Document 1qEXgpoZQx8aEBOo0Kqzn40Z

M U M - MILL dUatDuPont-13348 TRADE SECRET Study Title H-25914: Influence on Growth and Growth Rate of the Green Alga Selenastrum capricornutum Authors: Robert A. Hoke, Ph.D. -Barbra D. Ferrell, B.S. Study Completed on: August 18,2003 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 Laboratory Project ID: DuPont-13348 Page 1 o f 19 H-25914: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum DuPont-13348 CERTIFICATION We, the undersigned, declare that this report provides an accurate evaluation of data obtained from this study. Work Completed by: Associate Scientist 2003 UDate Issued by Study Director: Senior Research Ecotoxicologist and Manager / a h2o3 fate -2- Company Sanitized. Does not contain TSCA CBi H-25914: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum STUDY INFORMATION Submitter's Notebook Numberfs): Haskell Number: 25914 CAS Registry Number: DuPont-13348 A'"*''' Physical Characteristics: Amber liquid Stability: The test substance appeared to be stable under the conditions of the study; no evidence of instability was observed. Sponsor: E.I.- du Pont de Nemours and Company Wilmington, Delaware 19898 U.S.A. Study Initiated/Completed: July 22, 2003 / (see report cover page) -3 - Company Sanitized. Does not contain TSCA CBI H-25914: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum D u P o n t-13 348 SUMMARY A study was conducted to determine the effect of H-25914 on the growth and growth rate of the green alga Selenastrum capricornutum.a The algae were exposed to nominal concentrations of 0.1, 1, 10, and 100 mg H-25914/Liter of 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 of the test. The 72-hour EC50, and the 95% confidence interval (C.I.)bbased on nominal concentrations is as follows: Healthy Cell Count EC50 1.17 mg/L (95% C.I. = 0.75 to 1.59 mg H-25914/L) H-25914 exhibited medium concern under TSCA and was "toxic" to aquatic organisms (R51), classified as dangerous for the environment, and assigned the symbol "N" under the EU Directive in a 72-hour, acute test using the green alga Selenastrum capricornutum. a Also known as Pseudokirchneriella subcapitata. b The EC50 is defined as the "effective concentration" producing a 50% inhibition of growth relative to the control. - 4 - Company Sanitized. Does not contain TSCA CBi H-25914: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum DuPont-13348 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 of Botany - Culture Collection of Algae - The University of Texas at Austin - Austin, Texas 78713-7640. The culture method for Selenastrum capricornutum was based on published literature.'4,5j 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 of 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 Selenastrum capricornutum Nutrient Medium AAP Flask Volume Solution Volume Volume (mL) (mL) Ratio 250 50 5:1 C. AAP Nutrient Medium Preparation for Selenastrum capricornutum (Appendix A) AAP nutrient medium(4) was prepared by adding 1 mL of each of the 6 macronutrient stock solutions and 1 mL of the micronutrient stock solution to approximately 800 mL of Milli-Q (deionized) water, with mixing after each addition. The volume of the medium was brought to 1 liter with additional Milli-Q water. Appropriate proportions (1 mL of each stock solution for each 1 liter of medium) were used to prepare the larger volumes of 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 CBS H-25914: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum DuPont-13348 1. Test Solution Preparation Four nominal test concentrations (0.1, 1,10, and 100 mg H-25914/L AAP nutrient medium) were used in this study. Aliquots of 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. To achieve the desired nominal concentration of approximately 10,000 Selenastrum capricornutum cells/mL at test initiation, an approximate 0.340 mL (= 340 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 Photo- Shaking Illumination Period Speed Temperature (lumens/m2= lux) (hours) (rpms) (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 of each sample was loaded into the 2 wells of the hemacytometer and 8 squares from each grid area (16 total squares) were selected for counting. All cells located in the 16 squares were counted and recorded as healthy or unhealthy. The total number of 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 of healthy and unhealthy cells (e.g., deformed, scenescent, stunted) were recorded in the study records. The statistical calculation of the EC50 was based on mean healthy cell counts and nominal concentrations. -6Company Sanitized. Does no! contain TSCA CBf H-25914: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum DuPont-13348 RESULTS Based on visual observations, the blank control solution and the 0.1,1, 10, and 100 mg/L test solutions were clear and colorless before addition of 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 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 6885 lumens/m2 (= lux). The shaking speed was 98 revolutions per minute (rpm). The pH measurement of the culture medium was approximately 7.5 at study start. The pH measurements of the test solutions at test initiation ranged from 7.51 to 7.56, and at test termination ranged from 7.77 to 8.10. CONCLUSIONS The 72-hour EC50, including the 95% confidence interval (C.I.),abased on nominal concentrations is listed below: Healthy Cell Count EC50 1.17 mg/L (95% C.I. = 0.75 to 1.59 mg H-25914/L) H-25914 exhibited medium concern under TSCA and was "toxic" to aquatic organisms (R51), classified as dangerous for the environment, and assigned the symbol "N" 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. -7 Company Sanitized. Does not contain TSCA CBI H -25914: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum DuPont-13348 REFERENCES 1. Slob, W., (2002). Dose-response modeling of continued endpoints. Toxicol. Sci., 66, 298-312. 2. 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, ASTM STP1179. (W.G. Landis, J.S. Hughes, and M.A. Lewis, Eds.), pp 22-39. American Society for Testing and Materials, Philadelphia. 3. Official Journal of the European Communities, Volume 36, 4 May 1993, pp. 68, section 5.2.1. 4. 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. 5. American Society for Testing and Materials (ASTM). (1990). "Standard Guide for Conducting Static 96-h Toxicity Tests with Microalgae" in ASTM Annual Book o fStandards, E1218-90. Vol. 11.04, Philadelphia, PA. Company Sanitized. Does not contain TSCA CBI H-25914: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum DuPont-13348 TABLES -9 Company Sanitized. Does not contain TSCA CBf H-25914: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum DuPont-13348 TABLE 1 pH MEASUREMENTS OF TEST SOLUTIONS Nominal Concentration (mg/L) Test Initiation (0 hours) pH Test Termination (72 hours) pH Blank Control 0.1 1 10 100 7.56 7.53 7.53 7.51 7.51 7.77 8.10 7.98 7.78 7.80 -10- Conparry Sanitized. Dos not contain TS C CB i H -25914: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricomutum DuPont-13348 TABLE 2 CELL COUNT 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 2,600,000 2,740,000 0.1 mg/L Replicate 1 Replicate 2 10,000 10,000 2,980,000 2,720,000 1 mg/L Replicate 1 Replicate 2 10,000 10,000 1,730,000 1,280,000 10 mg/L Replicate 1 Replicate 2 10,000 10,000 240,000 210,000 100 mg/L Replicate 1 Replicate 2 10,000 10,000 230,000 340,000 -11 - C o m pm f Sanitized. Dees not contain TSC& C|| H-25914: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum D u P on t-13 348 FIGURE - 12- Company Sanitized. Does not contain TSCA CBI H-25914: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum DuPont-13348 FIGURE 1 72-HOUR HEALTHY CELL COUNT DOSE-RESPONSE CURVE OECD Model 2, Using MARQUARDT Method F OR MUL A 1 FROM CURRL I B . AWT I 4 7 8 9 r O R M U L A I = Ce 1 I _ C o u n 1 7 2 / 10 0 0 0 0 C O N C E N T R A T I D N S =0 0 . 1 I 10 1 00 OECD Model 2. Using MARQUARDT Method r O R U U L A I FROH CUR R U B . AWI H 7 B 9 F O R U U L A l = C e I I _ Co u n 1 72 / 1 0 0 0 DO CNCI K TUT I0NSMJ 0 . 1 1 10 100 C0 NC - 13 - Company Sanitized. Does not contain TSCA CBI H-25914: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricomutum DuPont-13348 /""'S APPENDICES - 14- Company Sanitized. Does not contain TSCA CBI H-25914: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum DuPont-13348 APPENDIX A AAP Nutrient Medium Constituents - 15 Company Sanitized. Does not contain TSCA CB! H-25914: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum DuPont-13348 AAP NUTRIENT MEDIUM CONSTITUENTS Stock Solutions" Component Concentration (g/L) Macronutrient Stock #1 NaN03 25.500 Macronutrient Stock #2 NaHC03 15.000 Macronutrient Stock #3 K2HPO4 1.044 Macronutrient Stock #4 MgS04*7H20 14.700 Macronutrient Stock #5 MgCl2-6H20 12.164 Macronutrient Stock #6 CaCl2*2H20 4.410 Final Prepared Medium Component Concentration (mg/L) Na 11.001 N 4.200 C 2.143 K 0.469 P 0.186 Mg 2.904b Ca 1.202 S 1.911 Stock Solutions" Component Concentration (mg/L) Micronutrient Stock H3BO3 MnCl2*4 H20 ZnCl2 CoCl26 H20 CuC12*2H20 Na2Mo04*2 H20 FeCl36 H20 Na2EDTA*2 H20 185.520 415.610 3.271 1.428 0.012 7.260 160.000 300.000 Final Prepared Medium Component Concentration (jig/L) 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 4. b Includes magnesium from both magnesium sulfate and magnesium chloride. - 16Company Sanitized, Does not contain TSCA CBt H-25914: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum DuPont-13348 APPENDIX B 72-Hour Healthy Cell Count Statistics - 17- Company Sanitized. Does not contain TSCA CB1 /-"N H -25914: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum DuPont-13348 72-HOUR HEALTHY CELL COUNT STATISTICS Results of Jonckheere test to determine NOEC Check for Number of Reps in FORMULA1 Maximum Number of Reps in Any Treatment Group or Control Maximum Number of Reps per Group is 2 Exact Permutation Methods Recommended Analysis o f | ^ ^ H H Q D a t a Test for TREffl^DnsiTHER DIRECTION in Response=FORMULAl ANALYSIS OF Cell Count at 72 Hours USING ALPHA=0.05 FULL DATA KEY _JT_ IS JONCKHEERE STATISTIC Z_JT IS STANDARDIZED JONCKHEERE STATISTIC XPR_JT IS P-VALUE FOR UPWARD TREND XPL_JT IS P-VALUE FOR DOWNWARD TREND P-VALUES ARE FOR TIE-CORRECTED TEST WITH CONTINUITY CORRECTION FACTOR SIGNIF RESULTS ARE FOR D ALTERNATIVE HYPOTHESIS Jonckheere Trend Test on Dose 0 + Lowest 4 Doses thru 100 PPM ___ JT____ Z_JT XPL_JT 8.5 -2.556039 0.0051146 XPR_JT SIGNIF DOSE . ** 5 Jonckheere Trend Test on Dose 0 + Lowest 3 Doses thru 10.PPM ___ JT____ Z_JT 5 -2.298393 XPL_JT 0.0123016 XPR_JT SIGNIF DOSE . **4 Jonckheere Trend Test on Dose 0 + Lowest 2 Doses thru 1 PPM ___ JT____ Z_JT XPL_JT 4.5 -1.192079 0.1666667 XPR_JT SIGNIF DOSE 3 Thus, the NOEC=l ppm. - 18- Company Sanitized. Does not contain TSCA CBI H -25914: Influence on Growth and Growth Rate o f the Green Alga Selenastmm capricormtum DuPont-13348 EC50 Estimatation OBSERVED PERCENT CHANGE FROM CONTROL MEAN FORMULAI FROM!" Obs CONC MEAN PCTCHNG N 1 0.0 26.70 02 2 0.1 28.50 72 3 1.0 15.05 -44 2 4 10.0 2.25 -92 2 5 100.0 2.85 -89 2 OBSERVED & PREDICTED PERCENT CHANGE FROM CONTROL MEAN FORMULAl=Cell_Count72 / 100000 Obs CONC N MEAN PRED PCTCHNG PREDPCTCHNG 1 0.0 2 26.70 28.2210 2 0.1 2 28.50 26.5972 3 1.0 2 15.05 15.6034 4 10.0 2 2.25 0.0753 5 100.0 2 2.85 0.0000 0 7 -44 -92 -89 0 -6 -45 -100 -100 EC5 0 using Method^=MARQUARDT, OECD Model 2 Covariance Matrix and Standard Error of Parameter Estimates _NAME_ yO EC 50 SE EC50 y0 -0.08165 0.81932 0.045333 -0.081648 0.21291 0.90516 Model 2 Parameter Estimates, EC50 and 95% Confidence Bounds yO EC50 LB UB 28.2210 1.16972 0.75241 1.58703 Model 2 Evaluation Measures SSREG SSERR SSTOTC SSTOTU RSQUARE 3195.49 21.5787 150.621 3217.07 0.85673 note Graph descriptions include the following: formulai: EC50 OECD MODEL 2 FIT BY MARQUARDT, - 19- Company Sanitized. Does not contain TSCA CBf