Document ppE7omX1D4VrL4B0vyOY7KGxk
_ AR126-0095
PFOS: A 96-HOUR STATIC ACUTE TOXICITY TEST WITH
`THE SALTWATER MYSID (Mysidopsis bahia)
FINAL REPORT WILDLIFE INTEMRNLAATBIROENLQTAUDLE,STPRNOOJ,E3C7T3N3UMBER: 4504-101
ScrUS150EOamPliTnoSeeNnusmtbeEPror8s5i0o1T0no3A5Ggearienes
AuORS at Dror
HenrOy. Krueger, Ph.D.
STUDY INITIATION DATE: Desir, 1998
STUDY COMPLETION DATE: June 24, 1999
AMENDED REPORTDATE: Apr 26,2000
1
Submitted to
3M Corporation
Envi5r3osnmBentualALavboeratory
St. Paul, MN 55144
Wildlife 4I5n9tCoemmrenrcae tivieonal Ltd. Easton, Maryland 21601 (410) 822-8600
Page 1of41
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PROJECT NO.: 454A-101
`GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT
SPONSOR: 3M Corporation
TITLE: PFOS: A 96-Hour Static Acute Toxicity Testwith the Saltwater Mysid (Mpysidopsis bahia) `WILDLIFE INTERNATIONAL LTD. PROJECT NUMBER: 454A-101 STUDY COMPLETION: June 24, 1999 AMENDED REPORT: April 26, 2000
`Thisstudywasconductedincompliancewith GoodLaboratoryPracticeStandardsaspublishedbythe
USS. Environmental Protection Agency in 40 CFR Parts 160 and 792, 17 August 1989; OECD Principles of GoodLaboratoryPractice,OCDE/GD (92) 32, Environment Monograph No. 45, Paris 1992;and JapanMAFF, 59 NohSan, Notification No. 3850, Agricultural Production Bureau, 10 August 1984, with the following exceptions:
Thetestsubstance wasnotcharacterizedin accordancewithfullGLPcompliance;however,the characterization wasperformedaccording to 3M StandardOperating Procedures and Meth andoalldras wda,ta arebeingmainit nthae 3iManrcheiveds. Thetestsubstanceisbeing recharacterized in accordancewith GLP.
Thestability of thetest substanceunder conditions of storage atthe test site wasnotdetermined in `accordance with Good Laboratory Practice Standards.
SEE STUDYDIRECTOR: Kurt R. Drottar `Senior Biologist
pee DATE
`SPONSOR APPROVAL:
garko
Sp
DATE
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QUALITY ASSURANCE STATEMENT
ThisstudywasexaminedforcompliancewithGoodLaboratoryPractice Standardsaspublishedbythe
U.S. Environmental Protection Agency in 40CFRParts 160 and 792, 17 August 1989; `OECD Principles of GoodLaboratory Practice,OCDE/GD(92) 32,EnvironmentMonograph No. 45, Paris 1992;and JapanMAFF, 59 NohSan, Notification No. 3850, Agricultural Production Bureau, 10 August 1984. The datesofall inspectionsandaudits andthedatesthatany findingswerereportedtotheStudyDirectorandLaboratory Managwee rem asfeoln lowts:
sin Te Ppt -- Sfosgt Shtoepmie AnalyticalDataand.
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Meads mes, 199 Am
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Ja H.Coleman. QualityAssurance Representative
tao
DATE
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REPORT APPROVAL
PROJECT NO: 454A-101
SPONSOR: 3M Corporation TITLE: (PMFyOsSi:dopAsi9s6-bHahoiuar)Static Acute Toxicity TestwiththeSaltwater Mysid WILDLIFE INTERNATIONAL LTD. PROJECT NUMBER: 454A-101
STUDYDIRECTOR:
GN SKeuariotrRBDirooltogtiasrt
MANAGEMENT:
DHieraerctyo0r., Aquatic,Toxicology and Non-Target Plants
_geeee
DATE
Hoste DA
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`TOA F CB ONL TENE TS THISACOVEEPABO.covcussrsseussrssarssrssessussess sssessssessossossusssssesessossessessassosssessossomsmrsssssssorss |
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OBJECHVE corres
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TABLES
Table 1 - Summary ofAalyCHtOMiistrcyDaBAl crn: 14
Table 2 - Temperature,DissolvedOxygenandpHof Waterinthe Test ChAMBETS........vcovumemsssss 15
`Table 3 - Cumulative PercentMortalityandTreatment-Related
BEECCtS..........ovwemmvsesssssmsssssses 16.
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`TABLE OF CONTENTS - Continued -
PROJECTNO.: 454A-101
APPENDICES Appendix 1- Salinityand pHofSaltwaterMeasuredDuringthe4-WeekPeriod
Appendix 11 AnalysesofPesticides, Organics,MetalsandOtherInorganics
AppendixIIT
The Analysis International
ofPROSin SaltwaterinSupportof Wildlife Ltd.ProjectNo.: 4S4A-I0] creme:
20
Appendix V - Personnel InVOIVed i the SWAY vss 39
ADPEOX V+ REDOTt AMEDD...
40
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SPONSOR SPONSOR'SREPRESENTATIVE: LOCATOFISTOUDNY, RAW DATAAND ACOPYOFTHE. FINALREPORT: "WILDLIFE INTERNATIONAL LTD. PROJECT NUMBER: TEST SUBSTANCE: stop:
MCEOANCNEMNTERAASTUIROENDST:EST TEST DATES:
LENGTHOFTEST:
TEST ORGANISM: SOURCEOFTEST ORGANISMS:
AGE OFTESTORGANISMS: S6HOURLGSO: 95%CONFIDENCELIMITS: CONNOCMEONRTTRAALTIITOYN: NO-OBSERVED-EFFECTCONCENTRATION:
SUMMARY "3M Corporation Ms. Susan A. Beach
`WildifeInternationalLtd. Easton,Maryland 21601
454A-101 PFOS (Perfluorodetans SulAfcidoPontasisicum Salt) PSaFlOtSwa:teAr9M6y-sHiodu(rMSytsaitdiocpAsciustbeaThoixai)city Testwiththe NegativeControl; 057, 1.1, 19and,S43 mga0 ii/L.
ExperiStmaret-nMatrcahl8, 1999 Biological Termina~tMiarocnh 12, 1999 Experimental Termination ~March 12, 1999 96Hours
Saltwater Mysid (Mysidopsis bahia) `EWaisltdoinf,eIManrytleanrdn2a16Lti0d1i.coulntuarels
Juveniles
T6mgaill 30md46mgaill
Limgaill
Llmgaill,
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INTRODUCTION
"ThisstudywasconductedbyWildlifeInternationalLtd.for 3MCorporationattheWildlifeInternational Ltd.aquatictoxicologyfacilityinEaston,Maryland.TheinlfephaseofthetestwasconductedfromMarch8, 19to9Mar9ch 12, 1999. RawdatageneratedbyWildlifeInternationalLud.and acopyofthefinalreportarc. fledunderProjectNumber 454A 101 inarchiveslocatedontheWillieIntemational Ltd. site.
OBJECTIVE
"TheobjectiveofthisstudywastoevaluatetheacutetoxicityofPFOS (PerfluorooctaneSulfonicAcid Potassium Salt) tothe salmtyswid,aMystidoepsris bahia, dura i96-nhogur exposureperiodunder static test conditions.
EXPERIMENTAL DESIGN
`Saltwater mysidswereexposedto ageometricsriesoffivetestconceatrationsand anegative (saltwater) control. Tworeplicatetestchambersweremaintainedineachtreatmentandcontrolgroup,with 10mysidsin eachtestchamberforatotalof 20mysidspertestconceatration. Nominal testconcentwreraesteliecotendisn consultationwiththe Sponsor,andwere basedupon theresultsofan exploratoryrengefindingtoxicitytest. Nominaltestconcentrsealetctiedownerse 1.1,1.8,3.0, 4.9and 8.2mgactiveingredient (a.YL. Meanmeasured. testconcentrationsweredeterminedfrom samplesofestwatercollectedfromcach treatmentandcontrolgroupot. thebeginningofthetes, tapproximately48 Boursandat esttermination.
`Themysidswereindiscriminatelyassigned toexposure chambersattestinitiation. Observatioonfs
`mortalityandotherclinicalsignsweremadeatapproximately4.5,24,48, 72, and96hoursaftetestiitition. Cumulative perocatmortality observed nth treatmentgroupswasusedto estimateorcalculateLCSOvaluesat 247,2an4d986 ho,urs.Thenomortalityconcentrationandno-observedt<ffectconcentration(NOEC)vere determinedbyvisualinterpretation ofthemortalityandclinicalobservationdata.
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MATEARNI DMA ETL HOS DS
Thestudywasconducted basedon the procedures outlined in the protocol,"PFOS: A 96-HourStatic
Acute Toxicity Test withtheSaltwater Mysid (Mysidopsis bahia)". The protocol was basedonprocedures
outlinnUe.Sd. EnvironmentalProtection AgencySeries 850EcologicalEffectsTestGuideliOnPePsT,S `Number 850.1035(1); U.S. EnvironmentalProtection Agency,StandardEvaluationProcedure, Acute Toxicity
Test forEstuarineandMarine Organisms(2);and ASTMStandard E729-88aStandard Guidefor Conducting
Acute Toxicity Tests with Fishes, Macrotnvertebrates andAmphibians 3).
`Test Substance
Thetestsubstancewasreceivedfrom 3MCorporationonOctober29, 1998andwasassignedWildlife Intemational Ltd.identificationnumber4675. Thetestsubstanwcaes desacsarwhiitb epoe wdedr. Itwas. identifiedas FC-95fromlotnumber 217(T-6295).Informationprovidedbythe Sponsorindicated apurity of 98.9%,andanexpirationdata of2008. `Thetestsubstance wasreanalyzedbythe SponsorandtheCertificateof Analysis dated March9, 2000 indicated a purity of 90.49%.Thetestsubstancewasstoredatambientroom temperature.
Prepoa f TerstaContcenitraotionns
`No testm conci entrn atioa nswel re 1.1,1.8,3.0,4.9 and 8.2 mg a.i/L, basedon a test substance purity of
90.49%. Allmaterialswhichcameintocontactwiththetestsubstanceduringpreparationoftestconcentrations.
`wereconstructedofplasticorstainlesssteel. A primary stock solution was prepared in dilution waterat & concentrationof 8.2mgai/L. Theprimarystocksolutionwasmixedwithan electricmixerforapproximately22 hourstoaidinthesolubilization ofthe testsubstance. `Aftermixing,theprimarystocksolutionwas `proportionallydilutedwithdilutionwatertopreparethefouradditionaltestconcentrations. Alltestsolutions.
* appcle earaandrcole orledss.
`TestOrganism
`Thesaltwatermysid, Mysidopsis bahia, was selected asthetestspeciesforthisstudy.`Thesaltwatermysid
isreprescatative ofanimportantgroupofaquatic marineinvertebrates andwasselected forusein thetestbased uponpasthistory ofuseandeaseofculturinginthelaboratory. Mysids usedinthetest wereobtained as
juveniles from cultures maintained by `Wildlife International Ltd, Easton, Maryland.
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Adultmysidswereheldatapproximatelythesame temperature as used duringthetest. Durthiehonldigng
andacclimationperiods,the adultsshowednosignsofdiseaseorstress.During the 14-day holdingperiod
precedingthetes, watertemperaturesrangedfrom25.010 25.5C. The pH ofthewaterrangedfrom7.90 8.1, salinity was maintainedat 20% (partsper thousand) anddissolved oxygen ranged from 7.0to 7.2 mg/L.
Instrumentationusedfor watermeasurementsisdescribedinthe Environmental Conditionssectionofthisreport.
Attestinitiation,thejuvenilemysidswerecarefullycollectedfromtheculturesand transferredtoglassbeakers.
`Mysidswere transferredfrom thebeakers tothe testcompartments using a wide-bore pipette.Juvenilemysids
ererelativelyfragileandwer,therefore, handledasltl aspossible. Thejuvenilemysidswerefedlvebrine
`shrimp(Artemia sp.)naupliidaily duringthetesttoprevent cannibalism.
Test Apparatus
"Testchamberswere 2-Lpolyethylene bucketsfilledwith approximately1000mLoftestwater.Thedepth ofthetest waterin arepresentativetestchamberwasapproximately 6.6 em.Test chawmerb e imeparr tias lly `positionedinanenvironmentalchamberdesignedtomaintain atemperature of252C. `Thetestchamberswere:
Iabeledwiththeprojectnumber, testconcentrationandreplicateandwerecovered with plexiglassthroughoutthe
test.
_----
`The water used for holding, acclimationand testingwas natural seawatercollected at Indian River Inlet, `Delaware, and dilutedto a salinity of approximately 20% with well water.`Salinity and pH measurementstaken.
duringthefour-weekperiodimmediatelyprecedingthe testarepresentedinAppendix I.
Thefieshly-ollestedseawaterwas passedtrough asand fille to remove prticles gratethan
`approximately25 wm, andpumpedinto a 37,800-L storage tank. The filteredseawaterthenwas diluted with freshwaterfrom awellontheWildlifeInternational Ltd.siteandacratedwithspraynozzles. Prtoiuso e,trhe `water again was filtered (0.45 um)to removemicroorganismsand particles. `Theresults ofperiodicanalyses `performedtomeasurethe concentrationsofselected contaminantsinsaltwaterusedbyWildlifeIntemationalLtd. are presie nApnpetnde ix IdL
Lightingused to illuminatetheculturesandtestchambersduringculturingandtestingwasprovided by fluorescent tubes that emitted wavelengths similar to natural sunlight (Colortone 50). A photoperiod of 16 hoofu lighr tans d8hoursofdarkness wascontrolledwith anautomatictimer. A 30-minutetransitionperiod
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of lowlightintensiwtaysprovidedwhenlightswentonand offtoavoidsuddenchangesinlighting. Light {nteasityattestinitiationwasapproximately437 huxa thesurfaceofthewater.Lightintewaas smeiasturyed using a SPER ScieLnudt.liighftmietecr.
Temperaturewasmeasuredincachtos chamberatthebeginningandend ofthetestsing aliquid-in-lass thermometer,Temperaturealso wasmeasuredcontinuouslyinonenegativecontrolreplicateusing a Fulscope ER/C Recorder, Thetargettest temperatureduringthestudywas252C. Dissolvedoxygenand pH measurementsweremadeonwatersamplescollectedfromallreplicatetestchambersofeach treatmentand control a estiitition,andatapproximately24-hourintervalsthereafer.Salinitywasmeasinutherdileutidon waterat testinitiation.
MeasurementsofpHweremadeusinga FisherAccumetModel 915pHmeter,anddissolvedoxygenwas `measuredusingYellowSpringsInstrumeatModel51Bdissolvedoxygenmeter.Salinity wasmeasuursiengda. Bio-Inc.mAquaaferunairefrnacteomet,er.
Obser
va
t
ions.
Observationsw
eremade
to
determine
t
he
numberofmortals.
Th
en
u
mof ibndievidurals
exhibiting.
clinics!signsoftoxicityorabnormalbehavioralsowereevaluated. Obsewerrevmadaeaptproiximoatenly4s.5,
24,48, 72and96hoursaftertestiiiaion.
Statist "The24,48,72and 96-hourLC50valuesandthe 95%confidenceintervalswerecalculatedwhenpossible
byprobitanalysis,themovingaveragemethodorbinomialprobabilitywithnos-linearinterpolation (45,6) sing thecomputersoftwareofC.E.Stephan (7). thisstudy,LCS0valuescouldnotbecalculatedat 24 nd48burs duetothelackofanadequateconcentration Tespoasepater,however,th probitmethodwasusedto cvaluate moratt7a2anld i96htourys.Thonomortalityconcentrationand NOECweredeterminedbyvisualinterpretation ofthe mortality and clinical observatdiatoan.
Analytic`aWlaCtheermsiasmtprlyeswerecollectedatmid-depthfromeachreplicatstestchamberofeachtreatmentand controlgroupatthebegianingofth test,at 48 hoursandattestterminationtomesureconceatratonsofhotest substance. Thesampleswerecollectedinplastic(Nalgene)vialsandanalyze assoon as possiblewithout storage. Analyticalproceduresusedintheanalysisofthe samplesareprovidedinAppeadixII.
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RESANU DDILSCUTSSISON
MeasoufTresteConmcenetrantiotns
Resultsofanalysestomeasureconcentrations ofPFOSinwatersamples collctedduringthetestare prescatedinTable 1andintheanalytical chemistryreport (AppendixI).Nominalconcentrsealetciteodfnosr useinthisstuwedrey1.1, 1.8,3an.d08.2,mg4a../9L. Samplescollectedattestnitionhad measuredvalues thatrangedfrom 52to71%ofnominalvalues.After22hoursofmixing, theprimarystocksolutionwas measuredtobe70to 71%ofnominalandwasprobably atthelimitofsolubliyinfilteredsaltwater. The, recoveries intheothertestconcentrations weresimilarbecause they wereproportional dilutionsof theprimary stock.Measuredvaluesforsamplestakeaat 48 hoursrangedfrom44 071%ofnominal.Measuredvaluesfor samplestakenat 96hoursrangedfrom36to 7o1 fnomi% nal. Whenmeasuredconceatoafttheisaompnless analyzedattestinitiation,approximately 48hoursandattestterminationwereaveraged,themeanmeasured. concenfotthrisastutdywieroe0n.5s7, 1.1, 19,3.0and 5.4mg./L. Meanmeasuredconcentrationswereused intheestimationorcalculationofLCS0 values.
ObseravndaMetasiureomenntss:
Measurementsoftemperature,dissolvedoxygenad pHarepresentedin Table2. Temperatureswere withinthe252C rang establishedforth test.Dissolvedoxygenconcentrationsremained26.6 mg/L (90%of saturation)throughoutthetest,MeasurementsofpHrangedfrom 8.1 to 8.2duringthetest.
Dailyobservationsofmortalityandotherclinicaligasoftoxicity observedduringthetestareshownin Table 3. Mysidsinthenegativecontrol,andthe0.57and1.1mg ../L treatmentgroupssppearednormaland healthyduringthetest.After96-hoursofexposure,mortalityinthe1.9,3.0and 5.4mg .i/Ltreatmentgroups was 10, 40and 75%respectively.LC50valuesand 95%confidencelimitsat 24,43,72and96hourswere estimatedorcalculatedfrom themortalitydata,andareshownin Teblo4.
CONCLUSIONS The96-hourLC50valueforsaltwatermysidsexposedto PFOSwas 3.6mg aL.The95%confidence Jimswere 3.0and 4.6mg .i/L,andtheslopeoftheconceatration-crurevsewpaasn4s.5e.The 96-hounor `mortality conoentrationandtheNOECwere1.1mg ../L.
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REFERENCES
1 `USO.PTESnvNiurmonbmeern8t5a0l.P1r0o3t5e:ctMiyosniAdgeAncuctye.To1x9i9c6i.tSyerTiesets. 850 ~Ecological Effects TestGuideilns(draf), 2 JUoSr.EEsntuvairrionnemeanntdalMParroitneectOirognaAngiesnmcsy.(Sh19r8i5m.pSt9a6n-dHaorudrEAvcaultueatTiooxnicPirtoyceTdesutr)e., AHcauztaerTdoxEivcailtuyatTieosnt
Division. OfficeofPesticide Programs. EPA 540/9-85-010. Washington, D.C. 3 MAaScrToiMnvSetratnedbarartdeEs7,2a9n-d8A8m5p.hi1b9i94a.nsS.taAndmaeridcGauniSdoecfioctryCfoornTdesuticngtAaicnundtgMeaTtoexriicailtsy.Tests with Fishes,
4 Thompson,WR. 1947.BacteriologicalReviews, Vol.Tl, No. 2. Pp. 115-145. 5 S"tAempehrainca,nCSEoc.ie1t9yf7o7.r "TeMsettihnogdasnfdorMaCtaelrciuallast.inPguabnliLcCaSt0io"n,NguumabteircSTTPo634x, pipan6d5c-H8a4oz.arldEvoaluagtioyns. 6 Finney, DJ. 1971.StatisticalMethods inBiologicalAssay. Second edition. Griffin Press, London. 7 Stephan, CE. 1978. USS. EPA, Environmental Research Laboratory, Duluth, Minnesota. Personal
communication.
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Table 1 `SummaryofAnalytical Chemistry Data
T `TDioluttiotn oWante:r: E__DFilSierede SeNltiwa,terMotolnsg Tbiinee V Conoeniion MCeonmcMenet] rwawtieonl PaOgfe
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000947
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Tae
Tempera,DissolvedOryenend pHof Wate ite TestChambers
-- C T Toek E OSte yiAptle
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WILDLIFE INTERNATIONAL Lm. -17-
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Table 4
LCS0 Values
-S_ponsor: Test Substance: TDeilsuttOirongaWnaitsemr:: _ Time 24 Hours
3M Corporation
PFOS
SFaillttwearetderSaMlytswiadte,rMysidopsis bahia
TCoonwfeird9en5c%e ~~UCponpfeirde9n5c%e
LCso gail)
Limits (mgail)
Limits (mgaill)
>54
=
E
-- Statistical Method Visual Interpretation
48 Hours
>54
-
=
`Visual Interpretation
72 Hours
44
36
62
Probit
96 Hours
36
30
45
Probit
-- Co"Cnofnifdideenncceelii-- mtitssccoouullddnnoottb-- beec calcualatetd w-- ith the mortalit-- y data obtzined.----
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APPENDI1X
rea------------------------ `Sponsor:
Salinity and pHof SaltwaterMeasured During the4-Week Period Immediately Preceding the Test
3M Corporation|
T`Teesstt OSurbgsatnainscme:: SPaFlOtwSater Mysid,Mysidopsis bahia
Dilution Water: Filtered Saltwater
--_--
Salinity(%6)
Mem
20N=4)
Ree
20-20
- pH. __B 82 (N=4)ee 8.1-s 82
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APPENDIX II
Analyses of Pesticides,Organics,MetalsandOtherInorganics
esi Si in Wildlife International Ltd. Saltwater!
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recess 20.
APPENDIX II
DPREOJSECOTTNO.O: 48584A0-01001)
`THE ANALYSIS OF PFOS IN FILTERED SALTWATER IN SUPPORT OF
`WILDLIFE INTERNATIONAL LTD. PROJECT NO.: 454A-101
00953
a WILDLIFE INTERNATIONAL Lm.
PROJECT NO.: 454A-101
REPORT APPROVAL
SPONSOR: 3M Corporation
"TITLE:
PFOS: A 96-Hour Static Acute Toxicity Testwith the Saltwater Mysid
(Mpsidopsis bahia)
`WILDLIFE INTERNATIONAL LTD. PROJECT NO.: 454A-101
PRINCIPALINVESTIGATOR:
4 SILL
"Van Hoven, Ph.D. Scientist
MANAGEMENT:
7/7072
`Willard B. Nixon, Ph.D.'
Manager, Analytical Chemistry
iow
DATE
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Introduction
Saltwatersampleswerecollectedfrom a staticacuteaquatictoxicitystudydesignedtodeterminethe effects of PFOS (Perfluorooctane Sulforic Acid Potassium Salt) to the saltwater mysid (Mysidopsis bahia). Thisstudywasconductedby WildlifeInternationalLtd.andidentifiedas Project No.: 454A-101 `TheanalysesofthesewatersampleswereperformedatWildlifeInternational Lid.usinghighperformance liquidchromatography withmassspectrometricdetection(HPLC/MS). Sampleswererecefoiravnaleysdis `onMarch8, 10and12,1999andwereanalyzedoncachsamplereceiptday.
`S TestubsantdIna ternn alStc ande ard
Thetestsubstance usedfortheanalytical portionofthisstudywasWildlifeIntemationalLtd. identification number 4675."Thetestsubstancewasusedtopreparecalibrationandmatrixfortification samples.
Theinternalstandardwasreceivedfrom 3MCorporationonJuly2,1998 andwasassigned Wildlife InternationalLtd. identification number 4526 uponreceipt.Theintemalstandard, agramlar material,was identified as: 1H, 1H, 2H, 2H Perfluorooctane Sulfonic Acid, ChemicalAbstractNumber: 27619-97-2. Thestandardvasstoredunderambieatconditions.
AnalyticalMethod
"Themethodusedfor theanalysisofthe saltwatersamples wasdevelopedat WildifeInternationalLtd andentitled"AnalyticalMethodforthe Determinationof PFOSinFreshwater, SaltwaantdAelrga,l Medium". ThismethodologywasincludedasAppendixIIofWildifeIntemationalLtd.protocol umber 454/011299/MVALISUB454. Itwasbaseduponmethodologyprovidedby 3M Corporation.
Samples werediluted in a 50%methanol : 50%NANOpure water solutioncontaining 0.mg14H0PF0OS (internal standard) and 0.05% formic acid (vA) so that they fell within the calibrationrangeofthe PFOSmethodology.
Conceatratonsofthe PFOSinthestandardsandsamplesweredeterminedbyreverse-phasehigh `performance liquid chromatography using a Hewlett-Packard Model 1100 High Performance Liquid
060955
AWR ILDLIFER INTE ERNAI TIONL AL L, o. eeeTPRROOJEECCTTNNOO.::d454S A-10, 1 23.
Chromatograph (HPLC) with a Perkin-Elmer API 100LC Mass Spectrometerequippedwith a Perkin Elmer TurbolonSpray ion source. HPLC separations were achieved using a Keystone Betasil Cy analytical column(100mum x 2mum LD., 3jumparticle size). The instrumentparametersare summarized inTable1. A methodflowchartisprovidedinFigur1e. Cafbration Limit of Quati
Calibration standardsof PFOS prepared in a 50% metha:n5o0l% NANOpurewatersolution containing 0.mg14H0PF0OS (internalstandard) and 0.05%formicacid (v/v), ranginginconcentration from 0.00229to 0.0457mga../L,wereanalyzedwiththesamples.Linearregressionequationswere generatedusingpeakarearesponseratios (PFOS :intemal stavenrsudstaherrespedcti)ve concentrations ofthecalibrationstandards.AtypicalcalibrationcurveispreseinaFitguered2. Theconcentratioofn PFOS inthesampleswasdeterminedbysubstitutingthepeakarea responseratiosintotheapplicableliar regressionequation.Representativeionchromoafltowaondghigrhcaalibmratsion stanardeaprersedntsed inFi3ganud4,rrespeectivsely.
`Themethodlimitofquantitation(LOQ) forthese analyseswassetat0.115mga.i/L calculatedasthe prodoufcthtelowestcalibrationstandardanalyzed (0.00229mg 2.i./L)andthedilutionfactorofthe `matrixblanksamples (50.0).
s
Fortification
`Threematrix blanksampleswereanalyzedtodeterminepossibleinterference. Nointerferenwceerse observedatorabovetheLOQduringsamplesanalyses(Table2). Arepresentativechromatogramof a `matrixblankispresiennFitguere5d.
Saltwater wasfortifiedat 0.457, 4.57and 9.15mg .i/Landanalyzedconcurrentlywiththesamples to determinethemeanproceduralrecovery(Table3). Sampleconceawertenrotacortreciteodfonrtshe meanprocraecodveruyorf97a.4l%. Arepreseatativechromatogramof amatrixfortificationispresented inFigur6e.
00956
AMENDED
WWIILLDDLLIIFFEE IINNTTEERRNNAATTIIOONNAALL LLm.. 24.
PROJEe CTNO.e4544-101
ExampleCaleultions Samplenumber 454A-101-3, nominalconcentration of 1.1mga.i/Linsaltwater.
Initial Volume: 0.500 mL Final Volume: 25.0 mL.
Dilution Factor: 50.0 PFOSPeakArea: 235466 InternalStandardPeakArea: 449012 PeakAreaRatio: 0.5244
Calibcurrvaeetquiatoionn.
Slope: 0.04044 Interopt: 0.05941 Curve is weighted (15). POS (aif stinsmumey
PFOS (mgai/l)insample
= PS eakareaoro attSilo"o--p(e e(t V-rinteprcep)t
0.5244 -005941
.
0.04044
= us
=
--PF--OS(4g
1/1)atinstrument xDilution 1000
Factor
_ 115x500 = Tio
= 0575
00957
AMENDED
WILDLIFE INTERNATIONAL Lm.
2s.
PROJECT NO.: 454A-101
PescntofrcentofNomNionmainal CeConcentratioon n = --PPRFOOSS ((omggoaa.1i/2L)) inonmsTianmEaplle |
Calculated recovery: 52.4%
Note:manual calculmaaytdiifofenr.
RESULTS
SampleAnalysis
|
Saltwater samples were collected from the static acute toxicity study with the saltwater mysid
(Mysidopsis bahia) at test initiation, March 8, 1999 (Hour 0), on March 10, 1999 (Hour 48), and at test
termination, March 12, 1999 (Hour 96). Themeasuredconcentrationsof PFOSinthesamples collected at
initiationofexposureofthetestorganisms(Hour 0)rangedfrom 52.4 to70.7% ofthe nominal
concentrations. Samples collected at Hour 48 had a measured concentration rangeof 43.5 to 71.0%of
nominalvalues. Samplescollectedattesttermination(Hour96)had ameasuredconcentrationrangeof
35t.o715.1% ofnomvali uesn (Taa blel 4). Areprche romas toge ramon fat testa samt pleii ssv howe nin
Figur7e.
00958
AMENDED
WILDLIFE INTERNATIONAL Lm. -26-
PROJECT NO.: 454A-101
Table 1
INSTRUMENT:
`Typical HPLC Operational Parameters
"Flewlet-Packard Model 1100 igh Performance Liquid eqCuihpperdwoithmaPwaeirttkhianoP-eErlgkmienrr-TEalumerprbAhPoIl1o0n0iLSoCnpsMorausarscyeS.peOctpreormeatteiernd selective ion monitoring mode (SIM).
ANALYTICAL COLUMN: OVEN TEMPERATURE:
KeystoneBetasil Cyscolumn (100mm x 2 mum 1D, 3 pumparticle
size)
30C
STOP TIME:
10.0 minutes
FLOW RATE:
0.220 mLminute
MOBILE PHASE:
72.0%Methanol :28.0%NANOpure Watercontaining 0.1% Formic Acid
INJECTION VOLUME: 2504
PFOS RETENTION TIME: Approximately 7.2 minutes
:
INTERNAL STANDARD RETENTION TIME:
Approximate4l9y minutes
PFOS MONITORED MASS: 498.6amu
INTERNAL STANDARD MONITOREA D MASS:
4e2e67ramu
.
00959
W_I--LDLIFE INTERNATIONAL Lo. 27.
ROPWROCJETCNTNOO.c:A454MA-1A01 L
`Table 2
`Matrix BlanksAnalyzed Concurrently During SampleAnalysis
Number (454A-101)
MAB-1
Sample
Type "Matrix Blank
Measured Concentration of PFOS'
(mgai/l) <LOQ
MAB-2
`Matrix Blank
<L0Q
MAB-3
Matrix Blank
<L0Q
"Thelimitofquant(iLtOQa)wtasi0o.11n5mga.iLbasedupontheproductofthe lowestcalibration
standard analyzed (0.00229 mg a.i./L) and the dilution factorofthematrixblank samples (50.0).
0960 AMENDED
T WILDLIN FE INN TERNAe TIONAL Lto. mPBRe OOJTEECTTNNO.O: E454AA-101 28.
`Table 3
Matrix Fortifications Analyzed Concuriently During Sample Analysis
-- Sa(m#p5l4eAN-1u0m1b)er
MAS-1
MAS4 MAS-7
`Concentrations of PFOS|
FForotilfiedd (mg a.i/L) MMeaaseuredd |
0.457
0.537
0.457
=
0457
0.396
RReePceorocvveenrete.dd u7 86.5
MAS-2
4.57
427
933
MMAASS-S5
445577
443463
9965.28
MAS-3
9.15
8.18
89.4
MAS-6
9.15
9.42
103
MAS-9
9.15
893
97.6
Mean =974
Standard Deviation = 9.41
CV =967
N=38
`Note:Resultsandcorrectionsfor newtestsubstancepurityweregeneratedusingMacQuanversion 1.5 sof andt manw ual ca alcur latie ons. Valueshavebeen rounded for reporting purposes.
"Result was0.942 mga.i./L.or 205%ofthenominalconcentration.Thisresultwas notusedtodetermine.
_the meanprocedural recoverySueto probable fortification and/or dilution error.
00961
AMENDED
WILDLIFE INTERNATIONAL Lo.
29.
PROJECT NO.: 454A-101
Table 4
--_-- `Nominal Test Concentration (mg a.i/L) 00
Measured Concentrations of PFOS in Saltwater Samples froam
Saltwater Mysid Static Acute Toxicity Test
Sample Number (454A-101)
T 2
13 2154
26
Sampling Time (Hours)
0
80
4%8
96
PFOS Measured Concentration'
(mg a.i/L)
<L0Q
<<LL00QQ
<<L1O0QQ
<LOQ
Percent of
Nominal
=
z-
--
-
11
3
0
4
0
15
48
16
48
27
96
28
96
0575
524
0.622
56.6
0.605
55.1
0.640
582
0391
355
0.580
529
18
5
0
6
0
17
48
18
48
29
96
30
9%
112
64.4
L19
683
L110
632
1.09
62.7
1.04
113
59.8
6s
|
30
7
0
192
65.7
8
0
199
619
19
48
192
65.8
:
20
48
191
65.2
31
96
179
612
3
9%
.
191
65.4
"Note:sRoefstuwlatrseaanndcdomrarencutailocnaslfcoulrantieownst.esVtsaulbuessthaancveepbuereintyrwouenrdeegdefnoerrraetpeodrutsiinngpguMrapcoQscusa.n version 1.5
The limitofquantitation(LOQ)was 0.115mg a.i/L basedupon the productofthelowestcalibration
standard analyzed (0.00229 mg 2.i./L) and thedilution factor ofthematrix blank samples (50.0).
Mean reosfduupllicatte redilutionsofthe original sample. Original result (12.0 mga.i/L)was not
included in the statistical analysis ofthe data.
Duplicate results for 454A-101-23are3.55 and 3.61.
000962
AMENDED
WWc HILODLLIFEEIINETEr RRNNAATTIOONNAALL TLDm..em PROJECe TNROO.: 4A54AP-10,1 "30-
"Table 4 (Continued)
--e Measured Concentrations of PFOS in Saltwater Samples from a Saltwater Mysid Static Acute Toxicity Test
`Nominal Test `Concentration
(mgai/L)
49
Sample Number
(454A-101-)
9 10
21 2 33 34
`Sampling Time (Hours)
0 0 48 48 96 96
'PFOS Measured Concentration" (mga.i/L)
3.05 266 2.96 335 3.11 31
Percent of
Nominal
616 538 599 67.8 63.0 629
82
1
0
582
707
2
0
5.78
702
223
4a8
355885
473150
35
96
522
633
36
96
5.86
JL]
"Note:Resultsand correctionsfornew testsubstance puritywere generatedusingMacQuan version 1.5
"The
`soanf dmat nuaw l caa lculr atioe ns.
limitofquantita(tLiOoQn) was
Valueshavebeen rounded
0.115mga.i/L basedupon
forreporting purposes.
theproductof thelowest
calibration.
`standard analyzed (0.00229mg 2.i./L)and thedilufacttoir oof tnhematrix blanksamples(50.0).
Meanresultofduplicateredilutionsoftheoriginalsample. Originalresult (12.0mga.i/L)wasnot
includedinthe statistical analysis of the data.
Duprelsulsiforc45a 4A-t 101e -23are 3.an5d3.5 61.
.
00963
AMENDED
> W--I_--LDLIFE INTERNATIONAL Lm. PPRROOJJEECCTTNNOO.:: 44s544AA-l1001l 31.
METHOD OUTLINE FOR THE ANALYSIS OF PFOS IN SALTWATER
`Preparematrixfortificationsamplesbyspikingtherequisitevolumeof PFOSstocksolutionsdirectly into filteredsaltwater using gas-tightsyringesand Class Avolumetricflasks.
+
inversions, Dilutematrixfortificationandtestsamplesintotherangeofthecalibrationstandardsby partiallyfilling Class Avolumetricflaskswith 50%methanol : 50% NANOpurewater solutioncontaining
0`.s1a0mp0lmeagnd4bHrPiFnOgtSh(eifnltaesrkntalosvtoalnduamred)w/iLtLhatnhed0d.il0u5ti%ofnosromlivcenatc.iPdro(vc/ev)s.stAhdedmtahteraixpblpanrkosapmpvrloileuuasmietnogef
the samedilutionandaliquotvolumeasforthe lowestfortificationlevel.Mixwellbyseveralrepeat
4
Ampulatesamplesand submitfor LCMSanalysis.
Figur1e. Analyticalmethodflowchartfortheanalysisof PFOS insaltwater.
00964
WWI UIDLLDLIN IFFEE T INTERE NATIR ONALN Lo. ATION RPRROJEA CTNO:L e 4544-e 1. 01 n-
250
200 x
Z 150
Ys
g
fio
050
000 000 0010 000 000 000 0050 Concentr(magtaii/oLn)
Figure2.
AtypicalcalibcrurvaeftoriPFoOSn.Slope =0.04044;Intercept =0.05941; r=0.9930.
Curves weighted (14).
00965
AMENDED
WILDI LIFE INTTEERNAATOIONNAAL LLm.,
ereemaeee PPREOOJEECCTENNO.O: S450440-1011 3
Intonsty: 3215cpa
100
us
90
80
70
a
so
"0
a /\ a0
20
181
J LvTTore2%SU3eNnm L aSRe S B6C% 17sEeaS nsm TSamne
.
Figure 3.
Arepresentativeionchromatogramof alow-level (0.00229 mga.i./L)PFOSstandard.
00966
AMENDED
WILDLIFE INTERNATIONAL Lm. 34
PROJECTNO.: 454A-101
"
175 Intent: 32743cps
0
a
7
o
5
"
5
151
.
2
2
25
127
05
.
:
S$T0 25en5 3LmABso 1sBe s7rs 2a1ss |TiSmeen
Figured.
Arepreseatativeionchromatogramofa high-level(0.0457mga.i/L)PFOSstandard.
00967
AMENDED
WILDLIFE INTERNATIONALLo.
-35-
~~ PROJECTNO:4s4adlol
100
90 80
20
Intensity: 1267 cps "2 :
1s
70
60
50
180
30
| 19A6
2o 0Je 2An AN alR |NSERW1 AN
|
T3er3h TSem ASor T6a% t7ars aait'm TSmano
Figur5e. Arepresentativechromatogramofamatrix blanksample (454A-101-MAB-1).
`Thearrow indicates thereteationtime ofPFOS.
660958
WIWILLDDLLIIFFEEIINNTTEERRNNAATTIIOONNAALLLLroo.. _36-
PPRROOWJECCTTNNOO.:: 445s4Ai-d10o1l
7s en: 11119 cps
10
.
o] -
PE
.
s
.
5
150
27 a
105
1
os 211s
ape
230
o To Oh Sh hw Ok T 7 oak Tseane
|
Figure6.
Arepreseatativechromatogramof amatrix fortificationsample (454A-101-MAS-1).
00969
WWIILLDDLLIIFFEEIINNTTEERRNNAATTIIOONALLLo.. -37-
PPRROOEJECCTTNOO.::445544A-10011
100 173 Intensity: 12998cs 90 80 70 60 s0 40
2s
150
20
ol ha a _o1_uiz Se oeNN s TTTSST 220
|
8 Hh he th BEeABWe 1W81r 2U11h | sTemn
Figur7e. Arepresentativechromatogramofatestsample (454A-101-3).
060970
WILDLIFE INTERNATIONAL Lm. 3.
PROJECT NO: 454A-101
APPENDIXIV
ChatnoPgroetocsol
`Thisstudywasconductedinaccordancewiththe approved Protocolwiththefollowingchanges:
1. Theprotocolwasamendedtoaddthe proposedexperimentalstartandterminationdates,testsubstance
2. Theprotocolwasamendedtoremoveannualfeed analysis. 3. Theprotocolwasamendedtospecifytheanalyticalmethodtobeused. 4. Thetest chamberswereplastic buckets. 5. pHwasmeasureddailyinalltestchambers.
00971
WWIILLDDLLIIFFEEIINNTTEERRNNAATTIONAL Lem.enpnpfpnionoo -39-
PROmJEnCT NOi.:l45m4A-101
APPENDIVX PersonnelInvolvedinthe Study
`ThefollowingkeyWildlifeInternational Ltd.personnelwereinvolvedintheconductormanagementofthis study:
1. HenrOy,Krueger, Ph.D,Director,Aquatic Toxicologyand NooTargetPlants 2. WillarBd.Nixon,Ph.D, Manager,AnalyticalChemistry 3. Mark A.Maak,Laboratory Supervisor 4, TimothyZ.Kendall,Laboratory Supervisor 5. KurtR.DroSentioraBiorlog,ist 6. RaymonLd.VanHPoh.Dv. Seciennti,st
000972
WILDLIFE INTERNATIONAL Lo.
40
PROJECTNO: 454A-101
APPENDIVXI
ReportAmendment
1. OriginalReport: Pa1-g 4,6e ands 21
Amendment: T`nheewpsiaggneastwureerseacnhdadnagteedstdouientcloutdheetahddeiatmieonnodefdthreerpeoprotrdattaem,ernevdimseednptaagsenAupmpbeenrdsi,xaVnId..
Reason:
Toreflecttheissuingofanamended report.
2. OriginalReport: Page2
Amendment: Thecompliancestatementwasrevised.
Reason:
Toclarify howthetestsubstancewascharacterized.
3. OriginalReport: Pag9e
`Amendment: Reason:
iInncflourdmiantgitohneprreoavniadleydsibsydtahteeaSnpdotnhseoprruerfilteyc,wtiangsatdhdeerdeatnoatlhyseiTseosfttShuebtsetsatnscuebssetcatnicoen,.
Toreflectthe currenttest substanceinformation provided by theSponsor.
4. OriginalReport: Entirereport.
`Amendment:
Alltest substance concentrationswere changedtoreflect thepurityofthetestsubstance. `as determinedby theSponsorin a reanalysis of thetestsubstance (FC-95, Lot217). Testconcentrations originallywerebasedonthe reported purityof 98.9%. The certofi anaf lysi isdcatead Mtarceh 9, 2000 indaipurcityaoft90e .49d%. Thereforael,l test substance concentrations, including nominal concentrations, measured concentrations, andLC50 values, were recalculatedand reported as mg a.i./L basedon `the 90.49% purity.
Reason:
To reporttheresultsofthetestbasedonthetestsubstancepurityof 90.49%atthe
reqoftuheeSposnsotr.
5. OriginalReport: Page?
`Amendment: `Experimental Termination --March 12, 1999
Reason:
To reportthe experimental termination date for thestudy.
000973
AMENDED
WILDLIFE INTERNATIONAL Lo.
ane
PROJECT NO.: 454A-101
APPENDIX VI ~ContinuedReport Amendment
AMENDMENT SIGNATURES
aml \ KStuurdtyRDiDrreoctttoarr
#/56/00
DATE
-
Direct7or,;AqA uatic Toxicology and Non-TargetPlants
a
REVIEWED BY: Gren
wa H Coleman `Quality Assurance
Y-26-00 DATE
00974
AMENDED
WILDLIFE INTERNATIONAL LTD.
PROJECT NO: 454A-101
Page 1 of2
AMENDMENT TO STUDY PROTOCOL
STUDY TITLE: PSFAOLS:TWAATE9R6-MHYOSUIRDS(TMyAsTiIdCopsAisCUbaThEia)TOXICITY TEST WITH THE
PROTOCOL NO.: 454/110998/MYS-96H1/SUB454
AMENDMENT NO.: 1
SPONSOR: 3M Corporation
PROJECT NO.: 454A-101
#
EFFECTIVE DATE: December 21, 1998
AMENDMENT: Page2
Add: Experimental Start Date: 3/8/99
Experimental Termination Date: 3/12/99
Test Concentrations: Negative Control, 1.2, 1.9,3.2, 5.4 and 9.0 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:TestOrganism, Page 5
Delete:
Feed provided to the mysidsinthe cultures will be analyzed at least once annually to
ensurethatthereare no contaminants at levels known to be capable of interfering with
the study.
REASON: HciosnttoarmiicanlanatnsalayrseepsroefseWaitldaltilfeeveIlnstekrnnaotwinontaol Lbted.caapqaubalteiocffientderhfaevreisnghwoiwtnhtthhaetsntuody.
AMENDMENT: APPENDIX II, Page 13
Add:
PLeirqfuiiudorCohocrtoamnaetSouglrfaoprihcy
AcMiads,sPoStpaescstiruommeStarlyt
(LCMS) (PFOS) in
Method for Freshwater,
FtihleteDreedteSramlitnwaattieornanodf
Algal Medium.
REASON: To add the analytical method to be used in the study.
00975
_
Reviewed] by OA 6 4
|
WILDLIFE INTERNATIONAL LTD.
LZ = \
|
STUDY DIRECTOR
4 fe LABOI Y MANAGEMENT
A. a. FasL
ONSOR'S REPRESENTATIVE
PROJECT NO.: 454A-101
Pa2gofe2
3/5177 DATE
D: fs /79
3/23/99
DATE
--
00976
PROJECT NO.: 454A-101
ILDLIFE INTERNATIONAL LTD.
DEVIATION TO STUDY PROTOCOL
STUDY TITLE: FEO,PFOS: Aion A 96-HOUR sma Ace STATIC ACUTE TOXICITY TEST WITH THE
PROTOCOL NO.: 454/110998/MYS-96H1/SUB454
DEVIATION NO.: |
`SPONSOR: 3M Corporation
oreo DATE OF DEFACTO DEVIATION: March 8, 1999
PROJECT NO.: 454A-101
DEVIATION: Ttheset cprhoatmobcoelrsstaatcetsuatlhlayt cthoentseissttcehdoamfbpelarsstwiicllbuccoknestiss.tofpolyethylene bottles. The
REASON: nBiootlaogdivsetrsoevelrysiagfhfte.ct tIhteisretshuel`tbsoesfttjheudstugdym.oefnthte Study Directorthat this deviation did
JZad)/ \
STUDY DIRECTOR
ra
LABORATORY MANAGEMENT
re
Spe3laq
DATE
alle
DATE
0977
WILDLIFE INTERNATIONAL LTD.
PROJECT NO.: 454A-101
Page 1 of 1
DEVIATION TO STUDY PROTOCOL
STUDY TITLE:
PFOS: A 96-HOUR STATIC ACUTE TOXICITY TEST WITH THE SALTWATER MYSID (Mysidopsis bahia)
PROTOCOL NO.: 454/110998/MYS-96H1/SUB454
DEVIATION NO.: 2
SPONSOR: 3M Corporation
PROJECT NO.: 454A-101
DATE OF DEFACTO DEVIATION: March 8, 1999
DEVIATION:
The protocol states that measurements of pH will be measured in alternate
`mreapldiecatneslolfetaescthcthraemabtemresn,tandcontrolgroup. Measurementsof pHwereactually
REASON: Biologistoversight. Itisthebestjudgmentof theStudyDirecthatttohirsdeviationdid `notadversely aftfheerecsulttsofthe study.
LZzEad\ STUDY DIRECTOR
2/25/79 DATE
LABORATORY MANAGEMENT
Jr--
DATE
000978
|
PROTOCOL
|
PFOS:
"WITH
TAH9E6-SHAOLUTRWSATTAETRICMYASCIUDTE(MTysOiXdIopCsIiTsYbaThEiaS)T
|
USS. Environmental Protection Agency
Series 850 - Ecological Effects Test Guidelines
(OPPTS Number 850.1035
|
ML RegsNo.02723
i 3MCorporation l
Submitted to
Environmental Laboratory
i
St.PauPl.,0.MiBnonxes3o3t3a3155133
1
WILDLIFE INTERNATIONAL LTD. HH
8598 Commerce Drive
i
Easton, Maryland 21601
(410) 822-8600
1
000979
`November 9, 1998
1
PROTOCOL NO.: 454/110998/MYS-96H1/SUB454
3M LAB REQUEST NO. U2723
1 WILDLIFE INTERNATIONAL LTD.
1
=
1
`PWFIOTS:HTAH9E6-SHAOLUTRWSATTAETRICMAYSCIUDTE(yTsOidXoIpCsIiTsYbaThEiSa)T
1
SPONSOR
1
E3aMviCroornpmoreanttiaonLaboratory SP.t0P.auBlo,xMi3n3n3e3s1ota 55133
1
SPONSOR'SREPRESENTATIVE: Ms. Susan A. Beach
TESTING FACILITY: 1
W8i5l9d8liCfoemImnteermcaetioDnrailveLd. Easton, Maryland 21601
1
STUDY DIRECTOR:
KSeunritoDrrAoqtuiaartic Biologist
1
LABORATORYMANAGEMENT: HDeniryrOeo. fcKrAutqeugaoetrir,cPTLDo. xic&oNolnoTagrgyet Plants
FOR LABORATORY USE ONLY
_
1
`Proposed Dates:
I
ExSptaerrtiDmaetn:tal
TExepmeirniamteinotnalDate:
pono: _AAA-JOL |
Test Concentrations:
--
1
TestSubstanceNo
Reference Substance No. (ifapplicable):
|
| empl eee I
PROTOCOLAPPROVAL
STUDY DIRECTOR
DATE
I
AGEMENT
a. i
PONSOR'S REPRESENTATIVE
BAT
30/92
DA'
000980
1
PROTOCOL NO: 454/110998/MYS-96HI/SUB4S4
3M LAB REQUEST NO, U2723
NILDLIFE INTERNATIONAL LTD.
"3.
INTRODUCTION
`Wildlife International Ltd. will conduct a static acute toxicity test with the saltwater mysid
(Mysidopsis bahia) for the Sponsor at the Wildlife Intemational Ltd. aquatic toxicology facility in
Easton, Maryland. Thestudywill be performed based on the guidelines provided in the U.S. Environmental Protection Agency Series 850 - Ecological Effects Test Guidelines OPPTS Number 850.1035 (1); U.S. Environmental Protection Agency, Standard Evaluation Procedure, Acute Toxicity
Testfor Estuarine and Marine Organisms (2);andASTMStandard E729-88a Standard Guidefor
ConductingAcuteToxicityTests with Fishes, MacroinvertebratesandAmphibians (3). Rawdata for all work performed at Wildlife InternatioLntadl. and a copy of thefinalreportwillbe filed byproject numberinarchiveslocatedontheWildlifeInternationalLtd.site,oratanalternative locationtobe
specified in the final report.
PURPOSE
`Thepurposeofthisstudyis todeterminetheacuteeffectsofatestsubstanceonthe saltwater
mysid (Mysidopsis bahia) during a 96-hour exposure period under statictestconditions.
EXPERIMENTALDESIGN
`Saltwater mysidswillbeexposedto ageometricseriesofatleast fivetestconcentrations and a `negative(dilution water)controlfor 96hours.Tworeplicatetest chambers will bemaintainedineach
treanadcotntrm olge roupn,witth 10mysidsincachchamberfor atotalof 20 mysidspertestconcentration.
`Nominaltestconcentrationswillbeselectedinconsultationwiththe Sponsor,andwillbebased
`upon the resultsofexploratory range-finding toxicity data, known toxicity data, physical/chemical
`propertiesofthetestsubstanceorotherrelevantinformation. Targetconcentneread ntoiteoxcneesd
1000mg/Lorthesolubilitylimitofthe testsubstanceinwater (whicheveris lower). Generally, each
testsubstanceconcentrationusedinthedefinitivetestwillbeatleast 60%ofthenexthigher
`concentration unless information conceming the concentration-effect curve indicates that a different
dilutionfactorwouldbemoreappropriate. Water samplesfrom cachtestchamberwillbecollectedat
`specifiedintervalsforanalysisof thetest substance.Resultsofanalyseswillbeusedtocalculate mean measured test concentrations.
PROTOCOL NO.: 454/110998/MYS-96H1/SUB454
3M LAB REQUEST NO. U2723
00951
WILDLIFE INTERNATIONAL LTD.
-4-
To control bias, juvenile mysids will be impartially assigned to exposure chambers at test initiation. Nootherpotentialsourcesofbiasare expectedtoaffecttheresults ofthe study. Each group
`of 10willthenbetransferredtothetestchamber. Observationsof mortalityandotherclinicalsignswill
be madethrotheu96-ghouhrteostpueritod. Cumulativepercentmortality observedinthetreatment
groupswillbeusetdo calcwuhelnpaostsiebl,e, LCSvaluesat 24, 48, 72and 96 hour intervals. The
`no-mortality concentration and the no-observed-effect concentration (NOEC) will be determined.
TestSubstance
MATEARNDIMEATHLODSS
Information on thecharacterization of test, control or referencesubstances isrequiredby Good
LaboratoryPractice Standards (GLP). The Spoisnressponosibrle for providing Wildlife International
Ltd.writtenverificationthatthetestsubstancehasbeencharacterized according to GLPspriortoitsuse
inthestudy. Ifwrittenverificationof GLPtestsubstancecharacterizationisnot providedto Wildlife
International Ltd., it will be notedinthecompliance statementofthefinalreport. Theattached form
IDENTIFOFITECSTASUTBSITAONCNE BY SPONSOR (AppendixI)istobeusetdo provide
info necer ssarm yfoa r Gt LPci omplo iann ce.
`TheSponsorisresponsibleforallinformationrelatedtothetestsubstanceandagreestoaccept.
anyunused testsubstanceand/ortestsubstancecontainers remaining attheendofthestudy.
PrepoafTerstaContcenitraotionns
`Thetestsubstancewilladministered to the test organism directlyintodilution water. This route
of administration wasselectedbecauseitrepresentsthemostlikelyrouteofexposuretoaquatic
organisms.
TestOrganism
The saltwater mysid (Mysidopsis bahia) has been selected as the test speciesforthis study.
Mysidsarerepresofeannitmpoarttanit gvroeupofaquatic marineinvertanedhbavrebaeetnseelesct,ed foruseinthe testbaseduponpastusehistoryandeaseofculturinginthelaboratory. Mystoibedussed
in the test will be less than 24 hours oldand will be obtained from cultures maintained at Wildife
GG0O9S2
PROTOCOL NO.: 454/110998/MYS-96H1/SUB4S4
3M LAB REQUEST NO. U2723
| WiLbLIFE INTERNATIONAL 1D.
|
-5-
|
IntemLda,Etastiono,Manryalanld.Theidentity ofthespecieswillbeverifiedbythesupplierofthe
original culofbtyuWilrdliefe International Ltd. personnel using appropriatetaxonomickeys such as
|
Price (4).
|
`Mysidswillbeculturedi waterfromth samesourceandatapproximatelythe sametemperature
aswill beusedduringthetest. Mysidsthat show signsofdiscaseorstressvillnotbe usedastest
|
organisms.
|
`Mysidsintheculturesandduringtesting willbefedatleastoncedaily. Thedietwillbelivebrine
shrimpnaupli(Artemsip.a).Feedingduringculturingmaybesupplementedwith saltwateralgacand
|
rotiasfneeedreds. Food providedto mysidsintheculturesvillbeanalyzeda leastonceannuallyto
ensurethatthere arenocontaminants atlevelsknown tobecapableofinterfering withthestudy.
|
Specification fo acoeptabelevelsofcontaminantsinmysiddietshavenotbeen established. However,
there arenoknown levelsofcontaminants reasonablyexpected to bepresentin thediet thatare
|
consideredtointerferewith the purposeorconductofthe test.
|
-- `Ntural seawatercollectedatIndianRiverInlet,Delawarewillbethesourceofthedilutionwater
|
`usedintheculturefacilityandtestsystem.The seawaterwillbeilteedthrough a sandfilepriorto
itsdeliveryto 37,800-Lhoklingtank.Thesalinityoftheseawatervillbedilutedto approximate2l0y
*/,, (partsprthousand)with WildlifeIntemationalLtd.wellwate intheholdingtank.The20 7,0
|
saltwaterwillbeaeratedusingspraynozzlesandfiltered(0.45yan)prior toitsdelivery tothe fst
|
`SalinityandpHvillbemeasure weeklytomonitortheconsistencyofthesaltwater. Meansand
|
rangesofthemeasuredparameters forthefour-weekperiodprecedingthe testwillbeprovidedinthe
finalreport. Analyseswillbe performedatleatonceannually todeterminetheconcentrationsof
|
selected organic and inorganic constituents ofthe seawater andresult of theanalyses willbe
|
`summairnithzefeindahl a
000983
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3MLABREQUENSOT, U2723
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| WILDLIFE INTERNATIONAL LTD.
|
"6
|
TestAoparatus
Test chambers will consist ofpolyethylene bottles. The sizeofbotleandthevolumeoftest
|
solution will be sufficient to mest the requirements of sampling for analytical verificationoftest
`substanceconcentrationsandroutine waterchemistryanalyses. Testchambers willbeindiscriminately |
||
`positionedin atemperatwuatrerbeat-hor<aonennvitronrmeontallclhamebedr.Testchambweilrlbse
Iabelled withthe project number,testconcentration and replicate.
`EnvironmentalConditions
|
Lightingusedtoilluminatethe culturesandtestchambersduringholdingandtesting willbe
provided by fluorescenttubesthatemitwavelengths simtionlataurral sunlight (c.g, Colortone 50).
1
A photoperiodof 16hours oflight and 8 hoursofdark will be controlled with an automatic timer. A 30-
`minutetransitionperiodoflowlightintensitywillbeprovided whe lights 80onand offtoavoidsudden
1
changesinlghtintensity.Lightintensity willbemeasa tuestriniteiatdion wiatSPEhR Scientific Lid.
lightmeterorequivalent.
|
"Thetargettesttempewirllabet25u+r2eC.Temperaturewillbe measuredtoeachtestchamber
1
atthebeginning andendofthe testusing aliquid-n-glassthermometer, Temperalasotwuilrbee
measuredwith continuousrecorderinonenegativecontrol replicate,Recordermeasurementswilbe
1
verifiedwith aliquid-n-glassthermometerprio to tetinitiation.
Dissolved oxygenwillbemeasuredinallreplicatesofth treatmentandcontrolgroupsa test
1
initiationandat approximately 24-hourintervalsthereafterusing a Yellow SpringsInstrumentModel
SBdissolvedoxygenmeter,orequivalent.Intheeventtha dissolvedoxygenlevelsfallbelow 60%
1
saturation,appropriateactionsvillbetakenafterconsultationwiththeSponsor. MeasuremeonftpsH.
willbemadein altematereplicatesofeachtreatment andcontrolgroupattestinitiation andat
|
approximately 24-hourinervalsthereafterusing aFisherAccumetModel915pH mete,o equivalent.
Ifatreatmentgroupreaches 100%mortality,dissolvedoxygen,pH,andtemperaturemeasurementswill
1
botakenatthenextsamplinginterval,thendiscontinued.Dilution watersalinitywillbemeasuredattest
initiation using aBio-Marine, Inc. Aquafiunarefractometer,orequivalent.
I
00984
I
PROTOCOL NO.: 454/110998/MYS-96HI/SUB4S4
3MLABREQUESTNO, U2723
1
1 WILDLIFE INTERNATIONAL LTD.
1
2
1
`Biolo
gical Measurements
`Observationsofmortality
and
clinical
s
ig
as
ofto
xi
ci
ty
will
be
mad
e
between
0-
24
hours,
and
at
1
24, 48, 72and 96hours + 1hour.Theeriteriafordeathincludelack ofmovement,absenceof
respiratory movements, andlackofreaction to gentle prodding. Sublethal effect, such as abnormal
1
locomotion, wil also be noted. Dead organisms will be removed from the test chambers at cach
observation time.
1
Surin
:
1
`Watersampleswillbecollectedfromeachtestchamberatthebeginning ofthetest,duringthe
{estand atheendofthetesttodetermineconcentrations ofthetestsubstance.Inthecveatthat 100%
1 `msoarmtpalliintgyiontceccuvrals.iSnaamnpyletrsveaitlmlebnet,otlhlecntsdamapt mliindgdoefptthhfarttormeeaatcmhenetswticlhlatmerbmeirn,ataenafloylzleodwiimnmgetdhieanteexlty
1
or placed in an appropriate storage contain (2g, polyethylenc or polypropylene bottle) and stored
underrefrigeunrtialatnailyozend.The sampleschemeissummaribezleowd:
1
PROPOSEDNUMBERS OF VERIFICATION SAMPLES
ExperimentalGroup. OHow __48Hows 96Hous
i
Control
2
2
2
i
Solvent Control(ifneeded)
2
2
2
Level 1-Low Concentration
2
2
2
1
Level 2
2
2
2
Level3
2
2
2
1
Level 4
2
2
2
Level 5-High Concentration
2
2
2
1
Totals
14 14
1
1
"Total Numberof Verification Samples 42
"Theabove numbersof samplsrepresentthosecollectedfromth testanddonotinclude quality control (QC) samples such as matrix blanks and fortifications prepared and analyzed during the
analytical chemistry phaseofthe study.
0098S
1
PROTOCOL NO.: 454/110998/MYS-96HI/SUB4S4
3MLAB REQUEST NO. U2723
|
1 - | WILDLIFE INTERNATIONAL LTD.
1
Analytical Chemistry
`ChemicalanalysisofthesampleswillbeperformedbyWildlifeInternational Lid. The analytical
1
`muesetdwilhlbeobasdeduponmethodologyprovidedbythe SponsorandidentifiedinAppendixIL.
"Themethodologyused oanalyzethetes samplesvil bedocumentedinthe raw dataandsummarized
1
in the final report.
1
`Data Analysis
`Whenthedose-responsepatternallowscalculation ofanLCS0value,thedatawillbe analyzed.
1
using thecomputersoftwareofC.E.Stephan (5). Theprogramwasdesignedtocalculatethe LCS0 value
andthe 95%confidenceintervalbyprobitanalysis, themovingaveragemethod,orbinomial probability
1
`with nonlinear interpolation (6,7,8). TheLCS0 value willbe calculated,when possible,using mortality
data collected at 24, 48, 72 and 96 hours. The no-mortality concentration and the no-observed-cffect
1
concentration (NOEC) will be determined by visually interpreting the clinical observation data.
REC TOBOEMR AIND TAIS NED
1
Recordstobe maintainedfordatageneratedby Wildlife International Ltd.vilinclude,butnot
1
belimited to:
1. A copyofthe signed protocol.
i
2. Ldentificationandcharacterizationofth tetsubstance,ifprovidedbythe Sponsor
3. Datesofinitiationandterminoaftthietoesnt.
4. Mysid history and culture records.
1
5. Stocksolutioncalcuanldapretpairatoionn ifapplicable
6. Observations
i
7. Water chemistry results (8, dissolved oxygen)
8. Themethodsusedtoanalyzetestsubstanceconcentrationsandtheresultsofanalytical
i
--
9. Statistical calculations,ifapplicable.
i
10. Testconditions(ightintensity,photoperiod, tc).
11. Calculationandpreparationoftest concentrations.
1
12. Copyoffinal report
00986
1
PROTOCOL NO: 454/110998/MYS-96H1/SUB4S4
3MLABREQUEST NO, U2723
|
.
| WILDLIFE INTERNATIONAL LTD.
|
"5
1
FINALREPORT
Afinalreport oftheresultofthestudy illbepreparedbyWildlifeIntemationalLud.Thereport
1
villinclude,butnotbelimitedtoth following,when applicable:
1. Nameandaddressofthefacilityperformingthestudy.
1
2. Dates uponwhichthestudywasinitiatedandcompleted an thedefinitiveexperimentalstartand
termination dates.
1
3. A statementofcompliance signedby the Study Director addressing any exceptions to Good
LaboratoryPractice Standards.
i
4. Objectivesandprocedures, as statedintheapprovedprotocol, incalllcuhadngeistnotghe
protocol.
1
5. Thetestsubstanceidentificationincludingname,chemicalabstract numberorcode number,
strength, purity, composition, and other information provided by the Sponsor
1
6. Stability and solubility ofthe test substance under the conditionsof administration,ifprovided
bythe Sponsor.
1
7. Adescription ofthemethodsusedtoconductthetest.
8. Adescriptionofthetestorganism,includingthesource,scientificname,ageo ifstage,feed
1
types, lightintensityandphotoperiod.
9. Adesoftcheprrepariatiopnoftte eistsoolutinons.
1
10. Themethodsusedtoallocateorganismstotetchambersandbeginthetest,thenumberof
organisms andchamberspertreatment, and thedurationofthetest.
1
11. Adescriptionofcircumstancesthatmayhaveaffectedthequalityorintegrityof thedata.
12. ThenameoftheStudyDirectorandthenamesof therscientist,professionals,andsupervisory.
personnelinvolvedinthe study.
1
13. A description of the transformations, calculations, and operations performed on the data, a
`sumnaaryandanalysisofthe iologicaldataandanalyticalchemistrydata,and astatementofthe
1
concdrlawunfrsomitheoananlysses
14. Statisticalmethodsusedtoevaluatethedata.
1
15. Thesignedanddatedreportsofeaocfthhe individual scioeronthetrpriofesssitonasls involved
inthestuifapdpliycab,le.
i
16. Thelocationwhere raw dataandfinalrepost ar 0besored.
000987
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3MLAB REQUEST NO, U2723
WILDLIFE INTERNATIONAL LTD. -10-
17. AstatementpreparedbytheQuality AssuranceUnitlistingthedate tha studyinspectionsand audits weremadeandthedates of anyfindingsreportedtotheStudy Director andManagement.
18. Ifitisnecessarytomakecorrectionsoradditionsto afinal reportafterit hasbeen accepted,such changesvillbemadeintheformofanamendmentissuedbythe StudyDirector.Theamendment will clearly identifythepactofthe finalreportthatisbeingamendedandthereasonsforthe amendment,andvillbesignedbythe StudyDirector.
CHANOFGPRIOTN OCG OL
Plannedchangestotheprotocolvillbein theformofwritenamendmentssignedbytheStudy DirectorandtheSponsor'sRepresentative. Amendmentswill be considereads pstoftheprotocol and illbe tachedtothefinalprotocol.Anyotherchangeswillbeintheform ofwrittendeviationssigned bytheStudyDirectorandfiledwiththerawdata. Allchangestotheprotocolwillbeindicatedinthe final report
GOOD LABORATORYPRACTICES "ThisstudywillbeconductedinaccordancewithGoodLaboratoryPractice StandardsforEPA (40CFRPart 160 and/or Part 792); OECD Principles ofGoodLaboPrractaicet(OoCDrE/GyD (92) 32, Environment Monograph No. 45); and Japan MAFF (59 NohSan, Notification No. 3850, `Agricultural ProductionBureau). EachstudyconductedbyWildlifeIntematioLnuadl.isroutinely examinedbythe Wildife InternationLtadl.QualityAssuranceUnitforcompliancewithGood Laboratory Practices, Standard Operating Procedures andthespecified protocol. A statement of compliancewithGoodLaboratory Practiceswillbepreparedforal portionsofthestudyconductedby Wildie Intemational Ltd. TheSponsorvil be responsible for compliance with Good Laboratory Practicesforproceduresperformedby therIaboratosies (cg, residueanaolrpyatsholeogys). Rawdata forallwork performedat Wildlife IntemationalLid.andacopyofthefinalreportvillbefiledbyproject number inarchiveslocatedontheWildlifeInternationalLid.sie, o at analterlnocaattionitvobee speicntiheffinialreepodrt.
00988
PROTOCOL NO.: 454/110998/MYS-96H1/SUB454.
3MLAB REQUEST NO. U2723
| WILDLIFE INTERNATIONAL LTD.
|
ne
1
REFERENCES
i
1 (UdSra.fiE)n,vOiProPnTmSenNtuamlbPerorte8c5t0i.o1n03A5g:enMcyy.si1d9A9c,utSeerTioexsic8i5t0y-TEecsotl.ogical Effcts Test Guidelines
2 US. Environmental Protection Agency. 1985. Standard Evaluation Procedure, Acute
Toxicity Testfor Estuarine andMarine Organisms (Shrimp 96-Hour Acute Toxicity Test)
|
D"HCa.zard Evaluation Division. OfficeofPesticide Programs. EPA-540/9-85-010, Washington,
|
3 AFiSshTesM, SMtacarnodianrvderEt7e2b9r-a8t8esa, 1a9n9d4.AmSpthainbdiaarndsG.uiAdmeefroircaCnonSodcuicettiyfnogrATceustteinTogxiacnidtyMaTteesrtisalwsi.th
|
4 tPhreiicre,EcWo.loWg.y. 19H8y2d.roKbeioyltoogitahe9S3h,a9l-l2o1w. WPart.eWrMyJsuikdaPucbelaisohfertsh,eTTheexaHsagCuoea.stPwriitnhteNdotinesThoen
Netherlands.
|
5
Stephan, CE. US. EPA. Personal communication.
1978.
Environmental Research Laboratory, Duluth, Minnesota.
|
6 Finney, DJ. 1971. StatisticalMethods in Biological Assay. Second edition. Griffin Press,
London.
|
7 Thompson, WR. 1947. Bacteriological Reviews. Vol IL, No. 2. Pp. 115-145.
|
8 Stephan, CE. 1977. "Methods for Calculating anLCS0,"Aquatic ToicologyandHazard
`Evaluations. American SocietyfToesting and Material. Publication Number STP 634, pp 65-
8
|
00989
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PROTOCOL NO: 454/110998/MYS-96HI/SUB4S4
3MLAB REQUEST NO. U2723
| WILDLIFE INTERNATIONAL LTD.
|
-12-
1
IDENTIFICATION OFATPESPTENSDUIBSXTANCE BY SPONSOR
|
TobeCompleted by Sponsor
1 Test Substance Identity (nameto be used inthe report): P(EOPS erfluo Sulr fonioc AocidcPot tasasiunmSaelt
|
Reference Standard (fapplicable): Analytical Standard: N/A
Internal Standard: LP1e2c2HfHlHuoroocSultfonaicAncied
|
TestSubstance SampleCodeorBatchNumber: __Lot217
Test SubPursity(t%AcativneIngrcedieent): __98.9
Expiration Date: _2008
| IL Test Substance Characterization dHeavetermtihrneeidodepnrtoiitoyr,ls1triengsTthue,slesBin ethipuritysstaub nddyciomnpe aocscaiotrni idoaonnocnren oweitthsherGcshLaaPnrdaaScttraedrnbidsaercd.s? Yes No X_
IL Test Substance Storage Conditions
|
Pleaseindicatetheresommendedstorageconditionsat WilleIntemationalLid.
--Ambietroomtemperawe
1
bHeiesnthestablyionfahcceorsdtanuceswtath GwLePSrtatnhdasrdsr? gcodons
Yes No X_
|
Other pertinent stability information:
| IV. TestConcentrations:
X__ Abdajsuesdtutpeosntcthoenpucreitnyt(r%)tag0it1vi0e0no%anboavie. DoanigTtesatdhietmtaetsetrciaolAnSeIcSt.iotno 100%
| V. Toxicity Information: Mammalian RatLDS0 25lmghkg MousoLDSO_NA
|
Aquatic:
Invertebrate Toxicity (EC/LCS0)
Fish Toxicity (LCS0)
D2 aphn7 iamam gna:gfle RaTiroutn:Libmeow
Other Toxicity Information (including findingsofchronic and subchronic tests):
1
_BessseMsDS
1
00990
PROTOCOL NO.: 454/110998/MYS-96H1/SUB454
3M LAB REQUEST NO. U2723
1
.
1 WILDLIFE INTERNATIONAL LTD.
i
.n-
ii Aeron Analytical Method to be Provided by Sponsor
i
i
i
i
i
i
i
f
I
#
B
f 000991
i
PROTOCOL NO.: 454/110998/MYS-96H1/SUBAS4
3MLAB REQUEST NO, U2723