to oifnitoiatciona. Nlooatheerpeotentiicaallsourcewsoiflbbieamsaaredexepegctoedtsoafhfesec9tthoeruesults PROTOCOLNO.: 454/110998/FAT-96H1/SUBAS4 SM acl SQUESTNO.UZIZS i i | ] | i i il ol H 1 ! 000590 i| | i a ' . Bwioure INTERNATIONAL LTD. |] `mortality observedinthetreatmentgroups willbeused tocalcuwhlenapotssiebl,e, LCS0valuesat24, 48,72and96hourintervals, Theno-mortlityconcentration andtheno-observedc-onecefntfreatciotn i (NwO ill bEe detCermi) ned. MATEARNDIMEATHLODSS i `TestSubstance Informationonthecharacterization of test, controlor referencesubstancesisrequiredby Good H LaboratoryPractice Standards (GLP). TheSponsorisresponsibleforprovidingWildifeIntemational Lud.writenverification thatthe testsubstancehasbeencharacterizedaccording to GLP priorto ifsuse i inthestudy.IFwritten verification ofGLPtestsubstance characterizationisnotprovidedto Wildlife InternationalLid.itwillbenotedinthecompliancestatement ofthefinal report,Theattachedform i IDENTIFICATION OFTESTSUBSTANCE BY SPONSOR (AppendixI)istobeusedtoprovide infnoecerssarmyfoar GLtPciomploiannce. un "TheSponsors responsibleforallinformation relatedtothetestsubstanceandagrees toaccept u anyumused testsubstanceandlortest substancecontainersremainingattheendofthestudy. `PreopfaTesrt Caonctentiraotionns i "Thetestsubstancevil beadministeredtothetestorganismdiretlyintodilutionwater, This routeofadministrationwas lectedbecauseit eprescatsthemostlikelyrouteofexposuretoaquatic a organisms. TestOrganism i "Thefheadmimow(Pimephalespromelas)hasbeenselectedasthetestspecies forthisstuds. Fatheadminnowsar representativeofanimportantgroupofaquaticvertebrates,endhavebeenselected nl foruse inthetest basedupon pastusehistoryinthelaboratory. Fishwillbefromthesame sourceand yearclass,andthestandard lengthofthelongestfishmeasuredwillbe 00morethan twicethat ofthe shortest.Fishwillweigh between 0.1 gand 3.0 eachandthetotalweightineachtestchambervillnot a exceed 0.8gramsish/Lofsolution.Totalengthsandwetweightsofth individualfishinonenegative controlreplicatewillbmeasured th en ofth testan willbeconsider representativeofthelength ol andweightofallfishusedinth study.Fish willbeobtainedfrom a commercial suppliesorhatchery, andtheidentityofthespecswillbeverifiedbythesupplierorbyWildifeInternationalLid. personnel I] usingappropriatetaxonomickeys,suchasEddy (4). 000591 ou PROTOCOLNO.: 454/110998/FAT-96HI/SUBAS4 3MLABREQUEST NO, U2723 i I 5 Jwioure INTERNATIONAL LTD. I `Fatheadminnowswillbeheldforatleast14daysprior tothetestinwater from thesamesource andatapproximatelythe sametemperatureasusedduringthetest. Anychianwantergtempeeratsure 1 illnotexceed3Cperday.Fatheadminnowswillbeheldatthetest temperaturefor aminimum of7 daysprior to testinitiation.Fatheadminnowsvillno beusedinthetestitheyshowsignsofdisease orstressorifmorethan 5%dieduringthe48hourspriorothetest.Attetinitiathteifaothnea,d 1 `minmows willbecollectedfrom holdingor seclimationtanksandtransferredtothetestchambers i Duringtheholdingperiod thoestfishwillbofdatleastoncedaily. Thediewillconsist of ive orfrozenbrine shrimpnauplii(Artemiasp),andlorcommercial food.Fishwillnotbe fodforatleast i awnnoudalalyystporieonrsturootthheatttehsetoreadrueriunogctohnettaemsitn.anFtescadtplreovveildsekdnoowtnhteofbisehcvaiplalbbleeaonfailntyezrefderaitnlgewaisttohntchee i Hostwuedyv.erS,ptehceirfeiacartcinonoskfonroawcncleepvtealbsloeflcvoenltsaomifncaonnttsarmeiansaonntasbilnyfeisxhpedciteetdstohbaveepnrostcbaetei etshteadbiletisthheadt. 1 areconsideredtointecferewiththepurposeorconductofth test. i Dilution Water Water used for theculturingendtestingoffatheadminnows willbe obtainedfrom awell approximately40metersdecplocatedontheWildlifeInternationalLid.site. Thewater willbepassed. i rough sandfilter ndpumpedito a37,800-L storagetankwherethewaterwillbeaeratedwithspray nozes. Priortousethewate illbefiltered10.045 aminordertoremovefineparticles. Waterused. forculturingandtesting icharacterize as moderately hard.Typicalvaluesforhardacss,alkalinitpyH,. i andspecific conduarceaptpraoxinmacteley: i AHlaarldinneistsy,,mmgg//LL2a5sCCeaCCOOy; oH 114950 81 1 SpecificConductance,pnhoslem 330 a conHsaridonfestshst,ewaeleklanllwianctiteyyr,. pMHeaanndssapnedcriafnigcecsoonfdtuhcetmanecaeswuirleldbpeamreamaestuerrsedfwoertehkelfyotuor-mwoeneiktpoerrtihoed I] tporedceotdeirnmgitnheetehsetcwoinlclebatzpraotviiodnesdoifnstehlefcitneadlorrepgoarnt.icAnaanldyisneorsvgainlilccbeopnsetriftouremntesdoaft tehaeswtoenlclewaantneuraalnlyd resusoftheanalyseswillbesummarizedinthefina report ou 000592 i PROTOCOL NO.: 454/110998/FAT-96H1/SUB454 3M LAB REQUEST NO. U2723 i MWiLoLiFE INTERNATIONAL to. NH -6- TestApparatus "Testchambersvillbe25-L,polyethylene aquariafilledwithapproximately 15Lof water, Test i chambers will be positioned in an eavironmental chamber of temperature-controlled water bath to maintain a temperature of 22 2C. Test chambers will be labelled with the project number, test i concentration andreplicate. |] Lightingusedtolluminatetheculturesandest chambersduring holding,acclimation,andtesting `illbeprovidedbyfluorescenttubesthatritwavelengthssimilartonaturalsunlight (c.,Colortone nl 50). Aphotoperiodof 16 hoursoflight and 8hoursof darkwillbecontrolledwithanautomatictimer. A30-minutetransitionperiodoflow lightintensitywillbeprovidedwhen lightsg0onandofftoavoid. i sudden changes in light intensity. Light intensity vill be measured at test initiation with a SPER Scientific Ltd. lightmeteror equivalent. i "Thetargetest teapevicllbaet22u. r2cC. Tempwilelbermeaasurtedalulrerplicaetes atthe beginningofthetestandatapproximately 24-hour intervalsthereafterusing a liquid-in-glass ul "thermometer. Temperaturealsowillbe measuredwith acontinuousrecorderinonenegativecontrol chamber, Recorder measurements will be verified with a liquid-in-glass thermometer prior o test i initiation. Dissolvedoxygenwillbemeasuredin llreplicatesofthetreatmentandcontrolgroupsattest ou initiationandatapproximately24-hourintervalsthezeaferusing a YellowSpringsInstrumentModel SIBdissolvedoxygenmete,oequivalent.Intheeveatthatdissolvedoxygenlevelsfllbelow 60% ol saturation,approprist actionswillb take firconsultationwiththeSpousor, MeasuremeonftpHs villbemadein llreplicates ofthetreatmentandcontrol groupsa estnitionandatapproximately od 24-hourintervalsthereafterusing a FisherAccumetModel 915pHmeter,orequivalent.Ifatreatment groupreaches 100% mortality,dissolvedoxygen,pH,andtemperaturemeasuremeatswillbotakenat a thenextsamplinginterval,then discontinued. Hardness,alkalinity,andspecific conductancewillbemeasuredinthedilution waterattest o initiation. Hardness andalkalinitymeasurementsvillbemadebytitrationusingproceduresbasedon methodsinStandardMethodsorthe ExeminationofWaterand Wastewater (5). Specific conductance ol willbemeasuredusingaYellowSpringsInstrumentModel 33Slnity-Conductviy-Temperaturemeter, orequivalent. 000593 ol PROTOCOLNO.: 454/110998/FAT-96HL/SUBAS4 3MLABREQUESTNO, U2723 Mwioure INTERNATIONAL LTD. 1 -7- a BiologicalMeasurements `Obseofrmovrtaalittyiandoclinnicsal signsoftoxicitywillbemadebetvween 0-24 hours, and at 1 24,48, 72and 96 hours 1 hour. Lethality is definedasthe lackofvisible movement (e.g. lackoffin oropercularmoveaneat)inth fishafergentleprodding. Al clinicalobserivnclaudtingiabononrmsal 1 behaviorwillbenoted i `Sampling for `Water AnalyticalMeasurements. samples willbe collectedfrom cach tetchamberatthebeginningofthetest,duringthe {estancatth cadofthetesttodetermineconcentrationsofthetestsubstance. Intheeventthat 100% i `mortalityoccursinanytreatment,then sampling ofthattreatmentwilterminatefollowingthenext sampling interval. Samples will be collected at mid-depth from cach test chamber and analyzed 1 immediatelyorplacedinanapproprist storagecontainer(cg. polyoerptolhypryopyllenesbonttlee) andstoredunderrefrigeratuintoinlanalyzed. The sampleschemeissummaribezleowd: |] PROPOSEDNUMBERSOF VERIFICATIONSAMPLES i Experimental Group a OHows - Hows Ho%us Control 2 2 2 n SolventControl (fneeded) 2 2 2 Level 1-LowConcentration 2 2 2 1 Level2 2 2 2 Level 3 2 2 2 1 Level 4 2 2 2 Level5-High Concentration 2 2 2 1 Totals 1 1 14 TotalNuomfVberifeicatrion Sampl=e4s2 1 "Theabovemubersofsamplesrepresentthosecollectedfromthetestanddonotinclude quality control (QC) samples such as matrix blanks and fortifications prepared and analyzed during the i anachlemisytrytphaiseocfthaestlay.AtthediscretionoftheStudyDirector, watersamplesfromone ormore appropriatetestchamberswillbecollectedandanalyzedifan analyticalerrorinsamplingor BR anailssyusspecitesd. Thereasonfortheadditionalsampleswillbedescribedbythe StudyDirectorand documentedinthe awdata andfinalreport. 000594 un PROTOCOLNO.: 454/110998/FAT-96HI/SUB454 3MLABREQUESTNO. U2723 | I . IwiLoLiFE INTERNATIONAL LD. I AnalytiCchaelrmCihcemailsatnralyysis ofthesamples willb performedby Wildlife IntemationalLtd.Theanalytical 1 `methodusedwillbebaseduponmethodologyprovidedbythe SponsorendidentifiedinAppendixII Themethodologyusedtoanalyzethetest samplesvil bedocumentedintherawdata sndsummarized i in thefinal report. i `DataA`nWahlyesnitshedose-responsepatie allowscalculation ofanLCS0value,thedatavillbeanalyzed. usin thecompute softwareofCE.Sicphan 6).Theprogramwasdesignedtocalculatethe LCS0 valu 1 awintdhtnhoenl9i5n%ecaorinnftiedrepnocleatiinotnerv7a,l8b9)y.prTohbietLanCaSly0svias,ltuhevmiollvbiencgaalvceurlaatgeedm,ewthheondp,oosrsbiibnleo,muisailnpgromboarbtialliittyy 1 datacolotedat24,48, 72and96hours,as ellastheincipientLCSO. Theno-mortlityconcentration andtheno-obscoencerntrvatieon(dNOE-C)owilflbefdeetercminted. 1 Records to bemaintaineRdfEorCdOatRaDgeSnTerOateBdEbyMWAiIldNliTfAeIInNteErnDationalLid.willinclude,butnot i belimitedto: : 1. Acopyofthesigacdprotocol. i 2 ldentianfdichcaraactteriizaotinonofth test substance,ifprobvytiheSdpoensodr. 3. Datesofinitiation endterminoaftthietoesnt. 4. Testorganism history,boldingandacclimation cords. 1 5. Stocksolutioncalcualndapretpairatoionn,ifapplicable. 6. Observations. |} 7. 8. TWhaetemrecthehmoidsstruysreedsutltosan(ca.lgy,zalektaelsintistuybasntdahnacrednceosnsc)e.ntrations andtheresults of analytical 1 `measurements. 9. Statistical calculiaftaippolicnabsle,. 1 10. Testconditions(ightintensity,photoperiotcd),. 11. Calculationandpreparationoftestconcentrations. . i 12. Co offip nalrey port. i 000595 i PROTOCOL NO: 454/110998/FAT-96H1/SUB4S4 3M LAB REQUESTNO, U2723 1 I - J WILDLIFE INTERNATIONAL tm. FINALREPORT 1 Afinalreportoftheresults ofthestudywillbepreparedbyWillifeIntemationalLid.Thereport i will include, but notbe limited o,tfhollowing, when applicable: 1. Nameandaddressofthe facilityperformingthestudy. 2. Datesuponwhichthestudywasiniistedand completed,andthedefinitiveexperimental startend 1 terminationdates 3. A statoefcmomeplinantce signed by theStudyDirector addressing any exceptions to Good 1 Laboratory Practice Standards. 4. Objectivesandprocedures, asstatedin the approvedprotocol,includingallchanges tothe a ty 1 protocol. 5. Tho test substance identification including name, chemical abstractumber or code number, 1 strength, pusity,composition,endotherinformation providedbytheSponsar. 6. Stabilityandsolubilityofth testsubstanceundertheconditionsofadministration,if provided bytheSponsor. 1 7. Adescription ofthemethodsusedtoconductthetest 8. A descriptionofthe test organisms, including the sourc, scientific name, age or Ife stage, i lengthsandweightsof arepresentativegroupoftestorganisms,fedtypes,lightintensiatndy photoperiod 1 9. Adescrofithpeprtepiaraotionnofth testsolutions. 10. The methodsusedto allocateorganismstotestchambersandbeginthetest,the numberof organismsandchamberspe treatment,andtheduration ofth tet. 1 11. Adescriptionofcircumstancesthatmayhave affectedthequalityorintegrityofthe data. 12. ThenameoftheStodyDirectorandthenamesofotherscientists, professionals, andsupervisory 1 personnelinvolvedin the study. 13. Adescriptionofthetransformations,calculations, andoperationsperformedonthedata, a 1 `summaryand analysisofthebiologicaldataand analyticalchemistrydata,and astatementofthe. concdrlawnufrsom tiheoananlysses. 1 14. Statisticalmethodsusedtoevaluatethedata 15. Agraphofthe concentraticournve-atmthoerentdoaflthiettesyt. 16. Thesignedanddate report ofeachoftheindividualscientistsorotherprofessional involved 17. Thelocationwhererawdataandfinalreportare tobestored. 5 18. AstatementpreparedbytheQualityAssuranceUnitlistingthedatesthatstudyinspectionsand `suitsweremad andthedatesof nyfindingsreportedtotheStudyDirectorandManagement. i PROTOCOL NO:: 454/110998/FAT-96HL/SUB4S4 000596 3MLAB REQUESTNO, U2723 IWWIILLDDLLIIFFEEIINNTTEERRNNAATTIIOONNAALL LvT0D.. I ~~ -10- I 19. Itsnosesarytomakecorrectionsoradditionstoa inal reportafterit hasbeenaccepted,such changesvillbemadeintheformofanamendmentissuedbythe StudyDirector.Theamendment I vil clearly identifythepartofthefinalreportthatisbeingamendedandthereasonsforthe amendment,andwillbesignedbytheStudyDirector. I CHANOFGPRIOTNOCGOL Plannedchangestotheprotocolwillbeinthe form ofwritenamendmentssignedbythe Study I DirectorandtheSponsor'sRepresentative. Amendments willbeconsidered os prtoftheprotocoland. illbeatached thofinalprotocol. Amyotherchangeswillbeintheform ofwritendeviationssigned. I by theStudyDirectorandfiledwiththeraw data.Allchangestotheprotocolwillbeindicatedinthe | finalreport. i GOODLABORATORYPRACTICES y "Thisstudywillbeconductedin accordancewithGoodLaboratory Practice Standards forEPA. I (40 CFR Part 160 and/or Part 792); OECD Principles ofGoodLaboPrractaicet(OoCDrE/GyD (92) 32, Environment Monograph No. 45); and Japan MAFF (59 NohSan, Notification No. 3850, I Agricultural Production Bureau). EachstudycondubycWtiledidfe InternLaidt.iisroountianelly examinedbythe WildifeInternatioLniadl.QualityAssuranceUnitforcompliancewithGood t LaboratoryPractices,StandardOperatingProceduresandthespecifiedprotocol. Astatementof `compliancewith GoodLaboratoryPracticesvil beprepared for allportionsofthestudyconductedby i `WildlifeIntemational Ltd. The Sponsorwil beresponsibleforcompliancewithGoodLaboratory Practicesforprocedurespecformedby therlaboratories(c,esidueanalysesorpathology). Rawdata forallwork performedatWii Intemational td.andacopyofthefinalreportwillbe filedby project numberinarchiveslocatedonthe Wildlife InternationalLid.site, ofatanalternativelocationtobe speicntiheffinialreepodrt. 000597 E1 PROTOCOLNO.: 454/110998/FAT-96HI/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL LTD. -1- | REFERENCES | 1 U(dSra.ftE)n,vOiProPnTmSenNtuamlPbreorte8c5t0i.o1n07A5g:encFiys.h1A9c9u6t.eSTeorxiiecsi8t5y0T-esEtc,olForgeischawlaEtfefercatnsdTMeasrtiGnuei.delines i 2 Organisation for Economic CooperationandDevelopment. 1993. OECD Guidelines for TesoftCheimincalgs. Guideline 203: Fish, Acute Toxicity Test. AdobypthteCoeuncdil on 12 | July 1992. .: 3 AFiSshTesM, SMatcarnodianrvderEt7e2b9r-a8t8eas.,a1n99d4.AmSpthainbdiaarnds.GuiAdmeefroircaCnonSdoucciettiynfgorATceustteiTnogxaicnidtyMaTteesrtisalwsi.th i 4 Eddy,S. 1974, TheFreshwater Fishes. Win. C. Brown Company Publishers, Dubuque, lowa. i 5 AWaPsHtAew,atAerW.WA16,1hWEPdiCtFio.n, A1m9e85r.icaSntaPnudblaircd HMeeatlhtohdAsssfoocriatthieonE.xamAimneartiicoann oWfatWearteWroraknsd \ Association. Water Pollution Control Federation, Neww York. i; 6 SPetrespohnaanl,coCmEm.uni1c9a7t8i.onUS. EPA, Environmental Researci h Laboratory, Duluth, Minnesota. i 7 Thompson, W.R. 1947.Bacteriological Reviews. Vol. II, No. 2. Pp. 115-145. | 8 ESvtaelpuhataino,nsC,EA.mer1i9c7a7.nSo"cMieettyhofdosTfeosrtiCnalgcaunldaMtiantgerainalL.CSP0u,b"liAcgautaitoincNTuomxbiecrolSoTgyPa6n3d4H,apzpa6r5d- 8. I 9 FLionnndeoyn,.D.J. 1971.StatisticalMethods inBiologicalAssay.Secondedition.GriffinPress, i 000598 i PROTOCOL NO.: 454/110998/FAT-96H1/SUBA4S4 3MLAB REQUEST NO. U2723 FwLiDoLuTreE I INTEN RNATA IONAL LTD. eee I -12- i -_ I IDENTIFICATION OFATPEPSTENSDUIBSXTANCE BY SPONSOR To be Completed by Sponsor |L K | KE Eo E IL I : i Beal a Ve No_X TestSubstanceIdentity (nametobeusdinthereport):E(QPS erfluSoulfronioeAocidcPottassaiumnSaelt Refeccace Standard(if applicable): Analytical Standard: NA Intermal Standard:_1.1.2.28LHLH.HPerfluoroostaneSulfonicAcid Test Substance SampleCodeor Batch Nomber: __Lot2l7 Test Substance Purity (% Active Ingredient): _989 ExpirainDate: 2008 Tet Substance Characterization `HSavteetmhioeeideepnrptiieotytt,yo aiseB useinth hiitysstnuuddbcyomripotascictaoironnddarnlocerroowntihcteshrGsctLhaaPnrSdatacardnebdraosrtcd.s? Yes__No X Test Substance Storage Conditions Pleaseindicat th recommendedstorageconditionsstWildeIntemationalLid. --A_ mbietroomtemperswee Hasthestabil substanceunderthesestorage conditions Otherpertinent stability information: -- IV. TestConcentrations: i _X_bAadsjeusdtutpeostncthoepnucneyn(4t)trgoia1v0et0ni%soboinv.e aD1oTgesdt tuhesmattsetricaolnAceSnItSr.atio0n 100% Hv roy nomi Mammalian: RatLDS0 251mghke MouseLDSO N/A i Aquatic: Invertebrate Toxicity (EC/LCS0) Fish Toxicity (LCS0) Duphni2a7mmgagne: Rai Troun t Lb impo, w 8 Other ToxicityInfor(imnclauditngifinodinngs ofcharnd soubchnroniio tecsts): PleseseMsps PROTOCOL NO: 454/110998/FAT-96H1/SUB4S4 000599 3M LABREQUESTNO, U2723 WILDLIFE INTERNATIONAL LTD. -i3- APPENDIX TI AnalyticalMethodtobeProvidedby Sponsor | | { . | | | | | . | | 000600 | PROTOCOL NO.: 454/110998/FAT-96H1/SUB454 MLABseus No.U2723