Document 3e4323mO8YLzeqy9pn8eN2QvJ
J .
AR12-Ol39 0BS-
PROTOCOL
'
`WIPFTOHS:DUACK7W-DEAEYD(TLOeXmInCaITgiYbTbEaSGT3)
i
U.S. EnvironmentalProtectionAgency
-Series 850 - Ecological Effects Test Guidelines 'OPPTS Number 850.4400
3M LabRequest No. U2723
Submitted to
.
3M Corporation
Environmental Laboratory 935 Bush Avenue
St. Paul, Minnesota 55106
Wildlife International, Ltd.
| 8598 Commerce Drive
*
\
`Easton, Maryland 21601
t
(410) 822-8600
September 17, 1999
04532
WILDLIFE INTERNATIONAL, LT ae
PFOS: A 7-DAY TOXICITY TEST `WITH DUCKWEED(Lemnagibba G3)
SPONSOR:
SPONSOR'S REPRESENTA~TIVE: TESTING FACILITY:
STUDYDIRECTOR:
LABORATORYMANAGEMENT:
3M Corporation
935Bush Avenue. `St. Paul, Minnesota 55106
Rochelle. Robideau WildeIntemational,Lid
$598 CommerceDrive `Easton, Maryland 21601
SKeairotBrrAotqteueiCBaroy lgSctheland WE AJoles DHieraeraytoOr.ofKrAoqgueart,icPThoDx.icology & Non-TargetPants
FORLABORATORY "USE ONLY
Proposed Dates:
Stat De:
TehminsionDate: _- .
Projecto: AGA 11]
`Test Concentrations: "TestSubstance No...
ReferenceSubstance No.(ifapplicable):
PROTOCOLAPPROVAL
to )Y DIRECTOR
2A -- RY MANAGEMENT --) eS EST ATIVE PROTOCOLNO: 454US1TS9/LEMIDISUBAS
__ifegfoo DATE fo Jee DAT
Bx
04533
MLABREQUESTNO. U2723
WILDLIFE INTERNATIONAL, LTD
3.
INTRODUCTION
`WildlifeInternational,Ltd.willconduct aseven-daytoxicitytestwiththe duckweed,Lemnagibba
G3,fortheSponsorattheWildlifeInternational,Lud.aquatictoxicologyfacilityin Easton,Maryland.The
study willbeperformed basedonproceduresin the U.S. Eavironmental Protection Agency Series850 `EcologicalEffects Test Guidelines OPPTS Number 850.4400(1). Rawdata forall workperformeadt `WildlifeInternational,Ltd.and acopy of thefinalreportwillbefiledbyproject numberinarchiveslocated onthe WildlifeInternational,Ltd.site,oratanalternativelocationtobespecifiedinthefinalreport.
PURPOSE
`The purpose of thisstudy isto determine the toxicity of PerfluorooctaneSulfonicAcid, Potassium `Salt(hereafter referred to as PFOS)to duckweed,Lemna gibba G3, understatictest conditions.
.
EXPERIMENTALDESIGN
Frondsofduckweed,Lemna gibba G3, willbe exposed to ageometricseries of at leastsix test
concentrationsand a negative (culture medium) controlforsevendays. Nominaltestconcentratwiilolnbse:
chosen in consultation with the Sponsor based uponinformation such as rangefinding toxicity data,known
toxicityinformationorphysical/chemicalproperties of PFOS. . `Targetconcentrations willnotexceed100
`mg/L orthe solubilitylimitofthetest substance inwater. Generally, eachconcenotfrPFaOStuiseodinn
the definitivetestwillbeatleast 50%ofthenexthighertreatment,unlessinformationconcerningthe
`concentration-effectcurveindicates that adifferentdilutionfactor wouldbemorappropriate.
Uniform, healthy looking plants willbe removed fromthe stockculture for testing. Fiveplants totalling approximately 15 frondswillbe addedtocach test chamber.Care willbetaken tocsurethatthe `numberoffrondsineachtestchamberis approximatelyequal.Three"biolrepolicgatei specrta realtm" ent `wbeitelsteld.Oneormoreadditional "analytical"replicateswillbeincludedinthetest, asneeded,toprovide: test solutionfor concentration verificationonDays 3, 5and 7 ofthe exposure. An abioticreplicateatthe `highestconcentration willbeincluded inthe test and will be sampled on Days3, 5 and7.Inorder tocontrol `bias,the position of thetestchambers willbe determinedby indiscriminate drawdaily duringthe exposure.
period. No other potentialsourcesofbias are expected to affecttheresultsofthe study.
Thetestresultswillbecalculatedbasedondirectcountsofthe numbersofduckweedfronds. These: `measurements willbe takenon Day3, Day 5andatthe end of thetest. Obserofvchlaort osiis,boreank-sup
ofduckweed colonies, changesin color, root destructionandother abnormal appearances will also be documented. An IC10, C50 and IC90values (e.g,the toxicant concentration that produces a 10%,50%
04534
PROTOCOLNO.: 454/091799/LEM7D/SUB454
3MLABREQUNOE.US27T23
WILDLIFE INTERNATIONAL, LTD -4-
aotrD9a0y%sr3e,du5catnidon7ionfftrhoendemxpuomsbuererpreerlaitoivde.tTohethec0oonbtsreorlvse)dwialdlvberesceaelfcfuelacttecdobnacsenetdroantitoonta(lNfOrAoEnCd)nu,wmhbiecrhs raepppreesaernatnscet,hweilhligbehedsettceornmciennetdrbaatisoendtuhpaotnienvdaulcueasti0o0naodfvtehresseteaftfisetcitcss]ornespulltasn,ttahnddofsreornedspproondsuecptaitonieo,r andotherobservedeffects.
MATER ANI DMA ETL HOS DS
TestSuIbnsftoarnmcaetiononthec"haracterizationof est,controlorreferencesubstancesisrequiredbyGood
LabPoracrticeaStatndaords(rGLyP).TheSpoasoriresponsibleforprovidingWildifs Internationa,Lid.
writtenverificationthat th testsubstancehasbeencharacterized accordingtoGLPspriortisuseinthe
Study:IfwrittenverificationofGLPtestsubstancecharacterization is notprovidedto WildlifeIntemational,
OLiFd,TiEtwSiTllbSeUnBotSeTd AinNtChEecBoYmplSiaPnOceNsStOatRem(eAnptpoefntdhiexfIin)alirteopboretu.sTehdetaottparcohveiddfeoinrfmorImDaEtiNoTnnIeFcIesCsAaTryIfOoNr
`GLP compliance.
-
"TheSponsor respoasibleforal informationrelate tothetestsubstanceandagreestoacceptany unusedtestsubstan and/ortestsubstancecontainersremaining ofth eadofthestudy.
`TestSolutionPreparation .
'
anddil"aTtihnegdtehsieraepdpcroonpcrieantteravtoilounmseooffPsFtOoScwksiolllubteioonbwtiatinhemdebdyipurme.pTahriengteastssionlgulteisotnoscmksaoylaultsioonbienpmreedpairuemd
`usingsecondarydilutionsoftheprimarystock,basedupon theconcentrationdesired.
Thetestsubstance willbeadumiristeredtothetestorganisminculturemedium. Thisroutsof administration wsselectedbecauseitrepreseatsthemostlikelyrouteofexposuretoducked,whichexist suspendedon awater surfscewithrootsextendingintothewatercolumn.
- `TestOr"gTahentisemtspecieswillbetheduckweed,LemnagibbaG3.Thisspcisirepresentatoifaven importantroupoforganismsandwasselectedfo ussinth testbaseduponpastuseand easeofhandling iAgnrtihcuelltaubroera,tWoarsyh.inOgrtiogni,naDl.Cd.uocrkswmeoetdhpelrsaunittsawbillelsbupepolbietra.inSetdocfkrocmulttuhreesUwniiltlebdeStmataesiDneptairatnimceunnltteuordfe
PROTOCOLNO: 454/091799/LEMTDISUB454 |
04535
3MLABREQUEST NO, U2723
.
Le
WILDLIFE INTERNATIONAL, LTD
-5-
`mediumat WildlifeInternationLatld,.Duckvweedusedintoxicitytestswillbeobtainedfromstockcultures thathavebeen activelygrowinginthismedium foratleast 2weeks. .
CulturCeuMletdurieummediumpreparedaccordingto WildlifeInternational,Lid.StandardOperatingProc.edures illbeusedasdilutionwater.Theconcentrationsofthemediumcomponentsareprescatedin Table1. Stock mutricatsolutionswillbepreparedbyaddingreageat-gradeorbeterchemicalsto Wildife International,Lid.wellwaterpurifiedbyreverse-osmosis. Apprvoloumepsofrtheistoackmtutrieent solutionsvilltheabedilutedwithpurifiedwellwaterto preparethetedium. The medwililbueGimtex sterilized (0.22jum)orautoclavedprior 1useinthetest.ThepHofthemediumshouldbe 5.0%0.1. Analysesvillbeperformedat least onceannuallytodeterminetheconcentrationsofselectedorganicand inorgnicconstituentsofthewellwater andresultsofthemostrecent GLPcompliantanalyseswillbe `summinathrefiinalzeepodrt.
Specificationsforacceptablelovesofcontaminantshave notbeen establishedforculturemedium. However,therearenolevel ofcontaminantsreasonablyexpectedtobepresentinthemediumthatare considered ointerferewiththepurposeorconductofthestudy.
TestApparatus
-
`Testchamberswillbe 250.aL. Nalgenebeakerscoveredwithdisposablepetri dishesandcontaining
100mLoftestsolution.The estchamberswilbeindiscriminatelypositionedon ashelfinanenvironmental
chamber. TestchamberswillbeLabeledwiththe projectnumber,testconcentration,andreplicate.
:
"Thetestwillbeconductedat25 2Cundercontinuous warm-white fluorescentlightingatan intoef5n00s0+i75t0hyux.Lightintensitywillbemeasureda fivelocationssurroundingthetestchambers: onDey 0ofthetest. Temperaturewillbemeasuredtwicedalyin acontainerofwateradjatcotehentetst Chaimnthbeenevirornmenstal chamberusing aliquidm-ericunry-thgerlmoamestesr,
ThepHdfeachtreatmentandthecontrolwillbemeasured a test iniision andtermination isin3g FisherAccumetModel915pHmeterorequivalent. Samplesfor pHmeasureefmteset ntintaso villbo colflroemcthetindeividdual batofcteshtsoelutisonpreparedforcachtreatmentandcontrolgroup.Attest termination, pHwillbemeasuredinpooledsamplesoftestsolutioncollectedfromeachofthethreereplicates ofcachrespectivetreatmentandcontrolgroup.
04536
PROTOCOLNO.: 454/091799/LEMTDISUBAS4
3MLABREQUESTUN2O72.3
.
i
WILDLIFE INTERNATIONAL, LTD
ge
`BiologicalMeasurements:
Growth,definedasanincreaseinthetotalnumberoffronds,willbe assessedonDay3,Day 5and atthe endof thetest.Additionally,thetotalnumber ofduckweedplantspresentineachtestchamberwill
bedetearttmestiternmineatidon. The numbersofchlorotic, necroticanddead frondswil beenumerated.
Chloroticfrondsarethosepossessing areasofbleachedcolorprogressingfromgreentoyellow. Necrotic fronds are those with localized regionsof deadordecaying tissue, usually surroundedbyhealthy tissue. `Frondswithnoapparentlivingtissue,usuallyallbrown, black, white,yelloworclear,willbeconsidered dead.Theduckweedplantswillbeobservedforthepresenceof rootdestruction(i.c.,deadorshortened roots in atreatmentgroupcomparedtothecontrols)andbreakupofduckweedcolonies(i.,anincreasednumber offree-floatingindividualfrondsin atreatmentgroupcomparedtothecontrols). Anyotherabnormalities infrondor plantappearancewillalsobedocumented.
`SamplingforAnalvtieal Measurements
`Samplesofthe exposuresolutionswillbecollectedonDays0,3, 5and 7and analyzedfortest
`substanceconcentrations. Thetermination daysamplesmaybefiltered (0.45jum),as necessary,toremove: `plantdebrispriorto analysis. Day 3 and 5 samples willbecollected from the extrareplicate test chamber(s) includedinthetestto provide samples for that analysisand fromthe abioticreplicates. Sampleswillbe
placedi aappropriatestoragecontainer (6.5, polyethylene,orpolypropylene botle)andvillbeanalyzed
immediately,orstored under refrigeration untilanalyzed. Thesample scheme issummaribezleowd:
[Ee|oi vo |m bus o |own s |os w | PROPOSED NUMBERSOFVERIFICATION SAMPLES
Control
SolventControl(if needed)
Level 1-LowConcentration Leve2l
Leve3l
Leve4l
Level6-HighConcentration
[o1e [] | |
PROTOCOL NO.: 454/091799/LEM7D/SUB454
604537
3MLABREQUENSOT. U2723
WILDLIFE INTERNATIONAL, LTD
-7-
`Theabovenumbersofsamplesrepreseatthosecollectedfromthetestanddonotinclude quality control (QC) samplessuchasmatrix blanksandfortificationspreparedandanalyzedduringtheanalytical cheaphnasieosfthtesrtudyy.
AnalyticalChemistry
Chemicalanalysisof thesamplesvillbeperformed byWillieInternational, Lid.The analytical `methodusedwillbebasedupon methodologyprovidedbytheSponsorand identifiedin AppendixIL. The `methodologyusedtoanalyzethetestsamples willbedocumentedi therawdata andsummarizedinthefinal report.
Statist - Percentinhibitionwillbecalculatedforcach treatmentandcontolgroupusingthe followingformula:
MeanControl -Mean Treatment PercentInhibition = FrMoenadnNCuomnbterrol FFrroonnddNNuummbbeerr X 100
: :
"TheDay 71CS0valuewillbedetermined,wheapossible,usinglinearinterpolation orothersuitable. techniques (3) withtreatmentresponse (frondmumber)andexposureconcentrationdata. Thepercentages ofdead,chloroticandnecroticfrondswil becalculatedfor cachtreatmentandcontrolgroupasthe mumber ofdead,chloroticornecrotic roadstoth total umberoffrondspresentineachtestchamber.
"Thedatawilbeevaluatedfornormalityand homogeneityofvariances.Ifthese assumptaireoments, Dunnett'sprocedureortheBonferronit-test (4) maybeusedtoevaluatethedata.Iftheassumptioonfs `normalityand homogeneityofvariancesarenotmet,non-parametricstatisticalmethodssucha theKruskal `Wallstest(4)maybeusedtoevaluat thedata. Anoobservedadverseeffectconcentration (NOAEC)will bedeterminedbaseduponevaluationofthestatistical results,thedose-responsepatternandoberdbserved effects (c.5,percentchlorosis,necrosis,ec).
.
REC TOO BE MR AIND TAIS NED
Records to be maintainedfordatageneratedatWildlifeInternational,Ld.willinclude,butnotbe
limitedto:
1. Copyofsigoedprotocol.
2. Mdentificationandcharacterization ofhetestsubstan,ifprovidedbySponsor.
3. Datesofinitiation andterminoafttheitoesnt.
004538
PROTOCOL NO: 454/091799/LEM7DISUB4S4
3MLABREQUENSOT.U2723
WILDLIFE INTERNATIONAL, LTD
-3-
4 Soueofplants.
5. Cultureconditions
.
pes
6. Growthmeasurements
7. Calculationandpreparation oftestconcentrations.
;
8. Observations.
9. Ifapplicable,themethodsusedtoanalyzetestsubstanceconcentrationsandth results ofanalytical
`measurements.
10. Statisticalcalculations.
IL Testconditionsandphysical/chemicalmeasuremeats.
12. Copyoffinalreport.
FINALREPORT
Afinalreportofth resultsofthestudywillbepreparedbyWildif Intentational,Ltd.Thereport
willinclude,butnotbelimitedtoth following,whenapplicable:
1. Name andaddressofthefacilityperformingthestudy.
-
2. Datesonwhich thestudywasinitiated andcompleted.ItistheresponosfithbeSiplonisotrtyo
providethefinaldatethatdataarerecordedforchemistrypathologyand/orsupporting evaluations
that maybegeneratedatotherlaboratories.
:
3. AstatementofcompliancesignedbytheStudyDirectoraddressingany exceptionsto Good
Laboratory PracticeStandards.
-
4. Objectives ndproceduresstatedintheapprovedprotocol,includingaaychangesintheoriginal
protocol.
5. Thetestsubstanceidentifiedbyname,cheanicalabstracts umberorcodenumber, strength,purity,
andcompositionorotherappropriatecharacteristics,if providedbytheSponsor.
6. Stabilityandthesolubilityofthetestsubstancesundertheconditionsof administration,ifprovided
bythe Spouse.
7. Adescription ofthemethodsusedtoconductthetest
8. Adescriptionofthotestsystem,includingth sourceofthetestorganismsandtheiscientificname.
9. Adescription ofthepreparationofthe estsolutions,methodsusedtoallocateorganisms t th test
= ===cohaamber}andbeginthetest, numbersoforganismsandchamber. spertreatment, anddurationofthe.
10. Adescription ofalcircumstancesthatmayhaveaffectedthequalityorintegityofthedata 11. ThonameoftheStudyDirector,thenamesofothe scicatistsocprofessioaals, odthenames ofall
suppeersornnelv, inivolvsedionthrestuydy.
04539
PROTOCOLNO.: 454/091799/LEM7DISUBAS4
MLABREQUESTNO. U2723
WILDLIFE INTERNATIONAL, LTD
"5.
12. Adescriptionofthetransformations,calculations,oroperationsperformedonthe data, asummary
"
`andanalysisof thebiologicaldataandanalyticalchemistrydata, andastatementoftheconclusions
drawnfromthese analyses.
13. Statisticalmethodsemployedforanalyzingthe data.
.
14. The signedanddated reportsofeach of theindividualscientistsorotherprofessionalsinvolvedinthe
study.
15. Thelocationwhereallrawdataandthefinalreportaretobestored.
16. A statementpreparedbythe QualityAssuranceUsitlistingthedatestha studyinspectionsandaudits `were madeandthedatesofany findings reportedtothe StudyDirectorandManagement.
17. Ifitisnecessarytomakecorrectionsoradditions to afinalreportafterithasbeen accepted,such
changesshallbemadeintheformofanamendmentissuedbythe StudyDirector. Theamendment
shallclearlyidentifythepartofthefinalreportthatisbeingamended andthereasonsforthe
alteration. AmendmentsshallbesignedanddatedbytheStudyDirector.
CHANGINGOFPROTOCOL
-
Plannedchangestotheprotocol willbeintheformofwrittenamendmentssignedbytheStudy
Directorandthe Sponsor's Representative. Amendmentswillbeconsideredas partoftheprotocolandwill
beatttoa thec finah lpre otocd ol. Anyotherchangeswillbeintheformofwrittendeviationssigned by the
`StudyDirector andfiled withthe rawdata.Allchangestothe protocolwillbeindicatedinthefinalreport.
GOOD LABORATORYPRACTICES
`ThisstudywillbeconductedinaccordancewithGoodLaboratoryPracticeStandardsforEPA (40 CFRPart160and/or Part792); OECDPrinciplesofGoodLaboratoryPractice (ENV/MC/CHEM(98)17); ad Japan MAFF (59 NobSan, Notification No. 3850, Agricultural Production Bureau). Each study conductedbyWildlifeInternational,Ltd.isroutinelyexaminedbytheWildlife International,Ltd.Quality AssuranceUnitforcompliancewithGoodLaboratoryPractices, StandardOperatingProceduresandthe `specifiedprotocol. AstatementofcompliancewithGoodLaboratoryPracticeswillbepreparedforall `portionsofthestudyconductedbyWildlife International, Ltd.TheSponsorwillberesponsibfloer `compliancewithGoodLaboratoryPractices forproceduresperformedbyotherlaboratories (c.g.,residue `analysesorpathology). RawdataforallworkperformedatWildlife International, Ltd.and acopy ofthe final reportwillbe filed byproject number in archives locatedon theWildlife International, Ltd.site, orat. `analternativelocation to bespecified inthe final report.
PROTOCOLNO.: 454/091799/LEM7D/SUB454
04540
3MLABREQUENSOT. U2723
WILDLIFE INTERNNAATIOONNAL, aLTDO
eee -10-
REFERENCES
eee
1 (UdSr.af)E,nvOiPrPoTnSmeNnutmablePrro8t5e0c.t4i4o0n0:AgAegnucayt.ic19P9la6n.tSTeorxiiecsi8t5y0Te-stEUcsolionggicLaelmEnfafecstpsp.TTeisetrGsuildaenlidnes
2 ASTMStaGn uid de1a 415r -91d E. 1991.StandardGuideforConductingStatic ToxicityTests with Lemna gibba G3.
3 Norberg-KinTgJ,. 1993. ALinearInterpMeo tholdfaortSuibleothanl Toxicity:The Inhibition
Concentration' (1Cp)
(Version 2.0). US. Environmental Protection Agency.
4
`West,Inc.and D. D. Gulley.
Cheyenne, Wyoming.
1996.
TOXSTATRelease 3.5.
Western EcoSystemsTechnology,Inc.
\
PROTOCOL NO.: 454/091799/LEM7D/SUB454
04541
3M LABREQUEST NO. U2723
A e WILDLIFE g INETERRNAATTIIOe ONNAALl,yLLTTDD stas u-
TABLE 1
.
M-HOAGLAND'S MEDIUM WITHOUT EDTA OR SUCROSE CONSTITUENTS
Compound
"Nominal Concentration
ZHa;SB0O6;THO
02826 mmgeLl
NaMoQy2H:0
012 mL
CMuaSClOr#4SHH,:00
008
362
mg
mg
+
KHPO,
068 gL
KNO;
,
Ca(NOs)H:0
1L511850 ggLL
$
FMegCSlOr,67HH;,00
504492 mglL
Tastaric Acid
30 mg
"ThepHofthe 10%HCL.
medium
will
bsadjusted,
as
necessary,
t
5.0
+
0.1
using
0.1
N
NaOF
or.
PROTOCOLNO: 454/091799/LEMD/SUBASS
04542
3MLABREQUEST NO. U2723
WILDLIFE INTERNATOIONNAALL,, LTD
eee
12
.
APPENDIXI
-
IDENTOFITEFST SIUBSCTAANCETBYISPOONSNOR
`TobeCanpletedbySpoasae
1 Test SubstanceIdentity(aametobeuse inthereport): PEQS(PerfluorooctaneSulfonicAcidPotassiumSalt ReferenceStandard(fapplicable): AnalyticalStandard: _N/A
InternalStanderd:_1,1.2.2H.HHHPerfluorooctaneSulfonicAcid
TestSubstanceSampleCodeorBatch Number:_Lot217
`Test SubstancePurity(% ActiveIngredient): __ 98.9
IL TestSubstanceCharactizton
`Expiration Date: _2008
Hap r y thedy eity ie ity acnS ompS oe crsoothihertdchR araiactetoristsnics Ys _x Mo IL TestSuSbtorsagteCaenndieonss
Pleasindicatetherecomend storageconditions tWile tematicnal,Lid.
Ae
Hab steab othe isubsttaneceeunddesrttheese ngscndiii ns
Le
Ys x No
IV. Test Concztratins:
Adjustestcoocetrationt 100% ai
----x__baseduponthe purity (%)given above.
|
Doocecomin10 100%
V. ToiciyInformation:
5
Mammglim: + R#LDSO 251mgMouwssLDSO_N/A
Adquiic:
Tovertebrate Tricity(EC/LCSO)
Fish Tawicity(LCS0)
E--
Daphniamagn:S0mg/l.
FatheadMinnow:38mg/l.
Ot Toxicity Inferno(ching fidingsofchronicandsubchronic ests:
Pleasesoo MSDS.
mn
en
PROTOCOL NO.: 454/091799/LEMTDISUBAS4
04543
`IMLABREQUENSOT.U2723
W-IL_D--LI--FE INTERNATIONAL, LTD 1.
APPENDIIXI AnaMel thoy dPrt ovii dedbc ySa ponl sor
Sampleswillboanalyzedbaseduponprocedures providedbytheSponsorin th followinganalytical L LPieqrfuliudoCrohorcotmaantgoSgurlafponhiyoMAacisdsPoStpaescstiruommeStarlyt((LPCFMOSS))IMneFtrheoshdwfaoterrt,hSealDtewtaetremriannatdiAolngoalf
Medium analyzAectoheptyeosftstahmepalbeosvvielmlebtehdoodcwuimlelnbteemdaiinntthaeirnaewdidnattahearnadswudmamtaar.iTzheedianctthuaeflimneatlhroedpoorlto.gyused to
PROTOCOLNO.: 454/091799/LEM7D/SUB454
04544
3MLABREQUENSOT. U2723
.
.
PROJECT NO.: 454A-111
Wr ILDLIE FE INTERe O NATIONAL Lo. ese P2age 12o1f22
AMENDMENT TO STUDY PROTOCOL
STUDY TITLE:
PFOS: A 7-DAY TOXICITYTEST WITHDUCKWEED (Lemna gibba G3)
PROTOCOL NO: 454/091799/LEM7D/SUB454
SPONSOR: 3M Corporation
AMENDMENT NO.: 1
PROJECT NO.: 454A-111
EFFECTIVE DATE: February 11, 2000
AMENDMENT: CultureMedium, Page 5
3MLAB REQUEST NO.: U2723
Change: The pHofthemediumshouldbe 5.0 +0.1.
Add:
ThepH ofthemedium shouldbe7.5 +0.1.
REASON: TochangetestmediumfromHoagland'sto 20XAAPmedium.
AMENDMENT: TABLE 1, Page 11
Remove: Entire table.
+
Add:
TABLE 1
20XAAP MEDIUM
[--
Noaioal Consentrion
MgCly6H,0
CeCly2H0
2432 mgl
880 mg
HBO,
3712 mg
MaCly4H,0
832 mgl
ZaCly
656 pg
FeCly6H,0
3.19 mg
CoCly6H:0
2856 pg
NazMoO,2H:0
1452 pg
CuCly2H,0
0240 pg.
Na;EDTA2H,0
600 mg
NaNO;
5100 mg
MgSO#TH,0
2940 mg
KGHPO,
2088 mg/l
reco,
wh sf
aThreipotnoHfLhemdmwillbesoi, eves,575 01 wigOTN NaOH
.
04545 v
al
WWI IILLDDLLN IIFFEE IT NTERNE ATIOR NAL LoN . ATIONPA ROJECTL NOP.: Pe4a5. gr4eA2-eo1f112
REASON: Tochange test mefd romi Hoagu lanm d'sto 20XAAPmedium.
LOh o STUDY DIRECTOR
LABO} "ORY MANAGEMENT
--=Sa--Le T7 hAin) SPONSOQRSRFPRESENTATIVE
ph DA
DA
33/40)
DA
004546
PROJECT NO.: 454A-111
WeILeODLmLImFEeNITcNETeEReNATeTIIOmONNmANLTmLDo.e. eesr stcsimmmserereePeDagesrlotf1
AMENDMENT TO STUDY PROTOCOL
STUDY TITLE: PFOS: A 7-DAY TOXICITY TEST WITH DUCKWEED (Lemna gibba G3)
PROTOCOL NO: 454/091799/LEM7D/SUB454
AMENDMENT NO.: 2
SPONSOR: 3M Corporation
PROJECT NO.: 454A-111
EFFECTIVE DATE: February 22, 2000
3M LAB REQUEST NO.: U2723
AMENDMENT: Page2
Add: Experimental Start Date: March 3, 2000
Experimental Termination Date: March 10, 2000 `TestConcentrations: Negative Control,13,25, 50, 100, 200and4m0 ga0 .i/L `Test Substance No.: 4675
REASON: Theaboveinformationwas notknownwhen theprotocolwassignedbythe StudyDirector.
BH
STUDY DIRECTOR
ler LAB \TORY MANAGEMENT
--_--A rs dls SFONSOIF REPREAENTATIVE
3/2/00
DATE
ie DA
. DaA pa)
04547
J
y0 e
E WILE DLIFENINETEORNAATTIIOONNAALLATLDo,. eerie
PROJECT NO.: 454A-113
Page lOoEfR2,
AMENDMENT TO STUDY PROTOCOL
STUDY TITLE: PcoFsOtSa:tuAm)96-HOURTOXICITY TEST WITH THE MARINE DIATOM (Skeletonema
PROTOCOL NO: 454/091799/SKE96/SUB454 SPONSOR: 3M Corporation
EFFECTIVE DATE: February 28, 2000
AMENDMENT: Page 2 <
Add: ExperimSetanrttDaatle:March3, 2000 Experimental Termination Date: March 7, 2000 `Test Concentrations: Negative Controland4.0 mg ai/L. `Test Substance No.: 4675
AMENDMENT NO.: 1 PROJECT NO.: `454A-113 3M LABREQUESTNO.: 12723
REASON: The above information wasnot knownwhen the protocol was signed by the StudyDirector.
AMENDMENT:ExperimentalDesign, Page 3
Change:
Themarine diatom, Skeletonemacostatum,willbeexposedto ageometric seriesofsixtest `concanedannetgatrivea(ct ulti ureo medniusm) confotr9r6 hoourls.
To:
`Themarinediatom,Skeletonemacostatum, willbe exposedto atest concentrationatthelimitof
`solubility and anegative (culturemedium)controlfor 96 hours.
REASON:Tochange experdeisimgnetoanltimiattelst.
.
AMENDMENT:ExperimentalDesign, Page 3
Change: Testsolutions willbeinoculated with approximately77,000cells/mL Test solutions willbe inoculated with 10,000 cells/mL.
To:
"Testsolutions willbe inoculatedwithapproximately 77,000 cells/mL.
REASON: Typographical error.
AMENDMENT: ExperimentalDesign, Page 3
Change: Three "biological"replicates per experimental group willbe prepared for evaluating celldensities.
To:
Six "biological"replicatesperexperimentalgroupwilbepreparedforevaluatingcelldensities.
REASON: Tclo change experii mental designdesignttooaa limit test.
004548
Reviewedby GhGB 3-1-00
:
W--I_L--DLIFE INTERNe ATIe ONLAo. L.
PROJECTNO.: 454A-113
WWNPwage2of2
AMENDMENT:Samplingfor Analytical MeasuremePnatgse7,
Change: Day 4 samples willbecompositedsolutionsfromthethreereplicatesremainingattheendofthetest.
To:
Day 4samples willbecompositedsolutionsfromthesixreplicatesremainingattheendofthetest.
REASON:Tochange analyticalsamplingbasedonadditionalreplicatesprovidedinthelimittest.
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( 53 Ho
STUDY DIRECTOR
X /| .
STAetn -------- LABORATORY |
JEMENT
SENSORSKEPRESENTATIVE
3/2/00
DATE
afro DA
SS7B)
DA
04549