Document 912ZBVDo3nwJOBV8abVY7Dm65
PFOS: A 48-HOUR STATIC ACUTE TOXICITY TEST
"WITH THE CLADOCERAN (Daphnia magna)
FINAL REPORT
`WILDLIFE INTERNATIONAL LTD. PROJECT NUMBER: 454A-104
3M LAB REQUEST NO. U2723
:
U. S Environmental Protection Agency
po Series 850 --Ecological Effects Test Guidelines
.
OPPTS Number 850.1010
`OECD Guideline 202
AUTHORS:
HenKu,rt RK.aDeront,tarPD
STUDY INITIATION DATE: December 10, 1998 STUDY COMPLETION DATE: June 4, 1999 AMENDED REPORT DATE: April 26, 2000
Submitted to
3M Corporation Environmental Laboratory
935 Bush Avenue St. Paul, Minnesota 55144
Wildlife I$5n9tCeomrenrsaDtriiveonal Ltd. eioy 224600 `Easton, Maryland 21601
Page 1 of 43
000656
AMENDED
WILDLIFE INTERNATIONAL Lo.
2.
PROJECT NO.: 454A-104
GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT SPONSOR: ~~ 3M Corporation TITLE: PFOS: A 48-Hour Static Acute Taxicity TestwiththeCladoceran (Daphnia magna) WILDLIFE INTERNATIONAL LTD. PROJECT NUMBER: 454A-104 STUDY COMPLETION DATE: June 4, 1999 AMENDED REPORT DATE! April 26,2000
USS. E"ThnisstvudyiwParsroctoenocdtuicontneAdgimenncceoymipnlni4a0ntcCeFwaRitPhalGrtosod16La0baonrdat7o9r2y,Pr1a7ctAiucgeuSsttan1d9a8r9d;saOsEpCuDblPriisnhceidpbleystohef `5G9ooNdoLhaSbaonr,atNoortyifPircaacttiiocen,NOoC. D3E8/50G,DA(g9r2i)cu3l2t,uErnavliPrroondmuecattioMnonBourgeraaup,h1N0o.Au45g,usPtar1s98149,92w;iatnhdtJhaepafnoMllAoFwiFn,g exceptions: character"iTzhaettieosntwassupbesrtafnocremweadsacncootrcdihnagrtacote3rMiSzteadindnaarcdcOopredraantcienwgiPtrhofceudlulreGs LaPndcoMmeptlhioadnsc,ea;nhdoawlelvrearw,dtahtea arebeing mainintthae3iManrcheiveds.Thetestsubstancei beingrecharacterizedinaccordancewith GLP. accord"aTnhceeswtiatbihliGtoyoodfLtahbeotreasttosruybPsrtaacnticceeunStdaenrdcaorndsd.itionsof storageatthetestsitewasnotdeterminedin
STUDYDIRECTOR:
oral ( Kurt R Drottar Senior Biologist
.
4/26/20 DATE
APP] :
5
yi
000687
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WILDLIFE INTERNATIONAL Lm.
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PROJECT NO.: 454A-104
QUALITY ASSURANCE STATEMENT USS. "ETnhviisrsotnumdenytwsa]sPerxoatemcitnieondfAogrecnocmyplinia4n0ceCwFiRthPGarotsod1L6a0baonradt7o9r2y,Pr1a7ctAiucgeuSsttan1d9a8r9d;sOasEpCuDbliPrsihnecdibplyetshoef `GoodLaboratoryPractice, OCDE/GD(92) 32,EnvironmeatMonograph No.45, Paris 1992;and JapanMAFF, 59 NohSan, Notification No. 3850, Agricultural Production Bureau, 10 August 1984. The dates of all inspectionsand auditsandthedatesthatanyfindingswerereportedtothe StudyDirectorandLaboratory Management werea follows:
ACTIVITY: Test SubstancePreparation PrAecpaalryaitcialonSemple MeWsasteurreChmeemaitsstry DBriaolfotgiRceaploDratta and DrAanafltytRiecpaolrDtaa and Final Repent AmendedReport
DATE CONDUCTED: February 15,1999
February 16,1999
February 17,1999
Ma17rand1c8,1h999
March23-25,1999 June4,1999 Ap20rmd2i5,2l000
DATEREPORTED TO: STUDY DIRECTOR: MANAGEMENT: February 15, 1999 February 16, 1999
February 17,1999
February 17,1999 .
March18,1999
February 19, 1999 February 19,1999:
Ma2r9,c19h99
March25,1999 Jue, 1999 Apri 25,2000
March26,1999 June 41999 April 25,2000
Se won WN. Colman
`JQuhds ualityHA.sCsourlaenmcaen Representative:
4-35-00
DATE
000688
AMENDED
Wo WM ILr DLI Ie FE E INTERR NATIN ONAL A Lro. TeI e ONA PROJEL CT NO.:L 454A-1. 04 -a-
REPORT APPROVAL
SPONSOR: 3M Corporation TITLE: PFOS: A 48-Hour Static AcuteToxicityTestwiththeCladocecan (Daphnia magna)
~ WILDLIFE INTERNATIONAL LTD. PROJECT NUMBER: 454A-104
STUDYDIRECTOR: edd \
KSeunritor BDiorlootgtiasrt
MANAGEMENT:
FeDiarrecyto4 r, Aquatic T oxicology and `Non-Target Plants
yps/oo
DATE
DATE 7
000689
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WILDLIFE INTERNATIONAL Lo.
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`TABLE OF CONTENTS
PROJECT NO.: 454A-104
`TABLES
`Table 1 - SumomfAa nalr ytiy calChemistryData.................
------------------
"Table2 - Temperature,DissolvedOxygen,andpHof Waterinthe TestChambers. ------
`Table 3 -CumPu ercel ntMa ortat lityi /lmmv obile ity andTreatment-RelatedEffects.....
---
Table 4 --EC10, EC50 80dECO
VAIUES.....oovervrresoressosos sesamin
18
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WILDLIFE INTERNATIONAL Lm.
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PROJECTNO.: 454A-104
`TOAFCBONTLENETS - Continue-d
FIGURE
Figur1e. ConcentratioCunrv-eR(4e8sHpOUonse DAta)..vvvvrmcnssnsnsns 19 v
APPENDICES
Appendix 1- SpecificConductance, Hardness, Alkalinity,andpHofWellWater MeasuredDuringthe4-WeekPeriodImmediatelyPrecedingthe Tet.
20
Appendix II Analyses ofPesticides,Organics, Metals andOtherInorganicsin
WifeIernaLtidi.Woelnl al WE.
21
AppendIiIx TheAnalysisofPFOS inFreshwaterin Supportof `WildifeInternLaitd.iProojnoctaNlo.: 454A-104 .......ccrvvnn 22
APPEDiXI=V ChA1D0 PGIEOSIOCOL...rrrrr
imme)
Appendix V - Personnel InviontlhevSeUdY......r i smn
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PROJECT NO.: 454A-104
SPONSOR. SPONSOR'S REPRESENTATIVE: LOCATIOFN STUDY, RAW "DATAANDA COPYOFTHE FINALREPORT:
WILDLIFEINTERNATIONAL 7 LTD. PROJECT NUMBER: TEST SUBSTANCE: stop:
MEANMEASUREDTEST CONCENTRATIONS: TESTDATES:
LENGTHOFTEST: TEST ORGANISM SOURCEOFTEST ORGANISMS:
AGE OFTESTORGANISMS:
T8HOURECS0: 95%CONFIDENCE LIMITS: NOMORTALITY/MMOBILITY CONCENTRATION: NCOO-NOCBESNETRRVAETDI-OENF:FECT-
SUMMARY 3M Corporation Ms.Susan A. Beach
`WildeInternational Lid Easton,Maryland 21601
454A-104 PFOS (Perfluorooctane Sulfonic Acid Potassium Salt) PFOS: A 48-Hour Static Acute ToxicityTestwiththe Cladoceran (Daphnia magna) NegativeControl, 11, 20, 33,56and 91mg ai/L.
ExperiStmarte-nFetbruaarly 16, 1999 Biological Terminatio-n February 18, 1999 Experimental Termination - February 18, 1999 48 Hours "Neonatecladocerans (Daphia magna) `EWaisltdoinf,eIMnatreyrlnaantidon2a1l6L0t1d. Cultures <24hours attestnition STmgaill 33nd 91mgail
33mgail
Bmgaill
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PROJECTNO.: 454A-104
|
INTRODUCTION
"ThisstudywasconductedbyWildifeInternationalLtd.for 3M Corporation a theWildlifeInternational Ltd. aquatictoxicologyfacilityinEaston, Maryland. Thein-ifephaseofthetestwasconductedfrom February 16, 19toF9ebru9ary 18, 1999.RawdatageneratedbyWillieInternationalLtd.and acopyofthe finalreportarefledunderProjectNumber454A-104inarchiveslocatedontheWildifeInternationalLtd. sie.
:
OBJECTIVE
`Theobjectiveofthisstudywastoevaluatetheacute toxicityof PFOS tothe cladoceran,Daphniamagna, during a 48-hourexposureperiodundertat testconditions
EXPERIMENTAL DESIGN
Dapwherenexipodsedsto ageometricseriesof ivetestconcentrationsand anegative(dilutionwater) control. Tworeplicatetestchambersweremaintainedincachtreatinentandcontrolgroup;with10daphnidsin
eachtestchamber for atotal of20daphnidspertestconcentration. Nominaltestconcentwreraetseileoctnedisn consultwaittihtohenSponsor,andwerebaseduponth resultsofanexploratoryrangefindingtoxicitytest. `Nominaltestconcentrationsselectedwere12,20, 33,55and 91mgactiveingredient (a. IL. Meanmeasured
testconcentrationsweredeterminedfromsamplesoftestwatercollectedfrom each treatmentand conrolgroupat: thebeginningofth tet, atapproximately24hours,anda testtermination.
Daphnidswereindiscriminately assignedtoexposurechambersattestinitiation. Observatioonfs
`mortality/mmobilit andotherclinica sigasoftoxiityweremadeatapproximately3,24 and48hoursaftertest initiation. Cumulativepercentmortalityandimmobility observedinthetreatmentgroupswerousedtoestimate.
orcalculateEC10, ECS0andEC90valuesat24and48hours.Thenomortality/immobiltyconcentration and the no-observed(N-OEeC)fwerfe deetcermtine-d bcy voisunalicnteerprnetattiorn ofathtemiortoalinty, immanodcblinicialolbserivatiotn dayta.
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PROJECT NO.: 454A-104
MATEARNI D MA ETL HOS DS
`Thestudywasconductedaccordingtothe proceduresoutlinedinthe protocol, PFOS: A48-HourStatic
AcuteToxicityTestwiththe Cladoceran(Daphnia magna). Theprotocolwasbasedonproceduresoutlinedin
US. EnvironmentalProtection AgencySeries 850 ~Ecological Eff TesetGc uidt elis nes OPPTS Number
850.1010(1), OECDGuideline for Testingof Chemicals, 202: Daphnia sp. Acute Immobilization Test and
Reproduction Test (2)and ASTM Standard E-729-88a, StandardGuideforConducting AcuteToxicity Tests with Fishes, MacroinvertebratsandAmphibians (3).
TestSubstonce
`The test substance was received from 3M Corporation on October 29, 1998 was assigned Wildlife
InteLtrd.n idena tift icati iono numn bera46l 75.Thetest substance wasdescribedas awhitepowder.Itwas identifiaesd FC-95fromlotnum 217b(T-e 629r 5). Informationprovidedby the Sponsorindicated apurity of 98.9%andanexpirdaatetofi2o00n8.ThetestsubstancewasreanalyzedbytheSponsorandtheCertificate of
Analysisdated March9, 2000indicated apurityof 90.49%. Thetestsubstancewasstoredatambient room
temperature.
`PreopfTaesrtCaonctentiraotionns
Nominaltestconcentratiweornes12, 20, 33, 55 and 91 mga.i/L,basedon atest substpuarintycoef
90.49%. Allmaterialswhichcameintocontactwiththetestsubstanceduring preparationoftestconcentrations
-
`wereconstructed ofplasticorstainlesssteel. Aprimarystocksolution waspreparedindilutionwaterat a
concentofr91amtgia.oi./nL.The primarystocksolutionwasmixedwithanelectricdrummixerwith astainless
steel paddleforapproximately 19.5hourstoaidinthesolubiolftiheztesatstubistaoncne.Aftermixing,the
`primary stock solution was proportionally diluted with dilution water to prepare the four additional test
`concentrations. Alltestsolutionsappearedclearandcolorless.
TestOrganism
`Thecladoceran, Daphniamagna,wasselected asthetestspeciesfor thisstudy. Depahrernepreisendtats ive
`ofanimportantgroupofaquaticinvertebratesandwereselectedforuseinthetest baseduponpasthistoryofuse. andeaseofculturinginthelaboratory. Daphnidneonatesusedinthetest were lessthan 24-hours oldandwere `obtainedfromculturesmaintainedby WildlifeInternationalLtd., Easton,Maryland. Identifoifcthaeotriigionanl `brood stockwasverifiedbytheAcademyofNaturalSciences, Philadelphia, PA.
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`Adultdaphnidswereculturedinwaterfrom thesamesourceandat approximatelythe sametemperatureas
useddurtihenteg st.Adultdaphnidsinthecultureswereheldforatleast28dayspriortocollection of the Juvefnoritlesteinsg. Theadultsshowednosignsofdiseaseorstressduringtheholdingperiod.During the
14-dayperiodprecedingthetet,water temperaturesrangedfrom 19.9 020.4C.The pHofthewaterranged
from 8.1to8.3,and dissolvedoxygenrangedfrom8.3 to 88mg/L. Instrumeunsetdfaotrwiaotenr
`measurements are described in the Environmental Conditions section ofthis report.
`Neonat daphidswerebisinedfortesting from individualadult aphidswhichwer observed 0haven
`neonatespresentlessthan 24hourspriortotestinitiation.Theprogenyfrom10adultswereusedinthetest.At testinitiation,the juveniledaphnidswerecollectedfromtheculturesandindiscriminately transferrteod 10-mL glassbeakers.Thedaphnids thenweretransferredfromthebeakerstothetest compartments.Alltransferswere performedbelowtheair/waterinterfaceusing awide-borepipet. Daphnidsinthecultureswerefed amixtureof
yeast, Cerophyll, and trout chow, as well as a suspension of the freshwater green alga, Selenastrum
capricornutum. Theadultswerefedpriortotestinitiation, butneonateswere notfed durtihentesgt.
Test Apparatus
`Testchambersconsisted of250-mLplastic(Nalgene)beakerscontaining 240mLoftestsolution. The
depthoftestsolutionin arepresentativetestchamber was6.4can. Testchamberswereindiscriminpalatceedliyn
`atemperatwau terr bate hse-ttc o maointnaintatrempoeralturleoe f20d+ 1C.Testchawm ereb labe eller d wisth
theproject number,testconcentarndaretpliicaoten.
Dilion Water
Thewaterusedforculturing andtestingwasfreshwater obtainedfrom awellapproximately40metersdeep locatedontheWildlifeInternatLitdo.nsiatle.Thewellwaterischaracterizedasmoderately-whaatrerd. The
`specific conductance, hardness, alkalinity, and pHof thewell water during thefour per- iodw imme edie atekly
preceding thetestarcpresentedin AppendixL
`Thewellwaterwaspassedthrough asandfilterto remove particlesgreaterthanapproximately 25 an,and
pumped nto a37,800-L storagetankwherethewaterwasaeratedwithspraynozzles.Prior us,thewater
againwasfiltered(0.45 um)to removemicroorganismsandparticles.Theresultsofperiodicanalysesperformed
000695
R WILDLR IFE OA INTERNATIONAL Lo. ese 1.
PRROOJECCTNNOO.:44540A-1M04 .
tomeasuretheconcentrationsofselectedcontaminantsinwellwaterusedbyWildlifeInternational Ltd.are presien Anpptenedidx II.
Environmental Conditions
Lighting used toilluminatethe cultures and testchambersduring culturing and testingwas provided by
fluorescenttubes thatemittedwaveleagthssimilartonaturalsunlight (Colorto5n0e). Aphotoperioofd
16 hoursof lightand 8hoursofdarknesswascontrolledwith anautomatictimer. A 30-minutetransitionperiod.
of low lightintensitywas providedwhenlightsweton andoffto avoidsudden changesinlighting. Light
intensityattestinitiationwasapproximately359 luxatthesurfaceofthe water.Lightintewnassmeiasturyed `using a SPERScienLtdt.liighftmietcer.
`Temperaturewasmeasuredincachtestchamberatthebeginningandendofthetestusing aliquid-in-glass
thermometer. Temperaturealso was measuredcontinuously in onenegativecontrolreplicate using a Fulscope: ER/CRecorder.Thetargettesttemperatureduringthestudywas 20: 1C.ThepHanddissoxoyglenvconetednt. `ofthe water weremeasuredineachtestchamberattestinitiation,atapproximately 24 hoursaftertestinitiation "andattheendofthe test.Hardness,alkalinity,specificconductanceandtotalorganiccarbon(TOC)were `measured in the dilutionwater attestinitiationandattest termination.
Measurementsof pH weremadeusing aFisherAccumetModel 915 pHmeter, anddissolvedoxygenwas
`measuredusing a YellowSprings InstrumentModel51Bdissolvedoxygenmeter.Specificconductancewas `meaussinugarYeelldow Springs InstruMmodeeln3t3Salinity-Conductivity-Temperaturemeter. Hardnessand alkalinitymeasurementsweremadebytitrationbasedonproceduresinStandardMethodsforthe Examination
ofWaterand Wastewat(e4)r.TotalorganiccarbonwasmeasuredusingaShimadzuModel 5000TOC analyzer.
Observations.
Observationsweremadetodeterminethenumbers of mortalities andimmobileorganisms.Thenumbersof individualsexhibiting clinicalsignsoftoxicity or abnormal behavioralsowereevaluated. Observationswere
`madeappr3,o24,x andi 48 hm oura saftt ertee stinl itiay tion.
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JWAILLoDLLiIcFeE IImNeTrEnRNAATTIIOONNAALLLLGo.. -12-
TPRROJOECOTNNO.: S45S4A-104
StatisticalAnalyses
`The 24 and 48-hourECS50 values andthe 95%confidenceintervals werecalculated whenpossiblebyprobit analysis, the moving average method or binomial probability with non-linear interpolation (5, 6, 7)using the computersoftwareof C.E. Stephan(8). Thenomortalict oncy ent/ rati ionmanmdNoOEbCwi erl edeitertminyed `byinspectionofthemortality,immobility andclinicalobservationdata. Inthisstudy,EC10andEC90 values werecalculatedwhenpossibleusingtheBruce-Versteeg Method(9)becausethere werelessthantwo
`concentrationswithpartial mortalityorimmobility(i.c.,theprobitmethodcouldnotbeused).
AnlyticalChemistry
Sampleswere collectedfrommid-depthofeachreplicatetestchamber atthebeginning ofthetest,at
`approximately24hoursandattestterminationtomeasureconcentrationsofthetestsubstance.Thesamples
`werecollectedinplastic(Nalgene)bottlesandanalyzedimmediately withoutstorage.Analyticalprocedures `usedinthe analysisofthesamplesareprovidedin AppendixIIL.
RES ANU DDIL SCUT SSIS ON
MeasoufTresteComnceentrantiotns
Resultsofanalysestomeasureconcentrations of PFOSinwatersamiplescollectedduring thetestare
presentedinTable 1andintheanalyticalchemistryreport (AppendixI). Nominalconcenstelrecatetdfiorousne:
inthisstudywere12,20, 33, 55and91 mga.i/L. Samplescollectedattestinitiation (0Hours)hadmeasured `valuesthatrangedfrom 85to112% ofnominal. Samplescollectedat 24hourshadmeasuredvaluesthatranged from92to 113%ofnominal,whereasmeasuredvaluesfor samplestaken at 48hoursrangedfrom92to 106%of nominal. Whenmeasured concentrations of thesamples collected at testinitiation, 24hours, and attest
terminationwereaveraged,themean measuredconcentrationsforthis studywere11,20,33,56and 91mga /L.
Meanmeasuredconcentrationswereusedintheestimationorcalculation ofEC10,EC50andEC90 values.
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DWE ILDLIFE INTERNATOINONAALL Lm.. 13-
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ObseravndaMteasiuroemnensts
Measurementsoftemperature,dissolvedoxygenand pHarepresentedin Table2. Temperaturesremained `withinthe 202 1 Crangeestablishedforthetest.Dissolvedoxygenconcentrationsremained 8.6mg/L(95%of saturation)throughoutthetest.Measurements of pHrangedfrom8.2to8.6duringthetest.
Dailyobservationsofmortality,immobility andotherclinicalsigns observedduringthetestareshownin `Table 3.Daphnidsinthenegative controlappearedhealthyandnormal throughoutthetest,although5%
`mowrasotbsearveldati48thouyrs.Daphnidsinthe 11and 20mga.i/Ltreatmentgroupsalsoappearednormal `withno mortalityimmobilityorovertclinicalsignsoftoxicity. After 48 hoursofexposure, mortality/immobility inthe 33, 56and91 mgai/L treatmentgroupswas0, 35and 100%,respectively. Althoughno
`mortality/immobility occurredinthe 33 mga.i/Ltreatmentgroup,onedaphnidwasobserved to be lethargic. Based on the 5% controlmortalityobserved,this lethargy wasnotconsideretod be treatment-related. EC10, EC50and EC90 values and 95%confidencelimitsat 24and48hourswereestimatedorcalculatedfromthe `mortalitdayta,/aindmarme sohobwniinlTiabtley4.
`CONCLUSIONS
The 48-hour ECS0 valueforDaphnia magnaexposed to PFOS was 61mga.i/L with 95%confidence
limitsof 33 and 91mga.i/L. Thenomortalicotnceyntr/atiionamndmNOoECbweire3l3migta.iy/L.
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REFERENCES
1 (UdSra.ft)E,aOviPrPoTnSmeNnutmablePrro8t5e0c.t10i1o0n.AgAegnucayt.ic i1n9v96e.rtebSreartieesAc8u5t0e -ToExciocliotgyTiecsatl,EFfrfeecsthswTaetsetrDGauipdhenliidness.
2
IOmrmgoabniilsiaztaitoinonfToerstaEcnodnRoempircoduCcotoipoenraTteisto.n
OaEnCdDDGeuviedleolpinmeenftor.Test1i98n4go. fChDeampihcanlisa.
sp. Acute Guideline
202. Paris.
3 MAaScTroMiSntvearntdebarradteEs7,2a9n-d88Aam.phi19b9i4a.nSst.anAdmearridcGaunidSoecfiocrtyCofonrduTecsttiinnggAacnudtMeTeorxiiacilt.yTestswithFishes,
4 A16PtHhAE,diAtiWonW,AA,merWiEcCaFn.Pub1l98i5c.HSetalatnhdaAsrsdoMceiatthioodn.sfoArmethreicEaxnamWiantaetrioWnoorfkWsatAesrsoacniadtWiaons.tewWataetre.r Pollution Control Federation, Nevw York.
5 Stephan, CE. 1977. "Methods for Calculating an LCS0," Aquatic Toxicology and Hazard Evaluations. American Society for Testing and Materials. Publication Number STP 634, pp 65-84.
6 Stephan, CE. 1978. U.S. EPA, Environmental Research Laboratory, Duluth, Minnesota. Personal communication.
7 Thompson, W.R. 1947. Bacteriological Reviews. Vol. Il, No.2. Pp. 115-145.
8 Finney, DJ. 1971. StatisticalMethods inBiological Assay. Second edition. Griffin Press, London.
9 EBnrvuicreo,nRmeDn.taalnTdoxDiJc.olVoegrystaenedgC.he1m99i2s.tyA, Vstoalt.isItLi,cpalpp1r4o8ce5d-u1r4e94f.ormodeling contimioustricitydata.
000699
WILDLIFE INTERNATIONAL Lm.
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Table 1
SummaryofAnalytical Chemistry Data
TI os Substance: PFOS
DTielsuttOiorngWaantesr:. CWlealdloWcaetrear,Daphniamagna
ToCmoncscaaitS rlTa)teoxn mlip ce l Tine g CoM gnoaeianm)tone MeComn(s mcgMenaetiraslt)ieondeTNeod moimfnal
Negative
B7
0
<<10Q
<
5A
u2
<p1r0]Q
B
Ws
63
2
AB
o0
11008 5
n
2
AB
22
1125s
AB
88
112009
2
AB
00
11871 2
2
100
BA
22
22186
AB
818
21813 4
.
5
BA
00
324012 3Tom
100
BA
22
33641 0
BA
u8s
331430
55
BA
00
95095
5
102
BA
22
85700
BA
88
Ss668 4
ol
BA
00
817026
0
100
BA
22
80414
5A
pr8i
982847
"The limitofquantitation (LOQ) we45s8 mg 8.1.
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Tabl2e
`Temperature, DissolvedOxygenand pHof Waterinthe TestChambers
TTeetSubstnee: MPFOeS me DTiovtiOomnmWiesemr: __CWlelolsVeer,Daphniamagna Cognantiani)on Rebs TCQ e ro kT; ST Y opo G 5 BSo 85 83 ssh 19 ss as
n
A5 Lvs4 a895 8833 8[TE ss m mses sass
A5 a4 889 8833 8ss8s wmo es81 wass
5
A5 aL4900 8833 88 88ss 2m1o 3s5s sess
6
A5 313 0911 8844 88s 88s 5:020 8877 w4ss
5
A5 BB3a3 s91 ss8s 88ss 885s 2210 88s7 ssss
VTi hourdivinwatermesmremets
CaCO, 315 uanhoslemand < | mgCIL,
froespeacrtiivenly,.Shah,poche
smshsanes
sodTOCwee
36mg
CaCO Togs
?Temperaturemeasuredcontinuouslyduringthetestrangedfrom approximately19.51020.5C.
> TA1h8d0eismssotlltvee1dosmxiCynogCoeOnncGo3nl1cue5ontnrmawtbaiotosenroimfmea5a.ns4dmrge</m1Lemtrsegpro,ersebenptaos6rc0idv%saslaaytl.uriattiyon,apt2c0iCicnofnrdeuschtwaatnecre.and TOCwer 140ml 1 CaCO,
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-- S Tork 2C 08 De p mp -- ,
RrCtesy tow RDieo T oo bee pee T Do bak pee mmibew har bhtae eae mfi s
T 3 6% 5 e bo um oo om
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u A PomRo80 owEey 0 00 wwwm ooLE o ww oo
mma oowm o wom opp mmo
Ba mo 0 mm o.o 0 wm ooo wm oo
" aPoowRm o80 0omwm A Bowmoo0 oooeumM
Si emtCy
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osHI o mu x 3 Bow oosoc m
83
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WILDLIFE INTERNATIONAL Lm.
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PROJECT NO: 454A-104
Tabled
EC10, EC50 and EC90 Values
`Sponsor: `Test Substance:
D`TielsuttOirongaWnaitsemr:: _
Time
24 Hours
3M Corporation
PFOS
CWlealdlocWeartaenr, Daphnia magna
EC10
CLoonfwiedre9n5ce%
(mg a.i/L)
2
Limits
81
CoUnpfpiedre9n5ce%
Limits
83
Statistical
Method
"Bruce-Versteeg
48 Hows T24imHeours
5
ECS0
(mg>oa.1i./L)
<u
Lower 95% Confidence
= Limits
>9
Upper 95% Confidence
LirmEi.ts.
BruceVersteeg
Statistical
VisuaMleItnshpoedction
48 Hours
-
Time
Aan
6
EC90
(mgaill)
>
3
"Tower 95%
Confidence ~~ .
Limits
=
9
Upper 95%
Confidence:
Limits
TT
Bisomisl
Statistical
Method
Visual Inspection
48 Hours
&
<u
>91 BruceVersiceg
Confidence limits could not be calculatedwiththe mortality/immobility dataobtained.
000703
AMENDED
WILDLIFE INTERNATIONAL Lm.
"194
Figure 1. Concentration-ReCsurpvoen(s4e8HourData)
PROJECT NO.: 454A-104
8
7
2 86
g35
ES4 5 5
=3
2
1 10
)
.
%
100
Mean Measured Concentration (mg a.i./L)
1000
000704
AMENDED
WILDLIFE INTERNATIONAL Lm.
20.
PROJECT NO.: 454A-104
APPENDIX
SpecificConductance, Hardness,Alkalinityand pH of Well WaterMeasured.
Duringthe 4-WeekPeriodImmediatelyPrecedingthe Test
Spoor
Women
`Test Substance: PFOS
Test Organism: ~~Cladoceran, Daphnia magna
Dilution Water: Well Water
x
Mean
~
Range
SpecificConductance
(mmhos/cm)
3IN=4)
310-315
Hardness
(mg/L as CaC0y)
132 (N=4)
128-136
Alkalinity
(mg/L as CaC0y)
178 (N=4)
176-178
pH
BIN=4)
82-83
000705
WILDLIFE INTERNATIONAL Lr.
"21.
PROJECTNO.: 454A-104
APPENDIX AnaliofnyPWesistilcedideesIs, nOrgtaneicms, aMLeittda.liWseaolnldnWOatatheelrrInorganics
ANALYSIS
MEASURED CONCENTRATION
`Miscellangoouls BMeaesurermenets ToAulCrnimeCeon'
. oa
me
:< i1r6]0
wwrr
EEEe OroABupehnaapeHsCnccdPiCgBs
:<i 0008006008s8
wira
SCiiomrnBeic Lindne)
:: 800638
4nt
DBioRdEd?E Endosun, A
:<< 085050080888 < 5008
a=irr ar
EEnxdtoonniinSBte
:: Gaors
ia
HEenpinseree
:< d0o0ss
l"or
HPoEe RiGrBria beim
Ls:< a005300%
rur4
: 03%
wr
BPoeBEian
:< 003%2
awrs
RFeEs E
< 8u%m
FFaa
MetalsandOther gorganics
Lo
Anpeer
E<E #8 =74
Cachan? |
358
gl.
Sam
<
io
=r
fV= oismions
<<: ii1309
naFrge
oe Nei
<< :
03300 a
ar Fe
Ha
<
3
"r
fl
< Bio
pid
:: pecfomebdyOShTEEnfvaimsaet,Gspesil,Fmlprl focosalpliieancn eNcoveeman ov3, e1b99e7r boughNovem 7, 197
000706
WILDLIFE INTERNATIONAL Lm.
2.
APPENDIX TI
PROJECTNO.: 454A-104
`THE ANALYSIS OF PFOS IN FRESHWATER IN SUPPORT OF
`WILDLIFE INTERNATIONAL LTD. PROJECT NO.: 454A-104
000707
WILDLIFE INTERNATIONAL Lo.
3
PROJECT NO.: 454A-104
REPORT APPROVAL
SPONSOR: 3M Corporation
TITLE:
PFOS: A 48-Hour Static Acute Toxicity Test with the Cladoceran
(Daphnia magna)
WILDLIFE INTERNATIONAL LTD. PROJECT NO.: 454A-104
PRINCIPAL INVESTIGATOR:
Scientist
1
-
. Van Hoven, Ph.D.
MANAGEMENT:
Jill in
`Willard B. Nixon, Ph.D:
Manager, Analytical Chemistry
:
425-00
DATE
Jon.
DA
000708
AMENDED
i WIELDO< LIFEE-- ENITNETo RERNNAe ATTIIOOe NNAALLe LLIrDo,. eeeeee TPRROe JOECLTNNe OO.;:d4Se 540A0-1A0n 4 24
Introduction
Freshwater sampleswerecollectedfrom astaticacuteaquatictoxicitystudydesignedtodeterminethe effectsof PFOS(PerfluorooctaneSulforicAcid Potassium Sal)tothecladoceran (Daphniamagna). This studywasconductedby WildifeInternational Ltd.andideatifiedasProject No.: 454A-104. Theanalyses ofthesewatersampleswereperformedat WildlifeIntemationalLtd. usinghighperformalinqcueid `chromatographywithmassspectrometricdetection(HPLC/MS). Sampleswerereceivedforanalysison February16,17and18, 1999andwereanalyzedoneachsamplereceiptday.
TeSstubsantd Ina ternn alStcande ard
Thetestsubstanceusedfortheanalyticalportionofthisstudywas WildlifeInternationalLid. identificationnumber 4675. Thetestsubstancewasusedtopreparecalibrationand matrix fortification
samples.
`Theintemalstandardwasreceivedfrom 3MCorporationon July2,1998andwasassigned Wildlife International Lid. identification number 4526 upon receipt.Theintemal standard, agranular material, was
identified as: 1H, 1H, 2H, 2H Perfluorooctane Sulfonic Acid, ChemicalAbstractNumber: 27619-97-2. `Thestandardwasstoredunderambientconditions.
"
}
`The method used fortheanalysis ofthe freshwater sampleswasdevelopedatWildlifeInternational Ltd.andcatitled "AnalyticalMethodfortheDeterminationof PFOS inFreshSalwtwaatert,aendrAlg,al Mediu".ThismethwaosidncloudeldasoAppgendyix IofWildiifeInternationalLtd.protocolnumber 4541011299/MVALISUBAS4, Itwasbased uponmethodologyprovided by 3M Corporation.
Sampleswere diluted in a 50% methanol : 50% NANOpurewater solution containing 0.mg14H0PF0OS (internal standard) and 0.05% formic acid (v/v) so that they fell within the calibration rangeofthe PFOSmethodology.
Concentrationsofthe PFOSinthestandardsandsamplesweredeterminedby reverse-phasehigh performanceliquidchromatographyusing aHewlett-PackardModel 1100HighPerformanceLiquid Chromatograph (HPLC) with aPerkin-Elmer API 100LC Mass SpectequrippoedmwitehatPeerkirn-
000709
WILLDLIEFENINETERRNNAATTIIOONNALtLrme.. 2s.
PRPROOJJCECTTNNOO.::d4s54tAA-1l044
Elmer TurbolonioSnpsoruarcye. HPLCseparationswereachievedusing a KeystoneBetasil Cy `analyticalcolumn (100mm x 2mm ID, 3pmparticlesize). Theinstrumentparametersaresummarized inTabl1e. AmethodflowchartisprovidedinFigure1.
c o tat ia
Calibration standardsof PFOS prepared in a 50% methanol: 50% NANOpure water solution
containing0.100mg4H PFO (intemalstandard)/L and 0.05% formic acid (v/v), rangingin concentration
from 0.00915 to 0.0457mga.i/Lwereanalyzedwiththesamples. Linear regression equations were generatedusingpeakarearesponseratios(PFOS :internal standard)versusthe respectiveconcentrations of thecalibrationstandards.AtypicalcalibrationcurveispresentedinFigure 2. Theconcentratioofn
PFOSinthesampleswas determinedby substitutingthe peak area responseratiosintotheapplicablelinear
regressionequation. Representativeionchromatogramsoflowandhighcalibrationstandardsarepresented inFig 3anu d 4, r respee ctivs ely.
"Themethod limitof quantitation(LOQ) for these analyseswas setat 4.58 mg a.i/L calculated as the
product of thelowestcalibration standardanalyzed (0.00915 mg 2.i/L)andthe dilutionfactorofthe
matrixblanksamples (500).
.
`MFatorixrBltankianfd icatiSamoplnes
`Threematrixblanksampleswereanalyzedtodeterminepossibleinterference. No interferenceswere observedatorabovetheLOQduring samplesanalyses(Table2). Arepresentativechromatogram of a `matrixblankispresiennFitguree5d.
Freshwaterwasfortified at9.15, 45.7and91.5 mg 2.i./Landanalyzedconcurrentlywiththesamples todeterminethemean procedural recovery(Table3). Sampleconcewnertenrota cortreciteo dfonrts he
inFigur6e. `meanprocre ecodveruyor f9a 6.2l%. Arepresentativechromatogramof amatrix fortificationispresented
000710
AMENDED
WILODLLIFEE IINNTTEERRNNAATTIIOONNAALL Lo.
ee -26-
TPRROOTJIECCYTRNOO.:: A45S4AA-104
`ExampleCalculations
`Sample number 454A-104-27, nominal concentration of 12 mga. in freshwater.
Initial Volume: 0.100 mL
Final Volume: 50.0 mL
Dilution Factor: 500
PFOSPeak Area: 302788
Internal Standard Peak Area: 585991
Peak Area Ratio: 0.5167
+
Calibration curve equation.
Slope: 0.02117 Intercept: 0.05525 Curve is linear,
: :
PROS(ugai/l)atinstmment
=+Pexakaorrecaa ratioreo =-(Vi(Vn4 terocp)
Slope. 3
05167-00525 002117
= us
PFOS (ngai/L)insample
=
--PFOS(4g
2.1L)
at
instrument 1000
x
Dilution
Factor
_ 218x500 Too
= 109
000711
AMENDED
WILDLIFE INTERNATIONAL Lm.
7.
PROJECTNO. 454A-104
PercentofNominal Concentration. = PrFoOsSWatiL) )mir sample PFOS(mg 2.i./L)nominal
Calculatedrecovery: 91.6% Note: manualcalculmaatydiifofenr.
~
RESULTS
`SampleAnalysis
Freshwatersamples werecollectedfrom theacutetoxicity studywiththecladoceran(Daphniamagna)
at test initiation, February 16, 1999 (Hour 0), on February 17, 1999 (Hour 24), and at test termination,
February 18, 1999(Hour48). Themeasuredconcentrationsof PFOS in thesamplescollectedatinitiation
ofexposureofthetestorganisms (Hour 0)rangedfrom 85.5 to 112%ofthe nominal concentrations. `SamplescollectedatHour 24had ameasured concentrationrange of92.2to 115%ofnominal values.
Samples collectedattest termination (Hour 48)had ameasured concentration rangeof91.6 to 106% of
`nominalvalues (Table4). Arepresentativechromatogram ofatestsampleisshowninFigure 7.
000712
AMENDED
WILDLIFE INTERNATIONNAAL Lto. 28
eeeTPRROOJTECCTTNNOO.:: S4554AA-I10044
Table 1
Typical LC/MS Operational Parameters e INSTRUMENT.eer ee "Hewlee tt-Pat ckard Mr odel 1100 High Performar nce Liquid
eCquihpperd woithmawaPietrthkainoP-eErlgkmienrr-TEalumreprbAhoPIl1o0n0iLoSCnMpsaorsurascSyep,ecOtpreomreatteerind selective ion monitoring mode (STM).
ANALYTICAL COLUMN: Keystone BetCayscoliumln (100mm x 2 mm LD.,3 um particle size)
OVEN TEMPERATURE: 30C
:
STOP TIME:
100 minutes
FLOW RATE:
0.220 mUminute
MOBILE PHASE:
70.21.90%%FMoertmhiacnAoclid: 28.0% NANOpure Water co:ntaining
INJECTION VOLUME: 500
:
PFOS RETENTION TIME: Approximately 7.1 minutes
INTERNAL STANDARD RETENTION TIME:
Approximately 4.9 minutes
PFOS MONITORED MASS: IMNOTNEIRTNOARLESDTMAANSDS:ARD
498.6 amu 4267 amu
000713
BWIE LDLIFE INTERNNAATTIIOONNAALLbLom., 29.
PPRROOJJEECCTTNNOO.::44s5e4AA-l1o0s4
Table2
MatrixBlanksAnalyzed ConcurrentlyDuring SampleAnalysis
(`3N5u4mAb-1e0r4) MAB-1
MAB2
Sample
Type "Matrix Blank
: MatrixBlank
MeasuredConcentration of
(mPgFaOiSl'l) <10Q
<L0Q
MAB-3
Matrix Blank
<LOQ
`Thelimitofquantitation (LOQ)was4.58mga.i/Lbaseduponthe productofthe lowestcalibration standard analyzed (0.0m0g9a.1i/5L)andthedilutionfactorofthematrixblank samples (500)
000714
AMENDED
WILDLIFE INTERNATIONAL Lo. 30-
PROJECT NO.: 454A-104
Table 3
Matrix Fortifications AnalyzedConcurreatlyDuring Sample Analysis
Sample Number
(US4A-104)
MAST MAS4 MAST
"Concentrationsof PFOS (mgai/L)
Fortified
915 "es
915
Measured.
542 07! 9.15
-- Percent
Recovered
920 106 100
MAS2
457
403
88.1
MASS
457
481
105
MASS
457
451
98.7
MAS3
91s
787
860
MAS6
915
840
918
MASS
915
897
980
Mean = 962
Standard Deviation =7.11
+
ov =739
N=
"Note: Results andcorrectionsfornewtestsubstancepurityweregeneratedusingMacQuanversion 15
softwareandmanualcalculations. Valueshavebeenroundedfo reportingpurposes.
* Mean resultofduplicate injectoifotnhseredilutedoriginal sample. Original result (5.48 mg a../L) was
notincludedinth statisticalanalysisofthedata.
:
Duplicate results for 454A-104-MAS-4 are 9.24 and 10.2.
000715
AMENDED
WILDLIFE INTERNATIONAL Lo. 31.
PROJECT NO.: 454A-104
Table 4
Measured Concentrationsof PFOS in Freshwater Samples from a Cladoceran Static Acute Toxicity Test
`Nominal Test Concentration (mgai/l)
00
Sample Number (454A-104)
| 2 113 25 2%
Sampling Time (Hours)
0 0 22 4 a
PFOS Measured Concentration'
(mgaisl) <L0Q <L0Q <<LL00QQ <L0Q <L0Q
Percent of
Nominal = --
2
3
0
105
885
4
0
106
895
15
2%
16
2
112L5s
913058
27
3
109
916
.
2
4
120
101
2
5
0
6
0
17
2
18
2%
29
8
30
ko
m2
855
181
902
28
13
216
107
214
106
188
9.6
3
7
0
302
91.7
8
0
341
104
19
2
340
103
20
2
36.1
110
31
8
313
95.1
32
48
340
103
"Note:Results andcorrectionsfornewtest substancepuritywere generated usingMacQuanversion 1.5
softwareandmanualcalculations. Values have beenroundedforreportingpurposes.
"The limitofquantitation (LOQ) was 4.58 mg .iL baseduponthe prodoufcthte lowest calibration
standard analyzed (0.00915 mg a../L) and the dilution factorofthe matrix blank samples (500).
Mean resultofduplicate injectionsofth rediluted original sample. Originalresult (113 mg a.i/L) was
not included in the statistical analysisofthe data.
Duplicate results for 454A-104-23 are 90.6 and 89.6.
AMENDED
000716
+ WILDLIFE INTERNATIONAL Lm. -n-
PROJECTNO.: 45A-104
`Table 4 (Continued)
Measured Concentrationsof PFOS in Freshwater Samples from a Cladoceran Static Acute Toxicity Test
Nominal Test Concentration
(mg a.i/L) 55
Sample Number (454A-104) NY]
10 21 2 3 3
Sampling Time
(Hours) 0 0 2 2 a8 48
PROS Measured, Concentration' (mgai/l)
505 499 570 60 568 564
Perocfent Nominal 21 09
104 ns 104 103
9
n
0
876
95.7
2
0
102
12
2
2
90.1?
9%5
2%
2
844
922
3s
48
87
969
36
48 . 924
101
"Note: Results and corrections for new test substance purity were generated using MacQuan version 1.5
software and manual calculations. Values have been rounded fo reporting pirposes
"Tshtaenldiamridtaonfalqyzeud a(0n.00t91(i5LtmOgQa)awt.a.isL)4o.a5nnd8mthge ad.iliu/tLiobnafsacetdoruopfotnhtehmeaptrtoidxubcltaonfktshaemplloewses(t50c0a)l.ibration
Mean result of duplicate injectionsofthe rediluted original sample. Originalresult (113 mg2./L)was
not included in th statistical analysisofthe data.
Duplicate results for 454A-104-23 are 90.6 and 89.6.
000717
AMENDED
WILDLIFE INTERNATIONAL Lm.
33.
PROJECTNO.: 454A-104
METHOD OUTLINE FOR THE ANALYSIS OF PFOS IN FRESHWATER
`Preparematrix fortificationsamplesbyspikingtherequisitevolumeof PFOSstocksolutionsdirectly intofreshwaterusinggas-tightsyringesand Class Avolumetricflasks.
~
l
Dilutematrixfortificationandtestsamplesintotherangeofthecalibrationstandardsbypartiallyfilling
Class Avolumetricflaskswith 50%methanol: 50% NANOpurewatersolutioncontaining
0.100 mg 4H PFOS (internal standarda)n/dL0.05%formicacid (v/v).Addtheappropvroiluametoef `sampleandbringtheflasktovolumewiththedilutionsolvent.Processthematrix blanksampleusing. thesamedilutionandaliquotvolumeasforthelowestfortification level. Mix wellbyseveralrepeat
inversions.
:
.
Ampulatesamplesandsubmitfor LC/MSanalysis.
Figure1. Analyticalmethodflowchartfortheanalysisof PFOS infreshwater. .
000718
WILDLIFE INTERNATIONAL Lm.
3.
PROJECT NO. 454A-104
120
100
om
060
'
040
:
00]
.}
000 0000 0010 000 0030 0040 0050
Concentration (mg a.i/L)
Figur2e. Apia allbcurreafoirPoFOnS. Slop= 0.02117; org= 005525, 1 =086 AMENDED
WILDLIrFEE INTRERNNAATOIONNAAL Lo.esses 37.
OPROCJETCTNNO0.2: 445548A0-010048
-
100 90 80 70 60 50 40 0
a
4 ntonsty: 1772cps 189
.
. -
.
-
EA TiU.
AACA GL 01 ME IL
a, A8TC
2002B1 R|
Sam
25 3m 56 6% 7 8m Tm
Figur5e. Areprescatative chromatogramof amatrixblank sample (454A-104-MAB-3).
"Thearrowindicatestheretentiontimeof PFOS.
000720
WILDLIFE INTERNATIONAL Lm.
-40-
PROJECTNO: 454A-104
APPENDIIXV ChatnoPgroteocsol
`Thisstudywasconductedin accordancewiththeapprovedProtocolwiththefollowingchanges:
.
1. Theprotocol wasamended toaddtheproposed experimentalstartandterminationdates,test
`conceanndtetstrsuabsttanicenoumnbesr. 2. Theprotocolwasamendedtodeleteannualfeed analyses. 3. Theprotocolwasamendedtoaddtheanalyticalmethod.
4. Theprotocolwas amendedtochangethestatisticalanalyses.
000721
WILDLIFE INTERNATIONAL Lr.
a2.
PROJECT NO.: 454A-104
APPENDIVXI ReportAmendment
1. OriginalReport: Pa1-g4,6eands23 Amendment: TnheewpSaiggneastwuererseacnhdadnagteedstdouientcloutdheetehdedaitmoenndoefdhreerpeoprtodrattaem,erenvdimseendptaagseAnpupmebnedrsi,xaVnLd. Reason: Toreflecttheissuingofanamendedreport.
2. Original Report: Poge2
Amendment: Thecompliancestatementwasrevised
Reason:
Toclarifyhowthetest substancewascharacterized.
3. OriginalReport: Paged Amendment: iInncflourdmiantgitohneprreoavniadleysdbisydtatheeaSnpdotnhseoprurreiftlyec,twiansgtahdedreedatnoaltyhseTiseosftStuhbesetsatnscuebssetcatinocne., Reason: "Toreflectthecurrenttestsubstanceinformationprovidedbythe Sponsor.
4. OriginalReport: Entire report
i
Amendment: AaTlseldsetttecesotrnsmcuiebnnsettrdaabntycieotcnhosneocrSeipngotirnnasatolirloyinnwsaweerrereaebncaahlsayensdgioseondtftothhreeerfeelspctotsrtuthbeespdutprauinrticyteoy(fFohCfe-9t95e8,t.Ls9ou%bt.s2t1aT7nh)ce.e
`fcceosntceensturbanstoitfoananisnc,aealnyfsdcioEsndCccaevntatelradautMeiseo,trnwcse,hr9e,rien2cc0al0ul0dciiunlngatdendioamncidnaarapetluproeirtcdtyoeondcfaesn9tm0r.ga4t9ii%on.Ls,Tbhaesmeredaeosfnuotrarehl,del
90.49%purity.
Reason:
Toreporttheresultsofthetestbasedon thetestsubstance purity of90.49%atthe reqoftuheeSposnsotr.
000722
AMENDED
WILDLIFE INTERNATIONAL Lo.
5.
PROJECT NO.: 454A-104
APPENDIVXI
Report Amendment
AMENI
Kan Droit
v
A
Director, Aquatic Toxicology and Non-Target Plants.
REVIEWED BY:
Sopp Coen H. Coleman `Quality Assurance
p/p /00
DATE
oxyou fee
_ueo0 DATE
000723
AMENDED
WILDLIFE INTERNATIONAL LTD.
PROJECT NO.: 454A-104
Page 1 of2
AMENDMENT TO STUDY PROTOCOL
STUDY TITLE: PFOS: A 48-HOUR STATIC ACUTE TOXICITY TEST WITH THE
i
CLADOCERAN (Daphnia magna)
a
PROTOCOL NO.: 454/120198/DAP-48H1/SUBA54
AMENDMENT NO.: 1
SPONSOR: 3M Corporation
PROJECT NO: 454A-104
i
EFFECTIVE DATE:December21, 1998
a
AMENDMENT: Page
Add: Experimental Start Date: 2/16/99
Experimental Termination Date: 2/18/99
id
Test Concentrations: Negative Control, 13, 22, 36, 60 and 100 mg a.i/L.
Test Substance No.: 4675
H
REASON: The above information was not known when the protocol was signed by the Study
Director.
AMENDMENT:TestOrganism, Page 5
.
ol
Delete:
Feed (YCT) provided annually to ensure that
to daphrids there are no
in the cultures wil be contaminants at levels
analyzed known to
at be
least once capable of
H
interfering with the study.
REASON: Historical analyses of Wildlife International Ltd. aquatic feed have shown that no
H
contaminants are present at levels known to be capableof interfering with the study.
uo
AMENDMENT: APPENDIX II, Page 14
H
Add: PLeirqfuliudorCohocrtoamnaetSouglrfaopnihcyAcMiads,sPoStpaescstiruommeStalrty((PLFCOMS)S)inMFeretshhowdateforr, FtihleteDreedteSramlitnwaattieornanodf
Algal Medium
oH
REASON: To add the analytical method to be used in the study.
u
000724
Hi o.
DtAiisneedd
I
B WILDLIFE INTERNATIONAL LTD.
I
' Aird
i
Los (0. IZ oA
I
SPONSOR'S REPRESENTATIVE
|
i
5
i
i
|
KE
I
I
Po
Paros fseton
2/1 [99
DATE .
. 000725
WILDLIFE INTERNATIONAL LTD.
PROJECT NO.: 454A-104 Page 1 of1
AMENDMENT TO STUDY PROTOCOL
STUDY TITLE: PCFLOAS:DOCAER4A8N-H(ODUaRphnSiTaAmTaIgCnaA)CUTE TOXICITY TEST WITH THE
i
PROTOCOL NO.: 454/120198/DAP-48H1/SUB454
AMENDMENT NO.: 2
`SPONSOR: 3M Corporation
PROJECT NO.: 454A-104
EFFECTIVE DATE: March 9, 1999
AMENDMENT: DataAnalysis, Page 8
Change:
`When the dose-response pattern allows calculation ofEC10, EC50 and EC90 values,
the data will be analyzed using the computer software of CE. Stephan (6).
- To: When thedose-responsepattern allows calculation of EC10, EC50 and EC90 values, the data
will be analyzed using the computer softwareof C.E. Stephan (6), or another appropriate `methodology.
REASON: The computer software of C.E. Stephan does not calculate EC10 or EC90 values.
ol \
STUDY DIRECTOR
JOARATORY MANA(
a (Oh.
SPONSOR'S REPRESENTATIVE
2/nl1
DATE
2 J/g
DATE
4/1)494
DATE
045106amend?
000726
hewn Shofor sd
a
PROTOCOL
a
PFOS: A 48-HOUR STATIC ACUTE TOXICITY TEST
a
"WITH THE CLADOCERAN (Daphnia magna)
USS. Environmental Protection Agency
n
Series 850- Ecological Effects Test Guidelines
(OPPTS Number 850.1010
Ho
and
`OECD Guideline 202
H
3M Lab Request No. U2723
HH
Submitted to
|
3M Corporation
i
Environmental Laboratory
.
St PauPl.,O.MBinonxes3o3t3a315133
i
WILDLIFE INTERNATIONAL LTD.
[]
590 ComersDive
Easton, Maryland 21601
ol
(410) 822-8600
=
000727
o
December 1, 1998
ot
PROTOCOL NO.: 454/120198/DAP-48H1/SUB454
3M LABREQUEST NO. U2723
n WILDLIFE INTERNATIONAL LTD.
a
H H Ho
|i]
uo Ho ||
1] |]
|]
>
PFOS`:WIATH48T-HHEOCULRASDTOATCIECRAACNU(TDEapThOniXaImCaIgTnYa)TEST
`SPONSOR:
En3viMrCoonrmpeonrtaatiloLnaboratory PSt..0.PaBuol,xMi3n3n3e31sota 55133
SPONSORSREPRESENTATIVE: Ms. Susan A.Beach
TESTINGFACILITY:
W8i5l9d8ifCeoImnmteemracteioDnrailveLid.
Easton,Maryland 21601
STUDYDIRECTOR:
KSeunritoDrrAoqtuaartic Biologist
LABORATORYMANAGEMENT: HDiernercytOor.KofruAeqgueart,icPThoDx.icology & Non.Target Plants
FOR LABORATORY USE ONLY
ProposedDates:
:
||ExSperwimeDntaul e________ `ETxpeemrismeinotanlue
|
| ProjectNo. _4SA = 10
| TestConceatrations:
I`Test Substance No.
Reference SubstanNcoe. (f applicable):
Wed |]
PROTOCOLAPPROVAL
STUDY DIRECTOR
pres
DATE
my nN
ml
TO! AGEMENT SPONSOR'S REPRESENTATIVE }
DAT2E Je,[1p 2 [7/4 2
DATE
000728
ml
PROTOCONOL: 454/120198/DAP-48HI/SUBASS
3MLABREQUESTNO, U2723
it]
WILDLIFE INTERNATIONAL LTD.
i|
INTRODUCTION
uo
`WideIntemational Ld.willcondaustcatticacute toxicity testwiththecladoceDarpahnni,a
ol
`maga,fortheSponsorattheWildife IntemationalLid. aquatictoricology faciinlEaisttony, Maryland.
"ThestudywillbeperformedbasedonproceduresintheU.S. Environments]ProtectionAgency Series
850 Ecological Effects Test Guidelines OPPTS Number 850.1010 (1) in OECD Guideline for Testing
mn
of Chemicals, 202: Daphnia sp. Acute Immobilization Test andReproduction Test (2) and ASTM.
Standard E-729-885,StandardGuidefor Conducting Acute Tosicity Tests with Fishes, Macroinver-
i]
tebanrdAmaphitbiaens(s3).Rawdataforallworkperformedat WildlifeInternational Ltd.anacodpy
ofthe final report will be filed by project numberin archives located on the Wilde International Lid.
nl
site oratanalternative location obespecifiedinthefinal report.
nl
OBJECTIVES
"Theobjective ofthisstudyis todeterminetheacuteeffectsofatstsubstanceonthe cladoceran,
om
`Daphniamagna,undec tat testconditionsfor aperiodof48 hours.
m
EXPERIMENTALDESIGN
Daphnidswillbeexposedtoageometricseriesofat leastfivetestconcentrationsandanegative
o
(lutionwater)controlfo 48hours. Tworeplicatetestchamberswillbemaintainedincachtreatment
andcontrolgroup,with 10neonatedaphnidsineachchambersothata toalof 20neonatedaphnidsaro
[|
exposedin eachtreatmentandcontrolgroup.
ol
Nominaltestconcentrationsvillbeselectedinconsultationwiththe Sponsor,andvillbebased
`uponinformation suchastheresultsofexploratory range-finding toricitydata,knowntoxiitydata,
ol
`physical/chemical propertiesofthe test subsorotthaernrelcevaent information.Targetconcentrations
neednotexceed 1000mg/Lorthesolubility limit ofthetestsubstanceinwater(whicheverislower)
|}
`Generally, eachconcentration oftestsubstanceusedinthedefinitivetestwilbeatleast 60%ofthenext
highertreatment,unlessinformationconcerning theconcentration-effectcurveindicatesthat adifferent
ol
dilution factorwouldbemoreappropriate. Watersamplesfromeachtestchamberwillbecollectedat
=
specifiedintervalsforanalysisofthetestsubstance. Resultsoftheanalyseswillbe sedtocalculate
mo
meanmeasuredtestconcentrations
000729
wm
PROTOCOL NO: 454/120198/DAP-48H1/SUB4S4
3MLABREQUEST NO. U2723
WILDLIFE INTERNATIONAL LTD.
-4-
|
To control bias, neonate daphnids will be impartially assignedtoexposure chambers at test
ntiaion. Nootherpotential soursofbiasareexpectedtoaffecttheresultsofthe study. EC10, ECS0
and EC90 values will be calculated, when possible, based on the number of dead or immobilized
I
aphnidsobservedineachtestconcentration afteeach24-hourinterval ofexposure.Theno-mortality
|
concentrationandtheno-observed-cffect concentration (NOEC) willbedetermined.
MATEARNDIMAETLHODSS
I
TestSubstance
Information on the characterizationoftest, control or reference substances i required by Good
i LLaibdo.rwartiottreynPvrearcitfiicceatSiotnantdhaartdset(eGsLtPs)u.bsTtahneceShapsbeoeinscnrheassrpaocnotseirbrilzeedfaocrcporrodviindgitngoWGilLdPlsifperiIonrtteroniattsiuonsael
i Innttheematstiuodnya.l LItfwdr,iittewnilvlebreinfiocattieoninofthGeLcoPmptelsitasnucbessttaantceemcehnatraocftetrhizaftiinoanl irsenpoortt.prTohveiadttetaodchWeidldfloirfme
1
IDENTIFICATION OF TESTSUBSTANCE BY SPONSOR (Appendix Iistobe ustoperovdide
i
information necessary for GLP compliance.
-
"The Sponsorisresponsibleforal information elatedtothe estsubstanceandagreest0accept
i
anyunusedtestsubstanceandortestsubstancecontainersremainingatthe ndofthestd.
Drspac
..
1
d
"Thetestsubstancewillbeadministeredtothetestorganism directlyintodilution water. This
routeofadministratwiaosnselected bocause irepresent themostlikelyrouteofexposuretoaquatic
organisms.
i
`TestOrganism
d
Thecladoceran,Daphniamagna,hasbeen selectedasthetestspeciesforthisstudy. Daphnids
=
arerepresentativeofanimportantgroupofaquaticinvertebrates, and havebeenselectedfor seinthe
i
testbaseduponpastuse historyandeaseofculturinginthe laboratory. Daphnidneonatestobeused
}
inth testwillbeles than24 hoursoldandwillbeobtainedfromculturesmaintained atWildlife
do
InterLind.,aEatstoin,oManrylaanld. Theidentityofthespecieswillbe verifiedbythesupplierofthe
000730
o
PROTOCOL NO.: 454/120198/DAP-48H1/SUB454
3M LAB REQUEST NO. U2723
d WILDLIFE INTERNATIONAL LTD.
d
=
d
original cultureorbyWildlife International Ltd. personnel using appropriate taxonomic keys such as
Pennak (4).
d
Daphnidswillbeculturedinwaterfromthe samesourceandat approximately thesame.
d
tempas iel rbe saeddturiungtrhe teest. Daphnidsintheculturesproducingneonatesforthetestwill
beheld foratleas 10daysprior ocollection oftheneonatesfortesting. Adultdaphinntheicudltusre
willproduceanaverageofatleast 3youngperadultperdayoverthe 7dayperiodpriortothetest.
}]
`Neonatesfrom daphnidsthatshowsignsof diseaseorstresswillnotbeusedastestorganisms.
u
Daphihidsinholdingthatproduceephippiaalsowill notbeusedtosupplyneonatesfo testing.
`Daphridsintheculturesvil befed oncedaily. Thediet il be amixtureofyeast,Cerophyll,
H
and trout chow (YCT), supplemented with a suspension of the freshwater green alga Selenastrum
capricornutum. Adultarfe duringthe 24-hour periodpriortotet initiation, but neonates will ot
ou
befeddurtihentesgt.Feed(YCT)providedtodaphnidsinthecultureswillbeanalyzedat leastonce
d
`iann,uallSytpo eonsourenthaot ttherpearetno colntaominanctshat ilevelskndowndtobdecapabvlesofintm erfering h withthe
However,therearenoknownlevelsofcontaminantsreasonablyexpectedtobepresentinth dietthat
ol
areconsidered tointerferewiththepurposeorconductofthetest.
ad
`Neonateswillbeobtainedfortestingfromatleastthreeindividual adult. Priortotestinitiation,
theneonates will be collectedfromculturesandtransfered to smallcontainers.Thedaphviildlbse
a
released ntoth testcompartmentsbelowthe watersurfaceusing awide-borepipete.
|]
DilutionWater
`Thewaterusedforculturingandtestingvillbeobtainedfrom awellapproximately 40 meters
dH
decplocatedontheWildlifeInternatLitod.nsaitle. Thewaterwill be passedthrough a sandfilterand.
pumpedinto a37,800-Lstoragetankwherethewatervill beaeratedwithspraynozzles. Pritoousre
d
thewater illbefilteredto 0.45saminordertoremovefineparticles. Waterusedforculturaingd
5
testing is characterizedasmoderately bard. Typical valuesfor hardness, alkalinity, pH and specific
o
condductuancce tanceaarree approximately::
000731
ol
PROTOCOLNO.: 454/120198/DAP-48H1/SUB4S4
3MLABREQUESTNO. U2723
m
.
WILDLIFE INTERNATIONAL LTD.
om
"
|
AHlkaarldinneistsy,,mmgg//LLaassCCaaCCOO,,
145 19
pH
81
o
SpecificConductance, mhoslcm 330
Hardness, alkalinity, pH and specific conductance will be measured weekly to monitor the
m
consiofsthteweelnlwcateyr. Meansandrangesofthemeasuredparametersforthefour-week period
precedingthetestwillbeprovidedinthefinal report.Analyseswillbeperformedatleastonceannually
om
todeterminetheconcentration ofslected organicand inorganicconstituentsinthe waterandresults
oftheanalyseswillbe summarizedinthefinalreport.
TestApparatus
Test chambers will coonfpsolyiethsyletns battles. The size of bottle andthevolumeof test
solution wil be sufficient to meet the requirementsofsampling for analytical verification of test
`Substanceconcentration androutine waterchemistryanalyses. Testchambweilrbse indiscriminately
|
positionedin atemperaturecontrolledwaterbathoran environmental chamber. Testchambveilrlbse
Tabelled withthe project number, test concentration an replicate.
&
Cea
|
Lightingusedtoilluminatethecultures andtestchambersduring culturingandtesting willbe:
providedby fluortuebesstchaetnemtitwavelengths simtionlataurral sunlight (.,Colortone 50).
=
Aphotoperiod of 16hoursof lightand 8hoursofdarknesswillbecontrolledwithanautomatictimer.
`A 30-minutetransitionperiodoflow lightintensitywillbeprovidedwhealightsgoonand offtoavoid
=
sudden changes in light intensity. Light intensity will be measured at test initiation and at test
termination with a SPER Scientific Lid.lightmeo tequievalernt.
`Thetargettettempewrillabet20u:+r1eC. Temperaturewillbemasuredineachtestchamber
atthebeginningandendofthstestusing ahand-heldthermometer. Tempaelsrowiallbtemuearsureed
withacontinuousrecorderinonenegativecontrolchamber.Recordermeasurweilmlbeevneritfised
=
with 8hand-heldthermometerpriortotestinitiation.
000732
PROTOCOL NO;: 454/120198/DAP-48H1/SUB4S4
3MLABREQUESTNO, U2723
fi
i WILDLIFE INTERNATIONAL LTD.
i
-7-
i
Dissolvedoxygenvillbemeasuredinallreplicates ofthe treatmentandcontrolgroupsattest
initiation and at approximately 24-hour intervalthercaferusing a YellowSpringsInstrumentModel
i
S1B dissolvedoxygenmete,orcquivalent. Intheeventtha dissolvedoxygen levels fallbelow60%
saturation,appropriateactionswill betakenafterconsuliationwiththeSponsor. MeasuremeonftpsH.
o
willbe madeinallreplicatesofthetreatmentandcontrolgroupsa testinitiationandatapproximately
24-hourintervalsthereaferusing aFisherAccumetModel915pHmeer,orequivalent.Ifatreatment
)
`groupreaches100%mortality, dissolvedoxygen,pH,andtemperaturemeasurementswillbetakenat
i
thenextsamplinginterval,thendiscontinued.
i
Hardness, pH,alkalinity,speificconductance and otalorganiccarbon(TOC)willbemeasured
inthodilutionwateatthebeginningand endofthetest.Hardnessandalkalinitymeasuremweinlltbse
i
made by titration using procedures baonsmeethoddsinStandard Methodsfor the Examination of
Water and Wastewat(e5r).Specificconductancewillbemeasuredusing aYellow SpringsInstrument
uo
Model 33Saliity-Conductivity-Temperature. meter, of equivalent. Total organic carbon will be.
a
`meoanasShiumadrzueModdel 5000TOCanaloyr zequeivarlen,t.
`BiglogicalObservations
d
Observationsofmortality,immobiliaznadctliiniocanlsignsoftoxicitywillbemadebetween 0-
24 hours, andat24and48hours & 1hour. Dapnothblentosiwimdvitshin 15secaofenrgdentsle
i
agitationofthetestchamberwill b consideredimmobile.
`Sampling forAnalyticalMeasurements
un
`Watersampleswillbo collectedfrom cachtestcharmeratthebeginningofthetestandat 24and
a
48hours(2 1 hour)todetermineconcentrationsofthetestsubstance. Intheeveatthat 100%mortality
-
occursinanytreatment,thensamplingofthttreatmentwillterminate followingthenext sampling
|}
interval. Sampleswillbecollected atmid-depthfromcachtestchamberandanalyzed immediately,or
:
placeinanapproprite storagecontainer (2,polyeylene orplypropylencbole) andstoredunder
a
refriguentrilaantaliyzoedn.The samplescheme issummaribzeleodw:
m
000733
mw
PROTOCOLNO.: 454/120198/DAP-48H1/SUB454
3MLABREQUENSOT. U2723
o
WILDLIFE INTERNATIONAL LTD.
o
*
d
PROPOSED NUMBERS OF VERIFICATION SAMPLES
Experimental Group
0 Hours
24 Hours
48 Hours
i]
Control
2
2
2
Solvent Control (ifneeded)
2
2
2
Hl
Level 1-Low Concentration
2
2
2
Level 2
2
2
2
oH
Level3
2
2
2
Leveld
2
2
2
oH
Level 5-High Concentration
2
2
2
Totals
1
1
14
a
"Total NumofVebrifeicatrion Sampl=e4s2
oH
`Theabovenumbersofsamples representthosecollectedfrom thetestanddo notincludequality ~
]
"control (QC) samples such as matrix blanks and fortifications prepared and analyzed during the
analytical chemistry phase ofth study.
ou
AnalyticalChemistry
Chemical analysisofthesampleswillbcperformedbyWildlife Intemational Lid. The analytical
Ho
methodusedvillbebaseduponmethodologyprovidedbytheSponsorand identifiedin Appendix I.
"Themethodologyusdtoanalyzethetestsampleswillbedocumentedintherawdataand summarized
|
in the final report.
H
[I =
da
= oi
oa
DataAnalysis
`Whenthedose-responsepattemallowscalculationofEC10, ECS0and EC90 valthuedcataswi,ll boanalyzedusingthecompute software ofC.E. Stephan (6). Theprogramwasdesignedto calculate theECvaluesandthe 95% confidenceintervalsbyprobitanalysis,themovingaveragemethod,or
binomialprobability withnonlinearinterpolation(7, 8 and9).. The EC values willbecalculautsiendg
mortalitdyat/acoillemctmedaot2b4iandl4i8htourys.The no-mortalityconcentrationandtheno-
Gbserved-effectconcentration (NviOllbEedetCermi) ned.
000734
PROTOCOL NO: 454/120198/DAP-48HI/SUB4S4
3MLABREQUEST NO. U2723
i
.
0 WILDLIFE INTERNATIONAL LTD.
9
i
x
REC TOO BE MR AIND TAIS NED
RectoboemraindtaisnedfordatageneratedbyWildlifeIntemational Lid. willinclude,butnot
o
be limited to
|
1. Acoopftyhe signed protocol.
2. Identification and charactoefrtheitezstasutbsitaoncne,ifprobvy ithedSpoensodr.
a
3. Daoftinietiatsion andtermination ofthe test.
4. Hiosfthtetesotorrganiysm,cultureandholdingrecords.
|]
5. Stock solution calculation and preparation.
6. Daily observations.
nm
7. Waterchemistryresults (c., hardanndalekalsinitsy).
8. If applicable, the methods used to analyzetestsubstance concentrationsandthe results of
H
analytical measurements,
9. Statistical calculations.
a
10. Testconditions (ight intensity,photoperiod tc).
11. Calculationand preparationoftest concentrations.
o
12. Copyoffinal report.
FINALREPORT
o
Afinal reportoftheresultsofthestudywillbepreparedbyWildif InternationalLid.Thereport
a
willinclude,butno belimitedtothe following,whenapplicable:
1. Name andaddressofthefacilityperformingthestudy.
ol
2. Dates uponwhich thestudywas initiatedandcompleted,andthedefinitiveexperimental startand
termination dats.
3. AstatementofcompliancesignedbytheStudyDirectoraddressinganyexceptions to Good
Laboratory Practice Standards. 4. Objectivesandprocedures, as statedinthe approved protocol, includingal changestothe.
-
protocol.
5. The test substance identification including name, chemicalabstractnumber of code number,
strength, purity, composition, and otherinformationprovided by the Sponsor.
6. Stabilityandsolubilityofthetest substanceunderthe conditionsof administration, if prov0i0d7e35
by the Sponsor.
PROTOCOLNO. 454/120198/DAP-4$H1/SUB454
3MLAB REQUEST NO. U2723
oa
.
on WILDLIFE INTERNATIONAL LTD.
_
-10-
.
7. Adescriptionofthemethodsusedtoconducttheest.
8. Adescriptionofthetestorganisms,includingthesource,scientific name,ageorlfstage, feed
d
types,results oflatestfeedanalyses,lightinteanndphsotiopetrioyd.
9. Adesoftc heprrepariatiopnoftth tiestsoolutinons.
.
10. Themethodsusedtoallocateorganisms otestchambersandbeginthetest,the numberof
organismsandchamberspertreatment,andtheduration ofthetest.
11. Adescriptionofcircumstancesthatmayhaveaffectedthe qualityorintegrityofthedata.
12. ThenameoftheStudyDirectorandthenames ofotherscientists,professionals,andsupervisory
* personnelinvolved inthe study. 13. A description ofthe transformations, calculations, and operations performed on the data, a
`summaryand analysisofthebiologicaldataandanalyticalchemistrydata, and a statementof the: concdrlawufrsomitheoananlysses. Agraphplottingtheconcentrationresponsecurvefor each
periodanECS0iscalculated,whensufficientdataexists.I the datias condtuoecvaliuavtieon
by probit analysis, th slopeofthe concentration-response curve will be reported.
14. Statisticalmethodsusedtoevaluatethedata.
15. Thesignedanddatedreportsof eachoftheindividualscientistsorotherprofessionals involved
-
inthestudy.
16. The locationwhererawdataandfinalreportaretobestored.
17. AstatementpreparedbytheQuality AssuranceUnitlistingthedatesthatstudyinspectionsand
i
`auditsweremadeandthedates ofanyfindingsreportedtotheStudyDirectorand Management.
18. Ifitisnecessarytomakecomectionsoradditions oa finalreportafeithasbeenaccepted,such
changesvillbemadein theformofanamendmentissuedbytheStudyDirector. Theamendment
=
willclearlyidentifythepartofthefinalreportthatisbeingamended andthereasons forthe
amendment, andwillbesignedby theStudyDirector.
CHANOFGPRIOTN OCG OL
PlannedchangestotheprotocolvillbeintheformofwrittenamendmentssignedbytheStudy
Directorand theSponsor'sRepresentative. Amendmentswillbeconsideredaspartoftheprotocoland
=
willbeattachedtothefinalprotocol. Anyotherchangeswillbeinthe formofwrittendeviationssigned
bytheStudyDirectorand filedwiththeraw data. Allchanges 1o the protocol willbeindicatedinthe
-
final report.
000736
PROTOCOL NO. 454/120198/DAP-48HI/SUB4S4
3MLABREQUEST NO, U2723
on
m WILDLIFE INTERNATIONAL LTD.
n
-n
GOODLABORATORYPRACTICES
ol
"Thisstudswillbeconductedinaccordancewith Good LaboratoryPractice Standardsfor EPA
(40 CFR Part 160 and/or Part 792); OECD PrinciplesofGood Laboratory Practice (OCDE/GD (92)
ol
32, Environment Monograph No. 45); and Japan MAFF (59 NohSan, Notification No. 3850,
Agricultural Production Bureau). Each study conducted by Wildlife Intemational Lud. is routinely
m
examined by the Wildife Intemational Ltd. Quality Assurance Unit for compliance with Good
i
Laboratory Practices, Standard Operating Procedures and the specified protocol. A statement of
compliancewith GoodLaboratoryPracticeswillbepreparedforallportionsofthestudyconductedby
`Wildiifs International Ltd. The Sponsor vill be responsible for compliance with Good Laboratory
Practicesforproceduresperformedbyotherlaboratories,(cg.residueanalorypatsholeogys). Rawdata
foralworkperformedatWildlifeIntemationalLid.andacopy ofthefinalreportwillbefledbyproject
`number in archives located on the Widife International Lid. site or a an alternative location to be
specified in the final report.
PROTOCOL NO: 454/120198/DAP-48H1/SUB454
000737
3MLABREQUEST NO. U2723
[| WiouFEINTERNATIONALWO.
E
-12-
E
REFERENCES
.
B
1 (UdSr.aftE)n,viOrPonTmeSntNaolamPrboetrec8t5i0o.n10A1g0e;ncyA.gu1a9r9i6c. ISnevreiretse8b5r0a-teEcAocluotgiecaTloxEifcfietcytsTeTsets,tFGrueisdhewlaitneers
Daphnids.
Bn
2 IOmrmgoabniilsiaztaitoinonfoTresEtcoannodmRiecpCrooodpcetriaotnioTnesta.ndODEeCveDloGpumideenlti.ne 1f9o8r4.TesDtainpghnoifaChspe.miAccaultse
u
Guideline 202. Paris.
i
3 PAiSshTesM, SMtacarnodfamrvderEt7e2b9r-a8t8eas., a19n9d4.ASmpthainbdiaarndsG.uiAdmeerfiocraCnonSdocuicettiynfgorAcTuetsetiTnogxaicnidtyMaTteersisalwsi.th
4 Pennak, RW. 1978. Freshwater InvrtcbratsoftheUnited States. 2ndEd. 365p.
H
5 AWaPsHteAw,atAerW.WA16,thWEPiiCtFio.n, A1m9e85r.icaSntaPnudbalircd HMeeatlhtohdAssfsoocriatthieonE.xamAimneartiicoann oWfatWearteWroraknsd
Association. Water Pollution Control Federation, New York.
i
6 SPterespohnaanl,coCmEm.unic1a9t7i8,on.US. EPA, Environmental Rescarch Laboratory, Duluth, Minnesota
u
7 LFionnndeoyn,. DJ. 1971. StatisticalMethods inBiologicalAssay.Second edition. Griffin Press,
H
8 Thompson, WR. 1974. Bacteriological Reviews. VolIL No.2. Pp. 115-145.
u
9 `SEtvealpuhaatnio,nsC.E.Ame1r9i77c.anMSeoctiheotdysofrorTeCsatliungltainndgMaatnerLiCalS.0,PAugbulaitcaitcioTnoNxiucmobleogrySaTnPd6H34a.zaPrpd
65-84.
ou
000738
-
EAT AT. Sea rAmama A Amrrs TAL
J,
| WILDLIFE INTERNATIONAL LTD.
|
-3-
|2
IDENTIFICATION OFATPEPSETNSDUIBXSTANCE BY SPONSOR
|
Tobe CompblySepotnseor
1. Test SubstanceIdentity (name tobeusedinthereport): PEOS(PerfluorooctaneSulfonicAcidPotassium Salt
|
Reference Standard(if applicable): Analytical Standard: _N/A
Intemal Standard: _P1.1e.2.f2HHlHuH oroocSultfonaicAncied
|
Test Substance Sample Codeor Batch Number: __Lot 217
'
Test SubsPurtitya(%nAcctieve Ingredient): _989 ExpirationDate: _2008
|
IL Test Substance Characterization
Have the identita y, streng BE and Dcompositiron oor onthserschaaranctaeribstaics
|
determinedprior o itsuseinthisstudyinaccordancewith GLPStandards? Yes No X_
IL Test Substance Storage Conditions
|
Pleaseindicatethe recommendedstorageconditionsatWildlife IntemationalLid.
|
Ambient room temperature
|
bHeaens dtheetesrtambiinlietsyoifntahcceotredsatnscuebswtiatnhcGeLunPdSetranthdeasredsst?orage condiions
Yes No_X
Otherpertinent stability information: --
- IV. Test Concentrations:
:I
I V. Toxicity Information:
_X_ Abdajsuedstutpeosnttcohnecpeunrittrya(t%i)otgoniv1e0n0%sbao.ve.
Dao5n"tTeatdjthuesmtatteesrticaolnAceLnLtSratioton 100%
=
Mammalian: ~~ RatLDSO_25Imghke MouseLDSO_N/A
|
Aquatic
Invertebrate Toxicity (BC/LCS0)
Fish Toxicity (LCS0)
DaphniSa0mmg/al gna; FatheadMinnow:38mg/l,
L
Other Toxicity Information includingfindingsofchronicandsubchronic tests):
I
Please sce MSDS
|
000739
L
PROTOCOLNO.: 454/120198/DAP-48HI/SUB4S4
3MLABREQUEST NO. U2723
WILDLIFE INTERNATIONAL LD.
-14-
APPENDIXTI | Analytical Method to be Provided by Sponsor
PROTOCOL NO: 454/120198/DAP-48H1/SUB4S4
000740
3MLAB REQUEST NO. U2723
