Document RJm1nL8jq3Km3pz8E4Zyvxnoa
cl
AR26-0187
OES -Y
PROTOCOL
PFOS: A96-HOURTOXICITY TEST `WITH THE FRESHWATER DIATOM (Navicula pelliculosa)
U.S. Environmental Protection Agency
`Series 850 - Ecological Effects Test Guidelines
`OPPTS Number 850.5400
-
' `3MLabRequesNot.U2723
`Submitttedo
:
3M Corporation
Environmental Laboratory
935 Bush Avenue:
St. Paul, Minnesota 55106
Wildlife International, Ltd.
\
8598 Commerce Drive
:
Easton, Maryland 21601
(410) 822-8600
September 17, 1999
04498
WILDLIFE INTERNATIONAL, LTD
2.
PFOS: A96-HOUR TOXICITY TEST WITH THEFRESHWATER DIATOM (Vavicula pelliculosa)
`SPONSOR:
`SPONSOR'SREPRESENTATIVE: IESTNGFACLITY:
STUDYDIRECTOR:
LABORATORYMANAGEMENT:
3MCorporation `Environmental Laboratory 935 Bush Avenue St. Paul, Minnesota 55106
RochellRe Robidean Wild InternatioLind,.
8598 Commerce Drive.
`Easton, Maryland21601
SarsDrotiar Cary Stbasland
`Senior Aquatic Biologist
HE thief
HDeniryrOe.oKfcrAutqeugoaetrri,cPTAoDx.icology & Non-TargetPlants
FOR LABORATORY USE ONLY
Proposed Dates:
'
`Experimental
`Experimental
Start Date:
ProjoctNo:
_OY4A-/12 `TerminationDate:
.
`Test Concentrations:
---
"TestSubstance No.:
`ReferenceSubstanceNo.(ifapplicable):
PROTOCOL APPROVAL
DEpo H STUDYDIRECTOR
x4
LABOI
RV,
\GEMENT
=S ,a A Fm Y .
SPONSORS REPRESENTATIVE
=
PROTOCOLNO: 454/091799/NAVI6/SUB4S4
fhe DATE
1/8foo
DATE
DA)
04499
3MLAB REQUESTNO.U2723
WILDLIFE INTERNATIONAL, LTD
3.
INTRODUCTION
`WildlifeIntemational,Ltd.willconduct afive-daytoxicitytestwiththefreshwaterdiatom,Navicula
pelliculosa,fortheSponsor attheWildlife International, Ltd. aquatictoxicologyfacility in Baston, `Maryland.Thestudywillbe performedbasedonproceduresintheU.S.EnvironmentalProtectionAgency
`Series 850 -EcologicalEffectsTestGuidelinesOPPTS Number850.5400(1). Rawdataforallwork
performedatWildlifeInternational,Ltd.and acopyof thefinalreportwillbefiled byprojectnumberin
archiveslocatedontheWildlifeInternational,Lud.sit,oratanaltemativelocation to be specifiedinthefinal
report.
"=
PURPOSE
"Thepurposeofthisstudy istodeterminethetoxicity ofPerfluorooctane SulfonicAcid,Potassium `Salt(hereafterreferred to as PFOS) to the freshwaterdiatom, Navicula pelliculosa.
EXPERIMENTALDESIGN
:
`Thefreshwaterdiatom, Naviculapelliculosa,willbeexposed to ageometricseriesofsixtest
`concentrations and anegative (culturemedium)controlfor 96 Bours.Targetconcentwrilalntotieoxcneesd
100mg/Lorthesolubilitylimitofthetestsubstanceinwater. Generally,thenominalconcentraotfciaocnh
testsubstanceusedinthe definitivetestwil beatleast 50%ofthenexthighertreatment,unlessinformation
`concerning the concentration-effectcurve indicates that adifferentdilutionfactor would be more appropriate.
Testsolutions willbeinoculatedwith 10,000celsfmL.Three"biorleploicatgespircexpaerilmen"tal
groupwillbepreparedforevaluatingcelldensities.Oneormoreadditional "analytical" replicates willbe
includedin the test, as needed, to provide test solution forconcentration verification onDay 3of the. exposure. Anabioticreplicateatthe highestconcentrationwillbeincludedinthetestandwillbe sampled onDay 3andDay4.Inordertocontrolbias,thepositionoftheflaskswillbedeterminedbyindiscriminate drawdailyduringthe exposureperiod.Nootherpotentialsources ofbiasare expectedtoaffecttheresults
ofthestudy.
`Theresponseofthealgae willbemeasuredintermsofcelldensity,biomassexpressedasarea under thegrowthcurve,andgrowthrate. AnECS0(i.c.,thetheoreticaltestconcentrationthatproduces a 50% `reductioninthemeasured parameter)valueforcelldensity willbe calculatedforeach24 hourinterval. ECI0,EC50 and EC90 valuesfor celldensity, biomass (EyC)and growthrate (E.C) willbe calculated,if
04500
PROTOCOLNO.: 454/091799/NAV96/SUB454
3MLABREQUENSOT. U2723
WILDLIFE INTERNATIONAL, LTD
4
possible,atthe72 and96 hourintervals.Thenoobservedadverseeffectconcentration (NOAEC),which isthe highesttestconcentrationthatinducesnoadverseinhibitoryeffectongrowth, willbedetermined relativeto cachparameterat72and 96hoursbaseduponevaluationofthestatisticalresultsandthe dose-
responsepattem.Attheendofthe96-hourexposure, algistaticeffectswillbe differentiatedfromalgicidal +
`effectsinthosetreatmentswhicharemaximally inhibited.
TestSubstance ~ MATEARNDIMAETHLODSS
Informationonthecharacterization oftest,controlorreferencesubstancesisrequiredbyGood LaboratoryPracticeStandards (GLP). TheSponsorisresponsibleforproviding WildlifeInternational,Ltd.
writtenverificationthatthe testsubstancehasbeen characterizedaccordingtoGLPspriortoifsuse the
`study.Tfwrittenverificationof GLPtestsubstancecharacterizationisnotprovidedto WildlifeInternational, Ltd,itwillbenotedinthecompliancestatementofthe finalreport.TheattachedformIDENTIFICATION
OFTEST SUBSTANCE BYSPONSOR (AppendixI)istobeusedtoprovideinformationnecessaryfor
GLPcompliance.
"TheSponsorisrespofornalslii nfobrmaltieonrelatedtothetestsubstanceandagreestoaccept any unusedtest substanceand/ortestsubstancecontainers remainingattheendofthestudy.
Fest Seton Brsgueals
"ThedesiredconcentrationsofPFOSwillbeobtainedbypreparing asinglestocksolutioninmedium
`anddilutingthe appropriatevolumeofstocksolutionwithmedium.Thetestsolutionsmayalsobeprepared
`using secondary dilutionsofthe primary stock, based upontheconcentration desired.
Thetestsubstancewillbeadministeredto thetestorganisminalgal medium. Thisrouteof
administration wasselectedbecauseitrepreseatsthemostlikelyrout ofexposuretoalgae,whichexist suspineawanterdcoelumdn.
`TestOrganism Thetestspecieswillbethefreshwaterdiatom,Naviculapelliculosa.Thisspeis creprieseentatsive
ofanimportantgroupofalgaeandwasselectedforuseinthetestbaseduponpastuseand caseofhandling.
inthelaboratory. Stockcultures,obtainedfromtheCultureCollectionofAlgaeattheUniversityofTexas
PROTOCOL NO: 454/091799/NAV96/SUB454
04501
3MLABREQUEST NO. U2723
WILDLIFE INTERNATIONAL, LTD
ose
atAustin oranothersupplier,willbemaintainedin culturemediumat Wildlife International,Ltd.for a
`minimumoftwoweekspriortousein atoxicitytest.Diatoms usedintoxicitytestswillbe inexponeatial
`growthphase,whichisdefinedastheperiodofgrowthwhenalgal celsaredividingat aconstantrate.
Justpricrtobeginningthetest,aninoculumofthestockculturewillbepreparedsothateachmilliliter
ofinoculumcontainsenougheelstoprovidean italclldensityofspproximately 10,000cells.incach
replicate.
-
Culture Mediom
CulturemediumpreparedaccordingtoWildlifeInternational,Ltd. StandardOperatingProcedures `willbe usedasdilutwiatoenr.Theconcentrations ofthecomponentsinthemediumare presentedin Table 1(2). Stocknutrientsolutionswillbepreparedbyadding reagent-grade orbetterchemicalstoWildlife International,Ltd.wellwaterpurifiedbyreverse-osmosis. Appr volo umespofr thei stoa ck nt utrie ent `solutionswillthenbe diluted with purified wellwaterto preparethe medium. Themed wililbeufilm ter `sterilized (0.22jum)orautoclavedpriortouse.Analyseswillbeperformedatleastonceannuallyto
determinetheconcentrationsofslectedorganic andinorganicconstituentsofthewellwaterand resultsof
themostrecent GLP compliant analyseswillbesummarizedinthefinalreport.
`Specificationsforacceptablelevelsof contaminantshavenotbeenestablishedforculturemedium. However, therearenolevels of contaminants reasonablyexpectedtobepresentinthemediumthatare
`consideredtointerferewiththepurposeorconductofthestudy.
TestApporats
`Test chambers willbe sterile, 250-mLpolycarbonateErlenmeyerflaskspluggedwithfoamstoppers
andcont100amLiofntesitsonlutigon. The testchamberswillbeindiscripmosiintiaontedeolnay
`mechanicalshaker tableinanenvironmentalchamberandwillbeshakencontinuously at approximately 100
rpm. Testchamberswillbe Labelledwiththe project number,test concentrationand replicate. .
Con
`Test flaskswillbe heldat24 +2Cundercontinuouscool-whitefluorescentlightingatanintensity
of4310 0% l0 ux.Lightintensitywillbemeasuredat fivelocationssurroundingthetestflaosnDkays0
004502
PROTOCOLNO.: 454/091799/NAV96/SUB4S4
3MLABREQUENSOT. U2723
~
Hs
W--I_L--DLIFE INTERe NATIOy NAL,e LD -6-
ofthetest. Temperaturewillbemeasuredtwicedailyin a containerofwateradjacenttothetestflasksinthe
cavironmentalchamberusing acalibrated,hand-heldmercurythermometer.
.
`ThepH ofcachtreatmentandcontrolgroupwillbemeasuredattetinition andterminatingusing. ~ aFisherAccumet Model 915pHmetero equivalent. SafmorppHmelasuerecseatattetinitiavtiilobne collectedfromtheindividualbatchesoftestsolutionpreparedforcachtreatmentand controlgroup.Attest termination,pHwillbemeasuredinpooled samplesoftestsolution collectedfrom cachofthethresreplicates ofeach respectivetreatmentandcontrolgroup. J
`Celldensitieswillbemonitodurreindgthetes byconductingcllcountsusingabemacytometaendr amicroscope. Onesamplewillbecollectedfromeachreplicateofthetreatmentandcontrolgroupsat approximate 24-hourintervalsduringtheexposureperiod.Thesampleswillbeevaluated immediatelyor
willbeheldinthedark at approximately 4Cuntil cellcountscanbeperformed. Eachsamwilplbeldilueted
usinganelectrolytesolution(1soton),asneeded,to maintaincounting accAusmarll aamocuntoyfe.ach samplevillbeloadedonto 8 hemacytometerandthetotal mumberofcellsin10gridswillbecounted. The celldensity ofthesamplewillbecalculatedbasedonthemean mumberof celspergrid.
Microscopic examination ofalgaewillbemadetoassesschangesingrossmorphology. Algaewill . `beobservedtoassesschangesincel shape,cllcole,andagbregation (clumopfithnecgel)l. Observations. willbeusedtodeterminewhethertreatment.elated effcts includethosechanges ingrossmorphology.
Saul
.
SamplesoftheexposuresolutionswillbecollectedonDays0,3and 4andanalyzedfo tetsubstance. `concentrations.Day 0sampleswillbetake fromeachbatchofestsolutionpriortotheirdistributionamong. thereplteistcchaambteres. Day 3samples willbecollectedfromtheextrareplicatetest chamber(s)included. inthetesttoprovidesampleforthatanalysisandfromtheabioti replicates.Ifmorethanonereplicates includedfortheDay 3 sampleanalysis,thenthesolutionsfromeachreplicatewillbecompositedto provide. thesafomrthp attrl eatme ent. Day 4sampleswillbecompossoiluttieondsfromthethreereplicates remaatthieenndoifthne tegst.
PROTOCOLNO: 454/091799/NAVI6/SUBAS4
604503
3MLABREQUESNOT, U2723 wast
WILDHLiIFeE NITNeTRENRaNAmTiIOONNAALL,,ULTBD 7.
`Samplescollectedon Days 3and 4maybecentrifugedorfilteredtoremovealgalcelsprior to analysis. Sampleswillbeanalyzedimmediatelyorplacedinanappropriatestoragecontainer (e.g,
`polypropylene or polyethylenebottle) and storeduntilanalyzed.`The sample scheme is summarizedbelow:
[vco|cou o|iono s mows e | PROPOSED NUMBERS OF VERIFICATION SAMPLES
Control
SolveatControl(fnccdd)
Level1-LowConcentration Level2 Leve3l Leve4l Level5
(| ws Level 6-HighConcentration
)
`The abovenumbersofsamplesrepresentthosecollectedfrom thetestanddonotincludequality `control (QC)samplessuchasmatrixblanksandfortificationspreparedandanalyzedduringtheanalytical
`chephmasieosftthesrtuydy.
ge Cn
Chemical analysis of the samples willbeperformedbyWildlife Intemational Ltd.`Theanalytical `method usedwill be based uponmethodology providedbythe Spoan ndis deno tifiredinAppendix IL.
-- mea. . `Modificationsmade totheanalyticalmethodwillbe documentedintheraw dataand described inthefinal
a
Biomassexpressedas areaunderthegrowthcurvewillbecalculatedforth treatmentandcontrol
roupsusingthe following formula:
04504
PROTOCOLNO.: 454/091799/NAV96/SUB4S4
3MLABREQUENSOT. U2723
TWHILDDULIrFEEIIMNeTRENRNAAITOINONAALLY, 210D,
.
ER
A= (ONYX HNN NY 2th) + AHNar Ne2NV2te))
Where:
AN==AMrecaasuunrdeedrnuthmebgerorwotfhcceulrsvfea.Latt,
:
NN;, ==MMeennaauummbssberoouuff cceerrllilssef/emmLLaaddtt,t;
N,==TimMeofefirssmtummebsaesuorufemcerenltsaemfLeardtbte,ginningoftest(hours)
ta==TTiimmesooffnSetchomnedamseuarseumreenmtaefnttearfbteergibnengiinngnoifngtoesftt(ehsotu(rBso)urs)
Growthrates)willbecalculatedfothe treatment ndcontrolgroupsusingthefollowingformula:
InN,InN
re
-- to
1N4 == AMvereagesapnecuimsfbiecgruroofwctrehlrsaietneLadtt,
:
Ntoo==TMimeeofbaemgasmibnenruooifftnceregtls(eihoauLrasd)t,
t=Timeof thmeasurementafterbeginningoftest (hours)
Pesocatinhibitionwillbecalculate forcachramentgroupbaseduponmeancel deasity,biomass: andgrowthrateusingthe followingformu:
PerccotInhibition =M_eRaenspConose n-MetRaensrTproenoastemlent X 100 `MeanControlResponse
ECvalues willbedetermined,wheapossible,usingincrinterpolation oothersuitabletechniques withtreameatresponse i.celldensitics,biomass an growth ate)and exposure onceatrationdatafor cach 24-intehrvalooftuetrest.
oT
"Ano observedadverseeffectconcentration(NOAEC)willbeseletedbasedon aevaluationofthe
dose responsepattemandtheresults ofthestatisticalanalysisusing individualreplicatevaluesofthecel
densitybioanmdgraowtsh raste. Statisticallysignificantdifferencesbetweenthecontrolan thetreat
groupswillbeidentifiedusinganalysisofvariance (ANOVA)and atestto comparetreatmentmean
PROTOCOL NO: 454/091799/NAVIG/SUBAS4
3MLABREQUESNOT.U2702304505
_ WILDLIFE INTERNATIONAL-- , LD . -9-
responsestothecontrolresponse (c.g.Dunestest orBonferroni's tes).Datawillbeassessfeodr c`noorrrmeacliatnyaycnodnhdiotmioogneonefitoynofnovrarmiaalnicteysorpurinoertquoapleerrfroorrmvianrigatnhceesA,NiOfpVoAss.ibTler.Iafnassolfveonrtcwmoinlatlrbtoleiiussouensdetsod inadditiontothenegativecontrol,theirrespoaseswillbecomparedusingStudent' ttst. Datafromthe 0cnirolgroupswillbepooledfor treatment velanalysesif nostatisticaldifferencesexist.Otherwise, allcomparisonswillbemadeto th solve coutrolgroup. Statistical ommparisonsforthe ANOVAandthe comparisonofmeanresponseswillbecadoutusingTOXSTATV3.5software(4) orequivalent.
`Records to bemaintainedfoR rdatE ageneC rTatOedO BaEt WMR iAldIliNfD eTIAntIeS NrnEatDional,Lid.willinclude, butnotbe
limitedto:
1. Copyofsignedprotocol.
2. Identificationandcharacterizationofthetes substance,ifprovidedbySponsor.
3. Datesofinitiationandterminoafteitoesnt.
:
4. Souccofalgae.
5. Cultureconditions.
:
6. Growth measurements.
7. Calculationandpreparation of testconcentrations. 8. Observations.
. ;
9. Ifappliable,themethodsusedtoanalyzetetsubstanceconcentrationsandtheresults of analytical
measurements
1101.. STteastitsctoincdailtciaolnuslaatnidopnhsy.sicallchemicalmeasurements. |
12. Copyoffinalreport.
Afinalreportof theresultsofthest[uFdyIwNiAlLlbReEprPeOpaRrTedby WildlifeInternational, Ltd.Thereport willinclude,but no belimitedto thefollowing,when applicable:
1. Nameandaddressofthefacilityperformingthestudy.
PROTOCOLNO.: 454/091799/NAVSG/SUBAS4
04506
SMLABREQUESTNO.UZ723.
W_I--LDLIFE INTERNATIONAL,aLo
-10-
-
-
2.
Datesonwhichthestudywasinitiatedandcompleted.Itisth responsibilityoftheSpoasorto providethefinaldatethatdata rorecordedforchemistrypathologyandlorsupportingevaluations
thatmaybegenerateda otherIsboratoties.
3. Astatement ofcompliance signedbytheStudyDirectoraddressinganyexceptionstoGood
LaboratoryPractice Standards.
4. Objectives andproceduresstatedintheapprovedprotocol,includinganychangesintheoriginal
protocol "
5. Thetest substanceidetiiedbynan,chemicalabsract umberorcode number,strength,puriy,
composition,andothercharacteristics povidedbytheSpousor.
6. Stabilityandsolubilityofthetestsubstancesundertheconditionsofadministration,ifprovidedby
the Spoasor.
7. Adescriptionofthemethodsusedtoconductthetest
8. Adescriptionofth testsystem,includingthesourceofthe estorganismsandtherscientificname,
9: Adescriptionofthepreparation ofthetestsolutions,methods sedtoallocateorganist tothetest
c-hambersandbeginthe est, numbersoforganismsandc hambers pertreatmeat,and duration ofthe
10. Adescriptionofcircumstancesthatmayhaveaffectedthequalityo itegrityofthedata.
11. ThenameoftheStudyDirectorandthenamesofotherscicatits,professionals,andsupervisory
personnelinvolvedinthe study.
12. Adescriptionofthtransformations,calculations,o operationsperformedonthedats,8summary
andanalysisofthebiologicaldata andanalyticalchemistrydata, and astatementoftheconclusions
drawn fromtheseanalyses.
13. Statisticalmethodsemployedforanalyzingthedata.
14. Thesigoedanddatedreportsof achoftheindividualscientistsorotherprofessionalsinvolvedathe
sud.
.
15. Thelocationwhererawdataandfinal reportarotobestored.
16. AstatementpreparedbytheQualityAssuranceUilistingthedatestha styinspectionsandmits
weremadeandth datsofanyfindingsreportedto the StudyDirectorandManagement.
17. Ifitisnecessarytomakecomectonsoradditionsto afinalreportaferithasbec acocpted,such
`changesshallbemadeintheformofan amendmentissiedbythe Study Director. Theamendment
PROTOCOLNO: 454/091799/NAVO6/SUBASS
3MLABREQUESTNO.
04507
U2723
_WI--LDLIFE INTERNATIONAL, 10 --
sahlatlerlactlieoan.rlAyimdeenadtimfeyntthsesphaarlltboefstihgeofeidnaalnrdedpaotretdtbhyattihesSbteuidnygDaimreecntdoer,dandthe reasonsfor the
Plannedchangestothe
CHANOFGPRIOTN OCG OL
protocalwillbeinthe formofwritenamendmentssignedbythe
Study
DirectorandtheSpoasar'sRepresentative. Amendmeatswillbeconsideredasprtoftheprotocolandvill
beatatotchefihnalpreotocdol. Anyotherchangeswillbe itheformofwritendeviationssignedbythe
Study Directorandfledwiththe raw data.All changes otheprotocolwillbeindicatedinthe finalrepart.
`GOOD LABORATORYPRACTICES
`ThisstudywillbeconductedinaccordancewithGoodLaboratoryPracticeStandardsforEPA (40 `CFRPert 160and/or Prt792); OECDPrinciplesofGoodLaboratoryPractice (ENV/MC/CHEM(98) 17); aod JapanMAFF(59 NobSen, Notification No. 3850, Agricultural Production Bureau). Eachstudy
`conductedby WildlifeIntemational,Ltd.isroutinelyexaminedbytheWildlife International,Ltd.Quality
AssuranceUnitforcompliancewithGoodLaboratoryPractices, StandardOperatingProceduresandthe.
specifiedprotocol. Astatementof compliancewith Good Laboratary Practiceswillbepreparedforall
portionsofthestudy conductedby WildifeIntemational,Ltd. The Spwiollbn eressponsoibler for `compliancewithGoodLaboratoryPractices forproceduresperformedbyotherlaboratories (e.g.residue anaolrpyathsoleogys). RawdataforalworkperformedatWildlifeIntemational,Lid.and acopyofthe finalreportwillbefiledbyproject numberinarchiveslocatedon theWildlifeInternational,Lid site,orat an altemativelocationtobespecified in thefinalreport.
PROTOCOL NO: 454/091799/NAV96/SUBAS4
04508
3MLABREQUEST NO. U2723
W-I_--LDLIFE INTERNATIONAL, LD 12-
REFERENCES
OOOO
1
US. Environmental ProtectionAgency. (draft), OPPTS Number 850.5400: Algal
199.Series 850- EcoloEgffiectcs aTelst `Toxicity, Tiers Iand IT.
Guidelines
2 TAeSstTsMwiStht MicraoaGlnguaied d.Aem1a 2e1r8ir -c9a0d nE.Soc19i9e0t.ySftoarnTedsatridnGguainddeMfaotrerCioanldsu.c`tPihniglaSdtealtpihci9a6,-PHenonusryTluvxainciiat.y
3 CNoonrcbeenrtgr-aKtiinong,(ITC,J).Ap1p9r9o3.acAhL(iVneerasriIonnt2e.r0p).olUaStSi.onEMxevitrhoondmefnotraSluPbrloettehctailToonAxigceintcyy:,TEhnevIinrhciobmienttiaoln
Research Laboratory, Duluth, Minnesota.
4 `WCehseyte,aInnec,.aWnyodmDi.nDg..Gulley. 1996. TOXSTATRelease3.5.`WesternEcoSystemsTechnology,Inc.
:
\
PROTOCOLNO.: 454/091799/NAV96/SUB4S4
04509
3MLABREQUEST NO. U2723
A WILDLIFE INTA ERNATIONAALy, LIDD
ee-- BER
TABLE 1
-
FRESHWATERALGALMEDIUMWITHSILICA CONSTITUENTS
--_-- Component
_-- MgClr6r0 CaCle2H0
Nominal Concentration
:
241460 mmLg
H;BO;
MaCls4H,0
ZnCl,
0.1856 mg/L.
0416 mg/L
328 gL
FeCly6H0
0.1598 mg/L.
CNoCelM#o6HO,20HO
"
CuCly2H,0
1428 gL
726 gL
0012 gL
Na:EDTA2H,0
0.300 mg/L.
MNgaSNOO;STHO
21545700 mmgg/tL
KPO,
1.044 mg,
-
NNaasHCSOi,O9H0
2105000 mmg/gL
NesSeby:SH0
0010 mg/L
"ThepH ofthemedium wilbe adjusted,asnecessary,to7.5 0.1using 0.1
_'--N'_N--aOH or 10%HCL
-
A tm rem cern
PROTOCOL NO.: 454/091799/NAV96/SUB454
04510
3MLABREQUENSOT. U2723
-- WILDLIFE INTE_ RNATIONAL, 10----y Sue
APPENDIXT . DENTOIFTEFSTSCUBSATANTCEBIYSPOONSNOR
TobeCompblySepotnsaor
L RTeefsetrScuabcsetSauncnedIadrednti(tfy a(pnpalmiectaoblbee:usAneadliynttihcaelreSptoarntd)a:`rdF: ONS(APerfluorooctane SulfonicAcid PotassiumSalt
TestSubstanceSampleCodoorBatchNuInmtbeerrna:l_St_aLnodatr2d1:_71,12.2HH HHPerfluorooctaneSulfonicAcid
TestSubstancePurity(%ActiveIngredient): _ 98.9
I TestSubseCharctrizaicn
ExpirationDate: _2008
SHp timoe yortsoi oongsBphtlsy nnmdoyeo posaiocnom coeee rrCdcFhrtSin ielsis?s Ye x Mo
IL TestSubsStotragaeConndictioens
Plas fit recommendedscragecodons aWide trata],Lid.
| HR iste in ertt oecodon
Yo
J --
_--
SER IV. TestConeenttions: .
I--
pr RE V. Tofxormation:
ont 100%
Memmalian:
Aqui:
RatLDSO 251mg/kg MouseLDSO_N/A
InvertToxicityGOLCS0) FishTnicy(C50)
: 226g
Raith Trou: 1
A
FT
Ot Todicty Information ochingfing ofcrcsc sbetrni5c):
x MN mat
PROTOCOLNO: 454/091799/NAVS6/SUBASS
04511
3MLABREQUNOE.US27T23
WILDLIFE INTERNATIONAL, 10
"15.
APPENDIIXI
-
AnalyticalMethod ProvidedbySponsor
`methodSsr:anples willbeanalyzed basedupon procedure provide bytheSpousorinthefollowinganalytical
L LRMeiecqlduliiuduormCoohcrioamoagtSouglrfaopnhiycMAacisdsPoStpaescstiruommeStarlyt((LPCROMSS))IMneFtrheoshdwfaotrert,hSealDtewtaetremrinaantdiAolngoalf analyAz tchoeptyeotfstahmepalbeoswvielmlebtehdoodcwuimlelntbeemdiainnttheairnaewddianttahaenrdaswudmamtaar,Tizheed aictthualfminealtrehpoortd. kulsoedgtyo
\
PROTOCOLNO.: 454/091799/NAV96/SUB4S4
I
04512
3MLABREQUENSOT. U2723
. WILDLIFE INTEO RNATIONt AL Lo.
PROJECTNO 454-112 (hPagee llofl1
AMENDMENT TO STUDY PROTOCOL
STUDY TITLE: (PNFaOvSi:culaApel9l6icHulOoUsaR) TOXICITY TEST WITH THE FRESHWATER DIATOM
PROTOCOL NO: 454/091799/NAVI6/SUB4S4 SPONSOR: 3M Corporation
AMENDMENT NO: 1 PROJECT NO.: 454A-112
EFFECTIVE DATE: February 23,2000
3MLABREQUESTNO: 12723
AMENDMENT: Page2 ~;
Add: EExxppeerriimmeennttaall STtearrmtiDnaattei:onFDeabtreu:arFye2b5r,ua2r0y0209, 2000
.
TTeesstt
SCounbcsetnatnrcaetiNoon.s:
4N6e7g5ative
Control,
70.0,
92.5,
125,
167,
225, .
300
and
395
mg
aL/L
REASON: Theaboveinformationwasnotknownwhen theprotocol wassignedbytheStudyDirector
AMENDMENT: ExperimentalDesign, Page 3
Change: Targetconcentwrilalntotieoxcneesd 100mg/L.orth solubility limi ofthetestsubstanceinwater. To: `Targetconcentrationswillnotexceed 1000mg/Lorthesolubilitylimitofth testsubstanceinater REASON: Nominaltestconcentrations exceed 100 mg/L.
Ero STUDY DI R
J LABORATORY MANAGEMENT :
yh DATE
Pa
-- A=LE Setitrne,
SPORSORS REPRESENTATIVE
] DA
004513
hthep00
-
W_ILDLIFE Ir NTERNATIOe NAL Lo.
PROJECTNO.: 454A-112
0 PPaegelLoaf |
AMENDMENT TO STUDY PROTOCOL
STUDYTITLE: (NPFaOvSi:culaApel9li6c-uHloOsUaR) TOXICITY TEST WITH THE FRESHWATER DIATOM
PROTOCOL NO: 454/091799/NAV96/SUB4S4 SPONSOR: 3M Corporation EFFECTIVEDATE:February 28, 2000
AMENDMENTNO.: 2 PROJECT NO.: 454A-112 3MLABREQUENSOT.: U2723
AMENDMENT:Biological MeasurementsPage 6
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Atthe eadofthetest,algistatic effects,whichresultintheinhibitionofcellgrowth,willbedifferentiated from algicidaleffects,whichresultinthedeath ofcells.Aliquots(0.5mL) oftest solution willbetaken fromcachreplicateofthosetreatmentgroups wheregrowthismaximallyinhibited.`Maximallyinhibited treatmentsarethoseinwhichit cannotbevisuallydeterminedif livealgalcellsarepreseat(e.g,absence. c`ouflctoulroermteodtihuemtetsotsoalcuotnicoenn)t.r`aTthieosneoafltiqhuottesswtilslubbsetpaonoceltehdabtythceoonrceetinctarlaltyiosnhaonudlddinlouttaefdfweictthgurnotwrteaht.eAd ``nmeLgwatiitvheccounlttruroelwmieldlbieupmr.`epGarroewdtbhyodfiltuhteisnegsaub0c.u5l-tumrLeasliwqiuloltbfermoomnointoerneedgaftoirveacmoantxriomlruemploifcat9edtayos1t0o0 determinewhetherinhibition of growth observed during the testisreversible.Sampleswillbecollected forcellcountsatrecovery phase initiation,termination andatapproximately3-dayintervalsbetween initiationandtermination. `Therecovery phasemay beterminated oncealgalgrowthissufficientto indicatethatthealgalcellscollectedfrom thetreatmentgroups havefully recoveredfromeffectsdueto thetest substance.This will be determinedthroughvisualexamination ofthe recoverytestsolutions.
REASON: Toadd arecoveryphasetotheprotocol.
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