Document Dv9011Ky6aj44K2aOkX8Yg42N

TRADE SECRET AR226-3170 DuPont-4488 Study Title H-24616: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum Laboratory Project ID: DuPont-4488 Author: Terry Lee Sloman, B.S. Study Completed on: November 16,2000 Performing Laboratory: E.I. du Pont de Nemours and Company Haskell Laboratory for Toxicology and Industrial Medicine Elkton Road, P.O. Box 50 Newark, Delaware 19714-0050 Work Request Number: Study Code Number M Company Sanitized. Does not contain TSC A CBf -1 - H-24616: Influence on Growth and Growth Rate o f the Green Alga Selenastmm capricornutum DuPont-4488 STUDY INFORMATION Substance Tested: 3 Svnonvms/Cojdes: H-24616 9 Haskell Number: 24616 Purity- Known Impurities ^ jj Physical Characteristics: Sponsor: E. I. du Pont de Nemours and Company Wilmington, Delaware 19898 U.S.A. Study Initiated/Completed: November 6, 2000 / (see report cover page) In-Life Initiated/Comnleted: November 6, 2000 / November 9, 2000 company Sanitized. Does not contain TSCA CBI -2 - H-24616: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum DuPont-4488 SUMMARY A study was conducted to determine the effect of H-24616 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-24616/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.), and the NOEC1234based on nominal concentrations are as follows:2,3,4 Healthy Cell Count EC50 NOEC 30 mg/L (95% C.I. not calculated) 100 mg/L H-24616 exhibited medium concern5 under TSCA and assigned "acute toxic" to aquatic organisms (R52)6 and classified as dangerous for the environment under the EU Directive in a 72-hour, acute test using the green alga Selenastrum capricornutum. 1 The EC50 is defined as the "effective concentration" producing a 50% inhibition o f growth relative to the control. The NOEC is defined as the highest concentration o f test substance that has no significant effect on the measured parameter relative to the control. 2 BB N Software Products Corp., 1988. An RS/1 RPL.procedure conducting W illiams' test and probit analysis. 3 McCullogh, L.P. and J.A. Nelder. 1989. Generalized Linear Models 2nd Ed. Chapman and Hall, London 511 pp. (See Chapter 9, p. 328). 4 Snedecor, G.W. and W.G. Cochren. 1980. Statistical Methods, 7th Ed. Iowa Stat. Univ. Press, Ames, Iowa. 507 pp. (See Table A 4, p. 469). 5 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 STP 1179. (W.G. Landis, J.S. Hughes, and M.A. Lewis, Eds.), pp 22-39. American Society for Testing and Materials, Philadelphia. 6 Official Journal o f the European Communities Vol. 36, 4 May 1993, p. 68, section 5.2.1. Company Sanitized. Does not contain TSCA CBI H -24616: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum DuPont-4488 CERTIFICATION I, the undersigned, declare that this report provides an accurate evaluation o f data obtained from this study. Issued by Study Director: Terry Lee Sloman, B.S. Associate Scientist lk-/0ov-aooo Date 1 company Sanitized. Does not contain TSCA CB1 -4 - H-24616: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum DuPont-4488 RESULTS Based on visual observations, the blank (normal culture medium) control solution and the 0.01, 0 .1,1,10, 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 slightly 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 8440 lumens/m2 (= lux). The shaking speed was 110 revolutions per minute (rpm). The pH measurement o f the culture medium was 7.45 at study start. The pH measurements of the definitive test solutions at test initiation ranged from 6.86 to 7.45, and at test termination ranged from 7.09 to 7.54. Company Sanitized. Does not contain TSCA CBI H-24616: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum DuPont-4488 TABLES Company Sanitized. Does not contain TSCA CB1 H-24616: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum DuPont-4488 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 7.45 6.87 6.87 6.86 6.86 6.86 6.86 7.18 7.09 7.28 7.38 7.25 7.49 7.54 Company Sanitized. Does not contain TSCA CBS -7 - H-24616: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum DuPont-4488 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 2,190,000 2,590,000 0.01 mg/L Replicate 1 Replicate 2 10,000 10,000 2,130,000 2,280,000 0.1 mg/L Replicate 1 Replicate 2 10,000 10,000 2,090,000 2,590,000 1 mg/L Replicate 1 Replicate 2 10,000 10,000 2,160,000 2,270,000 10 mg/L Replicate 1 Replicate 2 10,000 10,000 2,340,000 2,490,000 100 mg/L Replicate 1 Replicate 2 10,000 10,000 2,170,000 2,470,000 1000 mg/L Replicate 1 Replicate 2 10,000 10,000 680,000 770,000 Company Sanitized. Does not contain TSCA CB1 H-24616: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum DuPont-4488 FIGURES impany Sanft,'zetf- oes no! contain TSCA CBI -9 - H-24616: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricomutum DuPont-4488 FIGURE 1 72-HOUR HEALTHY CELL COUNT DOSE RESPONSE CURVE TOLERANCE DISTRIBUTION 01 BASED ON MAXIMUM QUASI-U |h m D J U !S T E D F O R B A C K G R O U N D R E S P O N S E ITIMjftlON O F N O H M I T C U R V E P A R A M E T E R S 72 Hr Healthy Cell Count (#cells/inL> Obs. Fitted Response Quasi-likelihood assumed: Variance = 1.11793e+12*Mean/`0. _ Nonnit{%) - 15.083342 + -10.248316*Logl0{Dose) with 95% confidence limits. Com pany Sanitized. Does not contain TSCA CB H-24616: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum DuPont-4488 APPENDICES Company Sanfflzefl. Dises no! contain TSCA CBI H -24616: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum DuPont-4488 APPENDIX A 72-Hour Healthy Cell Count Company Sanitized. Does not contain TSCA CB1 H -24616: Influence on Growth and Growth Rate o f the Green Alga Selenastmm capricornutum DuPont-4488 m I WILLIAMS TEST OF # @ A L C 3 A E S C R E E H | M H ^ 1 72 Hr Healthy Cell Count (ttceTTs/mL) * W.T. Rank o o 0 0 0 o 1 Dose 0.00 0.01 0.10 1.00 10.00 100.00 1000.00 Mean Response 2390000 2205000 2340000 2215000 2415000 2320000 725000 Std Error 200000 75000 250000 55000 75000 150000 45000 Transform Mean 6.376872 6.343157 6.366723 6.345240 6.382708 6.364578 5.859500 Transform Std Error 0.036428 0.014778 0.046577 0.010786 0.013492 0.028119 0.026991 nm 2 2 6.360481 2 6 *360481 2 6.360481 2 6.360481 2 6.360481 2 5.B595Q0 t 0.412269 0.412269 0.412269 0.412269 0.412269 13.013216 W.T. _ Rank t crit. 0 0 1.895 0 2.002 0 2.039 0 2.058 0 2.069 1 2.076 One-sided test at the 95% CL for a decreasing trend Common logarithm transformation, i.e. Y = loglO(X), applied to the data. MSE = 0.001581 with 7 d f . Ref: Williams, D. A. Biometrics 28: 519-531 (1972). Parameter Background Intercept Slope Statistic: Value QUASI-LIKELIHOOD ESTIMATES OF NORM IT PARAMETERS FO 72 Hr Healthy Cell Count (#cet Is/tnL) Est imate 2390000.000000 15.083342 -10.248316 Error 200000.000000 910,830377 420.889905 Corr. with Background 1.000000 Chi square df Significance 0 ispersion 1.11793e*13 10.000000 0.001 1.11793**12 Corr. wfth Intercept 0*000000 1.000000 t-value 2.228139 Corr. with Slope 0.000000 -1.029416 1.000000 <t*CVslope)A2 8373.682287 Quantile (%) 1 5 10 20 25 30 40 50 60 70 75 80 90 95 99 Estimate 49.978753 42.863207 39.521856 35.802244 34.482763 33.339324 31.369545 29.633801 27.994100 26,340131 25.466700 24.528133 22.219660 20.477997 17.570710 Lower 95HCI : * - - Upper 95XCI - * - - -. * Quasi-Likelihood assumed: Variance = 1.11793e+12*HeanA0 . Convergence achieved. Relative change _in_c|u.si-11 kel ihood 5.235931e-08 Variances and covariances have been ul'*t--*plied by the di**s*p*ersion parameter of 1.11793e +12* Company Sanitized. Does not contain TSCA CB1