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- 2-25) 413 TRADE SECRET M A ZG - 223 Revised Page DuPont-4488 Study Title H-24616: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricomutum Laboratory Project ED: DuPont-4488 Author: Terry Lee Sioman, B.S. Original Report Completed: November 16, 2000 Report Revision Number 1: February 13, 2002 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 W ork Request Number:! Study Code Number Company Sanitized. Does not contain TSCA CBt __ _ H-24616: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum STUDY INFORMATION DuPont-4488 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) Tn-T,ife Tnitiated/Completed: November 6, 2000 / November 9, 2000 Company Sanitized. Does not contain TSCA CB1 H-24616: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum Revised Page DuPont-4488 SUMMARY A study was conducted to determine the effect o f H-24616 on the growth and growth rate o f the green alga Selenastrum capricornutum. The algae were exposed to nominal concentrations o f 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. An inverse quadratic regression1was fit to the data. The fit o f the model to the data was good (R-square = 0.92). The estimated EC502was 870 mg/L, with 95% fiducial bounds o f 736 mg/L and 1004 mg/L. H-24616 exhibited low concern3 under TSCA in a 72-hour, acute test using the green alga Selenastrum capricornutum. 1 Draper, N.R. and Smith H. 1981. Applied Regression Analysis, Wiley. 2 Bruce, R.D. and Versteeg, D.J. 1992. Environmental Toxicology and Chemistry, 11, 1485-1494. 3 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. Company Sanitized. Does no? contain TSCA CBI -3 - 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 2-000 Date Company Sanitized. Does not contain TSCA CBl -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 of algal cells, the test and blank control flasks were placed on a shaker table in a chamber with a temperature o f 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 o f 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 capricomutum DuPont-4488 TABLES Company Sanitized. Does not contain TSCA CB!~ -6- 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 CB1 H-24616: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum DuPont-4488 TABLE 2 DATA SUMMARY Nominal ,Dav 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 -8- H-24616: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum DuPont-4488 FIGURES Company Sanitized. Does not contain TSCA GBI -9 - H-24616: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum Revised Page DuPont-4488 FIGURE 1 72-HOUR HEALTHY CELL COUNT DOSE RESPONSE CURVE Company Sanitized. Does not contain TSCA CBT H -24616: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum DuPont-4488 APPENDICES Company Sanitized. Does not contain TSCA CBI -11 - H-24616: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricomutum DuPont-4488 APPENDIX A 72-Hour Healthy Cell Count *rmpany Sanitized. Does no' contain TSCA CBt - 12- H-24616: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum PERCENT CHANGE PROM CONTROL DATA SET COUNT fron C:\AQUATICS\EGX\TEMF\STADLER Obs cone MEAN PCTCHNG N 1 0.00 239.0 0 2 2 0.01 220.5 -8 2 3 0.10 234.0 -2 2 4 1.00 221.5 -7 2 5 10.00 241.5 12 6 100.00 232.0 -3 2 7 1000.00 72.5 -70 2 Quadratic Regression on Concentration with IDENTITY Link Dependent Variable: count Source Sum of DF Squares Mean Square F Value Pr > F Model 2 Error 11 Corrected Total 13 43305.49213 3581.36501 46886.85714 21652.74607 66.51 325.57864 <.0001 R-Square 0.923617 Coeff Var 8.645215 Root MSE 18.04380 count Mean 208.7143 Source C CSQR DF T y p e III SS Mean Square F Value Pr > F 1 20.6074183 20.6074183 0.06 0.8060 1 525.4971774 525.4971774 1.61 0.2301 Parameter Estimate Standard Error t Value Pr > 1t 1 Intercept C CSQR 231.0159500 0.0397454 -0.0001983 5.84950152 0.15798021 0.00015606 39.49 0.25 -1.27 <-0001 0.8060 0.2301 Revised Page DuPont-4488 Nonlinear Model Estimation Sunniary EC50 using Method^HARQUARDT, Inverse Quadratic Regression with IDENTITY link Method Iterations R PPC RPC(u) Object Objective Observations Read Observations Used Observations Missing Marquardt 5 1.42E-10 1.71E-11 3.4 51E-6 8.19E-10 3581.365 1015 14 1001 Source Sum of DF Squares Mean Square Approx F Value Pr > F Regression Residual Uncorrected Total Corrected Total 3 653169 11 3581.4 14 656750 13 46886.9 217723 325-6 66.51 <.0001 Parameter Approx Std Error Approximate 95% Confidence Limits yo 231..0 5.8495 218.1 243.9 YOP 0 .0397 0.1580 -0.3080 0.3875 u 870 .1 68.3788 719.5 1020.6 Approximate Correlation Matrix yO YOP u yO 1.000 -0.452 -0.459 YOP -0.452 1.000 0.884 u -0.459 0.884 1.000 Approximate Covariance Matrix Y0 YOP u yO 34.217 YOP -0.418 u -183.456 -0.42 0.02 9.55 -183.46 9.55 4675.66 STDERR LB UB 68.3788 736. 029 1004.07 EC50 B70.051 Company Sanitized. Does contain TSCA CBt H-24616: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum Revised Page DuPont-4488 APPENDIX B Reason for Revision No. 1 Company Sanitized. Does not contain TSCA CB1 - 14- H -24616: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum REASON FOR REVISION NUMBER 1 Revised Page DuPont-4488 The report was revised as follows: Pages 3 10 Summary: Recalculation of test endpoints. Figure 1: Recalculation o f test endpoints. 13 14-15 Appendix A: Recalculation o f test endpoints. Appendix B: Reason for Revision No. 1 added. The following page(s)s were revised to reflect these changes. The original pages (along with those listed above) were removed from the report and are maintained in the Medical Research file. Pages 1 Title Page Study Director: Terry Lee Sloman, B.S. Z o o O, Date - 15 - mpany Sanitized. Does not contain TSCA CBI