Document pmqd0NmNGyDYbKjaJB6k3aKn6

AR226-2801 DuPont-3381 TRADE SECRET Study Title H-24215: Static, Acute, 96-Hour LC50 to Rainbow Trout, Oncorhynchus mykiss Laboratory Project ID: DuPont-3381 Test Guidelines: U.S. EPA Pesticide Assessment Guidelines Subdivision E, Section 72-1 (1982) OECD Guideline for Testing Chemicals Section 2: Fish, Acute Toxicity Test, No. 203 (1992) Commission Directive 92/69/EEC EEC Method C.l (1992) Author: Carl F. Muska, Ph.D. Study Completed on: December 20,1999 Performing Laboratory: E.I. du Pont de Nemours and Company Haskell Laboratory for Toxicology and Industrial Medicine Elkton Road, P.O. Box 50 Newark, Delaware 19714-0050 Work Request Number: Study Code Number: Page 1 o f31 Company Sanitized. Does not contain T3CA CBi H-24215: Static, Acute, 96-Hour LC50 to Rainbow Trout, Oncorhynchus mykiss DuPont-3381 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT This study was conducted in compliance with U.S. EPA FIFRA (40 CFR part 160) and/or 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 No. 3850). Submitter / Sponsor: E. I. Du Pont de Nemours and Company Wilmington, Delaware 19898 U.S.A. Study Director: Carl F. Muska, Ph.D. 30 Date f, . -2Company Sanitized. Does not contain TSCA CBl H-24215: Static, Acute, 96-Hour LC50 to Rainbow Trout, Oncorhynchus mykiss QUALITY ASSURANCE STATEMENT Haskell Sample Number(s): 24215 Dates of Inspections: Conduct: N ovem ber8,1999 Records, Reports: December 14, 1999 Dates Findings Reported to: Study Director: December 14, 1999 Management: December 17, 1999 DuPont-3381 Reported by: VaXC (J I Joseph C. HaHnamill Sr. Ql a iut nalliiHty# AA ssurance Auditor 17-t a c - it i f Date -3 - Company Sanitised. Doas net contain t sc a c b i j^242^5^Static^AUte^_96-jic)ur LC50 to Rainbow Trout, Oncorhynchus mykiss DuPont-3381 CERTIFICATION We, the undersigned, declare that this report provides an accurate evaluation o f data obtained from this study. Analytical Report by: y - - BogdanSzostek, Ph. D. ResearchChemist o~ p e c -- Date Issued by Study Director: Carl F. Muska, Ph.D. Director, Ecotoxicology >0 -fcec-- MT? Date -4Company Sanitized. Doss not contain TSCA COl H-24215: Static, Acute, 96-Hour LCS0 to Rainbow Trout, Oncorhynchus mykiss TABLE OF CONTENTS GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT QUALITY ASSURANCE STATEMENT.................................................. C ERTIFICA TIO N ........................................................................................ LIST OF TABLES........................................................................................ LIST OF FIG U R ES...................................................................................... STUDY INFORMATION............................................................................. STUDY PERSONNEL................................................................................... SUMMARY....................... .............................................................................. IN T R O D U C T IO N .......................................................................................... MATERIALS AND M ETH O D S................................................................. A. Test Substance...................................................................................... B. Test Solution Preparation.................................................................... C. Dilution Water............................................................. ........................ D. Test Organism Culture........................................................................ E. Test Methods......................................................................................... F. Sample Preparation and Chemical Analysis.................................... . 1. Sample Collection and Treatment.................................................................... 2. Instrument and Conditions................................................................................. 3. Quantitation........................................................................................................ G. Statistical Analysis................................................................................ RESULTS AND DISCUSSION.......................................... ......................... A. Analytical R eport.................................................................................. 1. Chromatographic Results.................................................................................. 2. Test Solution Results......................................................................................... B. In-Life Report......................................................................................... 1. Rangefinding Study............................................................ ................................ 2. Definitive Study....................................' ............................................................. C O N C L U S IO N .................................................................................................. RECORDS AND SAMPLE ST O R A G E............ .......................................... R E F E R E N C E S .................................................................................................. TA BLES............................................................................................................. FIG U R ES.... ................................................. DuPont-3381 Page .....4 ....6 .... 6 .... 7 ... 8 ....9 ..10 ..10 ..10 ..10 ..10 ..10 ,,11 .11 ...11 ...12 ...12 .13 .14 .14 ..14 ..14 .14 ..14 ..14 ' 15 15 16 17 27 -5- Company San.t cd. Doos ob tain TSCACBI 96-Hour LC50 to Rainbow Trout, Oncorhynchus mykiss DuPont-3381 LIST OF TABLES Page 1. CHEMICAL CHARACTERISTICS OF HASKELL LABORATORY WELL WATER....................................18 2. MEASURED CONCENTRATIONS OF H-24215 IN TEST SOLUTION SAMPLES...................................... 19 3. WATER CHEMISTRY OF THE DILUTION WATER CONTROL AT TEST START................................... 20 4. DISSOLVED OXYGEN CONCENTRATION (mg/L) OF H-24215 TEST SOLUTIONS............................... 21 5. pH OF H-24215 TEST SOLUTIONS.................................................................................................... 22 6. TEMPERATURE (C) OF H-24215 TEST SOLUTIONS........................................................................... 23 7. MORTALITY OF RAINBOW TROUT, ONCORHYNCHUS MYKISS, IN AN UNAERATED STATIC ACUTE, 96-HOUR TEST WITH H-24215................................................................................'............... '........24 8. SUBLETHAL EFFECTS IN RAINBOW TROUT, ONCORHYNCHUS MYKISS, IN AN UNAERATED STATIC, ACUTE, 96-HOUR TEST WITH H-24215................................................................................ .........25 9. MEAN, MEASURED CONCENTRATIONS OF H-24215 CAUSING MORTALITY TO 50% (LCS0) OF RAINBOW TROUT, ONCORHYNCHUS MYKISS, AT SPECIFIC TIME INTERVALS................................. 26 LIST OF FIGURES Page 1. REPRESENTATIVE ANALYTICAL CALIBRATION STANDARD CURVE FOR H-24215....... 28 2. REPRESENTATIVE HPLC CHROMATOGRAM OF A CALIBRATION STANDARD SOLUTION............29 3. REPRESENTATIVE HPLC CHROMATOGRAM OF A H-24215 TEST SOLUTION SAMPLE....................30 4. REPRESENTATIVE HPLC CHROMATOGRAM OF A WATER CONTROL SOLUTION SAMPLE...........31 -6-t contain TSCACBI Company Sanitised. Does Hjj42jj^_Statu^Acute, 96-Hour LC50 to Rainbow Trout, Oncorhynchus mykiss DuPont-3381 STUDY INFORMATION 9th Collective Nomenclature: Octanoic acid, pentadecafluoro-, ammonium salt Haskell Number: 24215 CAS Registry Number: 3825-26-1 Composition: Purity: Known Impurities: Physical Characteristics: Light colored liquid Stability: The test substance appeared to be stable under the conditions of the study; no evidence of instability was observed. Solubility: Highly soluble in water. Sponsor: E. I. du Pont de Nemours and Company Wilmington, Delaware 19898 U.S.A. Study Initiated/Completed: November 5, 1999"/ (see report cover page) In-LifeInitiated/Completed: November8, 1999/November 12, 1999 -7 Company Sanitized. Does not contain TSCACBt^ Acute, 96-Hour LC50 to Rainbow Trout, Oncorhynchus mykiss STUDY PERSONNEL Study Director: Carl F. Muska, Ph.D. Management: Robert W. Rickard, Ph.D. Primary Technician: Laurie D. Bouchelle Animal Culturist: Erica Flanders Analytical Chemist: Bogdan Szostek, Ph.D. Management: S. Mark Kennedy, Ph.D. Toxicology Report Preparation: Bonnie J. Bieber, B.S. DuPont-3381 -9 -8 - tscacbi SaniViio'1- Docs not con'a'n Company H-24215: Static, Acute, 96-Hour LC50 to Rainbow Trout, Oncorhynchus mykiss DuPont-3381 SUMMARY The acute toxicity o f H-24215 to unfed fingerling rainbow trout, Oncorhynchus mykiss, was determined in an unaerated, 96-hour, static test in accordance with the Organisation for Economic Co-Operation and Development (OECD) Guideline for Testing Chemicals: 203; the European Economic Community 92/69 Annex V - Method C .l; and the United States Environmental Protection Agency, Pesticide Assessment Guidelines Subdivision E, 72-1. The study was conducted with 5 concentrations of H-24215 and a dilution water control at a mean temperature o f 11.8C (range o f 11.6-12.1C). Test solutions were not renewed during the 96-hour exposure. Two replicate test chambers were used per test concentration with 10 fish in each chamber (20 fish per concentration). Fish in the water control averaged 2.8 cm in standard length and 0.21 g in wet weight, blotted dry, at test end. Exposure o f rainbow trout to mean, measured H-24215 concentrations o f 554,1090,2280,4560, and 9360 mg/L resulted in 0 ,0 ,0 ,7 5 , and 100% mortality, respectively, at the end of 96 hours. Mean, measured concentrations ranged from 87 to 94% of the nominal concentrations. No mortality or sublethal effects were seen in the water control fish. The highest mean, measured concentration causing no mortality at test end was 2280 mg/L. The lowest mean, measured concentration causing 100% mortality at test end was 9360 mg/L. Surviving fish exposed to 4560 mg/L exhibited effects such as swimming at the surface, labored respiration, dark coloration, lethargy, and erratic swimming. There were no mortality or sublethal effects below 4560 mg/L. Mean, measured H-24215 concentrations were used for calculation of LC 50 values. Since H-24215 is a 20% solution o ffp flH l^ n water, results are also expressed in terms o f mean m easured|j|pM |fioncentrations. The results are summarized as follows: Nominal concentrations of H-24215, mg/La Mean, measured concentrations of H-24215, mg/L 96-hour LC50 (95% confidence interval) for H-24215, based on mean, measured concentrations, mg/L Mean, measured concentrations o n H ta N m g /L 96-hour LC50 (95% confidence interval) foiW jBBM based on mean, measured concentrations, mg/L dilution water control, 625, 1250, 2500, 5000, and 10,000 554, 1090, 2280, 4560, and 9360 4001 (3327 to 4932) 111, 218,456,912, and 1872 800 (665 to 986) a Not adjusted for purity by analysis during preparation. -9 s not contain TSCA CB/ Company Sanitized. Do; H-242151_Static, Acute, 96-Hour LC50 toRainbow Trout, Oncorhynchus mykiss DuPont-3381 INTRODUCTION The objective of this study was to assess the acute toxicity of H-24215 to unfed fingerling rainbow trout, Oncorhynchus mykiss, during an unaerated, static, 96-hour test. MATERIALS AND METHODS A. Test Substance pernuoroocianoate; in water. The test substance contains B. Test Solution Preparation Test substance solutions were prepared by direct addition o f H-24215 to dilution water. All test solutions, including the water control, were stirred for 30 minutes prior to use. Test substance solutions were clear and colorless and there was no insoluble material present. C. Dilution Water Dilution \^$er'originated from the Haskell Laboratory well which is 378-feet deep and is cased and sealed into bedrock to prevent contamination. The hardness o f the well water is adjusted to approximately 110-140 mg/L as CaCC>3 by the flow-proportioned addition o f CaCL. The well water is then aerated, passed through a green sand filter to remove iron, and filtered through 50-, 10-, and 5-fim filters to remove particulates. The water is heated or chilled as appropriate and distributed through aged polyvinyl chloride piping. The dilution water is analyzed twice yearly for major anions and cations, metals, total organochlorine and organophosphate pesticides, and polychlorinated biphenyls (Table 1). The dilution water meets OECD(1) and ASTM(2) criteria and specifications. **&**,, D. Test Organism Culture Rainbow trout, Oncorhynchus mykiss, were purchased from Thomas Fish Company, Anderson, California. The fish were held in a 272-L, circular, fiberglass holding tank (84-cm diameter, 49 cm deep) for 25 days in continuously-flowing well water at Haskell Laboratory. During the' 7-day period preceding the test, water temperatures ranged from 10.3 to 13,2C (mean 12.1 C). The fish were fed freshly-hatched, live brine shrimp (Prime Artemia Inc., Midvale, Utah) and AquaMax Starter Fingerling 300 5D03 (PMI Nutrition International, Inc.) twice daily on weekdays and once daily on weekends and holidays. Sickness, injury, and abnormalities were -10;Snet contain TSCACBI Company Sanitized. Dees H-24215: Static, Acute, 96-Hour LC50 to Rainbow Trout, Oncorhynchus mykiss DuPont-3381 not present in fish in the holding tank nor in the fish used for testing. No mortality was seen during the 48 hours prior to use for testing. Fish were identified by labels on the holding tanks and test chambers. E. Test Methods^2,4'5^ Five nominal concentrations, and a dilution water control were used in this study. Nominal concentrations of H-24215 of 625, 1250,2500, 5000, and 10,000 were chosen for the definitive test based on the results of a preliminary rangefinding study (see Results and Discussion). The dilution water control contained no solvent. Test chambers were stainless steel aquaria [30 (length) x 14.5 (width) x 30 (height) cm] which held approximately 9 L of test solution (13-L maximum volume; 21.5-cm test solution depth). Two replicate test chambers were used per test concentration with 10 fish in each chamber (20 fish per concentration). Each chamber was covered with a glass plate to prevent fish from escaping. Random numbers were used to assign test concentrations to the test chambers and position o f test concentrations in the water bath. Rainbow trout used in this study were not fed approximately 29 hours prior to nor during the test, and were assigned to the test chambers using random numbers. Addition of fish to the test solutions was initiated approximately 41 minutes after mixing o f the test solutions was completed. Mortality and behavioral observations were made at test start, every 24 hours thereafter, and at test end. Dead fish were removed from the test chambers when observed. Criteria for death were the absence o f opercular movement and lack o f reaction to gentle prodding. At test conclusion, all surviving fish were humanely sacrificed using MS222. A recirculating water bath was used to maintain mean temperature in the test chambers during the 96-hour test at approximately 11.8C with a range of 11.6 to 12.1 C (Table 6). In addition, a continuously-recording thermometer was used to check for temperature variation in 1 replicate of the dilution water control. A photoperiod of 16 hours light (approximately 199-450 Lux) and 8 hours darkness was employed which included 30 minutes o f transitional light (11-157 Lux) preceding and following the 16-hour light interval. Dissolved oxygen, pH, and temperature were measured in all replicate chambers o f the control and test substance concentrations. These measurements were taken before fish were added at test start, every 24 hours'thereafter, and at test end or at total mortality in a concentration. Total alkalinity, EDTA hardness, and conductivity o f the water were measured before fish were added at the beginning of the test. Test solutions were not aerated during the test. F, Sample Preparation and Chemical Analysis 1. Sample Collection and Treatment One sample plus a back-up sample of each test solution were received from each replicate test chamber including the controls on Day 0 of the definitive study. One unscheduled sample plus a -11 - Com oanv Sanitizer*. Ccec- contain 1SCA CSi H-24215: Static, Acute, 96-Hour LC50 to Rainbow Trout, Oncorhynchus mykiss DuPont-3381 back-up sample for A and B replicates was also received on Day 1 because o f the total mortality in the 10,000 mg/L nominal test concentration. On Day 4, one sample plus a back-up sample of each test solution, except the 10,000 mg/L, were received from each replicate test chamber including the controls. Concentrations o f H-24215 were measured directly by high performance liquid chromatography/tandem mass spectrometry (LC/MS/MS). All scheduled samples were analyzed on the day o f receipt. The Day 1 samples were stored refrigerated till Day 4. The Day 1 samples were analyzed on Day 4 together with the Day 4 samples. 2. Instrument and Conditions HPLC Instrument: MS Instrument: Hewlett Packard Model 1100 HPLC Micromass Quattro II MS with "Z-spray" electrospray LC parameters: Column: Mobile Phase: Flow Rate: Column Temperature: Injection Volume: Column Switch: Zorbax RX-C18, 2.1 x 150 mm, 5.0 /im particle size 25% 2 mM ammonium acetate in water 75% methanol 0.2 mL/min 25C 50 iL 1.70 min MS parameters: Ionization mode: Capillary voltage: Source Temperature: Desolvation Temp: Collision Energy: Scan Function: electrospray (ESI), negative ions -2.8 kV 150C 300C 25 V MRM: 413.00 m/z (parent) to 168.80 m/z (daughter); time: 0-4 min. 3. Quantitation The test substance, H-24215, was used as the reference standard. A stock solution of the reference standard, H-24215 was made in HLWW with a target concentration o f 5000 mg/L. The calibration standards were prepared on each test day by diluting the H-24215 stock with HLWW to target 50 mg/L level. The 50 mg/L stock was used to obtain calibration standards by dilution with HLWW with concentrations ranging from 1.25 to 5 mg/L. Duplicate injections of test and calibration standard solutions were made and peak heights and areas were determined by electronic integration. The calibration curve was generated by regression analysis of the peak height or peak area data from chromatography o f the calibration solutions. A typical calibration curve is shown in Figure -12Company Sanitized. Does not contain TSCA CB/ H-24215: Static, Acute, 96-Hour LC50 to Rainbow Trout, Oncorhynchus mykiss DuPont-3381 1. Data for test solutions were compared to the working curve to determine concentrations of H-24215. Representative chromatograms o f a calibration standard solution, a test solution and a control solution are presented in Figures 2 to 4, respectively. The limit of quantitation (LOQ) and limit o f detection (LOD) were determined by calculating the signal to noise (S/N) ratios for selected chromatograms. Four chromatograms were examined for the S/N ratio. The LOD was calculated as the concentration equivalent to S/N ratio equal 3. The LOD was calculated as the concentration equivalent to S/N ratio equal 10. G. Statistical Analysis The 96-hour LC50 and 95% confidence interval was calculated by the moving average-angle m ethod^ based on mean, measured H-24215 concentrations. The highest mean, measured concentration causing no mortality at test end and the lowest mean, measured concentration causing 100% mortality at test end were assessed by visual observation. - 13Comp Oe oniaifi T5CACEM H-24215: Static, Acute, 96-Hour LC50 to Rainbow Trout, Oncorhynchus mykiss DuPont-3381 RESULTS AND DISCUSSION A. Analytical Report 1. Chromatographic Results H-24215 eluted as a well-resolved peak with a retention time of approximately 2.5 minutes. The LOQ and LOD were 5.2 and 1.6 /rg/L, respectively. 2. Test Solution Results Mean, measured values of H-24215 ranged from 87 to 94 % of the targeted nominal concentrations (Table 2). All measured values o f H-24215 were within 1.5 times the lowest value for all replicates within a concentration. These data indicate that H-24215 concentrations were maintained at acceptable levels throughout the definitive test. Control solutions showed no detectable concentrations of H-24215. B. In-Life Report 1. Rangefinding Study A static, rangefinding study with 5 fish per concentration was conducted using a dilution water control and nominal concentrations of 50,100, 500, 1000, and 5000 mg/L. At the end of 96 hours, the respective mortality values were 0, 0 , 0 , 0 , 0 and 60%. Test substance solutions were clear and colorless with no material present during the test. 2. Definitive Study Nominal concentrations of H-24215 for the definitive study were 625,1250,2500,5000, and 10,000 mg/L (10 fish per replicate, 2 replicate chambers per concentration). A dilution water control was used in this study. Mean, measured concentrations o f H-24215 were 554,1090, 2280,4560, and 9360 mg/L, respectively. Control solutions showed no detectable concentrations of H-24215. All test substance solutions were clear and colorless with no insoluble material present during the test. Dilution water quality was acceptable based on OECD(l) and ASTM(2) criteria, and no quantifiable concentrations o f pollutants and pesticides were present in the most recent semi annual dilution water analysis (Table 1). All chemical and physical parameters for the definitive test (Tables 3 - 6 ) were within expected ranges. Total alkalinity, EDTA hardness, and conductivity of the dilution water control were 49 mg/L CaCC>3, 122 mg/L CaCC>3, 240 /xmhos/cm, respectively. During the test, dissolved oxygen concentrations ranged from 7.5 14- ji-24215: Static, Acute, 96-Hour LC50 to Rainbow Trout, Oncorhynchus mykiss DuPont-3381 to 11.2 mg/L, pH ranged from 7.1 to 7.2, and mean temperature was 11,8 C with a range o f 11 6 to 12 .1C. At test conclusion, fish from the water control ranged from 2.5 to 3.2 cm in standard length (mean 2.8 cm) and 0.15 to 0.30 g in wet weight, blotted dry (mean 0.21 g). Standard length of the longest fish was not more than twice the length o f the shortest fish in the control. Loading in the water control was 0.23 g/L at test conclusion. Data on daily mortality and sublethal effects are presented in Tables 7 and 8 , respectively. No mortality or sublethal effects were seen in the dilution water control fish. Surviving exposed to a mean, measured H-24215 concentration o f 4560 mg/L exhibited sublethal effects such as swimming at the surface, labored respiration, dark coloration, lethargy, and erratic swimming. There were no mortality or sublethal effects below 4560 mg/L. The highest mean, measured concentration causing no mortality at test end was 2280 mg/L. The lowest mean, measured concentration causing 100% mortality at test end was 9360 mg/L. A summary o f the LC50 values and 95% confidence interval at specific time intervals is presented in Table 9. The 96-hour LC50, based on mean, measured H-24215 concentrations, was 4001 mg/L with confidence interval o f 3327 to 4932 mg/L. Since H-24215 is .a 2 0 % solution o f j p U j B n water, results are also expressed in terms of mean, m easuredpBBBPl concentrations. TKe 96-hour LC50 based on mean, m easuredfijp(3;oncentrations, was 800 mg/L with 95% confidence interval o f 665 to 986 mg/L. CONCLUSION H-24215 was assessed for acute toxicity to unfed fingerling rainbow trout, Oncorhynchus mykiss, in an unaerated, static, acute 96-hour test. The 96-hour LC50, based on mean, measured concentrations of H-24215, was 4001 mg/L. RECORDS AND SAMPLE STORAGE Specimens (if applicable), raw data, and the final report will be retained at Haskell Laboratory, Newark, Delaware, or at Iron Mountain Records Management, Wilmington, Delaware. - 15- .....r A. V O ** sn * c * cB> H-24215: Static, Acute, 96-Hour LC50 to Rainbow Trout, Oncorhynchus mykiss DuPont-3381 REFERENCES 1. Organisation for Economic Co-Operation and Development (OECD). Guideline for Testing Chemicals: 203, 17 July 1992. 2. American Society for Testing and Materials (ASTM). (1988). Standard Guide for Conducting Acute Toxicity Tests with Fishes, Macroinvertebrates, and Amphibians. E 729-88a. Annual Book of ASTM Standards, Vol. 11.04. 3. Zucker, E. (1985). Hazard Evaluation Division, Standard Evaluation Procedure: Acute Toxicity Test for Freshwater Fish. EPA-540/9-85-006. U.S. Environmental Protection Agency Office o f Pesticide Programs. Washington, D.C. 20460. 4. Official Journal of the European Communities. Annex to Commission Directive 92/69, Method C. 1. Acute Toxicity for Fish, 29-December-1992. 5. Peltier, W. H. and Weber, C. I., Eds. (1985). Methods for Measuring the Acute Toxicity of Effluents to Freshwater and Marine Organisms. EPA/600/4-85-013. United States Environmental Protection Agency, Environmental Monitoring and Support Laboratory, Cincinnati, Ohio. -16- H-24215: Static, Acute, 96-Hour LC50 to Rainbow Trout, Oncorhynchus mykiss DuPont-3381 TABLES - 17;om pany contain T5C % H-24215: Static, Acute, 96-Hour LC50to Rainbow Trout, Oncorhynchus mykiss________________________ # DuPont-3381 TABLE 1 CHEMICAL CHARACTERISTICS OF HASKELL LABORATORY WELL WATER a Parameter BOD, mg/L COD, mg/L DOC, mg/L TOC, mg/L Total Kjeldahl N, mg/L Ammonia N, mg/L Turbidity, NTU Phenolics, mg/L Color, apparent Co/Pt' Chlorine, Residual Solids, tot. susp., mg/L Aluminum, mg/L Antimony, mg/L Arsenic, mg/L Beryllium, mg/L Boron, mg/L Bromide, mg/L Cadmium, mg/L Calcium, mg/L Chloride, mg/L Chromium, mg/L Cobalt, mg/L Copper, mg/L Cyanide, mg/L Iron, mg/L Fluoride, mg/L Lead, mg/L MDLb 0.29 1.9 0.30 0.30 0.17 0.030 0.12 0.011 5.0 0.040 1.3 0.052 0.025 0.0010 0.0017 0.019 2.0 0.00011 0.038 3.00.00056 0.0011 0.0011 0.0040 0.016 0.40 0.023 Analytical Value 0.34 J* NDC NDd ND 0.36 J 0.038 J 0.16 J ND ND 0.041 J ND ND ND ND ND ND ND ND 44.4 59.4 ND ND ND ND ND ND ND Parameter Manganese, mg/L Magnesium, mg/L MBAS/LAS, mg/L Mercury, mg/L Nickel, mg/L Nitrite, mg/L Nitrate, mg/L Ortho-phosphate, mg/L Potassium, mg/L Selenium, mg/L Silver, mg/L Sodium, mg/L Sulfate, mg/L Sulfide, mg/L Zinc, mg/L Ca/Mg ratio Na/K ratio Volatile priority pollutants, /ig/L Acid extractable priority pollutants, /ig/L Base/neutral priority pollutants, /ig/L Pesticides/PCBs, /ig/L Organophosphorus pesticides, /ig/L MDLb 0.0016 0.040 0.024 0.000042 0.0030 0.40 0.030 1.8 0.36 0.0016 0.000066 0.093 1.5 0.011 0.0036 NA8 NA 1-40 1-16 1-21 0.0020-0.20 0.064-0.20 Analytical Value 0.0025 J 3.14 0.027 J ND ND ND ND ND 2.72 ND ND 7.49 6.2 ND 0.0048 J 14.1 2.75 ND ND ND ND ND *Sample analyses performed at Lancaster Laboratories, Lancaster, Pennsylvania, date of sample collection 11 February 1999 unless indicated otherwise, MDL = minimum detection limit, cND indicates not detected at the MDL, dBlank corrected, *Units based on cobalt/platinum reference,f A "J" follows analytical values which were greater than the MDL but less than the limit of quantitation, 8NA = not applicable. - 18- r.s,c o t,ta!n''SCRC9' SanUize^- Co'"s Compaq H-24215: Static, Acute, 96-Hour LC50 to Rainbow Trout, Oncorhynchus mykiss DuPont-3381 TABLE 2 MEASURED CONCENTRATIONS OF H-24215 IN TEST SOLUTION SAMPLES Nominal H-24215 Concentration Concentration Measured H-24215 Concentration (mg/L) (mg/L) (mg/L)a Day 0 Day 4 Mean, Measured H-24215 Concentration (mg/L)6 Mean, Percent Measmgd Recovery S in Concentration (mg/L) (%)c Water Control A Water Control B 0.0 0.0 ND d ND d ND d NDd _ -- __ -- 625 A 625 B 125 575 536 125 587 516 554 111 89 1250 A 1250 B 250 1180 969 250 1210 1020 1090 218 87 2500 A 2500 B 500 2570 2020 500 2560 1970 2280 456 91 5000 A 5000 B 1000 5150 4040 1000 5300 3760 4560 912 91 10,000 A 10,000 B 2000 2000 10,500 8470e 10,400 8070e 9360 1872 94 a The test substance (H-24215) is a 20 % solution o ^ H J E n water. b Mean, measured concentration was calculated as [(Day 0a + Day 0b)/2 + (Day 4a + Day 4b)/2]/2. Day 0 and Day 1 results were used to calculate the mean measured concentration for 10,000 mg/L dose level. c Based on nominal H-24215 concentrations. d ND denotes none detected. The limit of detection for H-24215 was 1.6 |ig/L. ' Total mortality was observed on Day 1. The concentrations of H-24215 in the samples submitted on Day 1 were . 8470 mg/L and 8070 mg/L for replicates A and B, respectively. -19Company Sanitized. Does not contain TSCA CBI H-24215: Static, Acute, 96-Hour LC50 to Rainbow Trout, Oncorhynchus mytciss DuPont-3381 TABLE 3 WATER CHEMISTRY OF THE DILUTION WATER CONTROL AT TEST START Total Alkalinity (mg/L as CaCOs) EDTA Hardness (mg/L as CaCOs) Conductivity (/imhos/cm) Dilution Water Control 49 122 240 -20i,,ompany Sanitized. Dees net contain TSCA CBI H-24215: Static, Acute, 96-Hour LC50 to Rainbow Trout, Oncorhynchus mykiss DuPont-3381 TABLE 4 DISSOLVED OXYGEN CONCENTRATION (mg/L)'1' OF H-24215 TEST SOLUTIONS Mean, Measured H-24215 Concentration (mg/L) Water Control 554 1090 2280 4560 9360 0 Hours A} B' 11.2 11.1 11.1 10.8 10.9 10.8 11.0 10.9 10.5 10.7 10.6 10.7 24 Hours At Bf 10.1 9.6 9.3 8.8 8.9 8.6 8.9 8.6 8.8 8.6 10.2 10.2 48 Hours3 A+ Bt 9.8 9.6 9.1 8.6 8.7 8.6 8.8 8.5 9.2 9.0 -- 72 Hours3 At Bt 8.7 8.6 8.3 7.8 8.0 7.8 7.8 7.5 8.2 8.3 "" 96 Hours3 At Bt 9.3 9.2 8.8 8.3 8.6 8.4 8.3 8.0 8.1 8.9 " The theoretical dissolved oxygen concentrations at saturation is approximately 10.8 mg/L at 12.0C. t A and B represent replicate test chambers containing 10 fish each (total 20 fish per test concentration) at test start, a Dash (-) denotes no reading taken due to total mortality on study day 1. -21 Company Sanitized. Doss net contain t s c a c b i H-24215: Static, Acute, 96-Hour LC50to Rainbow Trout, Oncorhynchus mykiss__________ DuPont-3381 TABLE 5 pH OF H-24215 TEST SOLUTIONS Mean, Measured H-24215 Concentration ________ (mg/L)________ Water Control 554 1090 2280 4560 9360 0 Hours A' Bt 7.2 7.2 7.2 7.1 7.2 7.2 7.2 7.2 7.2 7.2 7.1 7.2. 24 Hours At Bt 7.1 7.1 7.1 7:1 7.1 7.1 7.1 7.1 7.1 7.1 7.2 7.2 48 Hours8 A+ Bt 7.1 7.1 7.1 7.1 7.1 7.1 7.1 7.1 7.2 7.2 - 72 Hours8 At Bt 7.1 7.1 7.1 7.1 7.1 7.1 7.1 7.1 7.2 7.2 -- t A and B represent replicate test chambers containing 10 fish each (total 20 fish per test concentration) at test start, a Dash (-) denotes no reading taken due to total mortality on study day 1. 96 Hours8 A+ Bt 7.1 7.1 7.1 7.1 7.1 7.1 7.1 7.1 7.2 7.2 - -22- Qat\Ufcec- . VlO'- ;3Vikain T o C A C&V H-24215: Static, Acute, 96-Hour LC50 to Rainbow Trout, Oncorhynchus mykiss DuPont-3381 TABLE 6 TEMPERATURE (C) OF H-24215 TEST SOLUTIONS Mean, Measured H-24215 Concentration (mg/L) Water Control 554 1090 2280 4560 9360 0 Hours At Br 11.8 11.8 11.8 11.8 11.8 11.8 11.8 11.8 11.8 11.7 11.8 11.8 24 Hours At B1 11.9 12.0 11.9 12.0 12.0 12.0 12.0 12.0 12.0 12.0 11.9 11.9 48 Hours At Bt 11.6 11.6 11.6 11.6 11.6 11.6 11.6 11.6 11.6 11.6 -- 72 Hours At Bt 11.6 11.6 11.6 11.6 11.6 11.6 11.6 11.6 11.6 11.6 - 96 Hours At Bt 12.1 12.1 12.1 12.1 12.1 12.1 12.1 12.1 12.1 12.1 -- t A and B represent replicate test chambers containing 10 fish each (total 20 fish per test concentration) at test start, a Dash (-) denotes no reading taken due to total mortality on study day 1. -23- sssan* Santi: d. Doss ;ot contain TSCA C91  % 15: Static, Acute, 96-Hour LCsqto Rainbow Trout, Oncorhynchus mykiss DuPont-3381 TABLE 7 MORTALITY OF RAINBOW TROUT, ONCORHYNCHUS MYKISS, IN AN UNAERATED, STATIC, ACUTE, 96-HOUR TEST WITH H-24215 Mean, Measured H-24215 Concentration (mg/L) Water Control 554 1090 2280 4560 9360 24 Hours At Bt Number of Dead Fish / Number of Fish at Study Start 48 Hours 72 Hours A+ Bt a t Bt 0/10 0/10 0/10 0/10 7/10 10/10 0/10 0/10 0/10 0/10 1/10 10/10 0/10 0/10 0/10 0/10 8/10 10/10 0/10 0/10 0/10 0/10 3/10 10/10 0/10 0/10 0/10 0/10 9/10 10/10 0/10 0/10 0/10 0/10 5/10 10/10 96 Hours A+ Bt 0/10 0/10 0/10 0/10 9/10 10/10 0/10 0/10 0/10 0/10 6/10 10/10 t A and B represent replicate test chambers containing 10 fish each (total 20 fish per test concentration) at test start. -24Company Sanitizcd. ontasn TSCA. CB1 H-24215: Static, Acute, 96-Hour LC50 to Rainbow Trout, Oncorhynchus mykiss___________________________________________________________ DuPont-3381 TABLE 8 SUBLETHAL EFFECTS IN RAINBOW TROUT, ONCORHYNCHUS MYKISS, IN AN UNAERATED, STATIC, ACUTE, 96-HOUR TEST WITH H-24215 Mean, Measured H-24215 Concentration (mg/L) Water Control 554 1090 2280 4560 9360 24 Hours At b t Number of Fish with Effects / Number of Fish Alive 48 Hours 72 Hours At b t At b t 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 1ajk 1bk yaik 2 'jk l ijk, l ck/2 jaijk jajk l dij/l ^cdj ^adi ljk/3 l ck, 5ik/9 2jk,3 ck/7 l di/5 LLLLLL 96 Hours A+ bt 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 l dij/l 1adj Iaid l cd, l di/4 LL f A and B represent replicate test chambers containing 5 fish each (total 10 fish per test concentration) at test start. a at the surface b dark coloration c erratic swimming d labored respiration OBSERVATION KEY e hemorrhagic f other: g hyperactive h lying on the bottom i lethargic j partial loss o f equilibrium k rapid respiration L total mortality -25- Company Sanitized. Dess net contain TSCA CBI H-24215: Static, Acute, 96-Hour LC50 to Rainbow Trout, Oncorhynchus tnykiss DuPont-3381 TABLE 9 MEAN, MEASURED CONCENTRATIONS OF H-24215 CAUSING MORTALITY TO 50% (LC50) OF RAINBOW TROUT, ONCORHYNCHUS MYKISS, AT SPECIFIC TIME INTERVALS Time LC50 (mg/L)a 95% Confidence Intervals (mg/L)a Lower Upper 24 Hours 48 Hours 72 Hours 96 Hours 4802(960)b 4479(896) 4123(825) 4001(800) 4036(807) 3713(743) 3426(685) 3327(665) 5619(1124) 5606(1121) 5100(1020) 4932(986) a y-intercept are not calculated by the moving average-angle method, b J f lH H /a lu e s reported in parenthesis. Company Sanitized. Dess not contain TSCA OBI -26- H-24215: Static, Acute, 96-Hour LC50 to Rainbow Trout, Oncorhynchus mykiss DuPont-3381 FIGURES -27Compny Sar n -js s not cot ',ain TSC C Si H-24215: Static, Acute, 96-Hour LC50 to Rainbow Trout, Oncorhynchus mykiss DuPont-3381 FIGURE 1 REPRESENTATIVE ANALYTICAL CALIBRATION STANDARD CURVE FOR H-24215 0.0 1.0 2.0 3.0 4.0 5.0 6.0 H-24215 Concentration, mg/L -28Company Sanitized. Co contain TSCA C H-24215: Static, Acute, 96-Hour LC50 to Rainbow Trout, Oncorhynchus mykiss DuPont-3381 FIGURE 2 REPRESENTATIVE HPLC CHROMATOGRAM OF A CALIBRATION STANDARD SOLUTION Itj. Tone: 15:57; 12 D tc I2-N w -I999 Iratiun*oLOUATT2 Calibration standard solution contains H-24215 at concentration o f 2.5 mg/L. -29- Company Sanitizcd. Do- r.ct contain TSCA CB1 H-24215: Static, Acute, 96-Hour LC50 to Rainbow Trout, Oncorhynchus mykiss DuPont-3381 FIGURE 3 REPRESENTATIVE HPLC CHROMATOGRAM OF A H-24215 TEST SOLUTION SAMPLE Ini. Time: lfc4(hSS D tte 12-Nov-l 999 Iratium ercO U A T R Test solution sample contains H-24215 at nominal concentration of 625 mg/L. Sample was diluted 250 times before ' analysis. -30Company Sanilittsri. Dovo net contain TSCA CS H-24215: Static, Acute, 96-Hour LC50 to Rainbow Trout, Oncorhynchus mykiss DuPont-3381 FIGURE 4 REPRESENTATIVE HPLC CHROMATOGRAM OF A WATER CONTROL SOLUTION SAMPLE l o j . T m : 1S:24:25 D le 12-No-l999 lratm m arO U A T T 2 H-24215 would elute at retention time of approximately 2.5 minutes.