Document p2MMV7a3L7VojXDweRM0xwV7E

AR22C = 327] " -_-- --~ TRADE SECRET #Razg -- 327/ DoDuwPonnte11604 Study Tite H-25435: InflAulegnaceSoenleGnraoswtrtuhmacnadprGircoowrtnhuRtaumteofthe Green Laboratory Project ID: DuPont-11604 TEST GUIDELINES: OSeEctCiDonG2u:idEeflfiencetsfoonr TBeisottiincgSCyhsetmeimcs,alNsumber 201 (1984) ~~ Commission Directive 92/69/EEC EEC Method C3 (1992) AUTHOR: TerryLee Sloman, B.S. STUDY COMPLETED ON: March 31, 2003 PERFORMING LABORATORY: HEa.LskdelulPLoanbtodreatNoermyofuorrsHeaanldthCaonmdpEannvyironmental Sciences NEelkwtaornk,RoDaedl,aPw.aOr.e B1o9x71540-0050 work Request Nuveen: (IN service Cope Numer: (IR ~ TT Farias -- Company Sanitized. Does not contain TSCA Cal 1T 425035: nE onGn row and men _ -- - Duron 11604 `GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT `This study was conducted in compliance with U.S. EPA TSCA (40 CFR part 792) Good Laboratory Practice Standards, which are consistent with the OECD Principles of Good Laboratory Practice (as revised in 1997) published in ENV/MC/CHEM(98)17 and MAFF Japan Good Laboratory Practice Standards (59 NohSan Number 3850) except for the item documented below. The item listed does not impact the validityofthe study. The test substance is commercially available and was characterized by the supplier prior to the initiation ofthis study. An analysis was provided, although the characterization was not performed under Good Laboratory Practice Standards, based on our knowledge of the performing laboratory and its experience with analyzing this chemical, we. have no reasonto doubt the validityofthe analysis. Applican/t Sponsor: E.L du Pont de Nemours and Company Wilmington, Delaware 19898 ~ USA. StudyDirector: Ta Terry Lec Sloman, B.S. Associate Scientist 3)-mAR-2003 Date Applican/t Sponsor: DuPont Representative Date -- eeeee-- Compan-- ySantzsd-- , Doesot-- cottSta-- eGAiOnBI 1G2r5o4w3s5:te oetnhcee GonrGerlogwtaSnhldonasrm capricorn QUALITY ASSURANCE STATEMENT Haskell Sample Number(s): asus Datesof Inspections Protocol: January7, 2003 Conduct: January 7, 2003 Records, Reports: March 17, 19, 20, 2003 Dates Findings Reported to: Study Director: March 20, 2003 --_ Management: March31, 2003 Duon11608 Reporedby: Meuse Gurney Denise M.Janney `Quality Assurance Auditor 3PM 2203 Date. _-- CompanySaitize. oesnot contain TSCA Cal G1r12o5w4t3h5R:aIte fhle oGnurGernnoAwlcgaSseenldenacricontu _ Duron 11604 CERTIFICATION We, the undersigned, declare that this report provides an accurate evaluationofdata obtained from this study. AnataRepriedby: GnZ "Bogdan Saosick, PhD. `SeniorResearch Chemist bh "dap Date Statistical Reportedby: "JohnW. Green,Ph.D., Pa.D. Sltre-2003 Date. ~ `Principal Research Statistician usdby SyDictor: LL ca Seni `TearyLeeSloman, B.S. 2l-mAk-2003 Date. _-- Company Saniizsd. Dossnot contTSaCAiGanl 1G.r2o5w43h5:RaItnefolufetnhceeGorneGenroAwltghaaSnedlenastrum capricornutun --_ TABLE OF CONTENTS DuPont-11604 Page GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT snr? QUALITY ASSURANCE STATEMENT worse CERTIFICATION css LIST OF TABLES cssssssssmsssssssmsssssmssssssnon LIST OF FIGURES crc LIST OF APPENDICES rms STUDY INFORMATION sss] STUDY PERSONNEL cms) FE INTRODUCTION css0 MATERIALS AND METHODS cocoons10 A. TECHIE consmmmmmmmmmmmmmmmi--------0 ~ B. TeSt SUBSIANCE rss 10 SS |} F. EXDETMENtal DESIg...rrnrnrsssssssssssssssss | 2 Gi. TEStSOMOS rss | 2 H. Test Condi-tDiefionintisve and RECOVET TeStS |2 1. Selenastrum capricornutum Growth Measurement... SS K. AnalysisofTest Solutions - DEfIVe Test... 14 RESULTS AND DISCUSSION crn19 A. Environmental Conditions - Definitive and RECOVery TESS rrr 19 B. Test Substance Stability and Analytical Verification... 19 C.. Selenastrum Capricornutum GROWN... 20 CONCLUSIONS css2 RECORDS AND SAMPLE STORAGE corms21 REFERENCES cms 32 BN ------ J ~~ J ------| `Company Sanitzed. Does not contain TSCA C81 HGr2o5w4t3h5:RtIenfloufetnhceeGorneGernoAwltghaaSnedienasirum capricornutu Dupont 11604 --~ LIST OF TABLES Page 1. pH MEASUREMENTS OF TEST SOLUTIONS... 2. CHAMBER LIGHT INTENSITY, SHAKING SPEED, AND TEMPERATURE RANGE 3. MEASURED CONCENTRATIONS OF H-25435 IN TEST SOLUTIONS... 4. HEALTHY CELL COUNT DATA SUMMARY - DEFINITIVE TEST. 5. AREA UNDER THE GROWTH CURVE DATA SUMMARY - DEFINITIVE TEST .. 6. GROWTH RATE DATA SUMMARY-DEFINITIVE TEST... 7. 72:HOUR (DAY 3) STATISTICAL ANALYSES SUMMARY - DEFINITIVE TEST... -- aomnilth nil mal I" 30 31 LIST OF FIGURES Page 1. REPRESENTATIVE ANALYTICAL CALIBRATION STANDARD CURVE FOR H-25435: gE NE A, -3 --~ 2. REPRESENTATIVE CHROMATOGRAM OF A CALIBRATION STANDARD SOLUTION... 35 3. REPRESENTATIVE CHROMATOGRAM OF THE BLANK CONTROL SOLUTION... wi? 4. REPRESENTATIVE CHROMATOGRAM OF A TEST CONCENTRATION SOLUTION... 30 LIST OF APPENDICES Page A AAP NUTRIENT MEDIUM CONSTITUENTS... smmm-- B. 72-HOUR HEALTHY CELL COUNT STATISTICS FOR THE DEFINITIVE TEST... dd C. 72-HOUR AREA UNDER THE GROWTH CURVE STATISTICS FOR THE DEFINITIVE TEST..........48 D. 72-HOUR GROWTH RATE STATISTICS FOR THE DEFINITIVE TEST... 53 Ee Company Sanitized. Does not contain TSCA CBI 1G2r5o4w35FaItnefolfutehneceGarnoGenroAwlthsnSdlnasru cprcornuun STUDY INFORMATION Substance Tested: JN Smonyms/Codes: {prNzeNrrN Haskell Number: 25435 = "J Dupont 1604 Known Impurities: {NN `Physical Characteristics: Amber brown liquid Stability: cTohnedittesitonssuobfstthaencestaupdpye.ared to be stable under the Sponsor: WE.il.midnugPtoonnt, dDeelNaewmaoruer1s9a8n9d8 Company USA. Study Initiated/Completed: January 6, 2003 / (see report cover page) -- ----------e-- e-- ee-- ec-- ts CompanySarita. Doesnot contTSaOAiGonll G1r.2o5w4i3h5:RaItneoflfutenhceeGorneGenroAwltghaaSnedlenasrun capricormitm. --_ STUDY PERSONNEL Study Director: Terry Lee Sloman, B.S. Management: Robert A. Hoke, Ph.D. Primary Technician: Barbra D. Ferrell, B.S. Analytical Chemist: Bogdan Szostek, Ph.D. Management: S. Mark Kennedy, Ph.D. Statistical Analysis: John W. Green, Ph.D. Ph.D. Management: Janice L. Connell, M.S., B.A, CLH. Ecotoxicology Report Preparation: Wanda F. Dinbokowitz Management: Nancy S. Selzer, M.S. DuPont 11604 I `Company Sanitized. Does not contain TSCA CBI H`G2r5o4w3th5:RaItneoflfutenhceeoGnreGenroAwltghaaSnedlenasirum capricornutum DuPont 11604 ~~ SUMMARY AgresetnudaylgwaasSecloenndauscttreudm tcoapdreitceromrinnuetutmh.e eTffheectalogfaHe-w2e5r4e3e5xopnostehde gtoronwotmhinaanldcgornocwetnthrraattieoonfstohfe 6.25,12.5, 25, 50, and 100 mg H-254353liter of nutrient medium (ppm). tFeosrt tmheeddieufimnirteinveew(adlo.seT-hreesepfofnescet)wtaesst,etxhpereosrsgeadnaisspmesrcweenrte einxhpiobsietidonfoirn7g2rohwoturhsb(a3seddayosn)hweiatlhtohuyt cell count (cell density), area under the growth curve, and growth rate relative to the blank. (normal culture medium) control for the 72-hour (day 3) intervalofthe test. `The 72-hour results" based on nominal concentrations are as follows: Healthy Cell Count ECNyOoEC Area Under the Growth Curve ECyo NOEC > 100 mg H-25435/L > 100 mg H-25435/L > 100 mg H-25435/L > 100 mg H254351L Growth Rate ~ ECs NOEC > 100 mg H-25435L. > 100 mg H-25435/L. Noneofthe definitive test concentrations exhibited a 50% or greater growth inhibition based on healthy cell count relative to the blank control. Therefore,a recovery test was not performed. ab TAhlseoEkCnsownadsefPisneedudsosktihrec"hefnfeercitilvleacsounbcecnatpriataion" producing a 50% inhibition ofgrowh relative fo the -- ecfofnetcrtolonTthheemNeOasEuCreids dpaerfaimneetearstrheleathiivgehe{s0tthconccoenntrtorla.tionoftet subsiance hat has no satsicaly significant FEZ Company Sanitized. Doesnot contain TSCA Cal 1G.r2o5w4t3h5:RaItenoftfheloGnrueGerenowAnltghcaaSneedlenasirum capricormutun. Dupont 11604 --_ INTRODUCTION `This study was conducted to determine the effectofH-25435 on the growth and growth rate of the green alga Selenastrum capricornutum.* MATERIALS AND METHODS A. Test Guidelines `The study design complies with the following test guidelines: + Organisation for Economic Co-Operation and Development (OECD) (1984). 201 Effects on Biotic Systems. Guidelinefor Testingof Chemicals. + European Economic Communities (EEC) (1992). Directive 92/69/EEC Annex V. Part C23, Algal Inhibition Test. Methodsfor the DeterminationofToxicity. B. Test Substance ~~ The test substance, H-25435, was supplied by the sponsor as an amber brown liquid. It was. received October 17, 2002; the expiration date is April 17, 2003. C. TestSystem Selenastrum capricornutum, a freshwater, unicellular, non-motile, green alga, was used in this study. 1. Original Culture Source "The original culture source was the Departmentof Botany - Culture CollectionofAlga-e The Universityof Texas at Austin- Austin, Texas 78713-7640. 2. Culture Maintenance Ttohethceuslttuudrye,mSeetlheondasftorruSmeclaepnraisctorrunmuctaupmriccuolrtnuruetsiiwmewreasmabiansteadionendpuunbdliesrhtehdelsiatmeeraetnuvrier?.onmPerniotral conditions used in the study. Theorganisms were cultured in sterilized 250 mL Erlenmeyer flasks containing approximately 50 mLoffiltered (= filter-sterilized) AAP nutrient medium and were aseptically transferred to fresh medium every 3 to 7 days. The flasks were fitted with foam Stoppers to permit gas exchange. ~ + Also knownasPseudokirchnerillasubcapiata. - Company Saniized.DoesnotconTtSaCAiCnBI G1r2o5w4t3h5:RatIneoflfutehnceeGorneGenroAwltghaaSnedlenasirum capricornutum Dupont 11604 --_ D. Test Design . "The test design used for the Selenastrum capricornutum definitive and recovery tests is described below: Organism Nutrient Flask Volume Solution Volume Volume Medium (mL) (mL) Ratio Selenastrum capricornutum~~ AAP 250 50 si E. Test Preparation (Appendix A) 1 AAP Nutrient Medium Preparation for Selenastrum capricornutum AAP nutrient medium was prepared by adding 1 mLofeachofthe 6 macronutrient stock (sodleuitoinoinzseda)ndwa1temr,Lwoiftthhemimxiicnrgonaufterriecnatchstaodcdkitsioonl.utiTohnetovaoplpurmoexiomftahteelyme8d00iummLwoafsMbirloluig-hQt"fo liter with additional Milli-Q" water. Appropriate proportions (1 mLofeach stock solution for cach1 lier ofmedium) were used to prepare the larger volumes ofthe medium required for the definitive and recovery tests. "The medium pH wasadjustedto 7.46 with 0.1N hydrochloric acid. For both the definitive and ~ rceaccohvweirtyhteast0s.,2t2heummecdeilluumlowsaesacfeitlattere-sfitletreirl.izTedheuscionngtaCionmerisnwgithprteh-estreersiullitziendgfiflitlrtaetri-osntesryilsitzeemds AAP nutrient medium for use in the definitive and recovery tests were stored in the refrigerator inthe dark at approximately 4C and acclimated to room temperature prior o use. Any remaining unused medium was properly stored. 2. Test Solution Preparation Prior to the initiation ofthe definitive test, method development was conducted to determine the appropriate concentrations to be used in the definitive test. A primary stock solution was prepared by dissolving the test substance in 1000 mL filter`SAtewroilrikziedngAsAtPocnkustorlieunttiomnewdaisupmrfeopraraendobmyindaillutcionngcetnhterpartiiomnaroyfs1t0o0ckmsgolHu-t2io5n4w3i5t/hLf(i=lte1r0-0 ppm). Sterilized AAP nutrient medium to make a nominal concentration of 100 mg H-25435/L (= 100 ppm). Aliquotsofthe filter-sterilized AAP nutrient medium were used for the blank (normal culture medium) control solution. Test solutions were prepared using aliquotsofthe `nominal 100 mg/L working stock solution and diluting with filer-sterilized AAP nutrient `mediumtomake nominal concentrationsof 6.25, 12.5, 25, and 50 mg H-25435/L.. Aliquots of the nominal 100 mg/L working stock solution were used for the 100 mg H-25435/L test concentration solution and the abiotic (stability) control solution. -- EE `Company Sanitized. Does not containTSCA G8 HG-r2o5w4t3h5:RaItnefloufetnhceeGorneGernoAwltghaaSnedlenasirum capricornutun DuPont. 11604 -- 3. Test Culture Preparation For each definitive control and test solution, 3 aliquots (50 mL each) were placed in separate sterilized 250 mL Erlenmeyer flasks fitted with sterilized foam stoppers. Each flask, excluding the abiotic control, was randomly assigned a number to eliminate bias while counting. To achieve the desired nominal concentrationof approximately 10,000 Selenastrum capricornutum cells/mL at test initiation,an approximate 0.370 mL (= 370 iL) aliquotofalgal inoculum from a logarithmically growing stock culture was aseptically transferred to each flask, except the abiotic control, F. Experimental Design 1. Definitive Test `The organisms were exposed for 72 hours (3 days), without test medium renewal. Test flasks were arranged on a lightedshelfin a chamber in a non-systematic design and were re-positioned each working day. Each blank control, test concentration, and abiotic control was tested as 3 replicates. Cell counts were made approximately 24, 48, and 72 hours after the definitive test initiation (0-hour or day 0). 2. Recovery Test A Noneofthe definitive test concentrations exhibited a 50% or greater growth inhibition based on healthy cell count relative to the blank control. Therefore, a recovery test was not performed. G. Test Solutions Solutions used in the definitive test were as follows: Blank Control AAP nutrient medium containing no H-25435 Treatment nominal 6.25, 12.5, 25, 50, and 100 mg H-25435/lter nutrient medium Abiotic Control nominal 100 mg H-25435/liter nutrient medium; (no Selenastrum capricornutum) H. Test Conditions -Definitive and Recovery Tests The control and test flasks were placed in a chamber (Hotpack model 06084) and air temperature. in the chamber was recorded continuously with a continuous temperature recorder (Dickson Model SLA45C?7). The algae were incubated for 72 hours without test medium renewal for the definitive test. Illumination was supplied by cool-white fluorescent tubes. The target environmental parameters are described in the following table. -= Company Sanitized.Doesnotcontain TSCA CBI `FG-r2o5w4t3h5:RaIteonftfheloGnrueGeernoAnwlighcaaSeneldenastrun capricorniutum DuPont 11604 ~~ Photo- Shaking Initial Illumination Period Speed Temperature Test Population Qumens/m* =lux) (hours) (rpms) cq Definitive 10,000 cells/mL 6000010000 24 100 2x2 I. Selenastrum capricornutum Growth Measurement 1 Definitive Test Selenastrum capricorniutum growth measurement was determined by visually counting the numobfceelrls taken fromanapproximate 0.2 mL sample from cach flask at approximately 24, 48, and 72 hours from the definitive test initiation. The counts were conducted using a hemacytometer and a compound microscope. An aliquotofeach sample was loaded into the hemacytometer and 16 grids were selected for counting. All cells locatedinthe 16 grids were counted and recorded as healthy or unhealthy. The total numberofcells counted per grid was multiplied by 10,000 to determine the numberofcells per milliliter. Cells outside these 16 grids were not counted nor included in the total number. Observations and countsofhealthy and unhealthy cells e.g., deformed, senesced, stunted) were recorded in the study records. Most counts were made at approximately the same time cach day. Statistical calculationsofthe ECso and NOEC were based on mean healthy cell counts and nominal concentrations. ~ 2. Recovery Test Noneofthe definitive test concentrations exhibited a 50% or greater growth inhibition based on healthy cell count relative to the blank control. Therefore,a recovery test was not performed. J. Statistical Analyses `The mean healthy cell count, area under the growth curve, and growth rate at the 72-hour interval for each test concentration were expressed relative to the blank control. All calculations were based on the nominal concentrations. `The area under the growth curves for each interval were calculated according to the following formula: A=[0.5%1 X(N - Nol + [0.5% (12-1) X (Na +Ny -2N0)] Ht [05% (a= fa) X (Na +No = 2N0)] where f1, 3, f.1, and , are timesofobservation measured from the initiationofthe test and No, Ni, No.1, and N, are the initial and subsequent healthy cell counts (cells/mL) corresponditnog the observation times. `The growth rate (u) for each test concentration and for the controls were calculated according to --~ the following formula: - = `Company Sanitzed. Doesnot conTtSaCAiCnBI `1G.r2o5w4t3h5:RaItnefloufetnhceeGorneeGnroAwltghaaSnedlenasirum capricornutum DuPont 11604 ~~ Wo - InN) 0 winhietirael at,ndisstuhbesteiqmueeonftohbesaelrtvhaytcieollncmoeuanstusr(ecdelflrso/mmLt)hecoirnirteisaptoionndoifntghteo tthesetoabnsderNvoatainodn Ntimaer.e the `The percent growth inhibition, % I, was calculated according to the following formula: WI _== .c-T x100 `twhheebrleaCnkiscoenqturaoll taotathseemleecatnedhseaalmtphlyincegllinctoeurnvtal, aanredaTunidsetrhethmeegarnowhetahltcuhryvcee,llorcogurnotw,tahreraatuenfdoerr the growth curve, or growth rate for each test concentration ata selected sampling interval. Negative valuesof inhibition indicate stimulation of growth. iTnhheib"ietfifoenctoifvgercoowntchenrterlaattiivoen"to(EthCesoc)onwtarosl.defTihneedECasspthvealcuoencaenndtraastsioocniaptreoddu9c5i%ngcaon5f0id%ence cloimnictesntwrearteiodnetaegarimnisntedthbeymweeaisguhrteeddplaeraasmte-tseqru.areTshneoNn-OlEinCe,ardreefgirneesdsiaosntohfethhieghleosgtocfotnhceenttersattion ocfotntersotl,suwbasstadnecteetrhmaitnheaddfnroomsiagntirfeincdanttesetf(fJeoctncoknhetehreem-eTaerspusrterda poarrWaimleltiearmrse)laatpipvleiteodtihneabsltaenpk- --_ down manner. All tests of significance were at o= 0.05.0 K. Analysisof Test Solu-tDiefoinintisve Test 1. Sample Collection Tcohnetrcooln,caenntdrtaetsitocnosoncfenHt-r2at5i4o3n5(innomtihneawlor6.k2i5n,g1s2t.o5c,k25(,noamnidna5l0 1m0g0Hm-g25H4-3255/4L3)5s/oLl)u,tibolnasnkwere vceornicfeinetdraattitoenstsionliuttiiaotniosnw(adsayta0)k.enApnriaolritqouotthefradodmietiaocnhooffStehleenblaasntkrucmonctarpolriacnodrntuesttum. The pH was determined for eachofthe working stock, blank control, and test concentration solutions. T50h,eacnodnc1e0n0trmatgioHn-s2o5f43H5-/2L5)4,3a5ndinatbhieotbilcacnokntcroonltr(onl,omtienstalco1n0ce0nmtrga/tLioHn-(2n5o4m3i5n/aLl)6s.o2l5u,ti1o2n.s5,w2e5r,e Veearcihfoifedtahtetceostnttreorlmiannadtitoesnt(cdoanyce3n).trTathieosnessoalumtpiloenss waenrdetparkeinpgaraendablyiqpuoootlfirnogmalel3achroepfltihcaetepsoofolred samples. The pH was determined for eachofthese day 3 samples. The concentrations of in eachofthe test solutions were determined by high performance liquid chromatography/mass spectrometry (HPLC/MS). --~ -- `Company Sanitized. Does not contain TSCA CBI G12r5o4w3i5h:RaItneflofutehnceeoGnreGenroAwltghaaSnldenastrum capricornutum DuPont 11604 -- 2. Sample Treatment An aliquot from the blank control, working stock, and test concentration were sampled at the study start (day 0) and submitted for analysis. An aliquot from the blank control, test concentration, and abiotic control were sampled at the endofthe exposure (day 3) and submitted for analysis. Back-up solutions for each sample also were provided. All samples were analyzed `on the day ofreceipt. Day 0 samples were also analyzed on day I. Centrifugation and setting of algae were investigated as methods for analyzing day 3 samples. Settingofalgae was the preferred methodbasedon analytical recoveriesofthe test material. Theday 3 samples were placed in a refrigeanrdasttoorerd for 3 daysto facilitate the settolfailgnaeg. After 3 days of storage the supematantsofthe refrigerated day 3 samples were taken and analyzed. Aliquots of all test solutions were transferred to autosampler vials for analysis by LC/MS. The samples were diluted before analysis with AAP nutrient medium as follows: 6.25 mg/L (1:2), 12.5 mg/L(1:5), 25 mg/L (1:10), 50mg/L (1:25), and 100mg/L (1:50). -_-- `Company Saniized. Does not contain TSCA CBI `1G.r2o5w4i3h5:RaItneflofutehnceeGorneGenrAolgwaaSnedlenastrum capricorn DuPont. 11604 --_ 3. Instrumentation and Conditions HPLC Instrument: MS Instrument: Model 2795, Waters Quattro Micro, Micromass LC Parameters: Column: Mobile Phase: Luna 3u CI(2), 20 mm x 2mm A- 50 mM ammonium acetate in water C- methanol Gradient: `Time (sin) %C Flow (mL/min) 0 30 025 5 100 025 10 100 025 10.1 30 0.50 12 30 050 12.1 30 025 Column Temperature: Injection Volume: Column Switch: 30C 50uL 2and 10 min MS Parameters: ~ Ionization mode: Electrospray, negative ions Capillary voltage: 32kV Cone voltage: 30V Source Temperature: 120C Desolvation Temperature: ~~ 450C Data Acquisition Function: ~ C6-P-C6 bis ester ion: SIR of 789 m/z; 0-10 min C6-P-IPA mixedester ion: SIR of 485 m/z; 0-10 min C6-P mono ester ion: SIR of443 m/z; 0-10 min 4. Quantitation H25435 isa The resent in H-25435 can be subdivided into Stinct groups: `monitored by LC/MS though `One chemical species the study. The selected was selected from each group and species chemical structures, names, and abbreviations used were as follows: -_-- Company Saniized. Does not contain TSCA CBI GH2r5o4w3h5RaItenoffltuhenoGcreneeGnrAolgwaaSnedleasirun cpricomam ~ Duponc 1604 The smc SIs rcp. Tie sfepsetciseusbstthaantcweerHe-2m5o4n3i5tohraesdndoutrbienegntchhasrtaudcyt.eriTzheed tcoondceetnetrrmaitnieonasctoufalincdoinvtiednutaslocfhetmheictahlresepecies tHh2at54we3r5e monitored during the study were represented asthe total concentrations ofthe bAypdriismsaorlyvisntgoacpkpsroolxuitmiaonteolfyt6h0e rmegfeorfenHc-e2c5o4m3p5ouinndS,00S H-m2L5o4f35AAP nut-- rient mettum- was made ACaplpirborxaitmioantesltyan3d0armdisnwuetreesopfrespoanriecdatoinonewacahs treestqudiaryedbtyodciolumtpiloentweiltyhdiAsAsoPlvneuttrhieeentstmesudbisutamncaet. wcoenrceenmtardaetiaonnsdrpaenagkinagrefarsowme0r.e2d88etteor5m.i7n5edmeb/yL.elecDturpolniiccatienteignrjaetcitoino.ns ofcalibration solutions cThhreocmaaltiobgrraatipohniccuarnvaelsyswiesorefgtheenecraaltiebdrabtyiornegsorleustsiioonns.anaDlaytsaisofofrttehst psoelauktiaornesawdaatsafoibttteadintoedthferom appropriate calibration curves to determine the concentrationsof eachofmonitored species. The Te `Company Sanitized. Doss not contin TSCA GI 1Gr2o5w4t3h5:RaItnefolufetnhceeGorneGernoAwltghaaSnedlenastrum capricornutum DuPont11604 ~~ laivmeirtoagfedneotiescetiloenve(lLiOnDc)hraonmdatliomgirtaomfsqouafnbtliatnatkiocnon(trLoOlQs)amwpelreesdaentdercmoimnpeadrbiyngcatlhceulmattointghtehseignal `reolafatceadlipberaaktsionnesatratnhdearrdeotfekntnioonwtnimceoonfctehnteraatniaolny.te.TwTohechLrOoDmawtaosgrcaamlscuwleatreedeaxsa3mitnimeedsftohrenoiseconcentration equivalentofthe mean noise level. The LOQ was calculated as 10 times the concentration equivalentofthe mean noise level -- --~ J Tiss -- Company Sanitized. Doesnotcontain TSCA Cai 1.25435: Influence on Growh and SSeoew eRanteosfthseGmroum Amlgeo ppricorman_____PufouNicts --_ RESULTS AND DISCUSSION A. Environmental Conditions - Definitive and Recovery Tests (Tables 1-2) For the definitive test, the pH measurementsofthe test solutions ranged from 7.23 to 7.48 at the 0-hour and from 7.66 to 8.49 at the 72-hour interval. For the definitive test, light intensity fortheareas used in the chamber ranged from 8580 to 9440 lumens/m? (= lux). The mean light intensity was 9022 lumens/m?. For the definitive test, theshakingspeed was 107 revolutions per min (rpm). rFaonrgtehde fdrefoimni2t5i.v1e tteost2,5t.h3eCtefmopretrhaetudrueraftrioonmotfhethdeigsittauldyr.eadout on the temperature recorder B. Test Substance Stability and Analytical Verification (Table 3, Figures 1-4) 1 Chromatographic Results --~ RRo 2s awcll. resolved peak witha retention timeofapproximately 6.8 minutes. (EY |utcd 2s a well-resolved peak with a retention time of approximately 5.9minutes.GEESEeluted as a well-resolved peak with a retention time CE; omn2ogra ofa of approximately 5.3 minutes. Figure 1 showas representative calibration curve obtained for typical standard is shown in Figure 2. Figures 3 and 4 show chromatograms ofa blank control solution and test concentration solution, respectively. The LOD H-25435, and LOQfor respectively. The LOD ERG ere determined to be 0.001 mg/L and 0.003 and LOQforCMRro determined mtog/beL of 0.012 mg/L and 0.04 mg/L of H-25435, respectively. The LOD and LOQfori were determined to be 0.0036 mg/L and 0.012 mg/L of H-25435, respectively. 2. Test Solution Results `The mean, measured concentrationof(ESM in the day 0 working stock (= 100 mg H-25435/Ltest concentration, = abiotic control) was 85 mg/L. This represents 85% recovery of wthoerkacitnigvestioncgkre(di1e0nt0. mTgheH-m2e5a4n3,5m/eLatseusrtecdonccoenncternattriaotni,o=noafb( ioticS contS rol)S was R 94 mgi/nL.theTdhaiys 0 represents 94% recoveryofthe active ingredient. The mean, measured concentration ofl EE the day 0 working stock (=100 mg H-25435/L test concentration, = abiotic control) --_ was 102 mg/L. This represents 102% recoveryofthe active ingredient. These data indicated the -_-- `Company Sanitized. Does not contain TSCA CBI StGF-ro2ou5wt4t3h5R:RattIenofolffettnhhceeeGGroreneeeGnnroAAwlltgghsaSaSeneldolneansatsrruummccaapprriiccoorrnnuum DuDbuoPno-n1t116106044 rm stock solution was prepared at the desired concentration and confirmed the expected test concentration was achieved. The mean, measured concentrations. oF -- the d4a4ym0g/nLo,mirensaplec6t.i2v5e,ly1.2.T5,hi2s5,reapnrdes5e0ntmsg8/0L, t8e8s,t8c4o,nc8e8nt%rarteiocnovseorlyuotfitohnsewaecrteiv5e.0i,ng1r1e,di2e1n,t,and r6e2s5p,e1ct2i.v5e,ly2.5,Tahnedm5e0anm,gm/eLatseustrecdonccoenncternattriaotniosnosloutfioAns were 6.2, 15, 27, ir and the day 0 50 mg/L, nominal respectively. This represents 99, 120, 108, 100 % recoveryofthe active ingredient, respectively. `5T0hemgme/aLnt,esmtecaosnucreentdrcaotnicoennstorlauttiioonnssowfeG re 6.S 9, 13M , 26,S and 5t2hemdga/yL,0rneosmpeicntailve6l.y2.5,Th1i2.s5r,e2p5r,esaenndts s1t1o0c,k1a0n4,d t1e0s4t,co1n0c4e%ntrreactoiovnersoyolufttihonesawcetrieveprienpgraerdeidenatf,trheespdeecstiivreeldy.nomTihneaslecdoantcaenitnrdaitciaotnesd.theA.fter 3 days, 50,and the mean, 100 mg/L mteesatscuornecdenctornacteinotnrsa,tainondsao biotic cor ntrol son lutions twheerneo2m.i5n, a5l.96,. 2152,, 2102.,5 ,an2d5, 45 mg/L and 44 mg/L, respectively. After 3 days, the mean, measured concentrations of O abiotic E controlY solutionisrwtecren5o.m3i,na12l,62.42,5,541,2.a5n,d251,0050m,ga/nLd a1n0d09m5gm/gL/tLe,strcesopneccetnitvrealtyi.onsA,ftaenrd a3ndday1s0,0tmheg/meLatne,stmceoansceunrterdatcioonncse,ntarnadtiaobnisotoiQ c control soS lutionsiwnerthee1n.o6,mi7.n6a,l169.,245,3,1a2.n5d, 25, 50, c8o6ncmegnt/rLatainodns110E mg/L, respE ectively. The-- blank -- control-- soluti-- ons co-- ntained no detectbabolteh:day 0 and day 3. ~ UinsgirnegditehnetmoefaHn-o2f54t3he5,doafyt0heannodmdianyal3 6r.e2su5l,ts1,2.t5h,e2c5o,nc5e0n,tr1a0t0imongs/oLf( test cE oncenNtratS ion an<cdtitvhee arbeiporteiscenctosnt6r0o,l6s8o,lu6t6i,o6n4s,w6e5r,ea3n.d756,58%.45r,ec1o6v.e5r,yo32f,t6h3e, aacntdiv6e4.i5ngmregd/iLe,ntr,erspeescpteicvteilvye.ly.This aUcstiinvge ithnegrmedeiaennotfotfhHe-2d5a4y305,aonfdthdaeyn3omreisnualtls,6.t2h5e,c1o2n.c5e,n2t5r,at5i0o,ns10o0GmUgE/SLEtSeEstScGoGncNeGntGr.atGi,on and trheepraebsieonttisc9c2o,nt1r0o8l,s1o0l2u,ti1o0n4s,w9e7r,ea5n.d759,51%3.r5,ec2o5v.e5r,y5o2f,t9h7e, aacntdiv9e4.i5ngmrged/iLe,ntr,esrpeescpteicvteilvye.ly.This iUnsgirnegditehnetomfeaHn-o2f5t4h35e,doafyth0eanndomdianyal3 results, the 6.25, 12.5, concentrations oWSR 25, 50, 100 mg/L test concentration active and the arbeiporteiscenctosnt6r8o,l8s2o,l9u0t,io9n5s,w9e4r,ea4n.d251,0160.%3,re2c2o.v5,er4yo7.f5,t9h4e,aacntidve10i6ngmregd/iLe,ntr,esrpeescpetcitvievleyl.y.This C. Selenastrum capricornutum Growth (Tables 4-8, Figures 5-7, Appendices C-E) 1. Definitive Test eTxhpereosrsgeadniisnmtserwmesroefepxeprocseendt fionrhi7b2ithioonurisnwgirtohwotuhtbtaesstemdeodnihuemalrtehnyewcealll.coTuhnet,eaffreecatusnwdeerrethe ee ------ ee `Company Sanitzed. Does not contain TSCA CBI 1G.r2o5w4t3h5:RaItnefloufetnhceeGorneGenroAwltghaaSnedlenasrum capricornutun Dupont 11604 ~ `growth curve, and growth rate relative to the blank control for the 72-hour intervalofthe tes. The 72-hour results are as follows: Healthy Cell Count ECs NOEC Area Under the Growth Curve ENCOsEC Growth Rate ECs NOEC > 100 mg H-25435/L >100 mg H-25435/L > 100 mg H-25435/L > 100 mg H-25435/L > 100 mg H-25435/L > 100 mg H-25435/L 2. Recovery Test Nheoanletohyfctehlel cdoefuinntitrievleattievsettcoontcheenbtlraatnikocnosnterxohli.biTtheedraef5or0e%, oarrgerceoavteerrygtreostwtwhasinnhoibtipteironfobramseed.d on CONCLUSIONS ~ TSehleerneawsatrsunmocsaipgrniicfiocrannuttiunmhiwbhiteonryeexfpfoesctedontotnhoemgirnoawlthcoanncdengtrroatwitohnsroatfe6o.f25, 12.5, 25, 50, and 100 mgH-25435/L for 72 hours. Therefore, the ECsy and NOEC for ll3 endpoints are greater than 100 mg H-25435/L. RECORDS AND SAMPLE STORAGE All data and records for analytical characterizations conducted by the sponsor will be retained by the sponsor. `NSepweacrikm,enDs(eliafwaaprpel,ictahbeleD)u,ProanwtdCatrao,paPnrdottehcetfiionnalArrecphoirvtesw,ilNlebwearrket,aiDneeldawatarHea,skoerlaltLIarboonratory, Mountain Records Management, Wilmington, Delaware. -- `Company Sanitized. Does notcontain TSCA GBI H`G-r2o5w4t3h5:RaItnefolfutehnceeGorneGenroAwltghaaSnedlenastrun capricormitun Dupont 11604 ~~ REFERENCES 1. Miller, WE; Greene, 1.C.; Shiroyama, T. The Selenastrum capricornutum Printz Algal `Assay Bottle Test; U.S. Environmental Protection Agency; U.S. Government Printing Office: `Washington, DC, 1978; EPA-600/9-78-018. 2. American Society for Testingand Materials (ASTM). (1990). "Standard Guide for Conducting Static 96-h Toxicity Tests with Microalgac" in ASTM Annual BookofStandards, E1218-90. Vol. 11.04, Philadelphia, PA. 3. Snedecor, G.W. and Cochran, W.G. (1967). Statistical Methods, 6th edition. The lowa State University Press. 4. Tamhane, A.C., Dunnett, C.W., Green, JW. and Wetherington, J.D. (2001). Multiple test procedures for identifying the maximum safe dose. J. Amer. Statist. Assoc. 96, 835-843. --~ -- = Company Sanitzed. Does notcontain TSCA CBI H`G-r2o5w4t3h5:RaItneoflfuetnhceeGorneGenroAwltghaaSnedlenastrun capricornutun --~ DuPont 11604 TABLES --~ --~ Te Company Sanitized. Doss not contain TSCA CBI `HG2r5o4w3t5h:RaIteonftfheloGrnueGerenowAnltghcaaSneedlenasrun capricornutum. DuPont 11604 --~ TABLE 1 pH MEASUREMENTS OF TEST SOLUTIONS Nominal H25435 Concentration Exposure Initiated: pH 07-Jan-2003 0-Hour (Day 0) Exposure Ended: 10-Jan-2003 72-Hour (Day 3) Blank Control 748 7.66 625 mg/L 739 7.78 125mg 738 8.43 25 mg/L, 732 8.49 50 mg/L. 7.28 8.48 100 mg/L 723 8.47 Abiotic Control (100 mg/L) 723 7.66 ~ TABLE 2 CHAMBER LIGHT INTENSITY, SHAKING SPEED, AND TEMPERATURE RANGE Mean Light Intensity atTest Initiation Oscillations Temperature Test (Qumens/m* = lux) (rpms) ca Definitive 9022 107 25110252 -- Ee `Company Saniized. DoesnotconTtSaCAiGnBI G2r5ow4t3h5:RatIenoflfutehnceeGorneeGnroAwlthgaSnedlnastrum capricornutum DuPont 11604 _~ MEASURED CONCENTRATIOTNASBLOEF3H-25435 IN TEST SOLUTIONS a Ey Sample Description ToFtoarlm,uNloamtiinonal _G_ENoNminEal S (CmoncLent)ration Concentration Concentration" (mg/L) (mg/L) Day0 Day3 Mean % Mean Recovery Blank Control 0 Working Stock (100 mg/L) 100 625 mg/L 625 125mg 125 25 mg/L 25 -- 50mg/L 50 100 mg/L 100 Abiotic Control (100 mg/L) 100 0 ND ND ND NA 100 8 Ns NA NA 625 50 25 35 60 125 59 sas es 25 2 2165 66 50 "4 20 0 6 100 8 45 6s 6 100 85 44 6s 6s ab DHef-ine2dwi5natse4arsmss3oufm5medgHb2e510403%t5feorruatnialeyntitcamledpuirupmo.ses. MNeDanstoafnddsafoyr0naontdddetaeyc3tedc.oncTehnetraltiimiotnos,fdetection was calculated a 0.001 mg. FNNSAssttaannddss ffoorrnnoott sauppblmiictabelde.. & Ndaoy0sepaabriaottiecacnaolnytsoils wwearsecaolnidquucottsedofstinhceestahmeewosorlkuitinogn.stoTche,retfhordea,yt0he1m0e0amsugr/eLdtecsotncceonntcreanttiraotnioonb,saindethfeor the worsockk cianbneusgd forthe 100 mg/Ltetconcentratnditohenabiotic contr. Ge ---- `Company Saniized. Does not contain TSCA CBI HG2r5o4w3t5h:RaItenoftfheloGnruoGerenowAntlhgcaaSeneldenastrum capricornutun Dupont 11604 ~ MEASURED CONCENTRTAATBILOEN3S(COoFntHi-n2u5e4d3)5 IN TEST SOLUTIONS Mean, Measured DescSraimpptlieon Total, Nominal Formulation Conc(emngt/rLa)tion Nominal Con(cmegnt/rLa)tion" = Concentration (mg/L) Day0 Day3 Mean` RMeecaonve%ry Blank Control 0 0 ND* ND ND NA" Working Stock (100 mg/L) 100 100 9 Ns NA NA 625 mglL 625 625 62 53 515 125 mg/L 125 125 15 2 Bs Ie 25mg 25 25 27 4 255 102 ~ 50 mL 50 50 50 4 52 104 100 mg/L 100 100 ot 100 97 97 Abiotic Control (100 mg/L) 100 100 94 95 945s 95 4b DH25e43f5inwitaesrnmassoseufmmdegd tHo be-1002%tefo5rtania4elnyttic3maeldpi5uurmp,oses 4 MNeDanstoafnddsafyor0naontdddetaeyct3edc.oncTehnetraltiimoitnos.f detection vas calculated 5 0.001 mg. FNNSA ssatannddssffoorrnnoott saupbpmliictaebdl.e. & Naboisoteipcarcoantteraonlalwyesrieswalaisqucootnsdoufcttheedssaimnecstohlutwioonr.kinTgheretfocokr,et,hthdeamyea0su1r0e0dmgco/nLcenettractioonnobctaeinnedrfaoarntdthteiheodany,0 working stockcanbe usdfo the 100 mg/L test concentration and the biotic contol. ET -- `CompanySanitized.DoesnotcontaTSiCnA CBI `125G435Rr:atIeoonfwtfhetlGorneuhGerneoAwnltghcaaSneldenastram capricornuun DuPont 11604 ~~ TABLE 3 (Continued) MEASURED CONCENTRATIONS OF H-25435 IN TEST SOLUTIONS Sample Description' Total, Nominal __ Nominal Mean, Measured GENESEE Formulation MEMES Concentration Concentration (mg/L) Concentration" (mg/L) (mg/L) Day0 Day3 Mean -- Mean % Recovery Blank Control 0 0 ND' ND ND NA Working Stock (100 mg/L) 100 100 102 Ns" NA NA 625 mg/L 625 625 69 16 425 68 125mg 125 125 1376 103 82 25mg 25 25 6 19 25 % 50 mg/L 50 --~ 100 mg/L 100 50 2 4 as os 100 102 86 94 9% Abiotic Control (100 mg/L) 100 100 102 10 106 106 eee eee ba FDe-f2i5n4e3d5iwnatseramssosufmmedgoHbe.1200l%5efor4rnaunt3arliye5tnitcamledpuirupmo.ses. NMeDanstoafnddsafyor0naondddetaeyci3edc.onTchenetrlaitmiiotnso.fdetection NA sandsfo not aplicable. ws calculaast0.e00d1 mg. & dNNaoSysse0tpaaanbridaosttefiaocrncanolonyttsrsiouslbwmwaietsrteceodan.ldiuqucottesdosfitnheetshaemweorsokuitnognst.ocTkh,etrehfodra,yt0h1e0me0smsgu/rLedtecsotncceonntrcaetinontrobaiatainindoedtnhf,eor the Working sockcanbeusedforthe100mg/L est concenndtthrsabiotteicoontonl. --~ - `Company Saniized. Does not containTSCA CBI `HG-r2o5w4t3h5;RaIteonftfheloGnrueGeernoAnwltgchaaSeneldenastrum capricormutun DuPont-11604 --~ TABLE 4 HEALTHY CELL COUNT DATA SUMMARY - DEFINITIVE TEST FucTomuntion eB] 3 EE rimTei]yan GMB ExpoDsayPi1doumary 200 To woo] a Sd I emr e I -- --) somal-- ----7 1ee)l-- TseY 7e)nB-- --ET iaw|n) 5 1) --)-- | nse | 23 ou]| nnoooo stpope SSi2m0o0n [e CosteTT a ool mol r ems siansl ~ me 1 woeol wweeall soesol waes]l Toon] Sooo 0 on s meno i Tef e EeT e I |e om [|1]i2 Cement |1] Y 7S e II -- mgt oowwll moosnes] imsoanns]l wsaonlal smalne 3]--s ]E-- e wd]| 0hr0e0s 1s0o0o00] e0na0 sSooaotoon] ool man eeu uma -- 1TS S-- S--E 1]-- | lo10s0e0] 12n0o0o prseayoe]d SSaantmos 7 son T --r1o S seu al-- T mTeTsl Bs -- swnen|l --~ -- = Company Sanitized. Doesnotcontain TSCA CBI - TABL5 E AREA UNDER THE GROWTH CURVE DATA SUMMARY - DEFINITIVE TEST = -- = S T= e == r = p r == = e = eet = Simi 1 soomooo] al pr00o.0s0 sal sion] | il . E S E= e e = =e e = = [variancePA peeiol-- goveens esi] T = --_-- -- T r =--= Fsspe mee ma To aswomsnl -- susossol 0 od sel si C E S = S T E S= == =p == = e e o= e= = = = r = r oee e = = -- r= E= = E == [Varmnee A coven seen seen] ~ ~ ree - `GROWTH = RATE TABL6E mi e emen DATA SUMMARY - DEFINITIVE TEST E= ( = e =w -- &r = T &T == e = = S= =T=ET ]=8=oe F e-- r] =="-- aos ==4| CE= eEEfFr EE Eo .= e = S T S= === r F ==E-- = e= == EC = = S ECSNf = EEE=E E i -- == E -- = o = . C = = SF= L 5= = = = i = -- = == ~ 1.25435: InflonuGreownthcaned t eo iie ELH, ~~ TABL7E 72-HOUR (DAY 3) STATISTICAL ANALYSES SUMMARY - DEFINITIVE TEST NBom5in0al Mean Standard Concentration __ Response Error N_| "RTeexstpCoonnsceen(tmrga/tLi)on Lev Value CoHeuanltthy Cell B6la2n5kmCogn.trol 1225m5gml 5010m0mgg 3S4u40m00s0 3060055 33 [EC 10 3S7060607 s17a37s18 33 |NOEC S10 3S3e6s6s6a7 6201m3 33 AGrroewatUhnCduerrvtehe B6l2a5kmgCloLntol 1228m5pmlg S100m0myg.. $575200000000000 132620228421075 33 [ECy 10 671392200000000 4948137158146 33 [NOEC >100 06898600000000 S53a4T20a6s0 33 GrowhRate B6l2an5kmCgontrol - 1225m5pmlg So1m0g0mrg o0osmi0 0o0o0m%s 33 [ECs 10 00008833 0000000166 33 |NOEC 10 o000s207 0000003019 33 ab CEoCnfiwdaesnccaelncuelravteadl singProb analysis. --_ EE `Company Sanitized. Donotecontsain TSCA CBI H`G-r2o5w4t3h5:RaItneoflfuetnhceeGorneGenroAwltghaaSnedlenastrum capricornutun --~ DuPont-11604 FIGURES --_ -- Te Company Sanitzed. Doesnotcontain TSCA CBI G1r.2o5w4t3h5:RaItnefolfatenhceGorneGenrAolwtghaSaenldnastrur capricorn Dupont11604 --~ FIGURE 1 REPRESENTATIVE ANALYTICAL CALIBRATION STANDARD CURVE FOR 8 woo] % oom g m 32 moo |[ Exumion fh ited i: K-09 y=-1100.84' +20486.2x+6207.05 | | 4d r ovrr 2 resmegey 3 4s Pa H-25435 Concentration, gL Ji oo -- TLTsnwimortestimpesaremnin 23 om R=0.9999 a0 | go ZS ao] | = 200 1 || | olo ~ z s] &s 6 H-25435 Concentration, mg/L. -- -- Company Sanitized. Dos not contain TSCA CBI H-25435: Influence on Growth and. `GrowthRateof theGreenAlga Selenastrum capricornutum DuPont-11604 ~~ 14000 => zx 10000 i 3000 `Eqouftahetfiitteodline: y=2469.12 259.168 R=09992 ooo 5 won 2000 0 0 123456 H-25435 Concentration, mg/L. ~~ Company Sanitized. Donote contsain TSCA CBI G14254r35:aotneowfthuaGnrGnernoAcwlpeaSnedleasirum copricrnuram Duront1604 ~ FIGURE 2 REPRESENTATIVE CHROMATOGRAM OF A CALIBRATION STANDARD SOLUTION T0802 Soon ZG) 00 - ish 17668a 000i05 sTso6s7.e1s4 % 751 0 pa) pry Th ok 0%min] I 25933Y i copc: eaion of 0c.56 mion. meofspproximaely 8 mies, Caltrain sda solution ono [0803572 Soom 2%) 0 - 1o0z0s29s0 FSR AT10E450S40T.0o4 % 535720 poy pry oho Foy in S ois 2 Y12: oneenrattoniofo05n6 mLi.mofsprosiatly 5.9 mimes. Clin sand ston -- i--------ieee CompanySanitized. Doss not contain TSCA Gall G1r2o5w4t3h5:RaItnfloufetnhceeGorneeGnrAolgwaSaenldenastrum capricormitum DuPont 11604 ~ [070803072 Smooth25) 100 676 FSRof chanelT5ic 269904004 hg 1- 178.48 S285 go o 70 a 550 [1 min Q2O5435 at i: retention time ofspproximatly 5.3 minutes. concentrationof0.56 mL. Calibration standard solution contains --_ ' . --~ ee Company Sanitized.Doesnotcontain TSCA CBI G12r5o4w3t5h:RatInefolufetnhceeoGnreeGnroAwltghaaSnedlenasirun capricornutin DuPont-11604 FIGURE 3 REPRESENTATIVE CHROMATOGRAM OF THE BLANK CONTROL SOLUTION 070703615 Smooin23) 100- FISIRof 1 chan Ti5c 77301002 200 4% 650 550 MY4 cue a retention timeof approximately 6.8 minutes. --_ [076703575 Smooih(vn23) ] F2SIR of 1 chanelT5i.c 6.17204002 20 a5 550 500 1000 OY lt a rttion timeofspproximatly 5.9 minutes. E-- CompanySaniized. Doesnot contain TSCA Cal HG-r2o5w4t3h5;RaItnefloufetnhceeGorneeGnroAwltghaaSnedlenastrum capricornutun ~ [070703015 Smooth(in2:3) 100- DuPont 11604 FSRof 1 channelT5i.c 35904002 * o 200 300 500 500 min I 1clu at aretention timeofspproximately 5.3 minutes. ' . --_ EE CompanySanitized.Dossnot contain TSCA C81 `1G.r2o5w4t3h5:RatInefolfuetnhceeGorneGenroAwltghaaSnedlenasru capricormitun Dupont 11604 FIGURE 4 REPRESENTATIVE CHROMATOGRAM OF A TEST CONCENTRATION SOLUTION 01080324 Smooh(n23) 100, 44989696.807.66059 FSRof | hamelEmSe. 4.896e+005 % * o 751 min 75 io P17) 5% 950 Q 2343 aR 3 nominal: concentra:tionroeftent6i.o25nmtiLme.ofaTphpersoaxmipmlaetewlays6d.i8lumtiendut1e2s.beTfeosrtesaonlaultyisoins.sample contains H- --_ 010803623 Smoot 2%) FZSIR of hamnelES: oe n5m87 68330000ie4 ses1059175 *% Zh h% sho a0 min R 25435 at noM minal conccnt1r.aton of re6t25rtmiLon.tiTmehosfaspmpprloexwiamstedliylu5t.9ed m1i2nebesf.oreTessntalsyosliu.tion sample contsins H- --~ - Company Sanitized. Does not contin TSCA CB `25G4r3o5w:RaItnefloufetnhceeGorneGenroAwltghaaSnldenasirum capricornutin DuPont11604 aS 010803524 Smooth(#20) FESRof 1 chamel5. 100, - 676 742300i0c4 28502454 66505 * 68 77 o 250 50 550 5% min O at nominM al concent: ration of 6r.25imcg/aLn. tTimheeosfamapplperowxaismadtielluyte5d.31m2inbuetfeosr.e aTneaslytsisso.lution ssmple contains H-25435 -- Company Saniized. Does not contain TSCA CBI GH-r2o5w4t3h5:RaItneoflfuetnhceeGorneGenroAwltghaaSnedlenasirum capricornutuns --~ DuPont-11604 s APPENDICES --~ Company Sanitized. Does not contain TSCA CBI H`G2r5o4wt3h5:RaItneoflfutenhceeGorneGernoAwltghaaSnedl.enastrun capricornutum --~ DuPont-11604 -- APPENDIX A AAP Nutrient Medium Constituents `Company Sanitized. Does not contain TSCA CBI 2Gr5o4w3th5:RaItneflofutehnceeGorneGenroAlwghaaSnldenasiram capricornutin. -- AAP NUTRIENT MEDIUM CONSTITUENTS DuPont11604 Stock Solutions Component Concentration (gL) Final Prepared Medium Component _ Concentration (mg/L) Macronutrient Stock #1 NaNOs 25.500 Na 11001 No 4200 Macronutrient Stock #2 NaHCO; 15.000 c 214 K 0469 Macronutient Stock #3 K:HPO; 1.044 P0186 Mg 2.904" Macronutrient Stock #4 MgSO. 7H:0 14.700 ~ Macronutri.ent Stock #5 MgCl+6H:0 12.164 ca 1202 s 19m Macronutrient Stock #6 CaCly2H:0 4410 Stock Solutions Component `Concentration (mg/L) Final Prepared Medium Component _ Concentration (ug/L) Micronutrient Stock H:BOs MnCl H:0 ZnCl, CoClye6 H;O CuCl2H;0 Na:MoO2 HO FeClie6 HO NazEDTA-2 H:0 185.520 415610 3271 1428 0012 7.260 160.000 300.000 B 32460 Mn 115374 Zn 1570 Co 0354 Cu 0004 Mo 2878 Fe 33.051 --~ ab AInsclruedpeosrtmeadginneRseifuemrefnrcoem2.both magnesium sulfte nd magnesium chloride. -= `Company Sanitzed. Does not contain TSCA CBI GF-r2o5w4t3h5:RaItnefolfuetnhceeoGnreGenroAwltghaaSnedlenastrum capricornutun. DuPont 11604 --_ APPENDIX B 72-Hour Healthy Cell Count Statistics for the Definitive Test ee Company Sanilized. Does not contain TSCA CBI HG2r5o4w3t5h:RaItnefloufetnhceeGorneGenroAwltghaaSnedlenastrum capricornutum DuPont-11604 72-HOUR HEALTHY CELL COUNT STATISTICS FOR THE DEFINITIVE TEST RESOPBOSNESREV:EDCOPEURNCTENfTroCmHADANTGAESEFTR:OMQUCOENSTROLAVNRE1R1N604 obs R(magt/e1) wean pcrmNG n 21 6.250 3135434303030330 0 33 3a 12.255 373461606060.0607 EEss 5c 10500 33381563636363.3637 -2a 33 - Company Saritized. Doesnotcontain TSCA C81 `HG-r2o5w4t3h5:RaIteonftfheloGnrueGeernoAnwltgchaaSeneldenastrum capricormutum. DuPont 11604 --~ STATISTICAL ANALYSIS LIST FILE REECPOOTROTX MCERAESAUTREEDMEONNTS19:F0R0OMWEDDANTEASSEDTAYAWR2121J6A0N4O3 GROUP STATISTICS FOR Count BY DOSE dose do(smeav/La)l COUNT MEAN MEDIAN sm_pEv so_sRr 12 6.250 33 33814503030303.3030 33674100000000 1261244745969..4809 376001535359..1939 3a 12.255 33 347138060606060.0607 33777100000000 380808898878..5183 157133721577..5527 s5 15000 33 33381563636363..6373 33960000000000 11403689798261.9220 265339313223.3636 REPORTSTCARETAITSETDICOANL A1N9A:L0Y0SIWSEDNLEISSDTAYFILE220AN03 `SHAPIRO-WILK AQUATIC TSEESLETNAOSFTRNUOMR:MALFIUTLYL ODFATACount sm SKEW ORT SWSTAT BVALUE SIGNIF 699952.38 0.54723 0.46679 0.94643 0.37177 REPORTSCTRAETAITSETIDCOANL A1N9A:0L0YSIWSEDLNIESSTDAYFILE22JANO3 ANALLEYVSEINSE OTFESVTARFIOARNCCEounOtN F-ULFLULLDAMToAdeSlET ~ Bffect DF LEVENE P_VALUE SIGNIF Doss 5 0.26667 0.52276 2Taheadnaatlaysiwsasoffovuanrdiatnocebewinlolrmbaellyperdfiosrtmreidb.uted with equal variances. REPORTSCTRAETAITSETIDCOANL A1N9A:0L0YSWIESDNLEISSTDAYFILE220ANO3 DOASNEASLYS0,IS6O.2F5,SEL12E.N5A,STR2U5,M 5-0,FUL1L00DnAgT/AL ECOTOX OF AQUATIC SELENASTRUM obs Class Levels values 1 dose s 123456 Count FROM PULL DOAVTEAR,ALWLeiFg-ThEtS=TNSOWEFIOGRHATN,OVVaAriance Ratio=0.33 obs mefect NouF m. DeDnF Evalue ProbE 1 dose s 12 0.63 0.6838 Te CompanySanitized. Doesnot conTtSaCAiCBnI `HG-r2o5w4t3h5R:atIeonftfheloGnrueGeernoAnwlgta chaSeneldenastrumcapricornutun. DuPont-11604 LSCoMuEnAtN.SFR- ODMOSFEULMLEADNATSA,ADWJUeSiTgEhDtNFOORWETIHEGHTE,FVFaErCiTaSncOeF RSaEtLiEoN=A0S.T3R3UM obs LeveL Loman se oor 1 2 0eas 3ios1s 65 1s0o0 33118503030303.3030 448800999966..2277 1122 43178306060606..0607 448800999966..2277 1122 33381563626363106373 4488009999661.2277 1122 ESTIMATEUDSIDNOGSEALEPFHFAEC=T.S05&FDOURNNCEOTMTPARFIOSRONTSwo-TsOidCeOdNTARLOTLERNATIVE Count. FROM FULL DATA, Weight=NOWEIGHT,Variance Ratio=0.33 Estimate D2u-nsniedtetd Test Group SIGNIF p-value Mean DDOoSSEE QTuRAEDND DDOoSsEE 32--11 DDOOSSEE 45--11 DOSE 6-1 00..7610294085 :. 00ll7919904s62 341158303030303030 33 00ll9s89899892 33733166666666..6677 33 0l95650 385333333 3 ~ REPORTSTCARTEIASTTEIDCAOLN A1N9A:L00YSIWSEDLNIESSTDAYPILE22JANO3 ANALYSIS OF BCOTOX USING ALPHA=.05 FULTLESDTATA `JaonndckLhoeweesrte T5reDnosdesTestthroonughDoDseose0 100 ng/L KEY. Analysis of Count ZZCCCPIS1JSON3CCKHWEIETRHECOSNTTAITNIUSITTIYC CCOOMRPRUETCETDIOWNITFHACTTOIRE CORRECTION PPLIUDPNCCEE IISS PP--VVAALLUUEE FFOORR DUOPWWNAWRADRDTRTERNEDND SP-IVGANLIUFESREASRULETSFOARRETIEF-OCRORARETCWTOE-DSITDEESDTALWTIETHRNACTOINVTEINUHIYPTOYTHCEOSRIRSECTION FACTOR Jone zc zcce pivper pIDNCE SIGNIF 74.5 0.539566 0.5781065 0.2615961 0.6918234 NJootneckheere test results are included in the summary table. Gr0ouaplmienaenasr tsrheonudldbebfeoreexamtirneendd ttoestcherceksulftoer alraeck-acocfe-pftietd. ar Company Sanitized. Doesnotcontain TSCA CBI GH2r5o4w3t5h;RatInefolfuetnhceeoGnreeGnroAwltghaaSnedlenasirun capricorntuns DuPont 11604 APPENDIX C 72-Hour Area Under the Growth Curve Statistics for the Definitive Test ww Company Sanitized. Does not contain TSCA C81 `1G2r5o4w3t5h;RaItneoflfutenhceeGorneGenrAowltghaSaenldenastrum capricormutun. DuPont 11604 --~ 72-HOUR AREA UNDER THE GROWTH CURVE STATISTICS FOR THE DEFINITIVE TEST obs R(nagt/eL) MEA pcTomNG NM 21 6.25o 5s57020e00000000 4[RY3 a3 1.35s 6s7132200000000 i5 33 5s 10So0 6c1o8s8s0o000000 s 5a a --~ --_-- `Company Sanitized. Does not contain TSCA CBI G12r5o4w3t5h;RatIeonftfheloGnrueGeernoAnwltgchaaSeenldenastrum capricornutunt DuPont-11604 --~ STATISTICAL ANALYSIS LIST FILE ERCEOPTOORXT MCERAESAUTREEDMEONNTS19:F0R0OMWEDDANTEASSDEATYAWR2121J6A0N4O3 GROUP STATISTICS FOR Area BY DOSE dose do(snegv/La)l COUNT MEAN MEDIAN STD_DEV sT_gRr 21 6.250 33 5575204000000000 5695878600000000 216138008082580..3933 123262482249156..0768 33 12.255 33 6617532200000000 5589078600000000 18633619108651.59.7 94481371581954..6040 56 15000 33 6610898600000000 5695470600000000 99245820701080..5020 55437432208830.5553 REPORTSTCARETAITSETDICOANL A1N9A:0L0YSIWSEDNLEISSDTAYFILE22JANO3 SHAPAIQRUOA-TWIICLKSTELEESNTASOTFRUNMO:RMAFLUILTLYDAOFTAArea oss sm skew KURT SWSTAT VALUE SIONIF 18 10846462.15 -0.31113 -0.052253 0.96885 0.77585 REPORTSCTRAETAITSETIDCAOLN A1N9A:L00YSWIESDNLEISSTDAYPILE22JANO3 ANALLEYVSEINSE OTFESVTARFIORANACrEeaON-FUFLULLLDMAoTdAelSET --~ Effect DF LEVENE P_VALE SIGNIF Dose 5 0.26034 0.92624 orz - AThneandaaltyasiwsasoffovuanrdiatnocebewinlolrmbaellpyerdfiosrtmreidbuted with equal variances. --~ Sos -- Company Sanitized. DoesnotconTtSaCAiCBnI `H.25G435R;ratIenofolfutewhnceeGortneGernhoAwltgha aSnedl.enastrumcapricornuum DuPont11604 --~ REPORTSTCARETAITSETDICOANL A1N9A:L0Y0SIWSEDNLEISSTDAFYILE22JAN03 DAONSAELSYS0,IS6.O2F5,SEL1E2.N5A,STR25U,M 5-0,FUL1L00DAmTgA/L ECOTOX OF AQUATIC SELENASTRUM Obs Class Levels Values 1 dose s 123356 Area FROM FULL DAOTVAE,RALWLeigh-tT=ENSOTSWEFIOGRHTA,NVOaVrAiance Ratios0.33 obs Effect oNuFm DDeFn Evalue Probe 1 dose 5 12 0.33 0.8862 LASrMeEAaNSFRO- MDOFSUELLMEDAATNAS, ADWJeUiSgThEtD-NFOORWETIHGEHT,EVFaFErCiTaSnceOFRSaEtLiEoN=A0.S3T3RUM obs LEVEL LoMERN s= oor 1 2 0eas 5557020400000000 747543s531s8i1s1a8s 1122 ~ i 3 a1s 6671352200000000 77445533551188111188 1122 ss so100 6G1o8s8s00000000 77445533551188111188 1122 ESTIMATEDUSIDNOGSEALEPFHFAEC=T.S0S&FDOURNNCEOTMTPARFIORSONTSwoT-OsidCeOdNTRAOLLTERNATIVE Area FROM FULL DATA, Weight=NOWEIGHT,Variance Ratio=0.33 Estimate D2u-nsniedtetd Teesyt SIONIF p-value Mean DDoOsSeE TQURAEDND DDoOsSeE 23--11 DDOOSSEE 53--11 DOSE 6-1 00..5579747886 ; 00119890906915 5657030200000000 33 0o1l9o9s5s7i1l 6601936200000000 33 0lo88sa 61880000 3 --~ Ee `CompanySanitized. Doesnotcontain TSCAC81 G H.254r35R;ao tIeonfwtfheGlotrneueGnh reoAwnltghcaaSenedl.enastrum capricormutum ~~ DuPont-11604 REPORTSCTRAETAITSETIDCAOLN A1N9A:L0Y0SIWSEDNLEISSTDAFYILE22JANO3 ANALYSIS OF EFCUOLLTODXATUASING ALPHA=.05 Test aJnodnckLhoeweerset T5reDnodsesTestthroounghDoDseose0 100 ng/L KEY. Analysis of Area ZZCCCFISTJSON3CCKHWEIETRHECOSNTTAITNIUSITTIYC CCOOMRPRUETCETDIOWNITFHACTTIOER CORRECTION PPIIUDRNCCEE IISS PP--VVAALLUUEE FFOORR DUPOWWANRWDARTDRTERNEDND SPI-GVNAILUFESREASRULETSFOARRETIEF-ORCOARRETCWTOE-SDIDTEEDSTALWTIETHRNACTOINVTEINUHIYTPYOTHCEOSRIRSECTION FACTOR Jone ES Z0CF PIURCF PIDNCE SIGNIF 71.5 0.3085257 0.3470914 0.3642613 0.6064045 oJorneckheere test results are included in the summary table. tGoroauplmienaenasr tsrheonudldbbeeforeexamtirneendd ttoestchreecksulftosr alvaeck-aocfce-pftietd. -- --~ 52Company Sanitized. DoesnotcontainTSCA CBI `HG-r2o5w4t3h5;RaItneoflfutenhceeGorneGenroAwltghaaSnedlenastrum capricormutun, -- DuPont 11604 APPENDIXD - -- 72-Hour Growth Rate Statistics for the Definitive Test = `Company Sanitzed. Does not contain TSCA C81 `HG-r2o5w4t3h5;RaIteonffheloGnrueGeernoAnwltgchaaSeneldenasirun capricornutum DuPont-11604 --_ 72-HOUR GROWTH RATE STATISTICS FOR THE DEFINITIVE TEST obs Rate wn pore N (ng/L) 21 6.205 00..0087059066666677 2o 33 +3 12.255 00..00883623236373 F2R 3 65 10s00 0.0826606.6078 a 2 33 = -_-- CompanySanitized. DoesnotconTtSaCAiGanl H-25435; InflonuGeronwtchaend GrRaoteofwtheGtreenhAlga Selenastrum capricornutun. DuPont11604 ~~ STATISTICAL ANALYSIS LIST FILE ERCEOPTOORXT MCERAESAUTREEDMEONNTS19F:R00OMWEDDANTEASSEDTAYAWR2121J6A0N4O3 `GROUP STATISTICS FOR G_Rate BY DOSE dose do(nsge/vLa)l COUNT MEAN MEDIAN STD_DEV so_eRr 12 6.250 33 00..008709906677 00..00881292 .000062361227945200 .000031654146798431 33 12.255 33 00..008832623637 00..00882242 .000021815071155145 000001663459955799 65 10500 33 00.003820606070 001l008s219s 000015520814181813 000003806561626279 REPORTSTCARTEIASTTEIDCAOLN A1N9A:L00YSIWSEDLNIESSDTAYFILE220AN03 SHAPIARQOU-AWTIILCK TSEELSETNAOSFTRNUOMR:MALFIUTLYL ODFATGA_Rate oss sm EE KURT SWSTAT P_VALUE SIGNIF 18 .003186676 -0.90046 0.43426 0.91952 0.12677 REPORTSTCARTEIASTTEIDCAOLN A1N9A:L00YSIWSEDLNIESSTDAYFILE22JANO3 ANLAELVYESNIESTEOSFTVAFROIRAGN_CREatOeN F- ULFLULLDATMAodeSlET -- Bffect DF LEVENE P=_VALUE SIGNIF Dose 5 0.7763 0.85454 The dnatoatewas found to be normally distribi.ted with equal variances. An analysis of variance will be performed. --~ - = Company Sanitized. Does not contain TSCA CBI H-25435; InflonuGeronwtchaend Growth Rateofthe Green AlgaSelenastrumcapricornutuns DuPont 11604 --~ STATISTICAL ANALYSIS LIST FILE REP"OARNTALCYRSEIASTOEFD OSNELE1N9:A0S0TRWUENDN-ESFDUALYL DA22TJAANO3 DOSESECO0,TOX6.2O5F, AQ1U2.A5T,IC 25S,ELE50N,AST1R0U0Mmg/L obs Class Levels Values 1 dose s 123456 G_Rate FROM FULL DOAVTEAR,ALWLeiFg-ThEtS-TNSO WFEOIRGAHTN,OVVaAriance Ratio=0.33 obs Effect NoaFm DeDnF Evalue ProbF 1 dose. 5 12 0.62 o.c8se LG_SRMaEtAeNSFR-ODMOSFEULLMEADNATSA,ADJWeUiSgThEtDs=FNOORWETIHEGHTE,FVFaErCiTaSncOeF SRaEtLiEoN=A0S.T3R3UM obs LEVEL LeMEAN se oor 1 2 0eas 00..008709906677 0.0o2o1z81899883333 1122 5i 21ss o0llo0s823266373 00o2o1281899883333 1122 -- 5s s1o00 oollooseo2o6s6o7 .ooooz211m8s%e8s3s3 1122 ESTIMATEUDSIDNOGSEALEPFHFAE=C.T0S5 &FDORUNNCEOTMTPARFIOSRONTSwoT-OsidCeOdNTRAOLLTERNATIVE G_Rate FROM FULL DATA, Weight-NO WEIGHT, Variance Ratio=0.33 Bstimate Du2-nsniedtetd SIGNIF p-value TGreosutp. Mean N DpoossEs QTuRaEDND DDOOSSEE 23-.11 DDOOSSEE 45--11 DoSE 6-1 o0l.s5ez0s763.| . o0lls8sssa21e5 00ll007833066373 33 00..9999076586 00..008820206070 33 0.97113 0l0s2667 3 -- Te Company Sanitized. Does not contain TSCA CBI `FG-r2o5w4t3h5R:atIenoflfutehnceeGorneeGnrAowltgha aSenldenastrumcapricornutun DuPont11604 ~ STATISTICAL ANALYSIS LIST FILE REPOARNTALCYRSEATTSEDOFONECO1T9O:0X0USWIENDGNESADLAPYHA=.2025JANO3 FULL`TEDSATTA aJnodncLkohweeesrte TreDnosdesTestthroounghDoDseose0 100 mg/L Analysis of G_Rate K2CeyTS JONCKHESRE STATISTIC COMPUTED WITH TIE CORRECTION PCLCUFBCF131S4CPW-IVTAHLUECONFOTRINUUPIWTAYRDCOTRRREENCDTION PACTOR PP-IVDANLCUPESISARPE-VFAOLRUETIFEO-RCODRORWENCWTAERDD TTERSETNDWITH CONTINUITY CORRECTION PACTOR SIGNIF RESULTS ARE FOR A THO-SIDED ALTERNATIVE HYPOTHESIS Jone zc 2cce PluRCF PIDNCF SIGNIF 73 0.5013542 0.53992 0.2946261 0.676242 NJoontcekheere test results are included in the summary table. tGoroauplmienaenasr tsrheonudldbebeforeexamtirneendd tteostcherceksulftosr alraeck-aocfce-ptietd. ~ --~ 7 `CompanySanitized.Dossnot conTtSaCAiCanl