Document YrQBdg855b23Og3ZgVOMxvLY0

ARG 1336 MR 267555 REPORT ACUTE TOXICITY STUDY IN DAPHNIA MAGNA WITH T-4127 NONTOOTXOXSuPbrsotjeacntce331413346907 g =2 32832 == 38558 ~ a -Pagetor21- =) EY 7 Taz NOTOX Prac 3434s Se UBEYENOEi PCOMPNR...s ... ai NOTOX BV, ragentThoe sNetehrnian,ds Tion satmuoGstosrendtsintisnroport has ben corecty reported andvas onda in compliance TheOZ00 Pris of God LasrateyPracico hicharsseinnconaformlitywyi: Toe Uni Sates Food and DgAdmiisaon Tite 21 God of Fara Relais Prt 58. TRheegunaiteondsSFataers1E6n0.veanmena retin Agency(FIA).Tt40Cada o Fcaral nReengiuedaSPtaatone7sn6Es2vionmenil ProtectionAgency(TSCA) Tite 40 Goce of Fra SOry OrBoocieg)re // J owe g ey / M1F0aa23na.asgoefmvGeanentn:deetW.aEacrot5oxcsaony Y= ha 2 oul 2focglhont 2 T4127 (QUALITY ASSURANCESTATEMENT NOTOX B.V.,`s-Hertogenbosch, The Netherlands NOTOX Project 334349 "rTehsiuslrtespaocrctuwraastelayudfifftoecdtbtyhethreaNwOdaTtOa.X Quality Assurance Unit to ensure that the methods and TDuhreindgattehseoofn-QsuiatletiynsApsescutrioannsceprioncsepdeucrtieosnsapaplnidcaabuldeitos atrheisgyivpeenobfelsotwu,dy were inspected. DATES OF QAU INSPECTIONSIAUDITS onsite inspections) (Process) JMualyy0813--1351,, 22000022 ((EAcnaoltyotxiiccaolloSguyp)por) protocol inspection(s) (Study) January 31,2002 report audi(s) (Study) May 01,2003 REPORTING DATES JJuulyn1e70,42,0200202 Janu31a, 2r00y2 May01,2003 Head of Quality Assurance Cu. Mitchell B.Sc. Date: 12 - 5-03 3. Tat27 NOTOX Project 334349 SUMMARY Acute Toxiclty Study in Daphnia magna with T-4127. T63h4e1:st"uWdaylperroqcueadluitryes-- dDeestcerrimbiendatiinotnhiosfrtehpeoritnhwiebirteionbaosfetdheonmobtihleitIySoOfIDnatpemhantiiaonmaalgSntaanSdtarradus -- Athceutteesttoxmiectithyotdesst,anTdhivradlieddiittyiocnr,it1er9i9a6-o0f4t-h0e1.EEICn addidrietcitoinv,e t9h2e/6p9r,oPcaerdturC:esMweetrheoddsesfiogrntehed. to meet dDeatpehrnmiian"a,taionndotfheecOotEoxCiDctgyu,idPeulbilinceaNtoi.on2N0o2. PLa3r8t3,: "DDeacpehmnbiearsp1.,99A2c,uCt.e2.im"mAocbuitleisTaotxiiocnitTyesfto"r, Adopted Apri 4, 1984. TTr-i4p1h2e7nyislbaseanlztylcponhsoisspthionngioufmP(ecraftliounoircopoacrtt)a.neBsausleodnyolnmetthheyrleamsipdoens(eanoifontihce paanrito)niacndpart of the | te1s9t5s2u0b8staCn.ce water solubility of T-4127 was determined 0 be between 0.6 and 0.9 mgf at `aTnheunstfutdryesdtasrotleudtiwoint,hparelpimairtetdetateaxploosaidninggtrwaetnotyofd1a0p0hnmidgs paenrdcmoangcneenttircaatliloyn sttoirarefditfeorrefdoaurnd dthaeysf.iAtebreldanakncdonttheroulnfwiatseraeldssooilnuctliuodnesd.prAefpearre2d4athaourlsooadfinogxrpaotseuorfe a1l0l0ormgga/nibsemcsameex.posed to immobitsed. A1f0i0na%lotfesatWwaatserpeArcfcoormmemdodeaxtpoesdinFgratcwteinonty(dWaApFh)nipdrseppaerrecdoantcaentlroaatdiionng 0at4e.5o,f1100,020m,g45aanndda bwiltahnkdiccohnltorrooldfimoearthymiasxiliamneu.mAolfth4o8ughohurvsa.poAullrgplraesssswuarreewuassedreilnattihveelfyinlaolw,tebstasweadsopnret-htereHaotnerdy. cthoenssttaundtyewvaapsorpaetrifoonrmweadsinsilalira-tipgohstsviebslseeclsa.usSeamfoprldeesctraekaesne doufrtiensgt tchoencfeinntalratteisotnws.erTeheardedfeorde. directly to isopropanol at a ratio of 15 to prevent any additional loss. "nTohtebaenamlayitinctaalipnerdogartammobraestehdanon80th%e aonfitohreicInpitairatl scohnocwenetdratthiaotnt,heinasctpuiatlocfonalclepnrtercaatuitoinosncsotualkden 10 prevent adsorption to glass and voatization. Ianntdhteecmopnetrraotlunroe)morretmaaliitnyewdawsitohibnsetrhveerd.anFguersthperre,saclrlitbeesdt cboyndtihteipornosto(cpoHl,.oxygen concentration Diimlmutoibonislotofyfa Dwaapthenripahamsagenoaf.T-C4h1e2m7icoarligainnaaltyinsgisfrofomthaelaonaidoinnigc rpaatret ooff T1-0041m2g7/shionwdeucdedth1at00th%is c"oDarprhensipaomndaegdnwaitehxpinoistiealdTt-o4T1-2471c2o7ncweanstr<at1iomnsgdo(<wn0.100203.m0g2/m)glb.asHeedncoen,qtuhaent4ii8chat-iEonCooffthe anionic part and was already reached within 24 hours of exposure. Leinmsiutriendg.tThehearnoalyitiacarleasluisptpiocrptoossniboinlltyytthheaatnihoenaicctpuaarltcoofncTe-n4t1r2a7t,iovnalsidoiftythoef ctahteiotneitc cpoarutldwanortebe fhuirgthheerr arnedsemaorcrheasntdabldeectihdaendtthooaseccoefptthtehaenhiiognhilcyptaorxti.cHloawbeevlienrg,(tRhSe0)spboansseodrodnidtnhoetcaurgrreenetwith results. " T4127 NOTOX Project 334349 PREFACE Sponsor Study Monitor Testing Facilty S`AtquudaytiDciTroexcitcoorlogy: TAenaclhyntiiccaall cCoh-eomridsitnrayt:or Principal Scientist Study Plan a3MM CCeonrtpeorr,atBeuiTdoixincgo2l2o0g-y2-02 SPT..0.PBAoUxL,33M2I2N0NESOTA 55133-3220 USA 3S.MAEnBveiarocnhmental Technology and Safety Services. 9Bu3i5ldBiungsh2:A3vEe-n0u9e (StA.dPraeuls,sMtoNwh55i1c4h4.all communication is to be sen!) HNaOmTbOakXenBwVe.tering 7 T52h3e1NDetDhe'rsl-aHnedrstogenbosch IDnrgs..8M.. vBaongeWres.es Ir. MJ. Brekelmans S`StCtaoarmrtplPfierrotsjiteoecnxtp:loaJsstaurneeux:aproJysuul3ry0e,0:12,N0o02v20e0m2ber 13, 2002 `CCoommpplleettiioonn Apnroajleycsti:s:MNaoyv0e9m,b2e0r0313, 2002 TEST SUBSTANCE DIedsenctriifpitcaitoinon TDa4r1k2a7mber waxy solid D-2491 lot2 BCaotmcphosition 95 - 9292%% FNl-uMoertoheyllasPteormfelruocruoroatcitvaenesuiphonamide <<11%% EMtohtyhlyiAlAclochoohlol Test substance storage At ro<o1m%teImspoeprraoptyulreAlicnothhoeldark `Stabilty under storage conditions Expiry date Stable N31otMianrdcihca2te0d03 Stabilty in water The sponsor is responsible for al test substance data unless determined by NOTOX. PURPOSE The purpose of te toxicity test was to evaluate the influence of T-4127 on the mabilty of Daphnia magna. Tat27 NOTOX Project 334349 GUIDELINES T3h4e1:st"uWdaytperrouceadlurley.s dDeestcerrimbiendatiinotnhiosf rtehpeoritnhwibeirtieonbaofsetdheomnobtihleitIySoOf IDnatpehrnnaitaiomnaalgSntaanSdtarradus - `Acute toxicity test, Third edition, 1996-04-01. 1 addiion, the procedures were designed to meat the test methods and validy of the folowing g3Guideolipnees:an Economic Community (EEC), Geterminationof ecatoxicty, Publication ENoE.CL3di8r3e,ctDievece92m/b6e9,r P1a9r5t2,C: C.M2e.t"hAocdustefoTrotxhiecity for + OTDerasgptahinnniigzaao.tfioCnhefmoircEaclosn,ogmuiicdeCloin-eopNeor.at2i0o2n Paanrdt :De"vDealpohpnmieanstp.(,OAEcCuDt)e,IOmmEoCbiDligsuaitdieolninTeesstf'o,r Adopted April 4, 1984. ARCHIVING NfeOfTorOeXnceB.sV.amwpillleaacnhdivreawthdeatfao.loNwoindgatdaatwailfobreatwifleahsctra1w0nyeWairhso:uptrtothoecoslp,onrseopror'ts, wteistesnubstance consent. DEFINITIONS Itmesmtobvielsssealtiaorne:ctohnossiedearneidmatlosbenotimambalebiteo.swim within 15 seconds aftr gentle agitation of the Cus: the concentration estimated to immbilise 50% of the dapha afte a defined period of exposure. NC(oi.OobismnmeorbvicelidsEeaftfioennc)ttis rrecoradedt. i(NoOECnY:the highest tested concentration at which no effect TEST SYSTEM Species Reason for selection Validityof batch Characteristics Daphnia magna (Crustacea, Cladocera) (Straus, 1820) "aTchcisepstyesdtesmpehcaiess.been selected as an intemationally bDraopohdn,isdhsoowriingignantoedsifgrnosmofa shteraelstshysusctohcaks, 2morttoal5l"y >an2i0m%a,lpsraensdentcheeorefwmaaslenso,dseplhaiyppiinathoer dpirsocdoulcotuiroendof the first brood. <Fo2r4thheoutess.t selection of young daphnia with an age of " Ta127 NOTOX Project 334349 BREEDING Start of each batch pWliatchinngewa-bboourtn2a5n0imoafltsh,eim.inlteoss10thlaines3 dofaymsedoilud.rnby.in an all-glass culture vessel. Maximum age of the cultures. Renewal of the cultures. weeks wAfeteekr.7 days of cultivation half of the medium twice a Temperature of medium 18:22C, constant within = 1C. Feeding Daily, a suspension of fresh water algae. Medium MP.7,,1a9s90p:reSseclreinbieudmbydeDfri.ciEelnecnydiin-CSrcuhsnteaicdeear.(AEnlen, B.- uDlatprahsntiruactmuaraglnaappStrroaaucsh. tPoroanttoepnlnaaslmada1m54a,g2e5-i3n3). `SvCe loomwipnogocfsmheiadmtiicuiamsoMaInn:alytical rNaadceo)saaetsis(smlonv)e.d in1 Cwaat,erHpuOre by r2e8ve3r8se amosis (THRO): NMaoksoh,lyMO 1s2i3s3 Taco sements (mo: K8at ossx Fo 00s Mo GRbangs o0o0z12ss Ho 8 0006s oo0zs ou 00c0o8te3 Cseoat oooo0o1zs0 vNagorazio 0205003 Maconutter (raf). NNaa.NSOO, HO 1o00z . XK2P0O,, ose oe earn on) Tr5asrine 00 Bioin 01s Toeharcness: 250mg! expressed GACOgandthapH 80 0ater2 aan. 7. Tar27 NOTOX Project 334349 REFERENCE SUBSTANCE. "This report includes the results ofa reference test with potassium dichromate. PREPARATION OF TEST SOLUTIONS. TCvohpmaeprlseetitaoennldsya.rddiTsthseeostltvepesrdtoitcneogdsoufsrucebossncrteeaqnnutcireraetcdioongncesennettrhraaatttidioionsntsoufrobtretsshttasbtolelesuttaisnoyndss,theowmhmioscghheosnuhooluubedlsd pmciroxentvtuaerinentsedof(e.. fim of the test substance on the water surface). TTTro4pn1h2bo7nsytilsabanesncasleytlwcpoahnorssispsthoilonunbgiilouifmtyP(eocrfaltTui-oon4ri1oc2op7catwra)an.essBduaelstfeeodrnmyoiinmneettdhhetyolraebmseipdboeents(weaenoiefonnti0hc.e6paaanrni)doia0cn.d9pamrgtfolfatthe 10.5 = 0.6C (NOTOX Project 340842, using the column elution method). LIinmeHiotooatTnkehistoserolmiuintxiatounmreweaawssausrpirtenhpgeanfrlemaadsgkbn1yetlwiiecrieaglohlfiytntegsit1em0d0edmfiogumfoofuwTra-ds4a1yas2d.7deoAdnfetarogtolhbaelsassitnsruiarnflgaocapede.irnAigofdrrattnepeloafci1n0g0 ItaSnnusgihrieoPoahwretarsofactnlhdeearSmcibhxututuscrloen6wt0aa4is)netuodsfetedesmtaossvusebutschthanwlhcaierlgepeararntuconitdeihsse,srowplhaviretcdhwtaseesstttfslieudbrsaettdatnthcheerbopauatgtrhotamieposfapt(echaer., fi>ter {Sum). The final test solution was clear and colourless. F`fAoiltntaglsautsebasstwtaarneceusdeudeoitnnoatahdlesafosirnspalisotuenrsftatcowega.lsaAsfpstreesrturrpfleaaaccteiesnd.g wtAihteshtcdoaicrcrkhieslroolriuontdiaiomnmeewtaahsysulrspiiranengpeafrl0easdpkrb5eyviewrneotigolhfoistnsegsotf 5o F0o0mmwgapsoewfrapiTrso-ed4a,p1adt2rdh7oeeddsoitnlouttohibeotnasiapnrmeaeplaworaaedydinbaguttraawtlieotahodofiunt1g0a0dradmtioenlgo.ftSe1si0tm0uslmutgbas/nteawonuacssleyt(,rbalanansnofket-rhcreoenrdtre0la)3.siAfntegr the SSFeirparacictreniangottniroa(ntWifAounPns)newwleaasrndsperlpeeatprataroteesddtabfbyrioidsmiettifhooernc2eo%nfiirheoeuorWfsA.tFhS.eusbAeslpleaqfriuaneatnlitotlensytfutsnhoneleuWlt.ailLoenosrwewArecrtceeoscmtlmeoardaatnedd Foones. The test sohions originated from those used for the acute fish toxicy test (NOTOX Project 334338). LMT TEST APfliimoitretedstsowlaustiopnerpfroerpmaerdedexaptoasilnogadtiwnegnftaytedaopfh1n0i0dsmgp/elr acnondcaenbtlraantki-ocnonttoroal.fiNteoresdamapnldeasnwere taken during the mil test FINAL TEST: `TEST CONCENTRATIONS Ta127 4lo5a,d1i0n,g r2a0t,e4o5fa1n0d0 m1ol.0of0aW%AF prepared ata .8- T4127 NOTOX Project 334349 Control (TBelsatnkm-ecdointurmo)w.ithout test substance or other additives TEST PROCEDURE AND CONDITIONS Testtype static Test duration Tost vessels. 48 hours d1i0c0himoir,odaillm-egtlhayslss.iPlraen-et.reated with `oAnltthhoeugHhenvraypocuornpsrteasnstuerveapwoarastireolnatwiaveslysliolwa, bpaossseiodle. tchaeussetufdoyr wdeacsrpeearsfeoorfmteedstincoainrc-etnitghrtatvieossnesl.sT.herefore Medium 1S0, prepared in mill-RO water Number of daphnia 20 per concentration Loading 10 per vessel containing 80 mi medium Light 16 hours photoperiod daily Feeding No feeding Aeration No aeration of the test solutions. Introduction of daphnia Within 2 hours afte preparation of the test solutions. `SAMPLING FOR ANALYSIS OF TEST CONCENTRATIONS sDpuepcliifciactaetisoanmopfltehse woreirgeintoafkesnamfprloemsthteaksetnocdkursionlguttihoen tdeisrte.ctDluyraifntgertphreefpianraalttieosnt doufptlhiecaWtAe F as a tShaemp4.5laesnwdor1e0t%akdeilnutfiroonms aalfltecron2c4enhtoruartsioonfseaxnpodstuhreebalnandk-fcroonmtrtohlea4t.t5h%e dstialruttioofntahtetehsete,nfdroofm the test period. The method of analysis is described in the appended Analytical Report. Sampling:TVroelautmmeent Storage `5Smaimpflreosmwtehreeapapdrdoexdimdiarteectcleynttoreisoofptrhoepatneoslt vaetsaserlast.io oNfot1:a5pptloipcrabelvee,ntsaamnpylaedsdiwteiornealanlaoslsy.sed on the day of sampiing. MEASUREMENTS AND RECORDINGS immobility (including mortality) At24 hours and 48 hours. pH and dissolved oxygen cAtontcheentbreagtiinonnisnganadndthaetctohnetreoln(ds).of the test, for all Cosine [Vesa |Mabe Respro2h |responiashi| reeee TTEETrET E= rs The results of measurement of pH and oxygen concentrations (mg#) are presented in Table 2. -- [oe 9 [Powe] Einaltest: rE La i Serr------------ Taz H m eo= reI mt b:eH O E E3 Lee e bE: m=H r el ekr3eer (100mg) Vewsor | Nomoor | Responsesiaih |Responsesiaoh | exposed % rem n. ison [w |wo o] Tan27 NOTOX Project 334349 ACCEPTABILITY OF THE TEST 1. Inthe controls, not more than 10% of the daphnids became immobilised or trapped at the 2. sAlulrtfeasctecoofndtihteiwoantse(rp.H, oxygen concentration and temperature) remained within the 3. rTahnegaensalpyrteisccarlipbredogbryamthbeapsreotdocoonl.the anionic part showed that the actual concentrations cporuelcdauntoitonbsetmaakiennttaoinperdevaetnmtoardesotrhpatnio8n0t%ooglfatshseaninditivaollcatoinlcieznattiroantion, in spite of all CONCLUSION fUrnodmeratlhoeadcionngdirtaitoenosfo1f0t0hemgp/rlesienndtuacceudt1a0t0es%ts,imdmiolbuitliiotnys ooff aDawpahtneirapmhaagsneao.f CTh-e4m1i2c7aolriagnianlaytsiinsg Gofotwhne1a0ni0o.n0i2cmpgar.t oHfeTn-c4e1,2t7hseh4o8whe-EdCtshoatofthDisapchonrrieasmpaongdneadewxipthosineidtiatloTT4-1412277cwonacsen<tr1amtgi/olns h(<ou0r.s02o3f emxgpo)subrae.sed on quantification of the anionic part and was already reached within 24 eLinmsiutriendg.tThehaenralyistiacarleasluisptpiocrptososniboinlltyytthheatantihoenaicctupaarltcoofncTe-n4t1r2a7t,iovanlsidoiftythoef ctahteitoensitc cpoaurtldwenrotebe. fhuirgthheerr arnedsemaorcrhe asntdabdleectihdaendtthooaseccoefptthtehaenhiiognhiicyptaortx.icHloawbeevleinrg, (tRh5e0s)pboanssoerd doindtnhoet caugrrreenetwith resus. 2. Tater NOTOX Projct 33434 REFERENCE TEST mm] [ome SEtnaart:: NNoovveemmbbeerr004,,22000022 48hour Acute Toxicty Study in Daphnia magna wih K.CriOy NOTOX Prjec 356669). nB Seostae udyDparnoncaedsue raep.s Adec0su5ct1r5ei5b.IedmPoiabriitssa0.2rtoAopoonrutTtweeorsexAbdyaospeftdoerodDnA1parp14,501a9In8nd4e.rhaetiOoEnaClDStgauinddealridne TTh roefoarenEe ceEtosivwasas caxapr5adsmeoldaluftao0nrdc0mh.aecskibmtaunemkscoefonn4sr8lv.otuyTreosfnfhcoeaKpthertiOasywsectroeenmceaexsnptouassteeoddnpbsyaof 0.10, Concontaion. as"The raefberaenncefsoubmstMaenrccek,, pDoatramsastiaudm,dGiechrrmoamnayt.e (K:Cr:Oy, art. 4864, batch no. K28974764) AGceutnooimmmeo,biizationofdaphnia ato 2 and48 hours in he reference est wih potassium FP[ oKoarmn:e(mgh)| expos[ ed w T 0[ [oa r o [o |eW_ oman after 41|8 ho owu 0 ras*ar||| Toows [ww w |o [wm _ o [[o0 oo [[wwmm || EEoe n aT ntto tdpoeex oRnw0po0 581 5 ee [ww] epeeetsitnsaitesosrgopoapa sTmHs sSt5on0ot ir.Tes hp vas red TT ahE actaue aalrooasmpoonntrsweeishnnttinsctoonrrcfoiencrraaelinoGcnasts.atcHowotinehceKt.hoaCddtrsNOOeTanOoXsi.iionf thies rbaangiesofofDatpehnoixapemcaiegdna he 24n-EC,0 was 0.93 mo! wih 85% dual iis of 0.82 ~1.2 moh. himemo4b81i-yEaCis1w0asmos1.timalod 0 bo 0.63 mol wih 20% immobilty at 0.5 mg and 100% hTeeperostocoalh,orcaawwdaastapaonndoampoodrunofdenrisGLsPtycoanrdsoKnepstwnihea NQAO-TcOheXcakrrives. The est i RB ANALYTICAL REPORT ACUTE TOXICITY STUDY IN DAPHNIA MAGNA WITH T4127; DETERMINATION OF THE CONCENTRATIONS NONTOOTXOXSuPbrsotjeacntce331413346907 -Page 10f8- rar ---- TRE (Analytical Chemistry) it oe Praogt 19 T4127 NOTOX Project 334349 PREFACE Analytical study Start: Completed: 1113 NNoovveemmbbeerr 22000022 PURPOSE The purpose of the analyical study was to determine the test concentrations and to validate the `analytical method used. REAGENTS Mill-Q water Methanol 1S0-medium Ammonium acetate 2.0 mM ammonium acetate Isopropanol (2-propanci) Isopropanol containing internal standard `sTuabpsweaqtueerntpluyripfaiesdsebyd roevveerrascetdivoastmedosciasrbaonndand ionexchange cartridges; Millipore, Bedford, MA, USA P.a., Merck, Darmstadt, Germany see main report Fractopur, Merck, Darmstadt, Germany 154 mg ammonium acetate in1 live of Mll-Q water p.a., Merck, Darmstadt, Germany s1u4l0foynlaotfeainsomleutthiaonnoolf f2i8l1ed6umpg/tlop2e0r0f0iumoirwoioctthane isopropanol. INTERNAL STANDARD. NIademnteification number DeBsactrcihpntiuomnber PuErxipitryy Date CeSrttoirfaigeedconditions Supplier APeSr5f1u7orooctane sulfonate (FC-95) LWhoitt2e17solid (determined at NOTOX) 9210.F4e9b%ruary 2003 (determined at NOTOX) YAtesroom temperature in the dark M The sponsor i responsible for al test substance data unless determined by NOTOX. `SAMPLE PRETREATMENT PROCEDURE iEsaocphrospaamnpolleco(n5tamii)niwnagsinttreamnaslfesrtraenddqauradntwitaastiavedldyednt(oinate5r0nalmistvaolnudmaertdrciocnfcleanstkraatnidon2516m4i goff). T4127 NOTOX Project 334349 ANALYTICAL METHOD eT7.ov4pe1h2eo7nnyiyib2oeasnuaslatydicoponnnsoitssnpteihnaagnniooofnmiPc(ecrpaaftriuotonroifocoTac4rtt1a)n2e7UspuufosoinnnygrieHmqiuegethshtyPoiefarmftihoderemsa(panoncniesoonLrii,cquqpiuadarnC)ihlaranotdmivaeloagnraalpysheys wih Mass Spectrometric detection (LCMSMS). Anaivical conditions Coumn KBeetyasstionCe1)8,50 x 20mm; &=pm (Thermo Hypersi, MCoolbuimnptheamspeerature CFloolwumn temperature `IAnjdetcotsiaonmpvloelrumtee,mperature Detection Interface Monitored masses 25C (minutes 25 Amo%rim ace@te C CC eosT T w 5 w h | 11&]|| [Cr eoo| T s w w s [ || 1 0||| 32050Camin aS1C0cI4EX MSS system APL.300 mass spectrometer (oTApuperprblaoitefedodnBisinpornsaeyygsatatetim4vs5e,0fTooCnr:omnNotgdoef,.loCwanraatdeao)f 7000 mifmin. VMIRRM tiensttersnualbssttaanncdearmdizmz5124.919.=0>-->169898.0 `Standard and caibration soutons Standard soluoftT4i12o7nwerse prepared in methanol. Oenrmeaalchsdtaanydoafrdan(anltyseirsa,lcasltiabnrdaatirodncsoonlcuetnitornastiinon1516(44u)gI)SwO-emreedmiuamdieisuopprforpoamntawlocsonttaanidnairndg solutions. VALIDATION OF THE ANALYTICAL METHOD. fTohqeueLsGtMofStMhSe smpeotnhsoord,uvsaiedawlisonownayspnaorltcyovmaplliedtaetde.d curing NOTOX project 334338. Upon T4127 NOTOX Project 334349 DATA HANDLING -- SAMPLE ANALYSIS Calibration Response (R): RecPerakaarneaetesstrsubostanmce s Peak area internal standard Calibration curve: tTehsetsruebssptoannscee,waussincogrlrienleaatredrewgirtehsstihoencaonnacleynstirsat(iloenasotf fsaqcutoarr.es method) and a (1/concentration) weighting `Samples R = asC+d R = = croenscpeonntsreatciaolnibirnattihoen csaolliubtriaotnion solution [mg] ba == sinitoepreclepitmg] uOsnedeafcorh qduaayntoiffiacnaatliyosn.isB,ottwhoccaalliibbrraattiioonnssoolluuttiioonnsswwereer:e siinxjescatmepdl(eisn.duUpsliicnagtet)hebeffoourrereasnpdonasfteesr,aamcaalxiibrmautimonof curve was constructed. Concentration analysed: Recoveryof recoverysamples: C=de2R2-b2 (mon) R C = = rcoenscpeonntsreatciaolnibirnattihoencsaoilburtaitoinon a = ssoiloupteiolnim[gm]g] b= intercept Concentration analysed Concentration prepareede 100%] Concentration relative to nominal Concentration analysed ~------------------- 100 [%] Concentration nominal T4127 NOTOX Project 334349 RESULTS -VALIDATION OF THE ANALYTICAL METHOD See NOTOX project 334338 *96-hour acute toxicity study in zebra-fsh with T4127 (static). RESULTS - SAMPLE ANALYSIS Table 1 shows the analytical results for the samples from the ecotoxicity study'. Table 1 ConcentrationsofT4127 in test medium (Full test 1) sTaimmpeinofg | Dsaamtpelionfg | aDnaatleysoifs [Percentage| Expected| Analysed'| Reaive hours] | [d-mmeyy) | (ddmmyy)| offit1cate' | (moll | [mel expetcoted 0? 11-Nov-02| 11-Nov-02| 100 07170 100 0750 0? 11Nov02| 11-Nov-02| 110000 007766 007556 7 8 4P5y 003344 002240 7590 20 20 00.1155 00.1163 1a073 10 10 0076 | 0076 | 0020 | 0.064 38 8 45 003 | 0021 61 405 000 3 | 002% | nd. 69 na 0 0 ne. na 2 12Nov-02| 12:Nov-02 10 10 00007766 || 0000227 | 326 45 45 00003344| | <<LLooDD -- 8 13Nov-02| 13Now02 | 4455 0000334 || <<LLooDD -- 1 BPaesrecdenotangheoefmeSasuumredceornecdesnttaoonnp(r.e.pa0r7ed60amnoom)inianltceounncdeinutlaetdonvoafte1w0i0mtoouht kas. * M<e2a0n%ouflveipskcattheoarnwailsyasmsg.iTchaotemdaimum deviationbetweentheresponsescalculatedfor eachsamplewas. * CCoamlbcilnaeeddwbiythexNtrOaTpoOlaXtiporno.jeRcets3p3o4n2s3e8s. wero 570%of he lowest caibrston ove, 13. MNoatxdiemtuecmtedde.viTathioen biestowfedeenterctieon s(LpODwo)awsna2s3s0%.ea02ns3dmf27.%, repoctvel na. Not applicable. *WAhleetnacalcuvaatlnugeswheerreacatuesaalksusGiangnngotthouConndceandicroantcoennstara8tmioennst,iTohneerdelire5, soanmee.iforencesmightbe serve T4127 Er Bl=l) STD EBmirael hee NOTOX Project 334349 Er E BriE n nm = : standard | EnE m | i :3 ||| I22 f iz3 | 3z: | 3 Figure 1 HPLC-chvomatogramof ablank (1/5 (v/v) ISO-mediumyisopropanol containing internal standard). twOr h prBEE: | | Ei il EBiE sTeosmtstance Ei: : a7 i3 aIntnernacl ||| i il | Io | | {| Figur2e cHoPnLtCacihnirnogmiantteorngarlasmtanodfard0..500 mg T4127 in 115 (vv) ISO-mediumisopropancl Tatzr NOTOX Project 334349 ~~ I 7 :| ~~ I pd Fes RHegarenssadniiine.forRseauponnsesiag1ain(st4)conScOe-nmtreadtiomrsi.sopropanol containing