Document Zn34XyMrqj8yMqYE65VOZNJLL

AR226-3080 DuPont HLO-1997-00636 TRADESECRET g ta frjig s Toxicity o f H-22387 to the Freshwater Alga, Seknastrum capricormtom Haskell Laboratory Project IP Haskell Laboratory Outside Report No, 1997-00636 Tggg_ryij!ng 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 (FIERA) Subdivision J. 123-2 A nfisaa Timothy J. Ward Jeanne P. Magazu Robert L. Boeri Study Initiated On May 2,1997 Study Com pleted O n September 17,1997 T.R. Wilbury Laboratories, Inc. 40 Doaks Lane Marblehead, Massachusetts 01945 E:I. du Pont de Nemours and Company Wilmington, Delaware 19898 U.S.A. T.R. Wilbury Study Number 1 o f 41 mfiwinvoywHUBniwewujiiBfgentmrrTwgwi r DuPont HLO-1997-00636 THIS PAGE RESERVED FOR SPECIFIC COUNTRY REQUIREMENTS T.R. Wiibury Study Numbe 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) mid OECD Principles o f Good Laboratory Practice (OCDE/GD(92)32). Stability o f 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 Pont de Nemours mid Company Sponsor: DuPont Specialty Chemicals Company Representatives: Study Director atm (goaauutthhor Jeanne P. Magami T.R. Wilbury Laboratories, Inc. < ?-/W Date Sponsor Representative Guat-Lian C. Kreamer E. I. du Pont de Nemours and Co. Registration Manager Date E. I. du Pont de Nemours and Co. T.R. Wilbury Study Number 3 o f 41 Com pany S anitized. D oes n u tco n tain TSCA CBI DuPont HLO-1997-00636 QUALITY ASSURANCE STATEMENT Submitted by: T.R. Wilbtuy Laboratories, Inc. 40 Doaks Lane Marblehead, Massachusetts 01945 T R Witbury Laboratories, Inc., study number "J0XICIty 22387 to the Freshwater Alga, Selenastrum cqpriconmtm , was audited bv the T.R. Wilbuiy Laboratories Quality Assurance Unit for compliance with the protocol, standard operating procedures, and applicable Good Laboratoty Practices (U.S. EPA 1992 and OECD 1992). Qwhty assurance audits were performed and the findings reported to TJR Wilbury flw a tn ri management and the study director on the 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 Arthur P. Paradice Quality Assurance Officer T.R. Wilbury Laboratories, Inc. T.R. Wilbury Study Numbel 4 o f 41 Prepared by: CERTIFICATION DuPont HLO-1997-00636 < W 7 .. JeaWe P Magazu (j 0 Study Director and Coauthor Aquatic Toxicologist Jacqueline M. Nevius Biologist A fo r/P eter L. Kowal* Aquatic Toxicologist foci Jennifer A. Mi Biologist o p i-f? . 7^7 Robert L. Boeri Coauthor T.R. Wilbury Study Nui 5 of 41 DuPont HLO-1997-00636 table of contents SECTION: Title Page This Page Reserved for Specific Country Requirements Good Laboratory Practice Compliance Statement Quality Assurance Statement Certification Table o f 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 o f Records 7.0 Protocol Deviations 8.0 References APPENDICES: Appendix 1. Description o f the Freshwater Medium Appendix 2. Protocol and Amendments PAGE 1 2 3 4 5 6 7 8 10 11 11 11 11 11 13 15 16 17 24 25 26 27 30 T.R. Wilbury Study Numb 6 o f 41 DuPont HLO-1997-00636 INDEXof tables and figures Page Table I. Chemical characterization o f a representative sample o f deionized water used to formulate medium for the toxicity test with the freshwater alga, Selenastrum capricormtum, rad H-22387. Table 2. Cell growth data from the toxicity test with the freshwater alga, Selenastrum capriconmtum, and H-22387. Table 3. Average specific growth rate and percent o f control average specific growth rate from the toxicity test with the freshwater alga, Selenastrum capricormtum, and H-22387. Table 4. Effective concentrations (EC25s and ECSOs) fromthe todci^test with the freshwater alga, Selenastrum capricormtum, and H-22387. Table 5. Temperature measured during the toxicity test with the freshwater alga, Selenastrum capricormtum, and H-22387. Table 6. pH values measured during the toxicity test with the freshwater alga, Selenastrum capriconmtum, and H-22387. Table U . Description o f the freshwater medium used for the toxicity tea with test with the freshwater alga, Selenastrum capriconmtum, and H-22387. Figure I. Growth o f the freshwater alga, Selenastrum capriconmtum, exposed to H-22387 for 120 hours. 12 18 19 21 22 23 28 20 T.R. WHbury Study Numbe fi mM 7 o f 41 Test Substance: Synonyms/Codes: DuPont HLO-1997-00636 STUDY INFORMATION H-22387 Haskell Number: CAS Registry Number but indicates a word wrap. 22387 Composition: Known Impurities: Note: The equal sign (=) is not a part o f the chemical name, but indicates a wand wrap. T.R. Wilbury Study Number 8 o f 41 Comnany P-f-- DuPont HLO-1997-00636 STUDY INFORMATION (C 'tinned) Physical Characteristics: Stability: Clear, tari-colored liquid The test substance was expected to be stable under the conditions o f the study. Sponsor: E.I. du Pont de Nemours Mid Company Wilmington, Delaware 19898 U.S.A. Study Initiation/Compietion: May 2. 1997/September 17,1997 In-Life Phase Initiation/Compietion: Study Personnel: May 8, 1997/June9, 1997 See Certification page T.R. Wilbury Study Numbei 9 o f 41 1.0 SUMMARY DuPont HLO-1997-00636 The acute toxicity o f H-22387 to the freshwater alga, d Hmcrrihed in this report. The test was conducted for E.I. du Pont de Nemours and Tomnanv for 120 hours from May 30 to June 4,1997, at T.R. Wilbury Laboratone^Inc., in Marblehead Massachusetts It was conducted according to the protocol developed for by the sponsor. h ' w s 1 ' * drar' " ~ 0,ored - `upptod The a w u performed under sudic conditions with tin concentmtions o f J * and a dilution water control at 24 1C. Nominal concentrations o f H-221S7 (control), 6.5, 13, 25, 50, 100, and 200 mgrt,. No insoluble durhg the toxicity test. The dilution water was stenle ennched meihum adjtsted to a p H of 7.5 A lgae used in the test warn firm .. culture ongmajy procured ftom the Culture Collection o f Algae o f the Univettity o f T e a s at A uam ^m tas, ami ^ ^ , t0 conditions for more than 14 days at T.R. Wdbury U boratonea_ CsB rnrns u e m a daily using a hemocytometer. Nominal concentrations o f H-22387 were used fo .calculations. Exnosure of Selenasmm capricarmttm to H-22387 for 120 hours resulted in effective ^ . rrn e nnfi pfcn of 44 and 64 mg/L, respectively, when c&Icubted using S S S S S n i f TM ^ l ^ ^ e c u U calculaed ush foe averaee snecific growth rate. The 120-hour no observed effect concentration (NOEC) was 6.5 mg/L when calculated using the number of cells/mL, and 13 mg/L when calculated using the average specific growth rate. T.R. Wilbury Study Number ..Gnmnanvftaniti imri-ft 10 of 41 TOvnvDT I DuPont HLO-1997-00636 2.0 INTRODUCTION The purpose o f this study was to determine the 24, 48, 72, 96, and 120 hour effective concpntrflti"" (EC25 and EC50) and the 120 hour no observed effect concentration (NOEC) o f H-22387 on the freshwater alga, Selenastrum capricormitum, exposed under static conditions. 3.0 METHODS AND MATERIALS 3.1 TEST SUBSTANCE: The sample o f H-22387 used during the study (T.R. Wilbury sample n u m b erg B N delivered to T.R. Wilbuiy Laboratories on April 25, 1997. It was containedhna*100mL clear glass bottle that was labeled with the M owing information: H - 2 2 3 8 | H H 4'23' 97' 107 4 g NET> 262 3 g gr0SS " ThtSSt substance waS tested as rf c j ^ ^ wThnut correction for percent purity. The sample o f H-22387, a clear, tan-colored liquid, was shipped from ^ ^ e n M Research and Development, Haskell Laboratory for Toxicology and Industrial Medicme Elkton Road, P.O. Box 50, Newark, DE *9714^050 in temperature. Prior to use the sample was stored m the dark at room temperature (approximately 20 2C). Unused test substance is returned to the sponsor. 3.2 DILUTIO N WATER: Water was carbon-filtered tap water passed over a mixed resin bed deionizer (Tablel).^ Appropriate quantities of reagent-grade chemicals (Table 1.1 m Appendix 1) were added to the water to prepare a sterile enriched medium (U.S. EPA, 197; T . R i uiy Standard Operating Procedure number 6); pH o f the medium was adjusted to a target pH o f 7 5 with 0.1 N hydrochloric acid (VWR Scientific Products, lot# 9608091) prior to use. The medium was used for acclimation o f test organisms and for all toxicity testing. The medium contained less than 10 mg/L particulate matter and a total organic carbon concentration o f 4.1 mg/L. 3.3 TEST ORGANISM : Algae used for the test (Selenastrum capricormitum, UTEX 1648) were from a culture originally procured from the Culture Collection o f Algae at the University of Texas at Austin, Texas, and delivered to T.R. Wilbury Laboratories on January 30,1997. T.R. Wilbury Study Number 11 of 41 P om patiyiSnnitiiMimlii.ficirnrr IJUVBI DuPont HLO-1997-00636 Table 1. Chemical characterization o f a representative sample o f deionized water used to formulate medium for the toxicity test with the freshwater alga, Selenastrum capricomutum and H-22387. Panunstcr* Unitof Measurement Limit o f Quantitation Measured Value Metals Aluminum mg/L 0.20 ND21 Arsenic <ng/L 0.010 ND Boron mg/L 0.025 ND Cadmium mgdL 0.00050 ND Calcium mg/L 0.20 ND Chromium mg/L 0.0030 ND Cobalt mg/L 0.0020 ND Copper mg/L 0.0040 ND Iron mg/L 0.10 ND Lead mg/L 0.0030 ND Magnesium ng/L 0.050 ND Mercury mg/L 0.00020 ND j) Nickel Potassium mg/L mg/L 0.0050 0.15 ND ND Silver mg/L 0.0020 ND Sodium mg/L 0.60 ND Zinc mg/L 0.020 ND Chloride Fluoride Total Phosphorus mg/L mg/L mg/L 1.0 0.10 0.050 ND ND ND Organochlorine Pesticides Mg/L Mg/L 0.3 4 ND M3 Oiganophosphorus Pesticides Merphos Naled PCBs Mg/L Mg/L Mg/L Mg/L Mg/L 0.5 1.0 1.0 1.0 1.0 ND ND ND ND ND 1 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 of quantitation. T.R. Wiibury Study NumbeJ 12 of 41 mSamfmmf UUILj IILL UUIliHMI I'lMTObffll 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 subsample of algae used to inoculate medium at the start of the definitive test came from a 7-day old culture. Identification of foe 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 o f H-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 o f test substance to a total volume of 250 mL with algal medium. Appropriate amounts o f 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 o f the test, the number o f algal cells/ml at all tested concentrations was more than 90% o f the control growth. A definitive test was conducted from May 15 to 20, 1997 with a control and five nominal concentrations of H-22387 (65, 130, 250, 500, and 1,000 mg/L). A stock solution with a nominal concentration o f 1,000 mg/L was prepared by bringing 0.500 g o f test substance to a total volume o f 500 mL with algal medium. Appropriate amounts o f 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 o f the test, the number o f algal cells/mL was: 90% of the control at 65 mg/L, 32% o f the control at 130 mg/L, and <1% o f 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% o f 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 oftest 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 o f 500 mL with algal medium. Appropriate amounts of this stock solution were added to algal medium to prepare the test concentrations. Nominal concentrations o f the teat 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 ofapproximately 10,000 cells/mL. The test was performed in 250 mL glass Erfenmeyer flasks (typically approximately 80 mm bottom diameter x 130 mm height) T.R. Wilb. j tudy Number 13 o f 41 Company Raniti^oW DuPont HLO-1997-00636 that contained 50 mL oftest soiutioa The test solution depth was approximately 1.5 cm. Test vessels were loosely capped with inverted glass beakers and randomly arranged on a rotary shaker adjusted to lOOrpm in an incubator during the test (a random numbers table was used to select the location for each vessel and vessels were repositioned daily). A 24-hour light and 0-hour dark photoperiod was automatically maintained with cool-white fluorescent lights that provided a fight intensity ofapproximately 120 pEin/m2sec. The number o f algal cells/mL in each test vessel and die occurrence o f relative size differences, unusual ceil shapes, colors, flocculations, adherence o f cells to test containers, or aggregation o f cells were dteimined visually with a hemocytometer by means of direct microscopic examination. Cell counts were madeand recorded at 24,48,72,96, and 120 hours. The measured concentration of test substance in test medium was not determined analytically, hut 25 mL samples were collected from each solution prior to distribution to replicate test vessels at the stmt o f die final definitive toxicity test. These samples were placed in 40-mL amber glass bottles and stored in a refrigerator to be analyzed ifneeded to verify test substance concentration at the test start (samples were not analyzed). Temperature ofthe incubator was measured and recorded daily (Ever Ready Thermometer Company, Inc., West Haierson, New Jersey, thermometer number 2968), and pH (Beckman Instruments, Inc., Fullerton, California, model pHI 12 meter, instrument nun*-144 pHT5) was determined in each test vessel at the beginning and end o f the test and at 72 hours. The temperature in a representative vessel of water incubated with the test vessels was continuously recorded. A determination o f whether toxic effects were algistatic or algicidal was performed at the conclusion o f the toxicity test. After 120 hours o f testing, 0.5 mL o f 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 GROW TH EFFECT EVALUATION: The mean cell density values at each sampling inter al for each concentration are expressed relative to the control. The percent o f control growth is calculated according to the following formula: T %ControI = -----------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 Numbe 14 of 41 Gompny sanitized. Does not contain TSCA CBI DuPont HLO-1997-00636 The average specie b.owib rare for each time interval is calculated using the following formula: Average Specific Growth Rate! In NB-In No where t. is the time o f observation measured from the initiation of the test and No and N,, tho initial and subseauent densities (ceBs/ml) corresponding to the observation time. 3.5 STATISTICAL METHODS: Results o f the toxicity test were interpreted J y jw ^ a S regression technique described by Bruce and Versteeg (1992), when possible. TOeresuitt were calculated using both the number ofcells/mL and t h e s f S l ^ a n d S t l e 'S 'test chi-square test was used to determine if data were normally was used to determine if variances were homogeneous. The no observed efiaft concentration (NOEQ, the highest tested concentration at which * ? * ^ J ? * rate was not significantly different from the control (a = 0.05), was determined usmg aone- way analysis o f variance (ANOVA) and Dunnett's test (TOXSTAT 3.3; GuUey, U aL, 1990). It was Hng both the number of ceUs/mL and the average specific growth rme m each test vessel at the raid o f the test. Nominal concentrations of H-22387 were used for all ralcuiations. T.R. Wilbury Study Numbe 15 o f 41 wiipiiwy wufliUeU. BIM1HOTSonfamT5CS 'HIT DuPont HLXM997-00636 4.0 RESULTS AND DISCUSSION The * 1 poputado, g - - J .- 6,507,000 cells/mL in the control after 120 hours;( ' ^ aojuior -rr (Table 6). * - . J l .. * . b ^ * <f . . 2 and 3 and W growth during the 120-hour EC25 and EC50 calculatedusing EC50 and using the average specific growth !ate are iw an * fl ! t| adherence o f cells tNo .te--st conwtain-ers, or aggregation o f cells) were noiea 5 T * 1 * * NOEC is 6.5 mg. H-22387 when calculated using t h e n ^ e r f c e U ^ . " S' H-22387 when calculated using the average specific growth rate. Dunng the a lg i^ a lg jc id id aldcidal. was algistatic rather than T.R. Wilbury Study Number 16 o f 41 ompnny Oaiiili/Bg'; Does not contain TSCA CBI 5.0 CONCLUSION DuPont HLO-1997-00636 Hie 120-hour EC25 and EC50 calculated using the number o f cells/mL are 44 mg/L 64 mg/L H-22387, respectively, and the 120-hour EC25 and EC50 calculated using average specific growth rate are 100 mg/L and 110 mg/L H-22387, respectively, effects (size differences, unusual cell shapes, colors, flocculations, adherence o f cell _ test containers, or aggregation of cells) were noted (hiring the test. The 120-hour NOEC is 6.5 mg/L H-22387 when calcuia.ed using the number o f ceils/mL, and 13 mg/L H-22387 when calculated using the average specific growth rate. T.R. Wilbury Study Number 17 of 41 -Company Sanitized: Dutib ilui contain TSC~CB DuPont HLO-1997-00636 Table 2. Cell growth data from the toxicity test with die freshwater alga, Selenastrmn capricormtum, and H-22387. Nominal Concentration ofH-22387 (mg/L) Rep. Number ofCell per Milliliter _______________ Hour ofExposure----------------------- 0 24 48 72 96 120 0 (control) 6.5 13 25 50 100 200 1 2 3 mean 1 2 3 mean % ofcontrol 1 2 3 mean %ofc(HrtM>l 1 2 3 mean % ofcontrol 1 2 3 mean % ofcontrol 1 2 3 mean % ofcontrol 1 2 3 mean % ofcontrol 10,000 <10,000 10,000 <10,000 10,000 16,000 10,000 <16,000 10,000 <10,000 10,000 <10,000 10,000 <10,000 10,000 <10,000 100 <63 10,000 <10,000 10,000 <10,000 10,000 <10,000 10,000 <10,000 100 <63 10,000 <10,000 10,000 <10,000 10,000 <10,000 10,000 <10,000 100 <63 10,000 <10,000 10,000 <10,000 10,000 <10,000 10,000 <10,000 100 <63 10,000 <10,000 10,000 <10,000 10,000 <10,000 10,000 <10,000 100 <63 10,000 <10,000 10,000 <10,000 10,000 <10,000 10,000 <10,000 100 <63 120.000 112.000 80,000 104.000 40.000 18000 60.000 39,0) 38 10.000 12.000 11.000 11.000 11 <10,000 <10,000 <10,000 <10,000 <10 <10,000 <10,000 <10,000 <10,000 <10 <10,000 <10,000 <10,000 <10,000 <10 < 10,000 < 10,0 00 < 10,000 < 10,000 <10 968,000 6,020,000 7,200,000 544,000 5,480,000 6,180,000 844,000 5,420,000 6,140,000 785,000 5,640,000 6,507,000 712,000 4,520,000 5,220,000 922,000 5,940,000 6,520,000 798,000 4,700,000 5,560,000 811,000 5,053,000 5,767,000 103 90 89 808,000 4,260,000 6,180,000 748,000 4,540,000 4,920,000 780,000 4,610,000 5,140,000 779,000 4,470,000 5,413,000 99 79 83 422,000 4,520,000 4,660,000 308,000 4,200,000 4,780,000 322,000 3,880,000 5,200,000 351,000 4,200,000 4,880,000 45 74 75 110,000 136,000 146,000 131,000 17 1,240,000 3,800,000 1,620,000 3,700,000 1,440,000 3,220,000 1,433,000 3,573,000 25 55 <10,000 <10,000 <10,000 <10,000 <1 338,000 1,680,000 466,000 1,300,000 482,000 1,720,000 429,000 1,567,000 8 24 < 10,000 < 10,000 < 10,000 < 10,000 <1 < 10,000 < 10,000 < 1 0,0 00 < 10,000 <1 < 10,000 < 10,000 < 10,000 < 10,000 <1 T.R. Wilbury Study Ni 18 o f 41 Company Sanitized. Boes notcontairrTSC^CBI DuPont HLO~1997-00636 Table 3. Average specific growth rate and percent ofcontrol average specific growth rate from the toxicity trat with the freshwater alga, Selenasirumcapriconmtum, and H-22387. Nominal o f H-22387 imn/Ll 24 hour Average Specific Growth Rate 48 hour 72 hour 96 hour 120 hour 0 (control) 6.5 13 25 50 100 200 <0.020 0.000 0.000 0.000 0.000 0.000 0.000 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 0.054 0.053 0.052 0.052 0.049 0.042 0.000 Nominal Concentration o f H-22387 (tm/L) PercentofControl Average Specific Growth Rate 24 hour 48 hour 72 hour 96 hour 120 hour 0 (control) 6.5 13 25 50 100 200 0 0 0 0 0 0 - --57 100 98 98 4 98 97 96 0 80 95 96 0 59 79 91 0 0 59 78 0 00 0 T.R. Wilbury Study Numberl 19 o f 41 company sanitized. Does not contain YSA l DuPont HLO-1997-00636 - --Control -* -6 .5 mg(L 13 mg/L --*--50 mg/L -- 100 mg/L -- 200 mg/L 25 mg/L Figure 1. Growth o f the freshwater alga, Selenastrum capricomutom, exposed to H-22387 for 120 hours. T.R. Wilbury Study Ni 20 o f 41 I WfflJSSny^anTBzeSTBoes'noRontam'fSSWJSF DuPont HLO-1997-00636 Table 4. Effective concentrations (EC25s and EC50s) fiora the toddty test with Time (hours) Value Ghgfc) 95 Percent Confidence lim its G htfL ) C alculated Using the Number o f Ceils per M illiliter EC50 24 48 72 96 120 <6.5 -- <6.5 . -- 24 18 to 31 37 32 to 42 64 55 to 75 EC25 24 48 72 96 120 <6.5 <6.5 15 23 44 -- -- 10 to 21 19 to 29 35 to 56 C alculated Using the Average Specific Growth Rate EC50 24 48 72 96 120 EC25 24 48 72 96 120 <6.5 6.8 52 100 110 <6.5 <6.5 47 97 100 -- <6.5 to 7.9 50 to 55 <6.5 to >200 <6.5 to >200 -- -- 44 to 50 <6.5 to >200 <6.5 to >200 T.R. Wflbury Study Ni 1 21 o f 41 '0te' rito fcritin i SUATIBI' DuPont HLO-1997-00636 TaMe5. Temperature measured during the toxicity test with the fteshwater Seknastrum capricamtum, and H-22387. Ii T.R. Wiibury Study Number 1 22 o f 41 Tables. DuPuw H i 0-1997-00636 pH values measured during the toxicity test with the freshwater alga, Selenastrumcapriconmtum, and H-22387. Nominal Concentration o f 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. Wilbury Study Ni 23 o f 41 Company-Sanitized:-Boes not contain TOCA GDI DuPont H LO-1997-00636 6.0 RETENTION O F RECORDS The final report will be transferred to the sponsor and raw data wiU be initially ardiived at T R . Wiibury Laboratories, Inc., and then transferred to die sponsor. They will be im intain^ldther at Haskell Laboratory for Toxicology and Industrial Medicine (Newark, Delaware) or be archived at Iron Mountain (Wilmington, Delaware), formerly the DuPont T.R. Wiibuiy Study Numi 24 o f 41 Company Sanitized. Does not contain TSCA CBI DuPont H LO -997-00636 7.0 PROTOCOL DEVIATIONS Protocol D eviations The range-finding test was conducted with more than 1 replicate o f each concentration (to increase the accuracy o f the toxicity determination). This deviation did not affect die outcome o f the test and no other deviations occurred during the study. T.R. Wilbuiy Study Numbe 25 of 41 Company Sanitized. Does not contain TSCA CBI 8.0 REFERENCES DuPont HLO-1997-00636 Bruce, R.D., and D.J. Verateeg. 1992. A Statistical Procedure for Modeling Continuous Toxicity Data. Environmental Toxicology and Chemistry. Voi. 11. No. 10, pp. 1485-1494. EEC. 1992. Official Journal o f the European Communities, Commission ofEuropean Communities, Annex to Directive, Part C: Methods for the Determination o f Ecotoxicity. EEC 92/69 Method C.3. Algal Inhibition Test. Gulley, D.D., AM. Boelter, and H.L. Bergman. 1950. TOXSTAT Version 3.3. Fish Physiology and Toxicology Laboratory, University of Wyoming, Laramie, Wyoming. OECD. 1992. Hie OECD Principles o f Good Laboratory Practice. OECD Series on Principles o f Good Laboratory Practice and Compliance Monitoring. Number 1. Environmental Monograph No. 45. OCDE/GD(92)32. Paris. OECD. 1984. OECD Guidelines for Testing o f Chemicals. Section 2: Effects on Biotic Systems. Method 201, AJga Growth Inhibition Test. Adopted 4 April 1984. jj U.S, EPA 1978. The Selemstrum capricorrmtum Frantz Algal Assay Bottle Test. EPA600/9-78-018. Environmental Research Laboratory, Corvis, Oregon. U.S. EPA 1988. Pesticide Assessment Guidelines. Subdivision E, Hazard Evaluation: Wildlife and Aquatic Organisms. Ecological Effects 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 o fAquatic Plants - Tier 2. Ecological Effects Brandi, Hazard Evaluation Division, Office ofPesticide Programs, Washington, D C. U.S. EPA 1992. 40 CFR Part 160. Federal Insecticide, Fungicide, and Rodenticidi Act (FIERA); Good LaboratoryPractice Standards; Final Rule. U.S. EPA 1993. 40 CFR Part 797. Tone Substances Control A x Ten Guidelines; Final Rules: 797.1050. Alga! Acute Toxicity Test. T.R. Wilbury Study Number gr 26 o f 41 to n ta any,Saiait.iaflduUar. DuPont HLQ-1997-00636 A ^ n d ix 1 Description o fthe Freshwater Medium T.R. Wilbuiy Study Ni 27 o f 41 DuPont HLO-1997-00636 Table 1.1. Description o f the freshwater medium used for the toxicity test with the freshwater alga, Selenasirum capricortmlum, and H-22387. Culture medium is prepared as follows: add one mL o f each stock solution in 1 through 4 in the order given to approximately 0.9 L o f autoclaved distilled or deionized water and then dilute to one liter. Adjust final medium pH to 7.5 0.1 with 0.1 normal sodium hydroxide or hydrochloric acid ss sppropristc 1. Sodium Nitrate Stock Solution: Dissolve 25.5 gNaNOj. Magnesium Chloride Stock Solution: Dissolve 12.164 g MgPhtfMdO. Calcium Chloride Stock Solution: Dissolve 4.41 g C a C fe ^ O . Micronutrient Stock Solution: 0.1855 g H3BO3 (Boric Acid) 0.0014 gCoCfeefiHaO 0.4154 gMnCbMHiO 0.0073 g Na2Mo04*H20 0.0033 gZnCfe 0.000012 gCuCl2*2H20 f 0.3000 g Na2.EDTA2H20 [Disodium (ethylenedinitrilo) tetraacetic acid] 0.1598 g FeCI26H20 Dissolve these in 1 L autoclaved deionized water. 2. Magnesium Sulfate Stock Solution: Dissolve 14.7 g MgS(>4*7H20 in 1 L autoclaved deionized water. f Weigh out 0.1200 g of CuCl2*2H20 and dilute to 1000 mL. Take 1 mL o f this stock mid dilute to 10 mL. Take 1 mL o f this second dilution and add to the micrdnutrient stock. 3. Potassium Phosphate Stock Solution: Dissolve 1.044 g K2HPO4 in 1 L autoclaved distilled water. T.R. Wilbury Study Number 28 o f 41 Company Sanitized. Does not contain TSCA CBI DuPont HLO-1997-00636 Table 1.1. Continued. Description o f the freshwater medium used for the toxicity test with the freshwater alga, Selenastrum capricornutum, and H-22387, 4. Sodium Bicarbonate Stock Solution: Dissolve 1S g NaHCOj in 1L autoclaved distill! water. Final concentration of macronutrients as salts and elemental concentration (mg/L) in distilled or deionized water. Compound Concentration (mg/L) NaNQj MgCl26H20 CaCl2*2H20 MgS047H20 K2HP04 NaHCOj 25.5QC 12,164 4.410 14.700 1.044 15.000 Element N Mg Ca S P Na K C Concentration fme/L) 4.200 21..920024 1.911 0.186 11.001 0.469 2.143 T.R. Wilbury Study Number 29 o f 41 L inH iWBoniuaiTBUi DuPont HLO-1997-00636 Appendix 2 Protocol and Amendments T.R. Wilbury Study Number 30 of 41 nnnT any n " - mrl"PT,'Tltiffl[fn Tf?fli*i f?nt Protocol Title DuPont HLO-1997-00636 E l duPotcteNemand Conway Ha^Ubortocyfi>rTdeoloayBrfIndus>*IMSidne 1099ESktonRoad, PoatOffleeBos 50 New**, Delaware 19714 Testing Faefity T.1LWiBuyUbootoriei, be. 40 Doutaf-we ^iWdiMi, M im rtm rTr 01945 StudyDirector: PeterL. Kowalski Estimated T o t M u latta Dale: Eatmated Sperimenta! StartDate: Estimated Esperimenti! TerrataatiwsDate: Estimated Tert Completion Date: April, 1997 May, 1997 Juki 1997 July. 1997 Protocol Approval 6W k^ C I - Btftt TJLW ilbuiy Study Ni iw osfotf*1 Page 1 o f8 T.R. Wilbury Study Number 31 o f 41 Company Sanitized. Do r s not nnntair TCCArC-W- DuPont HLO-1997-00636 1.0 PURPOSE To detennine the 24, 48, 72, 96, and 120 hour flhetfweanrantratk* (EC2Sa and BCSOi) tod the 120 hour do observed cffbet eoosoatratk (NOEC) of 323*7 to &fewcfl!rvweqfooi7iKftflnexposeduaderadsoooditton. 2.0 TESTSUBSTANCE 2.1 Hie test eubstanoe and purity to&matkiu win bo mppSed by the sponsor. H223S7 wfll be stored in the dide at room tampantun is the ori^nel Safins w n .w Handling of the teat materiel win be In taoorienae with la&rmetkiw emariM! in fl spoMOMuppItadMatariil SafltyOeU Shoes ndTJL WObuty swndmit Operating Procedure Number 3. Al unused teat adbataaea win bo returnedto the sponsor. 2.2 >vKim! onBcrninalfcanagfrailoetofthetaat aubessacc. Teat aubatennitodc solutionswinto pripaiid iddidiiiai ordiluiionwaterwhhott theuseofa solvent,ifpossible. Ifawlv b r e t ^ d ^ needandtheconcentrationofs ` 3.0 TESTSPECIES 3.1 Theselectionoftestspade*endtherouteofedsilnirintkaredmenraaadbythe sponsor. This specie* repraeante e modal eystara gansnfy neaiidaad aa appropriatetostudyaquatictoxicity. Haa%WMaeierojiiPBaaifllflflC #1648}ftoma tinglesoireewillbeusedto fn&totsthetaL Itwfflbeobtained froma single, 5-10dayold, fo-houseeutare that hi* beentownto beactively growing(.e. capable oflogarithmicsmith withintbeteat period) in at least 2 subculturespriortotheatarioftoedeflaUmtori. 3.2 TheoriginalsoureaoftbealgalcultureistbeUaivenityafTeaasandthecuteae eriBbeidenliSadbylbeUaivenityofTeaaa. TbekSeetfcywOiboverfiedatT.R. WllburyLaboratoriesusingatmaxwfflfekey. 3.3 Pretestobservationdataconcerningto*source, haadUagprocedures,receiptdate, andhealthofteatossanismawfflbenoocdedandreposted. 3.4 0AiflsrafmadetuwwaatevrraiBndbaetmthaeinstaamineed(dunntodperarsitoadticancdootdaMmopneenpiuriorthtaottievislt!hbadtuisaetdoifeinr teatins- AlgaewiUbemaintained IndUuttonwaterat taat coodhlons (temperature andphctoperiod)foratleast !4dayspriortotesting. T.R. Wi&utyStudy Page2ofl T.R. Wiibury Study Numbe^ 32 o f 41 DuPont HLO -1997-00636 4.0 0PO8BR*CONmON3 FORTHTDEHNSIIVE TEST 4.1 wiDbe 42 OiiKioQ water will be sterile synthetic media riputed to t pH o f 7.5 0.1 with odium Igdronde or hydrochloric acid. Defeated water uaed to prepare the meda will be fite of mewnfafc canoaomdoa c f p e ria to , red 1 ttedcaB y dusaclerizri (at least twice yearly asalyriB)p to ,to use. 4.3 4.4 Cultures will be plaeedon a otbctal shaker adjusted to apjHwmnately 100 cycle* per minute during the teat. Aeratine-wiHnotbe employed. 4.5 Photoperiod will be lutomiticalljr eootreffled andedjustedt 2 4 h stn Bgbtand 01iouid*itw ithaaiittea% ofpptosim tely IWiiBs&Aec. Thaphotopcriod and tight inteniity will be snessiredand recorded d^. 4.6 Tfaetest vessels will be 250 ml glass Erieaneysr flasks sapped with inverted gi** oeakaci (all flasks will be the same volume). Vessels wifletwtiin 50 id ffO K cif their volume o f solution). Badi vessel 3D botowfed sn ft the wdytamiber,, concentration, and r e c a te number. 4.7 Teat media will be applied to the vessel only st the beginning o f the spostile period. 5.0 RANGEFINDING TEST 5.1 T baresdtoofdw ni^findivbiw atiiitioB aiidlorprarioutM tiagdataw iabe used to aefecttha concentration nnge needed fly to e re te tfe n o f t e d efitte ECSOa. The actual eooceotratioos fbr the defitte teat wifl be identified in a protocol 5.2 If a range finding test ia conducted, the algae will be exposed to a series o f concentrations o f teat aubatanoe phis a control (dDodoa water wnthait iest substance) and a advent control (if required) under static oonfitioaa. Each treatment group wiU initially eootiit o f approximately 10,000 eeOsAnl with ooe replicate per concentration. TJLWiBwiyStudyN u n i b e r ^ g ^ Bage3ofS T.R. Wilbury Study Number 33 o f 41 CmRpany Snnitiged. Duieji ituluuntalTfTaCWlgBT DuPont HLO-1997-00636 s-3 Algae will be in&ahtiiaBtefy is 'nndotnly tact enab (a random numbenuNe willbe used to choosethe test vend ponfien). 6.0 DEFINITIVE TEST -6.1 The slgse will be exposed&r 120boutsto a seriesofat teed fiveeoosHtmlfcmi ***" * *A!Sae5L eMte* *4 * *** <w*k* CfrajuawO rater attic eondaioB*. the dihitiouftctar betweenconcentrations win be ippm S otu ty 13 6.2 Etch non-control treatment group all initially consist o f igam daatdv fP P ro ^sM y 1,000edWail ia each o f 3 vends. The algae wifi be mdiicriminatdy assigned to mndoraiypositiaacdtest vessels TMftM" 30 ntiautes o f te*t substance addition. ft.ri-.qjtn 6 3 The pH will be meswred end recoded at <he bcgmnmg and end o f the test ami after 72 bourn (an additiondrepficaWvessdffliiybeestablistiedfbr the 72 hour pH measurement). 6.4 Total orgaaic cariicn (TOn) xndprfeil, mjKwr in th rti-rib. yr*k^/- w ll be measured at the beg&miag o f die teat These data wffl be faadoded la the anal report 6.5 The number ofedb/hdwfil be determinedand recorded after 24^48,72,95, and 120 hours during the test The number o f cdli/ml and any abnormalities will be determined microscopically wing a hemacytometer. 6A fathetestcooceim lka when grawth h trorinuAy itfrixted abradd mri ikiom ic 7.0 ANALYTICALCHEMKTRY 7.1 The measured enncfltri<wmn f w in tett n^t h / I - - -- analytically by T Jt. Wfflwiy Laboratories, Inc. Samples wifi be eachsohition prior to its dimibution to replicate vessels at the start o f the definitive ioxidty test. These scnplet will be placed in amber glass bottles and refrigerated for possible analysis. Page 4 o f8 T.R. Wifbuiy Study Number 34 o f 41 DuPont HLO-1997-00636 7.4 DATAANALYSISANDSTATISTICALMETHODS 8.1 Thsi 24. 48, 72; 96, and 120 hour EC2S and ECSO valuea end their 95% confidence intervals wffl 1 calculated by the weighted leeet equine non4meer regresses method, ifpossibfe. ^ ffcf mjs&erofceQi/mlindasecoadtimeusingtheaveragespecificgrowthrate. The 120 hour no observed eflbct coneentnto i (NOEC; the highest tested concentration dm is not significantly different from die control at the 9SH confidencelevd determinedwithstsodaid juristical techniquessuchas analysisof variance)wifibeprovided,ifpossible. 9.0 QUALITYASSURANCEANDQUALITYCONTROL 9.1 Thetest willberepealedifcomic!ceilconcent* does not increasebyst least . 16timesby72bounorisnotlogarithmicby 120hours. 9.2 The test will be conducted accordingto U.S. EPAand OECDGood Laboratory PracticeStandards. Thereportwincontaina statement attestingto tfais&cL 9.3 Thetesi andreportwillbeauditedbytheQualityAssuranceUnit. 9.4 TheStudyDirectorwillberesponsiblefiwreviewingall dataandfijrrecordingany changes to procedures outlined in this protocol in a protocol amendment. Protocol en'endmentswill be suppliedto the sponsorfor approval 9.5 Raw data and a copy of the final report will be archived at T.R. Wilbury Laboratories, Inc., forat least 10yearsor it mil be transferredto the sponsorfor archiving. . 10.0 10.1 Anoriginal reportwillbepreparedafterreviewofa draft rep :t faythe sponsor. The final report willbesifted bythe StudyDirector and staJfthat conductedthe study and prepared die report, rod by brepresentative of the Quality Assurance Unit. TiL WilburyStudy Pages of8 T.R. Wilbury Study Number 35 o f 41 flnm pany Sanitborl nTt fimtijn T S OA o p l DuPont HLO-1997-00636 10.2 lisefinalreport shouldconsistofat leastthefollowingafimnatioa: Title ptge, inrJiiAlng the itudy title, hu requirement, author, study catcpledonfate,tcatjngftcility,andsponsor Sponsor-suppliedconfidentialityclaim*(ifany) fin iv i T-Vm rtm y Prycrirji QualityAssuranceStatement TableofContents TntrodffetfO" an|t nm nm y n flH t [w im ib m i m l re<ail> MethodsandMaterial^ineksdiaga descriptionofteatmethod*,organisms, dilutionwater, and statistical tedmiquet Results,includingdatafromrange-findinganddefinitivetests,EC25and EC50values, andNOECvalues Reteoces Appendices, iiK%dfpgfWw?tcr qniltydita 11.0 RECORDSTOBEMAINTAINED 11.1 Recordsthatwillbemaintainedthroughoutthe studyinclude: Samplereceiptand Jnrinim wtf Waterqualitydata Page6 of8 T.R. Wilbury Study Number 36 o f 41 rtnmnam/ S a n i t i ^ n nag nnt r n n*~;" DuPont HLO-1997-00636 12.0 REFERENCES Since* R J),, and Verateeg. 1992. A Stsiisdcd Procedure ftr Modeling Continnou5 ToxicityData. Envin^ Toxicol,andCbem. VoL11. No. 10, pp. 1,485-1*094. EC. 1992. Freshwater algal growth S m y r n a su b ^ ca tu s and A Volume35^9 December1992. OECD. 1992. The OECDPrinciple! of Good Laboattxy Practice. OECD Series on PrinciplesrfGood LaboratoryPracticeandCompfiraceMonitoring. Number !. EnvironmentalMonographNo.45QECD/GD(92)32. Paris. OECD. 1984. OECDQniM m fo r TestingofChernies!. Session2: Bfl&MtsonBiotic System*. Method201,AlgtGrowthInhibitionTest. Adopted4 April 1984. U.S. ERvaonmcntsI Protection Aseacy 03PA* 1988. Podcide A m m a t Guhfcfines. ^dhirionE, P^Kminn- wadEfe and Aquatic Orgszisms. Esolofflcal Bflh-t Branch. Hazard Evaluation Diviiion, Office ofPadeide Program*, Wsdangtoo,D.C. Daft,Mush 1988. U-.S.EP.A1.9.89. 40CmFRPut792.Trad.AeSuhstanooaCoInt.t.r--oliA->st(ATllSMGMAd)I;IGloOoMdlsbontoiy U.S. EsvironmesstilPratection Agensy(EPA). 1989. Pesticide Assasment Gutddines. SuMHt-'"" J. 123-2: Growth rad Reproduction of Aquario Piente - Tier 2. Blanch, Hazard Evaiuation Divisori, Office of Pesticide D.C. U .S. EnwnunetdSl Protection Agency (EPA). 1992. 40CPRPW tl60. Federai Inaecticide, H npdei and Bodeeticide Act P PR A ); Good Uboratoty Prosfiee Stradrab; Farai Ride. U.S. EPA 1993. 40 CHI Part 797. Trade Substance*Contrai Act Tert Guidelines; Finsi Bules. 5797.1050.AgriAcuteTradchyTest TJR. WillxuyStudy Page7 of8 T.R. Wilbury Study Numbe 37 of 41 o a it^ a iiy^aiiiu/fcfUTTaoeyiiBr'eOTfamTjC'A'UBr DuPont HLO-1997-00636 SUMMARYOF TEST CONJMnTONS TeatT/pc: Test Duration: DilutionWater Culture AcclimationPeriod: Temperature: Photoperiod: Light Intensity: NumberofConcentrations: D&rtioiiFactor: NumberofReplicates: Initial Inocnhim: TestVend Size: Test Vessel Volume: Solvent: EffectMeasured: Test Concentration Analysis: S etenastntm capricom ttem OECDGiride&ne201 U.S. SPA(TSCA) 797.1050 U.S.EPA(HFRA)SubdivisionJ. 123-2 Static 120hours Sterilesyntheticmedia 14days 242*C 24 boutslightand0 hoursdark Approximately120pBn/m'sec Sormon Approximately1.5-2.0 3 Approximately 10,000 crib per ml 250ml SOad Hone,ifpossible Ifa solventis required, 100mg/L dimethylfixtzsamidewill be used Growthresponse Vfoftnnea pHat0,72,and 120hours;TOCand particulatematterinthedilution waterat0hours Control eeScoaceutnliouincrease of at lent 1than hy72houtsand logarithmic growthoccursby 120bows AeeeptibSetemperaturerange T.R. Wilbury Study Number 38 o f 41 Company Sanitizad. Does not contain TSCA CBI DuPont HLO-1997-00636 AMENDMENT t Toxicity o fH-22387to theFreshwaterAlgs, Sdenasavmccpricormmm E.I. duPontdeNemounandCompany 1099ElkionRoad,PortOfficeBcSO Newark,Delaware 19714 TestingFacility TU WilburyLaboratories, Inc. 40DesksLane Marblehead,Massachusetts 0194S StudyDirector: PeterL. Kowalski Changes: 1. ThehighesttestedconcentrationofH-223S7willbe 1,000mg/L. 2. ConcentrationsofH-223S7forthedefinitivetoxicitytestwlbeO(control), 65,130,250,500,and 1,000mg/L. JgufjfBfftn fftf i^JffTHWT 1) Qf tO ffKfftffffr fn^ww|tnn mtapmg origins!protocol(change2). EffectiveDatefor Changes: May15,1997. Effect ofChanges: None. ProtocolAmendmentApproval C lCfGquA&nr~ Date: feSSL StudyDirector:_ TestFacilityManagement: TM . WiiburyStudyNumber 4 Date:__a.g.f.1da. CjI/JfoP---------- Date: S/dfi' lofi T.R. Wilbuiy Study Number 39 o f 41 ouiupaiijl 5amuzeW"UflS rt firitSIH' ft>LK UBI DuPont HLCM997-00636 AMENDMENT 2 Toxicity o fH-22387 to the Freshw aterAlga, Selcmstnm capricamutum E.L duFoot de NemouixandCompany HtH r J^twinitfrty fo r T y rirrfo fly . ~ i tUf ^ frin 1099EUaonRoad,PostOfficeBox50 Newark,Delaware 19714 TestingFacility TJL WQbuiyLaboratories, Inc. 40DoaksLane Marblehead,Massachusetts 0194S StudyDirectors PeterL.Kowalski Change: TheStudyDirectorisJeanneP.Magazu. Reason forChange: PeterL. KowalskiisnolongeremployedbyT it WilbuiyLaboratories. EffectiveDateforChange: May30,1997. EffectofChange: None;JeanneP.Magazuisqualifiedandavailableto serveasStudyDirector. Sponsor. Protocol Amendment Approval I-- C Date: StudyPincctor0^fA>ii e Date: (kCZ.Q'1 Testing Fscility Management;. T it WilbuiyStudyNumber Date: /T S (5 0 ftl Pcge l o f i T.R. Wilbuiy Study Number 40 o f 41 Company Sanitized. 9 v^sJQ]J22it ! ! lIS d ^ i DuPont HLO-1997-00636 AMENDMENT 3 Toxicity ofH-22387 to theFreshwaterAlga,Selemsmtmcapricormstttm E.LduPontdeNemoursendCompany HaskellLaboratoiyforToxicologytad industrialMedicine 1099HktonSold, Post OfficeBox 50 Newark,DeUware 19714 TJl. WilburyLaboratories,Inc. MDoduLne Marbtehead.Massachusetts 01945 StodDirector: leanneP.Magazu ConcentrationsofH-223B7forthedefinitivetoxicitytestwiilbe0 (coatrol), 6.5,13,25,50,100, and200rng/L. Reason forChange: Toconedin errorin previousamendment. Effective Datefor Change: May29,1997. Effect ofChange: None. ProtocolAmendmentApproval Sponsor. ... .. ..................... Dite: StudyDirector Dte:. TestingFacilityManagement T.R. WilburyStudy 'flU ljtj Q f / l M " ------- Date: b b f o z ( $ l P sgelofl T.R. Wilbury Study Number 41 o f 41 PiffljaamLSatiififPfl, Hoffs.rrf -- f- :" Tcr>A C^ J