Document RJd3ZKkJOgbLGkrqanmdDGXEn

Protocol.PFOS: A 7-D (Lemna gibbaG3) PROTOCOL PFOS: A 7-DAY TOMCrrY TEST WrrH DUCKWEED (LemnagibbaG3) U.S.EnvironmentaPlrotectioAngency Series850 -EcologicaElffectsTestGuidehm OPPTS Nunber $50.4400 3M Lab RequestNo. U2723 Submittedto 3M Corporafion EnvirorumentaLl4ibomtory 935 Bush Avenue St PauL Minnesota 55106 WildlffcIntcrnational,Ltd. 8598 Commerce Drive Easton,Maryland 21601 (410)822-8600 September 17, 1999 WILDLIFE INTERNATIONAL, LTD -2. PFOS: A 7-DAY T03aCrrY TEST WrrH DUCKWEED (LemnagibbaG3) SPONS SPONSOR!S REPRESENTATIVE: TESTING FA STUDYDIRECT LABORATORYMANA 3M Corpomtion EnvironmentalLabomtory 935 Bush Avenue St.Paul,Minnesota55106 RochelleR. Robideau VrildliIfnttmmatiomdI,A& 8598 Commerce Drive Easton,Mwilmd 21601 J;m@@ CAO SeniorAquaticBio ol;@; Hemy 0. Krueger,Ph.D. DirectoorfAquaticT("dwlogy& Non-TargetPlants ProposedDates: Experimental StartDate: ProjectNo.: TestConcentratims: Ten Subsm= No.: FOR LABORATORY USE ONLY TaminafionDate: Rcf== SubstanceNo. (ifappucable): PROTOCOL APPROVAL SToby DMECTOR DATE LAB61KTbRY MANAGEMENT '::@SSPP@@@PREDS;@kAR#Tf@frSa@@@ATIVE PROTOCOL NO.: 454/091799/LEM7D/SUB454 DAl-5 4f4,wct-' 6/ 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL, LTD -3- MROP=O WildlifIenternatioInAa&lw,ilclonducatseven-datyoxicitteyswtithffidcuckweedL,emnagibba G3,fortheSponsoarttheWildlilfaetmuatimaLltd.aquatitcoxicolofgaycilintEyastonM,arylanT&he studywillbe performed based an proceduresin theU.S. EavirommentalProtectioAngency Series850 EcologicalEffectsTest GuidelinesOPPTS Number 850.4400 (1). Raw dataforallwork perfomiedat WildlifelatemationalLtd.and a copy of th-f.inalreportwillbe filedby projectmunbcr inarchiveslocated on theWildlifehitcrmtionalLtd.siteo,r atan altmmativelocationtobe specifieidnffi&cW report. PURPOSE The purposeof thisstudyistodetamiinedw toxicitoyf PerfluorooctanSeulfonicAcid,Potass' Salt(h=after refen-odtoasPFOS) toduckweed, Lenma gibba G3, understatitcestconditions. EXPERIMENTAL DESIGN Frondsof duckweed,Lemna gibba G3, willbe evwed to a geometricserieosf atleastsixtest concentrationasnd a negative(mdt= medimn) controlforsevendays.Nominal testconcentrationwsillbe chosen in consultatiownith theSponsor based upon informationsuch as rangefindingtoxicitdyata,known toxicityinformationorpbysicaychcmicalpropertiesofPFOS. . Targetconcentrationwsillnotexceed 100 mg/L or thesolubilitlyimitofthetestsubstancein water. Generally,each concentratioonf PFOS used in the definitivteestwiU be atleast50% of the next highertreatinm@unlessinformationconcerningffic concentration-effeccutrve indicatetshata differendtilutionfactorwould be more appropriate. Uniform,healthylookmg plantswillbe removed from thestockculturefortesting.Five plants totauingapproximately15 fim& willbe added toeach testchamber. Care willbe takcn.tcow= did ffic. number of frondsin eachtestchamber isapproximatelyequal Three "biologicar'r-eplicpaetrestreatment willbe tested.One ormore additiciw'wah*Al' replicatewsillbe includedinthetestasneeded,toprovide testsolutionforconcentratiovnadficatimon Days 3,5 and 7 oftheexposure.An abiotirceplicateat the highesct4mccatmdoa wiU be includedm the testand willbc sampled on Days 3,5 and 7. laorder to control bias,the positionof ffictestchambers willbe determined by ind@eriminate draw dailyduring fficexposure period. No other potentialsources of bias are expected to affectthe resultsof the study. The testresultswillbe calculatedbased on directcounts of the numbers of duckweed fmds. Ilm mewmetnents willbe taken on Day 3, Day 5 and atthe end of the test Observations of emomsis, break-up Of duckweed colomes,changesin color,rootdestructionand-otha abnormal appemm= willalsobe docuzncnted.An IC 10,IC50 and IC90 values(e.g.t,he toxicantconcentratiotnhatproduces a 10%, 50*/o PROTOCOL NO.: 454/091799/LEM7D/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL, LTD -4- or 90% reductionin frondnumber relativteo the controlsw)illbe calculatebdased on totalfrond numbers atDays 3,5 and 7 of theexposurepaiod. lle no observedadverseeffecctoncentratio(nNOAEC), which representdsw highestcon=tration thatinducesno advaw effectosn plantand frondproductionor appearance,willbe determinedbased upon evaluationof thestatisticraelsultst,he dose-respons@padmn, and otherobserved effects. MATERLUS AND WlrH@DS Test Sub Informationon the of test@controlor refermcesubstan= isrequiredby Good LaboratoryPracticeSMiidw& (GLP) Ile Sponsor ismponsi-bleforprovidingWildlifeInWnationaL lAd. writtenvenficatiotnhatthetestsubsta= has been dundamed sb*. IfwrittevnerificatoonfGLP testsubsta= accordmg toGLPs priortoitsuse inthe isnotprovidedtoWildlifeInternational, LtcL,itwillbe notedinthecompliaj= sw=mt ofdiefinarleport lle attachefdorm :RMFICATION OF TEST SUBSTANCE BY SPONSOR (Appeadix1)istobe used toprovideinformationnomsary for. GLP compliance. The Sponsor isresponsibleforallinformationrelatedto thetestsubstanceand agreesto acceptany unused testsubstanceand/ortestsubstancecontainersremainingattheend of the sh*.. Test SolutionPLe2aration The desiredcaxznuations of PFOS wM be obtainedby preparinga singlestocksolution*mmedun and dilutinfgt appropriatveolume of stocksoluttonwithme&unl The testsolutionmsay alsobe prepared' using secondarydilutionosf theprimary stock based upon the concentratiodnesut& The testsubstancevaU be administeredto the testorganismm culturemc&unl This routeof adnumsb-atiownas selectebdecauseitrepr=nts themost lgwlyrouteofnposure toduckweed,which odst suspendedon a watersurfacewithrootscyimding intothewatercolu= Test Orgggig Iletest'spedes willbe the duckweed, Lemna gibba G3. This speciesis representativoef an importantgroup oiorgsaism and was selectedforuse inthe testbased upon pastuse and caseof handug in the laboratory.Originalduckweed plantsvnU be obtainedfrom dw United StatesDepartment of Agdcdture, Washington,D.C. or anothersuitablesupplier.S.tockculturewsillbe maintainedincult= PROTOCOL NO.: 454/091799/LEM7D/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL, LTD -5- mediumatWildhfIrn,t=atioLmtdd,.Duckweeudsedm toxictietsywtislbleobtainferdomstocckultures thathave been activelygrowing in tinsmedimn for atleast2 weeks. Culture culturemedium preparedacmdmg to WildlifeIntemtionaL Ltd.StandardOperatingProcedures willbe used as chlutlownater The cmcmulfions ofthe medium components arepresentedinTable 1. Stock nutrientsolutionwsillbe preparedby adding mgcnt-gra& or betterdxmcals to Vrddhfc IntemationaLLtd.wellwatcrpurifiedby mvemo-mm@. Appropriatevolumes of the stocknutrient solutionwsillthenbe dilutewdithpudfiedwellwaw to prepareft modium Ile mc&mn willbe filter stmihzed(0.22tLm)or auto&vcd priorto use inthe test The pH of the medium shouldbe 5.0:L0.1. Analyseswillbe performed at leaston= annuallyto determinethe concentrationosf selectedorganicand morgamc constituentosf the wellwater and resultsof the most recentGLP compliantanalyseswillbe summarized inthe finalreport. Specificationfsoracceptablelevelsof cmummants have notbeen mubhshed forcult.ureme&wm However, thereareno levelsof contaminantsreasonablyexpectedto be presentin themedium thatare consideredtointeerewiththepurpose orconductofthesb*- Test Appm-atm Testchambers willbe 250@ipL Nalgm=V bcal=s coveredwithdisposablePetridishesand OmWn'ng - 100 mL oftestsoluticnThe testcbmbcrs vn-Ube ML%aklioat* positioneodn a shelfinan cnvhdmncdd chamber. Test chambers willbe labeledwith thePrOjcctnwnber, testconcentrationa-nd replicate- Conditions The testwillbe conductedat 25 * 2"C under continuouswarm-whitefluormmt lightingatan intensoift5y000:k750IwL ligbitiiwmitwyiUbemeasureadtfivleocatiosn=s=&g theteachambers on Day 0 ofthetest.Temperau= win be measured twicedailyina containerofwateradjacenttothetest chambers inthe=viromnental chamber using a liquid-in-glumsercury thermometer. The pH 40wh trea=cnt.ao&tbe controlwillbe measured attestinitiatiaond terminatiounsing FisherAccumet Model 915 pH meteror equivalent Samples forpH measurementsattestinitiatiwoinffbe collectefdrom the individuablatchesof testsolutionpreparedforeachtreatmentand controlgmW At test tominadon, PH willbe measured inpooled samples of testsolutioncollectefdrom each of the&= replicates of each respectivetreatinenatnd controlgroup. PROTOCOL NO.: 454/091799AEM7D/SLIB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONALP LTD -6- BiologicalMeasurments Growth, defmod asan increaseinthetotalnumber of fivnds,willbe assessedon Day 3,Day 5 and attheend ofthetest AdditimAy, thetotalnwnber of duckweed plantspresentineach testchamber Will be determinedattestWmmatLon. ne numbers of chloroticn,ecroticand dead frondswillbe enumerated. Chloroticfrondsarethosepossessingareasof bleachedcolorprogmsing from green to yellow.Necrotic fronds= thosewith localizerdegionsofdead or decayingtism, wually surroundedby healthytissue. Fronds with no apparentlivingtissueu,suallyallbrown, blad; white,yellow or clearw,illbe considered dead. The &wkwwd plantsi,@ibUe observedfordw presenceofrootdm-tn@ (Le.,dead or sborwned roots ina tub=t group compared tothecontrolsa)nd bre&W ofduckveod colonies(i.e.a.n increasednumber of f=-floatmg individuaflrondsina treatmentgroup compared tothecontrols)A.ny otherabnormalities in frondor plantappearancewillalsobe documcntecl Smp-h-n& forAn@13Lhc-amleasurements Samples of theexposuresolutionswillbe couertedon Days 0, 3, 5 and 7 and analyzedfortest substanceconcentrationsl.le terminatiodnay samples may be filtere(d0.45pm), asnecessaryt,qremove plantdebrispriortoanalysisD.ay 3 and 5 sampleswillbe collectefdrom ft extrareplicatteea chamber(s) includedinthetesttoprovidesamples forthatanalysisand from theabioticreplicatesS.amples willbe placedin an appropriatestoragecontainer(e.g.p,olyethyleneo,r polypropylenebottle)and willbe analyzed 'atelyo,r storedunderrefrigeratiountilanalyzed.The sample scheme.is.s6mmafizedbelow- PROPOSED EVerimental Group Control SolventControl(ifnwded) Level I-Low Concentration L@"el 2 Level 3 Level 4 NUMBERS OF VERIECATION Day 0 Day 3 I I I 1 1 1 1 1 1 SAWLES Day 5 Day 7 Level 6-IfighConcentration 1 21 21 21 Totals 'Includesabioticsamples. 8 9 9 9 PROTOCOL NO.: 454/091799/LEM7D/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL, LTD -7- The above numbers of samples representthosecollectedfrom thetestand do not includequality control(QC) samples such asmatrixblanks and fortific-afipornesparedand analy7M duringtheanalytical chemistryphase ofthesh*. And3iicalChemis Chemical analysisofthesamples willbe performed by WildlifeI,nternationaLlt,cL The analytical method used willbe based upon methodology providedby the Sponsorand id=tlfiedinAppendix M lU methodology used toanaly=Oe testsamples wffibe doam=tod in theraw dataand swnmuized inthefinal reporl StatisticAaglWyNes Percentmiubmm vnU be calculatefdoreach fteauncntand controlgroup usingthefollowingfammla. Mean Control- Mean Treatment PercentInhibition Frond Number Frond Number X. 100 Mean ControlFrond Number The Day 7 ICSO valuewillbe determined,vhen possible,usinglinearinterpolationr othersuitable techniques(3)withbmtment response(frondnumber) and exposureconcentratiodnata..me pcrc4.tftges of dead,chloroticand necroticftmds willbe calculatefdoreach treatmentand controlgroup as thenwnbcr of dead,chlorotiocr necrotifcim& tothetotalmnnber of frondspresentineachtestchamber. llw datawillbe evaluatedfornormalityand homogeneity of variances.Iftheseassumptionsareme@ Duanctes procedureor dc Bonferrom t-test(4)may be used toevaluatethedata. Ifdieassumptionsof normalityand homogmeity ofvariancesarenotmet,non-oarametdcstatistimceatlhods suchastheKhmW-... Wallistes(t4)may beusedtoevaluatheedata.A noobserveaddverseeffecctoncentrat(iNoOnAEC),@M be determinedbased upon evguationof the statisticraelsultst,hedose-responsepatternand otherobserved effect(se.g.p,ercentchlorosinse,crosise,tc.). RECORDS TO -BE MAINTAINED Records tobe maintainedfordataS=ated atWildlifeInternationaLlt,d.willincludeb,ut not be limitedto: I- Copy of signedprotocol. 2. Identificatiaonnd of thetestsubstance,ffprovidedby Sponsor. 3. Dates ofinitiatioand terminationofthetesl PROTOCOL NO.: 454/091799/LEM7D/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL, LTD 4. Source of plants. 5. Cultureconditions. 6. Growth measurements. 7. Calculationand preparationof testconcentrations. S. Observations. 9. ifapp'Ucable,themethods used to analyn testsubstancecmcmuiftons and theresultsof analytical measurements. 10. Statisticcaallaimom. 11. Test conditionsand physical/chemicanl2casurements. 12. Copy offinalreporl FINAL REPORT A finalreportoftheresultosfthesb* willbe preparedby Wildlifehftmtional LtcL The report willincludeb,ut notbe limitedtothefollowingw,hen applicable* 1. Name and addressofthefacilitpyerformingthesh*. 2. Dates on which thesb* was initiateadnd completed.Itistherespons*ibilitoyf the Sponsor to providethefine datethatdata= recordedforchemistrypathologyand/orsupportingevaluations thatmay be generatedat od= laboratories. 3. A statementof compliancesigned by the Sb* Directoraddressing-an@oweptions to Good LaboratoryPracticeStandards. 4. Objeefivesand proceduresstatedintheapproved protocolincludingany changesintheoriginal protocol. 5. The testsubstanceidentifiebdy name, chemical dsftcts number Or code number, strengthp,urity. and compositionor otherapprapdate.chanwtaistiesi,fprovidedby theSponsor. 6. Stabilitaynd thesolubilitoyf thetestsubstancesi-,] theconditionosf ailministratiiofnp,rovided 7. 8. 9. ---- by theSponsor. A descriptioonf themediods used to conduct thetest A descriptioonf ffictestrA-ten3i6ncludingthesourceofthetestorganisms and theirscienfifincame. A-descriptioonf thepreparationof thetestsolutionsm,ethods used to allocatoergmdmw tothe test chambe@ -Andbe& theWs@ numbers of organimns and chambers per treatment,and durationof the tesl 10. A descriptioonf allcircumstancesthatmay have affectedthequalityor integritoyf thedata. 11. The name of theSb* Director,the names of othersciciw@ or professionalsa,nd thenames of all supcrvisorpyersonnelinvolvedinthesb*. PROTOCOL NO.: 454/091799/LEM7D/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL, LTD -9- 12. A descriptioonfffiVc=formations, calculationosr,operationsperformedon thedata,a summary and analysisofthebiologlcadlataand mm@yfic4dchemistrydata,and a statementofdw conclusions drawn from &= analyses. 13. Statisticmaelthods employed foranalyzingthe data. 14. The signedand datedreportsof each of theindividuaslcientistosr otherprofessionalisnvolvedin the Sb*. 15. The locationwhcarlelraw dataandthefinarleporatretobe store& 16. A Mkment preparo.by the QualityAssurance Unit hsbm the datesffidSWY MsPect'Olasand audits were made and ffidcatesof any findingsreportedtodieSu* Directorand Management 17. Ifitisnecessarytomake correctionosr additionsto a finalreportafterithas been accepted,such changes shallbe made indm form of an amendment issuedby the Study Director.The amendment shallclearlyidentiftyhe partof the finalreportthatisbeing amended and the reasonsfor the alteratLorALmendments shdi be signedand datedby theSh* Director CHANGING OF PROTOCOL Planned changesto theprotocolwillbe inthe form of writtenamendments signedby the Study Directorand theSpmsoes RepresentativeA.mendments willbe consideredaspad of fficprotocoland will be attachedtothefnd protocol.Any otherchanges win be inffifcorm of writtendeviationsignedby the StudyDirectorand filedwiththeraw data-AU dianges totheprotocolwdl.beindicateidnthefmd report. GOOD LABORATORY PRACRICES This sb* vnffbe conductedinaccordancewith Good LaboratoryPracticeStandardsforEPA (40 CFR Part160 atLd/oPrart792);OECD Principleosf Good LaboratoryPractice(ENV/MCICHEM (98)17); and Japan MAFF (59 NohSan, NofifciafionNo. 3850, AgriculturaPlroductionBureau).-Each sb*' conductedby WdMc ktmmataong Lt& isroutineleyxamined by theWil&de Int=atLonaL Ltd.Quality Assurance unit forcompliancewith Good LaboratoryPracticesS,tandardOpm-atmg Proceduresand the spocfficdpmtocol. A statementof compliancewith Good LaboratoryPracticeswillbe preparedforall portionsof the sb* conductedby W-ddhfe internationaLlt,d. The Sponsor willbe responsiblefor compliancewith Good LaboratoryPracticesforproceduresperformedby otherlaboratorie(se.g.r,esidue analysesorpaffiqlogy)R.aw dataforallwork performedatWildlifeInte=tionaL Ltd.and a copy of the. finalreportwill@e filedby pmject number inarchiveslocatedon theWildlifeIntema6onal,Ltctsiteo,r at. an alternativleocationtobe specifiedin the finalreport. PROTOCOL NO.: 454/091799/LEM7D/SLTB454 3M LAB REQUEST NO. U2723 WILDUFE INTERNATIONAL, L-rD -10- REFEP.ENCES I US. EnvironmentalProtectionAgency. 1996.Series850 - EcologicaElffectsTestGuidelines (draftO)P,PTS Number 850.4400:AquaticPlantToxicitTyestUsingLemna spp.,Tiers.Iandff. 2 ASIM StandardGuide 1415-91E. 1991.StandardGiddeforConductingStaticToiddtyTests %ithLemna gibbaG3. 3 Norberg-lGngTJ. 1993.A LinearlnterpolatiMoenthodforSublethaTlo3jdty.Y-heInhibition Concentration(lCp)Approach (Version2.0). V.S. PmtectionAgency. RacarchLabomtory. Duluth,hmfinmnes@ot& 4 West@Inc.and D.D. GuUey. 1996.TOMTATRelease 3.5.West= EooSystem Technologyh,m. Chcyc=, Wyomin& PROTOCOL NO.: 454/091799/LEM7D/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE I.NTERNA-nONAL, L-rD M-HOAGLANIYS MEDIUM TABLE I WITHOUT EDTA OR SUCROSE CONSTITUENTS Compound Nom;nnl Concentration H3BO3 ZnSOe7H20 2.86 mgfl 0.22 mg/L 14a;tMO04-2H20 CuSO,e5H20 I&C]6-4H20 KH2PO4 KNO3 0.12 nii/L .0.08 @ mg/L 3.62 mg/L 0.68 9/L 1.515 g(L Ca(NO3)2-4H20 MgSO4-7H20 1.180 g/L 0.492 g/L FcCI3-6H20 TartariAccid 5.4 3.0. mg/L The PH of the medimn willbe adjusted,as necessmy, to 5.0 0.1 using 0.1 N NAOH or100/aHCL. PROTOCOL NO.: 454/091799/LEM7D/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL, LTD -12- IDE4TOCAnON @APPINDDCI OF TM SUBSTANCE BY SPONSOR To be Compkftd by Spmsx 1. TestSubstanceldca&y (n=c tobe used intherepart)P:FOS (PerfluorooctmSeulfonicAcid PotassiumS ReferenceStandard(ifapplicable)A:nalyticaSltandard: N/A hitmudstandard: 1.12.2HJLH.HPerfluorooctancSulfonicAci Test Substance Sample Code,or Batch Number.- Lot 217 Test Substance Purity /oActiveIngredient): 98.9 ExpiratioDnate: 2008 IL TestSubstance Have ffikckod g@M udlyand coropositiomrodierdhamcbmlsdm 4 Ctedsi aurdcf==sftnbd=ard toitsum inthissb* ina=rdm= mdffGiLP Sbndards? Yes x No a TcstSubsta= StorW CaiM= please de reoommmded gardp oon&ons atVRdl& hftma&md, lAi Ambient Elasd3csUb!Uy ofthetmtmbstanx undertb= sta bem ddmM3bi6d iaacomdmce withGLP Stmxbrds? OdxrpadoentsW)iUyido@-@ cmdificas .____@Ycs x No IV. TestCzooer&afim: AdjusttestcmcmhzfimtoIW/o &L x basedWmdcpurky (*/ag)i%m abom V. Tadcitybimmatim Do nota4iusttestconeech-afimtc)1000/o &L TcstftmatainIALI-S. Nlm=abm- RatID50 251 mg&g MouseLD50 N-/A Aquafic- kvmtcb@Tmi&yMOLC50) DVIMia mafma: 27 mg/L FishTad&Y aoo) Rainbow Trout: II mpJL DI&LA magna: 50 mgll FatheadMinnow: 38 mg! OdxrTwicity kkimafim Cmchxfinfgindippof&x6c andmbdmoic tmts): ---Pleasseee MSDS PROTOCOL NO.: 454/091799/LEM7D/SUB454 3M IAB REQUEST NO. U2723 WILDLIFE INTERNATIONAL, LTD -13APPENDIXII An*dcal Method Providedby Sponsor Samples willbe ana4-md based upon proceduresprovidedby the Sponsor m thefollowinganalytical methods: I- Liquid ChromatographyMass Spwftometry (LCMS) Method for the Detamination of Perfl SulfonicAcid Potassium SaIt(PFOS) In Freshwater,Saltwaterand Algd Meffion A COPY of the above method willbe maintained'm the raw datal The actualmethodology used to anab=. the testsamples willbe documented in theraw data and summmizod inthe finalreport. PROTOCOL NO.: 454/091799/LEM7D/SUB454 3M LAB REQUEST NO. U2723 WILDLIFE INTERNA-noNALLTD. PROJECTNO.: 454A-111 Page I of2 AMEENDMEENT TO STUDY PROTOCOL STUDY TITLE: PFOS: A 7-DAY TOMCITY TEST WITH DUCKWEED (LemnagibbaG3) PROTOCOL NO: 454/091799/LEM7D/SUB454 AMEENDMIENT NO.: I SPONSOR: 3M Corporation PROJEC-rNO.: 454A-111 EFFEMVE DATE: Februaiv11,2000 3M LAB REOUEST NO.: U2723 AMENDMENT: Culture Page 5 Change: The pH ofthemedium shouldbe 5.0 0.1. Add: T"nepH ofthemedium shouldbe 7.5 0.1. REASON: To changetestmedimn from Hoagland'sto20X AAP mediwm AMENDMENT: TABLE 1,Page11 Rmove: Entiretable. Add: TABLE 1 20X AAP BEDILJM' Component Mgcl2'6H20 CaCI2-2H20 H3B03 l@-bCI2-4H20 ZnCI2 FeCI3-6H20 COC12-6H20 Na2MO04-2H20 CUC12-2H20 Na2EDTA-2H20 NaNO3 MgS04-7H20 K2BP04 NaHC03 Nommal Concentration 243.2 mgL 88.0 mgtl 3.712 mg/L 8.32 mg/L 65.6 lig/L 3.196 mg/L 28.56 pg/L 145.2 . gg/L 0.240 gg/L 6.00 mgl 510.0 mg& 294.0 mA 20.88 mg/L 300.0 mg/L The pH ofthemedimn wiU be adjusteda,snecessaryt.o7.5 0.1using0.1N NAOH or 10% HCI. [Rif WILDLIFE INTERNA71ONALLTD. PROJECTNO.: 454A-111 Page 2 of2 REASON: To change testmedium from Hoagland'sto20X AAP medium. STUDY DIREcrOR LL,A0KFBdTOIiYMAfiAGEMIENT SPO RESEII" DAI'I OL DAT-E DAT& WILDLIFE INTERNA-noNALLTD. PROJECTNO.: 454A-111 PageIofI AMENI)MIENT TO STUDY PROTOCOL STUDY TITLE: PFOS: A 7-DAY TO)UCITY TEST.WITH DUCKWEED (LemnagibbaG3) PROTOCOL NO: 454/0917995"7D/SUB454 AMEENDMIENT NO.: 2 SPONSOR: 3M Corporation PROJECRNO.: 454A-111 EFFECRIVE DATE: Febnnrv 22,2000 3M LAB REOUEST NO.: U2723 AMEENDMIENT: -Page2 Add: ExperimentalStartDate: March 3,2000 Experin=talTerminatioDnate:March 10,2000 TestConcentrationsN:egativeControL13,25,50, 100,200 and400 mg A.LAL TestSubstanceNo.: 4675 REASON@ The above informationwas not known when theprotocolwas signedby theStudyDirector STUDY DIRECTOR @/" / e LABdk,A:TOR'k MANAGENEW XZrt7-lA]L.LvE C:3)?-/00 DATE -7/0? DAT]K 'nA 7@V WILDLIFE INTF-RNA-noNAL LTD PROJECTNO.: 454A-113 Page I of 2 AMEENDMENT TO STUDY PROTOCOL STUDY TITLE: PFOS: A 96-HOUR TOMCITY TEST VQTH THE MARM costatum) DIATOM (Skeletonema PROTOCOL NO: 454/091799/SKE96/SU]3454 AMEENDMIENT NO.: I SPONSOR- 3M Corporatim PROJECR NO.:,.454A-113 EFFECRIVE DATE: Febniary28,2000 3M LAB REOUEST NO.: U2723 AMENDMENT: Page 2 Add: Expmin=W StartDate:March 3,2000 ExperimentalTerminationDate: March 7,2000 TestConcentrationsN:egativeControland 4.0mg a.i/L Test SubstanceNo.: 4675 REASON The above infonnatimwas notknown when theprotocolwas sipzd by theStudyDirector. AMENDMENT: EUcrimmtalDesi Page3 Change: The marine diatom,Skeletonemacostabim,willbe exposed to a geometricseriesof sixtestconcentrationasnd a negative(culu= mediwn) contoolfor96 hours. To: The marine diatom,Skeletonemacostaum3@willbe exposedto a testcontc,ntratiamtthelimitof solubilitaynd anegative(culturmeedium) controlfor96 hours. REASON: To change experimentaldesignto alimittest. AMENDMENT: EneffinentaDlesi Page3 Change: Test solutionswillbe inoculatedwith approximatdy 77,000.c"n3L inoculatewdith 10,000ccUshnL. _ Test solutionswin be To: Testsolutionwsillbe inoculatewdithapproxim@y 77,000cclWmL. REASON: TypographicamlTw. AMENDMENT: Enmimental Desi Page3 Change: Three"biologiod'replicatpeesr experimentalgroupwillbe preparedforevaluatincgeU densities. To: Six"biologicar'rephcatpeesr experinnntaglroupwillbe preparedforevaluatincgelldensities. REASON: To change experimentaldesignto a limittest. P,,,'ewedhi OL6 WILDLIFE INTF-RNAnoNAL- LTD. PROJECT NO.: 454A-1 13 Page 2 of2 AMEENDMIENT: Sa=ling forAn-ab!LcaM-eIam=ents Page 7 Change: Day 4 sampleswillbe compositodsolutionsfrom dw threereplicatersemainingatthecmdofthetest. To: Day 4 sampleswillbe compositedsolutionfsrom thesixreplicatersemainingattheend ofthetest REASON: To change analyticaslamplingbased on additionarleplicatepsrovidedinthelimittest. STUDY DiiMcrOR LABORATORY NIANAGEMENT @@@@PRESE P]KESE T@IVE@@@ 7T"l A@T E DATE