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PFOS: `A 96-HOUR TOXICITY TEST WITH THE "FRESHWATER ALGA (Selenastrum capricornutum)
FINAL REPORT
`WILDLIFE INTERNATIONAL LTD. PROJECT NUMBER: 454A-103A 3M LABREQUESTNO. U2723
nd U.S. Environmental Protection Agency
Series
-
850O-PEPcTolSogNiucamlbEefrfe8c5t0s.T5e4s0t0Guidelines
(OECD Guideline 201
AUTHORS:
Kurt R. Drotar
Henry O. Krueger, Ph.D.
STUDY INITIATION DATE: December 4, 1998
STUDY COMPLETION DATE: June 4, 1999
AMENDED REPORT DATE: April 26, 2000
Submitted to
Envir3oMnmCeonrtp]orLaatbioornatory
935 Bush Avenue
St. Paul, Minnesota 55144
Wildlife I45n98tCoemrmenrcae Dtriiveonal Ltd. Easton, Vargiand 21601
(410) 822-8600
Page 1 of 58
0 00607
AMENDED
WILDLIFE INTERNATIONAL Lm.
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PROJECT NO.: 454A-103A
GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT
SPONSOR: 3M Corporation TITLE: PFOS: A 96-Hour Toxicity Test with the Freshwater Alga (Selenastrum capricormutum) WILDLIFE INTERNATIONAL LTD. PROJECT NUMBER: 454A-103A STUDY COMPLETION: June 4, 1999 REPORT AMENDED: April 26, 2000
Thisstudy wasconductedincompliancewith GoodLaboratoryPracticeStandardsaspublished by tPhreincUiSpSl.esoEnfvGirooondmeLnatbaolraPtroorteycPtriaocntiAcge,enOcCyDiEn/4G0DC(9F2R) 3P2a,rtEsnv1i6r0onamnednt M79o2n,og1r7apAhugNuos.t451,98P9a;risOE19C9D2 and Japan MAFF, 59 NohSan, Notification No. 3850, Agricultural Production Bureau, 10 August 1984 with the following exceptions: characteTrihzaettieosntwsaubssptearnfcoerwmeadsanccootrcdhianragcttoer3iMzedSitnaandcacrodrdOapnecraetwiintghPfruolcledGuLrePscaonmdplMieatnhcoed;sh,oawnedvaelrl,rtahs e dataarebeingmaintainedinthe 3Marchives.Thetestsubstance isbeingrecharacitneacrcoirdzaencde with GLP accordTanhceestwaibtihliGtyooofdLtahbeotreasttosruybsPrtaacntciecuenSdtearncdoanrddsi.tionsofstorageatthYetes steWasnotdeterminedin
ul STUDYDIRECTOR:
Kurt R_Drotaar
DATE
Seaior Biologist
SPONSOR:
4.
Ra f
AMENDED
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WILDLIFE INTERNATIONAL Lm.
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PROJECT NO.: 454A-103A
`QUALITY ASSURANCE STATEMENT
`Thisstudywas examinedforcompliancewithGoodLaboratory PracticeStandardsaspublishedby
the U.S. Environmental Protection Agency in 40 CFR Parts 160 and 792, 17 August 1989; OECD PrinciplesofGood Laboratory Practice, OCDE/GD (92) 32,Environment Monograph No. 45, Paris 1992; and Japan MAFF, 59 NohSan,Notification No. 3850, Agricultural Production Bureau, 10 August 1984. "Thedatesof allinspectionsandaudits andthedates thatanyfindingswerereported tothe StudyDirector `andLaboratoryManagementwereas follows:
sce
DATECONDUCTED: STUDYDIRDEACTTOER.REPORMTAENDATGOEMENT:
i oaion InitialTrial 454A-103 Fey 18,99
Fay18.199 Fehr 19,199
-
nS
FrayRud, 1999 Fay 1999 Februay24, 199
DutRepotasdDes Mash 9,159 Meh15,199 Marh30,1999
DnfRemiDe DTesetSufbsitTarnnicaeliPr4et5p4airAa-tv1i0one3A fAinalytimcail Data and Four AmendedRegn
Apil 9, 1999
MaylomdlL1% Moy Omi Je, 199 AS00
April9, 1999
Mayll, 9 May, 19 3 Sot, 199
ARR0
oy Apil 12,1999 Moy. Je,1999 A200
iLy Timothy A' Springer, Ph.D.
`Manager, Regulatory and Technical Support
Satfoo
DATE
AMENDED
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WILDLIFE INTERNATIONAL Lm.
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PROJECTNO.: 454A-103A
REPORT APPROVAL
SPONSOR: 3M Corporation TITLE: PFOS: A 96-Hour Toxicity Test with the Freshwater Alga (Selenastrum capricornutum) `WILDLIFE INTERNATIONAL LTD. PROJECT NUMBER: 454A-103A
STUDY DIRECTOR:
as, \
Kurt R_ Drotar Senior Biologist
:
Sf36/08
DATE
MANAGEMENT:
Henry 6. PhD. Director,Aquatic Toxicology `and Non-Target Plants
5/50Jee
DA
AMENDED
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WILDLIFE INTERNATIONAL Lo.
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"TABLE OF CONTENTS
PROJECTNO: 454A-103A
TIS ACE msmmpmmmmmnsesmmmmmmms---------- LE
:
TABLES
:
Table 1 - SummaryofAnalyticalChemistryData .............
-
19
Tabl2e - Temperature Measuremeas ...........
tts 20
Tabl3e - LightIfensityMeASuICmENts.......vovovevenevsrsoneevnen 21
BE
--
nll
Tabl5e - Pg MeanCellDensitiesand PercentInhibitionforEach
Tabl6e - M2e4a-HnoAUrreIansrUVnadleDrUtFhIeOGGrtohwetThECSurvear ndPerr cent r InhibitionforEach
28
Table 7- MeanGrowthRatesandPercentInhibitionforEach24-Hour Interval
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TA OFB COL NTEE NTS ~Continued-
Tabl8e-EEXCP1O0S,UTEEC5P0IaOnd.E.C.90ValuesBasedonCellDensityOverthe96-HourS--. Tabl9e- EtChe1096,-EHoCu5r0EaxnpdosEuCre90PVearliuoeds.B.a.sedonArea UndernthemGromwthmCurmveO--ver------
`Table 10- EC10,EC50and EC90 ValuesBased on GrowthRateOverthe 96-Hour
"Table 11 -Cell DensitiesfortheReCOVEryPHASE .............c.ccovrvsvvnsriurrsssssssmsssnsessssssnsssssesss 29.
FIGURES
Figu1r-e NegativeControlAlgalGrowth,Expressed inCellDensity, Duringthe96-HourExposue.......
men 30
Figure 2- Concentration-ResponseCurve,ExpressedinCellDensity.
Figu3re- RecoveryPhase ResponseCurve, ExpressedinCell DENSI
31
............oerrinncc: 32
APPENDICES
Appendix I~ Analyses of Pesticides,Organics, MetalsandOtherInorganics
in WildlifeInternationalLtd.Well Water...................
ermine: 38
Appendix II-I TheAnalysisofPFOS inFreshwaterAlgalMediumin Supporoft `WildlifeInternationalLtd. Project No.: 454A-103A........occcovve
--J
Appendix IV- CellDensityforEachReplicate Per TreatmentOver the
96-HourExposurePeriod.......... tt
----------
Appendix V-
AreaUndertheGrowthCurvefor EachReplicate Per TreatmentOverthe
96-HOUTEXPOSUTEPEriO.....rv rr ttre
53
AppendixVI - GrowthRateforEachReplicatePer TreatmentOverthe 96-HourExposurePeriod.54
APEXVIL+ CHA80DPIOGIOGEOL S rms 55
AppendixVIII - PersonnelInvolvedinthe
Study.
te sna 36.
APPERix TX = REPORtAMEIMEDE css
ST
AMENDED
000612
WILDLIFENINETERRNNAATOIONNAALLtLom.. 7.
PPRROOJJEECCTTNNOO.. 445544AA-l10033AA
Ey `SPONSOR'S REPRESENTATIVE: DLAOTCAATAINODN OAFCOSPTUYDYO,FTRHAEW FINAL REPORT.
WLTIDL,DLPIRFOEJIENCTTENRUNMABTEIRO:NAL TEST SUBSTANCE: STUDY:
MCEOANCNEMNETARASTUIROENDST:EST TEST DATES:
LENGTHOFEXPOSURE: TEST ORGANISM. SOORUGRACNEISOMFST:EST C7E2L-LHODUER ETCSY0: 95%CONFIDENCE LIMITS: 9956%:HCOOUNRFIECDIE0N:CELIMITS: 9956%:1COOUNRFIECDSE0N:CELIMITS: 9965:%HOCUORNFEICDOE0N:CE LIMITS: 72-HOURNOAEC: 96-HOUR NOAEC
SUMMARY
M Corporation Ms. Susan A. Beach 'EWaisltdoinf,eMInDtern2a1ti6o0n1al Ltd.
454A-103A
PFOS (Perfluoroocatane SulAfcidoPontasisicum Salt) P(SFeOlSe:nasAtr9u6m-cHaopurricTooxrincuittuym)Test with the Freshwater Alga NegContarol,t5.5i,11,v21,e44, 86and 179mg aL
EExxpepriomsentTaleSrtamrti- nAApripl 1a2, 1169,919999
.
Experimental Termination-April 21, 1999
96Hours
.
cshvwater Alga (Selenastrumcapricormam Wildsitofn,eIntemaMatrylianodn2al16L0td1. ~,
4740amngd a7i8lm,gai/L, 4439amngdaSiOlmgai/L 761m6g .7a3imalgna.id/L 110357amngda1i5/3lmgai /L 44mgaill A4mgaill,
AMENDED
000613
WILDLIFE INTERNATIONAL Lo.
-s-
PROJECT NO.: 454A-103A
C"AURREVAEUNDER THEE GROWTH 9T52%HOCUONRFEICDSE0N:CELIMITS:
96-HOUREC10:
195%CONFIDENCELIMITS:
9965-%HCOOUNRFEICDSE0N:CELIMITS: 96-HOURECS0:
95% CONFIDENCELIMITS:
T2-HOURNOAEC: 96-HOUR NOAEC:
SUMMARY (Continued)
5754.mangda8i2 mgai/L
49mgai/l
40and 50mg ai/L
67Im7gaTiaalmlngaidl 145 mgaill
125and 155 mgai/L
H4mgaill 44 mgaill
"GROWTHRATE 72-HOURECS0:
95%CONFIDENCELIMITS:
120mg ill .
103and 132mgai/L
96-HOUR EC10:
59mgai/L
95%CONFIDENCELIMITS: . 54and 63 mgai/l
i
9965-%HOCUORNEFCISD0E:NCELIMITS:
11261mga51i3/8almg0aid/L,
96-HOUR EC90: 95% CONFIDENCE LIMITS:
>179 mgai/l. Not Calculable
72-HOUR NOAEC:
44mgaill
96-HOURNOAEC:
4amgaill
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WILDLIFE INTERNATIONAL Lo.
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PROJECT NO. 454A-103A
INTRODUCTION
This studywasconductedby Wildiie InternationalLtd.for 3M Corporation atthe Wildiife Intemational Lid.aquatictoxicologyfacilityinEaston,Maryland. Thein-ifephaseofthetestwas conductedfromApril 12, 1t9oA9pril921, 1999. RawdatageneratedbyWildlifeInternationalLtd and a. copyofthe finalreportare filed under Project Number 454A-103A in archives located onthe Wildife IntematiLuod.nsiatle.
OBJECTIVE
The objectiveofthe study was to evaluate the toxicityof PFOS (Perfluorooctane Sulfonic Acid Potassium Salt) to the growth of the freshwater alga, Selenastrum capricormutum, during a 96-hour `exposureperiod.
EXPERIMENTALDESIGN
The freshwater alga, Selenastrum capricormutum, was exposed to a geometric seriesofsix test
concentrationsand anegative(culturemedium)control under static Conditions for 96hours. Three
replicatetestchambersweremaintainedforeachtreatment andcontrolgroup. Oneadditonalreplicatewas alsomaintainedforanalyticalsamplingonDay 3ofthetest.Inaddition, one"abiotic"replicate (test solutionwithoutalgac)waspreparedforthehighesttestconcentration. Notm estcoincenn tratiaonswlere selectedinconsultation withthe Sponsorandwerebasedupontheresultsofrangefinding anddefinitive tests. Thenominaltestconcentrationsselectedwere 5.7,11,23,46,91and183mgactiveingredient (aL. Meanmeasuredtestconcentrationsweredeterminedfromsamplesoftestmediumcollectedfrom eachtreatmentandthecontrolgroupatestinitiation,atapproximately72hours,anda testtermination.
Attestinitiation,aninoculumofthealgalcellswaspreparedat aconcentrationofapproximately 1.0X 10cells/mL. Theconcentrationofalgalcelsintheinoculum wasverifiedand 1.0mLvasaddedto eachtestchambertoachieve a nominalconcentration ofapproximately 1.0 X 10celisnL. Sampleswere
AMENDED
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collectedfromeachreplicatetestchamberatapproximately 24-hourintervalsduringthetestto determine.
cell densities. Celldensitieswere measuredforeachreplicateandwereusedtocalcarueaslunadetrtehe
`growth curveandgrowrattehs. Percentinhibitionvalues relativetothecontrolwerecalculated forcach `parameter overthe 96-hourexposureperiod. ECSOvaluesbased upon cell densities,areas underthe
growthcurveandgrowthrateswerecalculatedforeach24hourinterval. EC10and EC90valuweerse.
calculated, if possible, for the 72 and 96 hour intervals. The no-observed-adverse-effect-concentration
(NOAEC)wasdeterminedbaseduponstatisticalevaluationofthe 72-hourand 96-hourresults.Attheend
`of the96-hourexposure,algistaticeffectsweredifferentiated fromalgicidaleffects.
MATEARNDIMA ETL HOS DS
"Thestudywasconductedaccordingtotheproceduresoutlinedintheprotocol,"PFOS: A96-Hour
Toxicity Test with the Freshwater Alga (Selenastrum capricornutum)". The protocol was based on proceduresoutlined inthe U.S.EnvironmentalProtectionAgencySeries850-EcologicalEffects Test
Guidelines, OPPTS Number 850.5400: Algal Toxicity Tiers I and II (draft)(1) and OECD Guidelines for
TesoftChiemincalgs, 201 Algal, Growth Inhibition Test (2).
TestSubstance
:
:
The test substance was received from 3M Corporation on October 29, 1998 andwasassigned
`WildlifeInternationalLtd.identifniucmbaetr4i6o75n.Thetestsubstancewasdescribedas a white
powder. It was identified as FC-95 fromlotnumber 217 (T-6295). Information provided by the Sponsor
indicated apurity of 98.9%and anexpirationdateof 2008. Thetestsubstance wasreanalybyztehde
SponsorandtheCertificateofAnalysisdatedMarch9, 2000indicated apurityof90.49%. Thetest
substance wasstoredat ambientroom temperature.
PrepoafTerstaContcenitraotionns
Nominaltestconcentrawterieo5n.7s,11, 23, 46, 91 and 183mga.i./L,basedon atest substance
puorf9i0.t49y%. Allmaterials whichcameintocontactwiththe testsubstanceduringpreparationoftest
`concentrationswereconstructedofplasticorstainlesssteel. Aprimary stocksolutionwaspreparedinalgal
AMENDED
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WILODULITF:E NITNETRERNNAATTIIOONNAALLLLor.. 1.
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`mediumat aconceatrationof 183mga../L.Theprimarystocksolutionwasstirredwith a magneticstir plate for approximately24 hours 10aidinthesolubilizationofthe testsubstance. Afermixing,the primarystocksolutionwasproportionally dilutedwithalgalmediumtopreparethefiveadditionaltest concentrations. Allfinalestsolutionsappearedclearandcolorless.
TestOrganism
"Thefreshwateralga,Selenastrumcapricornutum,wasselectedasth tetspeciesforthis study. The species isrepreseatative ofan important groupoffreshwateralgae,and was selected for useinthe test basedupon apasthistoryofuseandcaseofculturinginthe laboratory. Orialggalciultnureaswelre obtainedfromUTEX -TheCultureCollectionofAlgaeattheUniversityof TexasatAustinandhavebeen maintainedinculturemediumat WildlifeInternational L1d., Easton,Maryland.Algalcellsusedinthi test wereobtainedfromWildlifeIntemationLadl.culturesthathadbeen activelygrowinginculturemedium foratleasttwoweeksprior totestinitiation.Thenegativecontrolorganisms wereexpectedtqexhibit exponentialgrowthoverthe 96-hourexposureperiod.Exponentialgrowth,definedastheperiodofgrowth wherethealgalcellsaredividingat aconstantrate,isindicatedbythelinearsectionofthegrowthcurve (Figure1). OECDguidelinesrecommendthatcontrolcellgrowthshouldhaveincreasedby afactor ofat east 16with3diayns.
CultureMedium
`Thealgalcellswereculturedandtestedinfreshwateralgalmedium (3). Stocknutrientsolutions werepreparedby addingreagent-gradechemicalsto WildlifeIntemationalLtd.wellwaterpurified by reverseosmosis.Thetestmedium waspreparedbyaddingtheappropriatevolumesofstocknutrient solutionstopurifiedwellwater (AppendiIx.ThepH ofthemediumwasadjustedto7.5 +0.1using 10% HCIandthemediumwassterilizedby filtration(0.22 um) priortouse. Arweareplerfyormsedaetlesast `once annually todeterminetheconcentrationsofselected organic andinorganic constituentsinthewell water. Theresultsofanalysesperformedtomeasuretheconcentrationsofselectedcontaminantsinwell waterusedby WildifeInternationalLid. arepresentedin AppeadixII
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TestApparas Test chambersweresterile, 250-mL polycarbonate Erlenmeyer flasks plwuithgfoagmsteoppders
`andcontained 100mLoftestorcontrolalgalmedium.Thetest chamberswerelabeledwiththeproject `number, concentration and replicate, and were indiscriminately positioned on a mechanical shaker table in anenvironmentalchamber designedto maintainthedesiredtesttemperature throughoutthetest.Thetest chamberswereshake continuato1u00srlpym.
Environmental Conditions Test flasks wereheldinancovironmentalchamberata temperatureof24+2C.Thetemperatuorfe
acontainerofwater adjacenttothetestflasksintheenvironmentalchamberwasrecordedtusicedaily duringthetestusing aliquid-in-glassthermometer.
`Thealgaewereheldundercontinuouscool-white fluorescentlightingthroughoutthetest. Thetarget lightintweasn430s0 +i43t0lyux. Lightintensitywas measuredatthefour comersandthemiddleofthe shakertabledaly.Lightintensity wasmeasuredusingan SPER ScientificModel 840006lightmeter.
`The pHofthemediumpreparedforeachtreatmentandcontrolgroupwasmeasuredattestinitiation andtermusiniganFishaerAtccuimetoModnel 915 pH meter. Samples forpHmeasurematetnestt initiationwerecollectedfromtheindividualbatchesoftestsolution preparedforeachtreatmentandcontrol group. Attesttermination,samplesoftestsolution werecollectedfrompooledreplicatesofthetreatment `andcontrolgroupsfor pHmeasurement.
AlgalGrowthMeasurements
Testmedium samples werecollectedfromthetreatmentandcontrol groupsforthedetermination of algalcelldensities. Singlesamples werecollectedfromeachofthethree"biological"replicatesper treatment andcontrolgroupat 24-hour intervalsduringthe96-hour exposure,andwere counted immediately. Cellcountswereconductedusing ahemacytometerand microscope. Eachsamplewas dilutedusinganelectrolytesolution(Isoton),asneeded,tomaintaincountingaccuracy. Asmallamount ofcachsamplewasloadedonto ahemacytometerand10gridswerecounted. Themean numobfecelrls
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pergridwasestimatedandthisvaluewasusedtocalculatethecelldensity ofthesample, Usingthis technique,theminimum quantifiableclldeasitywas 1.0 X 10 cells/mL.
All 96 ourcellcountsampleswereexaminedmicroscopicallyforatypicalcellmorphology(e, changesincellshape,sizeorcolor). Growthofcllin thereplicatetestchambersalsowasassessedfor `aggregationsorflocculation ofcellsandadherenceofthecelstothetestchamber.
`Statistical Analyses Celdensities,areaunderthegrowthcurvevalues,growthrates andpercentinkibitionvalueswere
calculatedusing "The SAS System for WindoRelweasse"6.,12 (4). Arcaunderthegrowthcurvewas calculatedforthecontrolandtreatmeatgroup usingthefollowingformula:
A= (ONY2EHNAN ZN)tH Mor NeNID)
where: A=Area
.
N, =Nominalnumberofcells/mLatt, .
N; =Meanmeasurednumberofcells/mLat t
N; = Meanmeasurednumberofcells/mLatt;
N.= Meanmeasured numberofcellsimLatt,
=timeoffirst measurementafterbeginningoftest(hours)
=timeof secondmeasurementaterbeginning oftest(hours)
ta = time of n measurementafier beginningoftest(hours)
Growth ateswerecalculatedforthecontrolandeachtreatmentgroupusingthefollowingformula
Grotto- ON)
where: N, = Meanmeasured numberofcells/mLatt, N, =Meanmeasured umberofcells/mLatt, ta =Timeofnmeasurementafterbeginningoftest(hours)
Percentinhibition valueswerecalculatedforeachtreatmentgroupasthe percentreductionincell density,areaunderthegrowthcurveandgrowthraterelative tothecontrolreplicates.Thefollowing formulavasused:
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WILDLIFE INTERNATIONAL Lm.
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PROJECTNO.: 454A-103A
PercentInhib=iMteainoCenllDensity + Mean CellDensity esos X 100
`MeanCellDensitycose:
Celldensities,areasunderthegrowthcurveandgrowthrateswereanalyzed statisticallytoestimate the EC10, EC50 and EC90 values (i.c., the theoretical test concentrations that would producea 10, 50or 90% reduction in each parameter, respectively) and 95% confidence limits at 72 and 96 hours. EC50 valueswerealsocalculatedforthe 24and 48hourtimeintervals. The ECvaluesand 95% confidence
limitswere calculatedbylineayinterpolation using TOXSTAT Version 3.5 (5). Cell densities, areas under thegrowthcurve and growthrates at 72 and 96hours wereevaluatedfor normality and homogeneityof variancesusingthe Shapirteost-anWdBairtlletkt's'tesst, respectively. Thetreagrtoupmsweerenthten comparedto thecontrolusing Dunnett'stest. Resultsofthestatisticalanalyseswereusedtodeterminethe
'NOAECvalues.
AnalyticalChemistry
A
Samplesof testmedium werecollectedfromthenegative controlandeach treatmentgroupat test
-initiation, atapproximately 72 hoursandat testterminationtomeasureconcentrations ofthe test
substance. Samplesoftestmediumcollectedattestinitiationweretakenfromtheindividualbatchesoftest
solutionpreparedforeachtreatmentandthecontrolgroup. Samplescollectedat 72hourswerecollected
from theadditional "analytical"replicates. Samplescollectedattesttermination were a compositeofthe
remainingreplicatesfor cachtreaa ndt them cone tron lgrotup. Thesamples wereplacedinplastic
(Nalgene)bottlesandwereanalyzedimmediatelywithout storage. Analytical proceduresusedinthe
`analysisofthesamplesarepresentedin AppendixIIL
RESULTS AND DISCUSSION
MeasoufTresteConmcenetrantiotns:
Resultsofanalysesto measureconcentrationsof PFOS inthetestsolutions arepresentedin Table 1
and AppendIiIxI. Nominalconcentrationsselectedforuseinthisstudywere5.7, 11, 23,46, 91and
183mga.i/L. Samplescollectedatthebeginningofthetesthadmeasuredconcentrations thatrangedfrom 82to98%ofnominal. Samplescollectedat 72hoursandtestterminationhadrecoveries thatrangedfrom
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WILDLIFE INTERNATIONAL Lm.
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PROJECTNO.: 454A-103A
82 10 105%and 90to 103%ofnominal,respectively.Whenthevaluesobtainedfortest niiation, at 72hoursandattestterminationwereaveraged,themean measuredtestconcentrationswere5.5,11,21, 4420d, 179 8 mg a6 i JL.
ObseravndaMetasiuroemenntss.
Measurementsoftemperatureandlightintensityareprescotedin Tables 2and3,respectively. The temperaturesrangedfrom23.60 25.8Candwerewithintherangeestablishedforthetest(24:2C). The lightintensity rangedfrom 3870to 4540huxandwaswithinthedesiredrangeforth test (approximately 3870104730 hux)(Tabl3e).Measurementsof pH were7.4 t0 75onDay 0andrangedfrom7.5to 8.4at 96hours(Table 4).ThepHofthe "abiotic"replicateattestterminawtasi7o.4n.
TheeffofePcFOtS upon Selenastrum capriwacsdeoterrminnedbuyetvaluuatimng differenciesn celldensities,areas underthegrowthcurveandgrowthrates.Meanvalueswereusedtocalculategrowth inhfiorebachi24-thoiurpoerinod. Meancelldensities, areasunderthegrowthcurveandgrowthratesand theircorrespondingpercentinhibitionvaluesarepresentedinTables 5,6and7,respectively.Celldensity, areaunderthe growthcurve and growthratevaluesforeachindividualreplicatearepresentedin Appendices IV, V and VI, respectively. ECI0, ECS0 and EC90 values and 95% confidence limits
calculatedfor each 24-hourintervalbasedoncelldensity, arca underthe growthcurveand growthrate are
presentedinTables8, 9and10,respectively.
Changesincelldensityindicatedthat exponeatial growthoccurredinthenegativecontrol replicates (Figure 1).Thecoefficientofvariation forcontrolclldensitywas 4%.Aft 72hoursofexposurcel,l densitypercentinhibitioninthe 5.5,11, 21,44, 86and 179mg a.i/Ltreatmentgroupswas 5.2,-1.9,-5.5, 11,72and 97%,respectively. Dunnett'stestshowedthatcelldensitywassignificantlyreducedinthe 86 and 179 mg .i/Ltreatmentgroupsincomparisontothenegative control (p<0.05). Consequenttlhye, NOAECfor72hourcelldensitywas 44mg ai/L.After 96hoursofexposure,celldensitypercent inhibiintthie5o.n5, 11, 21, 44,86and 179mga.i/Ltreatment groupswas 11, -5.1, -18, -12, 77and 99%,respectively. Dunnett'stestshowedthatcelldensity wassignificantlyreducedinthe86and 179mg a.i/Ltreagrtoupms(eps0n.05t). ConsequtehneNtOlAyE,Cfor96hourcel densitywas44mga.i/L.
AMENDED
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WILDLIFE IINNTTEERRNNAATTIIOONNAALLULDo,.eee -16-
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`After72hoursof exposure,areaunderthegrowthcurvepercentinhibitioninthe 5.5, 11, 21, 44, 86 and 179mga.i/Ltreagrtoupmsweasn-2t.9, 1.3, -3.8, 5.8, 68and94%, respectively. Dummett' test showedthatareaunderthegrowthcurvewassignificantly reducedinthe 86and 179mga.i/Ltreatment groups(950.05). Consequently,the NOAECfor 72hourarea underthegrowthcurvewas44 img a.i/L. After96boursofexposure,areaunderthegrowthcurvepercentinhibition in the5.5,11,21,44, 86and 179mg 2/1treagrtoupmsweasn-8.t5,-3.3, -13, -53,75and 98% respectively. Dunnteesttstho'wesd thatareaunderthegrowthcurvewassigaificanlyreducedin the 86and 179mga. treatmentgroups (950.05). ConsequtehneNtOlAyE,Cfor96hourarcaunderthegrowthcurvewas 44mg a.i/L.
After72hoursofexposure,growthratepercentinhibitioninthe5.5,11,21,44, 86and 179mg ai/L treatment groups was -1.1, -0.68, -0.72, 3.4, 31 and 82%, respectively. Dunnett's test showed that growthratewas significantlyreduced inthe 86and179mgai/L treatmentgroups (ps0.05). Consequently,the NOAECfor72hourgrowthraewas44mg a.i/L.After 96hoursofexposugrroewt,h ratepercentinhibition inthe 5.5, 11, 21,44, 86and 179mga.i/Ltreatmentgroupswas -1.9,-0.84, -3.0 2.0,27and 79%, respectively. Dunnett'stest showedthatgrowthratewassignificantlyreducedinthe86 and 179 mg a.i/L treatment groups (ps0.05). Consequently, the NOAEC for 96 hour growth rate was H4mgail.
VisualandMicroscopic Observations
`After 96 hoursof exposure, therewere10 sigasofaggregation, flocculationoradherenceofthealgae 1thetest flasksinthenegativecontrolor any PFOStreatmentgroup.Inaddtheirewtereinoonotincea,ble changesincellcolorormorphology whencomparedtothenegativecontrol,although afewcellsappeared enlargedinthe 86and179mga.i/Ltreatmentgroup.
ReverosfGirobwithlInhiibittiyon The 179mga./Ltreatmentgroupwasmaximallyinhibitedattheendofthe96-hourexposure
period. Aliquotsofthetestsolution weredilutedwithalgal mediumandculturedforfivedays.Basedon thegrowth observedinthe recovery phase,theeffectonalgalgrowthwasfoundtoalgistatic. Celldensities fortherecoveryphase arepresentedinTable 11andar illustratedgraphicallyinFigure3.
AMENDED
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A WILDLIFE INTERNAe TIOe NAL Lo,. _ -17-
PRPORMOJCETCTNNOO.::d4S54AA-1G03AA
CONCLUSIONS "Theconclusionsofthisstudywerebasedonthemostsensitiveendpoint measured (i..,celldeasity, areaunderthegrowthcurveand/orgrowthrate). The 72-hourECS0,basedoncelldensity,ias 70mg a./Lvith95%confidencelimitsof44and 78mga.i/L. The96-hourEC10,basedoncelldensity,was 49mga../Lwith 95% confidence limitsof 43 and 50mg a.i/L. The96-hourECS0, basedoncelldensity, was 71mga.i/Lvith 95% confidencelimits of66and 73mga./L. The96-hour EC,basedoncell density,was 137mga../L with 95% confidencelimitsof 105and 153mga.i/L.The72-abndo96-uborur NOAECvalues,basedoncelldensity,areaunderthegrowthcurveandgrowthrate, were 44mga.i/L. Basedonthepresenceofvisiblealgalgrowthatrecoverytermination, PFOSwasconsideredtobe algistati, ratherthanalgicidal, attheconceatratiesotnesd.
AMENDED
000623
I WILDLIFE Ie NTER RNATIy ONAo L Lon . pm + -18-
PPRROOJEECCTTNNOO.::d455i4AA-M10033AA
REFERENCES
1 U(dSra.ft)E,nOviPrPoTnmSeNnutamlbePrro8t5e0c.t5i4o0n0:AgAelngcayl. To1x9i9c6i.ty, STeireircssIa85n0dI-I.Ecological Effects Test Guidelines
2 OOErgCanDiGzuaitdieolnionfefEocronToemstiicngCoofopCehremaitciaolnsa.nGduDideevleilnoep2m0e1n.t.Par1i9s8.4. Algal, Growth Inhibition Test,
3 wAitShTMiMcrSotaalngdaaer.d AGuuisdet11291980.-90E, Standard Guidefor Conducting Siatic 96-Hour Toxicity Tests
4
The SAS System for Windows. North Carolina.
1996.
Release 6.12, TS Level 0020.
SAS Institute Inc. Cary,
5 West, Inc. and D.D. Gulley. TOXSTAT Version 3.5. Copyright 1996. Western EcoSystems
`Technology, Inc., Cheyenne, Wyoming.
000624
ZWN LIOLLDLT IIPFEEE INTER RNATN IONAA L Lo.TIONAPPRRL OOJJEECCTTL NNOO.:.454TM 5344AA-1100. 33AA "19-
`Table |
_p--oner.
`Summary of Analytical Chemistry Data
TM Corporation
TTeessttSOurgbasnainscme:: FrPeFsOhSwater Alga, Selenastrum capricornutum
Dilution Water: __ "Nominal
Freshwater Algal Medium Sampling Fieasured
Viean Measured ==
E Concmgeantirlat)ion = Timel Concentraaitfi)on" Ce) onaceanltirla)t)ion NomoNiewfsnal
9"6
<o1q0Q
57
70
6a0m4
9%
584
5s
9%
n
70
111207
9%
21
u
100
2
0
198
2
91
%7
2237.1 -
1"
20
a2s7
:"
9%
%
63
9
70
88630
%
83
8
9
18
720
11896
9%
m
9
8
183
7
150
169
2
%
188
#`0L-ihmoituorsfaaQtmtepuslteaiswtneirtoenci(o.LltlOeQca)tewtdafsio0mo.1i1nn5dim vidauag il./bLa.tchesoftest solutionpreparedfo thetreatmentandcontrol JSoutsampleswerecollefom theaddon) replicate Gio). "$97c62o-anhtnooudlr9gs6rahomuoppul.essawmeprleeccoolmlpeocstietdefsroofmttehstesaodlduittiioonnaclollEFe)ctreedpflriocmaea(acbhiootifc)t.he threereplicatesper treatment and.
AMENDED
000625
WILDLIFEN INTERNE ATIONAL Lo.
iia YPRROOJNEOCTTNNOO.:: 54504A3-1A03.A -20-
Table 2
Temperature Measurements
_Spo--nsor:
3M Corporation
Test Substance: PFOS
Test Organism: ~~ Freshwater Alga, Selenastrum capricornutum
Dilution Water: __ Freshwater Algal Medium
Time ~
Temperature (0)
fu)
Measurement |
Measurement 2
o
26
22
1
us
u2
2
28
20
3
25.1
253 .
4
258
249
:
5
27
:
24
6
256
u9
7
20
20
E
8
29
208
9
29
239
000626
JWI IILLDDLLN IITFEE T INTERE NATIR ONALN Lr. ATIOPRN POROEJCECTA TNNOO.::4L 4554A-410. 03A a1.
Table 3
Light Intensity Measurements
STpeosntsSourb:stance: 3PFMOCSorporation
Test Organism: ~~ Freshwater Alga, Selenastrum capricornutum
nt Dilution Water: bFrese hwateb r Algal eMediuprme
Test
~
Day
No.1
No.2
No.3
No.4
Nos
01
33991400
3a88n0
44434000
31961000
0381700
2
4000
4290
4310
4540
4170
34
4212000
34949000
43385900
44017600
44214400
5
3910
a120
4180 4200
340
67
44138000
44128000
41392500
44240300
44307800
8
4010
4230
4180
4230
4450
9
4150
4090
4240
4350
4270
000627
"NWIILLDDLLIITFEE IINNTTEERRNNAATTIIOONNAALLLLom.. 2.
FPPRROOJJEECCTTNNOO.::454544AA--110033AA
Table 4
PH Measurements
---- Sponso-- r: 3M Corporation
Test Substance: PFOS
Test Organism: ~~ Freshwater Alga, Selenastrum capricormutum
Dilution Water, _ Freshwater Algal Medium
`Mean Measured
PH Measurements
Concentration ~
(mgai/L)
9 Hours!
"Negative Control
75
96 Hours 81
55
74
82
n
75
82
21
75
84
"4
74
83
8
74
78
1
74
75
169 (Abiotic)
=
:
74
0-hoursamples werecollectedfromthebatchesoftestsolution preparedforthe treatmentand
control groups at test initiation.
* 96-hoursampleswerecollectedfromthepooled replicatespertreatmentandcontrolgroup.
AMENDED
000625
a abies etn Desi ntPentonfiExch2s elDring heTe
FROCTNO: ssn
-- T[okCrpi riveA_ pSka cpcmm------
DiMCveorimmieersrtNo vTpseE ryi MCp E yote Ma-- l a e-- G pen
Cees Din wm Dw mme E Dme ume seeaaaume
ss
mw ow mm ox ase a seem a
w
ET
u
mow me as ese as sam a
"
we oa we se ssw ou mm wm
"
me ow ww 8 mw on aww
IV ncmpnT lSeSnswmVmpeis sscamhoe t in Dt
wer <L0%
vse
nAaesnn in tt 4 bigDt 045.
28$
82
mom
ue
PROJECTNO: 454i
Tubes
MeanAvesUnderthe ronsCurvesdPeco binforExch 2 Hour ealDuin heTs!
-- TowSham BFS ----
DTiosoOnpWia:trF_rFertoAAllbss,vSeesdleinca, eprom
TVeetmCMoeesiwmriwctn NZewitPoeat MenCmRen MemTenhem Maw n mPes
TT ewaiGne) Ave Gibin W Awa bin mAmm A bem wmAm le
ss mee 6 mee 43 ome 29 smo 4s
u mee 1s meme 89 0m 13 sms 33
n 0 A ewe 28 mow as saome
" Bw0 8 2omme 34 swe ss somew 53
w"
wae 3 eww s sme 6 name
Vioc i m ingS2e A0S6017 Manoin]clemayaG7tgusy oor oi womme wn
IIndincteetsnsssipiiggcaienddfiftlreonscceffroommr thheneoggpaatvtievoescoonuttroll3atW762iBhuorusrsvwiiinngggDDDeuumnentscsvcsp9zo<0t000y9).,.
233
a8
ot0
as.
FROECTNO: shin
Tae
Mtnrots 4Peo ii frFxch24 slDuro Te
TTokOopSi rw 70i8 teAn,crt cpm
DTe inCca.s_ rT eMomsA teePtene Moi m sPeet pM oeR Mai n ote
a avid) GowiRus lke Grau lin Gifu a bie Goes lane
5s wom omm oo sw au ams as
uI
.
a wasn ome ae own am oem ao
" wa ees a1 oo uo emma
" wm ow ems om ow a ww ow
TVsicsipogGw$e1s5 rCT
oratngi heeitmG8 ryt a
Drts
onor 009)
2
wwe
ieEcdoe he pie ar 43 otheDe 00)
388
RaB
Jere
a
FRORCTNO: ssa
aes
CIO, S020 ECOValesBt CllDensityOrr he9HosEposPosi
TTTeokeOpnie WF10r8oamAel,Scrat cpm
frst scotindrrfeor
min
ove
nwenn Tc-- an cwaen -- a--
TT
i
i
ET
ton amin
-
u
nen avant -
nom ow
come
Pe,
IN
Dowites
wtom
son
n
comin
w
reais
8
a8
moe
"ae
PROJECTNO: 454A-103A
Tables
CIO,ECS0aod ECD ValuesBasedcnAraUndertheGrom CurveOver he 86 HourExposure Period
-- TToonp F7-- 0r8 iAlp.Sens prion
Diver Feo AgMe,oC
Er
Tine s(cgaii0n)
mLaiisn)
(osgeesin)
Leien)
Teri TotDemin
=
wn
Ta 16
an Noten
-
"
Grutiis
tines "
wuss
sin
Stine
wis
2
en
(ogscesi0n) TotDeed NetDtriond
ws us
TgLaiiC mni) = -
sans istiss
8S
4[
AMENDED
a
PROTECTNO: A100
Teno
e T [EereeeCIO,ESD E90Vl BtnGre nR-- tOre S-- H x-- posPee d e
Toovte FmeteeAny irumecpr
sc
=
co
rToe
=a
cay cnn
tn
aso:n I ra=ca
I
=
hn arent
.
=
Sy 4
@
es dems
-
i we2hnenrrr spoaos t sweiinn R oomm ee-=
Tee
sg8
88
mom
WILDLIFE INTERNATIONAL Lm.
29.
PROJECT NO.: 454A-103A
`Table 11
Cell Densities for the Recovery Phase
STeet Substance: TDoisltutOirognanWoitoemr::_
WPMFCOS. FFrreesshhwwaatteerrAAllggal, SMeeldenisutrum capricormutam
Mean Measured
Cell Densities (Cells/mL"
`Test Concentration
(mg ai/l)
Dayo
Day3
"Negative Control Rep. A.
10,000
1,220,000
Days
6,420,000
179"
1,000
35,000
1,180,000
T Thetreatmentgroupwasdilutedto aconcentrationofthe testsubstance thattheoreticallywould
not inhibit growth.
:
:Due tothemethodusedto prepare recoveryphasetestsolutions,initialcelldensities werenot
equivalent throughout the treatment groups.
000635
AMENDED
WILDLIFE INTERNATIONAL Lm. -30-
PROJECT NO.: 454A-103A
Figure 1. NegativeControlAlgalGrowth,ExpressedinCellDeasity,Duringthe96-HourExposure.
10000000 1000000 100000
10000 1000
Mean Cell Density (Cells/mL) ~=--Negative Control
2
48
72
%
-
000636
AMENDED
WIL LDLE IFENINETERRNAO TIONY AL Lo. enfin -31-
Figur2e. ConcentratCuirveo,Enxp-reRsseedsinCpelolDneassiety
PROi JECT NO.: s 454A-103A
10000000
1000000 100000 .
1000
Mean Cell Density (Cells/mL)
-
:
--a--Negative Control --e--5.5 mall ----11.mgnL 21 mgt --o--44 mon 1----7868momltg
2
8
2
9%
.
Exposure Duration (Hours)
i
;
000637
AMENDED
WILDLIFE INTERNATIONAL Lm. "32
Figu3re-RecoveryPhaseResponseCarve, ExpressedinCel Deasity
PROJECTNO: 454A-103A
10000000 1000000 100000
`
1000 100 0
Coll Density (Cells/mL)
:
' --8--Nogativ-e Control
--a--179 mg
.
.
.
12345
BN
Exposure Duration (Days)
=
000638
AMENDED
WILDLIFE INTERNATIONAL Lo. .3-
PROJECT NO.: 454A-103A
APPENDIX1
Freshwater Algal Medium'
Sponsor.
SM Corporation
Test Substance: PFOS
Test Organism:~~FreshwaterAlga, Selenastrum capricornutum
Dilution Water: _ Freshwater Algal Medium
"Nominal
Compound
Concentration
MsCLO6H0
216 mg
CaCho2H:0
440 mgL
Ry
0185 mg/L
MaCLo4H:0 ZC;
0342186 mglg
FeCle6H:0
01598 mg/L
CoCLO6H:0
128 ug
Na:MoO,#2H:0
726 ug
CuCh92H:0
002 ug
Na;EDTA2H,0 NaNO,
02350500 mmgg/L
.
MgSO TH:0
KHPO,
1870 1044
mmgg
NaHCO.
150 mel
ThepHvasadjus1t0e7.d5 + 0.1 using 10% HCL
000639
WILDLIFE INTERNATIONAL Lto.
a
PROJECT NO.: 454A-103A
APPENDIX II
AnalysesofPesticides, Organics, Metals and Other Inorganics
saeesswvcovoEDmCon umoy in Wildlife International Ltd. Well Water'
T HEET
. &A =
SEp e
: om om
= 2 EEBTeHC.--
<o PPPoooswml0008 3gL
BuBtE,
:
i Poem =
BHBEEuE.
PPHoE ol =
BF HotterBoonie
PCB.1221
i BBHE PCB-1232
PCB-1242
cote ng Or rics Amiay=a
Coolcga? | f= ELoa?w
PE 3
0.005
=wl
<
0260
ri
- i <iPoBm00m .276200 Fg=oaLFL
)
3i 48 aEF
Peo EB Y] omg.
<Doo10m90 awl
BFe=
B mF Z
M3E AleBemby WaiteIcmstooLnil.forspi oie onNovember Shough7, 1997.
000640
WILDLIFE INTERNATIONAL Lm. 35.
APPENDIX Il
PROJECT NO.: 454A-103A
`THE ANALYSIS OF PFOS IN FRESHWATER ALGAL MEDIUM IN SUPPORT OF
WILDLIFE INTERNATIONAL LTD. PROJECT NO.: 454A-103A
000641
WILDLIFE INTERNATIONAL Lm.
-36-
PROJECT NO.: 454A-103A
REPORT APPROVAL SPONSOR: 3M Corporation TITLE: PFOS: A 96-HourToxicity Test with the Freshwater Alga (Selenastrum capricornutum) "WILDLIFE INTERNATIONAL LTD. PROJECTNO.: 454A-103A
PRINCIPALINVESTIGATOR: Rayna I Van Hoven, Ph.D. Scientist
MANAGEMENT:
stn 5}ip
Willard B. Nixon, Ph.D. Manager, Analytical Chemistry
. He 4-00
DATE
sith
DATE
000642
AMENDED
WILDLIFE INTERNATIONAL Lm. -37-
PROJECT NO.: 454A-103A
Introduction Freshwater algalmedium sampleswerecollectedfromanacutetoxicitystudydesigoedtodeterminethe.
effects of PFOS (Perfluorooctane Sulfonic Acid Potassium Sal) to the freshwater alga (Selenastrum capricornutum). This study was condubycWitldelidfe International Ltd. and identified as Project No.: 454A-103A. TheanalysesofthesewatersampleswereperformedatWildlifeInternationalLtd.usinghigh performanceliquidchromatography withmassspectrometricdetection (HPLC/MS). =Samples were receivedforanalysisonApril12, 15and 16,1999andwereanalyzedoneachsamplereceiptday.
`S Testubsantd Ina ternn alStcande ard
Thetestsubstanceusedfortheanalyticalportionofthisstudywas WildlifeInternationalLtd. identification number 4675. Thetestsubstancewasusedtopreparecalibrationstandardsandmatrix fortificationsamples.
Theintemalstandardwasreceivedfrom 3MCorporationon July2, 1998andwasassignedWiklife IntemationalLid.identification number4526 uponreceipt. Theinternalstandard, agranularmaterial,was identified as: 1H, 1H, 2H, 2H Perfluorooctane Sulfonic Acid, Chemical Abstract Number: 27619-97-2. `Thestandard wasstoredunderambieatconditions
AnalyticalMethod
"Themethodusedfortheanalysisofthefreshwateralgalmediumsampleswasdevelopedat Wildlife IntemationalLtd.and entitled "AnalyticalMethod Validation for the.Determination ofPFOSin Freshwater, SaltanwdaAlgtaleMerdia,". ThismethodologywasincludedasAppendix I of Wildlife Intemational Lid. protocol mumber 454/011299/MVAL/SUB454. It was based upon methodology probyv3MiCordporeatidon.
Sampleswere diluted in a 50%methanol : 50%NANOpurewatersolution containing 0.mg14H0PF0OS (intemal standard)/L. and 0.05% formic acid (v/v) so that they fol within the calibration rangeofthe PFOSmethodology.
Concentrations of PFOS inthestandardsand sampleswere determinedbyreverse-phasehigh performance liquid chromatography using a Hewlet-Packard Model 1100 High Performance Liquid
000643
WILDLIFE INTERA NATIONAL Lm.
itm 38.
TPRROOJEERCTTNNOD:;S45S4AS-10A3A
Chromatograph (HPLC)with aPerkin-Elmer API 100LC Mass Spectrometerequippedwith a PerkinElmer TurbolonioSnpsoruarcye. HPLCseparationswereachievedusing aKeystoneBetasil Cio analytical column (100mm x 2mm LD., 3 umparticlesize). Theinstrumentparametersaresummarized in Tabl1e. Amethodflowchartisprovidedin Figure1.
Calibration standards of PFOS prepared in a 50% methanol : 50% NANOpure water solution containing 0.100mg4H PFOS (internalstandard) and0.05%formicacid (v/v), ranginginconcentration from 0.00229to0.0457mg a.i/L wereanalyzed withthesamples.Lincarregressionequations were. encratedusingpeakarea responseratios (PFOS intemalstandard)versustherespectiveconcentration ratios(PFOS : internalstandard)ofthecalibrationstandards. Atypicalcalibrationcurveispresentedin Figure2. Theconcentrationof PFOSinthesampleswasdeterminedbysubstitutingthepeakarea responseratiosintotheapplicable linearregressionequation. Repreisonechrnomtatoagrtamsiofvloew. `andhighcalibration standardsareprescatedinFigures 3and4,respectively.
`Themethodlimitof quantitation (LOQ)fortheseanalyseswassetat0.115mga.i/Lcalculatedasthe prodouftchte lowest calibrationstandardanalyzed (0.00229 mg ./L)andthedilution facotfothre matrix blank samples (50.0).
`Threematrixblanksampleswere analyzedto determinepossibleinterference. Nointerferenceswere GbservedatorabovetheLOQduring samplesanalyses(Table2). Arepresentativechromatogramof a matrixblankispresiennFitguere5d.
Freshwater algalmedium wasfortifiedat0.915, 45.7and 220mg 2:/Landanalyzedconcurrently viththesamples todeterminethemeanproceduralrecovery(Table3). Sampleconcentrawteireonnost correctedforthemeanproceduralrecoveryof99.1%. Arepresentativechromatogramof amatrix fortiispfreiscacteadintFiiguoren6.
000644
AMENDED
WILDLIFE INTERNATIONAL Lm.
-39-
PROJECT NO.: 454A-103A
ExampleCalculations
`Sample number 454A-103A-2, nominal conceatration of 5.7 mg a../L in freshwater algal medium.
Initial Volume: 0.100 mL Final Volume: 25.0 mL DilutionFactor: 250 PFOS Peak Area: 164427 Internal Standard Peak Area: 240533 Peak Area Ratio: 0.6436
Calibration curve equation.
Slope: 3.43926
Intercept: 0.03327 Curveis weighted (15x).
PROS (1gai/L)atinstument
= o( ekaka2r5i e2aarat0ion = (Y-t intercept))r x151S,.o CCoonncceenntt) rraattiion
Slope
(0.6836 ~0.03327) x 100pgai/L 343926
= 189pgaill
Note: LS. = internal standard. PFOS (mgai/l)insample
= --Po FOS(ug .i/L)o atinstrument x" DilutRionAFaActoTr
1000 ug/mg
189x250 a 1000
000645
AMENDED
WILDLIFE INTERNATIONAL Lm.
40
PROJECT NO.: 454A-103A
= ATmgaill
Percentof Nominal Concentration = PP2FF0OO0SS(C(mrmgegnaa..Lii//ILL)) niRnomisnaBamlple
Calculated recovery: 82.0% Note: manual calculation may differ.
:
RESULTS
`SampleAnalysis, Freshwater algal riedium samples were collected from the acute toxicity study with th freshwater alga
(Selenastrum capricornutum) attest initiation, April 12, 1999 (Hour 0),onApril 15, 1999 (Hour 72), and at test termination, April 16, 1999 (Hour 96). The measured concentrations of PFOS in the samples collcted at niioaftexpoosunreofth testorganisms(Hour0) ranged rom 82.0 0 8.1% ofthenominal concentrations, Samples collected at Hour 72 had a measured concentration range of 91.7 to 105% of
nominal values. The Hour-72 abiotisamplewas 81.9%ofthe nominal concentration. Samples collected attest termination (Hour 96)had a measured concentrationrangeof 90.3 to 102%ofnominal values. The
Hour-96 abiotic sample was 103%ofthe nominal concentration (Table 4). A representative chromatogram ofa testsample is showninFigure 7.
000646
AMENDED
WILDLIFE INTERN NATO IONALILr.
ssimmenTPRRIOJBEECTTNNOO.::S4S54AA-A103A a1
Table 1
`Typical LC/MS Operational Parameters E INSTRUMENT: Hewlet-Packard MoS del 1100 High Performance Liquid
eCqhuriopmpaetdowgirtahpahPweirtkhina-PEelmrekriTnu-ErlbmAoePrIl1o0n0iLoSCnMpsaorusrascyeS.peOctpreormeatteeridn selective ion monitoring mode (SIM).
ANALYTICAL COLUMN: Keystone Betail Cys column (100 mm x 2 mm LD. 3 pum particle size)
OVEN TEMPERATURE: : 30C
STOP TIME:
10.0 minutes
FLOW RATE:
0220 mL/minute
MOBILE PHASE:
72.0% Acid
Methanol
:28.0%
NANOpure
Watercontaining0.1%
Formic
INJECTION VOLUME: 2504
PFOS RETENTION TIME: Approximately 6.6 minutes
INTERNAL STANDARD RETENTION TIME:
Approximately 4.5 minutes
PFOS MONITORED MASS: 498.6amu
INTERNAL STANDARD
MONITORED MASS:
4267 amu
000647
WILDLIFE INTERNATIONAL Lt.
a2
PROJECT NO.: 454A-103A
`Table 2
Matrix Blanks Analyzed Concurrently During Sample Analysis
(@`5N4uAm-b1e0r3A)
MAB-1
MAB2
Sample
Type
Matrix Blank
Matrix Blank
Measured Concentration of
(mPgFaOiSl'L)
<LOQ
<L0Q
MAB-3
Matrix Blank
<L0Q
`Thelimitofquanti(tLOaQt)wiaos n0.115mga.i./Lbaseduponthe productofthelowestcalibration standardanalyzed (0.00229 rig a.i/L)andthedilutionfactorofthe matrixblank samples (50.0)
900648
AMENDED
WILDLIFE INTERNATIONAL Lo.
a.
PROJECT NO.: 454A-103A
Table 3
Matrix Fortifications Analyzed Concurrently During Sample Analysis
Sample Number
(454A-103A7)
MAS MAS4 MAST
"Concentrationsof PFOS
mg ai/L)
Fortified
Measured
0915
0884
0915
110
0915
102
Percent
Revered
%6 120 12
MAS2
457
a3
902
MAS-5
Lo4s7
484
106
MASS
toast
414
906
MASA
20
210
952
MAS3B
220
200
91.7
MAS3C
20
209
950
MAS
220
21
101
MAS
220
202
919
Mean = 99.1
Standard Deviation =9.75
ov =98
N=
"Note: Results and corrections for new test substance purity were generated using MacQuan version 1.5
soafndtmanuwalcaalcurlatieons. Valueshavebeen roundedforreporting purposes.
000649
AMENDED
WILDLIFE INTERNATIONAL Lm. 4
PROJECTNO.: 454A-103A
Table 4
Measured Concentrationsof PFOS in Freshwater Algal Medium Samples from a Freshwater Alga Acute Toxicity Test
`Nominal Test Concentration
(mgai/L) 00
Sample Number (454A-103A7)
T 8 15
Sampling Time (Hours)
0 7 %
POS Measured, Concentration'
(mgai/) <10Q <10Q <10Q
Percent of
Nominal =~
57
2
0
9
7
1
%
an
820
604
105
584
101
un
3
0
107
898
10
7
12
96
n
9
121
102
2
4
0
198
862
n
7
231
101
18
9%
207
903
46
5
0
12
7
19
%
27
934
419
91.7
463
101
1
6
0
13
7
20
9%
3
910
0
940
83
%5
183
7
0
14
7
2
9%
1
98.1
186
102
mn
94.0
183
14A
7
150
819
(Abiotic)
2
9%
188
103
Note: Results and corrections for new est substance purty weregeneratedusing MacQuan version 1.5
software and manual calculations. Values have been rounded for reporting purposes.
"The litof quantitation (LOQ) was 0.115 mg a.iL based uponthe productofthe lovist calibration
standard analyzed (0.00229 mg .i/L)andthe dilutionfactorofthematrblianxk samples (50.0).
000650
AMENDED
A WILDLIFE INTRERNNAATOIONNAL Lo. re
FPRROOEJECCTTNNOO.: 445M4AA-d1003AA
METHOD OUTLINEFORTHEANALYSIS OF PFOS IN FRESHAW LGA ALT MEE DIR UM
Preparematrixfortification samplesbyspikingtherequisitevolumeofPFOSstocksolutionsdirectly intofreshwateralgalmediumusinggas-tightsyringesandClass Avolumetricflasks.
i
Dilutematrixfortificationandtestsamplesintotherangeofthe calibrationstandards by partiallyfilling Class Avolumetric flaskswith 50%methanol:50% NANOpurewatersolutioncontaining
0.100mg 4H PFOS (internal stan andd0.a 05%r ford mic) aci/ d(vL /v).Add the appropvroiluametoef sampleandbringthe flaskto volumewiththedilutionsolvent.Processthematrix blank sampleusing thesamedilution andaliquotvolumeasforthelowestfortification level.Mixwellbyseveralrepeat
inversions.
v
e
Ampulate samplesandsubmitforLC/MSanalysis.
Figure1. Analytical methodflowchartfortheanalysisof PFOSinfreshwateralgalmedium.
000651
A WILDLIFE INTERNAATIOA NAL Lo.renter a6
TPRROJOETCTHNOO.:S45A4A-N103SA
2.00
:
:
150
5
10
050
0.00
0.000 0.100 0200 0.300 0.400 0.500
Concentration (Ratic) ~*~
Figure2.
Atypical calibration curve for PFOS. Slope = 3.43926; Intercept = 0.03327; r = 0.9986.
Curve is weighted (1/x).
000652
AMENDED
WWIILLDDLLIIFFEEIINNTTEERRNNAATTIIONALLLLDm. ar.
PPRROOJJEECTNNOO.: 44s5t4aA-1l003sAA
100
20
%
80
0
60
ntonsity: 2849cps
3
23,
"50
40
30
2o0 u
A138
3T1a e2l5% 9a1m 1S3o1s 1651 178517 2811s | TSmane
Figure3.
Arepresentativeion chromatogram of a low-level (0.00229 mg a.i/L) PFOSstandard.
000653
AMENDED
W--I_L--DLIFE INTERNATIONAL Lo. ae
PROJECI TNO.: 454A-m103AA
To
o
i
185 Intensity: 32143 cps ne
8
.
.
o
5
4
3 2
P
135
'
:
|:
24
1s
Ai 3$13 6215 9a1m 1S2e1s 1651m 17815 2181s TScmaon
Figured.
Arepresentativeionchromatogram of ahigh-level (0.0457mga.i./L) PFOS standard. 000654
AMENDED
WWIILLDDLLIIFFEE IINNTTEERRNNAATTIIOONNAALLLLoo..
snormcrnos PROJECT NO.: assaisa 454A-103A __
100
3
----
w90
:
:
T
w
.
-50
"
-
"
wl Li Redes TAL
rae
TY a
Th
WW
wm
Figur5eS. Arepresentativechromatogramof amatrixblanksample(454A-103A-MAB-1). `Thearrowindicates theretentiontimeof PFOS.
000655
SWIULDDULIPFEEIINNTTEERRNNAATTIIOONNAALLALTmS.,
mmmePRROTJEEECETNNOO.L:44A54AAI-1B03AA -50-
To
187
intensity: 9104 cps
9
-
8
.7
&
.
5
4
=
8
137
2
1 24
11s
o=
es
o 31Tw 6215% 9a1m 1S21e 1651s 178815 2161s TSeiamne
Figure 6.
A representative chromatogramof a matrix fortification sample (454A-103A-MAS-1).
000656
WILDLIFE INTERNATIONAL Lm.
sie
PROJECT NO. 454A-103A
To
:
.
{
161 intensity: 11161 cps
8 7 s 5 4
#
140
2
1 25
118
~
:
S1%161255 9a11 153016 1651% 178517 28118'3 | TSiamne
Figur7e. Arepresentativechromatogramofatestsample (454A-103A-2).
000657
WILDLIFE INTERNATIONAL Lm.
52.
PROJECTNO.: 454A-103A
APPENDIX IV
CellDensity forEachReplicatePerTreatmentOverthe96-HourExposurePeriod
E TesSubsmce: PROS E -- TDeisltuiOorngWmetieor:_ ~~ FFrreesbawtaetre AAllgga,a MSeeldenisutramcapricorn A Conocnttion Col Dees Cala
TR(mgaC ill)
ReplAiBcate c
24MBHOo0uD0rs. 31.000
418I3H0o0Du0r0s 125000
7200H50o0u00rs0 95000
9o263H6oo0u0rm0s0 2640000
ss
a5
223900000 11386000000 5000000000 23.010800000000
35000 134000 25000 2540000
un
AB
320700000 112045000000 665600000000 22764800000000
c
31000 134000 aso 320000
2 -
a5 2360000000 110312.000000 846500000000 33304800000000
c
2500 128000 0000 3300000
"
AB c
230000000 115316.000000 27000 13500
5010500000 330000
332043220000000000000
"
AB
3178000000 67500000 211365000000 576000.00000
c
Zo
6000 162000 470000
m
BA
11580000 Z35o0o0 2w1.w000 53000000
c
1000
26000
500
26000
"TocheambmeirlocyilelldGeneelydoafsihteysofoacpkprcoitzartewlays10d,00elvrait etdiieo v(o0lhouumrse)w.as SATS] to eachVt
000658
AMENDED
WILDLIFE INTERNATIONAL Lm.
5.
PROJECT NO.: 454A-103A
APPENDIX V
AreaUndertheGrowthCurveforEachReplicate PerTreatmentOverthe96-Hour ExposurePeriod
`TSTeposontusOSorru:gbsamne: PF3RMOrCSoreporsatAilohgna.,oStlesr capricorn
DiluiMoenaWnaMtere_adFreerAle Medium
Co foe Um nieBt eGrowe Gore
Conogcantianti)on Replise --0 2--4ous 0_48Hows R_0E.72HoSws R __0-96Hous
TewCmm
A5
B26R0O00 2109020000 I0T5S200L00 5S19m12e00e0
c
252000 TROD 90800 46464000
ss
a5
21586000000 T19B608000. 2B0TB0AM0D 27780440000000
N0000 2000 1095600 S460
n
A5 204000000 1I62T00O00 1903904000000 4581050004000000
c
252000 92000 1080000 36760000
2
AB
21902,00000 2T0S00O0 BBEsmA0K 6752840000
:
c
216000 TRO 163400 36304000
"
BA 2604000000 2129020000 195I6Z4R000 45961504200000
c
204000 Toso 70 se4s000
5
BA
5264000000 1F06800000 24488m0K000 11433364800000
c 144000 TOON 2900 10320000
mw
BA
9600000 326000000 A S260000 1$4102400000
c
o
192000 40000 58000
000659
AMENDED
WILDLIFE INTERNATIONAL Lm.
san
PROJECT NO.: 454A-103A
APPENDIX VI
GrRoatw efot r Bh achReplicate PerTreatmentOverthe 96-HourExposure Period
`STTpeeoatnsSOowgrbu:sniasmmc:e FP3reMOsShCowraptoerartAiloan:, Seles capriconutim
Dilu"tiMoenaWneeMre.am_redFreshwaterAla Motion
Grow a
Congceantirant)ion Replicate w 0-24Mows _ 0_de 8Hows 0.7Howt s 0-9 Hous
C B 00457 T 005% 00570 rey
c
oom
00526
o0se2
aos
ss
AB
o00u37
0o0s54s4 . 0a0o60s8
a0o0s6s0s
oo
00541
00574
aos
n
AB
00u4s1s4 000054205 0o0o5s5e92 aa0ossss
c
oom
00541
00574
ave
2
AB
o0o0s3sss 0000555318 00006s1
o000s509s7
c
00m 00531 00s) 0060
"
AB aaooss 0000554646 a0o0s5e%r o00o8s9s5
c
oon ose 00509 00608
AB oaaotsiss [0r0r6 aoonaes aooots
c
00
aos
aos
0001
1m
AB
o00n16s9
ocoulse i 0o0m10e3
o00o1s174
c
0000 0019 oons 00010
000660
AMENDED
WILDLIFE INTERNATIONAL Lm. Zs
PROJECTNO.: 454A-103A
APPENDVIIXI `ChatonPrgoteocsol
`Thisstudy wasconductedinaccordancewiththeapprovedProtocolwiththefollowingchanges:
1. Tcheoprontoccoelwanandsttaemsretsnuadbesttdatnioceaondudmnbtehsre.proposedexperimentalstartandterminationdates,test 2. Theprotocol wasamendedtospecifytheanalyticalmethod. 3. Theprotocolwasamendedtorepeatth testwith ahigher estconcentration.
000661
WILDLIFE INTERNATIONAL Lo. -56-
PROJECTNO.: 454A-103A
APPENDIX VIII PersonnelInvolvedintheStudy
`Thefollowingkeypersonnelwere involvedintheconductor managementofthisstudy:
1. HenryO.Krueger, Ph.D,,Director,AquaticToxicologyandNon TargetPlants 2. WillarBd. Nixon,Ph.D.,Manager,AnalyticalChemistry 3. RaymondL. VanHPoh.Dv,,Seciennti,st 4. KurtRDroStenioarBirolog,ist 5. MarkA.Mank,LaboratorySupervisor
000662
WILDLIFE INTERNATIONAL Lm. L574
PROJECTNO.: 454A-103A
APPENDIIX ReportAmendment 1. OriginalReport: Pa14g,6eands36 Amendment: Tnhumebpeargs,esawnedrneewchsainggneadtutroesinacnludddeattheesamdeuendteodtrheepaodrdtidtaitoen,orfevtihseedrpepaogret amenasdApmpeendinxItX. Reason: Toreflecttheissuingofanamendedreport.
2. OriginalReport: Page2
Amendment: Thecompliancestatementwasrevised.
Reason:
To clarifyhowthetest substance wascharaclerized.
3. OriginalReport: Page 10
Amendment: Informationprovidedbythe Sponsorreflectingthereanalysisofthetestsubstance, siencctliuodni.ngthereanalysisdateandthepu-rity, wasaddedtotheTestSubstance
Reason:
Toreflectthe currettestsubstanceinformationprovidedbytheSponsor.
4. OriginalReport: Entirereport
Amendment:
sAulblstteasntcseuabssdteatnecrecmoinnceednbtyrtathieonSspownesroercihnaanrgeeadnatloyrseifsolefcttthheteepsutrsiutbysotafnctehe(tFeCs-t 959,89L%o,t21T7h)e.ceTretsitfciocnacteenotfraatniaolnyssoirsidgaintaeldlyMawrecrhe'b9a,se2d0o0n0tinhdeirceaptoerdteadppuurriittyyooff 9co0n.c4e9n%tr.ationTsh,ermeefoarseu,redallcontcesetntrsautbisontsa,ncEe Ccovnacleunetsr,atainodns,NOiAncElCudivnagluensomwienrale recalculatedandreportedas mg 2.1L.basedonthe90.49%purity.
Reason:
"Toreporttheresultsofthetestbasedonthetestsubstancepurityof 90.49%atthe reqoftuheeSposnsotr.
000663
AMENDED
WILDLIFE INTERNATIONAL LTD.
PROJECT NO.: 454A-103 Page 1 of
AMENDMENT TO STUDY PROTOCOL
STUDY TITLE:
PFOS: A96-HOUR TOXICITY TEST WITH THE FRESHWATER ALGA
(Selenastrum capricornutum)
PROTOCOL NO.: 454/110998/SEL-96H1/SUB454
AMENDMENT NO.: 1
SPONSOR: 3M Corporation
PROJECT NO.: 454A-103
EFFECTIVE DATE: January 4, 1999
AMENDMENT: Page2
Add:
Experimental Start Date: 2/19/99 Experimental Termination Date: 2/23/99 "Test Concentrations: Negative Control, 6.3, 13, 25, 50 and 100 mg a.i/L `Test Substance No.: 4675
REASON: The above information was not known when the protocol was signed by the Study
Director.
AMENDMENT: APPENDIX II, Page 16
-
Add: Liquid Chromatography Mass Spectrometry (LCMS) Method for the Determination of
Perfluorooctane Sulfonic Acid, Potassium Salt (PFOS) in Freshwater, Filtered Saltwater and
Algal Medium.
REASON: To add the analytical method to be used in the study.
Sort]
STUDY DIRECTOR
aL LABORATORY
(AGEMENT
do 6 ond
SPONSOR'S REPRESENTATIVE
I
al9(9
DATE
aff
DA
2/16/19
DATE
000664
I iui sby fi 2h
[EllWILDLIFE INTERNATIONAL LTD.
PROJECT NO.: 454A-103
Page of |
H
AMENDMENT TO STUDY PROTOCOL
oH
STUDY TITLE: P(SFeOlSe:nastArum9c6a-prHiOcUorRnuTtOumX)ICITY TEST WITH THE FRESHWATER ALGA
PROTOCOL NO.: 454/110998/SEL-96H1/SUB454
a
SPONSOR: 3M Corporation
AMENDMENT NO.: 2
PROJECT NO.: 454A-103
EFFECTIVE DATE: April 8, 1999
a
AMENDMENT: Page2
H Experimental TerminationDae: 2/23/99 Change:
Experimental Start Date: 2/19/99 `Test Concentrations: Negative Control, 6.3, 13,25, 50 and 100 mg a.i/L.
Ho
To: Experimental Start Date: 4/12/99
`Experimental Termination Date: 4/16/99
I
Test Concentrations: Negative Control,6.3, 13,25, 50, 100and 200 mga.i./L.
-
REASON: Tmhiecrotsecsotpwei.llAbneardedpietaotneadlbteesctacuosnceecnetlrlactoiuonntwsawseardedendoitnamnadaetweimtpht tahoebmtascinytEoCm9e0tevrlaunesd. a
AMENDMENT: EXPERIMENTAL DESIGN, Page 3
H
Change: a`tTaerrg.et concentrations will not exceed 100 mg/L or the solubility limit of the test substanicne
H
To: Targetconcentwrilaltnoiteoxcneesd 1000mg/Lorthe solubilitylimitof thetestsubstancein water.
i
REASON: Thenewnominalconcenetxcereda1t00imgoain/Ls.
u PESAN
STUDY DIRECTOR
2/8177 DATE
Ho
wd A
m Ja Reds nu
SPONSOR'S REPRESENTATIVE
H cusatonenents
4 fo
Yla1 24 DA
000665
Qf lorebsy JB. 34pTrd
{
a
PROTOCOL
=
PFOS: A96-HOUR TOXICITY TEST
`WITH THE FRESHWATER ALGA (Selenastrum capricornutum)
i
US. Environmental Protection Agency
HH
Series 850O- PEcPolSogNiucamlbEefrfe8c5ts0.T5e4s0t0Guidelines
`OECD Guideline 201
mu
ML RsNe U3
Submitted to
Envir3oMnmeCnotraplorLaatbioonrsory
P.0. Box 33331 St. Paul, Minnesota 55133
WILDLIFE INTERNATIONAL LTD.
8598 Commerce Drive Easton, Maryland 21601
(410) 822-8600
November 9, 1998
PROTOCONOL.: 454/110998/SEL-96H1/SUB454
000666
3M LABREQUEST NO. U2723
u - JA WILDLIFE INTERNATIONAL ro.
H
WITH THE FPRFEOSS:HWAA9T6E-RHAOLURGATO(XSIelCeInTasYtTruEmScTapricornutum)
H
SPONSOR:
3M Corporation
`Environmental Laboratory
H PSt..0.PaBulo,xM3i3n3n3e1sota 55133
ul
`SPONSORSREPRESENTATIVE: Ms Susan A. Beach
TESTING FACILITY:
Wilde Intemational Lid
Ho
`8E5a9st8onC,oMmamreyrlcaendDr2i1v6e01
STUDY DIRECTOR:
Kurt Drottar
Senior Aquatic Biologist
Ho
HH
LABORATORYMANAGEMENT: HDeirnercytoOro. fKrAuqeugaetri,cPThoDx.icology & Non-Target Plants
FORILABORATORYUSEONLY
_
mu
| Proposed Dates:
|
|]
| Eoxperoimebntalme_TommmoEnxperDimenutals
| prec: 4544-1e 03 s
|
||
|]
Test ConcentrA ations:
1
| TestSubstanceNo:
ReferenceSubstanceNo, (ifapplicable):
|
=
[1
ml
||
ma
A or
AN
STUDY DIRECTOR
/2/4(78
DATE
LAB {TORY {AGEMENT SPONSOR'S REPRESENTA
pfye
DATE
2/1
DAT
000667
PROTOCOL NO.: 454/110998/SEL-96H1/SUB454
3M LAB REQUEST NO. U2723
i
J WILDLIFE INTERNATIONAL Lo
1
=
INTRODUCTION
1
Wildlife Interational Ld. will conduct a 96-hour toxicity test with the freshwater alga, `Selenastrum capricomutun ox the Sponsor atthe Wife Intemational Lid. aquatic toxicology facility
1
iPnrEotaescttoionn,AMgaernyclyanSde.iTeshe8s5t0udEycvoilolglibcoalpeErfffeocrtmseTdebsatsGeudidoenlipnreosceOdPurPeTsSiNntuhmebUeSrS8.30E.n5v4ir0o0n(m1e)ntaanld
i
OwoErCkDpeGrufioderlmiendae tfoWriTledsltiifnegoInftCemhaetmiiocnaallsL,i2d0.1a:ndAlagac,opGyroofwtthheIfnihniablitrieopnorTtewsitl(l2)b.efRialweddbaytaprfoorjeacltl
1
umber in archives located on the Wildlife Intemational Ltd. it or at an atemative locattiooben.
speintcheiina frepoirt.
||
"Thepurposeoftisstudy istodetermiPnUetRhePtOoxSicEity of estsubstance othefreshwatergreen
un
alga,Selenastrum capricornutum, during a 96-hour exposure period.
i
"Thegroenalga,SelenasinuEmcXapPrEicRorImMutEuNm,TwAilLlbDeEcxSpIosGedNto ageometricseriesofat leastfive
{estconcentrationsand a egative (culemedium)controlfor 96hours. Targetconcentrawtiilolnnost
||
exceed100 mp/L orthesolubility limitofthetestsubstanceinwater. Generthaelnolmiyna,l
concentration ofcachtest substan usodinthedefinitive tetvillbeatleast S0%ofthenexthigher
a
`reament,unlessinformation conosminth coneentrtion-cffoctcurveindicates thatadiffrentdilution
factorwouldbemoreappropriate.
1 1
Tes solutions vil be inoculated with 10,000 cclsfmL. Three "biological" replicates per experimentalgroupvil beprepare forevaluatingcelldensities.Oneormoreadditonal "analytical"
replicateswillbeincuded thetest needed, oprovide est solutionforconcetrationverification
onDay3oftheexposure.Anabioticreplicatsot thehighestconcentrationvill be included nthetest
a
andvillbe sampledon Day 3and Day4.Inordertocontrolbis,tepositionoftheflaskswillbe
detrminedbyindiscriminatedrawdiyduringth exposureperiod. Noothe potesonurctesiofabials
i
`areexpectedtoaffecttheresultsofthestudy.
a
"Theresponseofthealgaewillbemeasuredintems ofceldeasity,biomassexpressedas area
undethegrowthcurve,andgrowth te.AnECS0(ic,th theoretical est concentrationthatproduces.
a
a 50% reductioninthemeasured parameter)valueforcell deasitywillbecalculatedforcach 24hour interval. EC10,5050 andECv9al0u forcl deity,ity,bibiomes (50) aodgow rie GOwillbe 000
a
PROTOCOLNO. 454/110998/SEL-S6H1/SUB454
3MLABREQUESNOT.U2723
a
MW WiLoLirE INTERNATIONAL LTD.
un
oun
calculated,
(NOAEC),
if possible,atthe 72and 96 hour intervals. Theno
which sthe highest test concenttrhaattiniduocens
observed
no adverse
adev ffece tconr cens trate ion
inhibitoryeffecton growth,
u
wrielslubletsdane d tt he dre oeslea-r trievesm tpoonci saechpn patatree armne.d teArtatthe7e2nadndo9f6thheou9r6s-hboausreedxuppoosnuervea,laulagtiistoantoicfetfhfeecsttsatwiisltlicbael
differefnrotmialagitciedadl effectsinthosetreatmentswhich aremaximally inhibited.
MATERANDIMEATHLODSS
ou inti Informationonthe characterizationof test,controlorreferencesubstancesisrequiredby Good
Laboratory Practice Standards (GLP)."The Sponsor isresponsible for providing WildlifeInternational
|]
Ltd writtenverificationthatthetestsubstancehasbeencharacterizedaccordingto GLPspriortoitsuse
inthestudy. IFwrittenverificationof GLP test substancecharacterizationisnotprovidedtoWildlife
ou
Intemational Lid.,itwillbenotedinthecompliancestatementofthefinalreport.The attachedform
IDENTIFICATIOOFN TEST SUBSTANCE BY SPONSOR (Appendix I) is to be used to provide.
mm
information necfe ors GLPscoa mplr iany ce.
A
"The Sponsori responsiblefor ll informationrelated to thetest substance andagrees to accept
anyunusedtestsubstanceand/or testsubstancecontainersremainingaftheend of thestudy.
PrepaofrTeasttSoliutoionns: ThetestsubstanwcilelbeadminitosSteleenrasetrduminanaqueous `medium.Thisroute of
administrationwasselectedbecauseit epreseaisthemost likely routeofexposuretoalgae,whichexists.
inaquaticenvironments.
Testsolutionswillbepreparedbydilutingoneormorestocksolutionswithalgalculturemedium
toachievethetargetconcentrations. `Stocks will be preparedin algal culturemedium. Allweights, volumesanddilutions used to preparethetestsolutionsw.illbedocumentedintherawdata.
TestOrganism `The fres greehnaw lga,aSelt enae strr um capricwoillrbenusueditn tuhismte, st. Thisspecies.
isrepresentativeofanimportantgroupof algacand wasselected foruseinthetetbasedupon pastuse
andeaseofhandlinginthe laboratory. Stock cultures, obtained from theCulture Collection ofAlgae.
atthe UniversityofTexasatAustinor anothersupplier ilbe maintainedinculturemedium at Wildlife
International Lid.for
a minimum oftwoweeks priorto use in
atoxicitytest. Algac usedin toxicity tests
000669
PROTOCOL NO.: 454/110998/SEL-96H1/SUB454
3MLAB REQUEST NO. U2723
|
[Ml WILDLIFE INTERNATIONAL to.
un
=
om
willbe inexponentialgrowthphase,whichisdefinedastheperiodofgrowthwhenalgalcelsarc
divatiacdonsitanntrtge.
|:|
Justpriortobeginnitnhge test,aninoculumofthestockculturewillbeprepsoathratecadch
milliliterof inoculumcontainsenoughoels to providean ital cell density ofapproximately 10,000
ou
celinleac/hrepmlicLate
om
CmCuletutremnediumpreparedsecordingto WldifeIntemational Ltd. Standard Operating Procedures
willbeused asdilutionwater.Theconcentofrthaectomipoonenntss inthe medium ar presentiend
|
Table 1 (3). Stockmutientsolutionsvil bepreparedbyaddingreagent-gradechemicalsto Wildlife
International Led.well waterpurifiedby reverse-osmosis. Appropriate volumes ofthestocknutrient
om
solutionsvillthenbodilutedwithpurifiedwellwatertopreparethemedium."Themedwiillbue fimlter
sterilized(02 amorsutoslpraiovrteodus)e.Thefinal pHofthemediumshouldbe7.5.4.1. Analy-
o
seswill beperformedatleas once annuallytodetermine theconcentrationsof selectedorganicand
inorganicconstituents ofthewellwate andresultsofteanalyseswillbesummarizedinthe inal report.
ou
Specifications for acceptable levels of contaminants have not been established for culture
`medium.Howes,ther ar novel ofcontaminantsreasonablyexpectedtobepreseatinthe medium
ol
thatareconsideredtointerferewiththepurposeorconductofthestudy.
HH
TestApparatus
Test chambers will be sterile, 250-mL polycarbonate Erlenmeyer flasks plugged vith foam
stoppers. Eachtestchamberwillhold 100mLoftestsolution.Testchawilm lbeb indie scrir minas tely
mn
positionedon amechanicalshakertablein anenvironmentalchamberandvillbeshakencontinuously
atapproximately 100 rpm. Test chambers wibellabelled with theprojectnumber, testconcentration,
oH
andreplicate
`EnvironmentalConditions
ol
"Test flasks illbe eldat24 +2Cundercontinuousfluorescentlighting (c..colwhitelight
tubes)atanintensityof approxima4t30e0ly 10% lux.Lightintensity willbemeasuredatfive
ol
locationsontheshakertabledailyduringthetest. Temwpillbeemerasuaredttwiceudalryinethe
environmental chamber usinga liquid-in-glass thermometer.
nm
000570
oH
PROTOCOLNO.: 454/110998/SEL-96H1/SUB4S4
3MLAB REQUESNOT.U2723
WILDLIFE INTERNATIONAL LTD.
-6-
"ThepHofcachtreatmentan thecontrolwillbemeasureda estinitiationandterminationusing aFisherAccumetModel 915pHmeterorequivalent. Samples willbecolflromethecbattchoeftdest solutionpreparedat est nition fo cachtreatmentandcontrolgroupforthemeasuremoefpnHt. At testtermipHnwialltbemieaosurned,in potoestlsoelutdion samples colfrolmtehethcreetrepleicatdes ofcach treaandtconmtreolgnroutp.
`BiologicalMeasurements `Celldensitieswillbemonitoredduringthetestbyconductingcellcountsusing 8 hemacytometer
and amicroscope.Onesamplevillbecollectedfromeachreplicateofth treatmentandcontrolgroups at approximately 24hourintervalsduringtheexposureperiod. The sampleswillbe evaluated immediately. Each samplewillbedilutedusinganelectrolytesolution (Isoton),asneeded, tomaintain
`countingaccuracy.Thecelldensityofthesamplewillbecalculatedbasedonthemeannumberofcells
:
pergrid.
Samplesoftestsolutionswillbecollectedfromeachreplicatepertreatmentandcontrolgroup attheendofthetest.Eachreplicate samplevillbeexaminedmicroscopicallyforatypicalcell `morphology(eg.changesinshape,sizeorcolor). Growthofcellintheflasksalsowill beassessed foraggregationsorflocculation ofcelsandadherenceofthecllstothetestchamber.
Attheendofthetest, algistatieffects,whichresultintheinhibitionofcelgrowth,willbe differentiated from algicidal effets, which result in the deathofcells. Aliquots (0.5 mL) of test solutionswillbetakenfromcach replicate testchamber where growthis maximally inhibited. Maximallyinhibitedtreatmentsar thoseinwhich itcannotbevisuallydeterminediflvealgalcellsare preseat (eg. absenceofcolortothetestsolution).Thesealiquotswillbecombinedanddilutedwith algalmedium to aconcentrationofthetestsubstancethattheoreticallyshouldnoteffectgrowth. A negativecontolwillbeprepare bydilutinga0.5Laliquotfromonenegativecontrolreplicateto 100 `mLwithalgal medium.Growth ofthesesubcultureswillbemonitoredforup to 9daystodetermine whetherinhibitionofgrowth observedduringthetest ireversible. Samplesvil becollcftorceedll countsat recoveayphassinitiation,terminationandatapproximately3-dayintervalsbetweeninitiation andtermination. Therecoveryphasemaybeterminatedoncealgalgrowthissulfcenttoindicatethat thealgalcells havefully recoveredfrom effetsductothetestsubstance.Thiswillbedetermined through visualexaminationoftherecoverytest solutions,
PROTOCONOL.: 454/110998/SEL-96H1/SUBA454
000671
3MLABREQUENSOT. U2723
1MWioLFe
INTERNATIONALLO.
.
.
1
substan`cSeamcponlceesontfratthieonesx.poDsauyre0soslaumtpiloensswwiillllbbeectoalkleeacfterdoomneaDcahysbat0c,h3oafntedst4asnolduatnioanlpyrzieodrfoo ttheesitr
1
distributionamongthereplicateestchambers.Day 3sampleswillbecollected romtheextrareplicate
est chamber(s) included in the test to provide sample for that analysis and rom the abiotic replicates.
|]
1 more than one replicate is included for the Day 3 sample analysis, then the solutions from cach replicatewillbcompositedtoprovid thesampleforthat treatment. Day 4sampleswillbecomposited
ul
solutionsfromthethree replicatesremainingatthe endofthetest
1
SamplescollectedonDays 3and 4 maybecentrifugedorfilteredto removealgal cellspriorto
apnaolyslis.ypSarmpoolreppsoylwyilleltehbyenleanenealbyzoetd )imamneddsitaoterleyduonrtipllaacneadlyizneda.n
appropriate
The sample
storage
scheme
ciosnstuaimnmearr(icz.egd,
i
below:
PROPeOSEr D NUm MBERS OF VERIFICATION SAMePLtES
Control
|
i
Solvent Control (ifneeded)
l Level 1-LowConsentration
a
Level2
Level 3
|
a
Levels
I
a |LLovel 5-HighConcentration L111 1
a
"Theaboveumberofsamples representthosecollectedfromthetestanddono includequality control (QC) stmples such as marx blanks and fortifications prepared and analyzed during the
i
analytical chemistry phaseofthestudy.
a
000672
a
PROTOCOL NO.: 454/110998/SEL-S6HI/SUB4S4
3MLAB REQUEST NO, U2723
[Ell WILDLIFE INTERNATIONAL Lm.
|
_8-
AnalyticalChemistry
Chemicalanalysisofthesampleswillbeperformbeyd WildlifeInternationalLtd. The analytical `method used will be based upon methodology provided by the Sponsor and identified in Appendix IL.
Modificationsmade tothe analyticalmethodwillbe documenitntehde rawdataanddescriibntehde final report.
StatisticalAnalyses Bioxnassexpressedas areaunderthegrow curvewillbocalculated orthetreatmentandcontrol
groups using the following formule: A= (ONAND INRE ) HNNNI) Where:
"AN=,= MArcaeundaerntuhmsebgersuoowftchrcclusr/veem.L adtt, NN;, ==MMeeaassuurreedd nnuummbbeerrooffcceelllsl/mmLL aatttt,
'N, =Measurednumber of cells/mLatt, : t, = Timeoffirstmeasurementafterbeginningoftest(hours) 1, =Timeofsecondmeasurementafterbeginningof test(hours) t, =Time of nthmeasurementafterbeginning oftest(hours)
Growthrates(x) willbecalculated forthe treatmentandcontrol groups usingthe following
formula:
pm --_IN, InN, tb
N1,==AMveearsaugreedspnecuimfbicergorfocwethllrsat/emL att, N, = Measured mumberofcelisfmLat
to=Time ofbegionftn esti(hn ourgs) t, =Timeofnthmeasurementafterbeginningoftest(hours)
PROTOCONOL.: 454/110998/SEL-96H1/SUB454
000673
3MLAB REQUEST NO. U2723
1
1 = fl WILDLIFE INTERNATIONAL LTO
A
Percent inhibition will be calculated for each treatment group based upon mean cell density, n
`biomass and growth rate using the: following formula:
ii `Percent Inhibition =_ `MeRaens`pCMooennatsnreCooln-trMoelaRRneessTpprooenanstsmeeent X 100
EC values will be determined, when possible, using. linear interpolation or other suitable
i
techniques with treatment response (i. ocll densities, biomass and growth rate) and exposure
u
concentration dataforeach24-hourintervalofthetest.
||
{hedoseA-noresopbosnesrevepdaattdevmerasnedeftfhectrceosncuesnotfratthieosnta(tNisOtAicEalCa)nawliylslibsuessienlgectienddbiavisdeudalornepalinceavtaelvuaaltuicosnooff
the cell density, biomass a
he treatment groups will
nd growth rate
be identified
. Stat
using
istically
analysis
significant d
of variance
ifferences b
(ANOVA)
etween the
and a test
control and
to compere
Mm
treatmentmean responsestothecontrol response(. cg , Dunnett's test or Bonferroni'st-test).Datawill
om
boassessedfornormalityandhomogeneityofvariancesprior toperforintheANOVA. Transform-
om
taisoonlsvweinltlcboenutsreodlitocuosrerdeicntaadndyictoinodnittoiotnhoefnengoant-invoercmoanltirtoylo,rtuhneeirqrueaslpeornosersvwariilalnbceecs,omifppaosrseidbules.inig
`Student's t-test. Datafrom thetwocontrolgroupswillbepooledfortreatment levealnalysesifno
statisticaldifferencesexist.Otherwise, allcomparisonswillbemadeto thesolventcontrolgroup.
Statisial comparisonsfor ANOVAsnd hecomparisonofmean responsesvill be cared outusing
TOXSTAT V3.5 software (4) or equivalent.
REC TOBOEMR AIND TAIS NED
RecordstobemaintainedfordatageneratedatWildlifeInternational Ltd.vil include,butnt
be limited to:
1 Copy of signed protocol. 2. Identificationandcharacterization of thetest substance,ifprovidedby Sponsor.
4 Souseofalgae. 3. Datesofinitiation andterminoaftthietoesnt.
000674
5. Culture conditions.
PROTOCOL NO.: 454/110998/SEL-96H1/SUB45
3M LAB REQUEST NO. U2723
8
Jl WILDLIFE INTERNATIONAL LTD
a
-10-
i ou
["]
ou
u
|| om ou
ou Mm mn
6. Growthmeasurements. 7. Calculationandpreparationoftestconcentrations.
8. Observations.
measurements 9. The methods usedto analyzetestsubstanceconcentrationsand theresultsofanalytical
10. Statistical calculations.
11. Testconditionsandphysical/chemicalmeasurements.
12. Copyof finalreport.
FINALREPORT
Afinalreportoftheresultsof thestudy villbepreparedbyWildlifeIntemational Ltd.Thereport.
willinclude, but notbe limited tothe following,whenapplicable:
1. Na andm addre essofthefacility perforthmesitundyg.
2. `Datesupon which thestudywasinitiatedandcompleted, andthedefinitive experimentalstartand
3.
terminationdates.
.
Astatementof compliancesignedbytheStudyDirectoraddressinganyexceptionsto Good
`LaboratoryPractice Standards.
4. Objectivesandprocedures,asstatedinthe approvedprotocol,includingallchanges tothe
protocol.
5. Thetestsubstanceidentification including name,chemical abstract numberor code number, strength,purity,composition,and otherinformation providedbytheSponsor.
6. Stabilityandsolubilityofthetestsubstance under theconditionsofadministration,ifprovided.
`bytheSponsor.
7. A descriptionofthe methods usedto conductthetest.
8. Adescorf tihetpesttorgianoismns, incluthde siounrcge, scineamen,atgeoir lfifesitagce, light
intaendnphsotoipetrioyd. 9. Adescriptionofthepreparationof thetestsolutions.
10. Themethodsusedtoallocateorganismstotestchambers andbegin thetest,the number of
organismsandchamberspertreatment,andthedurationofthetest.
11. A descriptionofcircumstancesthatmayhaveaffectedthequalityorintegrityofthedata. 000675
PROTOCNOOL.: 454/110998/SEL-96H1/SUB454
3MLABREQUENSOT. U2723
1
J WILDLIFE INTERNATIONAL L1D.
1
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1
12. ThonameoftheStdDirectorandthe namesof otherscieatists,professaindosnupaerlvisso,ry
personnelinvolvedin thestudy.
a
13. A description ofthe transformations, calculations, and operations performed on the data, a
summaryan analysisofthe biologicaldata ndanalyticalchemistrydata,andastatement ofthe
a
conclusionsdrawfrom tahnalyses. Agraphplottingtheconcentrerspaontseicuorvne.
14. Statisticalmethodsusedtoevaluat thedata.
a
15. Thesignedanddatedreportsofeach ofthe individual scientistsorother professionals involved
inthe study.
n
16. 17.
TAhsetaltoecmaetniotpnwrheeparreerdabwydtahteaQaunadliftiynAalsrseuproartncareeUtotbleissttionrgetdh.edatesthastudyinspectionsand
a
itsweremadeand hedates ofanyfindingsreportedtothe StudyDirectorand Management.
18. Ifitisnecessary tomakecomostionsoradditionsto afinalreportafer ithasbeenaccepted,such
au
changeswillbemadeintheform ofanamendmentissuedbytheStudyDirector. Theamendment - willlealy identifythepat oftefinal reportthatisbeingamendedandthereasonsforthe
om
amendment,andvillbe signedbythe StudyDirector
ou
CHANOFGPRIOTNOCGOL Plannedchangestotheprotocolwill be in the formofwritenamendmentssignedbytheStudy
Hl
DirectorandtheSponsar'sRepresentative.Amendmentsvillbeconsideredaspartoftheprotocol and
illbeattache to theinlprotocol.Anyotherchangeswillbe i theformofwritendeviationssigned
a
bythe StudyDirectorandfledwiththerawdata. Allchangestotheprotocol illbeindicatedinthe
=
final report.
GOODLABORATORY PRACTICES
H
"ThisstudywillbeconductedinaccordancewithGoodLaboratoryPracticeStandardsforEPA
(40 CFR Part 160 andlor Part 792); OECDPrinciplesofGoodLaboratoryPractice (OCDE/GD (92)
32, Environment Monograph No. 45); and Japen MAFF (59 NohSan, Notification No. 3850,
Agricultural Production Bureau), Each study conducted by Wildlife Intemational Lud. is routinely
examined by the Wilde Intemational Lid. Quality Assurance Unit for complisncs with Good
Laborstory racic,StandardOperating Proceduresandthespecifiedprotocol. Astatemenotf
PROTOCONOL; 454/110998/SEL-96H1/SUB4S4
3M LAB REQUEST NO, U2723
000676
WILDLIFE INTERNATIONAL LTD.
-12compliance with Good Laboratory Practices vill be preparedfo all portionsofthe study conducted by Wildife International Ltd. The Sponsor will be responsible for compliance with Good Laboratory Practice for procedures performbeyd other laboratories (e.g. residue analyses or pathology). Raw data forallworkperformedatWildifeInternationalLid.and acopyofthefinalreportwillbe filedbyproject number in archiveslocatedon the Wildlife International Ltd. site of at an altemative location to be: specified in the final report
PROTOCOL NO.: 454/110998/SEL-96H1/SUB4S4
000677
3MLAB REQUEST NO. U2723
J WILDLIFE INTERNATIONAL LTD.
1
-13-
1
RERINCES
1
1 US. Environmental ProtectionAgency. 1996. Series 850-EcologicalEffects Test Guidelines
(drafi), OPPTS Number 850.5400: Algal Toxicity, Tiers I and IL
1
2 OECD Guideline for TesoftChi emincalgs. 1984. 201: Alga, Growth Inhibition Test.
3 ASTM Standard Guide 1218-90E. 1990. StandardGuide for Conducting Static 96-Hour
1
Toxicity Tests with Microalgae.
4 West, Inc. and D.D. Gulley. 19%. TOXSTATRelease 3.5. Western EcoSystems Technology,
||
Inc. Cheyenne, Wyoming.
a
000678
a
PROTOCOLNO.: 454/110398/SEL-96H1/SUB4S4
3MLAB REQUEST NO. U2723
i WILDLIFE INTERNATIONAL LTD.
a
14
a
TABLE
FRESHWATER ALGAL MEDIUM CONSTITUENTS
ou
Component
Nominal Concentration
n
MCEaCCLl62HH,,O00
4142016 mmgg
MHa,CBl0o,4H,0
00.4118656 mmg/gL
ou
FeZCCl,i6H,0
302.81598 gmgl/L
i
CNoeC,lM6oHO,,20H,0
7124268 uugg
CNau,CELD2THA,20H,0
00031020 mugl
i
MNgaSNOO,,7H,0
21545700 mmegl
"]
KNaHHPCOO,,
1044 mglL 1500 mpl
"The pHofthemediumwillbeadjusted,asnecessary,t07.5%0.1using0.1
ua
NNaoOr1H0% HCL
ou
000679
i|
PROTOCONOL.: 454/110998/SEL-96H1/SUB454
3M LABREQUEST NO.U2723
J WILDLIFE INTERNATIONAL LTO.
1
-15-
1 Ju IDENTl IFICATION OFi ATPEPSETNSDUIBXSTAa NCE BYaSPbOaNn SOR a
I-
TobeCompletebdy Sponsor
I Test Substance dent (nametobeused nthereport): E(QPS erfluoSulrfonoicAocidsPottasasiunmSaelt
1
Reference Standard (f applicable): AnaIlnytteircnaall SSttaannddasr:d_NP1.1e.A2.r2F1fHHlHuorooSculftonaicAncied
1
Test Substance SampleCodeorBatch Number:_ Lot 217
Test Substance Purity (% Active Ingredient): _989
Expiration Dot: 2008
1
IL Test Substance Characterization
Havethe identity,bstyrdefing D ietytanudcbompaositatnidoirnoernforthoaecrcShtaarnadcatrerdibseticcns
1
Hetcrminedprior tisuseinthisstudyin accordancewith GLPStandards? YesNo X_
IL Test Substance Storage Conditions
i
_PA l_easem indicb atetheirece omment dedstr orageo condio tionsamtWilt dlifeIe ntemam tionp alLide . rsee
i
HHaes tehesa tabilityofthe testeseuwabl sCuLntFdeSrautahnnedsaecrsdtseo]rage condifions
YesN_o_X__
1
O-- ther p-- ertinen-- t steabi-- elite y inf-- oe rmatio-- ns ----------------------
1 IV. TestConceatrations:
_X_Adbajsuesdtupesotnctohnecpeunrittrya(t%it)ogoniv1e0n0%ebnoiv.e.
Dao1noTtesdttshte mastetricaolnAeSsmISp.ionto 100%
|] V. Toxicity Information:
Mammalian: ~~ RatLDS025lmghke MouseLDSO NA
i
Aquatic:
InvertebrE ateToxicZ ity (BC/LCS0)
2 Fish Toxic.ity (LC50)
Ha
Diphniamagna: Somg/l
Fathead Minnow, 38mg/l
Other Toxicity Information (including findingsofchronic and subchronic tests):
ul
HemMDS
000680
PROTOCOLNO: 454/110998/SEL-96H1/SUB4S4
3MLAB REQUEST NO, U2723
1
1 WILDLIFE INTERNATIONAL LTD.
1
-16-
APPENDIX I
1
Ansytical Method to be Provided by Sponsor
I
1
I
I
1
1
I
3
I
I
I
I
I
1
I
000681
[1
PROTOCOL NO.: 454/110998/SEL-96H/SUB4S4
MLAB REQUEST NO. U2723