Document zb5rpkG21GmzKYNbDxJvBdQ2R

M a te 441 TRADE SECRET M&M- 3Jf*? DuPont-11410 Study Title H-25434: Influence on Growth and Growth Rate of the Green Alga Selenastmm capricornutum Laboratory Project ID: DuPont-11410 Author: Terry Lee Sloman, B.S. Study Completed on: October 4,2002 Performing Laboratory: E.I. du Pont de Nemours and Company Haskell Laboratory for Health and Environmental Sciences Elkton Road, P.O. Box 50 Newark, Delaware 19714-0050 Work Request Number: Study Code Number: /,*<***`S, Page 1 o f 18 Company Sanitized. Does not contain TSCA CBI H-25434: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum DuPont-11410 CERTIFICATION We, the undersigned, declare that this report provides an accurate evaluation of data obtained from this study. W ork C om pleted by: iX X a J J J .------------------- Barbra D . F efrell, B .$. A ssociate Scientist 4 ocJvfa Im z. D ate Issued bv Studv D irector: *T 2yvuu 0 Terry Lee Slom an, B .S. A ssociate Scientist HO d t i&v 2 0 0 2 , D ate -2 Company Sanitized. Does not contain TSCA C8I H-25434: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricomutum STUDY INFORMATION Substance Tested: Svnonvms/Codes: H-25434 Haskell Number: 25434 DuPont-11410 Stability: The test substance appeared to be stable under the conditions of the study; no evidence o f instability was observed. Sponsor: E.I. du Pont de Nemours and Company Wilmington, Delaware 19898 U.S.A. Study Initiated/Comoleted: September 24,2002 / (see report cover page) In-Life Initiated/Completed: September 24,2002 / September 27,2002 Company Sanitized. Does not contain TSCA CBI H-25434: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum DuPont-11410 SUMMARY A study was conducted to determine the effect o f H-25434 on the growth and growth rate o f the green alga Selenastrum capricornutum. The algae were exposed to nominal concentrations of 0.01, 0.1, 1, 10, 100, and 1000 mg H-25434/Liter o f nutrient medium (ppm). The algae were exposed for 72 hours (3 days) without test medium renewal. The effect was expressed as percent inhibition in growth based on healthy cell count (also referred to as cell density) relative to the blank (culture medium) control for the 72-hour (day 3) interval o f the test. The 72-hour EC50, including the 95% confidence intervals (C.I.),a based on nominal concentrations are as follows:b,c,d Healthy Cell Count EC50 36.2 mg/L (95% C.I. not calculated) H-25434 exhibited medium concern under TSCA and classified "harmful" to aquatic organisms (R52)f under the EU Directive in a 72-hour, acute test using the green alga Selenastrum capricornutum. a The EC50 is defined as the "effective concentration" producing a 50% inhibition of growth relative to the control. b BBN Software Products Corp., (1998). An RS/1 RPL procedure conducting Williams' test and probit analysis, c McCullogh, L.P, and Neider, J.A. (1989). Generalized Linear Models, 2nd edition, Chapter 9, pp 328. Chapman and Hall, London. d Snedecor, G.W. and Cochran, W.G. (1967). Statistical Methods, 6th edition, Table A4, pp 469. The Iowa State University Press, Ames. e Smrchek, J., Clements, R., Morcock, R., and Rabert, W. (1993). Assessing ecological hazard under TSCA: methods and evaluation o f data. Environmental Toxicology and Risk Assessment, ASTM S T P 1179. (W.G. Landis, J.S. Hughes, and M.A. Lewis, Eds.), pp 22-39. American Society for Testing and Materials, Philadelphia. f Official Journal of the European Communities, Volume 36, 4 May 1993, pp. 68, section 5.2.1. -4 - Company Sanitized. Does not contain TSCA C8I H-25434: Influence on Growth and Growth Rate o f the Green A lga Selenastrum capricornutum DuPont-11410 MATERIALS AND METHODS A. Test System Selenastrum capricornutum, a freshwater, unicellular, non-motile, green alga, was used in this study. The original culture source was the Department o f Botany - Culture Collection o f Algae - The University of Texas at Austin - Austin, Texas 78713-7640. The culture method for Selenastrum capricornutum was based on published literature.(1,2) Prior to the study, Selenastrum capricornutum cultures were maintained under the same environmental conditions used in the study. The organisms were cultured in sterilized 250 mL Erlenmeyer flasks containing approximately 50 mL o f filtered { - filter-sterilized) synthetic algal-assay procedure (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. B. Test Design The test design used for the Selenastrum capricornutum study is described below: Organism Nutrient Flask Volume Solution Volume Volume Medium (mL) (mL) Ratio Selenastrum capricornutum AAP 250 50 5:1 C. AAP Nutrient Medium Preparation for Selenastrum capricornutum (Appendix A) AAP nutrient medium(1) was prepared by adding 1 mL o f each o f the 6 macronutrient stock solutions and 1 mL o f the micronutrient stock solution to approximately 800 mL o f Milli-Q (deionized) water, with mixing after each addition. The volume o f the medium was brought to 1 liter with additional Milli-Q water. Appropriate propprtions (1 mL of each stock solution for each 1 liter o f medium) were used to prepare the larger volumes o f the medium required for the study. The medium pH was adjusted to approximately 7.5. The medium was filter-sterilized using Coming pre-sterilized filtration systems each with a 0.22 pm cellulose acetate filter. -5 Company Sanitized. Does not contain TSCA CBI H-25434: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum DuPont-11410 1. Test Solution Preparation Six nominal test concentrations (0.01, 0.1,1, 10, 100, and 1000 mg H-25434/L AAP nutrient medium) were used in this study. Aliquots o f the filter-sterilized AAP nutrient medium were used for the blank (normal culture medium) control solution. 2. Test Culture Preparation For each blank control and test solution, 2 aliquots (50 mL each) were placed in separate sterilized 250 mL Erlenmeyer flasks fitted with sterilized foam stoppers. Each flask was labeled with a study number, test substance, concentration, replicate number, date, and experimenter's initials. To achieve the desired nominal concentration of approximately 10,000 Selenastrum capricornutum cells/mL at test initiation, an approximate 0.208 mL (= 208 pL) aliquot of algal inoculum from a logarithmically growing stock culture was aseptically transferred to each flask. D. Experimental Design and Test Conditions The test flasks were placed on a shaker table in a chamber in a non-systematic design and were re-positioned each working day. Air temperature in the chamber was recorded continuously with a continuous temperature recorder. Each blank control and test concentration was tested as 2 replicates. The algae were incubated for approximately 72 hours without test medium renewal and cell counts were made. Illumination was supplied by cool-white fluorescent tubes. The target environmental parameters are described in the following table. Initial Population 10,000 cells/mL Illumination (lumens/m2= lux) PhotoPeriod (hours) Shaking Speed (rpms) Temperature (C) 6000 to 10,000 24 100 24 2 E. Selenastrum capricornutum Growth Measurement Selenastrum capricornutum growth measurement was determined by visually counting the number of cells taken from an approximate 0.2 mL sample from each flask at approximately 72 hours from study initiation. The counts were conducted using a hemacytometer and a compound microscope. An aliquot o f each sample was loaded into the hemacytometer and 16 grids were selected. All cells located in the 16 grids were counted and recorded as healthy or unhealthy. The total number o f cells counted was multiplied by 10,000 to determine the number of cells per milliliter. Cells outside these 16 grids were not counted nor included in the total number. Observations and counts o f healthy and unhealthy cells (e.g., deformed, senesced, stunted) were recorded in the study records. The statistical calculation of the EC50 was based on mean healthy cell counts and nominal concentrations. -6 Company Sanitized. Does not contain TSCA CB! H-25434: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum DuPont-11410 RESULTS Based on visual observations, the blank control solution and the 0 .0 1 ,0 .1 ,1 ,1 0 , and 100 mg/L test solutions were clear and had no color before addition o f algal cells. However, the 1000 mg/L test solution was cloudy before addition o f algal cells. After the addition o f algal cells, the test and blank control flasks were placed on a shaker table in a chamber with a temperature of approximately 25C. The algae were incubated for 72 hours (3 days) without medium renewal. Illumination was supplied by cool-white fluorescent tubes. The mean light intensity in the chamber was 7534 lumens/m2 (= lux). The shaking speed was 100 revolutions per minute (rpm). The pH measurement o f the culture medium was approximately 7.5 at study start. The pH measurements of the test solutions at test initiation ranged from 7.29 to 7.63, and at test termination ranged from 6.72 to 7.05. CONCLUSIONS concentrations are as follows :(3,` Healthy Cell Count EC50 36.2 mg/L (95% C.I. not calculated) H-25434 exhibited medium concemb under TSCA and classified "harmful" to aquatic organisms (R52) under the EU Directive in a 72-hour, acute test using the green alga Selenastrum capricornutum. a The EC50 is defined as the "effective concentration" producing a 50% inhibition o f growth relative to the control. b Smrchek, J., Clements, R., Morcock, R., and Rabert, W. (1993). Assessing ecological hazard under TSCA: methods and evaluation o f data. Environmental Toxicology and Risk Assessment, A STM STP1179. (W.G. Landis, J.S. Hughes, and M.A. Lewis, Eds.), pp 22-39. American Society for Testing and Materials, Philadelphia. c Official Journal o f the European Communities, Volume 3 6 ,4 May 1993, pp. 68, section 5.2.1. -7 Company Sanitized. Does not contain TSCA CBI H-25434: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum DuPont-11410 REFERENCES 1. Miller, W.E.; Greene, J.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 Testing and Materials (ASTM). (1990). "Standard Guide for Conducting Static 96-h Toxicity Tests with Microalgae" in ASTM Annual Book ofStandards, E l218-90. Vol. 11.04, Philadelphia, PA. 3. BBN Software Products Corp., (1998). An RS/1 RPL procedure conducting Williams' test and probit analysis. 4. McCullogh, L.P. and Nelder, J.A. (1989). Generalized Linear Models, 2nd edition, Chapter 9, pp 328. Chapman and Hall, London. 5. Snedecor, G.W. and Cochran, W.G. (1967). Statistical Methods, 6th edition, Table A4, pp 469. The Iowa State University Press, Ames. 6. Smrchek, J., Clements, R., Morcock, R., and Rabert, W. (1993). Assessing ecological hazard under TSCA: methods and evaluation of data. Environmental Toxicology and Risk Assessment, A S T M S T P 1179. (W.G. Landis, J.S. Hughes, and M.A. Lewis, Eds.), pp 22-39. American Society for Testing and Materials, Philadelphia. 7. Official Journal of the European Communities, Volume 36, 4 May 1993, pp. 68, section 5.2.1. -8 Company Sanitized. Does not contain TSCA CBI H-25434: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum DuPont-11410 TABLES -9 Company Sanitized. Does not contain TSCA C8I H-25434: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum DuPont-11410 TABLE 1 pH MEASUREMENTS OF TEST SOLUTIONS Nominal Concentration (mg/L) Test Initiation (0 hours) pH Test Termination (72 hours) pH Blank Control 0.01 0.1 1 10 100 1000 7.63 7.39 7.33 7.35 7.32 7.32 7.29 6.72 6.93 6.98 7.03 7.05 6.97 6.92 - 10Company Sanitized. Does not contain TSCA CBi H-25434: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum DuPont-11410 TABLE 2 DATA SUMMARY Nominal Day 0 Healthy Cell Count Day 3 Healthy Cell Count Concentration (cells/mL) (cells/mL) Blank Control Replicate 1 Replicate 2 10,000 10,000 2,640,000 2,110,000 0.01 mg/L Replicate 1 Replicate 2 10,000 10,000 2,820,000 2,800,000 0.1 mg/L Replicate 1 Replicate 2 10,000 10,000 2,810,000 2,580,000 1 mg/L Replicate 1 Replicate 2 10,000 10,000 2,680,000 2,360,000 10 mg/L Replicate 1 Replicate 2 10,000 10,000 2,610,000 2,020,000 100 mg/L Replicate 1 Replicate 2 10,000 10,000 50,000 240,000 1000 mg/L Replicate 1 Replicate 2 10,000 10,000 0 0 -11 Company Sanitized. Does not contain TSCA C8I H-25434: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum DuPont-11410 FIGURE -12Company Sanitized. Does not contain TSCA CBi H-25434: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum DuPont-11410 FIGURE 1 72-HOUR HEALTHY CELL COUNT DOSE-RESPONSE CURVE TOLERANCE DISTRIBUTION OF #H25434_C72 ADJUSTED FOR BACKGROUND RESPONSE BASED ON MAXIMUM QUASI-LIKELIHOOD ESTIMATION OF NORMIT CURVE PARAMETERS 72 Hr Hoalthy Cell Count (&cells/mL) Obs. Fitted Dose (mg/L) Quasi-likelihood assumed; Variance = B .630834e+10*Meano0 . Normit{%> = 5.456969 + -3.501484*Logl0(Dose) with 95% confidence limits. -13 Company Sanitized. Does not contain TSCA CBI H-25434: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum DuPont-11410 APPENDICES - 14Company Sanitized. Does not contain TSCA CBI H-25434: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum DuPont-11410 APPENDIX A AAP Nutrient Medium Constituents - 15Company Sanitized. Does not contain TSCA CBI H-25434: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum___________________________________________________ DuPont-11410 AAP NUTRIENT MEDIUM CONSTITUENTS Stock Solutions" Component Concentration (g/L) Macronutrient Stock #1 NaN03 25.500 Final Prepared Medium Component Concentration (mg/L) Na 11.001 N 4.200 Macronutrient Stock #2 NaHC03 15.000 C 2.143 K 0.469 Macronutrient Stock #3 K2H P 0 4 1.044 P 0.186 Mg 2.904b Macronutrient Stock #4 M gS04*7H20 14.700 Ca 1.202 S 1.911 Macronutrient Stock #5 MgCl2-6H20 12.164 Macronutrient Stock #6 CaCl2*2H20 4.410 Stock Solutions" Component Concentration (mg/L) Final Prepared Medium Component Concentration (pg/L) Micronutrient Stock H 3B O 3 MnCl2*4 H20 ZnCl2 CoC12*6 H20 CuC12*2H20 Na2M o 0 42 H20 FeCl36 H20 Na2EDTA*2 H20 185.520 415.610 3.271 1.428 0.012 7.260 160.000 300.000 B 32.460 Mn 115.374 Zn 1.570 Co 0.354 Cu 0.004 Mo 2.878 Fe 33.051 a As reported in Reference 2. b Includes magnesium from both magnesium sulfate and magnesium chloride. - 16Company Sanitized. Does not contain TSCA CBI H-25434: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum DuPont-11410 APPENDIX B 72-Hour Healthy Cell Count Statistics - 17Company Sanitized. Does not contain TSCA CBI H-25434: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum DuPont-11410 72-HOUR HEALTHY CELL COUNT STATISTICS WILLIAMS TEST OF #@ALGAE@SCREEN@H25434 72 Hr Healthy Cell Count (#cells/mL) W.T. Rank Dose Mean Std Response Error Transform Transform Mean Std Error n m t 0 0.00 0 0.01 0 0.10 0 1.00 0 10.00 1 100.00 2 1000.00 2375000 2810000 2695000 2520000 2315000 145000 0 265000 10000 115000 160000 295000 95000 0 6.372943 6.448704 6.430163 6.400523 6.360996 5.039593 -0.301030 0.048661 0.001546 0.018543 0.027611 0.055645 0.340619 0.000000 2 2 6.448704 -0.404801 2 6.430163 -0.305735 2 6.400523 -0.147366 2 6.360996 0.063836 2 5.039593 7.124321 2 -0.301030 35.660201 W.T. _ Rank t crit. 0 0 1.895 0 2.002 0 2.039 0 2.058 1 2.069 2 2.076 One-eided test at the 95% CL for a decreasing trend. Common logarithm transformation, .e. Y loglO(X + 0.5), applied to the data. MSE = 0.035027 with 7 d . Ref: Williams, D. A. Biometrics 28: 519-531 (1972). Parameter Background Intercept Slope Statistic: Va lue Q U A S I -LIKELI HOOD E S T I M A T E S OF NORM 1T P A R A M E T E R S FOR #H25434_C72 72 Hr Healthy Cell Count {#eells/ml! Estimate Standard Error Corr. with Background Corr. with Intercept 2375000.000000 265000.000000 5.456969 3.053138 -3.5014B4 1.650424 1.000000 0.000000 1.000000 Ch isquare df Significance Dispersion t-value 1 .0357e+t 2 12.000000 0.001 8.630834e+10 2.178813 Corr. with slope 0.000000 -01..090707000104 <t*CVs lope)*2 1.054695 Quant fie {X) 1 5 10 20 25 3.0 40 50 60 70 75 80 90 95 99 Estimate 167.056252 106.716769 84.038052 62.9266B4 56.377166 51.078573 42.739333 36.180413 30.628047 25.627620 23.219016 20.802340 15.576542 12.266325 7.835817 Lower 95&CI - - - - -' - - - - Upper 95CI Suasi-11kel 1hood assumed: variance - 8.630B34e I0*fiean',o . Convergence achieved. Relative change in quaei*liketihood = 2.43SS83O-08 Variances and covariances have been multiplied by the dispersion parameter of 8.630834e10. - 18Company Sanitized. Does not contain TSCA CBI DuPont Haskell Laboratory for Health and Environmental Sciences November 4, 2002 cc: Quality Assurance TO: FROM: MR FILI [POCKET H-25425 J. C.Maslanka TEST SUBSTANCE STABILITY OF H-25425 Medical Research Project Number: Haskell Sample Number: Haskell Reference Standard Number: Analytical Test Code: Analytical Report Number: Notebook References: m HA-2002-094 Attached is the analytical report for the study identified above. This analysis was performed to satisfy protocol requirements for test substance stability but may also be used for other purposes. tcQ' ^ 00* sa*** TEST SUBSTANCE STABILITY OF H-25425 Medical Research Project Number: Haskell Sample Number: Analytical Report Number: HA-2002-094 A sample of H-25425 was submitted on October 14, 2002 anjj^nalyzed oi^Qctober 15, 2000;^, The percentage of active ingredient (a.i.) was measured to b eH P H M B R L ith a r.ange o H M b f nominal for replicate analyses (n = 3). The sponsor reported a purity oi when tie sample was submitted. ACKOWLEGEMENTS Sample preparation by Sheila A. Riley (Chemistry Associate). Sample analysis by Bogdan Szostek (Senior Research Chemist). SIGNATURE Report by: Date Issued: Janet C. Maslanka Analytical StaffChemist 4- Date pO' HA-2002-094 page 2 of 2 METHODS Analysis for the percent of a.i. in a sample of H-25425 was by high-performance liquid chromatography (HPLC) with a Gel Permeation Column (GPC) and refractive index detector according to the following method. Sonication was used to completely dissolve the test substance and the analytical standard stock solution. SAMPLE PREPARATION & ANALYSIS Aliquots (0.0413,0.0407 and 0.0412 grams) of H-25425 were dissolved in tetrahydrofuran (10 mL) to give nominal concentrations of 4130, 4070, and 4120 ppm, respectively. CHROMATOGRAPHIC CONDITIONS Instrument: Column: Mobile Phase Flow Rate: Injection Volume: Column Temperature: Detection: Hewlett-Packard Model 1100 HPLC Styragel HR 4; 7.8 mm x 300 mm (GPC column) 100% tetrahydrofuran (THF) 1.0 mL/min 50 pi 35C Refractive Index CALIBRATION & QUANTITATION A separate sample of H-25425 test substance was designated as the analytical reference standard (93.17% pure). A stock solution of H-25425 in tetrahydrofuran was used to make calibration solutions that bracketed the test solutions. Peak heights from replicate HPLC/GPC/RI analysis of each calibration solution were used to construct a calibration curve by least-squares regression. Measured concentrations for each test solution were determined by applying respective peak heights to the calibration curve. RESULTS H-25425 eluted from the HPLC column as a resolved p>qea^ witjj^ ri etention time o f about 8.582 0.01 minutes. The sponsor reported a purity o t r hen the sample was submitted. Table I. The percent of a.i. in the H-25425 sample analyzed October 15,2002. ppm H-25425 Percent Aliquot Nominal Measured Nominal 1 4130 2 4070 3 4120 Average % Nominal Standard Deviation Coefficient of Variation 3730 3720 3790 90.3 91.4 92.0 91.2 0.85 1% noti Saw t e a 0*