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AR226-010 | PFOS: `A FLOW -THROUGH LIFE-CYCLE TOXICITY TEST WITH "THE SALTWATER MYSID (Mysidopsis bahia) FINAL REPORT `WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454A-107 3M LAB REQUEST NO. U2723 U.S. Environmental Protection Agency Series 850 -- Ecological Effects Test Guidelines - (OPPTS Number 850.1350 AUTHORS: HeaKu0rt KR.eDrgot,tarPAD. STUDY INITIATION DATE: May 21, 1999 STUDY COMPLETION DATE: April 26,2000 Submited to | 3M Corporation `Environmental Laboratory 935 Bush Avenue. St. Paul, MN 55144 Wildlife International, Ltd. 1598 Commerrive Easton, Maryland 21601 (410) 822-8600 Pgs ofc 01191 WILDLIFE INTERNATIONAL, LTD. 2. PROJECT NO.: 454A-107 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT SPONSOR: 3M Corporation TITLE: PFOS: A Flow-Through Life-Cycle Toxicity Test with the Saltwater Mysid (Mysidopsis bahia) `WILDLIFE INTERNATIONAL LTD. PROJECT NUMBER: 454A-107 STUDY COMPLETION: April 26, 2000 `Thisstudy wasconductedincompliancewith GoodLaboratoryPractice Staans pdublaishredbdy tshe US. EnvironmentalProtection Agencyin 40CFRParts 160 and 792, 17 August 1989; OECD Principloefs `Good Laboratory Practice (ENV/MC/CHEM (98) 17); and Japan MAFF, 59 NohSan, Notification No. 3850, Agricultural Production Bureau, 10 August 1984, with the following exceptions: chaTrheatecswtastsupbeesrtafrnocreimewdzaasccanoorttdicnihgatroaoc3tneMriSzteadnidnaradcOcpoerrdaatnciengwPirtohcefduulrleGsLaPndcMoemtphloiadnsc,ea;ndhoalwlevaewr,datthae are beingmainit nthae3iManrcheivdes.Thetestsubstanceisbeing recharacterizedinaccordancewith GLP. Thestabilityofthetestsubstanceunderconditionsofstorageatthetestsite.wasnotdeterminedin `accordance with Good Laboratory Practice Standards. STUDY DIRECTOR: | 2a] \ Kurt R. Drottar Senior Biologist PON : waeso DATE Sponsor DATE 01192 WILDLIFE INTENRNAATTIOANLAL,, LLTTDD.. 3. PRPROOJJECCTTNNOO.::44554A4-A1077 QUALITYASSURANCE STATEMENT USS. E"nTvhiisrsotnumdenytwaalsPerxotaemcitnioendAfogrceomnpilnci4ayn0cCeFwiRthPaGrotsod1L6a0baonrdat7o9r2y,Pr1a7ctAiucgeuSsttan1d9a8r9d;sOasEpCuDbliPsrihnecdibplyetshoef `AGoodgLraiPrbodcuPcortaiucotrnicBleuar(eEatuNt,V1/uo0MCAru/rgCuHasEytM19l8(49.8)Th17e);daantedsJoafpalaniMnAspFeFct,io5n9saNnodhaSuadni,tsNoatnidfitchatdiaotnesNot,hat38a5n0y,. findingswerereportedtothe StudyDirectorandLaboratory Managementvereasfollows: acm: Protocol TestSubstancePropanion. LAinaglhyttiMceateSaRmeplaidngianngd Salinity Measurements CParleipbarraattiioonn Standards DBrioalogRiecpaolrDtaaand DAanatlyitiRcealpDtota end. FinalReport DATE CONDUCTED: May 24,1999 June 10, 1999 June16,1999 June 17,1999 July 21,1999 Octo1, b4nedr 5.1999 October 4-7,1999 April 20,2000 DATEREPORTEDTO: STUDYDIRECTOR: ~~ MANAGEMENT: May 24,1999 iy 16,1999 June14,1999 June 16,1999 June17,1999 June 18,1999 June 18,1999 June18,1999 Jy21,1999 October,1999 October, 1999 July 21,1999 Oc> tober 7,199 October, 1999 Apel 20,2000 Agri 24,2000 iyA. DATE `QualityAssuranceRepresentative: 01193 WILDLIFE INTERNATIONAL, LTD. 4 PROJECT NO.: 454A-107 REPORT APPROVAL SPONSOR: 3M Corporation TITLE: PFOS: A Flow-Through Life-Cycle Toxicity Test with the Saltwater Mysid (Mysidopis bahia) WILDLIFE INTERNATIONAL LTD. PROJECT NUMBER: 454A-107 dl STUDYDIRECTOR: KSeuaritoRr.BiDorlootgtiasrt . MANAGEMENT: Y4 DHierae?ct)o0r., AquatGriuccger,ToxPhi.cD.ology and `Non-Target Plants gees DATE 7%, fo DBA 01191 WILDLIFE INTERNATIONAL, LTD. 5. `TOA F CB ONL TENE TS GoodLaboratoryPracticeCompliance Statement. . PROJECT NO.: 454A-107 coresserserserermreeed abl OfCOBDS... . 5 Experimental Design... . onan TABLES Tabl1e- SummaryofAnalyticalChemiStryDBR ........cecusesctosesscosesiisnrine: 18 Table2- Salinityof WaterintheNegativeControlandHighest TreatmentGroupTest Chambers......19 Tabl3e- pHof Waterinthe Test CRAMDETS.......c.c.ooorivserininsnsssesssnses soso 20 Tabl4e- Temperature (C)of Waterinthe Test Chambers...................... - 21 Tabl5e DissolvedOxygenContent(mg/L)of WaterintheTest CADETS. . 22 G1195 WILDLIFE INTERNATIONAL, LTD. age PROJECT NO: 454A-107 `TABLE OF CONTENTS - Continued - Table 6- Survival of Juvenile Mysids Exposed to PFOS (Day 0 Through Pairing on Day 20)... Table 7- SurvivalofAdult Mysids Exposedto PFOS. (Day20ThroughTest Termin08aDatyion 35)......cowwurrsrsssorssson Table 8 - Mean Numberof Young Produced PerReproductive Day ..... Table9- MeanTotal LenogfAtduhlt Mysids atTest Termination. `Table 10 - MeanDry WeiogfAhdutlt Mysidsat Test Termination. - Table 11 - Survivalof Second-Generation Mysids Exposed to PFOS During a 96-StaHticoEXPuOSUTrE............... tp 23 -- - ores 28 -- ------ ------l APPENDICES Appendix I- D4i-lWuetieoknPWeartieodr ISamlmiendiityatMeelaysPurreecdedDiunrgitnhgetTheset... eS Appendix I~ AnalysesofPesticides, Organics, Metals and Other Inorganics in Wildlife International, Ltd. Saltwater. snes --L Appendix Ill- ~The Analysisof PFOS in Saltwater inSupportofWildlifeInternational, Ltd. Project No.: 454A-107 ............ -- Appendix IV+MysidReproduction... wmm---- Appendix V- MysidTotalLength (mm). Appendix VI Mysid Dry Weight (mg) senses rsrsrsenrsnse: 53 sr-------- `Appendix VII~ ChatnoPgrOte OCOLs..........c..cvwsuerivessonserse anne esseens -- AppendixVIII - PerInsvolo vedn intn heSe tudyl... essa -- 01196 WILDLI= FE TIN-TETRNoAmTIAONLAL,, LTTDD.. 1. PRPROOJJEECCTTNNOO.::445544AA-110077 SUMMARY SPONSOR: SPONSOR'SREPRESENTATIVE: LOCATIOFN STUDY, RAW DATAAND ACOPYOFTHE. FINAL REPORT: 3M Corporation Susan A. Beach `EWaisltdoinf,eIMnatreyrnlaatnidona2l1,6L0t1d. WILTLDD,LPIRFOEJIENCTTENRNUAMTBIEORN:AL TEST SUBSTANCE: stupy: NOMINAL TEST MCEOANCNEMNETARSAUTRIEONDS:TEST | concentrations: TEST DATES: LENGTHOFFRSTGENERATION EXPOSURE: 454A-107 PFOS (Perfluorooctane Sulfonic Acid Potassium Salt) PSaFlOtSw:ateAr MFylsoiw-dT(hMryosuigdhopLsiifse-bCayhcilae)Toxicity Testviththe: NegativeControl,0.086,0.17,0.34, 0.69, 1.4 and 2.7 mg ai/L. NegativeControl,0.057,0.12,0.25,0.55, 1.3 and 2.6 mgai/L EExxppeerrSSittaaimrrtt((emEOPnEeAC)tDn~)aJ-utnlMeaay126l,6,19199999 Biological Termination-July 25, 1999 . Experimental Termin-aJtuliy2o5n, 1999 35 Days TEST ORGANISM: SOOFUTESR TORGC ANISEMS: AGE OFTESTORGANISMS: Saltwater Mysid (Mysidopsis bahia) Wildlife International, Lid. Cultures Easton, Maryland 21601 Juveailes <24 hours old [NoEC: 025mg ail pLaoaErCe:: 00.3575 mmggaaiilll G1197 WILDLIFE INTERNATIONAL, LTD. -8- PROJECTNO.: 454A-107 INTRODUCTION "This studywas conductedbyWildlife InternationaLtld,.for3M Corpato therWila dlift e Inti emao tionn al, Ltd.aquatictoxicology faciinlEaisttony, Maryland.Thein-life phoftahets estweasconductedfrom June 16, 19 toJ9 uly9 25, 1999.RawdatageneratedbyWildlifeInternationLatld,.and a copyofthefinalreportarefiled `underProjectNumber 454Ai-na1rch0iv7es locatedontheWildlife International, Ltd. site. . `OBJECTIVE "The objectiveofthis study wasto evaltuha effectsof Perflurooctane Sulfonic Acid Potassium Salt (PFOS)onthesurvival,growth andreproductionofthesaltwatermysid(Mysidopsis bahia)underflow-through test conditions. EXPERIMENTAL DESIGN Mysidopsis bahia neonates, less than 24 hours old, were exposed 10 a geometric series of six test concentrationsand anegative (saltwater) controlfor35days.Nominaltest concentwerraesteliecotend isn consultationwith the Sponsor,andwerebaseduponknowntoxicitydata. Nominaltestconcentratiwoenrse 0.086, 0.17, 0.34, 0.69, 1.4 and 2.7 mg active ingredient (a.i/L. Meanmeasuredtestconcentrations were determinedfrom samplesoftestwatercollectedfromeachtreatmentandcontrolgroupatthebeginning of the test,atweeklyintervalsduringthetestandattesttermination. `Deliveryofthetestsubstancewasinitiatedapproximately 51 hourspriortotheintroductionof theneonate `mysidsto thetestwaterin order to achieve equilibrium ofthe test substanceinthetestchambers. Four replicate test chambers, each containing one compartmweintth 15mysids, were maintained for each treatmentandcontrol group. To beginthetest,neonate mysids wereimpartially distributedingroupsofoneortwoamong glass beakersuntil eachbeakercontained 15mysids.Themysidswerethentransfered thetestcompartments. A totalof 60mysidswereexposedineachtreatmentandthecontrolgroup. OnDay 20ofthetest,female andmaleadultswerepaired,andthereproductionofthepaired mysidswas `monitoredthroughDay35. Observoaftmoirtoalnitsy,clinical signsof toxicity, and reproductionweremade 01193 WILDLIFE INTERNATIONAL, LTD. So. PROJECT NO.: 4544-107 daily. Attest termination, the lengths and dry weightsofall survivingfirst-generationmysidsweremeasured.All `youngproducedin thetestwereremovedto aseparatetestchamberathe same tes concentrationon adaily `basis. The second-generation mysids were exposed for 96 hours under static test conditions. `The no-observed-effiect-concentration (NOEC) and lowest-observed-effect-concentration (LOEC) were determined by examinationofthe mortality, growth and reproduction data. The maximum acceptable toxicant `concentration (MA wasT calC eula) tedas thegeometric moe ftha eNOn EC and LOEC. MATERIALS AND METHODS `Thestudy was conducted based on the procedures outlinedintheprotocol, "PFOS: A Flow-ThroughLife- Cycle Toxicity Test with the Saltwater Mysid (Mpsidopsis bahia)". The protocol was based on procedures outlined in U.S. Environmental Protection Agency Series 850 - Ecological Effects Test Guidelines, OPPTS Number 850.1350 (1); andASTMStandard E1191-90 StandardGuidefor Conducting Life-Cycle Toxicity Tests withSaltwaterMysids 2). TestSubstance The testsubwsasrteceaiven dfrcom3eM CorporationOctober29, 1998andwasassigned Wildlife International, Ld. identification number 4675. The test substance was described as a white powder. It was identifiaesd FC-95from lotnumber217(T-6295). Informationprovidedbythe Sponsorindicated apurityof 90.49%, andanexpirdaatetoif2o00n8,Thetestsubstanwacse storedatambrooim teempen ratutre. PrepofaTerstaContcenitraotionns Onestock solwuastpriepoarned for eachofthesixconcentrations tested. Aprimarystockwaspreparedby dissolvingthe testsubstanceindilutionwaterat aconcentratoifon 0.0895 mg 2./mL. Theprimarystockwas stirred with an electric stainless steel top-down mixer for approximately 24 hours to aid in the solubilization of thetest substance. Aftermixing,theprimarystockappearedclear andcolorlesswith afoamonthesurface. `Aliquotsoftheprimarystocksolutionwereproportionallydilutedwithdilutionwatertopreparefiveadditional stocksatconcentrationsof0.0447, 0.0224, 0.0112, 0.00559and0.00 mg2a.i8/m0L. Thesix stocks were injected intothedilutermixing chambers (at arateof 4.60mL/minute) wheretheyweremixedwithdilutionwater 01199 WILDLIFE INTERNATIONAL, LTD. "10+ PROJECTNO.: 4544-107 (at a rate of 150 mL/minute) to achieve the desired test concentrations. All test solutions appeared clear and colorless. TestOrganism "The saltwatermysid,Mysidopsis bahia (recentlyrenamed Americamysis bahia), was selecteda the test speciesforthisstudy. Thesaltwatermysidisrepresentative ofanimportantgroup ofaquaticorganismsand was `selectedforuseinthetestbased upon pasthistory ofuseandeaseofculturinginthelaboratory. Mysidsusedin thetestwereneonateslessthan 24hoursoldandwereobtained fromculturesmaintainedbyWildlife International, Ltd., Easton, Maryland. Adultmysidswereheldinwaterfromth samesourceasusedduringthe tes. Adultmyswerie fdedlsive `brineshrimp (Artemia sp.)twoor threetimesdaily duringholding. Brine shrimpwereperiodicallyenriched with a fatty acid supplement (ALGAMAC-2000, Aquafauna Bio-Marine, Inc., Hawthome, Californiz). During the 14-dayholdingperiodprecedingthetest,watertemperaturesrangedfrom25.0 t025.4C. ThepHofthewater `rangedfrom8.0to8.1, salinityrangedfrom 20to 23%o (partsperthousand),anddissolvedoxygenrangedfrom 68107.4 mg/L. Instrumentation used for water measurements is described in the Environmental Conditions `section ofthis report. Attestinitiation,theneonatemysids werecarefullycollectedfrom theculturesand transferredoneandtwo atatigmlasesbetakeors. Mysidsthenwere transferredfromthebeakerstothetestcompartments.Alltransfers `were made using awide-borepipettebelow the surfoafcthee water. Themysids were fed live brine shrimp naupliithreetofourtimes adayduringthetestto preveatcannibalism(exceptonthelastdayofthetest). Test Apparatus A continuous-flow diluter was used to deliver each concentration of the test substance and a negative. (saltwater) control. Aperistaltic pump (Cole-Parmer Instrument Company, Chicago, IL) wasusedtodelivtehre six test substance stock solutions into mixing chambers assignedtoeach treatment group. The stock solutions weredilutedwithdilutionwate inthemixingchambersinordertoobtainthedesiredtestconcentrations. The flowofdilution watertothemixingchamberswascontrolledbyrotameters. Rotawem rece alit brateedpr riorsto testinitiatianodnatweeklyintervalsthereafterduringthe test. Theflow of testwaterfromeachmixingchamber was spit and allowed to flowinto replicate test chambers. Theproportionoftest water that was split nto cach, G1200 SWAILLDDLLIIPFEEIINNTTEERRNNAATTIIOONNAALL,,"L7TDD.. o&ohfeeeee u- TPRROOIJEECCTTNNOO:: 445540A-01077 replicatewascheckedpriotothetestandatweeklyintervalsthereafterduringth testto ensuothat flow rates variedbynomorethan +10%ofthemean fo thefourreplicates. "Thediluterwasadjustedsothaeachtestchamberreceivedapproximately 11volumeadditions festwater ev2e4rhouyrs. Thedeliverypump wascalibrate beforethetestandatapproximatelyweeklyintervalsduring thetest.Thegeneraloperationofthediluerwascheckedvisuallya easttwotimesper dayduringthetestand once ateatdofhtheteest. + Priortosexualmaturity,mysidswereheldinonecompartmentplacedi cach replicatetestchamber (1S/compartment). Thecompartmentswer glassbeakerswithnylonmeshscreen attachedtotwoholeson opposite sides. Afler mysids attained sexual maturity, reproductive pairs were placed in reproductive comp(aonrepatirpmerceompnarttmenst). Thereproductivecompartmentswereglassperi dishesithsidesof nylonmeshscreenattachedwithsiliconeadhesive.Thetestcompartmentswereplacedin9-Lglass aquariatest chamberscontainingapprox5iLmofatesttsoelultiyon.Pri topairing,th depthofwate in arepresentative testcomparwtasm6e.2ncmt.Afterpairing, thedepthofwaterin representative testcompartmentwas 5.5cn. "The test chambers were impartially positioned in a temperature-contrlled environmental chamber designed to maintain a temperature of 252C. Test compartments were uniquely identified and the test chambers were labeled withthe project umber, tet concentarndartepliicoatne. Thetest chambersforthe secondgeneration `exwepreo2-Ls,beaukerrswieth 1 Loftestsolution which wasdippedoutof estchamberfromtheappropriate treatmentgroup. Dilution Water "Thewaterusedforculturingandtesting wasnaturalseawatercollcted atIndianRiverInet,Delaware,and was diluted to asalinityofapproximately 20%swithwell water. Salinity measurements dutrhefiourn-wegek periodimmediatelyprecedingthetestar presentedin Appendix I Thefreshly-collectedseawaterwaspassedthrough asandfiltertoremoveparticles greaterthan approximate2ly5pum,andpumpedntoa37,800-L storagetankwherethewaterwasacraedwithspraynozzles. Priortodeliverytothedilute system,thewateragainwas fered (0.45pm)toremovemicroorganiasmnsd partcis.Theresults ofperiodic analysesperformedtomeasuretheconcentroaftsileocniesd contaminantisn saltwaterused by Wildife Intemational, Lud. arc presented in Appendix II. 01201 WILDLIFE INTERNATIONAL, LTD. n- PROJECT NO.: 454A-107 B " Lightingused oilluminatetheculturesandtestchambersduringculturingandtestingwasprovidedby fluorescent tubes that emilted wavelengths similar to natural sunlight (Colortonc 50). A photoperiod of 16 hoursoflight and8 hoursof darknesswas controlled with an automatictimer. A 30-minutetransitionperiod of low light intensity wasprovidedwhen lightswenton andoffto avoid sudden changes in lighting. Light intensityrangedfrom 623to815 luxoverthesurfaceofthenegatcointvroel, replicaAtetestchamber. Light intensity was measuredweeklyysing a SPER Scientific Model $40006C light meter Temperaturewasmeasuredincachtestchamberatthebeginningandendofthe test anda weeklyintervals duringthetestusing aliquid-n-glassthermometer. Temperaturealsowasmeasuredcontinuouslyin onenegative control replicate using a Fulscope ER/C Recorder. The target est temperature during the study was 25:2C. Dissolved oxygen and pH measurementsweremeasured in altenat replicatesofeach treatment and control groupa thebeginningandend ofthe testandatweeklyintervalsduringthetest. Sawlasimeansurieddtailyyin alernate replicatesofthe negative control and the highest treatment group with surviving mysids. Dissolved oxygen was measured using a Yellow Springs Instrument Model $B dissolved oxygen meter, and measurementsofpHweremadeusing aFisherAccumetModel915 pHmeter.Salinitywasmeasuusrinegda Bio-Marine, Inc., Aquafauna refractometer. BOiolobgicsal ervaantdMieasourenmensts Observationsofthe survival and behaviorofeach first-generation mysid were made daily throughotuhte test.Thecriteriafordeath includedlackofmovement, absenceofrespiratorymovements, andlackofreactitoon gentleprodding Atthetimeofpairing(Day20),thesexandmaturityofeachmysid wasdeterminedby `microscopic examination, and, when possible, male/female pairs were made for each replicate test chamber. Anyimmaturemysidsorextrafemaleswerediscardeda thi time. Sexually maturemales,which were letover afterpairingweremaintainedin aseparatecompartmentwithinthatreplicate. After mysidswerepaired,the numberofsecond:generation mysidswerecountedandrecorded dailyuntil test termination. Second-genmeyrsiadtsiweornealso observed forabnormal development andaberrantbehavior. Afer cach observation, second-gencration mysids were collected and exposed atthesame test concentration under static test conditions for 96 hours. Ifa male in amale/female pair died, itwas replaced with a male, if 041202 WADLIZE INTERNATIONAr TD,eee 1. SBOTEOTIO:$0030 available, from the poolofmales maintained inthesamereplicate. Attest termination,thesexofeach surviving first-generationmysidwasconfirmed and the lengthofeachmysidwasmeasuredusing calipers. Esch surviving first-generation was then placed in a drying oven at approximately 60C for approximately 24 hours. The dry weightofeach surviving first-generation mysid was then determined using an analytical balance. `Statistical Analyses Statistical analyses were-performed on survivalofthe first and second-generation mysids, the number of `young releasedperreproductive day, and the length anddryweight ofcach surviving first-generation mysid `Survival wasevaluated prior topairing(Day 0 throughDay 20) andafterpairing (Day 20 throughDay 35). Survivaldatawereanalyzedusing 2 x 2contingencytablesan theChi-squaretesttoidentifytreatmentgroups, thatwerestatisticallydifferent romthecontrolgroup. "Theanalysesofreproduction (umbofelvreyoungpropderreuprodcucteivedday)andgro(drwywetighhts and lengths) data included those treatments which did not exhibit astatistical reduction in survival. Analyses included the evaluation ofhomogeneityofvariances using Bartlet est and the assessmentofnormality using theShapiro-Wiltekst'. Whendataweredeemednormalandhomogeneous,ananalysisofvarianceestwasused todetermine whetherornotstatisticallysignificantdifferencesexistedamongexperimentalgroups. Those treatments statistically different from the control group were identified using Dunnetts test. The resultsofthe statisticalanalyseswereusedtoaidinthedeterminationofthe NOECand LOEC.The MATCwascalculataed5 thegeometricmeanoftheNOECand LOEC. Allstatistical testswereperformedusingapersonalcomputerwith SPSS/PC Version 2.0 (3) or "TOXSTAT Release 3.5" statistical software (4). AnalyticalChemistry Priortotes initiation,twosetsofpretestwater sampleswerecollectedfromtworeplicatetestchambersof boththelowandhighleveltreatmentgroupstodetermine ifnominalconcentrationshad been achieved. Water sampleswerealsocollectedfromtwoalternatingreplicatesonDays0,7,14,21, 28and 35. Water sampleswere collectedfrommid-depthofthetestchamberandplacedinplastic(Nalgenc)bottles. Samwerpealnaleyzedsas soon as possible without storage. Analytical procedures used in the analysis of the samples are provided in Appendix IL. 01203 Z WILo DLIF=E ITNETRERNNAATTIIOONNAALL,,=LTTDD.. 14 PPRROOJJEECCTTNNOO.::445544AA-l10077 RESANUDDILSCUTSSISON MeasoufTresteConmcenetrantiotns Resultsofanalysesto measureconcentraotfiPoFnOsSin water samplescollectedduringthetestare preseinnTtabeled1andtheanalyticalchemistryreport (Appendix I.Nominalconcentruastediiontnhiss studywere0.086,0.17,0.34,0.69, 1.4and2.7 mga.i/L.Whenmeasuredconcenoftsarmpaletscoilloectnedson Deys 0,7, 14,21,28and 35wereaveraged,the meanmeasuredconcentrationswere 0.057, 0.120,.25, 0.55, 1.3 and 2.6 mga../L,whichrepresented 66, 71, 74, 80, 93and 96% ofthenominal concentrations, respectively. Meanmeasuredconcentrationswereusedtoexpressthe NOEC, LOEC andMATC. Pretestsamples collected to verifydilutrperformance (AppendixII)werenotusedinthecalculationofmeanmeasuredconcentrations. `PM hysiecala andsCheu micr al emenoftWas ter Measurementsof salinityinthenegativecontrolandthehighesttreatmentgrouprangedfrom19 to 21%, throughout the test (Table 2), Measurements of pH ranged from 8.2 to 8.4 (Table 3) and temperature was `maintained within the 25+2C range established for th test (Table 4). Dissolved oxygen concentrations remained 5.8 mg/L (79%ofsaturation) throuthgtehsto(Tuablte 5). Survival Asummofasurrvivyalfromtestinitiation opairingonDay20ispresentedinTable 6. Ingeneralal,l surmyvsidisapvpeairednnormgal.Afer 20daysofexposure,survival i th negativecontrolgroupwas 78%. SurvivalinthePFOStreatmentgroups<0.55mga.i/Lrangedfrom 75to 92%andwerenotstatisticallydiffrent. fromthenegativecontrol.Survivalinthe 1.3and2.6mg .i/Ltreatmentgroupswas 32 and0%,respectively, andwasstatistically diffrent rom thenegativecontrol ( < 0.05). `Observoaftsuirovinvaslaferpairing (fromDay 2010testterminationonDay 35)arepresentedinTable7. In general, all surviving mysids appeared nommal. Survival in the negative control was 92%. Survival pericntehePnFOSttreaatmgentgerousps <0.55mga./Lranged from 90to 97%andwerenotstatistically differeatfromthenegatcointvroel.Survivalinthe 1.3 mga.i/Ltreatmentgroupwas 57%andwasstatistically differentfromthenegative control(7 < 0.05). 0120% WILDLIFE INTERNATIONAL, LTD. -15- PROJECT NO.: 4544-107 Reproduction Asummaryofthemean number ofyoung producedperreproductivedayi presentedin Table 8. Young `production for individual test compartments is prescated in Appendix IV. For cach female, the number of reproductivedayswasdefinedasthe mumberofdaysthatthefemalewasalivefromthedayoffirstbroodrelease ofany femaleinthetesttotheendofthetest.The dayoffirstbroodreleasein thisstudy was Day 22. Themean `numberofyoungproducedperreproductivedayinthenegativecontrolgroupswas 0.315. Reproductionratesin the 0.057, 0.12, 0.25 and 0.55.mg a../L treatment groups were 0.261, 0.361, 0.252 and 0.0559 young per reproddauy,rcestpecitivveley. Dunnett' estshowedthatreproductionwas significantlyreduced nthe0.55mg ai treatmentgroupwhencomparedtothenegativecontrol(7 < 0.05). The 1.3and2.6mg a../Ltreatment groups were not included in the statistical analysisofthe reproduction data du to a statistically significant diffeirnseurnvicvael. Growth . `Summariesofthelengthsanddryweights ofthesurvaiduvltimynsigds arepresenteidn Tables 9.and 10, respectively. Individual measurementsareprovidedinAppendices Vand VI.The mean leanndgmeatndhry weight in the negative control group were 6.43 mum and 0.63 mg, respectively. Mysids exposed to PFOS at concentrations <0.25mga...showedno statisticallysignificantreductionsi lengthordryweight(p>0.05) Mysidsexposedto0.55mg 2/1showedstatisticallysignificantreductionsinboth lengthanddryweight (950.05), The 1.3and2.6mg .i./Ltreatmentgroupswerenotincludedinthestatistical analyses ofgrowthdue: to:statsiignifsicatntdiiffecrenaceilnsulrvivyal. `SGecoend neraAcutteiExpoosunre "Theresults ofthesecondgenerationexposurearepresentedin Table 11. After96-hcoontruolrsursviv,al was 96%.Survivalinal PFOS treatmentgroupswas 295%andwasnotstatistically diffroemtrheceontnrolts. Allsurviving mysidsinth secondgeneration exposureappearednormalwithnoovertsigasoftoxicity. CONCLUSIONS There were no statistically significant effcts on survival, reproduction or growth of mysid shrimp (Mysidopsis bahia)exposedto PFOSatconcentrationsof 0.25mg ../Lfor 35days.Reproduction,lengthand 01205 WILDLIFE INTERNATIONAL, LTD. BTR PROJECT NO.: 454A-107 dryweightwerethemostsensitivebiologicalendpointsinthisstudy.Mysidshrimpexposedto 1.3 and2.6 mg 2/1 hadsignificantlyreducedsurvivalincomparison tothenegativecontrol.Mysidsexp10o0.5s5mgeaid/L had significantly reduced reproduction, length and dry weight in comparison to the negative control Consequently,the LOEC,basedonreproduction,lengthanddry weight, was0.55mg a:/L. The NOEC was 0.25mg a./LandtheMATCwascalculatedtobe0.37 mg ai/L.SecondgenemysridsaexptoseidtooPFnOS duringastatic 96-hourexposureshowednoadverse effects. 01206 WILDLIFE INTERNATIONAL, LTD. 17. PROJECT NO. 454A-107 REFERENCES 1 `UOSP.PTESnvNiurmobnmeern8t5a0l.1P3r5o0t:ecMtyisoindAgCehnrcoyn.ic19T9o6x.iciSteyriTeesst850- Ecological EffectsTestGuidelines (draft), 2 ASTM Standard `SaltwaterMysids, E1191-90, 1991. Standard American Society for Testing Guidefor Conducting and Materials. Life-Cycle Toxicity Tests with 3 SPSS Inc. 1988. SPSS/PC+ Version 2.0. Chicago,linois 4 West, Inc. and D. D. Gulley. 1996. TOXSTAT Version 3.5. Western EcoSystems Technology, Inc. Cheyenne, Wyoming, 04207 58 2 8 8 2 3 ge Ef 8% 45 8% 22 : 31 S| ifs 32 62 BA 8 8x f IF $3 25 59 39 25 13 5% 53 2 3 &% =a } i ose 85 53 8% se] I Him il PN EIiH kisi ! He 01203 So WILDLIFE INTERNATIONAL, LTD. 194 PROJECTNO.: 454A-107 Table 2 `Salinityof Water in the Negative Control and Highest Treatment Group Test Chambers STpeosntsSourbstance: P3FMOCSorporation DTielsuttOirongaWnaitsemr:: _ SFiallttewraetderSaMlytswiadt,erMysidopsis bahia -Day Replicate NeSgaaltiinvietyC(o3n%t)rol 01 T`A8 2210 32 cD 220 4 A 5 B 2200 5 c 7 D 220 8 A 9 B 02 10 c 1 D 2200 132 AB 2200 151' cD 2210 1s A 7 B 2200 18 c 19 D 2200 2210 AB 2200 223 Dc 220 225 AB 2200 2% c 27 D 220 239 AB 2200 3310 cD 2200 3 A 3 B 2200 34 c 35 D 2200 1O0nD0%amyosrt1a3l-i3t5y,isnatlhinei2t.y6wamsgm8e/a1stureiratetmhedent 1g.r3omupg.a.1/L 26mgaSail/inLi/ty1(3360m)gaill 2210 2109 2200 2200 2200 2200 . 2200 22 2200 2200 2200 2200 p2r0) 2200 220 2200 2200 2200 reatguromupedune tto 61203 WILDLIFE INTERNATIONAL, LTD. -20- PROJECTNO: 4544-107 Table3 `pHof Water in the Test Chambers. STepsM tmSubstC ance: op PFOS owion DTileusttiOorngaWnaitsemr:: SalMytsidw,Mysaidotpsiesbarhia _FiltredSaltwater TMeestaCnonMceeantsruarteidon TestDay (gail) "Replicate 0 7 14 2 2 35 NemwCom AB M-4 8-2 -- -- -& 83 bc -- -- 8-3 8-3 -z =- 0051 AB 84- 8-2 -- - 83 - - 83 Dc -- -- [-5 8-3 -- -- on BA c 8-4 8-2 -- -83 -- 8-3 =< 8-3 = D -- - 83 - z 025 BA 8-4 82- -- = - =83 8-3 Dc -- -- 8-3 8-3 -- ~z oss AB c 8-4 -8~2 -- - 83 -- 8-3 -z E=H D -- - 83 - - 13 AB 8-4 8-3 -- -- 8- 83 Dc -- -- 8-3 . 83 -- =zz 26 AB 84 - - -- - - 83 - -- - cD -= -= 83= -= -= =- ire Seefeo TOO mony 01210 WILDLIFE INTERNATIONAL, LTD. 21. PROJECT NO.: 454A-107 Table 4 Temperature (C)ofWater in the Test Chambers TTedoSrubsance: WoPmReOS DTielttOorngWnaitme:r |_ FSittrl dSaMbyva saied,rMtpidoepis bai MTeestaCnoMnecesnsreidon Tobey (gain) Repfeae 0 1 122 35 Tema AB B2O0 B25I1 muss m2y9 m2522 B2O9 c> 3300 23510 muss uass x512 225500 00s? 5BA 030 22523 ums0 22:00 2a11 22505 c> 3300 2222 mms0 22::00 2312 2210 on AB 3u0s 2m11 mmss ams1i a2s:0 2299 c> 2300 21522 auss 22310 01 2299 025 AB uuss o25z0 wurs m211 5210 22550 0 c> 2u9s 23500 umss 2a511. 2a10 2us50 oss AB u#s7 m2800 mwss a2s00 m2:10 22590 c5 u#s7 22500 uMss 22:500 22:500 22099 13 AB u#s7 u2s9 w7ma mmss uuss m2s8 c> #315 3310 ua1s 720 u25s0 2299 26 AB uuss m2510 m=zp 2z 3: -z r EcD rr ruuss me E31S0Le 2m11mr n33mmmS:- m :. *eMmespmeurreomresntsasdsournenceodndiesty10u0r%inmg o(raayn.gelfrom2451033 3-C. 0411 WILDLIFE INTERNATIONAL, LTD. "22 PROJECT NO.: 454A-107 Table Dissolved Oxygen Content (mg/L)of Water in the Test Chambers" STpeseteSurbsunce: MPCFOeS.pemien DTielsuttOirognanWaitsemr:: ___ FiSltearedSlalMtytwsaitdew,rMysaidotpsieabarhia TeMsteCnonMceenstrsateidon Teby (mgail) Replicas o 7 14 2 28 3s "~ TewComa AB 6-1 6-4~ - - - 63 Dc -- -- 6- 60- -- z- 0057 AB 6-1 63 - - 60 - - 62 Dc -- -- 6-0 6-0 -- -- on AB 6-1 6-1 -- -- 6-0 6-3 bc -- -- 6-3 6-0 2= -= 025 BA 6-1 6-1 -- -- 3x8 6-3 c - - 62 - = oD. - - - 58 - - oss AB 6-1 62 - = 60 - - 62 Dc -- - 62 - z - - 60 - = - 13 AB 6-1 6-2 -- -- 6-0 6-3 Dc -- -- 6-2 5-9 -- -- 26 AB 6-1 6-2 -- -- Dc =- -- 6 = = TRGoivedoxygenconeeneationof 4. mLrepresen 60% sarin a125Cin ? M2ea0s%u.rementsdiscontinueddue to 100% mortality z- z- - z = = water wilh a ly of 01212 WILDLIFE INTERNATIONAL, LTD. a3. PROJECT NO.: 454A-107 Table 6 Survivalof Juvenile Mysids Exposed to PFOS (Day 0ThrPaioringuonDgayh20) TSesrSubstance: WPMRGOS TDeiltsOirognaWneisemr:_FS ierel d SMeyo swidn,Mw yidoepsibria TMeset wCoMnecaesnuareidon ~ SoroDays30 Fy (mgain) Repliste No AlveNo Exposed Tow Fegeive Con Bx 1T0a1s5 60 c> 1a1nss 0057 AB 113s0 560 c> 11831155 on A5 1n00s5 asso 5c 1on1ss 025 AB 11031155 we c5 111ss oss BA 1w3i1s5 soso cb 11331155 13 AB anss 1960 c> uwss 26 - Tete AB 1ass 60 >c eatss ent rcs om sega conewig7 3contnblgesen0e05y) Percent Suvi 7 2 7s 5 20 o 01213 WILDLIFE INTERNATIONAL, LTD. 2. PROJECT NO: 4544-107 Table? (DaSuyrv2iv0aTlhorfouAgduhltTeMsytsTides rExmpoisendoantoDtPaiFyOo3S5n) "SpToesntsSoursstace: ~~PIMCFoOrSporation DTielsuttOirognaWantiesr.m: _SFuillttwroetdeSraMlytswiadt,erMysidopsis bahia pMiowMesso wed|_-__ s _e _Sue v_ ilsE lTe_ sm TE erminatioe mn msmmed memmm--- (mg ail) Replicate No_Alive/No Exposed. Toul Percent Negative Control Bx T7i1 36059 % Dc 180m 0057 BA 11221023 ane 9% Dc 11001110 012 AB 1m202 36140 0 bc 1701"0 02s AB Dc 10310 3657 El 91o0n : 055 BA Dc 170"10 3557 9 1[0310 13 AB pns ane sv Dc o3n "indi+cSgeuicsant Gren fomGe begative controlusing a 7 X 2 contingencyWhe = 005 01214 WILDLIFE INTAERNNATAIOLNA, L, LTD. ee "25 TPRROOWJECCTTNNGO:: 44554-01077 ) Table 8 MeanNumberofYoungProducedPerReproductive Day r Eo SMe Corporion me--m TTeetSOurbgstnainecme:: PSROaS ltMyswid,aMstidoeparis bahia Dilution Weer. _Fitred Saltwater TTMTees mConecesntration ReNpaomdubceiove NaYowuasl YoMuewgmRNepermodbuectave Ove (again) Replicate NegativeControl A Dos 70 Produced Ts Day 0257 Man + 0321005525 Bc 05 214 00226846 > 2 1 0452 0057 BA i "1 0022080 0261 : 00873 c> o56 123 00138765 on AB awo 22 00340708 0361 20101 cb 5wo4 218 00420577 025 AB 506 129 o02m14 ozsmeoom cD 5a6 210 00314749 055 AB s5e6 36 "000.510576 00559 1 00376 cb o56 31 000012799 13 AB 2i 00 00000000 000010000 cD i0 00 00000000 dTifStreneceainmsugnrivoaulpwasmotTeiodedTneSisielanalyes ofeproduciondueto satelSpE indicate significantdifferencefrom henegativecontolusingDunnett's es p< 0.05). 01215 e WILDLIFE INTERNAATO IONA ALe , LTD. e IPRROOJTEECOTTNNOO:: 45540A-01007 -26- Tabl9e MeanTotalLengthofAdult MysidsattheEndofthe35-DayTest Period TeSseteSurbsance: PWROS.ee DTielsuttiOorngaWnaitsemr:: _FSialttewraetdeSr aMlystied,rMpidopbsaihisa TTMCeoancMenetrwatwieond (gain) egatve Contal Replicate x cB > 0057 AB 5c 012 AB Dc 02s BA bc -R--eplicateMesa Gum) 6354 s6a4s6 s6a3s8 663562 663655 s8a2 663488 663386 Overal(laMme)an : + 100657 643 20075 63610105 . 64000548 035 BA s1o1s >c 56126 13 BA s5o93 Dc 5265 Thdiiefstfmiernntsougrrvciovuaeplwasnoteladedi Ge Gaal wnalyaes of allen dueTo 8 Indicate significantdiffrencefrom thenegativecontrol singDunnett'sest(p 0.05) 6140 200794 58520178 siealySgmEeant 01216 HPWILLDILIEFE ITNTEERRNNAATTIIOONNAALL,, LLTTDD. oe 27. TPRROOIJEECCTTNNOO.::445540A0-10077 Table 10 Mean Dry Weightof Adult Mysids athe Endofthe 35-Day Test Period S--_p--orn IM Corporation TTeesstt SOurbgsatnainscme:: PSaFlOtwSaterMysid,Mysidopsis bahia Dilution Water. Fired Saltwater "Me`aConnceMnetsrsatuiroend 3 --ReplicateMea-- n OverallMean = 5 (again) 7 _ Replicate (ng) (mg) NegativeControl AB 00579096 0634 2 0.0510 Dc 005653%6 0057 AB 00662176 0599200276 Dc 00356%5 012 A B 00664674 0641: 00241 Dc 0066047 025 .BA Dc 00660414 0622: 00227 o06s3s9 oss BA 00555586 062 = 000624 Dc 00556730 13 AB 00444708 0436.2 00441 c ~ --"_Th--is reatmentgroupwas not included D i (he statisticalanalysesoofd0rw3y w%eight us 0Satstcaly SgmEcant Idnidffiecreanitcsniegenifsiscuanrtvival, diffrencefromthenegativecontrolusingDuanet's est p < 0.05 01217 WILDLIFE INTERNATIONAL, LTD. 28. PROJECT NO: 454A-107 Table 11 `Survivalof Second-Generation Mysids Exposed to PFOS During 96-Hour Static Exposures TSepsSeuebsrtance: ~~ PMFCOoSpemien -- DTielsuttOirongaWneiesrm:. S_aFlitlwiaetderSaMlytswiatde,rMysidopsis bahia: TeestmCondceantration Toul 9 HNoeusoAf EexpAosrure Tega(tmigveaiClo)niral No. 7Exposed. (ObserEvaItio)ns' o0s7 os mn on 5 798) 025 @ EI) AN= Ap0p5e5ws Normal 5 BEN) Percent Sur5v%ival 1 9s os 100 01213 WIL LDLIE FENINETERRNNAATIOONNAALL,, ELTTDD.. 29. PPRROOEJECCTTNNOO::4455444-01077 APPENDIXI Dilution Water Salinity Measured During the 4-Week Period Immediately Preceding the Test -- Sponsor: 3M Corporation Test Substance: PFOS Test Organism: Saltwater Mysid,Mysidopsis bahia DiDliuluttiioonnWWaatteerr:,_ FFiieltreereddSSoabltwwoatteerr Mean Range Salinity(%:) 00-4 20-20 01219 WILDLIFE INTERNATIONAL, LTD. "30. PROJECT NO.: 454A-107 APPENDIX AnalysesofPesticides, Organics, Metals and Other Inorganics vA Tasmess Di--u d----nimnWildc life Io nternatin onal, Lc td. Salv twater'inmam momay JouignmsnaCuomr' <3 8 om3 Orsagchortes ad PCBs Cem wm `Alpha BHC BetaBHC [oo-- Chlordane: PBoBpE. FBFrroimSnen HHEn wte `Heptachlor Eporide Toxaphene PCB-1016 PCB-1221 ffPPjCCeBBei--is11i22tn4322 Me"NlmaandpOger organ ffroe] gae= fare fbr es Hewt oe SSaii A < 0.005 wr < 0.005 gL 0025 "gL << 3050%0s aprs <SI ompureemd aorm T TToas mse a om < 0005 gl < 0.500 gL < 0260, gL < 0260: gL ;SD<< oomuu0072m2600 aooHHmmLLm < 100 wh :i < F3I33 hi = :3:fsw ii omaa P3E SE . B= =< edi5 Eato Aeprtormedby Wit mont, Lure al clic Nov 5,199 TepeedyQTEionmenalGunes,FondaTosuns colionNaver 3 BroughNovember 7. >AnalysesperformedbyWildlifeIntemational, Lid. for samples collected on November $ through 7, 1997. G12Z0 A D WILDLIFE INTEe RNATIONAL, LTe D. r IPRROOTJEEe CCTTNNO.O: 4534040-210077 31. APPENDIX II `THE ANALYSES OF PFOS IN SALTWATER IN SUPPORT OF `WILDLIFE INTERNATIONAL PROJECT NO: 454A-107 o12z1 WILDLIFE INTERNATIONAL Lm. 3. PROJECTNO.: 454A-107 REPORT APPROVAL SPONSOR: 3M Corporation TITLE: P(MFyOsSi:doApsFilsobwa-hvTiahar)ough Life-Cycle Toicity Test with the Saltwater Mysid "WILDLIFE INTERNATIONAL LTD. PROJECT NO.: 4544-107 PRINCIPALINVESTIGATOR: dy 4 ieAage HK A. MacGregor, BS. Scientist MANAGEMENT: add `MWainlalagredr,B.ANnailxyotni,caPlh.CDh:emistry oorpor DATE ly DATE 0122 SWIeLDLIFE INTERNATIONAL Lm. 33. PPRROOEJECCTTNNOO.: A45S4AA-I107 Introduction Saltwater sampleswerecollectedfrom aflow-through life-cycletoxicitystudydesignedtodetermine the effectsof PFOS (Perfluorooctane Sulfonic Acid Potassium Salt) to the saltwater mysid (Mysidopsis aia). This study was conductedby Wii Interational Ltd. and dentificdas Project No.: 454A-107. `Theanalysesofthese water samples were performed at Wildlife Intemational Ld. usinghighperformance liquid chromatograwpihtyh raass spectrometric detection (HPLC/MS). Sampleswerereceived for analysis between June 15,1999and July 21,1999andwereanalyzedbetween June 15, 1999and July 22, 1999 TSestubsantdInaternnalStcandeard "ThetestsubstanceusedforthisstudywasWildifeInternationalLtd.identificationnumber 4675. The test substance was ustoperepdare calibraandtmaitroinx fortification samples `Theinternalstandardwas receivedfrom 3M Corporationon July 2, 1998andwas assignedWildlife International Ld. identification number 4526uponreceipt. Theintemalstandard, agranularmaterial,was identified as: 1H, 1H, 2H, 2H Perfluorooctane Sulfonic Acid, Chemical Abstract Number: 27619-97-2, `Thestandardwasstoredunderambientconditions. B AnalyticalMethod : `Themethodusedfortheanalysisofthesaltwatersamples wasdevelopedatWildifoIntemational Ltd. andentitled "AnalyticalMethodfortheDeterminationof PFOSinFreshwater, Saltwater,andAlgal Medium". This methodology was included as Appendix IIofWilde Intemational Ltd. protocol umber 454/011299/MVAL/SUB4S4. Itwasbasedupon methodology provibyd3eMd Corporation. Samples were diluted in a 50% methanol : 50% NANOpure water solution containing 0.100mg 4H PFOS (ternal standard). and 0.05% formic acid (v/v) so that they fll within the calibration rangeofthe PFOS methodology. Concentrationsofthe PFOSinthestandardsandsamplesweredeterminedbyreverse-phasehigh performance liquid chromatography using a Hewlett-Packard Model 1100 High Performance Liquid Chromatograph (HPLC) vith a Perkin-Elmer API 100LC Mass Spectrometer equipped with a Perkin- 01223 A WILDLIFE INTERNATOINONAALL Lo0.. 3. PPRROOJJEECCTTNNOO.::d45544AA-d10077 Elmer TurbolonSpray ion source. HPLC separations were achieved using a Keystone Betasil Cpe analyticalcolumn (50mm x 2mm LD. 3pmpastilesize). Theinstrumentparametersare summarizeind| Table1. Amethodflowchart sprovidedinFigure1. : CalCi urvb eanr dLia mitt ofQuianto itatn ion Calibration standardsof PFOS prepared in a 50% methanol: 50% NANOpure water solution `containing 0.100mg 4H PFOS (internal standard). and 0.05%formicacid (v/v), ranincgoncientnratigon from0.000915to0.00915mg a.i/L,wereanalyzedwiththesamples.Thesame andmost prominpeeaakt. responsefor PFOS wasutiltoimzoneitdor PFOSinallcalibration,qualityconantdstrudyosamlple,s. No. attemptwasmadetoquantify PFOSonthebasisofindividualisomericcomponents. Linear regression equationsweregeneratedusingpeakarearesponseratios(FOS : internalstavnersdustaherrespdect)ive concentrartiaost(iPFoOSn: intemalstandard)ofthecalibrsatatnidaordns. Atypicalcalibration curveis presiennFitguered2.Theconcentration ofPFOSinthesampleswasdeterminedbysubstitutingthepeak arearesponseratiosintotheapplicablelinearregressionequation. Represioencnhrtomaatotgriamvsoef lowandhighcalibrationstandardsarepresentedin Figures 3and 4, respectively. "Themethodlimitofquantitation(LOQ)fortheseanalyseswassetat 0.0458mga.i/Lcalculatedas theproductofthelowestcalibrationstandardanalyzed (0.000915mg 2./L)andthedilutionfactorof the `matrix blank samples (50.0). MaFtroixrBltankianfd icatSiamoplnes Sixmatrix blanksampleswereanalyzedtodeterminepossibleinterference. Nointerferenwceerse observedatorabovethe LOQ during samplesanalyses(Table 2). Arepresioen cnhrtomaatotgriamvofea `matrixblankispreseinnFitguered5. Saltwater was fortified at 0.0823, 0.366 and 3.66 mg ./L and analyzed concurrently with the samplestodeterminethemeanproceduralrecovery(Table 3). Sampleconcentwerreanottciororenctsed forthemeanprocreecodveruyorf9a2.8l%. A representativeionchromatogramofa matrixfortification is presienaFitguered6 01224 WILDLIFE INTERNATIONAL Lo. 35. PROJECT NO.: 454A-107 `ExampleCalculations Sample number 454A-107-3, nomial concentrationof0.086 mg a.i/L in saltwater. PeakAreaRatio =AnalytePeakArea/IntemalStandard PeakArea Concentration Ratio = ConcentrationofAnalyte/ConcentrationofInternal Standard Internal StandardConcegtration: 0,100 mg/L Initial Volume: 0.500 mL Final Volume: 25.0 ml. Dilution Factor: 50.0 PFOS Peak Area: 12963 Internal StandardPeak Area: 145150 Peak Area Ratio: 0.08931 Calibration curve equation. Slope: 5.76026 Intercept: 0.00940 Curve is weighted (I). PFOS (mg a.i/L) at ia nstrument = Peak area raStloe-me(V-intercept) x Internal Standard Concentrati.on 008931 -000940 =" sass *0100 =0.001387 PFOS(mg a.i/nsLamp)le = PFOS (mga.1/L)atinstrument xDilution Factor =0.001387 x 50.0 =0.06935 01225 W_I--LDLIFE INTEe RNATe IONAL Lm. 36. PRPOROJJEECCTTNNOO.:d4s5i4AA-l10077 PercentofNominal Concentration =_ PpFrOoSc(m(gSa.Li/LL)insample 100 =_0006098365 x 100=80.6% - RESULTS SampleAnalysis Saltwater samples were collected from the flow-through life-cycle toxicity study with the saltwater mysid (Mysidopsis bahia) at pre-test, June 15, 1999,attest initiation, June 16, 1999 (Day 0), and weekly duringth testthrough testtermination on July21, 1999(Day35). Themeasured concentrationsofPFOS in the samples collected at pre-test ranged from 57.3 to 99.3%ofthe nominal concentrations (Table 4). The measured concentrations of PFOS in the samples collected at initiation of exposureof the test organisms (Hour 0) ranged from 67.1 to 103%ofthe nominal concentrations (Table 5). Samples collected at Day 7, Day 14, Day 21 and Day 28 had measured concentration ranges of 45.8 to 95.6%, 70.5 to 99.6%, 57.1 to 87.9% and 59.9 t0 99.3% of nominal values, respectively. Sarl collected at test termination (Day 35) had a measured concentration range of 59.8 to 90.0% of nominal values. A representative ion chromatogram ofa test sample is showin Figure 7. 01226 W--I_--LDLIFE INTERNATeIOeNAL Lm, 37. PRPOROKJCETCTNNOO.:: 44S5A4AA-10077 Table 1 I--N_--STRUMENT: `Typical HPLC/MS Operational Parameters `CHhorwolmeattt-oPgarcakpahrdviMtohdealPe1r1k0i0n-HEilgmherPeArPfIor1m0a0nLceC LMiaqsuisdSpectrometer ~ seeqlueicptipveediwointmhonaiPteorrkiinng-mEolmdeer T(SuIrMb)o.lonSpray ion source. Operated in ANALYTICAL COLUMN: KeystoneBetaslCiscolumn (50mm x 2mum LD. 3umpasticesize) OVEN TEMPERATURE: 30C STOP TIME: 5.00 minutes FLOW RATE: 0220 mUminute MOBILE PHASE: 07.21.90%%FoMremtihcanAocli:d28.0% NANOpure Water co"ntaining INJECTION VOLUME: 2501 PFOSRETENTIONTIME: Approximatel3y.8 04.5 minutes INTERNAL STANDARD RETENTION TIME: Approximately 2.6 103.0 minutes PFOS MONITORED MASS: INTERNAL STANDARD MONITORED MASS: 498.6 amu 4267 amu 0427 LWLILDLIFE INTERNATIONAL Lro. eeiveneeeeeeeraeennn JPEROOTJEECCTINNOO.;:d48504A0-010077 38. Table 2 Matrix Blanks Analyzed Concurrently During Sample Analysis _-- Sample Measured Concentration of Number FOS (854A-107) Type (mgai/l) MAB-1 = `Matrix Blank <I0Q MAB2 Matrix Blank <LoQ MAB3 Matrix Blank <L0Q MAB4 Matrix Blank <LoQ MAB-S Matrix Blank <L0Q MAB-6 Matrix Blank <10Q "Thelimitofquantitation(LOG)was0.0458msa.L/Lbasedupon theproduct ofthelowestcalibration standardanalyzed (0.000915mga.i/L)andthedilutionfactorofthematrixblanksamples (50.0). 01223 --W_I--LDLIFE INTERNe ATIOe NAL n Lo. 39. PPRROOJEECCTTNNOO.:: 445548A-A10077 Table 3 Matrix Fortifications Analyzed Concurrently During Sample Analysis --_--_-- `Concentrationsof PFOS `Sample Number (gail) Percent --_-- (4M54AA-S10T7-) SF0Foormt0 iiffiiedd MMeeaa00ss7uw4r8eedd ReRceoc5vo0vee9rreedd MAS-4 MAS-7 0.0823 00823 0.0828 00673 101 818 MAS-10 MAS-13 0.0823 0.0823 0.0662 00693 804 842 MAS-16 0.0823 00787 956 MAS2 MAS-5 0366 0366 0350 0359 956 98.1 MAS MAS-11 0366 0366 0314 0303 858 828 MAS-14 MAS-17 0366 0366 0360 0287 984 84 MASS MAS-6 366 366 329 354 L899 96.7 MAS-9 366 MAS-12 366 339 388 926 106 MAS-15. 366 --MMAASS18S 36666 380 a3988 104 d10o9 Mean = 928 Standard Deviation = 9.19 cv =990 --_--_-- NN==1s G1R2I A WILDLIFE INTERNATOINONAALLvLeo,. 40- PPRROOJJEECCTTNNOO.::445544AA-10077 Table 4 Measured ConcentratSiaolntswoatferPFMOySsidinLPirfeet-eCsytclDeilTuotreirciVteyriTfeisctation Samples from a -- "Nominal Test Sample-- eSaemplieng PFO-- S Measured Percent Concentration (mg a.i/L) Number (454A-107:) Time. (Hours (Comngceanitr/a)ti?on of Nominal 0.086 PTI 24 PT2 24 0.0581 00493 676 513 PTs 24 PT 24 0.0627 79 0.0606 05 27 PT3 24 PT4 24 221 819 234 367 PTT 24 PTS 24 256 948 268 93 "Tsthaendlairmditaonfalqyuaznetdit(a0t.i0o0n0(91L5OQm)gwaa.si/0L).0a4n5d8tmhegdai.luit/iLonbafsacetdoruoftphtheemo partordinuxcbtloafntkhseamlpolweesst(5c0a.l0i)b.ration * Measured valuesofmatrix blanks samples were <LOQ, 83.0, 95.6, and 100, matrix fortification <LOQ, 93.4, 85.0 samples analyzed and 106 perceat concurrently with pre-test ofnominal concentrations for pre-test 1 and pre-test 2, respectively. 01230 A WILDLIFE INTERA NATIOONNAAL Le to. ee -a1- PERPOOJEECCTTRNOO: 445540A-11007] Table 5 Measured Concentrationsof PFOS in Saltwater Samples from a Saltwater Mysid Life-Cycle Toxicity Test `Nominal Test `Concentration (mg0a.0i/L) Sample `Number T (454A-107-) 2 Is 1P6y pa3e0 5565 7 & Sampling Time. (Da0y) 07 7 14 2114 21 2 2 35 35 PFOS Measured Concentration (m<g1a.0i/QL) ! <<1L00QQ <<LLo0QQ <<LL0OQQ <L0Q <<LL00QQ <L0Q <L0Q Percent. of = Nominal -z z --- 0.086 34 00 00.00567984 8601.27 7 7 00478 556 3118 174 2 as 2114 6 21 00.00660169 772005 0000651544 76444 00509 592 57 28 58 28 0.0515 59.9 0.0569 662 69 35 0.0580 674 70 35 0.0514 598 "The limitofquantitation (LOQ) was 0.0458 mg a.1/L based upon the productofthe lowest calibration standard analyzed (0.000915 mg a.i./L) andthe dilution facotfothre matrix blank samples (50.0) 01231 -- WILDLIFE INTe ERNATIOe NAL Lo. a2 PRPOROJJEECCTTNNOO::44554AAl-10077 Table (continued) Measured Concentrationsof PFOS in Saltwater Samples from a Saltwater Mysid Life-Cycle Toxicity Test Nominal Test Concentration (mg0a1i7l) Sample Number 5 (454A-107-) 6 210 334 a 4589 6701 7 Sampling Time (Day) 0 0 7 147 14 21 221 2385 35 PFOS Measured Concentration (m0g 1a.2is/L))! 000171748 00.112245 0.127 0.0970 00.11122 00..112248 0.119 Percent of No7mi5nal 4675.81 7ns9 77 57.1 7658.9 77593 70.0 034 7 0 8 0 0289 0286 8845.01 21 7 0231 679 2 7 0.197 57.9 335 1144 00227563 78142 5290 21 21 0227 0212 668 24 621 228 00.227612 7797..71 73 35 0.278 818 74 35 0251 738 `The limitofquantitation (LOQ) was 0.0458 mg a.i/L based upon the productofthe lowest calibration standard analyzed (0.000915 mg a.i/L) andthedilutionfactorofthematrixblanksamples (50.0) 01232 I WILDLIFE INTERNATO IONALN Lm. , a. PPRROOEJECCTTNNOO.::44454AAd-10077 Table (continued) Measured Concentrationsof PFOS in Saltwater Samples from a Saltwater Mysid Life-Cycle Toxicity Test . "Nominal Test Concentration (mg a.i/L) 0.69 ; Sample Number (454A-107-) 9 2130 2% 3378 5521 6 64 77%5 `Sampling Time (Day) 0 70 7 1144 221 2% 28 335 PFOS Measured Concentration (mg a.i/L) ! 0.562 00.568519 0.450 00.556423 00552186 03529 0.544 00.558536 Percent of Nominal 814 8952.5 652 7786.7 77458 767 788 8804.56 14 1 0 12 0 25 7 26 7 39 14 123 879 132 943 113 80.7 120 85.7 135 96.4 40 14 53 21 127 90.7 123 879 54 21 65 28 66 28 7 35 78 35 LIS 82.1 139 99.3 139 99.3 126 90.0 120 85.7 "The limitof quanti(tLaOtQ)iwoans.0.0458mga.i./L.based uponthe productofthe lowestcalibration standard analyzed (0.000915 mg a.i./L)) andthe dilution factorofthe matrix blank samples (50.0). 001233 TWIILLDOLLIFFEE IINNTTEERRNNAATTIIOONNAALLtLoo.. aan PPRROOEJECCTTNNOO.:: 44554AA-l10077 Table 5 (continued) Measured ConcentraotfiPoFOnSs in Saltwater Samples from a _---- Saltwater Mysid Life-Cycle Toxicity Test . . Nominal Test Concentration (mgai/L) Sample Number (454A-107-) Sampling Time (Day) PFOS Measured Concentration (mg ai/L)' Percent. of Nominal 27 13 0 2174 07 2a8 174 42 14 2.56 948 225789 9150.36 225340 984512 2.69 99.6 T The limitof quantitation (LOQ) was 0.0458 mg a.i./L. based upon the productof the lowest calibration standard analyzed (0.000915 mg a.i./L) and the dilution faocf tt hemo atrrix blank samples (50.0) 012341 WILDLIFE INTERNAO TIONAA L Lr., re PPRROOEJECCTTNNOO::445544A-01077 METHOD OUTLINE FOR THE ANALYSIS OF PFOS | IN SALTWATER Prepare matrix fortification.samples by spiking the requisite volumeof PFOS stock solutions directly intofilteredsaltwaterusinggas-tightsyringes andClass A volumetric flasks. + Dilutematrixfortificationandtestsamplesintotherangeofthe calibrationstandardsbypartiallyfilling Class A volu flasm ks we itht 50%rmei thancol : 50% NANO watep rsolu utior ncone tain ing 0A.p1p0r0omprgia4tHe vPoFlOuSmeo(inftesranmalplsetaannddabrrd)i/nLgtahnedf0la.s0k5t%ofvoorlmuimceawciidth(tv/hv)e.diAludtdiotnhe solvent.Processthe matrixblanksampleusingthesamedilutionandaliquot `volumeas forthelowestrfepoerattiinfviercsaiotlneisv.oenl.Mixwebyl sevel ral + Ampulate samples and submit for LCMS analysis, Figure1. Analytical methodflowchartforthe analysisof PFOSinsaltwater. 01235 --WN I--LD--LT I--FE E INTER RNATN IONAA L Lo.T ~ I~ ONAPPRL ROONJEECCt TTNNOO..:4m 45544A-01. 077 -t6- 0707 0.60 0.50 3 040 Zon 020 0.10 0.00 0.000 o ~ 0.020 0.040 0.060 0.080 0.100 Concentration (Ratio) Figure2. Atypicalcalibcurrvae ftoriPFoOSn.Slope = 5.76026;Inter=0c.0e09p40t; 1=0.9974. Curve isweighted (1/x). 01236 WILDLIFE INTERNATIONAL Lm. a- AVAILABLE BE PROJECT NO.: 454A-107 BEST COPY AVAILABLE RET Figure 3. Arepresentativeionchromatogramof alow-level (0.000915mga.i/L) PFOSstandard. 001237 WIWLIDLDLLIIFFEEIINNTTEERRNNAATTIIOONNAALLLLmo.. ~~~ _as- PPRROOEJECCTTNNOO.::s45a44l-1e07? BESTCOPY AVAILABLE Figured. Arepresentativeionchromatogramofa high-level (0.00915mg .i./L)PFOSstandard. 01233 WILDLIFE INTERNATIONAL Lm. 49 PROJECTNO.: 454A-107 BEST COPY AVAILABLE Figure5. Arepreseionncthraomtatiogvraemof a matrixblank sample (454A-107-MAB-1). `The arrowindicates theretentiontime ofPFOS. 01239 YWIILLDDLLIIFFEE IINNTTEERRNNAATTIIOONNAALL4L0m.. 50- PPRROOEJECCTTNNOO..:445444a-10?7 BESTCOPY AVAILABLE Figure 6. A representative ion chromatogram ofa matrix fortification sample (454A-107-MAS-1). 001240 ILWIDLLDILITFEE NITNETERRNNAATTIIOONNAALLLLom.. si PRPROOJJEECCTT NNOO.:: 44s544AA-l10077 BESTCOPY AVAILABLE Figure7. A representative ion chromatogramof a test sample (454A-107-3). 01211 e W--I_--LDLIFE INTERNATIONAL, LTD. p PPRROOJJEECCTTNNOO.::44S544AA-N10077 52. APPENDIIXV Mysid Reproduction DiTuEiOoorWeger.__FaloiweeartseSrateMsi, iMdidopsis bibahia T"MTeestmCoVnceSenUtmrDaetidon Replies Teasrmen NRaep1bDo.iexive Caste . 32 iiis v hi5 11 B 321 11u i it ProNadbYeoudmcsts 32 2 284 5 : c 321 1i1"i i36 3i 1ii :5 D 321 11ia 9 0057 A 321 i11i 383 31 1i 33 5 321 l11i4 552 5i 114 $3 c 321 i1ini 333 53 11a i3 b 521 11144 i72o ThembofreproductivedaysTsBemaimeofdayshathieiTealewas iv2eTom Br enddoffthe test. ve! YoungNRhaeyoperodcive 0264 0.286 o4s2 0283 0200 0186 0375 broodReese 01242 ZWIILLDDLLIIFEE NITNETRERNNAATTIIOONNAALL,,LLTTDD..~~ 53. PPRROOJJEECCTTNNOO.::d4s5i4Aa-110077 APPEIVN(CoDntiInueXd) Mysid Reproduction Be imine Tem Mowe r is: i var i M"TeestoCmopneceesr"iion Rojee comTemker NRTeepHoedwidve pNYauoeiuss 02s odees:e ; 2i55 5 321 3 c 231 i 251 3i A 321 5i B 231 i c 321 i5 D 251 i3 11ii"d 033i uhIs 7$i i 2 1iui 735 i 2 mrinti 92 11 :$ 11i4i 23i i1 m1ini 47i i 0 1i1i5 2ii 1i1i 031 1 3 YounogThNeEeproductve - ours 0407 0257 oan 0214 0344 orm 01243 -- WILDLIFE INTERNATIOANA, L, LTTDD.. sae PPRROOEJECCTTNNOO.::44S54AA-01077 APPENDIX IV (Continued) `MysidReproduction _Sp--on SM Corporation TestSubstace: ~~ PFOS D`DiTMielesouttaOinnorngMWaeWnaeatiesesru_rm_r:ed FFS iilletea rreeddl SSaMllywt osswinedw e,rrMysaidot psie s bar hiNaamberor Test Conoenration 3 Test Reproductive (mg0a5i5/L) A Replicate Compa2rT tment Day1Tsse' i3 112 B 21 11 33 11s4 c 21 1mn h3 11a 5 1 NYaombuasrof Prod3Tuced 0 00 2 00 30 YougNReaprboeduorcftive TaDa5y56 0107 00429 D 321 1114 000 "oom i 1 1 13 A 21 3 i3 00 8 0 0000 B 1 1 0 0000 c - - - - Temabe ofreproductivDeday he b1 erofday al heufalewasalivef0om paring toBe0en0d0Fes 0612441 3 - : g g5332e38s S| 5al5n3g3n8g8w88 ff| C513 o 283533383(33 aB a 9& 3 Ql3ne2ne9cg3y 8383 Z 3388585388 [3 533338588839 Y3l-mvmereeszaly ) 01245 3 g Aariify ga 18 en - | Hesagog Hagg oo [y 33458833fy | : of Heazszansasf.s ER 8 Haelrsessnzegzn azsf.gn H3forereneosaay 01216 i : of s3sssasszs| 3g Joel sammameef Je ssmsszs| 3 4 gsassezzsnsy Co f32 5 2: 52 eo] sassnzssss. fg or sssmsazen| 4 a -| 3333339585 : sesssensesas 5 i i3 jr i lf ail | I1H asf) H fs I sessmasan| 3 5s3sss of 8 ssssssansss| 8 -eeemermenz } 004247 : 3 :: [go] Biv | 38 Fool s333000000 | 8 led s5300| 8 SEER CEE i= [Te] essen | fi { smsssm| o ssasmsesn| Hi E re] ssszszSassE CelAl ) ssn [5 !) SEE ih Hl oom| 01248 WILDLIFE INTERNATIONAL, LTD. -59- PROJECTNO.: 454A-107 APPENDIVIXI Chanto Pgroteocsol Thisstudywasconductedin accordancewiththeapprovedProtocolwitth folowingchanges: 1. The proposedexperimental startandteruintiondateswereamendedtotheprotocol. 2. Analysisoffoodfor PFOSwasdeletedbyamendment. 3. Theproportionofwatersplittoeachreplicatewasnotcheckedatthe endofth test 4. Temperaturewasnotmeasuredi theBreplicate ofthe2.7mga.1/Ltreatmentgroupon Day14ofthetest. 5. Nominal testconcewneret0.r086a,0t.17i,0.o34,n0.6s9,1.4and 2.7mga.i/L. 04249 --WILE DLIFEE INTERNATIA ONAL, LTD. eee -60- PPRROOJIECCTTNNOG.;: 445340A3-10007 APPENDIX VII Personne Involved in the Study "ThefollowingkeyWildiieInternationLatld,.personnelwereinvolvedintheconductormanagement ofthsstudy: 1. Heary O. Krueger, Ph.D, Director, Aquatic ToxicologyandNon-Target Plants 2. Willard B. Nixon, Ph.D, Manager, Analytical Chemistry 3. Jon A MacGregor, Scientist 4. Mark A. Mank, Laboratory Supervisor 5. KuntR Drottar, Senior Biologist 01250 PROTOCOL PFOS: A FLOW-THROUGH LIFE-CYCLE TOXICITY TEST `WITH THE SALTWATER MYSID (Mjysidopsis bahia) U.S. Environmental Protection Agency ~Series 850 - Ecological Effects Test Guidelines (OPPTS Number 850.1350 3M LabRequestNo. U2723 `Submitted to 3M Corporation `Environmental Laboratory 935 Bush Avenue St. Paul, Minnesota 55144 WILDLIFE INTERNATIONAL LTD. 8598 Commerce Drive Easton, Maryland 21601 fr (410) 822-8600 May 13,1999 01251 r WILDLIFaE INTEe RNATIONAL, LTe D se ER PFOS: A FLOW-THROUGH LIFE-CYCLETOXICITY TEST `WITH THE SALTWATER MYSID (Mjsidopsis bahia) SPONSOR 3M Corporation Environmental Laboratory 935 Bush Avenue St. Paul, Minnesota 55144 SPONSORSREPRESENTATIVE: Ms. Susan A. Beach IESTINGFACIITY: `Wildife Intemational,Lud 8598CommerceDrive Easton, Maryland 21601 STUDYDIRECTOR: KurtDrottar `SeniorAquatic Biologist LABORATORY MANAGEMENT: Heaxy O. Krueger, PhD. Directorof Aquatic Toxicology & Non-Target Plants FOR LABORATORY USE ONLY Proposed Dates: Experimental `Start Date: Experimental "Termination Date: Project No.: -- cee Test Concentrations: 0Neg0ati9veC4ont,rol0, .19,03L8Sa,nd300m.ga7i/l5, , `Test SubstanNco.e: 4675 ReferenceSubs No.t(ia fappn licc able e): 4526 PROTOCOL APPROVAL STUDY DIRECTOR DATE JORATORY MANAGEMENT J o a. iach SPONSOR'SREPRESENTATIVE PROTOCOL NO.: 454/051399/MYS-LC/SUB454 DATE DA5/18/99 012252 3M LABREQUESTNO. U2723 HWIE LDLIFENINeTERNATIONAL, LTr D eeveseeeesmmmereeeeeeeemsn. 3. INTRODUCTION `Wildlife International, Ltd.willconduct a flow-through life-cycle toxicity testwith the saltwater `mysid,Mysidopsis bahia,forthe Spoatnths e Wio ldlr ife International, Ltd. aquatic toxicology facility in Easton,Maryland.The studywillbeperformedbasedonproceduresinthe U.S. EnvironmentalProtection AgencySeries 850 -EcologicalEffects TestGuidelinesOPPTS Number850.1350 (1);andASTMStandard E1191-90StandardGuidefor Conducting Life-Cycle ToxicityTests with Saltwater Mysids (2). Rawdata forall work performed at Wildlife International,Ltd.and a copy of the finalreport will be filed by project `numberinarchiveslocatedontheWildlife Intemational,Ltd.site, oratan alternative location tobespecified inthefinalreport. PURPOSE `Thepurposeofthisstudyis to determinethe effectsof atestsubstance on the saltwater mysid, Mysbaihiad ,duro ingcp hrons icexiposusre. Effectsongrowth,survival,andreproduction willbeevaluated. EXPERIMENTALDESIGN Mysidswillbeexposedto aseries ofsixtestconcentrations, anegative(dilutionwater)control `beginningatthejuvenilestage (<24hoursold)andcontinuingforatleast 7daysafterthemediantimeoffirst `brood release inthe control treatmeat. The testwill notbe terminateduntilatleast 28 daysandmaybe `continuedatthe requestofthe Sponsor. Testconcentrations willbechosen inconsultationwiththe Sponsor andwillbe baseduponknowntoxicitydata. Eachtestconcentrationwillbe 50% ofthenexthigher treatment. Four replicatetest chambers willbe used for each treatment and control group, and each test chamber `willcontain onecompahorldtingm15emynsitds.Inordertocontrolbias,mysidswillbeindiscriminately assignedtoexposurechambersattestinitiation. Nootherpotentialsourcesofbiasare expectedtoaffectthe: resofuthlesttudsy. Mysidswillbeindiscriminatelydistributedingroupsof oneto threeamongglass `beakersrepreseatingeachtestcompartmentuntileachbeakercontains 15 mysids.Themyswiil thdensbe `gently transferred into the test compartments. Thus, atotalof 60 mysids willbe exposed in each treatment `andcontrolgroup.After mysidsattain sexual maturity (usually 10 to 14 daysafterthe beginning ofthetest), femaleandmaleadultmysids willbepaired,andthereproductionofthepairedmysidswillbemonitored. 01253 PROTOCOLNO.: 454/051399/MYS-LC/SUB4S4 3MLABREQUENSOT. U2723 e WILDLIFE INTERNATIONAL, LTD e whe Observationsformortalityandclinicalsignsoftoxicitywillbemadedaily. Thenumoflbveeyourng `producedbycachparvillbecounted, recorded,andremovedtoaseparateestcompartmentatthesametest `concenontardaaitlyibaosins. Theyoungwillbeexposedunderstaticconditonsfor96hours.Thedry weightandlengthof ist generationmysidsaliveatthe endofthetestwillbedetermined. Daonsutrvivaal, `growth,andreproductionvillbeanalyze todetermineth effctsofthetestsubstanceonthese parameters. ~ MATEARNDIMA ETL HOS DS Test Substance Informationonthecharacterization oftest,controlorreferencesubstancesisrequiredbyGood LabPoracrticeaStatndaordsr(GLyP).The SponsorisresponsibleforprovidingWildlifeInternational,Lid. writen verificationthatth testsubstancehasbeencharacterizedaccordingtoGLP priortoitsuseinthe study.IfwritenverificationofGLPtest substancecharacterization inotprovidedfo WidieInternational, Ltd, twillbenotedinthecompliancestatementofthefinal report.TheattachedformIDENTIFICATION OFTEST SUBSTANCE BY SPONSOR(AppendixI)istobeusedtoprovideinformationnecessaryfor `GLPcompliance. `TheSponsorisresponsibleforal informationrelatedtothetestsubstanceincludingtheretention of areservesampleofthelotorbatchofth testsubstanceusedinthisstudy.TheSpoalnsosagroeesrto acceptanyunusedtestsubstanceand/ortestsubstancecontainersremainingatthe endofthestudy. Prepoaf TerstaContcenitraotionns "Thetestsubstancewillbeadministeredto th testorganism inwater. Thisrouteofadministration `wassclociod becauseitrepresents themostlikelyrouteof exposure to aquaticorganisms. TestOrganism `Themysid,Mysidopsisbahia, willbeusedinthistest. Thisspecieisrepresocfaantimapoirtvaent `groupoforganismsandwasselectedforus inthetestbasedupon pastuseandeaseofhandlinginthe Iaboratory. MysidswillbeobtainedfromWidifeInternational,Ltd.cultures. Allmyussediintdh tsest willbe romthesamebroodstock. 01251 PROTOCOLNO.: 454/051399/MYS-LC/SUB4S4 3MLABREQUENSOT. U2723 _ WILDLIFE INTERNATIONAL, LTD -5. Testswillbeinitiatedwithmysidslessthan 24-hoursold Guvenikes). Culwiltbeumairntaeinedsin arecirculatingsaltwatersystemkeptatapproximat2e5lCy.Saltwater willbe filteredthrough abiofilter andUV sterilizedpriortoenteringtherecirculatingsystem.Allmysidsvillbehel inthelaboratoryforat least 14daysbefore juvenilesarecollectedfortesting.Mysidswillnotbesubjectedtomorethan a 3C changeintemperatureor a 3 changeinsalinityinany 12-hourperiodduringthe14daysbeforetest initiation. Mysidswillbe fedlivebrineshrimpnauplii(Artemia sp)ad libitumdsily.Brineshrizpwillbe periodicallyenrichedwithALGAMA(faCtya-cid2sup0ple0men0t).Feed(brineshrimp)provided to the mysidswillbeanalfyorzPFeOdS. Excessfoodwillberemovedfromthetestchambersdaily. Mysidswillbehandledasinfrequeatlyaspossiblebutwhenhandlingisnecessary itwillbedone gently,carefully, andquickly. Asiphoncollectorsystemwillbeusedtocollectneonatemysidsdirectlyfrom broodtanks. Youngmysidswillbetransferredusinglarge-borepipettes. DilutionWater NaturalseawatercollectedatIndian RiverInlet,Delawarewillbethesourceofthedilution waterused intheculturefacilityandtestsystem.Theseawaterwillbefilteredthrough asandfilterpriorto tsdelivery 1037,800-Lholdingtank.Thesalinityoftheseawaterwillbe dilutedto approximately20 %s,(partsper thousand) ithWildlifeInternationalLid.wellwatetintheholdingtank.The20 7s,saltwwillabetaereatred usingspraynozzlesandfiltered (0.45pm)priortoitsdeliverytothetestsystem. Salinityvillbemeasuredweeklytomonitortheconsistencyofthesaltwater. Meanadranngessof themeasuredparametersforthefour-weekperiodpreceding th testvil beprovidedinthefinalreport. `Analysesvil beperformedtodeterminetheconcentrationsofselectedorganicandinorganicconstituents oftheseawaterandresultsoftheanalyseswillbe summarizedinthefinalreport. TestApparatus Acontinuous-flowdiluterwillbeusedtoprovideeachconcentrationofthetestsubstanceand a negative(dilwautetr)cionotronl. Asyringe pump,peristalticpump,or asimilardevice willbeusedto deliverthetestsubstancetomixingchamberswherethetestsubstancewillbemixedwithdilution water.The flowofdilution watentoeachmixingchamberwillbecontrolledusingrotameters.Therotamewtiellrbse: calibratedpriortoth testandverifiedand/orcalibratedatleastonce aweekduringthetest.Aftermixing, PROTOCOLNO.: 454/051399/MYS-LC/SUB4S4 3MLABREQUESTNO. U2723 01255 e WILDLIFE INTERNATA IONALe D , LTD e fe testsolutionswillbe split tocachreplicate chamber. Theproportion of water splitto each replicate will be. checkedpriortothetest,atapproximatelyweeklythereafterandattheendofthetesttoensurethatthese. flowratesvary bynomorethan +10%ofthemean. `Thediluter willbeadjustedsothateachtestchamberreceivesat least Svolumeadditionsoftest `soelveuryt24ihoourns.Peristaltic pumpswillbe calibrated prior totestinitiationand at approximately `weekly intervalsthereafterand syringe pumpswill be calibrated priorto testinitiation.The delivoefrthye testsubstancetothe testchamberswillbeginatleast48hourspriortothe testinorder toestablish `equilibriumconcenotftrheatestt siubostanncse. The general operationofthe diluterwillbe checked visually atleasttwotimesperdayduringthetest. `Test chamberswillbe9-L glass aquariafilled withapproximately 5 L oftestsolution.Eachtreatment `andcontrolgroupwillhavefourreplicatetestchambers.Testchamberswillbeimpartially positionedinan `environmentalchamberdesignedtomaintain atemperature of25 +2C.Testchawim llbb elabe ellr edwisth theprojectnumber,testconcentarndaretpliicoatne. `Priortosexualmaturity,mysidswillbe heldinonecompartmentplacedineachreplicatetestchamber (15/compartment).Thecompartmentsconsistofglassbeakerswithnylonmeshscreenattached totwoholes. `onoppsio dess ofi thebt eake er. Allattachmentsofthenylonmeshscreenandglasswillbe madewith silicone adhesive. Aftermysidsattainsexualmaturity(usually10to 14daysaferthebeginningofthetest),upto 5 `maleand 5femaleadultmysidswillbepaired perreplicate testchamber. Indipaivrsiwildlbeuhealdlin `reproductive compartments (onepairpercompartment)placedineachtestchamber.Thereproductive `compartments will be glasspetridisheswith sides of nylonmesh screen attachedwithsiliconeadhesive. Immature andadditionalfemaleswillbediscarded.Additionalmalemysidswillbemaintainedin aseparate testcompofa eachrreplticatmetee stchnambter. Additionalmaleswillbeusedtoreplacedeadmalesinthe `samerepdluriingtcheeaxpotsuree.Thetestwillnotbeterminated before 7dayspastthemediantimeof firstbroodreleaseinthe control(s). Thetestwillcontinueforatleast 28 daysandmaybeextended longer at the reqofu thee Sposnsotr. 001256 PROTOCOL NO.: 454/051399/MYS-LC/SUB4S4 3MLABREQUENSOT. U2723 WILDLIFE INTERNATIONAL, LTD 7. - . `Ambientroomlightwillbe used toilluminatethetestsystems. Fluoligrhtteubs es(c Coleorn tonte 50orequivalent)thatemitwavelengthssimilartonaturalsunlight willbecontrolledbyanautomatictimer toprovide aphotoperiodof 16 hoursoflight and 8 hoursofdarkness. A15-to 30-minutetransitionperiod `willbeprovidedwhenlightsgoon andofftoavoidsuddenchangesinlightintensity.Lightintensitywill bemeasuredat testinitiationandatweeklyintervalsthereafter with a SPERScientificLtd.lightmeteror equivalent. N Thetargettesttempewilrlbae2t5u#r2eC. Temwpille bemr onia toretdanudrer core ded continuously in anegativecontrolusing a FulscopeER/C Recorder (1900 JSeries ModelA)orequivalent `duthereni tirn eteg st.Recordermeasurementswillbeverifiedwith aliquid-in-glassthermometer priorto testinitiationandatleastweeklythereafter. Temperaturewillalsobemeasuredincachtestchamberatthe `beginning andendofthetestandatweeklyintervalsduringthetestusing aliquid-in-glassthermometer. Dissolvedoxygenand pHwillbemeasuredat thebeginningandtheendof the testandatweekly intervalsduringthetestinalternatingreplicatetest chambersof cach treatment level. In the event that dissolvedoxygenlevelsfallbelow 60%saturation,appropriateactionswillbetaken afterconsultationwith theSponsor. Salinitywillbemeasureddailyinalternatingreplicatesofthenegativecontroltreatmentand. thehighest PFOStreatmgeronupt.If100%mortalityoccursinany treatment,then dissolvedoxygen, pHand temperaturemeasurementswillbe made at thattimeandthendiscontinued. Dissolvedoxygen willbe `measuredusing aYellowSpringsInstrumentModel 51Bdissolvedoxygenmeterorequivalent,water pH. `will bemeasuredusing aFisherAccumetModel915 pHmeterorequivalent,andsalinitywillbemeasured using arefraocreqtuivoalemnteinsttruemenrt. Bicogi Observations ofmortality andclinicalsigns oftoxicityinfirst-generationmysidswill bemadedaily. `Thecriteriafordeath include lack ofmovement, absence of respiratorymovements, andlack ofreactionto gentleprodding. Liveyoungproducedineachcompartmentwillbecounted,recorded,andremoveddaily. Liveyoungwillbe exposedtothe sameconcentrationofthetestsubstance understatictestconditionsfor 01257 `approxim9a6htoeurlsy.Attheendof thetest,thedryweightandtotalbodylengthofeachfirst-generation PROTOCOLNO.: 454/051399/MYS-LC/SUB4S4 3MLABREQUENSOT. U2723 e W--_I--LDLIFE INTERNATIONAL, LTD s -8. `mysidwillbedetermined. Observations of abnormaldevelopmentandaberrantbehavior willbemade for first-andsocond-generationmysidsthroughoutthetest. Sumi . Water sampleswillbe collected fromtwo replicate test chambers of both the low andhigh level `concentrationspriorto exposure.Watersamplesalsowillbecollectedfrom twoaltematingreplicatetest chambersofeachtreatmentgroup and the controlatthebeginningofthe test,atweekly intervals during the test,andatthe endofthetesttodetermineconcentrations of thetestsubstance.Intheeventthat 100% `mortalityoccursinanytreatment, then samplingoftha treatmentwill terminatefollowingthenextsampling. interval. Sampleswillbecollectedatmid-depth fromeachtestchamber,andanalyzed immediately. The `sample scheme is summaribezleodw: -- PPRROOPPOOSVEIDDNNUUMMBBERRRSSOOFFVVEERRWICFAITOCNASSTMAMMIPPOLLEENSS enEExpePrinRcattO arorp E Pees' DD0y0 DDuy?? DDyuiMs DDuuamDDuyads EEww Control - 22 2 2 a2 2 Level 1-LowConcentration Lowi Levis Lewis 2 2 2 2 2 2 2 - 2 2 a2 2a 2 - 22 2 2 Ta S22 2 2 a2 2 Level 5 Level 6High Concentmion 2 2 2 2 2 2 22 2 2 2 a 2 TM Teous M 6 4 e www aoew iPmrnaoyhteebsreterseamqtplsen,swddielubpecedlnlcipednheahtieeScoppniooorns.inago,fthneddaidtde,iMnoreptainnagnaendpreofess swmipllindgomrenailod "OnesumplefomtheAand Bresetschimbere . ee Tou NumberofVerfeSamaplieso=8n8 `Theabove numbersofsamplesrepresentthosecollectedfrom thetestand donotincludequality control (QC)samplessuchasmatrixblanks andfortificationspreparedandanalyzedduringthe analytical chemistryphaseofthe study. At thediscretionofthe StudyDirector,Watersamplesalso wilbecollected fromatleastoneappropriatechamberwhenever a malfunctionisdetectedinanypartofthetestsubstance deliverysystem. 01258 PROTOCONOL.: 454/051399/MYS-LC/SUB454 3MLABREQUNOE.US27T23 hWIi LDLIi FE INeTiEdRNATIONAL,BLsTD 9. AnalyticalMeasurements `Chemicalanalysisofthe sampleswillbeperformedbyWildifeInternational, Ltd.Theanalytical methodusedvil bebased uponmethodologyprovidedbytheSponsorandidentifiedinAppedix IL. Modificationsmadetothe analyticalmethodwillbedocumentedintherawdataanddescribedinthefinal report. Datatobeanalyzedincludethesurvivaloffirst-generation mysids,the numberofyoungreleasedper reproductiveday,thedryweightand lengthofeachfirst-generationmysidalive a theendofthetest. If deemednecessary,clinical signsoftoxicityintheadultmysidsmayalsobeanalyzedstatistically. Concentration-responsecurveswilbefittedtotheadultsurvival, reproductionandgrowth data. Toevaluatewhetherornotsurvivalisaffectedbyth treatments, 2 X 2contingencytablesandthe chi-squaretestwillbeusedonsurvivaldata(i,numbersof aliveanddeadmysids). Analyseofs reproduction (umberofyoungproducedpersurvivingadult) andgrowth (weightandlength)datavillbe `evaluatedfornormalityand homogeneityofvariances.Ifthedataaredeemednormalwithhomogeneous variances,hypothesis testing singanalysisof variance(ANOVA)andmliplemeanstests (e.g,Dunes', `Bonferroni,Scheffe)wil beused. Ifthedatafailthetestfornormality orhomogeneity,thendata transformationsvillbe triedinanattempttocorrect thecondition. Whenthedatatransformatfiaiolntso correctfor non-normalityor heterogeacityof variances,nonparametricprocedureswillbeusedtoidentify statistically sigaificant differencesamongtheexperimentalgroups. `Thestatisticalanalysesof survival,growthandreproductiondata willidbutmaynotbeexclusively usedinthedeterminationofthe noobservedeffectconcentration (NOEC)andlowestobservedeffect concentration(LOEC). Themaximumacoeptabletoxicantconcentration(MATC)willbecalculatedasthe geometricmeanofthe NOECandLOEC. Allstatisticalevaluationswillbedoneusingcommercially available computer software programs such as TOXSTAT (3) OR SPSS/PCH (4). REC TOBOEMR AIND TAIS NED Recordstobemaintained fordatageneratedbyWildlifeInternational Ltd.willinclude,butnotbe limitedto: 0159 PROTOCOL NO.: 454/051399/MYS-LC/SUB4S4 3MLABREQUEST NO, U2723 WT LIeLDLIFE INTERNATIONAe L, LTD o -10- 1. Acopyofthesignedprotocol. 2 Identificationandcharacterizationofthetestsubstance,ifprovidedby theSponsor, 3. Datesofinitiation andterminoaftthietoesnt. 4. Historyofthetestorganism. 5. Testorganismweightand lengthmeasurementsatthe endofthetest. 6. Stock solutioncalcualndparetpairatoionn. . 7. Dailyobservations., 8. Waterchemistryresults (c.g, salinity and pH). 9. Ifapplicable,themethods measurements usedto analyze testsubstanceconcentrationsandtheresultsofanalytical 10. Statisticalcalculations. 11. Testconditions(ightintensity, photoperieotdc),. 12. Calculationandpreparationof testconcentrations. 13. Copyof finalreport. : FINALREPOR] Afinalreportoftheresultsofthestudy willbepreparedby WildlifeInternational,Ltd. "Thereport `willinclude,butnotbelimitedtothe following,whenappropriate: } 1. `Nameandaddressof thefacilityperformingthestudy. 2. Datesuponwhichthestudywasinitiated andcompleted. ItistheresponsofithbeiSplonisotr tyo `providethefinaldatethatdataarerecordedforchemistrypathology and/orsupportingevaluations that maybe generated atother laboratories. . 3 Astatementofcompliance signedbythe StudyDirectoraddressinganyexceptionstoGood Laboratory Practice Standards. 4. `Objectivesandproceduresasstatedintheapproved protocol,includingany changesintheoriginal protocol. S. `Thetestsubstance identification,includingname,chemicalabstractmumberorcodenumber,strength, purity,composition, andothercharacteristics providedbythe Sponsor. 6. Stability andsolubilityofthetest substanceundertheconditionsofadministration,ifprovidedbythe Sponsor. 7. Adescriptionofthemethodsusedtoconductthetest. 8. Adescriptionofthetestorganisms,includingthesourceof thetestorganisms,scieatificname,age, if stage, meansandrangesofweightsand dures, andfeeding regime. lengths,observeddiscases,treatments,holding proce- 01260 PROTOCOLNO.: 454/051399/MYS-LC/SUB454 3MLABREQUENSOT. U2723 r WILDLItFT E INTERNATIONAL, LTD r a. 9. Adescription ofthepreparationofthetestsolutions,themethodsusedtoallocateorganismstotest chambersandbeginth tet,thenumberoforganismsandchamberspertreatmeat,andtheduration ofthetest. 10. Adescriptionofcircumstancesthatmayhaveaffectedthequalityorintegrityofthedata. 11. Thenameofthe StudyDirectorandthenamesofotherscicatists,professionals,andsupervisory personnelinvolvedinthe study. 12. Adescriptionofthetransformations, calculations,andoperationsperformedonthedat,asummary `and analysisofthebidlogicaldataandanalyticalchemistrydats,end astatementoftheconclusions drawn romtheanalyses. 13. Statisticalmethodsusedtoevaluatethedata. 14. Thesignedanddatedreportsofeachoftheindividualscientistso otherprofessionalsinvolved ithe study. 15. Thelocation whererawdataandfinalreportaretobestored. 16. AstatementpreparedbytheQuality AssuranceUnitlistingthedatesthatstudyinspectionsandaudits weremadeandthedatesofanyfindingsreportedtotheStudyDirectorand Management. 17. Ifitisnecessarytomakecorrectionsoradditionsto a finalreportafterithasbeenaccepted,such changesvillbemadeintheformofanamendmentissuedbytheStudyDirector.The amendmentwill clearlyidentifythepart ofth finalreport thatisbeingamendedandthereasonsforthealteration. Amendmentswillbesignedanddatedbythe StudyDirector. : CHANOFGPRIOTNOCGOL Plannedchangestotheprotocol willbeintheformofwrittenamendmentssignedbytheStudy DirectorandtheSponsor'sRepresentative.Amendments willbeconsideredaspartoftheprotocolandwill beatatotchefihnalperotodcol. Anyotherchangeswillbeintheformofwrittendeviationssignedbythe StudyDircctoran filedwiththerawdata.Allchangestotheprotocolwillbeindicatedinthefinalreport. GOODLABORATORY PRACTICES `ThisstudywillbeconductedinaccordancewithGoodLaboratoryPracticeStandardsforEPA (40 (CFRPart 160and/orPart792); OECDPrinciples ofGoodLaboratoryPractios(ENV/MC/CHEM(98) 17); and JapanMAFF ($9 NohSan,Notification No.3850, Agricultural Production Bureau). Eachstudy conductedbyWildifeInternational,Ltd.isroutinelyexaminedbytheWildlifeInternational, Ltd.Quality AssuranceUnit for compliancewithGood Laboratory Practices, Standard Operating Procedures and the 001261 PROTOCOLNO.: 454/051399/MYS-LC/SUB4S4 3MLABREQUNOE.US27T23 e WILDLIFE INTERNATIONAL, LTD e 12- `specifiedprotocol. A statementofcompliance withGoodLaboratory Practiceswill be prepared for all portionsof thestudyconducted byWildlife Intemational, Ltd. TheSponsor willberesponsibfloer `compliance with Good Laboratory Practicesforproceduresperformedbyotherlaboratories(c.g,residue `analysesorpathology).Raw data for allwork performed at WildlifeInternational, Ltd.and a copy ofthe final reportwillbe filed by project numberinarchives located onthe Wildlife International,Ltd.site, orat `an alternativelocationtobespecifiedin thefinalreport. PROTOCOL NO.: 454/051399/MYS-LC/SUB4S4 001262 3MLABREQUENSOT. U2723 W_I--LDLIFE INTERNATIONAL, LTD BER REFERENCES 1 (UdSr.afEt,nvOiPrPoTnmSeNntuamlbePrro8t5e0c.t1i3o5n0A:gMeyncsyi.dC1h9r9o6n.icSeTroixsic8i5ty0-TeEscto.logical Effects Test Guidelines 2 A`SSalTtwMaStteraMnydsairdds,EA1m1e9r1i-c9a0n.So1c9i9e1t.ySftoarnTdesatridnGguainddefMaotreCrioalns,ducLitfeiCynclge To:xicity Tests with 3 West, Inc andD.D.Gulley. 1996.TOXSTATRelease 3.5. Westem BooSystems Technology, Inc Cheyenne, Wyoming. 4 Norusis, MJ. 1988. SPSS/PC+V2.0 base manual for the IBM PC/XT/AT and PS/2. PROTOCOL NO.: 454/051399/MYS-LC/SUBAS4 04263 3MLABREQUEST NO. U2723 W_I--LDLIFE INTERNATIONAL, LTD 14. APPENDIX IDENTOFITEF ST SIUBC STAA NCET BYI SPOONSN OR "TobeComplbyeStpoensdor I. TestSubstanceIdatity(nametobeusdi th rept): P(EQPS efluaSwrlfoonicAocisdPoitasasiunmSeal Reference Standard (fapplicable): Analytical Standard: NA `TestSubstanceSampleCodeorBatch Number:_Lot2l7 Test SubstancePurity (%ActiveIngredient): _989 ExpirationDate: _2008 IL TestSubstanceCharacterization Hove thedet,song purty snd composition afr chris `whichappropriately substance and refestranedarnd bceeen em pce101505 nhsSynor wsCL i? -- Ys _xNo IL TestSubs StotragaeConndictioens `Please indicatethe recommended storage conditionsat WildlifeInternational,Lid. Aube 0000000000 the aniity oolfthheettetstssbubstpunadeenrtfhceeseees:one conditions Ys _x No a IV. Test Concentrations: testconcentrtaot10i0o%n EEE EE E Do test tion to 100% V. TondcityInformation: Mammalizn: RtLDSO 251 mg/kg MouseLDSO_N/A Aquatic: Invertebrate Toxicity(EC/LCS0) `Fish Taxicity(LCS0) Duphni2a7mmgagns: _RaTroiut:mllbmgol w Daphnia magna:SOme/l Fathead Minow;38mg `Other Toxicity Information(includingfindingsofchronicand subchronictests): B0 lesse0 scoMs0 Ds 0000000000 : 01261 PROTOCOLNO.: 454/051399/MYS-LC/SUB4S4 3MLABREQUENSOT. U2723 W_I--L_D--L--I--FE INTERNATIONAL, LTD BTR APPENDITXI AnalyticalMethodProvidedby Sponsor metho`dSsa:mpleswillbeanalyzedbaseduponproceduresprovidedbythe Sponsorinthe followinganalytical 1. LiPqerufiludcCrhoorcotmaanieoSgurlafopnhiycMAacisdsPSopteacstsrioummetSraylt(L(PCFMOSS))MIentFhreosdhwfaotrert,hSeaDlettweartmerinaantdioAnlgoafl Medium `analyzAetchoeptyeostfstahmepalbesovwielmlebtehdoodcwuimlelnbteemdaiintthaeirnaevdidnattaheanrdaswudmamtaa.riTzhedeianctthueaflimnael rtehpoortd.oulsoedgtyo PROTOCOLNO: 454/05 1399/MYS-LC/SUB4S4 01255 3MLABREQUESTNO. U2723 -- WILDLIFE INTERNATIONAt L LTe D. PePPaRlgOeJolEoCfTf1tNO.: 454A-107 AMENDMENT TO STUDY PROTOCOL STUDY TITLE: SPAFOLS:TWAAFTLEORWM-YTSHIRDO(UMGysHidLoIpFsEis-CbaYhCiLa)E TOXICITY TEST WITHTHE PROTOCOL NO.: 454/051399/MYS-LC/SUB454 SPONSOR: 3M Corporation EFFECTIVE DATE: June 16, 1999 AMENDMENT: Page2 AMENDMENT NO.: 1 PROJECT NO.: 454A-107 Add: PExrpoeproismeedntDaalteSst:art Date: 6/16/59 Experimental Termination Date: 7/21/99 REASON: The above Director. information was not known when the protocol was signed by the Study AMENDMENT:TestOrganism, Page S Delete: Feed (brine shrimp) provided tothe mysids will be analyzed for PROS REASON: The feedhasalready been screened for PFO. Ra 4 \ STUDY DIRECTOR 2/7077 DATE LAB( \TORY' AGEMENT lela DA dr A Lpact, SPONSOR'S REPRESENTATIVE 2/27/99 DATE 01256 aesatonenesis Reviewed! by GAGD T1692