Document ZJ5pZB4OgwG8R5Y6M4D5zvenO

1 -------------------------------------------------------------------------------------------------------- IU A 6 - 3 -2 7 / 453 A M - /327 DuPont-11604 TRADE SECRET Study Title H-25435: Influence on Growth and Growth Rate of the Green Alga Selenastrum capricomutum Laboratory Project ID : DuPont-11604 Test Guidelines: OECD Guideline for Testing Chemicals Section 2: Effects on Biotic Systems, Number 201 (1984) Commission Directive 92/69/EEC EEC Method C3 (1992) Author: Terry Lee Sloman, B.S. Study Completed on: March 31,2003 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 Work Request Number: Service Code Number: Page 1 o f 57 Company Sanitized. Does not contain TSCA CBI H-25435: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum DuPont-11604 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT This study was conducted in compliance with U.S. EPA TSCA (40 CFR part 792) Good Laboratory Practice Standards, which are consistent with the OECD Principles o f Good Laboratory Practice (as revised in 1997) published in ENV/MC/CHEM(98)17 and MAFF Japan Good Laboratory Practice Standards (59 NohSan Number 3850) except for the item documented below. The item listed does not impact the validity o f the study. The test substance is commercially available and was characterized by the supplier prior to the initiation o f this study. An analysis was provided, although the characterization was not performed under Good Laboratory Practice Standards, based on our knowledge o f the performing laboratory and its experience with analyzing this chemical, we have no reason to doubt the validity of the analysis. Applicant / Sponsor: E.I. du Pont de Nemours and Company Wilmington, Delaware 19898 U.S.A. Study D irector je. Terry L ee Slom an, B .S . A ssociate Scientist 3 l~ /r\A # ~ 2 o o 5 D ate Applicant / Sponsor:__________________________________________ __________ DuPont Representative Date -2Company Sanitized. Does not contain TSCA CBI H-25435: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum QUALITY ASSURANCE STATEMENT Haskell Sample Number(s): 25435 Dates of Inspections: Protocol: January 7, 2003 Conduct: January 7, 2003 Records, Reports: March 17, 19, 20, 2003 Dates Findings Reported to: Study Director: March 20, 2003 Management: March 31, 2003 DuPont-11604 Reported by: Denise m. Janney ^ Quality Assurance Auditor Date -3 Company Sanitized. Does not contain TSCA CBI H-25435: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum DuPont-11604 CERTIFICATION We, the undersigned, declare that this report provides an accurate evaluation of data obtained from this study. ! Bogdan Szostek, Ph.D. 1 Senior Research Chemist D ate S tatistical R eported by: r John W . Green, P hD ., PhD . Principal R esearch Statistician 2hM *s~ 7x> 3 D ate Issued by Study D irecto r: 0 Terry Lee Sloman, B.S. Associate Scientist 3 f'/nA fr xooJ D ate .4. Company Sanitized. Does not contain TSCA CBi H-25435: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum DuPont-11604 TABLE OF CONTENTS Page GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT..................................... 2 QUALITY ASSURANCE STATEM ENT.........................................................................................3 C E R T I F I C A T I O N .................................................................................................................................. 4 LIST OF TABLES..................................................................................................................... LIST OF FIGURES................................................................................................................................6 LIST OF APPENDICES.............................................................................. STUDY INFORM ATION................................................................................................... STUDY PERSONNEL........................................................................................................................... 8 SUM M ARY..............................................................................................................................................9 INTRODUCTION............................................................................. MATERIALS AND M ETHODS....................................................................................................... 10 A. Test G uidelines.......................................................................................................................... 10 B. Test Substance ............................................................................................................................ 10 C. Test System................................................................................................................................. 10 D. Test D esign................................................................................................................................. 11 E. Test Preparation......................................................................................................................... 11 F. Experimental Design..................................................................................................................12 G. Test Solutions.............................................................................................................................12 H. Test Conditions - Definitiveand Recovery Tests................................. 12 I. Selenastrum capricornutumGrowth M easurement.................................................................13 J. Statistical A nalyses.................................................................................................................... 13 K. Analysis o f Test Solutions - Definitive Test.........................................................................14 RESULTS AND DISCUSSION................................................................................... A. Environmental Conditions - Definitive and Recovery T ests.................................................19 B. Test Substance Stability and Analytical Verification............................................................ 19 C. Selenastrum capricornutum Growth........................................... 20 C O N C L U S IO N S ............................................................................................ RECORDS AND SAMPLE STORAGE.........................................................................................21 REFERENCES................................................................................................................ TABLES............................................................................................................................ FIGURES................................................................................................................................................ 32 APPENDICES....................................................................................................................................... 41 6 7 19 22 23 -5 Company Sanitized. Does not contain TSCA CBI H-25435: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum DuPont-11604 LIST OF TABLES Page 1. pH MEASUREMENTS OF TEST SOLUTIONS......................................................................................................... 24 2. CHAMBER LIGHT INTENSITY, SHAKING SPEED, AND TEMPERATURE RANGE.................................24 3. MEASURED CONCENTRATIONS OF H-25435 IN TEST SOLUTIONS............................................................25 4. HEALTHY CELL COUNT DATA SUMMARY - DEFINITIVE TEST................................................................. 28 5. AREA UNDER THE GROWTH CURVE DATA SUMMARY - DEFINITIVE TEST .......... ............................. 29 6. GROWTH RATE DATA SUMMARY - DEFINITIVE TEST...................................................................................30 7. 72-HOUR (DAY 3) STATISTICAL ANALYSES SUMMARY - DEFINITIVE TEST....................................... 31 LIST OF FIGURES Page 1. REPRESENTATIVE ANALYTICAL CALIBRATION STANDARD CURVE FOR H-25435: WItt/KiKKt^...................... 2. REPRESENTATIVE CHROMATOGRAM OF A CALIBRATION STANDARD SOLUTION........................ 35 3. REPRESENTATIVE CHROMATOGRAM OF THE BLANK CONTROL SOLUTION.................................... 37 4. REPRESENTATIVE CHROMATOGRAM OF A TEST CONCENTRATION SOLUTION............................. 39 LIST OF APPENDICES Page A. AAP NUTRIENT MEDIUM CONSTITUENTS..........................................................................................................42 B. 72-HOUR HEALTHY CELL COUNT STATISTICS FOR THE DEFINITIVE TEST.........................................44 C. 72-HOUR AREA UNDER THE GROWTH CURVE STATISTICS FOR THE DEFINITIVE TEST................48 D. 72-HOUR GROWTH RATE STATISTICS FOR THE DEFINITIVE TEST......................................................... 53 -6 Company Sanitized. Does not contain TSCA CBI H-25435: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum STUDY INFORMATION Substance Tested: Svnonytns/Codes: . H-25435 DuPont-11604 Known Impurities: Physical Characteristics: Amber brown liquid Stability: The test substance appeared to be stable under the conditions o f the study. Sponsor: E. I. du Pont de Nemours and Company Wilmington, Delaware 19898 U.S.A. Study Initiated/Comnleted: January 6,2003 / (see report cover page) -7 Company Sanitized. Does not contain TSCA CBJ H-25435: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum STUDY PERSONNEL Study Director: Terry Lee Sloman, B.S. Management: Robert A. Hoke, Ph.D. Primary Technician: Barbra D. Ferrell, B.S. Analytical Chemist: Bogdan Szostek, Ph.D. Management: S. Mark Kennedy, Ph.D Statistical Analysis: John W. Green, Ph.D., Ph.D. Management: Janice L. Connell, M.S., B.A., C.I.H. Ecotoxicology Report Preparation: Wanda F. Dinbokowitz Management: Nancy S. Selzer, M.S. DuPont-11604 - 8Company Sanitized. Does not contain TSCA CB1 H-25435: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum DuPont-11604 SUMMARY A study was conducted to determine the effect o f H-25435 on the growth and growth rate of the green alga Selenastrum capricornutum.a The algae were exposed to nominal concentrations o f 6.25, 12.5, 25, 50, and 100 mg H-25435/liter o f nutrient medium (ppm). For the definitive (dose-response) test, the organisms 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 (cell density), area under the growth curve, and growth rate relative to the blank (normal culture medium) control for the 72-hour (day 3) interval o f the test. The 72-hour results'3based on nominal concentrations are as follows: Healthy Cell Count e c 50 NOEC > 100 mg H-25435/L > 100 mg H-25435/L Area Under the Growth Curve e c 50 > 100 mg H-25435/L NOEC > 100 mg H-25435/L Growth Rate EC50 NOEC > 100 mg H-25435/L > 100 mg H-25435/L None o f the definitive test concentrations exhibited a 50% or greater growth inhibition based on healthy cell count relative to the blank control. Therefore, a recovery test was not performed. a Also known as Pseudokirchneriella subcapitata. b The EC5ois 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 statistically significant effect on the measured parameter relative to the control. -9 Company Sanitized. Does not contain TSCA C8I H-25435: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum DuPont-11604 INTRODUCTION This study was conducted to determine the effect of H-25435 on the growth and growth rate o f the green alga Selenastrum capricornutum.a MATERIALS AND METHODS A. Test Guidelines The study design complies with the following test guidelines: Organisation for Economic Co-Operation and Development (OECD) (1984). 201 Effects on Biotic Systems. Guidelinefo r Testing o f Chemicals. European Economic Communities (EEC) (1992). Directive 92/69/EEC Annex V, Part C.3, Algal Inhibition Test. Methodsfo r the Determination o f Toxicity. B. Test Substance The test substance, H-25435, was supplied by the sponsor as an amber brown liquid. It was received October 17, 2002; the expiration date is April 17, 2003. C. Test System Selenastrum capricornutum, a freshwater, unicellular, non-motile, green alga, was used in this study. 1. Original Culture Source 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. 2. Culture Maintenance 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) 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. Also known as Pseudokirchneriella subcapitata. - 10Company Sanitized. Does not contain TSCA C8I H-25435: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum DuPont-11604 I). Test Design The test design used for the Selenastrum capricornutum definitive and recovery tests is described below: Organism Selenastrum capricornutum Nutrient Medium AAP Flask Volume (mL) 250 Solution Volume (mL) 50 Volume Ratio 5:1 E. Test Preparation (Appendix A) 1. AAP Nutrient Medium Preparation for Selenastrum capricornutum AAP nutrient m edium ^ was prepared by adding 1 mL o f each of 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 proportions (1 mL o f each stock solution for each 1 liter o f medium) were used to prepare the larger volumes o f the medium required for the definitive and recovery tests. The medium pH was adjusted to 7.46 with 0.1N hydrochloric acid. For both the definitive and recovery tests, the medium was filter-sterilized using Coming pre-sterilized filtration systems each with a 0.22 pm cellulose acetate filter. The containers with the resulting filter-sterilized AAP nutrient medium for use in the definitive and recovery tests were stored in the refrigerator in the dark at approximately 4C and acclimated to room temperature prior to use. Any remaining unused medium was properly stored. 2. Test Solution Preparation Prior to the initiation of the definitive test, method development was conducted to determine the appropriate concentrations to be used in the definitive test. A primary stock solution was prepared by dissolving the test substance in 1000 mL filtersterilized AAP nutrient medium for a nominal concentration o f 100 mg H-25435/L (= 100 ppm). A working stock solution was prepared by diluting the primary stock solution with filtersterilized AAP nutrient medium to make a nominal concentration o f 100 mg H-25435/L (= 100 ppm). Aliquots o f the filter-sterilized AAP nutrient medium were used for the blank (normal culture medium) control solution. Test solutions were prepared using aliquots o f the nominal 100 mg/L working stock solution and diluting with filter-sterilized AAP nutrient medium to make nominal concentrations o f 6.25,12.5, 25, and 50 mg H-25435/L. Aliquots o f the nominal 100 mg/L working stock solution were used for the 100 mg H-25435/L test concentration solution and the abiotic (stability) control solution. -11 Company Sanitized. Does not contain TSCA CBI H-25435: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum DuPont-11604 3. Test Culture Preparation For each definitive control and test solution, 3 aliquots (50 mL each) were placed in separate sterilized 250 mL Erlenmeyer flasks fitted with sterilized foam stoppers. Each flask, excluding the abiotic control, was randomly assigned a number to eliminate bias while counting. To achieve the desired nominal concentration o f approximately 10,000 Selenastrum, capricornutum cells/mL at test initiation, an approximate 0.370 mL (= 370 pL) aliquot o f algal inoculum from a logarithmically growing stock culture was aseptically transferred to each flask, except the abiotic control. F. Experimental Design 1. Definitive Test The organisms were exposed for 72 hours (3 days), without test medium renewal. Test flasks were arranged on a lighted shelf in a chamber in a non-systematic design and were re-positioned each working day. Each blank control, test concentration, and abiotic control was tested as 3 replicates. Cell counts were made approximately 24,48, and 72 hours after the definitive test initiation (0-hour or day 0). 2. Recovery Test None of the definitive test concentrations exhibited a 50% or greater growth inhibition based on healthy cell count relative to the blank control. Therefore, a recovery test was not performed. G. Test Solutions Solutions used in the definitive test were as follows: Blank Control AAP nutrient medium containing no H-25435 Treatment nominal 6.25, 12.5, 25, 50, and 100 mg H-25435/liter nutrient medium Abiotic Control nominal 100 mg H-25435/liter nutrient medium; (no Selenastrum capricornutum) H. Test Conditions - Definitive and Recovery Tests The control and test flasks were placed in a chamber (Hotpack model 06084) and air temperature in the chamber was recorded continuously with a continuous temperature recorder (Dickson Model SL445C7). The algae were incubated for 72 hours without test medium renewal for the definitive test. Illumination was supplied by cool-white fluorescent tubes. The target environmental parameters are described in the following table. - 12Company Sanitized. Does not contain TSCA CBI H-25435: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum DuPont-11604 Initial Test Population D efinitive 10,000 cells/m L Illumination (lumens/m2- lux) PhotoPeriod (hours) Shaking Speed (rpms) Temperature (C) 6 0 0 0 to 10,000 24 100 24 2 I. Selenastrum capricornutum Growth Measurement 1. Definitive Test Selenastrum capricornutum growth measurement was determined by visually counting the number o f cells taken from an approximate 0.2 mL sample from each flask at approximately 24, 48, and 72 hours from the definitive test 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 for counting. All cells located in the 16 grids were counted and recorded as healthy or unhealthy. The total number o f cells counted per grid 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 o f healthy and unhealthy cells (e.g., deformed, senesced, stunted) were recorded in the study records. Most counts were made at approximately the same time each day. Statistical calculations o f the EC50 and NOEC were based on mean healthy cell counts and nominal concentrations. 2. Recovery Test None o f the definitive test concentrations exhibited a 50% or greater growth inhibition based on healthy cell count relative to the blank control. Therefore, a recovery test was not performed. J. Statistical Analyses The mean healthy cell count, area under the growth curve, and growth rate at the 72-hour interval for each test concentration were expressed relative to the blank control. All calculations were based on the nominal concentrations. The area under the growth curves for each interval were calculated according to the following formula: A = [0.5 x ti x (Ni - No)] + [0.5 x (t2 - h) x (A2 + M - 2A0)] + ...+ [0.5 x (tn - v 0 X (An + An.! - 2A0)] where t\, t2, V i, and tn are times o f observation measured from the initiation o f the test and Ao, Ai, An-i, and An are the initial and subsequent healthy cell counts (cells/mL) corresponding to the observation times. The growth rate (p) for each test concentration and for the controls were calculated according to the following formula: - 13Company Sanitized. Does not contain TSCA CBI H-25435: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum DuPont-11604 ,, = ln (N n/N 0) ^ tn where tn is the time o f observation measured from the initiation o f the test and N 0and Nnare the initial and subsequent healthy cell counts (cells/mL) corresponding to the observation time. The percent growth inhibition, % I, was calculated according to the following formula: % I = ..^ C T x 100 where C is equal to the mean healthy cell count, area under the growth curve, or growth rate for the blank control at a selected sampling interval and T is the mean healthy cell count, area under the growth curve, or growth rate for each test concentration at a selected sampling interval. Negative values of inhibition indicate stimulation of growth. The "effective concentration" (EC50) was defined as the concentration producing a 50% inhibition o f growth relative to the control. The EC50value and associated 95% confidence limits were determined by weighted least-squares non-linear regression o f the log o f the test concentration against the measured parameter. The NOEC, defined as the highest concentration o f test substance that had no significant effect on the measured parameter relative to the blank control, was determined from a trend test (Jonckheere-Terpstra or Williams) applied in a stepdown manner. All tests o f significance were at a = 0.05.(3_4) K. Analysis of Test Solutions - Definitive Test 1. Sample Collection The concentrations o f H-25435 in the working stock (nominal 100 mg H-25435/L), blank control, and test concentration (nominal 6.25,12.5, 25, and 50 mg H-25435/L) solutions were verified at test initiation (day 0). An aliquot from each o f the blank control and test concentration solutions was taken prior to the addition o f Selenastrum capricornutum. The pH was determined for each of the working stock, blank control, and test concentration solutions. The concentrations o f H-25435 in the blank control, test concentration (nominal 6.25,12.5, 25, 50, and 100 mg H-25435/L), and abiotic control (nominal 100 mg/L H-25435/L) solutions were verified at test termination (day 3). These samples were prepared by pooling all 3 replicates for each of the control and test concentration solutions and taking an aliquot from each o f the pooled samples. The pH was determined for each o f these day 3 samples. The concentrations o f in each o f the test solutions were determined by high performance liquid chromatography/mass spectrometry (HPLC/MS). - 14Company Sanitized. Does not contain TSCA CBI H-25435: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum DuPont-11604 2. Sample Treatment An aliquot from the blank control, working stock, and test concentration were sampled at the study start (day 0) and submitted for analysis. An aliquot from the blank control, test concentration, and abiotic control were sampled at the end o f the exposure (day 3) and submitted for analysis. Back-up solutions for each sample also were provided. All samples were analyzed on the day o f receipt. Day 0 samples were also analyzed on day 1. Centrifugation and settling o f algae were investigated as methods for analyzing day 3 samples. Settling o f algae was the preferred method based on analytical recoveries o f the test material. The day 3 samples were placed in a refrigerator and stored for 3 days to facilitate the settling of algae. After 3 days of storage the supernatants o f the refrigerated day 3 samples were taken and analyzed. Aliquots o f all test solutions were transferred to autosampler vials for analysis by LC/MS. The samples were diluted before analysis with AAP nutrient medium as follows: 6.25 mg/L (1:2), 12.5 mg/L (1:5), 25 mg/L (1:10), 50 mg/L (1:25), and 100 mg/L (1:50). -15Company Sanitized. Does not contain TSCA CBI H-25435: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum 3. Instrumentation and Conditions HPLC Instrument: MS Instrument: Model 2795, Waters Quattro Micro, Micromass DuPont-11604 LC Parameters: Column: Mobile Phase: Luna 3u C l 8(2), 20 mm x 2mm A- 50 mM ammonium acetate in water C- methanol Gradient: Time (min) %C Flow (mL/min) 0 30 0.25 5 100 0.25 10 100 0.25 10.1 30 0.50 12 30 0.50 12.1 30 0.25 Column Temperature: Injection Volume: Column Switch: 30C 50 pL 2 and 10 min MS Parameters: Ionization mode: Capillary voltage: Cone voltage: Source Temperature: Desolvation Temperature: Data Acquisition Function: Electrospray, negative ions 3.2 kV 30 V 120C 450C C6-P-C6 bis ester ion: SIR o f 789 m/z; 0-10 min C6-P-IPA mixed ester ion: SIR of 485 m/z; 0-10 min C6-P mono ester ion: SIR o f 443 m/z; 0-10 min 4. Quantitation is a H V 1116 esent in H-25435 can be subdivided into three distinct groups: One chemical species was selected from each group and monitored by LC/MS though the study. The selected species chemical structures, names, and abbreviations used were as follows: - 16Company Sanitized. Does not contain TSCA CBI H-25435: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum DuPont-11604 The test substance H-25435 used as the reference compound. The test substance H-25435 has not been characterized to determine actual contents o f the three species that were monitored during the study. The concentrations o f individual chemical species that were monitored during the study were represented as the total concentrations o f the H-25435. A primary stock solution o f the reference compound, H-25435 was made by dissolving approximately 60 mg o f H-25435 in 500 mL o f AAP nutrient medium. Approximately 30 minutes o f sonication was required to completely dissolve the test substance. Calibration standards were prepared on each test day by dilution with AAP nutrient medium at concentrations ranging from 0.288 to 5.75 mg/L. Duplicate injections o f calibration solutions were made and peak areas were determined by electronic integration. The calibration curves were generated by regression analysis o f the peak area data obtained from chromatographic analysis o f the calibration solutions. Data for test solutions was fitted to the appropriate calibration curves to determine the concentrations o f each o f monitored species. The - 17Company Sanitized. Does not contain TSCA C8I H-25435: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricomutum DuPont-11604 limit o f detection (LOD) and limit o f quantitation (LOQ) were determined by calculating the average noise level in chromatograms o f blank control samples and comparing them to the signal of a calibration standard o f known concentration. Two chromatograms were examined for noiserelated peaks near the retention time o f the analyte. The LOD was calculated as 3 times the concentration equivalent o f the mean noise level. The LOQ was calculated as 10 times the concentration equivalent o f the mean noise level. - 18Company Sanitized. Does not contain TSCA CBI H-25435: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum DuPont-11604 RESULTS AND DISCUSSION A. Environmental Conditions - Definitive and Recovery Tests (Tables 1-2) For the definitive test, the pH measurements o f the test solutions ranged from 7.23 to 7.48 at the 0-hour and from 7.66 to 8.49 at the 72-hour interval. For the definitive test, light intensity for the areas used in the chamber ranged from 8580 to 9440 lumens/m2 (= lux). The mean light intensity was 9022 lumens/m2. For the definitive test, the shaking speed was 107 revolutions per min (rpm). For the definitive test, the temperature from the digital readout on the temperature recorder ranged from 25.1 to 25.3C for the duration o f the study. B. Test Substance Stability and Analytical Verification (Table 3, Figures 1-4) 1. Chromatographic Results *eluted as a well-resolved peak with a retention time of approximately 6.8 minutes. eluted as a well-resolved peak with a retention time o f approximately 5.9 minutes. eluted as a well-resolved peak with a retention time of approximately 5.3 minutes. Figure 1 shows a representative calibration curve obtained for chromatogram of a typical standard is shown in Figure 2. Figures 3 and 4 show chromatograms o f a blank control solution and test concentration solution, respectively. The LOD and LOQ f o r d e t e r m i n e d to be 0.001 mg/L and 0.003 mg/L of H-25435, respectively. The LOD and LOQ f o r ^ ^ H H H H B H V were determined to be 0.012 mg/L and 0.04 mg/L of H-25435, respectively. The LOD and LOQ for were determined to be 0.0036 mg/L and 0.012 mg/L o f H-25435, respectively. 2. Test Solution Results The mean, measured concentration o f in the day 0 working stock (= 100 mg H-25435/L test concentration, = abiotic control) was 85 mg/L. This represents 85% recovery o f the active ingredient. The mean, measured concentration in the day 0 working stock (=100 mg H-25435/L test concentration, = abiotic control) was 94 mg/L. This represents 94% recovery o f the active ingredient. The mean, measured concentration o f i ^ U k the day 0 working stock (=100 mg H-25435/L test concentration, = abiotic control) was 102 mg/L. This represents 102% recovery o f the active ingredient. These data indicated the -19Company Sanitized. Does not contain TSCA CBI H-25435: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum DuPont-11604 stock solution was prepared at the desired concentration and confirmed the expected test concentration was achieved. The mean, measured concentrations of in the day 0 nominal 6.25,12.5, 25, and 50 mg/L test concentration solutions were 5.0,11, 21, and 44 mg/L, respectively. This represents 80, 88, 84, 88 % recovery o f the active ingredient, respectively. The mean, measured concentrations the day 0 nominal 6.25,12.5,25, and 50 mg/L test concentration solutions were 6.2,15, 27, and 50.mg/L, respectively. This represents 9 9 ,1 2 0 ,1 08,100 % recovery o f the active ingredient, respectively. The mean, measured concentrations o f ( H H H H N V i n the day 0 nominal 6.25, 12.5, 25, and 50 mg/L test concentration solutions were 6 .9 ,1 3 ,2 6 , and 52 mg/L, respectively. This represents 110,104, 104, 104 % recovery o f the active ingredient, respectively. These data indicated the stock and test concentration solutions were prepared at the desired nominal concentrations. After 3 days, the mean, measured concentrations ^ nominal 6.25,12.5,25, 50, and 100 mg/L test concentrations, and abiotic control solutions were 2.5, 5.9, 12, 20, and 45 mg/L and 44 mg/L, respectively. After 3 days, the mean, measured concentrations of { f l H H H H H H H i in the nominal 6.25,12.5, 25, 50, and 100 mg/L test concentrations, and abiotic control solutions were 5.3,12, 24, 54, and 100 mg/L and 95 mg/L, respectively. After 3 days, the mean, measured concentrations o f f l | B H H H H | i n the nominal 6.25,12.5, 25, 50, and 100 mg/L test concentrations, and abiotic control solutions were 1.6, 7.6,19, 43, and 86 mg/L and 110 mg/L, respectively. The blank control solutions contained no detectable concentrations bth day 0 and day 3. Using the mean of the day 0 and day 3 results, the concentrations o f f l I H B H M H V ' active ingredient o f H-25435, o f the nominal 6.25,12.5, 25, 50,100 mg/L test concentration and the abiotic control solutions were 3.75, 8.45, 16.5, 32, 65, and 64.5 mg/L, respectively. This represents 60, 68, 66, 64, 65, and 65% recovery o f the active ingredient, respectively. Using the mean o f the day 0 and day 3 results, the concentrations of active ingredient of H-25435, o f the nominal 6.25,12.5, 25, 50, 100 mg/L test concentration and the abiotic control solutions were 5.75,13.5,25.5, 52, 97, and 94.5 mg/L, respectively. This represents 92, 108,102, 104, 97, and 95% recovery o f the active ingredient, respectively. Using the mean of the day 0 and day 3 results, the concentrations o f ^ g ^ g g g 0 H M > active ingredient o f H-25435, o f the nominal 6.25,12.5, 25, 50, 100 mg/L test concentration and the abiotic control solutions were 4.25,10.3, 22.5,47.5, 94, and 106 mg/L, respectively. This represents 68, 82, 90, 95, 94, and 106% recovery o f the active ingredient, respectively. C. Selenastrum capricornutum Growth (Tables 4-8, Figures 5-7, Appendices C-E)' 1. Definitive Test The organisms were exposed for 72 hours without test medium renewal. The effects were expressed in terms o f percent inhibition in growth based on healthy cell count, area under the - 20Company Sanitized. Does not contain TSCA CBI H-25435: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum DuPont-11604 growth curve, and growth rate relative to the blank control for the 72-hour interval o f the test. The 72-hour results are as follows: Healthy Cell Count E C 50 NOEC > 100 mg H-25435/L >100 mg H-25435/L Area Under the Growth Curve e c 50 > 100 mg H-25435/L NOEC > 100 mg H-25435/L Growth Rate EC50 NOEC > 100 mg H-25435/L > 100 mg H-25435/L 2. Recovery Test None o f the definitive test concentrations exhibited a 50% or greater growth inhibition based on healthy cell count relative to the blank control. Therefore, a recovery test was not performed. CONCLUSIONS There was no significant inhibitory effect on the growth and growth rate of Selenastrum capricornutum when exposed to nominal concentrations o f 6.25, 12.5, 25, 50, and 100 mgH-25435/L for 72 hours. Therefore, the EC50and NOEC for all 3 endpoints are greater than 100 mg H-25435/L. RECORDS AND SAMPLE STORAGE All data and records for analytical characterizations conducted by the sponsor will be retained by the sponsor. Specimens (if applicable), raw data, and the final report will be retained at Haskell Laboratory, Newark, Delaware, the DuPont Crop Protection Archives, Newark, Delaware, or at Iron Mountain Records Management, Wilmington, Delaware. -21 Company Sanitized. Does not contain TSCA CBI H-25435: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum DuPont-11604 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 fStandards, E 1218-90. Vol. 11.04, Philadelphia, PA. 3. Snedecor, G.W. and Cochran, W.G. (1967). Statistical Methods, 6th edition. The Iowa State University Press. 4. Tamhane, A.C., Dunnett, C.W., Green, J.W., and Wetherington, J.D. (2001). Multiple test procedures for identifying the maximum safe dose. J. Amer. Statist. Assoc. 96, 835-843. - 22Company Sanitized. Does not contain TSCA CSI H-25435: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum DuPont-11604 TABLES 2 3- - Company Sanitized. Does not contain TSCA CBI H-25435: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum DuPont-11604 TABLE 1 pH MEASUREMENTS OF TEST SOLUTIONS Nominal H-25435 Concentration Exposure Initiated: 07-Jan-2003 0-Hour (Day 0) pH Exposure Ended: 10-Jan-2003 72-Hour (Day 3) Blank Control 7.48 7.66 6.25 mg/L 12.5 mg/L 25 mg/L 50 mg/L 100 mg/L 7.39 7.38 7.32 7.28 7.23 7.78 8.43 8.49 8.48 8.47 Abiotic Control (100 mg/L) 7.23 7.66 TABLE 2 CHAMBER LIGHT INTENSITY, SHAKING SPEED, AND TEMPERATURE RANGE Mean Light Intensity at Test Initiation Oscillations Temperature Test (lumens/m2= lux) (rpms) (C) Definitive 9022 107 25.1 to 25.2 2 4- - Company Sanitized. Does not contain TSCA CBI H-25435: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricomutum DuPont-11604 TABLE3 MEASURED CONCENTRATIONS OF H-25435 IN TEST SOLUTIONS Sample Description3 Total, Nominal Formulation Concentration (mg/L) Nominal Concentration15 (mg/L) Concentration Day 0 Day 3 Mean % Meanc Recovery Blank Control 0 0 NDd ND ND NAe Working Stock (100 mg/L) 100 100 85 NS1 NA NA 6.25 mg/L 6.25 6.25 5.0 2.5 3.75 60 12.5 mg/L 12.5 12.5 11 5.9 8.45 68 25 mg/L 25 25 21 12 16.5 66 50 mg/L 50 50 44 20 32 64 100 mg/L 100 100 85g 45 65 65 Abiotic Control (100 mg/L) 100 100 85g 44 64.5 65 a Defined in terms o f mg H-25435/liter nutrient medium, b H-25435 was assumed to be 100% for analytical purposes, c Mean o f day 0 and day 3 concentrations. d ND stands for not detected. The limit o f detection was calculated as 0.001 mg/L. e NA stands for not applicable, f NS stands for not submitted. g No separate analysis was conducted since the working stock, the day 0 100 mg/L test concentration, and the day 0 abiotic control were aliquots o f the same solution. Therefore, the measured concentration obtained for the working stock can be used for the 100 mg/L test concentration and the abiotic control. - 25Company Sanitized. Does not contain TSCA CBI H-25435: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum DuPont-11604 TABLE 3 (Continued) MEASURED CONCENTRATIONS OF H-25435 IN TEST SOLUTIONS Mean, Measured Sample Description3 Total, Nominal Formulation Concentration (mg/L) Nominal Concentrationb (mg/L) Blank Control 0 0 Concentration Day 0 Mean % Day 3 Mean0 Recovery NDd ND ND NAe Working Stock (100 mg/L) 6.25 mg/L 100 6.25 100 94 NSf NA NA 6.25 6.2 5.3 5.75 92 12.5 mg/L 25 mg/L 12.5 25 12.5 15 12 13.5 108 25 27 24 25.5 102 50 mg/L 50 50 50 54 52 104 100 mg/L 100 100 94g 100 97 97 Abiotic Control (100 mg/L) 100 100 94g 95 94.5 95 a Defined in terms o f mg H-25435/liter nutrient medium, b H-25435 was assumed to be 100% for analytical purposes. c Mean o f day 0 and day 3 concentrations. d ND stands for not detected. The limit o f detection was calculated as 0.001 mg/L. e NA stands for not applicable, f NS stands for not submitted. g N o separate analysis was conducted since the working stock, the day 0 100 mg/L test concentration, and the day 0 abiotic control were aliquots o f the same solution. Therefore, the measured concentration obtained for the working stock can be used for the 100 mg/L test concentration and the abiotic control. - 26Company Sanitized. Does not contain TSCA CBI H-25435: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum DuPont-11604 TABLE 3 (Continued) MEASURED CONCENTRATIONS OF H-25435 IN TEST SOLUTIONS Sample Description8 Total, Nominal Formulation Concentration (mg/L) Nominal Concentration (m g/L ) Mean, Measured Concentration ____(m g/L)____ Day 0 Day 3 Mean Mean % Recovery Blank Control 0 0 NDd ND ND NAe Working Stock (100 mg/L) 100 100 102 NSf NA NA 6.25 mg/L 6.25 6.25 6.9 1.6 4.25 68 12.5 mg/L 12.5 12.5 13 7.6 10.3 82 25 mg/L 25 25 26 19 22.5 90 50 mg/L 50 50 52 43 47.5 95 100 mg/L 100 100 102g 86 94 94 Abiotic Control (100 mg/L) 100 100 102g 110 106 106 a Defined in terms o f mg H-25435/liter nutrient medium, b H-25435 was assumed to be 100% for analytical purposes, c Mean o f day 0 and day 3 concentrations. d ND stands for not detected. The limit o f detection was calculated as 0.001 mg/L. e NA stands for not applicable, f NS stands for not submitted. g No separate analysis was conducted since the working stock, the day 0 100 mg/L test concentration, and the day 0 abiotic control were aliquots o f the same solution. Therefore, the measured concentration obtained for the working stock can be used for the 100 mg/L test concentration and the abiotic control. - 27Company Sanitized. Does not contain TSCA CBI H-25435: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum DuPont-11604 TABLE 4 HEALTHY CELL COUNT DATA SUMMARY - DEFINITIVE TEST Nominal Total Formulation Concentration Blank Control Mean Standard Error Std. Dev. Variance 6.25 mg/L Mean Standard Error Std. Dev. Variance % Inhibition 12.5 mg/L Mean Standard Error Std. Dev. Variance % Inhibition 25 mg^L Mean Standard Error Std. Dev. Variance % Inhibition 50 mg/L Mean Standard Error Std. Dev. Variance % Inhibition 100 mg/L Mean Standard Error Std. Dev. Variance % Inhibition Exposure Initiated: Exposure Ended: _____________ P ay 0: 7 January 2003 Day 3 :1 0 January 2003 _________________________ Healthy Ceil Count (cells/mL) by Exposure Period Rep. 0-Hour 24- H o u r 48-Hour 72-Hour i 10,000 80,000 470,000 3,940,000 2 10,000 100,000 640,000 2,740,000 3 10,000 60,000 720,000 3,640,000 10,000 80,000 610,000 3,440,000 0.00 11,547.01 73,711.15 360,555.13 0.00 20.000.00 127,671.45 624,499.80 0.00E+00 4.00E+08 1.63E+10 3.90E+11 1 10,000 70,000 700,000 3,990,000 2 10,000 70,000 960,000 3,710,000 3 10,000 80,000 340,000 1,760,000 10,000 73,333 666,667 3.153,333 0.00 3,333.33 179,752.92 701,339.99 0.00 5,773.50 311,341.18 1,214,756.49 0.00E+00 3.33E+07 9.69E+10 1.48E+12 0.00 8.33 -9.29 8.33 1 10,000 110,000 520,000 3,770,000 2 10,000 80,000 500,000 3,570,000 3 10,000 90,000 920,000 5,200,000 10,000 93,333 646,667 4,180,000 0.00 8,819.17 136,788.56 513,257.57 0.00 15,275.25 236,924.74 888,988.19 0.00E+00 2.33E+08 5.61E+10 7.90E+11 0.00 -16.67 -6.01 -21.51 1 10,000 60,000 530,000 3,710,000 2 10,000 70,000 910,000 4,050,000 3 10,000 100,000 540,000 3,450,000 10,000 76,667 660,000 3,736,667 0.00 12,018.50 125,033.33 173,717.52 0.00 20,816.66 216,564.08 300,887.58 0.00E+00 0.00 4.33E+08 4.17 4.69E+10 -8.20 9.05E+10 -8.62 1 10,000 100,000 770,000 3,900,000 2 10,000 170,000 920,000 2,040,000 3 10,000 90,000 670,000 4,010,000 10,000 0.00 120,000 25,166.11 786,667 72,648.32 3,316,667 639,122.66 0.00 O.OOE+OO 0.00 43,588.99 1.90E+09 -50.00 125,830.57 1.58E+10 -28.96 1,106,992.92 1.23E+12 3.59 1 10,000 90,000 760,000 4,360,000 2 10,000 80,000 400,000 3,600,000 3 10,000 120,000 580,000 3,600,000 10,000 96,667 580,000 3,853,333 0.00 12,018.50 103,923.05 253,333.33 0.00 20,816.66 180,000.00 438,786.20 O.OOE+OO 0.00 4.33E+08 -20.83 3.24E+10 4.92 1.93E+11 -12.02 - 28Company Sanitized. Does not contain TSCA CBI H-25435: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum DuPont-11604 TABLES AREA UNDER THE GROWTH CURVE DATA SUMMARY - DEFINITIVE TEST Nominal Total Formulation Concentration Blank Control Mean Standard Error Std. Dev. Variance 6.25 mg/L Mean Standard Error Std. Dev. Variance % Inhibition 12.5 mg/L Mean Standard Error Std. Dev. Variance % Inhibition 25 mg/L Mean Standard Error Std. Dev. Variance % Inhibition 50 mg/L Mean Standard Error Std. Dev. Variance % Inhibition 100 mg/L Mean Standard Error Std. Dev. Variance % Inhibition Exposure Initiated: Exposure Ended: Pay 0: 7 January 2083___________________________________ Day 3:10 January 2003 Area Under the Growth Curve Based on ____________________ Healthy Cell Count (celis/mL) by Exposure Period Rep. 0-24 Hour 0-48 Hour 0-72 Hour 1 840,000 7,200,000 59,880,000 2 1,080,000 9,720,000 50,040,000 3 600,000 9,720,000 61,800,000 840,000 8,880,000 57,240,000 138,564.06 840,000.00 3,642,416.78 240,000.00 1,454,922.68 6,308,850.93 5.76E+10 2.12E+12 3.98E+13 1 720,000 9,720,000 65,760,000 2 720,000 12,840,000 68,640,000 3 840,000 5,640,000 30,600,000 760,000 9,400,000 55,000,000 40,000.00 2,084,610.28 12,228,295.06 69,282.03 3,610,650.91 21,180,028.33 4.80E+09 1.30E+13 4.49E+14 9.52 -5.86 3.91 1 1,200,000 8,520,000 59,760,000 2 840,000 7,560,000 56,160,000 3 960,000 12,840,000 86,040,000 1,000,000 9,640,000 67,320.000 105,830.05 1,623,822.65 9,417,515.60 183,303.03 2,812,543.33 16,311,615.49 3.36E+10 7.91E+12 2.66E+14 -19.05 -8.56 -17.61 1 600,000 7,440,000 58,080,000 2 720,000 .12,240,000 71,520,000 3 1,080,000 8,520,000 56,160,000 800,000 9,400,000 61,920,000 144,222.05 1,453,822.55 4,831,894.04 249,799.92 2,518,094.52 8,369,085.97 6.24E+10 6.34E+12 7.00E+13 4.76 -5.86 -8.18 1 1,080,000 11,280,000 67,080,000 2 1,920,000 14,760,000 50,040,000 3 960,000 9,840,000 65,760,000 1,320,000 11,960,000 60,960,000 301,993.38 1,460,410.90 5,473.280.55 523,067.87 2,529.505.88 9,480.000.00 2.74E+11 6.40E+12 8.99E+13 -57.14 -34.68 -6.50 1 960,000 10,920,000 72,120,000 2 840,000 6,360,000 54,120,000 3 1,320,000 9,480,000 59,400,000 1,040,000 8,920,000 61,880,000 144,222.05 1,345,808.31 5,342,059.53 249,799.92 2,331,008.37 9,252,718.52 6.24E+10 5.43E+12 8.56E+13 -23.81 -0.45 -8.11 - 29Company Sanitized. Does not contain TSCA CBI H-25435: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum DuPont-11604 TABLE 6 GROWTH RATE DATA SUMMARY - DEFINITIVE TEST Nominal Total Formulation Concentration Blank Control Mean Standard Error Std. Dev. Variance 6.25 mg/L Mean Standard Error Std. Dev. Variance % Inhibition 12.5 mg/L Mean Standard Error Std. Dev. Variance % Inhibition 25 mg/L Mean Standard Error Std. Dev. Variance % Inhibition 50 mg/L Mean Standard Error Std. Dev. Variance % Inhibition 100 mg/L Mean Standard Error Std. Dev. Variance % Inhibition Exposure Initiated: Exposure Ended: Day 0: 7 January 2003 Day 3:10 January 2003 Growth Rate Based on Healthy Cell Count (cells/mL) by Exposure Period Rep. 0-24 Hour 0-48 Hour 0-72 Hour 1 0.0866 0.0802 0.0830 2 0.0959 0.0866 0.0780 3 0.0747 0.0891 0.0819 0.0857 0.0853 0.0810 0.0061 0.0027 0.0015 0.0106 0.0046 0.0026 1.13E-04 2.11 E-05 6.90E-06 1 0.0811 0.0885 0.0832 2 0.0811 0.0951 0.0822 3 0.0866 0.0735 0.0718 0.0829 0.0857 0.0791 0.0018 0.0064 0.0036 0.0032 0.0111 0.0063 1.01 E-05 1.23E-04 3.99E-05 3.27 -0.47 2.35 1 0.0999 0.0823 0.0824 2 0.0866 0.0815 0.0816 3 0.0916 0.0942 0.0869 0.0927 0.0860 0.0836 0.0039 0.0041 0.0016 0.0067 0.0071 0.0029 4.51E-05 5.06E-05 8.16E-06 -8.13 -0.82 -3.29 1 0.0747 0.0827 0.0822 2 0.0811 0.0940 0.0834 3 0.0959 0.0831 0.0812 0.0839 0.0866 0.0823 0.0063 0.0037 0.0006 0.0109 1.18E-04 2.14 0.0064 4.11 E-05 -1.52 0.0011 1.21E-06 -1.61 1 0.0959 0.0905 0.0829 2 0.1181 0.0942 0.0739 3 0.0916 0.0876 0.0832 0.1019 0.0908 0.0800 0.0082 0.0142 2.02E-04 -18.82 0.0019 0.0033 1.09E-05 -6.41 0.0031 0.0053 2.79E-05 1.19 1 0.0916 0.0902 0.0844 2 0.0866 0.0769 0.0818 3 0.1035 0.0846 0.0818 0.0939 0.0839 0.0827 0.0050 0.0039 0.0009 0.0087 7.54E-05 -9.53 0.0067 4.46E-05 1.64 0.0015 2.25E-06 -2.10 - 30Company Sanitized. Does not contain TSCA CBI H-25435: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum DuPont-11604 TABLE 7 72-HOUR (DAY 3) STATISTICAL ANALYSES SUMMARY - DEFINITIVE TEST Nominal H-25435 Concentration Mean Standard Response Error .N Test Concentration Response (mg/L) Level Value Healthy Cell Count Blank Control 6.25 mg/L 12.5 mg/L 25 mg/L 50 mg/L 100 mg/L 3,440,000 360,555 3 3,153,333 701,340 3 EC50b 4,180,000 513,258 3 3,736,667 173,718 3 NOEC 3,316,667 639,123 3 3,853,333 253,333 3 >100 >100 Area Under the Growth Curve Blank Control 6.25 mg/L 12.5 mg/L 25 mg/L 50 mg/L 100 mg/L 57,240,000 3,642,417 3 55,000,000 12,228,295 3 e c 50 67,320,000 9,417,516 3 61,920,000 4,831,894 3 NOEC 60,960,000 5,473,281 3 61,880,000 5,342,060 3 >100 >100 Growth Rate Blank Control 6.25 mg/L 12.5 mg/L 25 mg/L 50 mg/L 100 mg/L 0.0810 0.0015 3 0.0791 0.0036 3 e c 50 0.0836 0.0016 3 0.0823 0.0006 3 NOEC 0.0800 0.0031 3 0.0827 0.0009 3 >100 >100 a Confidence Interval b EC50 was calculated using Probit analysis. -31 Company Sanitized. Does not contain TSCA CBI H-25435: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum DuPont-11604 FIGURES -32Company Sanitized. Does not contain TSCA CBI H-25435: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricomutum DuPont-11604 FIGURE 1 REPRESENTATIVE ANALYTICAL CALIBRATION STANDARD CURVE FOR -33Company Sanitized. Does not contain TSCA CBI H-25435: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum DuPont-11604 -34Company Sanitized. Does not contain TSCA CBI H-25435: influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum DuPont-11604 FIGURE 2 REPRESENTATIVE CHROMATOGRAM OF A CALIBRATION STANDARD SOLUTION 010803b12 Smooth(Mn,2x3) 100-1 m m6.76 I 16867.14 J 176386 F1 :SIR of 1 channel,ESTIC 1.766e+005 %- Tr 1 r 1 mjn 4.00 6.00 8.00 10.00 elutes at a retention time o f approximately 6.8 minutes. Calibration standard solution contains H-25435 at a concentration o f 0.56 mg/L. felutes at a retention time o f approximately 5.9 minutes. Calibration standard solution contains H-25435 at a concentration o f 0.56 mg/L. - 35Company Sanitized. Does not contain TSCA CBI H-25435: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum DuPont-11604 Ielutes at a retention time o f approximately 5.3 minutes. Calibration standard solution contains H-25435 at a concentration o f 0.56 mg/L. 36- - Company Sanitized. Does not contain TSCA C8I H-25435: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricomutum DuPont-11604 FIGURE 3 REPRESENTATIVE CHROMATOGRAM OF THE BLANK CONTROL SOLUTION would elute at a retention time o f approximately 6.8 minutes. ould elute at a retention time o f approximately 5.9 minutes. - 37Company Sanitized. Does not contain TSCA CBI H-25435: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum DuPont-11604 ould elute at a retention time o f approximately 5.3 minutes. 38- - Company Sanitized. Does not contain TSCA CBI H-25435: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricomutum DuPont-11604 FIGURE 4 REPRESENTATIVE CHROMATOGRAM OF A TEST CONCENTRATION SOLUTION 010803b24 Smooth(Mn,2x3) 100-j 6.76 49868.09 489066 F1 :SIR of 1 channel,ESTIC 4.896e+005 %- 7.51 n_ Aa 2.00 4.00 6.00 8.00 10.00 felutes at a retention time o f approximately 6.8 minutes. Test solution sample contains Hi25435 at a nominal concentration o f 6.25 mg/L. The sample was diluted 1:2 before analysis. 1elutes at a retention time o f approximately 5.9 minutes. Test solution sample contains H25435 at a nominal concentration o f 6.25 mg/L. The sample was diluted 1:2 before analysis. - 39Company Sanitized. Does not contain TSCA C8I H-25435: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum DuPont-11604 felutes at a retention time o f approximately 5.3 minutes, Test solution sample contains H-25435 at a nominal concentration o f 6.25 mg/L. The sample was diluted 1:2 before analysis. 3 4 0- - Company Sanitized. Does not contain TSCA CBI H-25435: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricomutum DuPont-11604 APPENDICES -41 Company Sanitized. Does not contain TSCA CBI H-25435: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum DuPont-11604 APPENDIX A AAP Nutrient Medium Constituents -42Company Sanitized. Does not contain TSCA CBI H-25435: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum DuPont-11604 AAP NUTRIENT MEDIUM CONSTITUENTS Stock Solutions3 Component Concentration (g/L) Final Prepared Medium 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 K 2H PO 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 CaCl22H20 4.410 Stock Solutions3 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 o04*2 H20 FeCl3*6 H20 Na2EDTA2 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. -43Company Sanitized. Does not contain TSCA CBI H-25435: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum DuPont-11604 APPENDIX B 72-Hour Healthy Cell Count Statistics for the Definitive Test -44Company Sanitized. Does not contain TSCA CBI H-25435: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum DuPont-11604 72-HOUR HEALTHY CELL COUNT STATISTICS FOR THE DEFINITIVE TEST OBSERVED PERCENT CHANGE FROM CONTROL MEAN RESPONSE : COUNT from DATASET: m p A W R 1 1 6 0 4 Obs Rate MEAN PCTCHNG N (mg/L) 1 0 3440000 03 2 6.25 3153333.33 -8 3 3 12.5 4180000 22 3 4 25 3736666.67 93 5 50 3316666.67 -4 3 6 100 3853333.33 12 3 -45Company Sanitized. Does not contain TSCA CBI H-25435: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum DuPont-11604 STATISTICAL ANALYSIS LIST FILE REPORT CREATED ON 19:00 WEDNESDAY 22JAN03 ECOTOX MEASUREMENTS FROM DATASET AWR11604 GROUP STATISTICS FOR Count BY DOSE dose dosevai COUNT (mg/L) MEAN MEDIAN STD_DEV STD_ERR 1 0 3 3440000.00 3640000 624499.80 360555.13 2 6.25 3 3153333.33 3710000 1214756.49 701339.99 3 12.5 3 4180000.00 3770000 888988.19 513257.57 4 25 3 3736666.67 3710000 300887.58 173717.52 5 50 3 3316666.67 3900000 1106992.92 639122.66 6 100 3 3853333.33 3600000 438786.20 253333.33 STATISTICAL ANALYSIS LIST FILE REPORT CREATED ON 19:00 WEDNESDAY 22JAN03 SHAPIRO-WILK TEST OF NORMALITY OF Count AQUATIC SELENASTRUM: FULL DATA STD SKEW KURT SW_STAT P_VALUE SIGNIF 699952.38 -0.54723 -0.46879 0.94643 0.37177 STATISTICAL ANALYSIS LIST FILE REPORT CREATED ON 19:00 WEDNESDAY 22JAN03 LEVENE TEST FOR Count - FULL Model ANALYSIS OF VARIANCE ON FULL DATA SET Effect DF LEVENE P_VALUE SIGNIF DOSE 5 0.26667 0.92276 The data was found to be normally distributed with equal variances. An analysis of variance will be performed. STATISTICAL ANALYSIS LIST FILE REPORT CREATED ON 19:00 WEDNESDAY 22JAN03 ANALYSIS OF SELENASTRUM - FULL DATA DOSES 0, 6.25, 12.5, 25, 50, 100 mg/L ECOTOX OF AQUATIC SELENASTRUM Obs Class Levels Values 1 dose 6 123456 OVERALL F-TESTS FOR ANOVA Count FROM FULL DATA, Weight=NO WEIGHT,Variance Ratio=0.33 Obs Effect Num . Den DF DF FValue ProbF 1 dose 5 12 0.63 0.6838 -46Company Sanitized. Does not contain TSCA CBI H-25435: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum DuPont-11604 LSMEANS - DOSE MEANS ADJUSTED FOR THE EFFECTS OF SELENASTRUM Count FROM FULL DATA, Weight=NO WEIGHT, Variance Ratio =0 .33 Obs LEVEL LSMEAN SE DDF 10 2 6.25 3 12.5 4 25 5 50 6 100 3440000.00 3153333.33 4180000.00 3736666.67 3316666.67 3853333.33 480996.27 480996.27 480996.27 480996.27 480996.27 480996.27 12 12 12 12 12 12 ESTIMATED DOSE EFFECTS & DUNNETT FOR Two-sided ALTERNATIVE USING ALPHA=.05 FOR COMPARISONS TO CONTROL Count FROM FULL DATA, Weight=NO WEIGHT,.Variance Ratio =0 .33 Estimate SIGNIF Dunnett 2-sided p-value Test Group Mean N DOSE TREND DOSE QUAD DOSE 2-1 DOSE 3-1 DOSE 4-1 DOSE 5-1 DOSE 6-1 0.60905 0.71144 0.99052 0.71946 0.98899 0.99982 0.95690 . 3153333.33 4180000.00 3736666.67 3316666.67 3853333.33 3 3 3 3 3 , STATISTICAL ANALYSIS LIST FILE REPORT CREATED ON 19:00 WEDNESDAY 22JAN03 ANALYSIS OF ECOTOX USING ALPHA==.05 FULL DATA TEST Jonckheere Trend Test on Dose 0 and Lowest 5 Doses through Dose 100 mg/L Analysis of Count KEY ZC IS JONCKHEERE STATISTIC COMPUTED WITH TIE CORRECTION ZCCF IS ZC WITH CONTINUITY CORRECTION FACTOR P1UPCF IS P-VALUE FOR UPWARD TREND P1DNCF IS P-VALUE FOR DOWNWARD TREND P-VALUES ARE FOR TIE-CORRECTED TEST WITH CONTINUITY CORRECTION FACTOR SIGNIF RESULTS ARE FOR A TWO-SIDED ALTERNATIVE HYPOTHESIS JONC ZC . ZCCF P1UPCF P1DNCF SIGNIF 74.5 0.539566 0.5781065 0.2815961 0.6918234 NOTE Jonckheere test results are included in the summary table. Group means should be, examined to check for lack-of-fit to a linear trend before trend test results are accepted. -47Company Sanitized. Does not contain TSCA CBI H-25435: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum DuPont-11604 APPENDIX C 72-Hour Area Under the Growth Curve Statistics for the Definitive Test - 48Company Sanitized. Does not contain TSCA CBI H-25435: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum DuPont-11604 72-HOUR AREA UNDER THE GROWTH CURVE STATISTICS FOR THE DEFINITIVE TEST Obs 1 2 3 4 5 6 Rate (mg/L) 0 6.25 12.5 25 50 100 MEAN PCTCHNG N 57240000 55000000 67320000 61920000 60960000 61880000 03 -4 3 18 3 83 63 83 -49Company Sanitized. Does not contain TSCA CBI H-25435: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum DuPont-11604 STATISTICAL ANALYSIS LIST FILE REPORT CREATED ON 19:00 WEDNESDAY 22JAN03 ECOTOX MEASUREMENTS FROM DATASET AWR11604 GROUP STATISTICS FOR Area BY DOSE dose dosevai COUNT (mg/L) MEAN MEDIAN STD_DEV STD_ERR 1 0 3 57240000 59880000 6308850.93 3642416.78 2 6.25 3 55000000 65760000 21180028.33 12228295.06 3 12.5 3 67320000 59760000 16311615.49 9417515.60 4 25 3 61920000 58080000 8369085.97 4831894.04 5 50 3 60960000 65760000 9480000.00 5473280.55 6 100 3 61880000 59400000 9252718.52 5342059.53 STATISTICAL ANALYSIS LIST FILE REPORT CREATED ON 19:00 WEDNESDAY 22JAN03 SHAPIRO-WILK TEST OF NORMALITY OF Area AQUATIC SELENASTRUM: FULL DATA OBS STD .SKEW KURT SW_STAT P_VALUE SIGNIF 18 10846462.15 -0.31113 -0.052253 0.96885 0.77585 STATISTICAL ANALYSIS LIST FILE REPORT CREATED ON 19:00 WEDNESDAY 22JAN03 LEVENE TEST FOR Area - FULL Model ANALYSIS OF VARIANCE ON FULL DATA SET Effect DF LEVENE P_VALUE SIGNIF DOSE 5 0.26034 0.92624 NOTE 'The data was found to be normally distributed with equal variances. An analysis of variance will be performed -50Company Sanitized. Does not contain TSCA CBI H-25435: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum DuPont-11604 STATISTICAL ANALYSIS LIST FILE REPORT CREATED ON 19:00 WEDNESDAY 22JAN03 ANALYSIS OF SELENASTRUM - FULL DATA DOSES 0, 6.25, 12.5, 25, 50, 100 mg/L ECOTOX OF AQUATIC SELENASTRUM Obs Class Levels Values 1 dose 6 123456 OVERALL F-TESTS FOR ANOVA Area FROM FULL DATA, Weight=NO WEIGHT,Variance Ratio=0.33 Obs Effect 1 dose Num Den DF " DF 5 12 FValue 0.33 Prob F 0.8862 LSMEANS - DOSE MEANS ADJUSTED FOR THE EFFECTS OF SELENASTRUM Area FROM FULL DATA, Weight=NO WEIGHT Variance Ratio==0.33 Obs LEVEL LSMEAN SE DDF 10 2 6.25 3 12.5 4 25 5 50 6 100 57240000 55000000 67320000 61920000 60960000 61880000 7453518.18 7453518.18 7453518.18 7453518.18 7453518.18 7453518.18 12 12 12 12 12 12 ESTIMATED DOSE EFFECTS & DUNNETT FOR Two-sided ALTERNATIVE USING ALPHA=.05 FOR COMPARISONS TO CONTROL Area FROM FULL DATA, Weight=NO WEIGHT,Variance Ratio=0.33 Estimate DOSE TREND DOSE QUAD DOSE 2-1 DOSE 3-1 DOSE 4-1 DOSE 5-1 DOSE 6-1 SIGNIF Dunnett 2-sided p-value 0.57778 0.59486 0.99961 0.80095 0.98811 0.99571 0.98854 Test Group Mean 55000000 67320000 61920000 60960000 61880000 N .. 3 3 3 3 3 -51 Company Sanitized. Does not contain TSCA CBI H-25435: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum DuPont-11604 STATISTICAL ANALYSIS LIST PILE REPORT CREATED ON 19:00 WEDNESDAY 22JAN03 ANALYSIS OF ECOTOX USING ALPHA=.05 FULL DATA TEST Jonckheere Trend Test on Dose 0 and Lowest 5 Doses through Dose 100 mg/L Analysis of Area KEY ZC IS JONCKHEERE STATISTIC COMPUTED WITH TIE CORRECTION ZCCF IS ZC WITH CONTINUITY CORRECTION FACTOR P1UPCF IS P-VALUE FOR UPWARD TREND P1DNCF IS P-VALUE FOR DOWNWARD TREND P-VALUES ARE FOR TIE-CORRECTED TEST WITH CONTINUITY CORRECTION FACTOR SIGNIF RESULTS ARE FOR A TWO-SIDED ALTERNATIVE HYPOTHESIS JONC ZC ZCCF P1UPCF P1DNCF SIGNIF 71.5 0.3085257 0.3470914 0.3642S13 0.6064045 NOTE Jonckheere test results are included in the summary table. Group means should be examined to check for lack-of-fit to a linear trend before trend test results are accepted. - 52Company Sanitized. Does not contain TSCA CBI H-25435: Influence on Growth and Growth Rate o f the Green Alga Selenastmm capricornutum DuPont-11604 APPENDIX D 72-Hour Growth Rate Statistics for the Definitive Test - 53Company Sanitized. Does not contain TSCA CBI H-25435: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum DuPont-11604 72-HOUR GROWTH RATE STATISTICS FOR THE DEFINITIVE TEST Obs Rate MEAN PCTCHNG N (mg/L) 1 0 0.08096667 03 2 6.25 0.07906667 -2 3 3 12.5 0.08363333 33 4 25 0.08226667 23 5 50 0.08 -1 3 6 100 0.08266667 23 -54Company Sanitized. Does not contain TSCA CBI H-25435: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum DuPont-11604 STATISTICAL ANALYSIS LIST FILE REPORT CREATED ON 19:00 WEDNESDAY 22JAN03 ECOTOX MEASUREMENTS FROM DATASET AWR11604 GROUP STATISTICS FOR G Rate BY DOSE dose 1 2 3 4 5 6 dosevai (mg/L) 0 6.25 12.5 25 50 100 COUNT 3 3 3 3 3 3 MEAN MEDIAN 0.080967 0.079067 0.083633 0.082267 0.080000 0.082667 0.0819 0.0822 0.0824 0.0822 0.0829 0.0818 STD_DEV .002627420 .006312950 .002857155 .001101514 .005284884 .001501111 STD_ERR .001516941 .003644783 .001649579 .000635959 .003051229 .000866667 STATISTICAL ANALYSIS LIST FILE REPORT CREATED ON 19:00 WEDNESDAY 22JAN03 SHAPIRO-WILK TEST OF NORMALITY OF G Rate AQUATIC SELENASTRUM: FULL DATA OBS STD SKEW KURT SW_STAT P_VALUE SIGNIF 18 .003186676 -0.90046 0.43426 0.91952 0.12677 STATISTICAL ANALYSIS LIST FILE REPORT CREATED ON 19:00 WEDNESDAY 22JAN03 LEVENE TEST FOR G_Rate - FULL Model ANALYSIS OF VARIANCE ON FULL DATA SET Effect DF LEVENE P_VALUE SIGNIF DOSE 5 0.37769 0.85454 NOTE The data was found to be normally distributed with equal variances. An analysis of variance will be performed. -55Company Sanitized. Does not contain TSCA CBI H-25435: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum DuPont-11604 STATISTICAL ANALYSIS LIST FILE REPORT CREATED ON 19:00 WEDNESDAY 22JAN03 ANALYSIS OF SELENASTRUM - FULL DATA DOSES 0, 6.25, 12.5, 25, 50, 100 mg/L ECOTOX OF AQUATIC SELENASTRUM Obs Class Levels Values 1 dose 6 123456 OVERALL F-TESTS FOR ANOVA GJRate FROM FULL DATA, Weight;=NO WEIGHT ,Variance Ratio= Num Den Obs Effect DF DF FValue ProbF 1 dose 5 12 0.62 0.6858 LSMEANS - DOSE MEANS ADJUSTED FOR THE EFFECTS OF SELENASTRUM G_Rate FROM FULL DATA, Weight=NO WEIGHT,Variance Ratio=0.33 Obs LEVEL LSMEAN SE DDF 10 2 6.25 3 12.5 4 25 5 50 6 100 0.080967 0.079067 0.083633 0.082267 0.080000 0.082667 .002189833 .002189833 .002189833 .002189833 .002189833 .002189833 12 12 12 12 12 12 ESTIMATED DOSE EFFECTS & DUNNETT FOR Two-sided ALTERNATIVE USING ALPHA=.05 FOR COMPARISONS TO CONTROL : FROM FULL DATA, Weight:=NO WEIGHT, Variance :Ratio: Estimate DOSE TREND DOSE QUAD DOSE 2-1 DOSE 3-1 DOSE 4-1 DOSE 5-1 DOSE 6-1 SIGNIF Dunnett 2-sided p-value 0.59763 0.82636 0.95528 0.85419 0.99068 0.99756 0.97113 Test Group Mean . 0.079067 0.083633 0.082267 0.080000 0.082667 N . 3 3 3 3 3 - 56Company Sanitized. Does not contain TSCA CBI H-25435: Influence on Growth and Growth Rate o f the Green Alga Selenastrum capricornutum DuPont-11604 STATISTICAL ANALYSIS LIST FILE REPORT CREATED ON 19:00 WEDNESDAY 22JAN03 ANALYSIS OF ECOTOX USING ALPHA=.05 FULL DATA TEST Jonckheere Trend Test on Dose 0 and Lowest 5 Doses through Dose 100 mg/L Analysis of G_Rate KEY ZC IS JONCKHEERE STATISTIC COMPUTED WITH TIE CORRECTION ZCCF IS ZC WITH CONTINUITY CORRECTION FACTOR P1UPCF IS P-VALUE FOR UPWARD TREND P1DNCF IS P-VALUE FOR DOWNWARD TREND P-VALUES ARE FOR TIE-CORRECTED TEST WITH CONTINUITY CORRECTION FACTOR SIGNIF RESULTS ARE FOR A TWO-SIDED ALTERNATIVE HYPOTHESIS JONC ZC ZCCF P1UPCF P1DNCF SIGNIF 74 0.5013542 0.53992 0.2946261 0.678242 NOTE Jonckheere test results are included in the summary table. Group means should be examined to check for lack-of-fit to a linear trend before trend test results are accepted. - 57Company Sanitized. Does not contain TSCA CBI