Document 3N5XmpoaNvwRykvzddgvOJMm3

AR226-3080 DuPont HLO-1997-00636 TRADE SECRET $ftHlym Toxicity ofH-22387 to the Freshwater Alga, Selenastmm capricomutum HMfceH Laboratory Project 8P Haskell Laboratory Outside Report No. 1997-00636 Teat Guidelines Organisation for the Economic Co-operation and Development(OECD) Guideline for Testing Chemicals: 201 EU Commission Directive 92/69/EEC, Method C.3 U.S. EPA (FIFRA) Subdivision J. 123-2 Autbon Timothy J. Ward Jeanne P. Magazu Robert L. Boeri Study Initiated On May 2,1997 Study Completed On September 17,1997 T.R. Wiibury Laboratories, Inc. 40 Doaks Lane Marblehead, Massachusetts 01945 Submitted lx El. du Font de Nemours and Company Wilmington, Delaware 19898 U.S.A. T.R. Wiibury Study Number) 1 of 41 "CWCST" DuPontHLO-1997-00636 THIS PAGE RESERVED FOR SPECIFIC COUNTRY REQUIREMENTS T.R. Wilbury Study Numbei1------| 2 of 41 Company Sanitized. Does not contain TSCA CBI DuPont HLO-1997-00636 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT This study was conducted in compliance with EPA FIFRA Good Laboratory Practice Regulations (40 CFR 160), EPA TSCA Good Laboratory Practice Regulations (40 CFR 792) and OECD Principles of Good Laboratory Practice (OCDE/GD(92)32). Stabilityof the test substance under exposure conditions was assumed but not verified. Deviations from the protocol are listed in Section 7.0. Submitter: E. I. du Font de Nemours and Company Sponsor: DuPont Specialty Chemicals Company Representatives: c ^ ^ y ^ ( . StudyDirector aro (yoauthor Jeanne P. Magazu T.R. Wilbury Laboratories, Inc. ^ w Date w^^y^ C. ^ir^u^f- fffli^l^ Sponsor RepresentativeDate Guat-Lian C. Kreamer E. I. du Font de Nemours and Co. Registration Manager Date E. I. du Font de Nemours and Co. T.R. Wilbury Study Number |^H---- 3 of 41 Company Sanitized. Duns nul contain TSCA CB) DuPont HLO-1997-00636 QUALITY ASSURANCE STATEMENT Submitted by: T.R. Wilbury Laboratories, Inc. 40 Doaks Lane Marblehead, Massachusetts 01945 9111 Wilbury Laboratories, Inc., study number "Toxicity ofH- T.R. 22387 to the Freshwater Alga, Selenaslrum caprscornufum", was audited by die T.R. WilbmystLaanbdoarradtoorpieesraQtiunagliptyroAcsesduurraensc,eanUdnitapfoprliccaobmlepGliaonocde wLaitbhortahteorpyroPtoraccotilc, es (U.S. EPA. 1992 and OECD, 1992). Quality audits were performedand the findingsreported to T.R. Wilbury assurance Laboratories management and the study director on die following dates: Audit Date Reported to Study Director Reported to Management Protocol: In-life: Raw Data/Draft: Final Report: 03/19/97 05/20/97 06/02/97 06/13/97 09/17/97 03/19/97 05/20/97 06/02/97 06/13/97 09/17/97 03/19/97 05/20/97 06/02/97 06/13/97 09/17/97 ^^/^L^. ^ ^ Arthur P. Paradice Quality Assurance Officer T.R. Wilbury Laboratories, Inc. T.R. Wilbury Study Numbe 4 of 41 S'W'^ROTrtair^reCACBr Prepared by: CERTIFICATION DuPont HLO-1997-00636 /A ^im .S-.t_u_d--y D-i.re-c---to.,,r_a--n.d-^Coia/ uthor Aquatic Toxicologist / h ^ A L C ^ ^ / / ^ ^acqueline -__^__-- M. Nevius Biologist M-n^z /^uMaa^ for:/Peter L. Kowal^O Aquatic Toxicologist W7 ^u^Ma^AA. w^? fon Jennifer A. Ma Biologist 7 I / ^ ^ Robert L. Boeri ^.p-^ Coauthor ^ Q ^ ^ - Ti&iothyJ. Ward Ahthor T.R. Wiibury Study Numbc^BBf 5 of 41 ^m^^fflffil.llliiiiaiiiiiiniiii.ii.ni.li TABLE OF CONTENTS DuPont HLO-1997-00636 SECTION: Title Page This Page Reserved for SpecificCountry Requirements Good Laboratory Practice ComplianceStatement Quality Assurance Statement Certification Table of Contents Index of Tables and Figures Study Information 1.0 Summary 2.0 Introduction 3.0 Methods and Materials 3.1 Test Substance 3.2 Dilution Water 3.3 Test Organism 3.4 Toxicity Testing 3.5 Statistical Methods 4.0 Results and Discussion 5.0 Conclusion 6.0 Retention of Records 7.0 Protocol Deviations 8.0 References APPENDICES: Appendix 1. Description of the Freshwater Medium Appendix 2. Protocol and Amendments T.R. Wilbury Study Numbef|||f PAGE 1 2 3 4 5 6 7 8 10 11 11 11 11 11 13 15 16 17 24 25 26 27 30 6 of 41 Company finiti7pd rinoc DuPont HLO-1997-00636 INDEX OF TABLES AND FIGURES Table Chemical characterization of a representativesample ofdeionized 12 1. water used to formulate medium for the toxicity test with the freshwater alga, Selenaslrum capricomutum, and H-22387. Table 2. Cell growth data from the toxicity test with the freshwater aiga, 18 Selwaslnimccipriconwtum,amdH-223Sf. Table Average specificgrowth rate and percent of control average 19 3. specificgrowth rate from the toxicity test with the freshwater alga, Selenaslrum capricomutum, and H-22387. Table Effective concentrations (EC25s and ECSOs) from the toxicity test 21 4. with the freshwater alga, Selenaslnm capricomutum, and H-22387. Table 5. Temperature measured during the toxicity test with the freshwater alga, 22 Selenasfnmi capriconmtwn, and H-22387. Table 6. pH values measured during the toxicity test with the freshwater alga, 23 Selenaslrum capricomutum, and H-22387. Table 1.1. Description of (he freshwater (nediuin used for Ae toxicity test with 28 test with the freshwater alga, Selenaslrum capriconwtum, and H-22387. Figure I. Growth of the freshwater alga, Selenastrum capriconwtum, exposed 20 to H-22387 for 120 hours. T.R. Wilbury Study Numbei 7 of 41 "ULIUL. IIUl fiontam I SCACBI" Test Substance: Synonyms/Codes: STUDY INFORMATION H-22387 DuPontHLO-1997-00636 HasteB Number: CAS Registry Number Note: The equal sips (=) Is oat & part ofthe chemical name, but indicates a word wrap. 22387 Composition: Known Impurities: Note: The equal sign (=) is not a part of the chemical name, but indicates a word wrap. T.R. Wilbury StudyNumber 1 8 of 41 DuPont HLO-1997-00636 STUDY INFORMATION (Continued) PhysicalCharacteristics: Stability: Clear, tan-colored liquid The test substance was expectedto be stable under the conditions of the study. Sponsor: E.I. du Pont de Nemours and Company Wilmington, Delaware 19898 U.S.A. Study Initiation/Completion: May 2. 1997/September17,1997 In-Life Phase Initiation/Compietion: Study Personnel: May 8,1997/June 9,1997 See Certification page T.R. Wilbury Study Numbeij 9 of 41 1.0 SUMMARY DuPont HLO-1997-00636 The acute toxicity of H-22387 to the freshwater alga, Setenastrwn capricornulum, is described in this report. The test was conducted for E.I. du Pont de Nemours and Company for 120 hours from May 30 to June 4,1997, at T.R. WUbury Laboratories, Inc., in Marbiehead, Massachusetts, it was conducted according to the protocol developed for T.R. Wiibury Study Numbefl----f|H-22387, a clear, tan-colored liquid, was supplied by the sponsor. The test was performed under static conditions with six concentrations oftes' substance and a dilution water control at 24 1C. Nominal concentrations of H-22387 were 0 mg/L (control), 6.5, 13, 25, 50,100, and 200 mg/L. No insoluble material was noted duringthe toxicity test. The dilution water was sterile enriched medium adjustedto a pH of 7.5. Algae used in the test were from a culture originally procured from the Culture Collection of Algae of the University of Texas at Austin, Texas, and acclimated to test conditions for more than 14 days at T.R. Wiibury Laboratories. Cell counts were made daily using a hemocytometer. Nominal concentrations of H-22387 were used for all calculations. Exposure of Selenastrum capricomuttim to H-22387 for 120 hours resulted in effective concentrations, EC25 and EC50, of 44 and 64 mg/L, respectively, when calculated using the number ofcells/mL, and 100 and 110 mg/L, respectively, whc.i calculated using the average specific growth rate. The 120-hour no observed effect concentration (NOEC) was 6.5 mg/L when calculated using the number of celIs/mL, and 13 mg/L when calculated using the average specificgrowth rate. T.R. Wiibury Study Number--------R 10 of 41 ^"r^r ^"^ii Bir.iiiiiiiini^iKH|||iifm)ff^gyir--im iiiiMBJi DuPontHLCM 997-00636 2.0 INTRODUCTION The purpose of this study was to determine the 24, 48, 72, 96, and 120 hour effective concentrations (EC25 and EC50) and the 120 hour no observed effect concentration (NOEC) ofH-22387 on the freshwater alga, Seletwstrum capriconwtwn, exposed under static conditions. 3.0 METHODS AND MATERIALS 3.1 TEST SUBSTANCE: The sample of H-22387 used during the study (T.R. Wiibury sample number^f^was "H-22381jmj|| delivered to T.R. Wlbury Laboratories on April 25,1997. It was contained ina100mL clear glass bottle that was labeled with the following information: A--------| 4-23-97, 107.4 g NET, 262.3 g gross." The test substance was tested as received, without correction for percent purity. The sample of H-22387, a clear, tan-colored liquid, was shippedfrom DuPont Central Research and Development, Haskell Laboratory for Toxicology and Industrial Medicine, Elkton Road, P.O. Box 50, Newark, DE 19714-0050, in a cardboard box at ambient temperature. Prior to use the sample was stored in the dark at room temperature (approximately 20 2C). Unused test substance is returned to the sponsor. 3.2 DILUTION WATER: Water was carbon-filtered tap water passed over a mixed resin bed deionizer (Table 1). Appropriate quantities of reagent-grade chemicals (Table 1.1 in Appendix 1) were added to the water to prepare a sterile enriched medium (U.S. EPA, 1978; T.R. Wlbury Standard Operating Procedure number 6); pH of the medium was adjusted to a target pH of 7.5 with 0.1 N hydrochloricacid (VWR Scientific Products, lot# 9608091) prior to use. The medium was used for acclimation of test organisms and for all toxicity testing. The medium contained less than 10 mg/L particuiate matter and a total organic carbon concentration of 4.1 mg/L. 3.3 TEST ORGANISM: Algae used for the test (Selenaslrum capricormitum, UTEX 1648) were from a culture originally procured from the Culture Collection of Algae at the University of Texas at Austin, Texas, and delivered to T.R. WIbury Laboratories on January 30,1997. T.R. Wilbury Study Number j f f t 11 of 41 aSatUjaanv^nffi>tiemi!iljii[j)i! "TflSBT" DuPont HLO-1997-00636 Table 1. Chemical characterization of a representative sample ofdeionized water used to formulate medium for the toxicity test with the freshwater alga, Selenastnim capricornnfum and H-22387. Parameter' Unit of Measurement Litnitof Q^^ntitetiQBi Measured Value Metals Aluminum Arsenic Boron Cadmium Calcium fjhnfwmim Cobalt Copper lion Lead Magnesium Mercury Nickel Potassium Silver Sodium Zinc Chloride Fluoride Total Phosphorus Organochlorine Pesticides Toxaphene Oiganophosphoms Pesticides Coumaphos Merphos Naied PCBs mg/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L Mg/L mg/L rog/L nig/L mg/L mg/L mg/L mg/L mg/L mg/L re^L P8/L P8/L M8^ pg/L pgfl.- p^L 0^0 0.010 0.025 0.00050 0.20 0.0030 0.0020 0.0040 0.10 0.0030 0.050 0.00020 0.0050 0.!5 0.0020 0.60 0.020 1.0 0.10 0.050 0.3 4 0.5 1.0 1.0 1.0 1.0 ND2 ND ND ND ND ND ND ia> ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ' Parameters were measured in deionized water that was collected during August, 1996 and analyzed by Lancaster Laboratories, Inc.. as part of routine water quality testing. 2 ND = not detected at or above the limit ofquantitation. T.R. Wilbury Study Numbe| 12 of 41 DuPont HLO-1997-00636 The algal culture was transferred to sterile enriched medium identical to medium used for this test and maintained at test conditions for at least 14 days before the definitive test. The subsampleof algae used to inoculate medium at the start of the definitive test came from a 7-day old culture. Identification of me original culture was verified using an appropriate taxonomic key. 3.4 TOXICITY TESTING: A range-finding test was conducted from May 8 to 13, 1997 with a control and four nominal concentrations ofH-22387 (0.10, 1.0, 10, and 100 mg/L). A stock solution with a nominal concentration of 100 mg/L was prepared by bringing 0.025 g of test substance to a total volume of 250 mL with algal medium. Appropriate amounts of this stock solution were added to algal medium to prepare the test concentrations. Test vessels during this test were loosely covered with glass beakers. Insoluble material was not observed at any time during the test. At the conclusion of the test, the number of algal cells/mi at all tested concentrations was more than 90% of the control growth. A definitive test was conducted from May 15 to 20, 1997 with a control and five nominal concentrations ofH-22387 (65, 130, 250, 500, and 1.000 mg/L). A stock solution with a nominal concentration of 1,000 mg/L was prepared by bringing 0.500 g of test substance to a total volume of 500 mL with algal medium. Appropriate amounts of this stock solution were added to algal medium to prepare the test concentrations. Test vessels during this test were loosely covered with glass beakers. Insoluble material (white particles) was observed at 250 and 500 mg/L at 72, 96, and 120 hours. No other insoluble material was noted during the test. At the conclusion of the test, the number of algal cells/mL was: 90% of the control at 65 mg/L, 32% of the control at 130 mg/L, and <1% of the control at 250, 500, and 1,000 mg/L. The test was repeated at lower concentrations because a NOEC could not be calculated and growth at all tested concentrations was <50% of the control growth at 24, 48, and 72 hours, making EC50 calculation impossible at those times. The final definitive test was conducted from May 30 to June 4, 1997, at 24 1C with six concentrations of test substance and a dilution water control. A 200 mg/L stock solution was formulated by bringing 0.100 g of test substance to a total volume of 500 mL with algal medium. Appropriate amounts of this stock solution were added to algal medium to prepare the test concentrations. Nominal concentrations of the test substance were 0 mg/L (control), 6.5,13,25, 50,100, and 200 mg/L. No insoluble material was seen in any test vessel during the test. Algae were randomly distributed among three replicates of the control and each test concentration at the rate of approximately 10,000 cells/mL. The test was performed in 250 mL glass Erienmeyer flasks (typically approximately 80 mm bottom diameter x 130 mm height) T.R.Wiib. y tucly Number g m ^ 13 of 41 Comn^ny RanitiT0^ n^"~ r-^imtain TSCA OB] DuPont HLO-1997-00636 that contained SOmL of test solution. The test solution depthwas approximately1.5 cm. Test vessels were loosely capped with inverted glass beakers and randomly arranged on a rotary shaker adjusted to 100 rpm in an incubator during the test (a random numbers table was used to select the location for each vessel and vessels were repositioneddaily). A 24-hour lightand 0-hour dark photoperiodwas automaticallymaintained with cool-white fluorescent lightsthat provideda fightintensityof approximately120 ^Em/n^sec. The number of algal cells/mL in each test vessel and die occurrence of relative size differences, unusual cell shapes, colors, flocculations, adherence of cells to test containers, or aggregation of cells were determined visually with a hemocytometer by means of direct microscopic examination. Cell counts were made and recorded at 24,48,72,96, and 120 hours. The measured concentration of test substance in test medium was not determined analytically, but 25 mL samples were collected from each solution prior to distribution to replicate test vessels at the start of die final definitive toxidty test. These sampleswere placedin 40-mL amber glassbottles and stored in a refrigeratorto be analyzedif needed to verifytest substance concentration at the test start (sampleswere not analyzed). Temperature of die incubator was measured and recorded daily(Ever ReadyThermometer Company, Inc., West Haterson, New Jersey, thermometer number 2968), and pH (Beckman Instruments, foe., Fullerton, California, model pffl 12 meter, instrument number 144 pHT5) was determined in each test vessel at the beginningand end of the test and at 72 hour's. The temperature in a representativevessel of water incubated with die test vessels was continuouslyrecorded. A determinatior of whether toxic effects were algistatic or algicidal was performed at the conclusion of the toxicity test. After 120 hours of testing, 0.5 mL of solution from each 200 mg/L test vessel were combined into one vessel containing 100 mL of fresh medium. The resulting solution was incubated under test conditions for 120 hours. 3.5 GROWTH EFFECT EVALUATION: The mean cell density values at each sampling inter* al for each concentration are expressed relative to the control. The percent of control growth is calculated according to the following formula: %Control = T -------- x 100 C where C is equal to the mean count in that interval's control sample and T is the mean count in that interval's treatment sample. Numbers greater than 100 indicate stimulation of growth. T.R. Wilbury Study NumbeAflHB^ M of 41 Company sanitized." Does not contain TSCA CBI DuPont HLO-1997-00636 The average specie biowili raic for sacli time interval is calculated using the following formula: Average SpecificGrowth Rate' In N-In No 4 where t is the time of observation measured from the initiation of the test and No and Nn are the initial and subsequentdensities (cells/mi) correspondingto the observation time. 3.5 STATISTICAL METHODS: Results of the toxicky test were. interpretedby using a weighted least squares non-linear regression technique described by Bruce and Versteeg (1992), when possible. The results w^ecaiojdMedu^ both the number of (^U^niLai^ the average specificgrowth rate. A chi-squere test was used to determine if data were normally distributed and Bartlett's test was used to determine if variances were homogeneous. The no observed effect concentration (NOEC), the highesttested concentration at which the cell densityor growth rate was not significantlydifferent from the control (a = 0.05), was determined usinga one way analysisof variance (ANOVA) and Dunnett's test (TOXSTAT 3.3; GuHey,rt al., 1990). It was calculated usingboth the number ofcells/mL and tile average specificgrowth rate in each test vessel at the end of the test. Nominal concentrations ofH-22387 were used for all calculations. T.R. Wilbury Study Numbel--------1 15 of 41 W\l UihIUieU. mkm contain iyUA6&l DuPontHLO-1997-00636 4.0 RESULTS AND DISCUSSION The algal populationgrewcownetlrloal nadftewra1s2i0nhloougrsph(Taasbelger2o)wdthu,rirnegsutillteinfginianl danefianviteirvaegteesot.f limits. The incubator 6,W50a7t,e0r00qcueaHlisty/mtLhrinouthgehout the test w(Taasbwlei5th).inThaeccpeHptoafbtleest medium was decreased temperature ranged from 23.2 to 23.6C 13 mg/L at the beginningof die test by the test substance at concentrations above (Table 6). Biological data generateddbuyrtihneg athceutdeetfoinxiitcivitey ttoexsitcwitiythteHst-2is23ill8u7stararetepdrienseFnigtuerdein1T. abTlhees ceUs/mL are 44 mg/L and 212a0n-dho3uranEdCa2lg5aalgnrdowECth50 calculated using the nuEmCb2e5r aonfd EC50 calculated using the 64 mg/L H-22387, respectively,and the 120-hour 100 mg/L and 110 mg/L H-22387, respectively(Table 4 ) . average specificgrowth late are unusual cell shapes, colors, Hoccuiations, adherence of cells No effects (size differences, aggregationof cells) were noted during the test. The 120-hour to test NOEC containers, is 6.5 mg/L or H-22387 when calculated using the number ofceUs/mL. and 13 mg/L H-22387 when calculated using the average specific growth rate. algistatic/algicidtaelst set up for 120-hours at the termination of the final During the definitive algae increased from <10,000 ceUs/mL to 308,000 cells/mL, indicatingtAhaant the test, effect of H-22387 at the highest tested concentration was algistatic rather algicidal. T.R. WiSbury Study Number ( 16 of 41 T: uoes not contain TSCA CBI 5.0 CONCLUSION DuPontHLO-1997-00636 Hie 120-hour EC25 and EC50 calculated using the number ofceUa/mL are 44 mg/L and 64 mg/L H-22387, respectively, and the 120-hour EC2S and ECSO calculated using the average specific growth rate are 100 mg/L and 110 mg/L H-22387, respectively. No effects (size differences, unusual cell shapes, colors, flocculations, adherence of cells to test containers, or aggregation of cells) were noted duringthe test. The 120-hour NOEC is 6.5 mg/L H-22387 when caicuiu.ed using the number of ceils/mL, and 13 mg/L H-22387 when calculated using the average specificgrowth rate. T.R. Wilbury Study NumbergM^^ 17 of 41 -Company Sanitized. Duub not contain TSCA UBI DuPontHLO-1997-00636 Table 2. Cell growth data from the toxidty test with the freshwater alga, Selenastrwn ccpricommwa, and H-22387. Nominal Number ofCelb per MUmitor Concentration of H-22387 Hour of Exposure (mg/L) Rep. 0 24 48 72 96 120 0 (control) 6.5 13 25 50 100 200 1 2 3 mean 1 2 3 mean % of control 1 2 3 mean %ofcoat-"l 1 2 3 mean % of control 1 2 3 mean % of control 1 2 3 mean % of control 1 2 3 msan % of control 10,000 <10,090 10,000 <!0,000 10,000 16,000 10,000 <16,000 10,000 10,000 10,000 10,000 100 00,000 <10,000 <10,000 <10,000 <63 10,000 10,000 10,000 10,000 100 <10,000 <10,000 <10,000 <10,000 <63 10,000 10,000 10,000 10,000 100 <10,000 <10,000 <10,000 <10,000 <63 10,000 10,000 10,000 10,000 100 <10,000 <10,000 <10,000 <10,000 <63 10,000 10,000 10,000 10,000 100 <10,000 <10,000 <10,000 <10,000 <63 10,000 10,000 10,000 10,000 100 <10,000 <10,000 <10,000 <IO,000 <63 120,000 112,000 80,000 104,000 968,000 544,000 844,000 785,000 6,020,000 5,480,000 5,420,000 5,640,000 7,200,000 6,180,000 6,140,000 6,507,000 40,000 18,000 60.000 39.000 38 712,000 922,000 798,000 811,000 103 4,520,000 5,940,000 4,709,000 5,053,000 90 5,220,000 6,520,000 5,560.000 5.767,000 89 10,000 12,000 11,000 11,000 11 808,000 748,000 780,000 779,000 99 4,260,000 4,540,000 4,610,000 4,470,000 79 6,180,000 4,920,000 5,140,000 5,413,000 83 <10,000 <10,000 <10,000 <10,000 <10 <10.000 <10,000 <10,000 <10,000 <10 422,000 308,000 322,000 351,000 45 110,000 136,000 146,000 131,000 17 4,520,000 4,200,000 3,880,000 4,200,000 74 1,240,000 1,620,000 1,440,000 1,433,000 25 4,660,000 4,780,000 5,200,000 4,880,000 75 3,800,000 3,700,000 3,229,000 3,573,000 55 <10,000 <10,000 <10,000 <10,000 <10 <10,000 <10,000 <10,000 <10,000 <1 338,000 466,000 482,000 429,000 8 1,680,000 1,300,000 1,720,000 1,567,000 24 <IO,000 <10,000 <10,000 <IO,000 <10 <10,000 <10,000 <10,000 <10,000 <1 <10,000 <10,000 <10,000 <i0.000 <1 <10,000 <10,000 <10,000 <10,000 <1 T.R. Wilbury Study Nur 18 of 41 Company Sanitized. Does nol-contatrrT'SCA'CBr Table 3. DuPontHLO-1997-00636 Average specificgrowthrate and percent of control average specificgrowth rate from the toxicitytest with the freshwater afga, Selenastrumcapricorsmttim,and H-22387. Nominal Concentration of H-22387 0 (control) 6.5 13 25 50 100 200 0.020 0.000 0.000 0.000 0.000 0.000 0.000 Averafie SpecificGrowtiliRate 96hour 0.049 0.028 0.002 0.000 0.000 0.000 0.000 0.061 0.061 0.060 0.049 0.036 0.000 0.000 0.066 0.065 0.064 0.063 0.052 0.039 0.000 i20heur 0.054 0.053 0.052 0.052 0.049 0.042 0.000 Nominal Concentration of H-22387 Percent of Control Average SpecificGrowth Rate 120 hour 0 (control) -- 6.5 0 13 0 25 0 50 0 100 0 200 0 - - 57 100 98 98 4 98 97 96 0 80 95 96 0 59 79 91 0 0 59 78 0 0 0 0 T.R. Wilbugy Study Numberi 19 of 41 company sanitized. Does not contain TSCA CBI 10,000.000 DuPont HLO-5997-00636 g 1l..u0u0u0,,000 u 8 100.000 10.000 --Control -- 50 mg/L --6.5 nig/L -*-13mg^L -^-100 mg^L ---- 200 mg/L -N-25mg^L Figure I. Growth of the freshwater alga, Selenastrum capricomutum, exposed to H. 22387 for 120 hours. T.R.WUbury Study Nun 20 of 41 ^!118R^?y^SS5?^"^SBM"SR5a^B!l'TSISCT?lB DuPont HLO-1997-00636 Table 4. Efiective concentrations (EC2S8aQdEC50B)fiom the toxkatytest with the fieshwattf alga, Selenastrum capriGonaifum, and H-22387. lime (hours) Value (tas/L) 95 Percent Coofidence Luoiti (mgtt.) Calculated U>ing die Number ofCeib per MlUUiSer EC50 24 48 72 96 120 <6.5 <6.5 24 37 64 EC25 24 48 72 96 120 <6.5 <6.5 15 23 44 Caleulated Using the Average SpecificGrowth Rate EC50 24 48 72 96 120 <6.5 6.8 52 100 110 EC25 24 48 72 96 120 <6.5 <6.5 47 97 100 -- -- 18 to 31 32to42 55 to 75 -- -- 10 to 21 19 to 29 35 to 56 -- <6.5to7.9 50 to 55 <6.5to>200 <6.5to>200 -- --. 44 to 50 <6.5to>200 <6.5to>200 T.R. Wlbury Study Numbe 21 of 41 LUUItJcinyJc^Lmd. Db^ riAt^htain ibLAbtJl TableS. DuPont HLO-1997-00636 Temperature measured during the twdcity test with die freshwater aiga, Selenastnim capricomutum, and H-22387. Hour of Exposure TemperBtureof Incubator (C) 0 23.2 24 23.5 48 23.3 72 23.6 96 23.5 120 23.4 T.R. Wilbury Study NumberI 22 of 41 Company Sanitized. Does not contain TSCA CBI Table6. DuPoiuM 1-1997-00636 pH values measured during the toxidty test with the freshwater alga, Selenoestmrn copriconmtum, and H-22387. Nominal Concentration of H-22387 (mg/L) 0 (control) 6.5 13 25 50 100 200 Replicate 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 Initial 7.5 7.5 7.5 7.5 7.5 75 7.5 7.5 7.5 7.3 7.3 7.3 7.1 7.1 7.1 6.7 6.6 6.6 5.9 5.8 5.7 PH 72 Hours 7.6 9.5 9.5 9.0 9.6 9.0 9.6 9.2 9.1 7.9 7.9 7.9 7.6 7.6 7.6 7.5 7.5 7.5 7.4 7.4 7.5 Final 7.9 8.9 9.2 9.8 9.6 9.8 9.6 9.3 8.5 9.8 9.9 9.4 10.1 8.8 9.9 10.0 9.3 8.4 7.9 7.8 7.9 T.R. Wilbusy Study Numfc 23 of 41 Company Sanitized. D( i TSCA CBI DuPontHLO-1997-00636 6.0 RETENTION OF RECORDS The find report will be tnnsfend to die ipomor and raw data will be initially archived at T.R. Wiibury Laboratories, Inc., and then traniforred to the ipomor. They will be mftintaiaed either at Hukdl Laboratory for Toxicology and InduBtrial Medicine (Nwrk. Delaware) or be archived at Iron Mountain (Wilmington, Delaware), fonneriy the DuPont Records Management Center (Wilimngton, Delaware). T.R. WiSbi.y Study Numbefl 24 of 41 Company Sanitized. Does not contain TSCA CBI DuPont HLO-1997-00636 7.0 PROTOCOL DEVIATIONS Protocol Devutioni The range-findingtest was conducted with more than 1 replicate of each concentration (to increase the accuracy of the toxidty determination). This deviation did not affect die outcome of the test and no other deviations occurred duringthe study. Company Sanitized. Does not contain TSCA CBt 8.0 REFERENCES DuPont HLO-1997-00636 Bruce, R.D., and D.J. Vsrsteeg. 1992. A Statistical Procedure for Modeling Continuous Toxicity Data. Environmental Toxicology and Chemistry. Vol.11. No. 10, pp. 1485-1494. EEC. 1992. Official Journal of the European Communities, Commission of European Communities, Annex to Directive, Part C: Methods for the Determination of Ecotoxicity. EEC 92/69 Method C.3. Algal Inhibitiora Test. Guuey, D D, AM. Boelter, and H L. Bergman. Physiology and Toxicology Laboratory, Wyoming. i&9J. TOXSTAT Version 3.3. Fish University of Wyoming, Lanunie, OECD. 1992. The OECD Principlesof Good Laboratory Practice. OECD Series on Principlesof Good Laboratory Practice and Compliance Monitoring. Number 1. Environmental Monograph No. 45. OCDE/GD(92)32. Paris. OECD. 1984. OECD Guidelines for Testing of Chemicals. Section 2: Effects on Biotic Systems. Method 201, A/gaGrowth Inhibition Test. Adopted4 Apri?1984. US. EPA 1978. The Selenastrum capricomutum Pasta. Algal Assay Boole Test. EPA- 600/9-78-018. Environmental Research Laboratory, Corvaliis, Oregon. U.S. EPA 1988. Pesticide Assessment Guidelines. Subdivision E, Hazard Evaluation: Wildlife and AquaticOiganisms. EcologicalEffects Branch. Hazard Evaluation Division, Office ofPesticide Programs, Washington,D.C. Draft, March 1988. U.S. EPA 1989. Pesticide Assessment Guidelines. Subdivision J. 123-2: Growth and reproduction ofAquaticPlants - Tier 2. EcologicalEffects Branch, Hazard Evaluation Division, Office of Pesticide Programs, Washington,D.C. U.S. EPA 1992. 40 CFR Part 160. Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA); Good Laboratory Practice Standards;Final Rule. U.S. EPA 1993. 40 CFR Part 797. Toxic Substances Control Act Test Guidelines;Final Rules: 797.1050. AlgalAcute Toxidty Test. T.R. Wilbury Study Number f B P 26 of 41 llllClffln^palnyllSg"^ti^a^~c^-^nt,.lfl^infa^?ftT<ilfllffiH^ DuPont HLO-1997-00636 Appendix 1 Description of the Freshwater Medium T.R. Wilbury Study Numbeijfl------fe| 27 of 41 DuPontHLO-1997-00636 Table I.I. Description of the freshwater medium used for the toxicity test with the freshwater alga, Selenastrum capricomutum, and H-22387. Culture medium is preparedas follows: add one mL of each stock solution in 1 through 4 in the order given to approximately 0.9 L ofautoclaved distilled or deionized water and then dilute to one liter. Adjust final medium pH to 7.5 0.1 with 0.1 normal sodium hydroxideor hydrochloricacid aa appropriate. 1. Sodium Nitrate Stock Solution: Dissolve 25.5 g NaNOa. Magnesium Chloride Stock Solution: Dissolve 12.164 gMgCla^SHzO. Calcium Chloride Stock Solution: Dissolve 4.41 g CaCI^HzO. Micronutrient Stock Solution: 0.1855 g HsBOa (Boric Acid) 0.0014 gCoCi;6H20 0.4154 gMnCl^HzO 0.0073 g Na2Mo04*HzO 0.0033 gZnCh 0.000012 gCuClz2H20t 0.3000 g Na2.EDTA*2HzO [Disodium (ethylenedinitrilo)tetr&acetic acid] 0.1598gFeCh6H20 Dissolve these in 1 L autoclaved deionized water. 2. Magnesium Sulfate Stock Solution: Dissolve 14.7 g MgSC^THzO in I L autoclaved deionized water. f Weigh out 0.1200 g ofCuClz^HzO and dilute to 1000 mL. Take I mL of this stock and dilute to 10 mL. Take 1 mL of this second dilution and add to the micronutrient stock. 3. Potassium Phosphate Stock Solution: Dissolve 1.044 g K.2HP04 in 1 L autoclaved distilled water. T.R. Wdbury Study Number! 28 of 41 Company Sanitized. Does not contain TSCA CBI DuPont HLO-1997-00636 Table 1.1. Continued. Description of the freshwater medium used for the tosicity test with the freshwater alga, Selenastrum capricwmitum, and H-22387. 4. Sodium Bicarbonate Stock Solution: Dissolve 15 g NaHCOa in 1 L autoclaved distilled water. Final concentration of macronutrients as salts and elemental concentration (mg/L) in distilled or deionized water. Compound Concentration fma/L) NaNO, MgClr6HzO CaCl22H20 MgS047H20 K2HP04 NaHCOa 25.500 12.164 4.410 14.700 1.044 15.000 Element N Mg Ca S P Na K C Concentration fnig/L) 4.200 2.904 1.202 1.911 0.186 11.001 0.469 2.143 T.R. Wilbury Study Number | 29 of 41 DuPontHLO-1997-00636 Appendix 2 Protocol and Amendments T.R. Wilbury Study Number i 30 of 41 DuPont HLO-1997-00636 Protocol Hde To EX duPoot donflooom 00 OxDnmy Huk^Libontuy Sir Toxicoiosy^Iului^ Medicine 1099 Biftoii Roil, PoROffieeBcK SO NewBk.Odwe 19714 TaOibcMy T.R.WiBxi^Ijboaioriei.Inc. 40Df)daLcne MuttdMid.llAMKteSEitB OI94S Study DbeetaR PelerLKowiIdd EittarttdTfitldllltlMltete: BitlmatfdExpateicati! Start Dfte: EstlmatfdExperfaBeateJTeralntisRBcte: Estimated Tert ComplcKoQDate; April, 1997 Miy.1997 Am^l997 July.1997 protocol Appmal SpOMQK V^^aH^O^s. C. ^WOw^- Study Dteter ff.h /f/^^l-^ .^ ^Vp- .Dtfe:__jea&dL2. T<stFalitvMtMgqneifc ^m^Q^^^-----' Ptfe: i9S/eZ/47 TA. Wilbury Study Kainbeifl------fe if^^^^^^^^R Pge 1 of8 T.R. Wlbury Study Number | 31 of 41 Company Sanitized. P"fs nnt nnntaip T'^'^/VCliH- DuPont HLO-1997-00636 1.0 PURPOSE To detenntoe the 24, 48. 72. 96. lad !20 hour efftedw eoaolnlia (EC2f and BCSO*) god the 120 hour no ^wxnA ffiKt ooaottntioa (NOBC) ofH423C7 to &bafma^amiftana90ieduadw^eoafitf^ 2.0 TESTSDB5TANCX 2.1 file test ib(taooe ud purityla&nattou wffl be iwSnd by 60 ipooMr. H22387 wffl be (tend in the duk t room t(Mn(un h tir ori^ul riffles coafiner. Htndtinj of the tett mgteriil wffl tr B MBoriiBM TMth iufiximtton eoatdiMd in (be voaiaMi^llB^ MUttUI SifttyOilte abut 4TJL Wlbmy Studttd Opflntinc Procedure SfoBter 3. Al uouMd tt ubMBM wffl bo Kitunied to the spixuor. 2.2 CdfiuIi&iM^BebuedwMNriBdegaeiiiin&iwrf&BtMtwlMUoee. Tut iubstuiMtocfciolulks^^bepnpdiB<liid(fiMdord^^ tkeuieofaioivaii.lfponaie. Ifafahmteiwii^t&aetisWxnKiudewilibe uiedind the conceotntiofioriotveai will aotoieMNi 100 mft&. 3.0 TEST SPECIES 3.1 IlM!ectSoaofeABpscteeadlberoute(tfdBilaintioeind(i!iiadbytba sponsor. Thb ipedei repreMnei modd Qitioi giMnQy ncofnizid u cppropritotoKiM3y!|Uitktoxioity. Hdlby&toMiMirfSprtaanwflNtt(UTBX ^64S)&omiii^toutce wffl to filed to UtitaUttoteiL b wffl (ie obulaed Sxim *ingl^ 5-10 djy old. i^bocw cuftwe fl bu bew cbinro to be ccfiveb' growing (i.e. ccpibto oflopritlnfe irowth whUo the tut period)bi ct teut 2 ubcuItiifeepriortotheftutoftbadi&Jtivetait 3^ TheoriiMwumctftbei^ou&untetlMUiiwii^afTaciuddMOulture wffllMldioahdIydttlUvailyoritaH. TtoltaftyabiflidTJL WObmyLilKKitoliM uibK tnaaaale fciy. 3.3 Pret^obsa^nd<id<tiC4Mise3^tbciouroe,hsadUfleprocedurei,Fe(x^d*!g, MdhctithoftMtoftmitnitwiIlbtitoofdtdgfldMportjd. 3.4 Algdcultumw^bemilitfdi^uaderiti^eoedEtieMiiriortoteitb&ii^ te (una witer nd it the OM iriiotop^Mi ud tiiiyenlim thit wB! hi uMd flar letting. Algte wifl be milnlglnedbdilutioawter at tettcaoditioM (temperature ind photoperio<0filr it leut 14 day prior to teltinf. T.R.Wi&my StudyNimbe^mi Pfge2ofC comjjaqY ganitized. qpRfi nnt nnnfairjJSfi^.iQgiliin DuPont HLO-1997-00636 4.0 ZXIOSirnKCONBITIONSEORTaXflXfINfSSVETEST 4.1 Ttotu>^.inieonte^aiidgitaeafflioiim(I^tMoBnba^Bbe ti'^^tfft^m^^wMn^ntfimtMrft^iiAitOT^iH^Mq^ 4.2 IXlutioawater^besteritesynt!Mticme&^ii^toBpHof7.Sl:0.1wA KiduB liydronde or hydrochloriccdd. Daknaed witcr uied to pcpue the (t BIflW^ ^nB 00 096 (u BIBUUmblfi COOOOBlXWOItt QapMbGMtol^Ifld iM WWItty dwMlerifBd Icut txrice yeuiy--fyJO fiiartooM. 4.3 Wcter teoipeiture vail be 24 2C. 'niatoB^eretiireoflbBiacubttorwiabe mauui^ uid recorded duly cod tha tampecstiire ffl & bofcor iocuiMied with (be tettveudfwiDbecosEiiBuotitlyresoBded. 4.4 Cu!tiituwfflbepl^oaaaort)itd<hitoa(5uitedtocpproximc^i00cy^ periaiauteduriugthett. AM6iiiiBfflaotbeo(iloynL 4.5 Photoperiodv^ beiutOffliticflfycoamiil^ adcdyu^to24lKHai^u^ Ohauisdiifcmthiaiitteaiityofcppfianmit^IZfitiEaitof'wc. Tha pbatopcffild adiigbtiBlen&tywillbenieaiureduidiecwfleddtfy. 4.6 neteftisidid!lbe2SOol^^Edam(^flula<^i^^immSed.^in 6eclcen(*UfluIawillbetbeuE!Nvo!uine). ViMbRDaoaUin SO01(2096 of E^ their volume of Boiutioo). vafef wiil be lifxial with <be Kudyluiabar. cooooBtN&cux, nd icpucttc ounffltfi 4.7 Text raedia will be nippUedto the vesedc oaSyat 6ft t^n^ng of the aqpoiuxe pQIOd. 5.0 RANGE HNDING TEST 5.1 Ttun!w!ljoftiie(u^fiii(^imalif6i(xiad^orpmua*ti^dtaiab6 ECSOi. Ilie etud eoacealiioai fbr tto debitiw tnt iria be iifaalffied h protocol inuodnicii& 5^ If & renga finding teft n coaducted, ibe dgu wiS be aqKoed to a ieriei of cooceaUBtioos of left abflinee D&K u. cootrol (d&ifi wxter without tot lutanoe) snd Mfoent coatnd (if required) under itctie eonfi&Mi. Bach UMttnent group win imtully comilt of cpproxifflfteiy 10,000 eeiliAal with ooe wfiacttc per coiccfllfBtiofl. TJLWi6my StudyNuaiber^BIIB P*flo3oft T.R. Wilbusy Study N-jmber^BHt 33 of 41 Company Sanitiged. DURA i IUL LUI lUll I TOSA CBI DuPont HLO-1997-00636 S3 f t MSU wM be iodiKriaiiiltc^ udgaid to 'naiiomly poiitiaMd niidora laiaben ute win be u*ed to doofe the tat veiid pcMidu^. web ( 6.0 DEFINITIVE TEST 6.1 Tliets*ewiabeacpoii6ri20bountoafriecoftlsafivBco!iffati^oac ^|m^^ of test lubriuc^ control, cat Miwat oooteol Gf nq^d) unfar itetic eoaditiou Ifce dOiilioB ftctor lMwaea eooeeati^olu'^ be u2.0. 6^ Etch Daiwoatod tiestzaest groiQ) MO axtiiliy tKnnnt of tpptwiiinntdly 10.000 ceilitalpertripEcateteitYBueL Eu2i.eeatnifFoupwilliailiiIlyeoi^or ppioxinittdy 10.001)cdistel ia cdi of 3 veueb. Tte lgu wffi be vaiSacdaaMtdf rvaswH to nadoa&* vwUoaeSteit veudi m(bb 30 "ff*" of tott subituce u^oo& TogmauaB^patUMidiBijMOOB^niHCT^TOlpeffBppirfBMiBfl duly. 63 ThepHwiUbeaicuu<^cz^ico(xkditiheb^iaoiEgBiideadaftiiete(tal after 72 houn (u uWSosaH afSsste vend auy be ect^lished for the 72 hour pH 6.4 Totdc!giieeBbaD(rOC)^pntiGiditeiiMawutlieitenle'Qii&elieiaei6iiB winbeiiieciuicdittbebegnmiBgoftbetett. 7fe*e dot wD be bduded ia die MBUCB|K)Xt. 6.5 TlieiuD6erofcdli^^bedc(cnmwdttidiecoidedifier2^48,72,96,ud 120 houn during the tat. The inate itfeDi/Bil ud ai^ dMaifities wBl be deteiinined imaascopicdiy uang i beaii^toffleter. 6.6 IntheteitcoacfalnliMwhaesra^itiBBau^iidib^dp^ wwSUk Vw be ttnBroinilfld ty ffffii^F11*^OlS OH OKtatt Mitrtioft fioal OMB BBpficite wiiiifiedimediaiaBBewteitvaM!MxiiDajtlfl1tocuthicfbrupto9dqx. Alter 2^d^(uioaaucro^()ocun^thaAureifaccmb^to*eeifdMi!g&te!ic efibct BU bocB XWBKB. 7.0 ANALY2ICAE-CBEMISIKY 7.1 '"lft nl^aIII1^ W^Cflt^tygng Wttf P^fftffI*^ r" If** inania ttqH Bflt ^ n^^fpin^q nlytic*By by TJL Wilbmy Lfbontofie*. Inc. Saoiple* wffl be aSaabed Sam wh lohition prior to fti dutriixition to replicate veueb t the stut of the definitive toxicity tat. Tbeie (tmplei wili be piwed in ttnber gteu t)0tt2es od refiigereted for pouible uufysa. TJLWiIfMiy StudyNumber l | f Pcge4ofC T.R. WiSbury Study Nijmberff 34 of 41 lOTiitM^ Bymmuu. uuyi, IIUL LUIIIJIII 1rcw'pot DuPont HLO-1997-00636 7.4 Nominal eoncenlraaoM ofteg tubaai^w^bau^ for ralcalgtOTfls. 8.0 DATA ANALYSIS AND STATB-nOULMETBOaS 8.1 The 24. 48. 72, 96;. and 120 hour EC25 and EC50 values end thar 95% cmifufanflo iatavdi wffl bo calculated by the weighted teut (({uucs noa^Mr i^wuioamelliodiifposabte. VdnM win bff ^'"I'^ml twifft. vww v^v fw immberofcdliMii)daMC(^tteMuiiBgtheve!Sgeip8dfi5KroitlirKt& The 120 hour BO oiMenwd effict ooaecaln&m (NOEC; the laziest tatod <ideacekvddeiefniiiidwithauid*nl8tluticdteshin<)uaB^escBa^ vfuoce) win be prowled, ifpouifde. 9.0 QUAIOTyASSUBANCE AND QUALnY CONTROL 9.1 The lest wiU be repeatedif coBlrotc^concentreika does ooiiaae^ by fit kf 16 tunes by 72 IKMBI aril flat togiri6mic by 120 lioan. 9.2 The test mB be conducted accordingto U.S. EPA and OEC0 Good LeboniOly PncticeStuiduds. 'nieiqMXtwiOeoiitiinidtteineattteiiingtoau&cL 9.3 The lest nd report will be aidited by the Quality AjiuruiceUait. 9.4 The Study Director wiU be reiponiibleibr (dewing cUdttasndfi^itesofidinaeigr changes to procedures outlined in this protocol in a protocol asaaaSiasfS. Protocol iT'endoMots wffl be tuppliedto the iponsor for pyrovd. 9.5 Raw data and & copy of the Old report will be archived at TJL WIlairy Labotatoiies,Ioc.,foratfea5t lOyeusoritwinbetfaiisfenedtothefpoanrfor archiving. 10.0 REPORT 10.1 An orin*l report will be prepitfedafter review of a draft rer 1 by the tponaor. The &! report wH be signedby the Study Director and staif that conducted the study and prepared the report, tad by represeatalne of die Quality Assurance Unit. TJLWiIbury StudyNumber|lf PcgeSof T.R. Wilbury Study Number | 35 of 41 r'r.mpc.ny g-^nitiTBTi Ponif.i.net ela-^-n'frT'?'^ CEL DuPont HLO-1997-0063 6 10.2 The^KpoHibouUcoB^aF*tle*atb661towiiifiiifim!ufioa: Tite pise. including 60 itudy title, dta requirement, author, stwty Spoiaor-HipplisdasSidaeaiSSjfcUrai Ci?ny) Good UKaitoly Pnc&e Stitoawt QiuIUy Auunace Sttiemeat Title ofContent* IfltixiductlOB md cuounuy octGit yooswsses 9W wsuSs Method* adMiteri^iii^idii^Bdeicriptioa of t method ofBUUCBi. lUgBiiti, incMinettott from mBg-fiqifag^A-feiiihietaan, B5 unil EC50vluc*.BidNOBCvalue Tcbubr nd ps{ibiBdjireseotttoia ofrtmlti KCsCSWCttS Appendices, inciuduigD W3tcr qmlity data 11.0 RECORDS TO BE MAINTAINED 11.1 Recordithu^ be miffitu^thnnifibout the study include: S*iq)Ie recent ad uufieiafiiniulioa Biolopcslobfefvxtions Witer qualitydita TJLWitbiuy StudyNumberfBy Ptgo6of8 T.R. Wlbuty Study NuniberlfV 36 of 41 nnmnany RanitiTorl nonr nr.t ^^^^ip TCJ;^ A ^^^ DuPont HLO-1997-00636 12.0 REFERENCES Bmce.ILD..ndVer8teeg. 1932. A St^cdProcsdure fix-Modeling ContioulU! ToridtyData. Bomroa.ToiAsaLiadaia. VoL 11. No. 10, pp. 1.485-1,494. t^ EC. 1992. Freih wxter ^ erowtb mhibhioa with &w?Md^m<x ai&^caftif uid Sslawstnwcaprtivnaitum. Officullounid ofthe European QmmKdiiet.L 383 A. Volume 35.29 December 1992. OECD. 1992. The OECD Pzinciplciof Good Libontmy Practice. OECD Series on Prindpla of Good Ltboratoty Practice and Compiiinge MoaltonBg. Number I. EaviroBmefltiMoi!OgripbNo.450ECEVGD(92)32. Pirii. OECD. 1934. OECD GuideluusibfTeituis ofOleniicil*. Soe&m2: EfiEestsoaBiotu: Synemt. Method 201. Alg* Growth Inhibition TeiL Adapted4 April 1984. U.S. Emiionnaitel Piotection Aseacy (EPA). 1988. fa&i4B AuueoKot GuideSoei. SubdbuioaE, Hutrd Ewbutioa: WildSA ial Aquciic Osysisiaa. Eootoffltal PKfnfpVCntfS* JPtrmBmDCl& iUtHtZ--U--uI aDXvMU&uBUB*l!O----u TSlJlawJrfWSOjMOk^ fUUO8IfCjB <GrKf" VrCtl^KilTSifSSS^Cu PI.SiijvuffMlUmBmSMSf WidBOgtoo,D.C. Dnfi. MHsh 1988. U.S.EPA1989. 40 CniPBt 792. Toxic Sije*t*a^CoaEro! Act (TSCA); Good Ldxintoly PfKtice Staagaitis; FiaaIRule. Feden! Reciter, Thiniity. Angmt 17,1989. U.S. EaviroBineate! Protectioa Agency (EPA). 1989. Pecdside AMegtment Guideline*. Subdivision J. 123-2: Growth md ReproductioB ofAautic Piantt - Tier 2. Ecological Effect Bonch, Husud EvahulioQ Diviaon, Office of Pesticide Prograns*. Washington,D.C. U.S. EauiOiimeBtdProtestionAgeacy (EPA). 1992. 400E>8.fUtieO. Fected Inectiiade,, aB^ai^tndRodtttic^ Act (HntA); Good Lri)Oto>yPtfa fide. U.S. EPA. 1993. 40 Cni POT 797. Toxic SubitensaCoBtMl Act THtGuideanes;RMl Bula. $797.1050. AigtIAciittToidiatyTwfc TJtWiBxay StudyNuinberf^Hf Pgc7of8 T.R. Wilbuty Study Nur 37 of 41 Oolnpaiiy oaiiiu^u."poeB"na'i'eantairt ibUAUBl' DuPont HLO-I997-00636 SUMMARY OF TEST CONamONS Accepttuto Species! VtsSssecoK Testt-fs: TestDwiausa: Dilution Water: Oultuie Acclimation Period: Texx^MndxifK rtotoperiod: NuafcerfCoaceatiioiB: DibiiioiirFector NuinberafRqriiutes: Tmt;l InfyiiliitnTestVeud^EK TestVeadVoiumf: Solvent: B5ectMeaBd; Test Concentration Analysis: Water Quality Measurements: AccepiflHiity Criteria: SeknastrvMcapricomitum OZCDQmde&M201 U.S. EPA(ISCA) 797.1050 U.S. EPA (FBIKA) Stdidhuion J. 1234 Stttic IZOiuun Sterile lyittheticmedia !4d*y* 24yc 24hounB^tafidOboundaric ApprozimiteiyiWpEia/m'iec SariDon AaiioiiUBSSeiyt.5-2.0 3 Appioximitely 10,000 ceilf per Enl 250 ml SOnil None,ifpois&Ie IfafoIventusequued.^lOOms/L ^QSBttSSfflEsXSSSSSSS'VfaLvSWSQ Gnywtfk xexpoDie None pHtt 0.72, and 120 houn.TOC and partiailrte iimtirf in the dilution wtertOboun CoalroIodlaaccatn&!oaBeueoF ttiettt 16 times by 72 houri and logarithmic growth occun by 120 bixa* Acceptabletempenlureruise TJL WilbiKySfaiayNmiAerj Page 8 of 8 T.R. Wlbury Study Number j 38 of 41 Company SanitizRf), noes not contain TSCA CBI DuPont HLO-1997-00636 AMENDMENT 1 T<xicityofH-22387tothePreihwttrA!gt.&fenannonc(pria EJ.du Pout deNoDMin and Coinii*ay Hikell laboratory for Toxicology aodladustriil Media 1099 EUcton Hold. Pott Office Box SO Newufc.Deiiwue 19714 Te<tiae facility TJl WilbiayLBbottonea, Inc. 40DodLiae MttOtehud, MiiMchutrttt 01945 Stuiiy Director: Peter L. Kowlria Changes: c ^ I. The highesttested concentration ofH-22387 ^ be 1/100 2. Concentralions ofH-22387 for the definitive toxicity test will be 0 (control), 65,130,250,500, and 1,000 mg/L BexxoB for Changes: Sponsor request (dunge 1) or to bclude infonnadoa mimifi fi^ the EffecUve Date for Changes: May 15,1997. Effect of Changes: None. Protocol Amendment Approval Sponsor WW^^i(ftwL^> CC ff^^egaai^w^t^^y---___________DDtfgKte sS"'/SQ/W. StudvDirector O^/i/^t^lJLC________ Pate: aS-ll.^ TestFaavM^pnenf ^^f QW^------ TJELWiIbuy Study Number tfHH| D^:^S^2------ hgelafl T.R. Wilbury Study Numbers------I 39 of 41 c.uiii|jyiiy syniiasa. uaesnai&Ariiain ihLALiBi DuPont HLO-1997-00636 AMBNOMENT2 Toxieity ofH-22387 to (be Fteshwater Alfia, Selenastrum apria Sponsor EJLduPoBtdeNefflouniad Company HaskeaLAbontotyforToncoiogyuidIaduitriiIMedicia 1099 EauwI&Otd.Pott Office Box 50 Newd^Ddnnre 19714 Tatine facility TJL Wilbmy Labonfories, Inc. 40D&>Lme MBbldead.MisndBBCtU 01945 Study Director: Peter L.Kowtlila Chinee: The Stwiy Director isJemne P. Mg2u. Re*fonforChnge: Peter L.Kowclddte oolong employed by Til Wlbutyl^ritorio. Eneetive Bate for Ctiange: May 30,1997. Effect of Change: None; Jeanne P. Nfsgizu u qualifiedand nubble to serve as StudyDirector. Protocol Amendmeal Approval Sponion G*\dt|flL. C.^fw^f__________ Pate: tifiS/9f. Study laMtorO^Ae^M?^f. / TO Date:_d^Zfl2. Testing FcilityManageniem: WW/^{/LS^---- Date: ^fSff/f) TJLWiBxily Study Nun&ia-l Page I of 1 T.R- Wiibury Study Number I 40 of 41 Company Sar|fe^, J^^s npt COIllgJnIgSi^^BL m DuPont HLO-1997-00636 AMENDMENTS ToxidtyofH-22387totlieFrethwterAlg,&&suflT<nayricooa((i( KJL, duPostdoNexQQuffiaDdCoinmxy HasicellLaboritoiyferTorico!ogyadlndutMrie*dl icine 1099 Eikton Road, Post Office Boic 50 Newark. Ddawu 19714 ^utiof l^acllfily T.R.WiIbuiyLabontories.Inc. 40Dod!iLue MmMdiad, MiMndinneff* 0194S Study OiKetor: JesfuieP.Ktegazu Change: Concentrations ofH-223E7 for the definitive toxioty tea wUl be 0 (cootrol), 6.5,13,25. SO, 100. md 200 nsg/L. Reason for Chsage: To coirest la COOT in & previous(imendnieat. Effective Date for Change: May 29,1997. Effect of Change: None. Protocol Aaendmeat Approval Sponsor.. ^lo^_ Ct^rtou^^ Dte:-ji^f3- Study Director Qp^fiMM^U Dte:_^eA&32- Testing Facffity Muagement: ,M!affi^^{i^-----. Date: 66/02^7 TJLWIbuy Study NiiinbeJ1fl|| Page 8 of 1 T.R. Wilbury Study Numberi 41 of 41 company gawaaeri..iiinnftiBiiiHait.Bfliiiitam.riiiM?iftiiiBiia^