Document 2wMr6xOMXNB68NOxe3JKkrk7
et
)
PROTOCOL
AR126-0183
0ES-S
PFOS: A96-HOUR TOXICITY TEST "WITH THE MARINE DIATOM (Skeletonema costatum)
"SerieUs.S8.50En-vEicroolnomgeinctaallEfPfreocttescTtieosntAGgueidneclyines
"
`OPPTS Number 850.5400
.
3M LabRequest No, U2723
Submitted to
-
3M Corporation Environmental Laboratory
St. Pa9u3l5, BMiunsnhesAovtea.nu5e5106
Wildlife International, Ltd.
Ne
:
Eas8t5o9n8C,oMmarmyelracndeD2r1i6v0e1
(410) 822-8600
September 17, 1999
:
CE
04515
ts SE
We ILDLE IFENINTEA e RNATIONAALL, LTv D ee oe
PFOS: A96-HOUR TOXICITY TEST `WITHTHEMARINE DIATOM (Skeletonema costatum)
SPONSOR:
3M Corporation
`Environmental Laboratory
935 Bush Avenue St. Paul, Minnesota 55106
SPONSOR'SREPRESENTATIVE:
TESTINGFACILITY:
RochellRe. Robidesu
Wi8l5d9l8iCfoeImnmteerrncaetDiorniavl,eLid. Easton,Maryland 21601
STUDY DIRECTOR:
KertBrotar Cary Suthertend Jpofee
Senior AquaticBiologist
LABORATORYMANAGEMENT: ~~HearOy.Krueger,PhD.
DirectorofAquatic Toxicology & Non-Target Plants -
`Proposed Dates:
FORLABORATORY USE ONLY
[Experimental Start Date:
Projecto:
_4SYA-[[3
Experimental TerminationDate: __ ~~ __
`Test Concentrations: `Test SubstanceNo.:
ee. ReferenceSubsNot .(ia fappnlicc able e): _________
PROTOCOL APPROVAL
(UH 2
STUDY DIRECTOR
Z
Z "DORSe REPREUNTAa TIVE
PROTOCOL NO.: 454/091799/SKE96/SUB454
1/2 Joo
DATE
4 4 loo
DATE
Ba fw
c04516
3MLABREQUESNOT. U2723
[I
WILDLIE FE INO TERNAN TIONAALy, LTD
errr
ade
INTRODUCTION
`WidifeInternational,Lid.willconducta 96-hourtoxicitytestwiththemarinedistor,Seletonema costatum,fortheSpoasarattheWildife Inermtions,Lid.aqueictoxicologyfacilityinEaston,Maryland. "Thestudywillb performed basedonproceduresinthe US. EnvirProtoectnionmAgeencnySetrias85l0EcolEffeoctsgTesitGcuidaelilnesOPPTS Number 850.5400 (1).Rawdataforallworkpecformeadt `WiiIntemational,Ld.sndacopyofthe final reportwillbe filedbyprojectruberinarchiveslocated `ontheWildifeInternational,Ltd.site, or at an alternativelocationtobespecifiedinthe ina rport.
PURPOSE
"Thepurposeofthisstudy i todeterminethetoxicityofPecfluorooctaneSulfonicAcid,Potassium
Salt(hereafterreferred 0asPFOS)tothemarinediatom,Skeletonemacostatum.
:
EXPERIMENTAL DESIGN
Themarine distom,Skeletonemacostatun,willbeexposedto ageometricseriesof sixtet concentrationsand anegative (culturemedium)controlfor96 hours. Targetconcentwrilalntotieoxcneesd 100mg/L orth soluilsylimitof thetestsubstancein water. Generally,thenominalconcentraofteiaocnh testsubstanceusedinthedefinitive testwillbeatleast 0%ofthenextighertreatmentunlessinformation concemingtheconocatration-effectcurveindicate that adifferent dilutionfactorwouldbemrsppropriste.
Testsolutions willbeinoculatedwith approximately77,000cells/mL,Testsolutions willbe `ienvaolcwcuietlauhdt1el0in,s0na0eg0.cteOlnlese/omdxLm. oTrhreaedd"ibtiioolnoagli"csamla"lyrteipclailcarteepslpiecarteexspweirlilmebnetianlcghreoudpinwitlhlebeesptr,eapanreeeddfeodr, 10provideestsolutionforconcentrationverificationoaDay3oftheexposure,Anabioticreplicasat the highestconcearaion il beincludedinthetestandwillbestmpledonDay 3andDay4.Inextdocoentrrol bias,thepositionofthe flaskswillbedeterminedbyindiscriminatedrawdlyduringthe exposureperiod. `No therpotential sourcesofbiasareexpectedtoaffecttheresults ofthestudy.
"Theresponseofthe algae willbemeasuredintermsofclldesit,biomassexpressedasaccaunder thegrowthcurv,andgrowthrate. AnECS0(ic,thetheoretical testconcentrationthatproduces 8 50% ~ reductioninthemeasure parameler)valueforceldensitywillbecalculatedforcach24bourinterval. EC10,ECS0and EC90valuesforcel deusity,biomass(EC)andgrowth ate(EC)willbecalculated,if possiblea the 72and96hou interval.Theno observedadverseeffectconcentration(NOAEC),which
PROTOCOLNO: 454/091799/SKES6/SUB454
3MLABREQUEST NO.U2723
04517
A WILDLIFE INTERNAATTIONAL, L(9D0 4
isthehighesttest conceatrationthatinduces noadverseinhibitoryeffectongrowth, willbedetermined rreelsaptoiuvseetpoactatcehmp.aAratmettheerenadt7of2tahned9966-hhoouurresxbpaosseurdeu,paolngesvtaltuiateifoencotsfwtihlelsbtaetdiisftfiecraelnrteisautletdfsarnodmtahlegidcoisdael effectsinthosetreatmentswhich aromaximallyinhibited.
1
.. MATEARNDIMEATHLODSS
Informationonthecharacterizationoftest control of referencesubstancesisrequired by Good LabPoractriceaStatndaords(rGLyP).TheSponsorisrsponsibl forprovidingWildlifenternationa,Lid. `writtenverificationthatthetestsubstancebasbecacharacterizedaccordingto GLP priortoisuseinthe
study. IFwritenverification ofGLPtestsubstancecharacterizationisnotprovidedto WideInterntional,
Lad,itvillbo notedinth compliancestatementofthefinareport.TheattachedformIDENTIFICATION. `OFTEST SUBSTANCE BYSPONSOR (AppendixI)i tobeuse oprovideinformation necessaryfor `GLPcompliance.
`TheSponsorisresponsibleforallinformation elatedtothetestsubstanceandagreestoacceptany
musedtest substanceandlor testsubstancecontainersrecautaheciadnof ith sntagd. ction Prepuat "Thedesired concentrationsof PFOS willbeobtainedbypreparinga singlestocksolutionin medium
`nddilutingtheappropriatevokumeofsocksolutionvithmedium.The estsolutionsmayalsobeprepared usingsecondarydition oftheprimarystock,basedupantheconcentrationdesired
PFOSwillbe aduinisteredto th testorganisminalgalmedium.Thisroutsofadministratwiaosn selectedbecause trepresentsthemostlikelyrouteofexposure to slgae,which existsuspendedin awater column.
PROTOCOLNO: 454/091799/SKE96/SUBAS4
04518
MLAB REQUESTNO.U2723
WILDLIFE INTA ERNAN TIONAAL, LEDD
eeer-- .5-
Test Organism
"Thetest species wil bethemarinealga,Seletonemacostarum Thisspecsisrepresentaotfivaen. importantgroupofalgaeandwasselectedfo use i thetestbaseuponpastusoand cas of handling i the laboratory. Stockcultures,obtained romtheCultureCollectionofAlgaeattheUniversityof Texasat Austinoranothersupplier willbemaintainedinculturemedium undertestconditionsatWildlifeInterna. tional,Ld.foraminimumoftwoweeksprior tousein toxicitytest.Algaeusedintoxitesctsiwiltlbyo inexponcatialgrowthphase,whichisdefine astheperiodofgrowthwhenalgalcellsaredividingat a constantrat. 3
Just pricetobeginningthe test,aninoculumofthestockculturewillbepreparedsothatcachmilter ofinoculumcontainsenoughcellstoprovideaninitalol densityofapproximately 77,000cellu.incach replicate.
Culture Medion
.
Culturemedium preparedfromnaturaloeartificialseawateraccordingto WifeIntersational,Lid.
`Standard OperatingProcedureswillbeused asdilutionwate. Thoconcenoftthercoampotneintos inntshe
mediumarepresienTanblte1e(d2).Stockmutriatsolutidusvel bepreparedbyadding reagent-grade
chemicalstoWildif Intemational,Ltd.wel waterpurifiedbyreverse-osmosis."Appropriatevolumesof
thestocknutrientsolutionswil theabedilutedwithartifical ornaturalsavertopreparethemedium,The
`medium willbe iter sterilized(0.22 pum)orautoclavedpriortouse. Analysesvillbeperfoartmleeadst
nce sunualy to determine the concentrationsof selectedorganicandinorganicconstituentsofthe wellwater
0dresults ofthemostrecent GLPcompliantanalyseswillbesummarizedinthefinalreport.
`Specificationsforacocptablelevelsofcontaminantshavenotbeen establishedforculturemedium. However,there arenolevelsofcontaminantsreasonablyexpectedtobepreseatinthemedium thatare consideredto interferewiththepurposeorconduct ofthestudy.
J
TestApparatus
"Testchambersvillbesterile250-mLpolycarbonateErlameyer asks pluggedwithfoam stoppers
andcon1t00amLiofntesitsonlutigon.The test chambers willbe indiscripmosiintiaontedeoln y2
PROTOCOLNO: 454/091799/SKES6/SUB454
04519
3MLABREQUEST NO. U2723
W_ ILDLIFE INTE-- RNATIONAL, L_ D -- OOO w O Ge
`mechanicalshaker ableinanenvironmentalchamberandwillbeshake continuouslyatapproximately 100 Pm. Testchamberswilbelabeledwiththeprojectnumber,tes conocatraion andreplicate.
EnvironmentalConditions
"Testflaskswil be heldat20 2Cunder14hoursofcoolihitefluorescentlightingatanintensity 04300: 430 xan10 hoursofdarkness.Light tensitywillbemeasuredatfivelocationssumounding thetest flasksonDay 0oftheest.Texnperatur willbe recordedtiodaily in acontainerofwateradja toth testflasksusing aliquidin-glassthermometer.
`The pHofcachtreatmentandcontrolgroupvillbemeasuredat tes nifiationandterminationusing
aFisherAccumetModel 915 pHmeterorequivalent. Samplesfor pH measuattrestieiamtieonwnilltbe
collectedfromtheindividualbatchesoftestsolutionpreparedfo eachtreatmentandcontrolgroup.Attest
termination,pH wilbemeasured ipooledsamplesoftestsolutioncollectedfroneachofthethre replicates
ofeachrespective treatandcontrolgroup.
.
ital
(Celldeasiticswillbemonitoredduringthetestbyconductingcelcountsusinga bemacytometerand 2microscope. Onesamplewillbecollectedfromeachreplicateofthetreatmeitandcontrolgroupsat `approximate24-hourintervalsduringtheexposureperiod.Thesampleswillbeevaluatedimmedinteolry
willbeheld inthedark tapproximately 4C untilcll countscanbeperformed.Eachsavaillpbeilteed
usinganelecrolytssolution(lsoten),as necded,to maintaincounting accuracy. Asmallamountofeach samplewillbeloadedonto ahemacytomeserandthetotalnumberofcellsin 10 gridsvillb counted. The celldensityofthesamplewill becalculatedbasedonthemeanmumberofcelspergrid.
Microscopicexaminationofalgaewillbe madeto assesschangesingrossmorphology. Algaewill beobservedtoassesschangesin cel haps,cellcolor,andaggregation (clumping)ofthecells Observations willbeusedto,determinewhethergrowth treatmentrelatedeffectsincludethosechangesingross
PROTOCOL NO: 454/091799/SKE96/SUBAS4
04520
3MLABREQUENSOT, U2723
-- O WILDLIFE INTERNATIOeNAeO L, LTD O 7.
.
.
`SamplesoftheexposuresolutionswillbecollectedonDays0,3 and 4andanalyzedfortestsubstance
eonceatations.Day 0samplesvillbetaken from cachbatch of test solutionprior otei istribution mang. therepltesit cchamaberts. Day 3samples wilbecollectedfromtheextrareplica testchamber(s) included inthetest toprovidesample orthat analysisand from the abiotic replicate.Ifmorethanonereplicaties
includedforthe Day 3sampleanalysis,thenthesolutionsfrom eachreplicate wilbecompositedtoprovide: the sampleforthattreatment.Day 4samples willbe composited solutions fromthe three replicates remaining attheeadof thetest.
`Samplescollected onDays 3and 4maybecentrifugedor filteredto removealgalcellspriorto
analysis. Sampleswillbeanalyzedimmediatelyor placedin anappropriatestoragecontainer (e.g,
`polypropylencorpolyethylene bottle) andstored until analyzed.The sampleschemeis summaribzeleowd:
[me | Dum o | oo ws s mmr] [PROPOSEDNUMBERSOFVERIFICASATMIPOLENS
Control
1
1
`SolventControl(if needed)
Level 1-Low Concentration
1
1
1
1
Level2
3
.
1
Level 3
1
1
Level 4
1
1
Level 5
1
1
[[ row ws Level 6-HighConcentration
2
2'
`Theabovenumbersofsamplesrepresentthosecollectedfrom thetestanddonotincludequality
`control (QC)samplessuch as matrixblanksand fortifications preparedandanalyzedduringthe analytical
rw mnSRAPRO IY,
.
eon son ers
a
Chemis
.
methodCuhseemdiwciallalnbaelbysaisseodfutphoensmaemtphloedsowlilolgbyeprpoevrifdoermbeydtbyheWiSlpdolnisfeorInatexmiadteinotniafliLetdid.n"ATphpeaennadliyxtIicTal
PROTOCOLNO.: 454/091799/SKE96/SUB454
3MLABREQUENSOT. U2723
04521
:
SHR
W_I--LDLIFE INTERNATIONAL, to
OO
-3-
.
Modificationsmadetothe analyticalmethodwillbedocumentedintherawdataenddescribedinthe ioal
.
repo
Si
}
Biomassexpressedaarea underthegrowthcurvewillbecalulatedforthetreatmentandcontrol
groupsusingthe following formula:
A= ONYYHNAN IND2t) HON rNZN 2X er)
Where: Nd
NAo==AMreeasauurneddermtuhmebgerroofcweclutlrsvh/emLatt,
NNyi ==MMeennaauummbbsseerrooouffcceerrlllissf/eeamLL.aaddttt,;
tN.=T=imMeofefirmsatummebasesruorufecmeernltalef.eradbtte,ginningoftes (hours)
ta==TTiimmeeooffnstehcmoenadmseuarseumreenmteanfterfbeergibnengiinngnoinfgtoefstt(ehsotu(rhso)urs)
'
(Grow ates)willbecalenlt fo thetreatmentandcontrolgroupsusingthefolowingformula: PE aN,InN, tet
:
N4,== MAveearsaugreesdpensuimfbiecgrroofwctehllrsa/tmeLatt,
tNo.=T=iMmeoefbaemugmsbeirouonfftcenresltil(sh/eonmulr.gsad)tt,
t=Timeofnth measurementafterbeginningof test(hours)
Perotinhibitionwillbe calalatdforeachtreatmentgroupbased uponmeancel deasity,biomass andgrowth rate usingthefollowingformula:
. MeanCon-MteanrTreoatmlent =r ere PercentInhibition = ReMsepaonnsCeon_t_roRleRsesppoonnssee: ~~ X 100 sme cera
PROTOCOL NO: 454/091759/SKE9G/SUBAS4
04522
3MLABREQUEST NO: U2723
_ WILDLIFE INTERNATIONAL-- , LD r OOO "5.
EC valueswillb determined,whenposible,usinglinerinterpolation o othersuitabletchiques. withtreatmeatrespouse(.c. elldensities,biomassandgrowthrate) andexposureconcentrationdatafor ach24-hourintervalofthetest.
A 20observedadversefe concentration(NOAEC)willbeselectedbasedon anevaluationofthe doseresponsepattemandtheresultsofthestatisticalanalysisusingindividual replicatevaluesofthecll density, bioanmdgraowtshraste.Statisticallysignificantdifferencesbetwosa th controlandthe treatment `groupswillbeidentified singanalysisofvariance (ANOVA) and atest compar treatmentmean responsestothecontrolresponse(cg,Dunnet'stestorBonferroni's tes),Datawilbeassessfeodr noanalityandbomogeociyofvariances prior operformingtheANOVA. Transformwialltbeiuosendtso `correctanyconditionof on-normaltyorunequalerrorvariances,ifpossibleIf solventcontrol iused
imaddition fo th negativecontrolthei responseswillbecomparedusingStudent'stest.Datafom the
twocontrolgroupswillbepooledfortreatment levelanalysesif nosaistial differencesexis.Otherwise, llcomparisonswillbemadetothesolve controlgroup. Statistical compfoartrheAiNOsVAoann sthe `comparisonofmeanresponseswillbecarried utusing TOXSTAT V3.5software(4)orequivalent.
.
REC TOBOEMR AIND TAIS NED
Recordstobemaintainedfordatageneratedat WildifeInternational,Lud willinclude,but otbe
limitedto:
.
.
1. Copyofsignedprotocol.
2. Mdentficationand characterizationofthetestsubstance,ifprovidedbySponsor.
.
3. Datesofinitiationandterminoaftthietoesnt.
4. Sourceofalgae.
5. Cultureconditions.
6. Growthmeasurements.
7. Calculationandpreparationoftestconceatrations.
8. Observations.
9. Ifapplicable the methodsusetoanalyzetestsubstanceconcentrationsand the results of analytical
`measurements
10. Statisticalcalculations.
11. Testconditionsandphysicalcheanicalmeasurements.
04523
PROTOCOL NO: 454/091799/SKESG/SUBAS4
3MLABREQUESTNO, U2723
W_IL--DLIFE INTERNATIONAL, LTD "10.
12. Copyoffinalreport.
OOO
--
FINALREPORT
A final report ofthe resultsofthestacywill be preparedbyWildife International,Lid. Thereport . `willinclude, butnotbelimitedtothefollowing,whenapplicable:
3 `Nameand addressofthefacilityperformingthestudy.
2. `Dateson whichthestudywasinitiated andcompleted.It istheresponsibilityoftheSpoasor to
providethefinaldatethatdataarerecordedforchemistrypathologyand/orsupportingevaluations
`thatmaybegenerated atother laboratories.
3. A statementof compliancesigned by the StadyDirectoraddressing any exceptionstoGood LaboratoryPractice Standards.
4. `Objectives andproceduresstatedinthe approvedprotoco,including anychangesintheoriginal protocol.
5 Thetest substance identified byname,chemical abstractnumberorcode umber, strength, pusiy,
`composition,andothercharacteristicsprovidedbythe Sponsor.
6. Stabilityandsolubilityofth testsubstances undertheconditions of sdministatio,ifprovidedby
the Sponsor.
:
7. Adescriptionofthemethods used to conductthe test.
. test. 8. Adescriptionofthetestsystem,includingthesourceofthetestorganismsandtheirscicatific name. 9. `A descriptionofthepreparationofth testsolutions,methodsusedtoallocateorganismstotho test `chambers andbeginthetest,numbersoforganisms andchamberspertreatment,anddurationofthe
10. Adescriptionofcircumstancesthatmay haveaffected thequalityorintegrityofthedata. 1. `The nameoftheStudyDirectorandthe namesofotherscientists,professionals,andsupervisory
`personnelinvolvedinthestudy.
12. Adescriptionofthetransformations,calculations,o operationspecformedonthedata, asummary
andanalysisofthebiologicaldat
drawnfom thesesmalyses.
aand
a
nal
ytic
alchemistry
data
,and
:
astat
e
me
ntofthec
.
o
n
cl
usi
ons
13. Statisticalmethodsemployedforanalyzingthedata.
14. study. Thesigned anddatedreportsofeachoftheindividualscientistsorotherprofessionalsinvolvedin the
PROTOCOLNO.: 454/091799/SKE96/SUB4S4
- 604524
3MLABREQUEST NO.U2723
W_IL--D--LIFE INTERNATIONAL, Lo
1.
15. Thelocationwhererawdataand inalreportar bestored.
16. A statementpreparedbytheQualityAssuranceUntisting th date thatsyinspectionsandaudits
17.
`weremadeandthedatesofany findingsreported othe StudyDirectorandManageme. itis necessary tomake orrctionsoradditions 0a finalreportaferithasbooaccepted,such+ _
changesshallbemadeintheformofanamendmeatissuedbythe StadyDirector,Theamendment
shallclearlyidentifythepartofthe finalreportthati beingamendedandthereasoasfor the
alteration. Amendmentsshal besignedanddatedbytheStudyDirector,
Plannedchanges
1
CHANOFGPRIOTNOCGOL theprotocolwillbeinthe ormofwritenamendmentssignedbythe
Study
DirectorandtheSpoosar'sRepresentative, Ameadimentsvillbe consideredaspartofthe protoeoanwill
bSetotdyDiarectooctrhaenh dffinla eprwoittodhcothle. Aranwydoatthae.rAclhancgheasngiesltlobtehienptrhoetfoocromlwioflwlrbietednidaetveidtonntshsiiganledbreyptohret.
`Thisstudywill be conducGteOdOinDsLccAorBdOanRcAewTiOthRGYoPoRdLAaCboTrIatCoErySPractice Standardsfor EPA (40 `aCnFdR PJaarpatn1M60AanFdFlor(P5a9rNto7b9S2a)n;,ONEoCtiDfPirciantciiopnleNsoo.f3G8o5o0,dALgarbiocrualttourraylPrParcotdiuocst(iEonNVBu/rMeCa/u)C.HEEMa(c9h8)st1u7d;y `conductedbyWilife International,Lu. routinelyexamine bytheWildifeInternationalLtd.Quality AssuranceUnitforcompliancewithGoodLaboratoryPractice, StandardOperatingProceduresandthe specified protocol. AstatementofcompliancewithGoodLaboratoryPracticeswil bepregiedforall portionsofthestudyconductedby WildlifeInternational, Lid. TheSpoasorwillberesponsibfloer `compliancewithGoodLaboratoryPracticesforprocedurespecformedbyoberlaboratories (6.5, residue. analyseso pathology).Rawdatafo llworkperformedatWildife nterationa,Lid. and acopyofthe inal reportwillbe ledbyprojectnumberin archiveslocatedonthe WidieIntemational,Lid.sit, orst anatemative locationtobespecifiedinthefinal report.
PROTOCOL NO. 454/091795/SKES6/SUBAS4
'
04525
3MLABREQUEST NO. U2723
-W--I_L--DLIFE INTERNATIONAL, LD 12-
REFERENCES
1 (UdSra.fEtn),viOrPoPnTmSenNtuamlbPerrot8e5c0t.i5o4n0A0:geAnlcgya.l To1x9i9c6i,ty,SeTriieerss8I5a0n-EdcoILl.ogicalEffects Test Guidelines
2 ATeSsTtsMwS itht Mica roaGn lugaied d.eA1a m2e1r8r i-c9a0dnE.Soc1i9e9t0y.SftoarnTdeasrtidnGg uadndeMfaotreCroianldsu.cPthiinlgaSdtealtpihci9a,6-PeHnonusryTlovxainciitay.
3 NCorobenrgcKieInTCgn,J),.Atp1p9r9ro3a.caAhL(tiVnereisairoIon2n.n0)t. UeSr,EpMeaothlovdafoirtSruPirbolotceechntainlonToAm xgiecine tcyy:,n TEhaveitIrnchaiambictailtoanl
Research Laboratory, Duluth, Minnesota.
4
`West,Inc.andD. D.Galley. Cheyenne, Wyoming.
1996. TOXSTATRelease3.5.
Wester EcoSystems Technology, Inc.
PROTOCOL NO.: 454/091799/SKE96/SUB4S4
04526
3MLABREQUENSOT. U2723
BEWILDLIFE INTERNATIONAL, 11D
13-
TABLE1 SALTWATERALGAL MEDIUM CONSTITUENTS ~
_--_--
--_--TomComipanneatalConcNomeinalnContcenrtrataionin
MFaeCClhu46HH::00
027126 mmgg
2CSu0S,0.u7SHH0O
.
CoChu6H0
0263765mpggll 606 pg
HBNuOE,DTA.2H0
1175.01 mmgg
;
KNPaONO,,
30 500
mpl mL
`NTahsiSaimOine9H5y,d0rochloride
200 mL 025 mg
Biotin --_-- Ba
005 gl 05 wgelb
:
Tihmeataebloyv3e0cpoamtpsopneerntthsouwsialnlbd.eadded to instant saltwateratasalinityofapprox.
u`sT--ihne_pg--H0.o1fNthNeamOeHdiourm1wi0l%lHbCeaLdjusted, asnecessary, tobetween7.8a5 8.1
PROTOCOLNO.: 454/091799/SKES6/SUB4S4
04527
3MLABREQUESTNO, Uz723
W-I_L--DLIFE INTERNATIONAL, rurp ER
OOOO
APPENDIX
IDENTOFITEF ST SIUBC STAA NCT EBYI SPOONSN OR.
TobeComplbeyStpoensodr
1 RTeefetcaSucbestaSntcaenddardt((apnpalimceab0lbe)e:wAenadlyitnitchaeleSptoannd)a:rd:PENSA(PerflurooetsnSule Aci Pass Slt. TestSubstanceSumpleCode BatchNumtbeernra:l_SLtaentda2r1d:7_P11.e2.f2HHhHuHaroocStulfaosincAceid
`TestSubstance Purity:(%ActiveIngredient): --989 ExpirationDate: 2008 IL TestSubstanceCharacterization
SEEEEE-------- propre
bse main
IL TestSubsStotragaeConndictioens
Pleas indicatetherecommend storageconditionsatWillieIntemational,Ltd
en et bom ess
Ye x
RRs. eS
IV. TestContains:
x bese pA atiedpeusrtcti oyr0eegnt mrtieona9t1.00i
V. Toseformation:
Mali:
Aquatic
RatLDSO 251 mg/kg MowseLDSO_NA
InvertebrateToxicity (BCLCS0) FishToxiy(LC50)
daa, 27
Reacts
.
DuphoiSa0mmg/al gns:_Fatheed Minnow: mall,
`OtherToxicyInformation(inchingfindingsofchronicandsubhrcic tests):
t PROTOCOL NO.: 454/091799/SKE96/SUBA4S4
04528 3MLABREQUENSOT. U2723
W_ ILDLIFE INTERNATIONA-- L, 10 r OOO BTR
APPENDIIXI
*
AnaMelthoy dPrt ovii ded bcySa ponl sor
Seanpcs willbeanalyzedbaseduponproceduresprovide bytheSponsor ithefollowagaalytcal
1. Liquid ChromatographyMassSpectrometry(LCMS)Method fortheDetermination
ofPerfluotooctaneSulfgicAcidPotassium Sat(PROS)InFreshwater, Saltwater andAlgalModiam nalsActohpetyeotfstahmepalbeosvwielmlebtehdoodcwuimlelnbteemdaiintnthaeirnaedidnatthaearnadswudmamtaar.i`zTehdeianctthueaflimneatlhroedpoolrot.gyusedto
\
PROTOCOL NO.: 454/091799/SKES6/SUB454
04529
3MLAB REQUENSOT. U2723
Muivervy ve.99 LAA 81V B2Z 0832
WILDLIFE INTERNATIONAL
@oos
PROJECTNO: 454A.113 A WILDLIm FE INT-- ERNATIO-- NAAL Lo.------------P-- olo?
STUDYTITLE:
AMENTOD STUMDYPERON TOCTOL PcoRsOtSa:nmA)96-HOURTOXICITYTEST WITHTHEMARINEDIATOM(Skeletonema
PROTOCOL NO: 454/091799/SKE96/SUB4S4
SPONSOR:: 3M Corporation
DR AFT
EFFECTIVE DATE: February 28,2000
AMENDMENT NO: 1
PROJECT NO.: 454A-4s14A-1LL3
3m
2 723
AMENDMENT: Page2
Add: ExEpxerpimeerntiamlSTteeaenrmtitDnaaattleio:nDMatae:r3M,c2a0h0r07,c20h00
~
T`Teesstt SCounbcsetnatnrcaetiNoon.s:: 4N675eg Contarolatnd4i.0mvga.e/L
REASON: Theaboveinformationwas notknownwhentheprotocolwassigacdbytheStyDirector.
AMENDMENT: Experimental Design,Page 3
.
Change: Tcohnecemnatrraitnieondsiaantodm,a Snkegealtcitveo(nceumlatcuroesmteadtuimu,mw)iclolnbtreoelxfpo o9s6edhotuorsa.geometrioseries ofsixtest
To: s"oTluhbmilairtiynaenddiaatnoegma,tSikveele(ctuolnteumreamceodsitautmu)mc,ownitlrlolbfoore9xp6osheodurtso, 8testconcestrationa th litof
REASON:Tochangsexperdiesimgnetonltimitateslt.
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AMENDMENT:ExperimentalDesign,Page 3
. Changs: Tiestnsoolutciwointushw1il0,l0ba0e0ctienlolecsu/lmdaLt.edwithapproximately77,000 allah Testsolutionswillbe To: `Testsolutionswillbeinoculatedwithapproximately77,000celal. REASON: Typographicalcor,
AMENDMENT: Experimental Design,Page 3
.
Change: Three"biological"replicatespercxperimentalgroupwillbepreparedforevaluatingcelldensities.
To: Six"biological"replicatesperexperimentalgroup willbepreparedforevaluating elldensiies.
REASON:N:TToo,changeexperdiesimgneto nlitmitatelst.
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AMENDMENT: SamplingforAnalyticalMeasuremePnatgse7, Chango: Day 4 sampleswillbocompositedsoutionsfromthethreereplicates remainingattheendofthoes, To: Day 4sampleswillbocompositedsolutionsfom theixreplicasremainingattheendofthetest. REASON: Tochangemalytcalsamplingbasedcnadditonalreplicatesprovidedinthelimit cst.
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STUDYDIRECTOR LABORATORY MANAGEMENT
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