Document DvGEbVLeg1vKdkbEm1OopqYBd

ARI26-0093 AN ACTIVATED SLUDGE, RPFEOSSP:IRATION INHIBITION TEST Jrap-- WILDLIFE INTERNATIONAL LTD, PROJECT NUMBER: 4546-101 3M LAB REQUEST NUMBER: U2723 aursons I, R. Scott Flages STUDY ITI DATE: Das 11, 98 STE CONTIN IN: His 900 AMENDED REPORT DATE: Ap20,2i00 SUBMITTETOD Eoicp omenalALabor SP ware S517 Wildlife International, Ltd. 8598 Commerce Drive Easto(n4,10M)ar8y2l2a-n8d60201601 Page of 49 00857 AMENDED WILDLIFE INTERNATIONAL LTD. "2. PROJECT NO.: 454E-101 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT SPONSOR: 3M Corporation TITLE: PFOS: An Activated Sludge, Respiration Inhibition Test 3M LAB REQUEST NUMBER: U2723 WILDLIFE INTERNATIONAL LTD. PROJECT NUMBER: 454E-101 STUDY COMPLETION: March 23, 1999 REPORT AMENDED: April 28,2000 "Thi study wasconductedincompliancewiththe U.S. EnviPrroteoctinonAmgenecyinn40tCFaRPlart Mo16n0o;gr1a7pAhuNgou.st45;19P8a9r;isO1E99C2D; aPnrdinJcaipplaensMoAfFGFoo5d9 NLoabhoSraant,orNyotiPfriaccattiicoens N[oO,C3D8E5/0G,DA(g9ri2c)u3l2tu]raElnPvriordouncmteinotn Bureau, with the folowing exceptions: characterTihzeedirenfceormepnlcieanscuebwsitatnhcGeo,oodbLtaabionreadtofrryoPmraAcltdicreicShtanCdhaermdsi.caAlcerCtoifmipcaatneyof(aMniallwyasuiksehea,sbWeIe)n, swuapsplnioetd by Aldrich. Stability, homogeneity and verificationofthe test and reference substances in the mixtureswerenot determined. characte`rTizhaettioenstasusbpsetrafnocremewdaasccnoortdcihnagrtaoct3eMriSzteadnidnaarcdcOopredraantciengwPirtohcfeduulrleGsaLnPcdoMmeptlhioadnsc,ea;nhdoawlevrearw,dtahtea arcbeingmainitnthae3iManrcheiveds.Thetestsubstance sbeingrecharacterizedinaccordancewithGLP. "Thestability ofthetestsubstanceunderconditionsofstorageatth test site wasnotdeterminedin accordance with Good Laboratory Practice Standards STUDYDIRECTOR: EBdwal rCdJ. ScOohaferJJoAy Manager, Biodegradation DATEfare 000868 AMENDED WILDLIFE INTERNATIONAL LTD. PROJECT NO.: 454E-101 3. QUALITY ASSURANCE STATEMENT 160, 1"7ThiAsusgtuusdtyw1a9s8e9x;amOiEneCsDfoPrrcionmcippllieasncoefwiGtohotdheLUa.bSo.raEtorynPrvactiicesPrr[oOtCeoDcEti/noGnDA(mg9e2n)ec3y2i]nn4E0ntvCirFaoRnPmlaenrtt PMroodnuocgtriaonpBhurNeo.au4;5Wi(ldOlCiDfeE/IGntDe(m9at2i)o3na2l],Laindd.JSatpaanndarMdAOpFeFr,ati5n9gPNroohcSeadnu,reNsoatinfditcahteisotnuNdoy,pr3o8t5o0c,olAg,rTihceudltautreasl ofallinspectionsandauditsandthe datsthat anyfindingswerereportedtothe StudyDirectorandLaboratory Management were as follows: AcTvITy: TestChamberPearson DOMeasurements RawDatsandDea Report FilReport Amended ial Report DATECONDUCTED: December 2, 1998 December22,1998 Jiouay 7404 28,1599 Mach23,1599 April 28,2000 DATE REPORTED TO: STUDYDIRECTOR: MANAGEMENT: December22,1996 December 23,1998 December22,1999 Decem2b,1e99r8 Jeary28,1999 February 3,1999 March23,1999 April28,2000 March 25,1999 Api 28,2000 . Hynson A Assurance Program Sf DATE free 000869 AMENDED WILDLIFE INTERNATIONAL LTD. 4 REPORTAPPROVAL SPONSOR: 3M Corporation TITLE: PFOS: An Activated Sludge, Respiration Inhibition Test 3MLAB REQUEST NUMBER: U2723 WILDLIFE INTERNATIONAL LTD. PROJECT NUMBER: 454-101 PROJECTNO.: 454E-101 : STUDYDIRECTOR: od 0 ff. Edward C_ Schaefer, BS. Manager, Biodegradation foo es : DATE MANAGEMENT: i) Ln HenryOKrueger, Ph.D. Director, Aquatic ToxicologyandNon-TargetPlants DAT!YP 5joe Loa ot S3ushdMCorpA_orBaetaicohn s)i)od) DATE 000870 AMENDED WILDLIFE INTERNATIONAL LTD. ER STUDY INFORMATION StudyInitiation Date: Experimental Start Date: Experimental Termination Date: `StudyCompletionDate: Amended Final Report Date: December 11, 1998 December 22, 1998 December 22, 1998 March23, 1999 April 28,2000 PROJECT NO.: 454E-101 `Study Director: - Sponsor: `Sponsor's Representative: `Study Personnel Edward C. Schaefer B.S. 3M Environmental Laboratory St. Paul, Minnesota 55133 SusanA. Beach Edward C. Schaefer, B.S., Manager, Biodegradation Heary O. Krueger, Ph.D, Director, Aquatic Toxicology nd Non-Target Plants Abul Siddiqui, B.S., Scientist, Biodegradation Sheri Trumbull, Technologist, Biodegradation R Scott Flaggs, B.S., Biologist, Biodegradation 000871 AMENDED WILDLIFE INTERNATIONAL LTD. "6`TOAFCBONTLENETS PROJECT NO.: 454E-101 Title Page RR Pagel Good Laboratory Practice Compliance Statement ce Pagel, QualityAssuranceStatement .............................. cer Pagde ReportApproval Pagel Study Information LL Pages TableofContents Page Abstract i Page ItrOduCtion Page OBJOCVE cece Page Experimental Design ee PagE MaterialsandMethods sestanasarrcasaresesssssrararensacs BOGS: ReTfesetreSnucbestSaunbcset.an.c.e : ._...._._........c.o.s..e.m.a..m.s.r.a..s.a.r.c..r.t.a..r.e.s.eseacssireranPaEgeY10 Route of ExpanodDusratuionre ................... Page 11 TestSystem I rage TTeessttChnoocunlumdanid.At.p.p.ai.r.a.t.ou.s..n..s ..................e.c.a.r.e.s.e..r.c.o.s.n.s.r.e.r..e.r.e.a.s.e.r.e.. PAagRe Hll SampleAnalysis o.oo... SCtaaltciusltaitciaolnAsnalyses... . L Pagen2 ICPargeeel23 ResanduDilscutssison .............. SR oro Pagel3 Conclusion - sessarsassscansrasesnscasarareres BOGEIE References . ee Page 1S 000872 WILDLIFE INTERNATIONAL LTD. PROJECTNO.: 454E-101 -7- `TABLE OF CONTENTS - Continued - TABLE Table 1. Respiration RaantdPeercsent Inhibitions ........ Page 16 FIGURES Figure 1. Dissolved Oxygen Concentration Over Time, Control 1... Page 17 Figure 2. DissolvedOxygen Concentration OverTime, Contro2l caressa ere reseea DEER 1B Figure 3. Figure 4. Dissolved Dissolved Oxygen Oxygen Concentration Concentration Over Over Time, Time, 3,5-Dichlorophenol, 3,5-Dichlorophenol, 3mg/L 1 mg/L ...................PPaaggee 2109 Figure 5. Figure 6. DDiissssoollvveedd OOxxyyggeenn CCoonncceennttrraattiioonn OOvveerr TTiimmee,, P3e,5r-fDliucohrlooorcotpahneenSoull,fo5n0icmgAc/iLd, Page 21 Figure 7. DPiostsaoslsvieudmSOaxlytg(enPConFce0nOt9r0aStmigo)naOiv,ler.T.i.me, Perfluorooctane Sulfonic Acid, Page22 Figure. DPissoolvetdSOaxalytg(ePsnFOCoSsn)c,e2nit7rmatugionmOver Timaie/L,...P.e..r..f.l..u.orooctane Sulfonic Acid, .Page23 Potassium Salt (PFOS), 9.0 mg/L ........... o.oo... Page24 Figure9. Dissolved Oxygen Concentration Over Time, Perfluorooctanc Sulfonic Acid, PotSaalt(sPFOsS),2i7mugaim/L ....................... Page25 Figure 10. Dissolved Potassium OSxalytg(ePnFOCSon)c,e9nt0ramtiognaOvier/TLim, .P.e.r..f.l.u.o.r..o.o.c.t.a..n.c..S.u.l.fonic Acid, oo. Page26 Figure 11. PDoitsassoslivuemd OSxalytg(ePnFOCSo)nc,e2n7tr1atmigonaiO/veLr .Ti.m.e.,.P.e.f.l.u.o.ro.o.c.toa.n.e.S.ulfonic Acid, oo. Page2] Figure 12. PoDtiasssoslivuemdSOaxlytg(ePnFOCSo)nc,e9n0t5ramtigon/OvLer Time,..P.e.r.f..l.u.o.r.o.o.c.t.a.n.eo.S.u.lfonic Acid, Lo. Page28 APPENDICES Appendix I MeasuredDissolvedOxygen(DO)Concentrations(mgO4L).. oo. Page29 Appendix IL. PGortaapshsiicuamlSPraelsten(tPaFtOiSon)oCfoPnecrecnetnrtaInthiiobnit.i.o.n andPerfluorooctane Sulfonic Aovcers.id, Page3l AppendiILx. Protocol, AmendanmdDeevinatiton.. ........................ Page 33 AppendiIVx. ReportAmendment .................ooo0. Lo. Pages AMENDED 000873 WILDLIFE INTERNATIONAL LTD. PROJECTNO.: 454E-101 -8- ABSTRACT "Theeffectofthetestsubstanceonactivatedsludgemicroorganismswasassessedby theActivated Sludge RespirationInhibitionTestMethod (OECDGuideline209). Thetet contained acontrol, referenceandtreatment groups. Thecontrolgroupwasused to determinethebackground respirationrateofthe sludgeandwasnotdosed withth testorreferencesubstance. The refgreourpwaesdonsecdwieth 3,5-dichlorophenol, known inhibitor ofrespiration,a concentrations o3f, 15 and 50 mg/L. Thetestsubstwaasndocseed at 090, 27, 9.0,27, 90, 271and 905mg aL. Aferanexposureperiodof 3hours,therespiration ratesofthetest solutionswere: measuredusing adissolvedoxygenmeter.Therespirationratesin thetwocontrolswere 39.8and 41.1mg O/oLadiifferhenceorf3.,2%.Thedifferenceincontrolrespiration rates waswithinthe 15%differencelimit establfoitshheteesdt.Thevalidityofth test wasfurthersupportedbytheresults romthe3,5-dichlorophenol referencegroup,which resultedinanECS0of 9.0 mg/L. TheECSOwaswithinthe 5to 30mg/Lrange considered acceptable for the tet, Difference inO;respirationratsbetweenthecontrolgroupandtestsubstancetreatmentswereobserved atalltestsubstanceconcentrations. Folisl@suommawryoifthenregss. "Treatment `Control | Control 2. 35-dichlorophencl 3mg/L 35-dichloropheaol 15 mg/L P3,e5f-lduiocrholoocrtoapnheenSoullf5o0nimcgA/cLid, Potassium Salt (PFOS) 0.90mg ai/L. PPefeluorroofctlaneuSSuulolffoonrniiccoAAcciiodd,,PcPootttaasssasiiuunmmSaSealltt ((PPFFOOSS)) 29..70mmggaa...i//LL. PePrefrlfulourooroooccttaannecSSuullffoonniiccAAcciidd,,PPoottaassssiiuumm SSaalltt ((PPFFOOSS)) 9207mmggaai./i/LL PerfluorooctaneSulfonic Acid, PotassiumSal (PFOS) PerfluorooctaneSulfonicAcid,PotassiumSat (PFOS) 271mga.i/L 905mg ai/L RespirationRate Percent. mgOyL/hour _ Inhibition 396 NA a1 NA 313 29 146 638 51 874 389 36 351 130 35 170 32779 16910 281 304 247 388 000874 AMENDED WILDLIFE INTERNATIONAL LTD. -9- PROJECT NO.: 454E-101 INTRODUCTION "This study was conductedbyWildife International Lid. for 3M Corporation attheWildife Intemational Lid. biodefagcilrityainEdastaont, Miaryolannd.OriginalrawdatageneratedbyWildlifeInternational Lid. and acopyoftheoriginalfinal reportar fileunderProjectNumber434E-101 inthe archiveslocated ontheWildife International Lid. ste. OBJECTIVE The objective of this study was to assess the effects of the test substance on activated sludge `microorganisms by measuring the respiration rat. EXPERIMENTAL DESIGN "Thetest containsdcontrol,reference,andtreatmentgroups. Thecontrolgroupwasusedtodeterminethe backgroundrespirationrteofthesiudgeand wasnotexposedtoth tetorreferencesubstances.Thereference group was dosed with 3,5-dichlorophenol,a known inhibitorofrespiration, at concentrations of 3, 15, and SO mg/L. Thetestsubswtasatenstcedaet0.90, 2.7, 9.0, 27, 90, 271and 905 mg aL. MATERIALS AND METHODS "Thisstudywasconductedaccordingtotheproceduresoutlinedintheprotocol, "PFOS:AnActivatedSludge,Respiration Inibiton Test,"(AppendixIV).Theprotocolwasbasedontheproceduresspecifiedinthe `OECDGuidelinefor TestingofChemicals,Method 209(1 andCounciloftheBuropeanCommunities,Guideline C11, Activated Sludge, Respiration Inhibition Test (2). TestSubstance `The test substance, Perfluorooctane Sulfonic Acid, Potassium Salt (PFOS), was received at Wildife Intemational Lid onOctober29, 1998andwas assigned Wildlife International Lid. identification number 4675, AMENDED 000875 WILDLIFE INTERNATIONAL LTD. "104 PROJECTNO.: 4S4E-101 "Thetestsubstancewasstoredunderambient conditions. Following is description ofthetest substanceusedin this study: Name: Perfluorooctane Sulfonic Acid, Potassium Salt (PFOS) Lot Number PY Physical Description: White powder Expiration Date: 2008 Purity: 90.49% `Water Solubility: Slight Storage Conditions: ~~ Ambient Room temperature Onthedayofadminitshetterstasubtstianocenwa,saddedtothe appropritaetset flasksviadirectweight addition. Thetest substancewasreanalyzedbytheSponsorandtheCertificate ofAnalysisdated March9, 2000 indicated a purityof 90.49%. `RefersnceSubstance A stock solutionofth reference substance, 3,5-dichlorophenol was prepared by dissolving 500 mg in 10mL of INN4andOthHen iting to 30 mL with NANOpurewater. While sing, cnough IN H,SO, was addedto reachthepointofincipientprecipitation.The solution of3,5-dichltohernwoapsdhileutnedotol1L with NANOpure water.ThepH ofanaliquotofthereferencesubstancestocksolutionwasmeasured (pH=7.18). Following s adescription ofreferencesubstanceusedi thisstudy. Cover Name: Manufacturer Lot Number: Physical Description: Handling Precautions: Expiration Date: Purity: `Water Solubility: Storage Conditions: ass 3,5-dichlorophenol `Aldrich Chemical Co., Milwaukee, WI 00410KZ White crystal Standard laboratory precautions March 13,2000 7% 73991 Ambient 000576 AMENDED WILDLIFE INTERNATIONAL LTD. u- PROJECTNO.: 454E-101 REoutxeofposanudDurrateion `The reference substance was administered by volumetric addition. Volumetric addition is the most * appropriteroutsofadminiosftasrubsatatncieionann aqueoussolution.Thetetsubstvaians administered by direct weight addition. Direct weight addition was selected as the most appropriate routeof administration basedontheobservedsolubility ofthe est substanceinwater. Stock solutionsthatwerepreparedatnominal concentrationsof 1g/L.and0.5g/L.in NANOpurewaterandsonicatedforapproximately20minutescontained testmaterial that wasnotinsolution.Thedurationofthe exposwuars3e hours TestSystem "The iologicaltestsystemwas aconsortiumofmicroorganismscommontotheactivatedsludgetreatment process. The organisms responsible forthedecomposition of organic materials are principally aerobic, and facultative bacteria,Thetestsystem was chosen becauseitis representative ofa treatment process that may. receive the test substance "TestConditionsandApparatus - `Control, reference, andtreatmenttestmixtureswereincubatedat 20+2Candacratedfor 3hoursat arate sufficient to provide acrobic conditions andmaintainsolidsinsuspension. Themixtures werepreparedand acrated in 500 mL Erlenmeyer flsks end then transferred into 300 mL Biochemical Oxygen Demand (BOD) bottlestoconduc the dissolved oxygen (DO) measurements. Test mixtures were identified by project number, test substance ID,testconcentration and bottle number, when appropriate. TestInoculum, Activated sludge was collected from the Prospect Bay Wastewater Treatment Facility, Grasonville, Maryland on December 21, 1998. The Prospect Bay facility receives wastes from predominately domestic sources. Thesludgewassievedusing a 2mmscreenandthensetled forapproximately 30minutes. The supemmatantabovethesettledsolids wasdrainedandthetotal suspendedsolids(TSS)concentrationofthe settled sludge was determined. Based onth result the concentrationofthe sludge was adjusted 10 4000 mg/L (+ 10%) by diluting with municipal water. Approximately 50 mLofsynthetic sewage (Protocol, Appendix IV) was added to cach liter ofactivated sludgeandthesludgewasaeratedovernightat 20 + 2C.Beforeuse,thepH andtotalsuspendedsolids concentration ofthe activated shudgs were determined. 000877 WILDLIFE INTERNATIONAL LTD. "12. PROJECT NO.: 454E-101 Procedure Testmixtureswerepreparedat 15minuteintervalsstartingwiththefirstcontrol. Thecontrolcontained 9.6 mLofsynthetic sewage, 120mLofinoculum,andenough municipal watertobringthetotalvolumeupto 300mL.Themixturewaspromptlyaeratedat aratesufficienttoprovideaerobic conditionsandkeepthesolids insuspension. Subsequentmixturescontained9.6mL ofsynthetic sewage, 120mLofinocthuealppu ropmria,te `amountoftestsubstanceorvolumeofreference substancestocksolution,and enoughmunicipal watertobring the totalvolumeup to 300 mL. Finally, a second control was prepared. All mixtures were aerfaorttherede hours. SampleAnalysis After three hoursofaeration, the contentsofthe first vessel weretransferred to a BOD bottleandthe `respirationratewasmeasured over aperiod ofupto 10minutes.Dissolved oxygenreadingswererecordedevery 10secondsfor 10minutesoruntiltheDOdroppedbelow 1.0mg/L, whichevercamefirstusing a YSI Model S0B DissolvedOxygen Meter.Therespirationratein subsequentvessols was determinedinanidenticalmannerat 15 minute intervalssothatthe contaticmteofthe test substancewiththeactivated slwaustd hreeg houe rs. Caleuations Arespiration ratewascalculatedforeachtestmixtureandexpressedinmgO,/L/hour. Theratewas calculatedusing DOvaluesbetweenapproximately6.5mg O,/Land2.5mgO,o/r oLver,a 10minute period iftheDOdid notreachapproxi2m.a5mtgeOl,/yL.Therespiration ratewascalculatedusingthefollowing equation: Respriater(magt O/Li/hoounr) =Oxygen uptakeoverthecalculation period/durationof calculationperiod `Thepercentinhibitionforeachtestsubstanceconcentration wascalculatedusingthefollowingequation andplotedagainstthelogarithm oftestsubstanceconcentration (AppendixI): 000878 WILDLIFE INTERNATIONAL LTD. PROJECT NO.: 454E-101 13 Peercent InhiIbn:hibition == 11 = ge28,r X 100 where: R, = oxygen consumption rate at a given concentrationofthe test substance RC, = oxygenconsumptionrate, Control 1 RC, = `oxygen consumption rate, Control 2 Statistical Analysss `Wheathedoseresponsepatter allowsforthecalculationofan ECS0value,thedataareanalyzedusing thecomputerprogramofC.E.Stephan(3).Theprogramwasdesignedtocalculatethe ECS0valueandthe95% `confidenceintervalby probit analysis,the moving averorabignoemia,l probawbitih nlonilitneyar interpolation (4,5,6). In this study, probit analysiswasusedtoevaluate inhibitioninthereference group. An ECSO value `couldnot becalculatedfor thetest substance. RESULTS AND DISCUSSION "The pHofthe reference stock solutionwas 7.18.Respirationrates andpercentinhibitions are presented inTabl1e. Dissolved oxygen readings recorded during thetestarepresentedintabular and graphical formsin AppeTannddII,irespcecteivesly. Thetemperaturerangerecordedtheday ofthetestwas 19-21C and was withinthelimitsspecifiedinthetestguideline.Themeasuredtotalsuspended solids(TSS)concentratainond pHofthesludgeonthe dayoftestingwas4380mg/Land7.87, respectively. The respiration rates inthe two controlswere 39.6 and41.1mgO/L/hr,or adifference of 3.7%. The `validityofthetest was further supported by the results from the 3,5-dichlorophenol reference group, which resultedinanECS0of9.0mg/L.The ECS0waswithinthe to 30mg/Lrange consideredacceptableforthe test. `The95%confidencelimitsfortheEC50 were6.9and 11.4 mg/L. 000879 WILDLIFE INTERNATIONAL LTD. PROJECT NO.: 454E-101 ae "The inhibitory effects upon respiration at test substance concentrations analyzed (090, 2.7,9.0,27, 90, 271 and 905 mg 2../L) ranged from 3.6% to 38.8%. Respiration ratesofthe test substance concentrations evalustedrangedfrom24.7mg L/h (at905mga:./L)to 38.9mg O/Luh (at 1 mg/L). CONCLUSION `The maximum inhibitory cffect upon respiration from Perflucroostane Sulfonic Acid, Potassium Salt (PwaF sobsOerveSdata)testsubstanceconcentrationof 905 mg../L,butonlyreached 38.8%. The ECS0 is thereforegreaterthan 905mg aL. 000880 AMENDED WILDLIFE INTERNATIONAL LTD. "15. REFERENCES PROJECT NO.: 434-101 1. Organisation for Economic Cooperation and Development. 1989. Activated Sludge Respiration Inhibition Test. OECD Guideline 209. 2. SClouudncgielRoefstphireaEtuironopInehainbiCtoiomnmuTensitt.ies. Directive 67/548/EEC. Annex V. Guideline C.11, Activated 3. Stephan, CE. 1978. US. EPA, Environmental Research Laboratory, Duluth, Minnesota. Personal communication. 4 Finney, D.J. 1971. StatisticalMethods inBiological Assay, second edition. Griffin Press, London. 5. Thompson, W.R. 1947. Bacteriological Reviews, Vol. Il, No. 2: 115-145. 6. `SAtmeeprhiacna,nCSEo.cie1t9y7f7o.r"TMesettihnogdasnfdorMaCtaelruilaaltsi,ngPaunblLiCcSa0ti,o"nANquumatbiecrTSoTxiPc6o3l4o,gypapn6d5-H8a4z.ardEvaluations. 000881 WILDLIFE INTERNATIONAL LTD. -16- PROJECT NO.: 454E-101 Table | Respiration Rates and Percent Inhibition Treatment Control 1 Control 2. 3,5-dichlorophenol 3 mg/L 3,5-dichlorophenol 15 mg/L 3,5-dichlorophenol 50 mg/L Pefluorooctanc SulAfcido,PontasisicumSalt(PFOS) Perfluorooctane Sulfonic Acid, Potassium Salt (PFOS) Perfluorooctane Sulfonic Acid, Potassium Salt (PFOS) Perfluorooctane Sulfonic Acid, Potassium Salt (PFOS) Perfluorooctane Sulfonic Acid, Potassium Salt (PFOS) Perfluorooctane Sulfonic Acid, Potassium Salt (PFOS) Pesfluorooctane Sulfonic Acid, Potassium Salt (PFOS) 0.90mgai/L 2.7 mg ai /L 9.0 mg ai/L 27 mg ai/L 90 mg ai/L 271 mg a.i/L 905 mg ai/L. Respiration Rate. mg O/Lhour 96 411 311 146 51 389 351 3s 379 27 281 27 Percent Inhibition NA NA 2.9% 68% 87.4% 36% 13.0% 17.0% 61% 19.0% 304% 388% 000882 AMENDED - PROIECTNO: 43-101 Contro1l so Jeo Ero 525600 Sao iw i 820 a 1v.o0 PRN -- 10 20 m0 40 so ew Time (seconds) FreT. D--ev_in----rg CrcassionOv Tee, Core 1.Ans ste esd diedrenremowd ocle 888 &8 J "ie PROIECTNO. 4101 Contro2l IE5so | 82z 60 S40 1 32 20 a 0.0 o At 100 200 300 400 500 600 Time (seconds) 3 Figure. Dpisonn OarypenConeOnvetrTai,iCoon n2.Aros istemean)dished yncontinwdtocules 82@ 2 AMENDED "5. PROIECTNO: 454101 g5 E80 82 60 s3 40 32 20 Eo 0 3,5-Dichlorophenol 3mgiL 1 . cs rt ts femme 100 200 300 400 500 600 Time (seconds) 28 Figure. DoicsloredlOtyreenpCiotrooinma OveTe 3.5 Dicbrophn, gl.Aron dctmere dished yenonecnetions wid 2&@ @ AvEDED "20. FROJECTNO: 454101 3,5-Dichlorophenol 15mglL 35 |! Eso < | $2x 60 O3 40 3$ 20 3200 -- + 0 100 200 300 400 500 00 Time (seconds) 2 Figwr4e. DiesdctclOevieneCpnsietroatmieo.nOveTi,.-Diheoph15emidl,.Aron dtesddissolvedoesconcn 32 2& AMEDD are FROIECTNO: 456.101 3,5-Dichlorophenol 50mgiL. eo Tk | Jeo E5 r6o0 50 S40 Ea $20 210 ! 00 100 200 300 400 500 s00 Time (seconds) Fae. DSeatiolclOorvpeenrCeopreon.OveTe, 3. Deen, 50ml.Arovsinesmesnrddsohvd ypecocirions. g8& 2 AoE [a ---- 300 3Ef87e00o a 8SzEa5io0 5 20 21.0 200 ow+ PFOS 0.90 mg a.i/L | . me aw-- we wm eo Time (seconds) NN Pere i Do CemOT gg22 i Aoi570%0 A ro ne Jo me FROIECTNOS 4S4E101 50 3Br8o0 ! seo $50 Siano 2a 2100 00 100 PFOS 2.7 mg a.ilL . . --p _-- t 20 300 "00 s00 s00 Time (seconds) 28 Figure. eDissnavedOsryoaCcooyrtooRnOveTid e,PC ocuspSTli.Aci,PossumSull(FOS)27mg aL. Arows nits 23@ ENDED ane PROECTNO: 4siE101 PFOS 9.0 mg a.i/L JE580 ol 52 60 8340 3s 20 23 00 | +eet 100 200 300 400 500 600 Time (seconds) 2 Figures. DeissisveeddOryhaCyonnsaotiovnOiesTed,PoelronlcaitanepSillocineAck,PassionSt PFOS), 90mg 1.Aros nics 2&2 2 AED SA oo 3 8.0 E70 FS ee6oo.0 E N2S0 a o1o0 o PFOS 27 mg a.iL , } 10 20 se ao so soo Time (seconds) o3 re D[ etOmr Conm Ov Tea P rose en s eFl otoSs FO,1m 11 re $2 " AMENDED - mocTyo. sion PFOS 90mg a.i/L IZ3oot | Sgouo . 3S 2.0 a 0.0 o ao aw -- sw aw so eo Time (seconds) . FeeDDiedtOOmpeCCoomrirnOOovTTomNPteseSSles A0 Fotos 709, 0m 11. rn nn 8g 2 ohne 2 PRoCTNO, 4siE101 LTosoo Eo 3$56P.0r0 2aa E i20 00 PFOS 271mg/ a.i.L : | . ------ 100 200 30 40 so soo Time (seconds) 2 Fr1. Dire Oere CvoenienmOrittTie,eolongospealslmeiAc.k FossaSI(E08) 7 mgi. Aro nies 8e8 " AMENDED ES FROJECTNO: 4848101 PFOS 805 mg a.liL se Joo | Eno goo Feo Sao 1 ffau a 0 --_-- o 100 20 300 w EY 0 Time (seconds) g Figure 12. DeissivredsOiynednCyonnsrtoiocneOavenrsTiee,dPoefCoUroRctEaoepSiulneAcid, PosiSalt(PFOS),905mg a1. As cle 3@ = AMENDED WILDLIFE INTERNATIONAL LTD. PROJECT NO.: 454E-101 29. Measured Dissolved OxyAgPePnE(NDDOI)XConcentrations (mg OL) --_---ie in e SSDigtlomhenal 35 e DTlreosripmehneDnilsavE e3d5 OrEyDggehner(onhegn)ol e FOS Pros ? Comal Sat SORE SORE oodmpain _avmesin oowkno 6673 i75s50 00 $3 3 a883 Io i&52 I186 i773d 85 8 & ooooes 885 37i3i 577s3 jaaoss] a&& &6&8 oaaokx 558 ii2 i773ss ii88 85 aa4) oaofel fio 6613] G0 3 & 7i17] 7 i8i 585 a98 a +] 85 GoGnxa 444333 &e1s ax a 3 178s 73 28444 E8&o F5e2) 5 be) 2 68a%:n 33i3 a&a fe I. G0 77id4s 73 888334 iai 5st0 3 is pe] oG85pl0 333%% 815) Gaz 33 5 233 88533 a3i iii 73 82 a is agonwk 3332 F4+e]] 0500 29 5 fAi7 7 - 888222 8 33i98 37 a3i a ogoknN 33x 282 uo 3% be] &EH1] a35 3334%3 33o33 4 2 3 o0o0l%0 1333 aii 6x. 31 i &&& sI8i 32i9 333%48 & 0 13 33 o663sa0 31i93 8i3 oe 11 2 4&s] 4] i880 i3fs%+] 33i12 80 3% 35 aG olRe131133 a@i aR 13 i 8i i 37a33 23i 713]3 73 20 2s aa al% 011s3 3337%3 an 3 82&1 a 7373 1iiss7 i3i$ 73 is 2 waaa3 33334 om = 3 aa6l 777383 1iii3$ 2ii0s 73 12 is B3l%2% Z I 32350 oH I is &0@l &@ 77i1 0jir3} ii1$s7 ] = ii BTHw = 3377 85 f:4)] :z i13 Bold umber ninedissolvedoxygenconcentrnon sedtocleiespirtion is AMEN0D)00895 WILDLIFE INTERNATIONAL LTD. PROJECT NO: 454E-101 -30- APPENDIX I (Continued) Measured Dissolved Oxygen (DO) Concentrations (mg OL) J Tresimen-- tDisol-- ved Oxy-- gen (m-- L) --------M (onyy, -- SOomsgsi __F2Tomsgein SomTgsoisn, -- 2=mgain _ o0SFmoasail Contoi2 0821308 6238s 7&6411 1673 77763 76638 77739 00o35r08a a& 6e2l &1I1 76&9 6868 67&7 oiiErzt $&5 5$857 666s 1i641 a63 16648 o8ot1aas 58%8 588 &6&i 6a4s 6a8 61l a88i23 532 235 858 8ae3l 88 &E8]@ 2 a500 s02i piai} 288 69&0 F5Fid 8s5i 330312300 iias iii 8523 Ba i i & 58a E3%d 228% 3 52 i d3sii5ae i3i pei2} 320 82 a 3 a 5388 +32] iiis 8 $ i %46a3% ii39 333575 aiiss - s81i s2i0 aai 00B55120 3336 33383 i3a3 Epiiod bti3} 3f3o 0o83a50 3338 3330 iaa iai iias 33374 oi%2ae0 33332 22237 3338 %38 3 35 38 ii3 priii 33332 8 a 3 80o65aa% i32s03 22338 33342 0710 is 2 33 aai aii 1j3xs 3 pe} 38 7o072r40i0 ii7s$ a3is 3331 33337 3332s 3323 o0088ar1s0 3223 11736 322 333%% 33376 33i3 a3 0824d 23i9s 111333 23285 33334 333%4% 11173 o88a158 11i37E 111332 2333 83 18 09 31 3330 33323 111332 30 31 i 208534 1i1s3a pezz 10%0 i2 : 31193 i23s9 33300 02=5 17 f+} fx} z Bodnumbers indie dissolved oxygen conenirions wedtocalle resprton es AMENDED 000896 WILDLIFE INTERNATIONAL LTD. PROJECT NO.: 454E-101 231. APPENDIX I Graphical PresentatioPnootfasPseirucemnStalItnh(iPbiFtOiSon)aCnodncPeenrtfrlautoiroonoctane Sulfonic Acid, 00897 WILDLIFE INTERNATIONAL LTD. 32. APPENDIX! PROJECTNO.: 454E-101 Percent Inhibition versus PFOS Concentration (mgiL) 2s i= fa i ds 1 5 o` 10 100 1000 PFOS Concentration (mg a.JL) Figure 1. Percent Inhibition versus Perfluorooctane Sulfonic Acid, Potassium Salt (PFO) Concentration 000898 AMENDED WILDLIFE INTERNATIONAL LTD. 33. APPENDIX IIl Protocol, Amendment and Deviation PROJECTNO.: 454E-101 000899 WILDLIFE INTERNATIONAL LTD. 34 APPENDIX III PROJECTNO.: 454E-101 : PROTOCOL PROS: AN ACTIVATED SLUDGE, RESPIRATION INHIBITIONTEST Ongaisstion fo E`cOoBnCoDmiGcuCiodoepleirnaet2i0on9sndDevelopment = `ConnellofBaop"eAasnCoocm,muGnuiidteileisneDiCc1i1ve 7/S4VEEC `3MLebRequesNot.U2723 8 Submite1d0 Bavi3roMamCeonrtpelLraatbioornsiry StPeP.,0M.iBnaexs3c3a33$15133 WILDLIFE INTERNATIONAL LTD. `E5as9onC,oMamrmyelrandD2i16v0e1 i0)s2.3600 3 Decabert 1598 PROTOCOLNO:: 454/12019ASRITISUBASS MLABREQUEST NO,U2723 000900 WILDLIFE INTERNATIONAL LTD. 23s. PROJECT NO.: 454E-101 APPENDIIXl WILDLIFE INTERNATIONAL uD. : 2 $E0S:ANACTIVATED SLUDGE,RESPIRATION INHIBITIONTEST SPONSOR: 3PEMOCoBropvoxr3a3t3ii 3oL1nabon SaiMiers $5133 SPONSOR'S REPRESENTATIVE: ~~Ms. SuA.sBeamch. TESTING FACILITY: WE2ia5ls9dtl8oiCnfe,IMonateyrmanantmdiDoni2sa1vl6eL0d1. STUDYDIRECTOR: EA Schaefer LABORATORYMANAGEMENT, HMainmseO.oKfAeceairPTD,.odNooogTagye Pa&ts: FORLABORATUSOEORNYL Proposed Dees: Sem joie SEE albe pToewCpeo:ntrA:H3E12T,0O0L0 uty S013 apa, COSislukt Tossubstmentto: _JoTS______ RoiptDus OIF "PROTOCOL APPROVAL QSTUoDYDaIREnCTlOR L Lo RMAtC NAGE R a EE) VE ATEsafufor soudT ofu AaE2 /a2 PROTOCOLNO: 4SUI2OISHASRITUBASA 3MLABREQUEST NO.UZ723 0C0S01 WILDLIFE INTERNATIONAL LTD. PROJECT NO.: 454-101 WILDLIFE INTERNATIONAL to "36 APPENDIIXII 3- INTRODUCTION "Thepaposeoftistestis 1 provid asrcingmethod0 dcdily ubszmes imay vel afcserimicdroio t tl bl poue nibiten ylessus bstan concotrerinabitodeiprdoailnyessts. ORECTIVE "Thebjctivof hesywill tossstheefototh etsbstnconactiveshud `icrby moesieagpeesipieisonese. A BCS0willbecaupos,se. `EXPERIMENTALDESIGN Thetetwill tain ctl,refrac,ed resentgro.Th cot groupiswed0 deem thebacked esi eeof hoshod ad illotbeexposedothe stsubstance. Lp concenotf3r,15a, tadi5omngs.The et stenowillbe tda 1,3, 10,30, 10,300,sod 100mg. - MATEARNDIMEATHLODSS Testmethodsi bas atheprocederesspecified ithoOBC Godel orTesting of `Chemicals,Method209 (1) sdCoun oftoEropesnConaouniics,Gude C.1,Acie Stade,RespiatriaontTiosot)n. Testun Ioanoth haactariaionoft,contolo elec bstancsis girdbyGood Labormory PracticeSanders(GLE),40CFR Pt 16031.ThoSpouse isponsfoixpbrovlidieng. Wid erst!Li writeveifcaiontet tet bsancbasboncharatcizndsoning 1012s cro ing athetet.TheatachdanIDENTIFOFITECSTASUTBSITAONCNE BYSPONSOR(AppendixI bewdtoprovidefocmstion secs forGLPcomple.If itnvonof GLP taedct i okprovidedtoWIAs enstions Li, twillbomtnthecomplsn sim ofhs ilspect. : PROTOCONOL: 454/1201SUASRITISUBAS4 MLABREQUESTNO.U2723 00302 WILDLIFE INTERNATIONAL LTD. PROJECT NO: 454E-101 3. APPENDIX WILDLIFE INTERNATIONAL to. : 4 ThoSpaspstfo llniin elatedtot tacedaresost ryesd stbane ne stbts esosrminin codof sy. `StockSoAlutlioocnkPlreptaratioofnt.hetssean willbe prep a inl conectionof 100g ce1.iighcmtwate e.g.Nope).Indion, okoionof3,-Gckcophcwcitll propiabydissolving 500g 10.of IN NeOFedtenting 030mL.withigh uty `cwaitpeir,nWtpirclpistriionn,gTaheusgolINoFoSfO,-(GpcopplethemnwLi)wlibl ibotdddd1torLavcithhtheipgohtloyf wr . "CTesot CnodrtioetsodicApop,arnaotuss:tt est crs will bat 204 2C odsrt fc3 1b2o5w0s0amlpratiuEfrceame0ymeastkisnnditdeeminrensefpereod.sTohe3m0i0tielsBiwoiclhlebmeircaelOrrpeondDetedd (BOD)bottle tothe conduct dissolvedcxygen(DO)measurements. TetIAnotciohruom d dg ProspectBeyWesterTres Pcwillwd th ocak forthe.ThePropetBey alyrecivewasnfenpeonydomstiowares. Tho apdesawtillsbheosveethvs wetitendgid2swmlledmessdtdehetad fsoprsepndeoidzsmitdlyT3SS0)moimntar.ioTnh oftested kodewillbodein.Bas anther thconectionof odgewil be td140m0L0 10%)by ditowithmiwles. 11 dgcmtbe sd ted of llectionoi he embec Fed bosd onsubsequentdays(msxismmfourdays),50mLofsyntheticsewage(AppendixIT)wil beaddedto e4a2ch7Ct.erBoefaictrivwatee,dsthiogpeasttdheteontdaol mf capchwdorksionlgiddescye.Tnhtreasthiuodgnewofilhlbeeaacratteidovveernsightwialtl2b0o trmiondmo nsey,wfodoH60- $0 4 idscontracof 000ml 10%), PROTOCOLNO: 4SU/IZDISASRITISUBASS MLAB REQUESTNO. 02723 000903 WILDLIFE INTERNATIONAL LTD PROJECT NO: 454E-10L WILDLIFE INTERNATIONAL (1D. "38 APPENDIX II os Pusat Testmireswillbopeepred 1mint trassarigwitthofrstcontr. Then. il contin mLofsyneswag, 120ml.of octcoughmunicipalwee obi th tava 0300.Th carwillbeprot ted t slitfpthslid: imap. Subaequctmixes willcia aofyt swag, 120mL.ofmca th opopist okamsofsa releasesocksti,mdcongh micewet ing ttlohano 10300mL.Final,ecodcontrol willboprepard.Altwil rbeest forties bore. `SammieAnais Af fe nesofserineomitsof fitveelwillboa BODbole reporewllbommr ove aperiodofpto10mits.Dissobedcypeneninge will rondovy 10 onde 10 ima owl5DOdup below L0mf, ichver comesft.Torespiration ti boseveelwillbedetemindi ninmac 15 mittral oth theconc ofthestsstan witteactvtdsadgis rohs. Calalsions Arospintioatwillbocultfrechtmish tndceed ng Lc Tho illbocelcltod singDOvasbetweensprit6.mgOL. 2.5mgOy crv 10mitpaso thoDOdocs ctecheppecitely2.5mgOL. Th petniinoccotmbt cocetrationwllbe canwingttheefllodwig equation ndpoi aginecration alo pepe: Percent Ihibitson= 1-- li X 100 PROTOCOLNO: 4SU12019WASRITSUBASS SMLABREQUESTNO. U2723 000904 WILDLIFE INTERNATIONAL LTD. PROJECTNO.: 454E-101 39. APPENDIIXI WILDLIFE INTERNATIONAL Lr. - "6 hare R= caypmai sctapoivencoonesctimon opf hetestmobstnancs RG, = oxy consumptCionotrnol,| RG, = comcomsumptioCnarrsoel2, ASECSDabowillbedvd pose,bas thopectnitionvers sk bts conocatration.Condenslit 951)forth BCS0wllbo determin singstandardsical procodares6). OualityContd. "Totsicsiderodvali cy teflowingales wr ck: : + thtwoctorepsrewitihin15n%oftcach sher; - wBCS0(aham)of 5 dcbropeooisfnthspiedrageof 030m. 'RECOBERMADINSTATINOED Recordstobomatin willnclde,botnt td1,thefollowing: 1 Acopyoltesigeedprotect 2 Meninocharacterizationof testsubstance aprovidedbySposa 3. Test eonsodrminatdisotn. 4. Epuokiisiono sftt iatiaondles. 5. Stock ohsion constrainclansdsectionprpuricn 6. Activatedsagesores sodrestdeasl. FE 5 Refoooombtancorouts. 5. AllGisivedoxygenmess. 10. Temprangeeesrdeaddatringufstrpereiod. 11. lnbbiioncrv md hdfo clio of BCS0. 12. clnBCSlOeda95%t confe ineldis 15. Acopyoftie astreport ~PROTOCOLNO: 45/12019UASRITISUBAS SMLABREQUESTNO. Uz723 00905 WILDLIFE INTERNATIONAL LTD. WILDLIFE INTERNATIONAL 1D. ~40- APPENDIIIXI a. PROJECTNO.: 454E-101 Nolreptoftheresofet[yFIwNlAlLbReEpePpORTbyWIL rion Lud Therept stoct,boti ied, Followingwbasplice 1. Ne enaddsofflypectin say. 2. Dats chicthsadywas sdend completo. 3. Astatement ofcomplesigebytheStyDict adeesinmyxposfoGood LaboPrracstStoardty. 4 Obfocvsmtpesrs todfn eprovdproces,ndingsychines ith gin 5. prtotaotni.onsodchitinofte st san tsprovidedbySpccing, CASmambo,patciv, athe christs, povidedbyteSponscc: 6 Adepoofthetrsfomatiossndlionsprtomedntodt. 7. Adesipionofthemets ed dees ogysade midcpl bt Adecipimoftots 5. Adeaipinofthepepe of he stslats, itingcoors) hertof sdnined horrieoniofnoe,. 10, Adoption oflcometstamayoface qualityoctetof din. 11. ToonamsotthsadGc,themesoftherectitsopoesionl,mdhsasof Al perviseyprom, vobednhsay. 12. Tope odaodeectsofeachof nfl cistsooh pros vd nthe oly, iplicable. 1. 14, Those Asme warethrwdtsmdf repelboss. propre bythQtyAssocUs ling iedecteoso ispocins enanit wer ade1 thedtsof eyGoingswer repaid toStdDicend 15. Maintiasgeememntm. yms cocci ans 0 i repo frihsbascp,mh hingevib sd thform of seme SeyDic,Th woos willlow eniytheptoft final epee it beingendedadthrss ox he mendes,co il be gadb toStyDic. PROTOCOLNO: 4SU120ISWASRITISUBAS4 MLABREQUESTNO. 723 060S06 WILDLIFEINTERNATIONALLD. ~~ PROWCTNO: siElol -a1- APPENDIIXI WILDLIFE INTERNATIONAL uo. : 2 Pochangs `CHAONF GPRIOTNOCGOL otsprotwillbe thfosofwit edssigedby hsSy DicedeSRpecpetitive,Aneodentswil becniderd1s ptof proces nd ibestchdoefdrtcn Anytchcgeswills fomofwidendevinsigned bythe SoyDiuctorsod ledwilhtheredit.Allchang spoon willboiictdne foal rpet. Thi stadywillbo dGcOOtDLsAoBxeOtRioAeTswOiRtYhGPoRoAdCLTiIbCrEaSryPrcics StandardsfoxEPA M4o0nCoAgRrPtN1o6.05;0,BsCoDPJrianpcsipMleAsFofFG(5o9dNLoakoSres,NeyoPtaicoensN(oO.C3D5E0/,GDAg0s)i2t,iEanPvrioocscion Biren).BachsycookedbyWILE tron Li.is vtieyccsmindbyteWilkie \OopeernsiinognPaLoi.QeuilmtydAthspsceiUsptfreco.mpAlsintcewnltGofoocdoLmabpoarsa wiPtrhaGctoiocedsL,Satbaindeayrd fProarct okipboedWieptirciscoefs LsouyecdoonyeobfyWioduereepmtstwoilelnolflLid.bRdyapiitcat amber activeoad heWM tration Lud.se, onlitive cain 0be spochefiolipeact. PROTOCOLNO: 45412019WASKITISUBASH 3MLABREQUESTNO.U2723 000907 WILDLIFE INTERNATIONAL LTD. PROJECTNO.: 454E-101 WILDLIFE INTERNATIONAL LD. -42APPENDIIXII -9- REFERENCES | ReOsrpgiarnaitsiaotnionniftoirEocnoTneosmt,iOcECoCoDpGeuriadteiloinnea2n0d9D.evelopment. 1989. ActedShudge 2 ACctoiuvnatcedofSttuhdegEeuRroepesanpColmmIrunhniiabtiittieosno.TeDnsirte.ctiveST/SHVEEC.AmexV.Guideline C11, 3 StBerpaahtaonn,sC.EA.rsz19i7o7m.S"cMeithofdosfToerstCiangemladtMnagtsiaaLsC.SP)"uAqubatilcNTuiumsbiceorlSaoTgyPta6n3id4,Hpoapz6anr5d-. ". PROTOCOLNO; 454/12019ASRIT/SUBASS IMLABREQUESTNO,U2723. C0908 WILDLIFE INTERNATIONAL LTD. -. Jpe----_ 0 WILDLIFE INTERNATIONAL LTD. PROJECT NO.: 454E-101 : _ emom`oer ri oeF AcESBTa ERCwAnNCEi eBYSo POnNn SORsos reste"eTo obteCeosmrplbtyeStpg oensdor i Sl fo Pn am. c BTooth n mi imi 1 a om--a-- n IL InNeESRt eeTCa oEnoaRneEnIinRViEi EsTiniox ee JCaosnfeppurbtli ofto y eo nobbpi pee ners_ ye NoX .STom mi ee utp Zits ownLDR02A teiGAn SI Depboiamagna:27mgfle -- beTr el m--i i-- rds -- ei -- Depboiamagos:SOmgfl, RaisboMwmetrout Eathead mimaow:3mpll FO -- SU 00909 WILDLIFE INTERNATIONAL LTD. PROJECT NO.: 454E-101 4a. APPENDIIXI WupuFe INTERNATIONALWO. ne APPENDIIX.SYNTHETICSEWAGE isso"liTnhgetssyothltilsoamgweeinpinrovgoifdeth ces micsrequiredfobacimab. Tspreparbeyd `boc i leofmnicwparl: 111600 pgosptcst 0307g5 wNeel 004258MCgCO,,2T5H0O 285 KIO, mownRtesconrnaadenyhceomtiamcinbmts thwialwleeredscwneabnleecveecl.eTfhobc e recao tenfd whon Kyneotwis fosbaegcaspe swbelecokf. tawit th he ady. PROTOCOLNO: 4SU1201SMASRITISUBASH MLABREQUESTNO,Uz723 00910 WILDLIFE INTERNATIONAL LTD. WILDLIFE INTERNATIONAL LTD. -as- Amp PROJECT NO.: 454E-101 TRORCTNO EL AMENTODSTUMDYEPRONTOCTOL. STUDYTITLE: PFS:ANACTIVATEDSLUDGE,RESPIRATION INHIBITIONTEST PROTOCOLINO: 4SUI2019WASKITISUBASH AMENDMENNOT: 1 SPONSOR: 3M Corporation PROJECTNO: 44-101 SMLABREQUESTNO: Uz23 AMENDMENT: PaTegstSuebsta;nce AM Thdietieont1stetamosdwsilplebpeatedroickeofbymdiisvseiwoeinghotflefstisou.bsDtanicetstwhitgrhto lily scewer REASON: Roooenoftoaddmeiesswieoinghvascihienngbocdkrsomsvolfumsetlroicwswieoensoclfubnily cfushseatcekt oe ESVECTIVEDATE: 21Deseaber 1998 AMENDMENT: Page4; StockScum repein De A1s0t0o0mckgsabcuttiovn.onfihgohtqesutaywtaatcew(i5lNleoporaerp)r.d 8ein cooomteion of REASON: RouttioonftaodGuriiesruiiognhwasdihoannbgdafrosmvolfitmoetliocwa.wddairnsocfsanlwyosfBe acskt base. ESVECTIVEDATE: 21Dceber 1998 Th Crowned anf 00911 WILDLIFE INTERNATIONAL LTD. PROJECT NO.: 454E-101 6 apENDIX MROBCTNO: Ssipiol WioureiNTeRnATONAL 0, MRR AMENDMENT: ug;Proce Chins TT v otmirs e il a praebp oofmlioeg hegtyowsa5s0,103a.0tiThneoitnhbetfiscpsdt rhTyhe ee iensrsiootewwytti,en1,3l0tmsd.oe fpidi nn yabptSrntgebeoiif pt ictFoi. om oivepes. imism vile To TT orTse e i aparepiBfoeaa1itbiesekoyskvSlpOOBo5sLt0.ionTgeoimfaheboeofovtislm1boedir.5rleTwhyolo afiesone13mLof hon, spotsosof e onve iewta i130e0as. Fol, ct wil testsubstanceorvolumeofreferencesubstancestocksolution,andenoughmunicipal REASON. R= oweofsisiawasssicgeenvofoBtbaviinbof ysonBsesock EFFECTIVEDATE: 21December 1998 SQTOoOTBJRELTORp is fue ow //-- ASHonbRs oTaES0 RTARTVeh i7 o2a [12 [9 ThCesena! >024 00912 WILDLIFE INTERNATIONAL LTD. PROJECT NO.: 454E-101 a7. : WILDLIFE INTERNATIONAL uD. APPENDIX II DEVIATIONTO STUDY PROTOCOL PROJECT NO: PEag1e o0f1 . STUDYTITLE: PFS: AN ACTIVATED SLUDGE, RESPIRATION INHIBITION TEST PROTOCOLNO: 454/1201S8ASRITISUBASA DEVIATIONNO: | SPONSOR: 3MCorporation PROJECTNO.: 4548101 DAOFDTEFAE CTO DEVIATION: Deciber 12,1998 a DEVIATION: Procedure, Page-5- Th mf.35-dclocopbeoomitrewas acridfre ors ndcoemite. REASON: Inder crocbystypes vac: "Tisdvi5n4 ctiimpoactnthe epoiftho sy. Ro fc. STUDY DIRECTO! %4 EC MANAGEMENT 2-399 DATE oxo far 00913 WILDLIFE INTERNATIONAL LTD. 48. APPENDIIXV Report Amendment PROJECT NO.: 454E-101 1. Original Report: Pages 1-5 an7d Amendment: Thepageswerechangedtoincludetheamendedreportdate, revisedpagenumbers,and ne signaturesenddatesduetotheadditionofthereportamendmentas Appendix IV. Reason: Toreflecttheissuingofanamendedreport 2. OriginalReport: Page2 Amendment: Compliance statementwasrevised. Reason "Toclarifyhowthetest substancewascharacterized. 3. Original Report Page 10 Amendment: Information provided by the Sponsor reflecting the reanalysis of test substance, includingthereanalysisdateandthepurity, wasaddedtotheTestSubstancesection. Reason Toreflectthecurrenttestsubstanceinformationprovidedbythe Sponsor. 4. OriginalReport: Entirereport Amendment: sAulblstteasntcesaubssdteatnecremiconnecdebnyttrahteioSnpsownseorriecnhaarneagneadlys0isroeffltechtetehsetspuubrsittayoncfe(tFhCe-t9e5s,t Lot 217).Testconcentrations oriwgerierenporatedlinlmg/yL. Thecertificaotfe sanuablsytsainsceDcaotnecdentMraartcihon9s,we20r0e0recinadliccualtaetdedaanpudrrietpyoorfte9d0a.s49m%g. aT.h.e/rLebfaosree,doanl ttehset 90.49% puriy. Reason "Toreporttheresultsofthetestbasedonth substancepurity of90.49%attherequest ofthe Sponsor. 000914 AMENDED WILDLIFE INTERNATIONAL LTD. -a- APPENDIX IV - Continued - Report Amendment PROJECTNO.: 454E-101 AMENDMENT SIGNATURES: ete Ly Edward C. Schaefer Manager, Biodegradation HeDinrercytor, Aquatic T, oPxh.iDc.ology and Non-Target Plants Vfoofoers DATE fete DA _ REVIEWED BY: Qualitf AsTs.uHryanncseonProgram Sufsevisor . 4/27). eta DATE 000915 AMENDED om on WILDLIFE INTERNATIONAL LTD. PROJECT NO: P4a5g4eEL-o10f12 on AMENDMENT TO STUDY PROTOCOL on STUDY TITLE: PFOS: AN ACTIVATED SLUDGE, RESPIRATION INHIBITION TEST ou PROTOCOL NO. 454/120198/ASRIT/SUB4S4 AMENDMENT NO. 1 SPONSOR: 3M Corporation PROJECT NO: 454-101 n 3MLAB REQUEST NO.: U2723 ol AMENDMENT: Page 3; Test Substance oi) Ade aTdhdeititoenstissutbhsetamnocsetwialplprboepriasdtmeinriosutteeroedfbaydmidniriescttrawteiionghotfatdedsitfosnu.bstaDncierstthwaetigahrte reltively insoluble in watr o REASON: Rsooluutteionoftoadndiisreicsttrwaetiiognhtwaasddcihiaonngebdecfaruosme vooflutmheetrliocwadwdaitteironsoofluabnilayquoefoutshesitoecskt a substance oi EFFECTIVEDATE: 21 December 1998 - a AMENDMENT: Page 4; Sock Solution Prepartion ol : Delete: A1s0t0o0cmkgsoaluctioinivoftehhieLgtheqsutasluibtsytwaantceerw(ie.llbNaenporpeupraer)e.dat anominalconcentrationof om REASON: Rsooluutteionoftaodmdiinriecsttrwateiiognhtwaasddcithiaonngebdecfaruosme vooflutmheetrliocwadwdaitteironsoolfubainlitayquoefoutshesttoecskt ol substance. ol EFFECTIVEDATE: 21 December 1998 a 000916 ot > TA Cro od vom 8% | PROJECT NO: P4a5g4eE2-010f12 |WD WB ILDLE IFE NINTTEe RNAATIOONAAL LTD.ets ee AMENDMENT: Page 5; Procedure | Change: Tceosnttrmol ivillxcwotinltluibnerp9r.e6epamrsLedoaft s1y5nmtihentuitcesienwtaergvea,ls1s2t0atimnLgwiotfhitnhoecuilsutmcoanntdroel.noTuhgeh 1 `ameurnaitceidpaalt awartaetreosubfrficnigntthetotokteael pvotlheumseoluidps tion 3s0u0spmenLs.ionT.he Smuibxsteuqrueewnitllmibxetpurroemspwtilly tecosnttoarinre9f.e6remncLesoufbsstyantnhceetisctoscekwasgoleu,ti1o2n,0anmdLeonfouignhocmuulnuimc,iptahle aaptperroptroibartienvgtohleutmoetaolf 1 `aevroaltuemdefuopr tthor3ee00homusL.. Finally, a second control will be prepared. All mixtures will be 1 To: "Tceosnttrmoilxtwuirlelscwoinltlaibnep9r.e6pamrLedaotf s1y5nmtihneutitceisnetweargvea,lss1t2a0timngLwiotfhitnhoecfuilrustmcoanntrdole.noTuhgeh | `amcurnaitceidpaaltaatreatretsoufbfriicnigentthetotokteaelpvothleumseoluidpstion3s0u0spmenLs.ioTn.heSmuibxsteuqrueevnitllmbixetpurroesmpwtillyl cteosnttasiunbs9t.a6ncmeLoorfvsoylnutmheeotifcresfeewraegnec,e 1s2u0bsmtaLnocefisntoocckulsuomlu,titohne, aapnpdroepnroiautgehammuonuicnitpaolf 1 wpraetpearredt.o Ablrilnmgixtthuerteostvaillvobleuameeratuepdtfoort3h0r0eemhLou.rs.Finally, a second control will be 1 REASON: RSooluutteioonftoadmdiinreicsttrawteiiognhtwaasddcithiaonngebdecfaruosme vooflutmheetrliocwadwdaitteironsoolfubainlitayquoefoutshesttoecskt 1 substance. 1 EFFECTIVEDATE: 21December 1998 - 1 pn olf STUDY DIRECTOR 1a [nls DATE 7 1 LABORATORY MANAGEMENT I Lo a Rich SPONSOR'S REPRESENTATIVE DA 2/9/79 2/12 la4 DA I 000917 K > Th Ceoensd! 12-29% | I] WILDLIFE INTERNATIONAL LTD. o DEVIATION TO STUDY PROTOCOL PROJECTNO: 4S4E-101 Page 10f1 STUDY TITLE: PFOS: AN ACTIVATED SLUDGE, RESPIRATION INHIBITION TEST PROTOCOL NO.: 454/120198/ASRIT/SUB454 DEVIATION NO.: 1 SPONSOR: 3M Corporation PROJECT NO.: 454E-101 DATE OF DEFACTO DEVIATION: December 12,1998 DEVIATION: Procedure, Pe 5: om `The 3 mg/L 3,5-dichlorophenol mixture was aerated for three hours and one minute. REASON: a Inadvertent ror by study personnel a IMPACT: `This deviation did not impact the integrityofthe study. Rode. 2-399 a STUDY DIRECTO DATE Bl U Cook mcaE a oan H 000918 0 PROTOCOL a PFOS: AN ACTIVATED SLUDGE, RESPIRATION INHIBITION TEST Organisation for Economic Cooperation and Development and a `OECD Guideline 209 a Councilof European Communities Directive 67/548/EEC Annex V, Guideline C.11 o 3M LabRequest No. U2723 mo Ey a | Hui a it Submitted to SM Copersion `Environmental Laboratory St. PauPl.,O.MiBmoexs3o3t3a31$5133 WILDLIFE INTERNATIONAL LTD. Eas8t5o9n8,CoMmymlearcne D2r1i6v0e1 (410) 822-8600 3 ~~ December 1, 1998 000919 PROTOCOL NO.: 454/120198/ASRIT/SUB454 3M LABREQUEST NO. U2723 1 WILDLIFE INTERNATIONAL LTD. I = a PFOS: AN ACTIVATED SLUDGE, RESPIRATION INHIBITION TEST SPONSOR: N i 3M Corporation Environmental Laboratory P.0. Box 33331 St. Paul, Minnesota 55133 SPONSOR'S REPRESENTATIVE: Ms. SusanA. Beach a TESTING FACILITY: W8i5ld9 CnoemrmmeartcieoDnrsilveLid Easton, Mayland 21601 1 STUDY DIRECTOR: Ed Schaefer a LABORATORYMANAGEMENT: HearyO. Krueger, PhD. `Managerof Aquatic Toxic&oNoln-oTagrgyet Plants i _ FOR LABORATORY USE ONLY a =Dates: - ul ExSrtpaeorrjiDmaoetne;t:al AalHlEyMOL ETuexorpmeyirnniamoteinnotnmalDatWe: o(2i/31T25_|| TetConcannon: 431020.100,300,0 oe (0%_12fufo ol LosSubstamoervo: 67S meceiptiwe, 1029.97 a [PROTOCOLAPPROVAL d arbL. STUDY DIRECTOR _fufir DATE : Mo 4 a \B( \TORY MANAGEMENT d SPONSOR'S REPRESENTATIVE DATE 1p [4 a)z]a2 DATE 000920 i PROTOCOL NO.: 454/120198/ASRIT/SUB454 3M LABREQUESTNO. U2723 1 WILDLIFE INTERNATIONAL LTD. I = INTRODUCTION I "Thepurpose oftistestistoprovide ascreening methodtoidentify substancesthat may adverselyafftaerobic microbialtreatmentplants and to indicate suitable non-inibitry test substance 1 concentrationsforus inbiodegradabiltiesttys 1 OBJECTIVE "Theobjectiveofthestudywil betoassesstheeffetsofthetetsubstanceonactivatedshudge 1 microorganisms by measth uresrpiraitionnrgae.An ECS0will becalculated, ipossible. i EXPERIMENTALDESIGN "Thetestwill conain acontrol,reference,andtreatmentgroup.Thecongrotupirsuosedlto 1 determinethebackgroundrespiration atofthe sheandwill notbeexposedtothetestsubstance. The reference group vill be dosed with 3,5-dichlorophenol a known inbibior of respiration, at 1 concentratoiofn3s, 15,and 50 mg/L.Thetstsubstance willbetesteadt 1,3, 10,30, 100, 300,and 1000 mg/L. : MATEARNIDMAETLHOSDS 1 `Testmethodsare basedonthe procedures specifiedinthe OECD Guideline for Testing of Chemicals, Method 209 (1) and Councilofthe European Communities, Guideline C.11, Activated [] Sludge, Respiration Inhibition Test (2). i ---- Informationonthecharacterizationoftest,controlorreferencesubstancesisrequiredbyGood i LaboratoyPractice Standerds (GLP), 40CFRPast 160.31. The Sponsor i respofnorspriovbidlineg `WiiIntemational Ltd.writtenverificationthtthetest substancehasbeencharacterized according i 10GLPpriortousinginthetest. Theattachedform IDENTIFICATION OF TEST SUBSTANCE BY SPONSOR (Appendix I)i to be sed to provide information necessary for GLP compliance If i writen vecifictionofGLEtestsubstancecharacterization i notprovidedto WidifeInternationalLtd, itwillbe notedinthecompliancestatementof te inalreport. i 00921 i PROTOCOL NO: 454/120198/ASRIT/SUB4S4 MLAB REQUEST NO. U2723 u WILDLIFE INTERNATIONAL LTD. . - u oy "TehdeSpostnseorsaresponnsioblefoortllsitnfonrmactioosnienlaetsedtmotiheitet substanndcoefand acgyroaeccespt u StockSolution Preparation , A stock solutionofth test substance vill be prepareadt a nominal concentrationof 1000 mg un civ.inigh ality at (e.gNanors). Tnadiion soc solutionof3-<iorophewnilc prepared by dissolving 500mgin 10mLof IN NaOH and then dilutingto 30 mL vith high quality o water, While siring, cnough IN H,S0, (approximately mL) will be added to reachthepoint of incipientprecipitation.Thesolutionof3,5~dichlorophenolthe vibedlilulted to 1Lvith high quality ou ater |] TCesonditsnid Apoparnatsus Controlreference,andtreatment testmixtureswillbeincubateda 20:2 2Candarated for 3 ol howsataratesufficienttomaintainsolidsinsuspension. Themixtureswill bepreparedandsersted ) in 500mLplasticErlenmeyerflasksandthenraisfrre into 300mL Biochemical OxygenDemand om (BOD)bottletotheconductdissolvedoxygen (DO)measurements. o Tet uActmivanted hdgefromtheProspectBay WastewaterTreatment lant il be used stheinoculum a forthetest. The Prospect Bay Facility receives wastes from predominantly domestic sources. The studgewillbesievedusing a 2mmsroenandthensettledforapproximately 30mines. The ol supeabmovmethaestctiaodnsoltids il bedrainedandth otal suspendedsolids(TSS) concentration " ofthesettledsludge willbedetermined.Basedontheresult,theconcentrationofthesludge wilbe ol adjustot40e0d0 mg/L (+ 10by%dilut)ing with municipal water. i 18thesludgecannotbeusedo thedayofcollectiono ifthe samebatch irequ tobeused onsubsequentdays (maximum ourdays),SOmLofsyntheticsewage (AppendixI)willbeaddedto ad cach teofactivate soattheendofcachworkingday. The shudgo willbe seatedovernightat 20 42C. Before us,the pH and otal suspendedsolidsconcenraotfiothne activated sludge will be a: determinedandif ecsary,dust toptsH 6.0. .0an asolidsconcentraoft4i0o0n0mg/L (+1 1800922 a PROTOCOLNO. 454/12019%/ASRIT/SUBAS4 MLABREQUEST NO, U2723 I WILDLIFE INTERNATIONAL LD. I " `Procedure I "Testmixtures illbepreparedat 15minuteinterval statingwiththe irs control. Thecontrol willcontain.6mLofsynthetic sewage, 120mLofinocanudlenuoumgh municipal wattoberirng the i totalvoluput03m00meL.Themixturewillbepromptlyaeratedata raesufficient okecpthesolids insuspension.Subsequentmixtureswill contain9.6mLofsyntheticsewage, 120mLofinoculutmh,e I appropriatevolumeoftetor referencesubstancestock solution,and enoughmuniwcatiertpobarilng thetotal volup uto 30m0meL.Finally,a second controlwillbeprepared, Allmixwtilulberaeeratsed 1 for threehours. 1 `SampleAnalysis Afterthree hoursofaeration,thecontentsofthefirstvesselwill betransferredto a BODbottle I andtherespirationraewillbemeasuredoveraperiodofupto 10minutes.Dissolvedoxygenreadings `willberecordedevery 10secondsfor 10minutesoruntiltheDOdropsbelow 1.0 mL,whichever 1 comesfirst. Therespiration rateinsubsequentvesselswillbedeterminedinanidenticalmannerat 15 minuteintervalssotha th contac imeofthetestsubstancewith th activatedshudge sthreehours. Calaalations 1 A respirationratewillbocalculatedforcachtestmixture andexpressedinmg Oy/L/hour. The ratevillbecalculatedusingDO valuesbetween approximately6.5mg OLand2.5mgOL,orover 1 210minuteperiodifthe DOdocsnotreachapproximately2.5 mg OL. 1 "Thepercent inhibitionfor eachtestsubstanceconcentrationwillbe: calcusiungltheafotlleowidng equationandplottedagainstconcentration on logpaper: 1 Peercent Znibition = 11- puRte o x 100 1 i PROTOCOLNO: 454/120198/ASRIT/SUB4S4 00923 3MLABREQUESNOT. U2723 n W_I--LDLIFE INTERNATIONaALeLD. . H " o where R= oxygen consumption rate at a given concentrationofth test substance: a RC, = oxygenconsumptionrte, Contro1l a RC, = oxygenconsumrapte,tCointoroln2 AnECS0value will bederived, ipf ossible, based on the percent inhibition versus test substance H concentration. Confidence limits (95%) for the ECSO will be determined using standard statistical ol procedures (3). `Quality Control i Thetesticonsidered validonlyifthefollowingcriteriaaremet: ~ theitwocontrol respirationratesarewithin 15%ofeachother; a ~ theECS0(3bows)of3,5dichloropbenol inth acsepte rangeof to 30 mg/L. a RECORDSTQ BE MAINTAINED Records to be maintained will include, but not limited to,th following: d 1 Acooftp hesigyned protocol. da 2. Identificationandcharactoeftrhietzesatstubistoannceas provbiySdponesodr. 3. Test initiation and termination dates 4. Experimental initaindatertmiinaotinon dates. d 3. Stock solution concentration calculations and solution preparation. - 6. Activatedsludge sourceandpretreatdmeteainlts. d 7. Test temperature and duration. 8. Referencesubstanceresults, a 9. All dissolvedoxygenmeasurements. 10. Temperaturerangerecduorinrgtedstpeeridod. i 11. Inhibitioncurveandmethodforcalculationof ECS0, . 12. IfcalEcCSu0anld95a%cotnfideencedlim,its, d 13. Acooftphefy inal report. 00924 a PROTOCOL NO:: 454/120198/ASRIT/SUBAS4 3M LABREQUESTNO,U2723 o i| WipeIIERNATIONA ORR (WW on " [FINALREPORT oa Afinal reportoftheresultsofthestudy il bepreparedbyWildife International Ltd. Thereport sto include, but is not limited to, the following when applicable: a 1. Name and address offacilityperforming the study. 2. Datesonwhichthestudywas nitiated andcompleted. a 3. A statementofcompliance signed by the Study Director addressing any exceptions to Good Laboratory Practice Standards. a 4. Objectivesandproceduressate inthe approvedprotocol, includinganychanges ntheoriginal protocol da 5. Identificationandcharacterization ofthetest substanceasprovidedbySponsorincludingname, (CAS number, percent active, and other characteristics,ifprovided by the Sponsor. oa 6. Adescription ofthetransformationsandcalculationsperformedonthe data. 7. Adescriptionof themethodsusedandreferencetoanystandardmethodemployed. oa 8 Adescriptonofthetest system. 9. A descriptionofthe preparationofthe testsolutions,the testing concentration(s), the route of da adminiansdtthedruraatitoniofothnete,st. 10. Adescriptionofaclircumstancesthatmayofaffectedthe qualityorintegrityofthedata. da 11. Thenameofthestudydirector,thenamesofotherscientistsorprofessionals,andthenamesof allsuppeersronnve,inivolsved ointhre stuydy. dl 12. Thesignedanddatedreportsofeachoftheindividualscientistsorotherprofessiionnvoallvesd in the study,ifapplicable. d 13. The locationwheretherawdataandfinal reportar tobestored. 14. AstatementpreparedbytheQualityAssuranceUnitlistingthedatesthath studyinspections d `ndauditswere madeandthedatesofany findingswerereportedtotheStudyDirector and Management. a _ 15. Ifitisnecessaryto makecorrectionsoradditionsto a inalreportafteritha boenaccepted,such. changesvillbemadeintheform ofanamendmentissuedbytheStudyDirector. Theamendment d wil clearly identifythepartofthefinalreportthat isbeingamendedandthereasonsforthe amendment,and willbe signedby theStudyDirector. [7| 000925 ol PROTOCOLNO.: 454/120198/ASRIT/SUBAS4 3MLAB REQUEST NO. U2723 | . e Re eA -------------- I * . CHANOFGPRIOTNOCGOL 1 Plannedchanges(0th protocol wilbeintheformofwrittenamendmentssignedbyth Study Dirstor and the Sponsor's Representative, Amendments will be considered as partofthe protocol and I illbe attached th ialprotocol.Anyotherchangeswillbeintheformofwritendeviationssigned 1 bpythe--StudyDirectorand fledwiththerawdata. All changestotheprotocol wil beindicatedinthe I GOODLABORATORYPRACTICES "Ths study vill be conducted in accordance with Good Laboratory Practics Standards for EPA 1 (40 CFRPart 160), OECD PrinciplesofGoodLaboratory Practices (OCDE/GD (92) 32, Bavironment Monograph No. 45); and Japan MAFF (59 NobSan, Notification No. 3850, Agricultural Production 1 Bureau). Each study condbyuWicldte Intermatonal Lu. is routinelyexaminedbythe Wildlife International Ltd.QualityAssurance Unit for compliance with Good Laboratory Practies, Standard 1 OperatingProceduresandthespecifiedprotocol. Astatementofcompliancevith GoodLaboratory Practices willbe preparedfoal portionsofthestyconductedbyWildife Intemational Lid. Rawdata 1 foral workperformedatWildifIntemationalLd.and acopyofthefinalreportwillbefledbyproject number in archives locaotnethde Wildife Intemational Lid. itoeat,a atemative location to be 1 speciintfhiefeindal report. 1 1 1 i 1 1 00926 L PROTOCOLNO: 454/120198/ASRIT/SUBAS4 MLABREQUEST NO, U2723 '] WILDLIFE INTERNATIONAL LTD. | " a REFERENCES . i 1 Organisation for Economic Cooperation and Development. 1989. Activated Sludge Respiration Inhibition Test. OECD Guideline 209. ul 2 ACcotuinvcaitledofSltuhdegEeuRreosppieraantiCoonmImnuhniibittiieosn.TeDsti,rective 67/S48/EEC. Annex V. Guideline C.11, Wl 3 ESvtaelpuhataino,nsC..EA.mer1i97c7a.n S"oMceitthyfoodrs TfeosrtCianlgcaunldatMiantgerainalLs.C50Pu,b"liAcqautaitoincNTumobexr iSTcPao6n3d4lH,aopzpga6r5yd- 8. i i PROTOCOL NO: 454/120 198/ASRIT/SUB454 000927 3M LAB REQUEST NO, U2723 ~ i WILDLIFE INTERNATIONAL LTD. 1 -10- 1 IDENTIFICATION OF APPENDIX | TEST SUBSTANCE BY SPONSOR TobeCoTm T pledbySpomor 1 YL Test Substance ett (ame beusdinh report): PsflySlcfonrieAocido,PocssaumnSaelt. TERE ex i SampleCodeorBatchNumber: Lot2l7 Purity (% Active Ingredient): 98.9% Expiration Date: _2008 1 IL Test Substance Characterization wHhaivcehtahpeipdreonptriity,strengthB, e1purityandScubstoancembeepnodoreottesrhmeiirncehdtaracpirtieorroistincs 1 IL Test Substance Storage Conditions 1 Pleaseindicatetherecomsmtoreagencodndietiodns at Wildife International Lid. Ambietoomtempenture. 1 Jia thestbaiblileof he tetsubstaununcdeertthesestoragecondiitions _-- Other pertinent stability information: | IV. Toxicity Information: Mammalian: RatLDSO 251mgkg Mouse LDSON/A. 1 Aquatic Invertebrate Toxicity (EC/LCS0) Fish Toxicity (LC50) Dephniamagns: 27mg. RainbowLitmgrllo, ut: Daphniamagna:S0me/l Eatbeadminnow 38mgll, 1 Other ToxicityInformation includingfindingsofchronic and subchronic tests): 1 SeeMsps. 0000000000000 1 L PROTOCOL NO: 454/120198/ASRIT/SUB454 00928 3MLABREQUEST NO. U2723 a We ILDe LIFE INTERNATIONAL LTD. a -1- APPENDIX II. SYNTHETICSEWAGE dissolv`iTnhgethseynftohleltoiwcisngewaamgoeupnrsovoifdes the necessary nutrients required for bacterial metabolism. It is prepared by substances in 1 literofmunicipal water: 160 g peptone i 110g meat extract 30g wea 07g NaCl i 04g CaCl, 20,0 02g MgSO, 7H,0 a 23g KHPO, a Known`tRoeacgoennttariandaencyhecmoinctaalmsionrabnetttsetrhwaillarbecruesaesdownhaebnlaeveaixlpaebclte.edTthoebceonpsrteisteunentatsnodfatrhecksynnotwhentitcosbeewcaagpeaabrleenooft interfering with the study. ol a PROTOCOL NO.: 454/120198/ASRIT/SUB454 00929 3MLABREQUESTNO, U2723