Document Z8b82Qn4nOLjpwoV1rvXKOOYd

AR226 - 3254 466 tHLt2eS7G6r7:omnAelpnSaaln Cmrsow ad row Rat capricorn STUDY INFORMATION Substance Tested;(m-- Synonyms/Codes: -- i) Haskell Number: 25767 = AR2R6- 3284 oDurwon e123n02 Known Impurities: None Physical Characteristics: White liquid dbserved. Stability: The test substance appeared to be stable under the conditionsofthe study; no evidence of instability was Sponsor: E.L du Pont de Nemours and Company Wilmington, Delaware 19898 USA. Study Initiated/Completed: May 27, 2003/(see report cover page) Company Saiz. oss not contain Tac ca T EE =>T -------- oHf2t5h7e6G7r:eeInnAfllguaencSeeloennGasrtorwumhcaanpdriGcroorwntuhtaRnate Dupont12592 SUMMARY A study was conductetdo determine the effect ofH-25767onthe growth and growth rateofthe green alga Selenastrum capricornutum.* The algae were exposed to nominal concentrations of 01,1, 10, 100, and 1000 mg H-25767/Literofnutrient medium (ppm). `The algae were exposed for 72 hours (3 days) without test medium renewal. The effect was expressed as percent inhibition in growth based on healthy cell count (also referred to as cell density) relative to the blank (culture medium) control for the 72-hour (day 3) intervalofthe test. `The 72-hour ECsq, including the 95% confidence intervals (C.1.)" based on nominal concentrations are as follows:* Healthy Cell Count ECs NOEC 46.0 mg/L (95% CL =208 10714) 10mgL t'oH-a2q5u7a6t7iceoxrhigbaintiesdmmse(dRSi2u)m* caonndccelrans'siffoireadqausatdiacnghearzoaursd ufonrdethreTeSnCviAroannmdenwtasunadsesrigtnheedE"Uharmful" Directive based on a 72-hour, acute test using the green alga Selenastrun capricornutum. -- ab ATlhseoEkCn,owins daesfPirneeuddaosktihrch"nefefreicetliavescuobncceanptiraattion" producinga 50% inhibitionofgrowdh relative (0 the cSloonbt,olW.,(2002). Doseresponse modelingofconnuous endpoins. ToxicologicalScience, V66; pp 298312. 4 `Smmeathcohdks.aJn,dCelveamleunattiso,nRo,fdaMtoar.eoEcnkv,irR.o,nmaenndtRaalbTeorx,iWc.olo(g1y9a93n)d.RiAssskeAssssinegsscmcelro,gAiaSlTMhaSzTarPd u11n7d9e.r TSCA: (PWh.iGl,adeLpahnidai, 1. Hughes,and M.A.Lewis, Ed.) pp 2-39. American Societyfor Testing and Materials, ~~ Official Journal ofthe European Communities, Volume 36,4 Mey 1993,pp. 6, section 5.2.1. Se SebemowermorseToa oar ofHt2h57e6G7re:enInAfllugeancSeolennGraoswitahcaapnrdiGcroorwmtuthuRrate Dupont 12692 MATERIALS AND METHODS A TestSystem Selenastrum capricornutum, a freshwater, unicellular, non-motil, green alga, was usedin this study. `The original culture source was the DepartmentofBotany -Culture CollectionofAlgae - The Universityof Texas at Austin - Austin, Texas 78713-7640. `The culture method for Selenastrum capricornutum was based on published literature. Prior to the study, Selenastrum capricornutum cultures were maintained under the same environmental conditions used in the study. The organisms were cultured in sterilized 250 mL Erlenmeyer flasks containing approximately 50 mLoffiltered (= iter-sterilized) synthetic algal-assay procedure (AAP) nutrient medium and were aseptically transferred tofresh medium every 3 to 7 days. The flasks were fitted with foam stoppers to permit gas exchange. B. TestDesign `The test design used for the Selenastrum capricornutum study is described below: Organism Nutrient Flask Volume_ Solution Volume Medium (mL) (mL) Selenasirum capricornutum ~~AAP 250 50 Volume Ratio sil C. AAP Nutrient Medium Preparation for Selenastrum capricornutum (Appendix A) AAP nutrient medium" was prepared by adding 1 mLofcachofthe 6 macronutrient stock. Solutions and I mLofthe micronutrient stock solution to approximately 800 mLofMilli-Q (deionized) water with mixing aftr each addition. The volumeofthe medium was brought to liter with additional Milli-Q water. Appropriate proportions (1 mLofeach stock solution for each1 literof medium) were used to prepare the larger volumesofthe medium required for the study. `The medium pH was adjusted to approximately 7.5 at the timeofpreparation. The medium was filte-sterilized using Coming pre-steilized filiration systems each with 2 0.22 pm cellulose. acetate filter. The medium was stored refrigerated and allowed to equilibrate to test temperature prior to use for testing. --~ Company Santizgq, Does not contain To amy - oHf2tSh7e6G7r:eeInnAfllugeancSeeolnnGarsotwtrhcnpdriGcroonuwthiRnate Dupont12692 --_ 1 TestSolution Preparation Five nominal test concentrations (0.1, 1, 10, 100, and 1000 mg H-25767/L AAP nutrient medium) were used in this study. Aliquos ofthe flte-sterilized AAP mutrient medium were used forthe blank (normal culture medium) control solution. 2. Test Culture Preparation For each blank control and test solution, 2 aliquots (50 mL each) were placed in separate sterilized 250 mL Elenmeyer flasks fitted with sterilized foam stoppers. To achieve the desired. nominal concentrationof approximately 10,000 Selenasirum capricormutum cells/mL at test initiation, an approximate 0.455 mL (= 455 pL) aliquotofalgal inoculum from a logarithmically growing stock culture was aseptically transferred to each flask. D. Experimental Design and Test Conditions "The test flasks were placed on a shaker table in a chamber in a non-systematic design and were repositioned each working day. Air temperature in the chamber was recorded continuously with 2a rceopnltiicnatueosu.s Ttehmepearlagtaeurweerreecoirndceurb.atEeadcfhorblaapnpkrocxoinmtraotlelaynd72tehsotucrosncweintthroauttiotnetwamsedesituemdraesnewal and cell counts were made. Illumination was supplied by cool-white fluorescent tubes. The target environmental parameters are described in th following table. PIonpiutliatailon Photo Shaking (umDelunmsiinma't=ilonux) (Pheoruirosd) (Sppmeesd) Temp(eCrQa)ture 10,000 celisfmL. 6000010000 24 100 2422 E. Selenastrum capricornutum Growth Measurement Selenastrum capricornutum growth measurement was determined by visually counting the numberofcells taken from an approximate 0.2 mL. sample from cach flask at approximately 72 hours from study initiation. The counts were conducted using a hemacylometer and a. `compound microscope. An aliquotofeach sample was loaded into the two welsofthe hemacytometer and 16 grids (8 from each well) were selected for counting. All cell located in the 16 grids were counted and recordedas healthy or unhealthy. The total numberof cells counted was multiplied by 10,000 to detemnine the numberof ells pr miller. Cells outside these 16 grids were not counted nor included inthetotal number. Observations and counts of healthy and unhealthy cell (.g., deformed, scenescent, stunted) were recorded inthe study nreocmoirdnsa.l cTohnecesnttartaitsitiocnasl.calculationofthe ECso was based on mean healthy cell counts and -- CompanyPanSaniSatnitzizeedd,. Doesnotcontain sc cay oHfAt2h5e7G6r7:eenIAnlgfalSeoulenenGansriocruwemhcapnrdiGcroorwntuhtiRna.te Dupont12052 RESULTS Based on visual observations, the blank control solution and the 0.1, 1, and 10 mg/L test solutions were clear and colorless before additionofalgal cells. The 100 mg/L test solution was slightly cloudy before additionofalgal cells. The 1000 mg/L test solution was cloudy before additionofalgal cells. After the additionofalgal cells, the test and blank control flasks were placed ona shaker table in a chamber with a temperatureofapproximately 25C. The algae were incubated for 72 hours (3 days) without medium renewal. Illumination was supplied by cool`wThhietsehfalkuionrgesscpeentedtuwbaess.1T03heremveolauntiloingshtpienrtmenisniuttyein(rtphme)c.haTmhbeeprHwamsea6s9u0r8emleunmteonsf/tmh?e (c=ulltuuxr)e. medium was approximately 6.8at study start. The pH measurementsofthe test solutions at test initiation ranged from 6.80 to 7.02, and at test termination ranged from 7.47 to 7.82 (Table 1). `Countsofhealthy algal cells on da0(eyst start and day 3 (test termination) are presented in Table 2. Cells counts for the blank control increased by greater than 16X over the courseofthe study. CONCLUSIONS . `The 72-hour ECs, including the 95% confidence intervals (CL) and NOEC based on nominal `concentrations were as follows: Healthy Cell Count EC NOEC 46.0 mg/L (95% CL =208 1071.4) 10mgL 'H-25767 exhibited medium concern for aquatic hazard under TSCA and was assigned "thheaErUmfDuilrte"octaiqvueatbiacseodrgoanniasm72s-(hoRu5r2,)"a"cuatnedtecsltausssiinfgiatehsde dgarnegeenraolugsa fSoerltehneasetnrvuimrocnamperinctournmduetrum. --_ cThoentE.Cs defineda the "ffective concentration" producing @ 0% inibiton ofgrowth relative to the Cokipany Sanitzad. Doo net caotain TSCA CBI Hof-t2h5e76G7r:eeInnAfllgueanSceleonnaGsrtrouwhcaepnrdicGorrnouwtaRmate DuPont.12052 --~ REFERENCES 1. Miller, WE; Greene, J.C.; Shiroyama, T. The Selenastrum capricornutum Printz Algal AWassshaiynBgottotnl,e TDeCs,t;1U9.7S8.; EEnPvAi-r6o0n0m/e9n-t7a8l-0P1r8ot.ection Agency; U.S. Government Printing Office: 2. AmericanSociety `Conducting Static for Testingand Materials (ASTM). (1990). "Standard Guide for 96-h ToxicityTests with Microalgac in ASTM AnnualBookofStandards, E1218-90. Vol. 11.04, Philadelphia, PA. 3. Slob, W., (2002). Dose-response modelingofcontinuous endpoints. V66; pp 298-312. Toxicological Science, 4. uSnmdrecrheTkS,CJA,:Clmeemtehnotds,s Ra.n,dMeovraclouactki,onRo.,fadnatdaR.abEenrvti,rWon.me(1n9t9a3l).ToAxsisceoslsoignyagnecdoRliosgikcal hazard Assessment, ASTMSTP 1179. American Society for Testing (W.G. Landis, 1S. Hughes, and Materials, Philadelphia. and M.A. Lewis, Eds), pp 22-39. 5. Official 52.1. Journalofthe European Communities, Volume 36, 4 May 1993, pp. 68, section ~~ --_ E-- E Company -Sanz. Doesnot ria vc opy -- Sn pm emer on onthand Gow Ras i Duponm t 12992 TABLES ee Company`Sanitizeq, Does not contain TSCA oy ER 2oSH0f-t2sh5e7e6GG7rr:leeeennIenfAllgugenpancSeealoennGassrtorwuttmhcaaundpGrmriocwotehrnRapte: ricormuten__D_DuuPbo_onatt._11220_9992 TABLE 1 PH MEASUREMENTS OF TEST SOLUTIONS `Nominal Concentration (mg/L) Test Initiation (0 hours) pH Test Termination (72 hours) pH `Blank Control 680 7.62 01 688 7.82 1 693 7.78 10 697 7.74 100 7.01 7.47 1000 7.02 7.47 een --_ -_-- Company Sanit.Dossnot contain soacar Thos tH2h5e767Gr:eeInnAfllugeancSeeonlGernowahceanpdrGircowotmhaRnate -- Dupont12992 CELL COUNTTADBALTEA2 SUMMARY ConcNeonmtirnaatlion Day 0 H(ecaelltlhsy/mCLe)ll Count Day 3 He(acletllhsy/mCLe)ll Count BlRaenplkiCcoatnetr|ol Replicate 2 1010000000 33,,349200,,000000 R0e.p1limcgat/eL1 10000 Replicate 2 10000 33,,032300,,000000 RepIlimcagte 1 Replicate 2 1010000000 23,291900,000000 Re1p0limcga/tLe,1 10000 3,150,000 - Replicate 2 10000 3,640,000 R1e0pl0imcga/teL.1 10000 40,000 Replicate 2 10000 30,000 1000 mg/L, RReepplliiccaattee 2| 10000 10000 0 0 -_-- pe `Company Santized. Dass not contain TSCA 0B -_ 5 ---m ofH-t2h5e76G7r:eeInnfAllg uSeencleeonnaGsrrouwtmhcsanpdriGcroronwuhtuRmate Dupont-12992 --_ FIGURE ~ Company Sanitized.Dossnot contain TSCACBI e AD fbieeGGreeL nMAlopsSelnoansaus-- nmceprriiecoomutien -- Dourwane129s% FIGUR1 E 72-HOUR HEALTHY CELL COUNT DOSE-RESPONSE CURVE er ---- lA A | A \ \ 20: \ spn ~ . EE J | olTTTRE www -- ee w 0R 2 ttl 3.m g, o es wi . -- S| " \ | eeTed Tm p- CompanySantzed.Dos not conan 150AOBI ofHt-2h5e76G1r7e:enInAfllugeanSceeloennaGsrtorwutmhcaapnrdiGcorronwutthumRate DDuPuonwt1i299;2 APPENDICES -YY `Company Saniized. Dossnotcontain TSCA CBI S r oHf-t2h5m e76G7m r:eenInAflle ugeanSceeeloenear Gsrtorwutmhci aanpdrGire ocwoahrnRao te muten_D_ DuuPoonwt_ 1i2z69n2 APPENDIX A AAP Nutrient Medium Constituents ~~ - company Sa"jtz0d. Do" greta TSOACE! R oHf-t2h57G6r7e:enInAfllugeanSceeloennaGsrtorwutmhcaapnrdiGcroe orwnth Rate: e DDupuoontw12i05m2 --_ --~ --A --P--TAU AP NI UTRIR ENTE MEDIN UMCCT OONNSSTTM IITTUUE EENNTTSD S ~I~UM ComponenSttock SolutCioonncsentration (/1) CompoFniennaltP_rCeopnacreendtMraetdiiounm(mg/L) NMaacNrOonsutrient Stock #1 25.500 Na 11001 No a200 Macronutrient Stock #2 NaHCO; 15.000 c 24 K 0469 Macronutrient Stock #3 KaHPO, 1.044 P0186 Mg 2.904 Macronutrient Stock #4. MgSO:7H,0 14.700 r~ Macronutrient Stock #5 MgCl+6H,0 12.164 CG 12m s 1.911 CaMaCclraorn2utHr;i0ent Stock #6. 4410 Stock Solutions Component Concentration (mg/l) Micronutrient Stock HBO; MnCled HO 2ZaCly CoCle6 yO CuCl 2H,0 FNeeCslMyo+6OsH-,20HyO N&EDTA2 H,0 -_-- 185.520 415.610 3271 1.428 0012 7.260 160.000 300.000 CompoFniennaltP_reCpoanrceedntMreatdiiounm(i/L) B 32460 Mn 11537 wm Co 1570 0354 Cu Mo 0004 2878 Fe 33051 a IAnsclruedpeosrmeadginneRseifuemrefnocem2b. ot magnesium sft and magnesium chioride, - (Gump Sanitized. Doss not contin TSCA CEI Lo oHLt2fh576G7r:eeInnAfllugeancSeeloennaGsrtorwutmhcaandprGircoowrnRate --~ Dupont12992 --_ APPENDIX B 72-Hour Healthy Cell Count Statistics Po mpany Seize. Oocr contanTSCACE! -Y ll oHf2t5h7e6G7r:eeInnAfllgueancSeloennaGsrrouwtmhcaanpdrGirocwotrh nRate: Dupont12092 72-HOUR HEALTHY CELL COUNT STATISTICS MMCaahxxeiicmnkuumnfoNrNuummNbbueemrrbeoOrffoRRfeeppRssepspienriAnnGyrRoEuTSprPeO1aN5tSmEe5nt Group or Control Exact permutation Methods Recommended ATANenAsaLtlYySsfTioSsr ODofFECORNEIAOSNINGEUXTPREaEtRNaIDMENinT FULL OATA URSeTsNpGonAsLeP=HFAOLRDM-U0L%AL XZIKPE0TYRTITIISSISSJTOAPNN-CDVKAAHLERUEDERIEZEFDOSRTJAUOTPNIWCSAKTRHIDECETRREENSDT.ATISTIC SXETLPGVLNAOILTFUESRIESSAURPLE-TVSAFLOARUREETIFEOF-RORCODORORMAEWLCATTREERDDMATTTERISEVTNEDWIHYTPHOTCHOENSTIIS.NUITY CORRECTION FACTOR Jonckheere Trend Test on Dose 0 + Lowest 5 Doses thru 1000 Ppa --TLS -2.928Z0I0T2 0.0X1P0L3I58T XPTLHsiourr poses Jonckheere Trend Test on Dose 0 + Lowest 4 Doses thru 100 pes -- J TTF 2.00Z8I36T 0.0X2P6L9O5T5 XRT S=ion doses Jonckheere Trend Test on Dose 0 + Lowest 3 Doses thru 10 Ppa ar TT -0.766Z1I3T1 0.X268P23T4 XRT! Stow osei ~ -_-- -------------------- e-- GompanySaniized. Do0ssnnottconconttaina TSCACBI o1f2t5e7G6r7o:eInnAfllugeanSceeoln eGrcotwthcoanpdGiroouwtshRate: DuPont12092 ~ OBSERVED & PFROERDMIVCLTAELD FPREORMCEFNETRRC1E OL MEAN FORMULAL=Cell_Count BMC 21 0019 NW 22 34.05 mw 33.000 rome 0 sevrcremo 0 15 w1o10o.00223 aallos 3o3l1z5ps 3333095807 25g.0n099 7= o 10 "1 61000 2 00 glg0s i-s5e9 -1E0n0 Covariance MEaCtS0rixusainndg MSettahnodda=rNdEWSTrOrNo,f oOfECPDarMaomdeeter2 Setimates ES wo ses se E050 4.3a29e0s3 16l6.e707 1I2.E01E15 Hodel 2 Parameter Estimates, ECS0 and 95% Confidence Sounds v0 cso = ws 33.6006 46.0739 20.7670 71.3805 --~ Model 2 Evaluation Measures REG sSER ssn ssrom asus nGfororatmpeahladle:scr01i52p06t.i6Oo9nEsCDiMnOc9Dl9Eu.Ld3e4269thFeirfBo4Yl6l9No.wW5iR2n6g: 8226.00 0.70801 GE, --_-- & Santee.Doesntont rch