Document 7Ob9LJXYBx3LLzDJqYRaonep6

- h t Z k - 3 A S if. 466 H-25767: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum_____________________________ STUDY INFORMATION Substance Tested: Svhonvms/Codes: Haskell Number: 25767 AR2 Z 6 - te g p DuPont-12992 Known Impurities: None Physical Characteristics: White liquid 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/Completed: May 27,2003 / (see report cover page) Comp Sanito*. a*, TgCA ^ -3 - H-25767: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum DuPont-12992 SUMMARY A study was conducted to determine the effect o f H-25767 on the growth and growth rate of the green alga Selenastrum capricornutum.a The algae were exposed to nominal concentrations o f 0.1,1,10,100, and 1000 mg H-25767/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 o f the test. The 72-hour EC50, including the 95% confidence intervals (C.I.),b based on nominal concentrations are as follows:0 Healthy Cell Count . EC50 NOEC 46.0 mg/L (95% C.I. = 20.8 to 71.4) 10 mg/L H-25767 exhibited medium concern11for aquatic hazard under TSCA and was assigned "harmftd" to aquatic organisms (R52)e and classified as dangerous for the environment under the EU Directive based on 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 o f growth relative to the control. c Slob, W., (2002). Dose-response modeling o f continuous endpoints. Toxicological Science, V66; pp 298-312. d 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 ST P 1179. (W.G. Landis, J.S. Hughes, and M.A. Lewis, Eds.), pp 22-39. American Society for Testing and Materials, Philadelphia. e Official Journal o f the European Communities, Volume 3 6 ,4 May 1993, pp. 68, section 5.2.1. H-25767: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum DuPont-12992 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.(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 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 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(I) was prepared by adding 1 mL of 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 of the medium was brought to 1 liter with additional Milli-Q water. Appropriate proportions (1 mL o f each stock solution for each 1 liter of 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 at the time o f preparation. The medium was filter-sterilized using Coming pre-sterilized filtration systems each with a 0.22 pm cellulose acetate filter. The medium was stored refrigerated and allowed to equilibrate to test temperature prior to use for testing. Company Sanitized, Does not zm fsm TSCA Cm -5 - H-25767: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum DuPont-12992 1. Test Solution Preparation Five nominal test concentrations (0.1,1,10,100, and 1000 mg H-25767/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. To achieve the desired nominal concentration of approximately 10,000 Selenastrum capricornutum cells/mL at test initiation, an approximate 0.455 mL (= 455 pL) aliquot o f 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 two wells of the hemacytometer and 16 grids (8 from each well) were selected for counting. 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 o f 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. Sanitized^ D o es m f confafn T SC 4 CBI H-25767: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricomutum DuPont-12992 RESULTS Based on visual observations, the blank control solution and the 0.1,1, and 10 mg/L test solutions were clear and colorless before addition o f algal cells. The 100 mg/L test solution was slightly cloudy before addition of algal cells. The 1000 mg/L test solution was 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 of approximately 25 C. The algae were incubated for 72 hours (3 days) without medium renewal. Illumination was supplied by coolwhite fluorescent tubes. The mean light intensity in the chamber was 6908 lumens/m2 (= lux). The shaking speed was 103 revolutions per minute (rpm). The pH measurement o f the culture medium was approximately 6.8 at study start. The pH measurements o f the test solutions at test initiation ranged from 6.80 to 7.02, and at test termination ranged from 7.47 to 7.82 (Table 1). Counts of healthy algal cells on day 0 (test start) and day 3 (test termination) are presented in Table 2. Cells counts for the blank control increased by greater than 16X over the course of the study. CONCLUSIONS The 72-hour EC50, including the 95% confidence intervals (C.I.),8and NOEC based on nominal concentrations were as follows: Healthy Cell Count EC50 NOEC 46.0 mg/L (95% C.I. = 20.8 to 71.4) 10 mg/L H-25767 exhibited medium concern1'4'1for aquatic hazard under TSCA and was assigned "harmful" to aquatic organisms (R52) and classified as dangerous for the environment under the EU Directive based on a 72-hour, acute test using the green alga Selenastrum capricomutum. a The EC50 is defined as the "effective concentration" producing a 50% inhibition o f growth relative to the control. C_-o7m-pamy Sanitized. Does not c'vifam TSCA CBI H-25767: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum DuPont-12992 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 o f Standards, E 1218-90. Vol. 11.04, Philadelphia, PA. 3. Slob, W., (2002). Dose-response modeling o f continuous endpoints. Toxicological Science, V66;pp 298-312. 4. 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 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. 5. Official Journal o f the European Communities, Volume 36 ,4 May 1993, pp. 68, section 5.2.1. - 8 Company Sanitized Does not contain T SC A C gf H-25767: Influence on Growth and Growth Rate o f the Green Alga Selenastmm capricomutum DuPont-12992 TABLES Company Sanitized. -9 - contain TSCACSf H-25767: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum DuPont-12992 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 1000 6.80 6.88 6.93 6.97 7.01 7.02 7.62 7.82 7.78 7.74 7.47 7.47 Company Sani,ted . Doas no, cow ,,,, TSCA c | - 10- H-25767: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricomutum DuPont-12992 TABLE 2 CELL COUNT DATA SUMMARY Nominal Day 0 Healthy Cell Count Day 3 Healthy Cell Count Concentration (ceils/mL) (cells/mL) Blank Control Replicate 1 Replicate 2 10,000 10,000 3,420,000 3,390,000 0.1 mg/L Replicate 1 Replicate 2 10,000 10,000 3,330,000 3,020,000 1 mg/L Replicate 1 Replicate 2 10,000 10,000 3,210,000 2,990,000 10 mg/L Replicate 1 Replicate 2 10,000 10,000 3,150,000 3,640,000 100 mg/L Replicate 1 Replicate 2 10,000 10,000 40,000 30,000 1000 mg/L Replicate 1 Replicate 2 10,000 10,000 0 0 Company Sanitized. D o e s not contain TSCA CBl H-25767: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum DuPont-12992 FIGURE Company Sanitized. Does not contain T SC A CB1 - 12- H-25767: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum DuPont-12992 FIGURE 1 72-HOUR HEALTHY CELL COUNT DOSE-RESPONSE CURVE OECD Model 2. Using NEWTON Method FORMULA 1 FROM F E R R E L .AWR14641 FORMULA1=CeIl_Count72 / 00000 C O N C E N T R A T IO N S ^ 0. I 1 10 100 1000 O EC D Model 2, U sing NEWTON Method F0RMULA1 FROM F E R R E L .AWR14641 F O R M U L A 1= C eI I _ C o un t 7 2 / 1 0 0 0 0 0 CQNCENTRAT 10NS=D D.1 1 10 100 1000 CONC Com pany Sanitized. Does not contain TSCA CBi -13 - H-25767: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricomutum APPENDICES Company Sanitized. Does not contain TSCA CBS H-25767: Influence on Growth and Growth Rate o f the Green Alga Selenastnm capricomutum DuPont-12992 APPENDIX A AAP Nutrient Medium Constituents Compaq S a n iti - 15- H-25767: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum DuPont-12992 AAP NUTRIENT MEDIUM CONSTITUENTS Stock Solutions" Final Prepared Medium Component_______ Concentration (g/L)______ Component Concentration (mg/L) Macronutrient Stock #1 NaN03 25.500 Na 11.001 N 4.200 Macronutrient Stock #2 NaHC03 15.000 C 2.143 K 0.469 Macronutrient Stock #3 K2HPO4 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"________________________ Final Prepared Medium Component Concentration (mg/L) Component Concentration (pg/L) Micronutrient Stock H3BO3 MnCl24 H20 ZnCl2 CoCl2*6 H20 CuC12*2H20 Na2M o04*2 H20 FeCl3*6 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. gedteed. D<*8not dentilo TSCA CB - 16- H-25767: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum DuPont-12992 APPENDIX B 72-Hour Healthy Cell Count Statistics - 17- H-25767: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricomutum D u P o n t-12992 72-HOUR HEALTHY CELL COUNT STATISTICS Check for Number of Reps in RESPONSE 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 f f l H H H s a t a Test for DECREASIN&TREND in Response=FORMULAl ANALYSIS OF ONION EXPERIMENT USING ALPHA=0.05 FULL DATA KEY _1T_ IS 10NCKHEERE STATISTIC Z_1T IS STANDARDIZED 10NCKHEERE STATISTIC XPR_1T IS P-VALUE FOR UPWARD TREND XPL_1T IS P-VALUE FOR DOWNWARD TREND P-VALUES ARE FOR TIE-CORRECTED TEST WITH CONTINUITY CORRECTION FACTOR SIGNIF RESULTS ARE FOR D ALTERNATIVE HYPOTHESIS lonckheere Trend Test on Dose 0 + Lowest 5 Doses thru 1000 ppb ___IT____ Z_JT XPi-- IT 11.5 -2.928002 0.0010358 XPR_IT SIGNIF DOSE . ** 6 lonckheere Trend Test on Dose 0 + Lowest 4 Doses thru 100 PPB ___IT_____ Z_IT XPI IT 11.5 -2.008316 0.026455 XPR_IT SIGNIF DOSE . ** 5 lonckheere Trend Test on Dose 0 + Lowest 3 Doses thru 10 PPB ___IT_____ Z_1T XPL_.1T 11 -0.766131 0.268254 XPR_1T SIGNIF DOSE .4 $*** Sanitized. Does not contain T S C A C S l - 18- H-25767: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricomutum DuPont-12992 OBSERVED & PREDICTED PERCENT C H A N Q ^ R M y j p N T R O L MEAN FORMULAI FROM FERREl/ S H I H } FORMULAI =Cel l_Count 7 Obs CONC N MEAN PRED PCTCHNG PREDPCTCHNG 1 0.0 2 34.05 33.6004 2 0.1 2 31.75 33.5499 3 1.0 2 31.00 33.0987 4 10.0 2 33.95 28.9073 5 100:0 2 0.35 7.4642 6 1000.0 2 0.00 0.0000 0 -7 -9 0 -99 -100 0 0 -1 -14 -78 -100 EC50 using Method=NEWTON, OECD Model 2 Covariance Matrix and Standard Error of Parameter Estimates ,NAME_ yO EC50 SE EC50 yO -4.32943 1.49259 166.707 -4.329 12.9115 1.2217 Model 2 Parameter Estimates, EC50 and 95% Confidence Bounds yO EC50 LB UB 33.6004 46.0739 20.7674 71.3805 Model 2 Evaluation Measures SSREG SSERR SSTOTC SSTOTU RSQUARE 8126.69 99.3469 469.526 8226.04 note Graph descriptions include the following: formulai: EC50 OECD MODEL 2 FIT BY NEWTON, 0.78841 w m m f Sanitized. Does noi contain c II - 19-