Document KRz2BK4Z7RZ4M0LBdGG3QDLD0

AR226-2801 TRADE SECRET Study Title H-24215: Static, Acute, 96-Hour LC5o to Rainbow Trout, Oncorhynchits 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.I (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: IHUx STUDY CODE NUMBER: jfl--i Page 1 of 31 Company Sanitized. Does no; contain T3CA COl 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 of 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 Font de Nemours and Company Wimiington, Delaware 19898 U.S.A. /k^^^_______ Study Director: CG^ 7-. Carl F. Muska, Ph.D. ^-/>C-^?7 Date f -2- Conipany Sanitized. L;oes not contain TSCA CBl QUALITY ASSURANCE STATEMENT Haskell Sample Number(s): 24215 Dates of Inspections: Conduct: Novembers, 1999 Records, Reports: December 14, 1999 Dates Findings Reported to: Study Director: December 14, 1999 Management: December 17, 1999 Reported by: ^^a^/J^1--Ih^J^ (j ' Joseph C. Hamill '-' SCr-. Q/t.u.a-ll:i^ty. A*,,s,,s.u..r.a..n-c,,,,e A. uditor /7-M^- /yy^ Date -3- S.n,,,^.Do.-notco^nTSCACm Company CERTIFICATION We, the undersigned, declare that this report provides an accurate evaluation of data obtained from this study. Analytical Report by: ^- Q 1^ ^ o^Q-l)^^'.-- ^ ^^^- ->'2-------___________ \Bogdan Szostek, Ph. D . D a t e Research Chemist Issued by Study Director: L 0^-^ TI /^-^^CL^Carl F. Muska, Ph.D. Director, Ecotoxicology ^)-^e^-m7 Date -4- Company San;ii=cd. Doas not contain TSCA CE-. TABLE OF CONTENTS Page GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT.................................. 2 QUALITY ASSURANCE STATEMENT...................................................................................3 CERTinCATION........................................................................................................................4 LIST OF TABLES........................................................................................................................ 6 LIST OF FIGURES ...................................................................................................................... 6 STUDY INFORMATION............................................................................................................. 7 STUDY PERSONNEL..................................................................................................................8 SUMMARY....................................................................................................................................9 INTRODUCTION....................................................................................................................... 10 MATERIALS AND METHODS ............................................................................................... 10 A. Test Substance................................................................................................................... 10 B. Test Solution Preparation.................................................................................................. 10 C. Dilution Water................................................................................................................... 10 D. Test Organism Culture..................................................................................................... 10 E. Test Methods.....................................................................................................................11 F. Sample Preparation and Chemical Analysis ..................................................................... 11 1. Sample Collection and Treatment..........................................................................................................11 2. Instrument and Conditions...................................................................................................................... 12 3. 12 Quantitation............................................................................................................................................ G. Statistical Analysis............................................................................................................ 13 RESULTS AND DISCUSSION................................................................................................. 14 A. Analytical Report.............................................................................................................. 14 1. Chromatographic Results .......................................................................................................................14 2. Test Solution Results.............................................................................................................................. 14 B. In-Life Report.................................................................................................................... 14 1. Rangefmding Study................................................................................................................................ 14 2. Definitive Study....................................^................................................................................................ 14 CONCLUSION............................................................................................................................ 15 RECORDS AND SAMPLE STORAGE................................................................................... 15 16 REFERENCES............................................................................................................................ 17 TABLES....................................................................................................................................... FIGURES..................................................................................................................................... 27 -...-o.^.--------"'"^61 Company Sa.--1- 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% (LC5o) 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 TSCACB1 Company Sanded. ,Do.e.-.tccontain STUDY INFORMATION 9th Collective Nomenclature: Octanoic acid, pentadecafluoro-, ammonium salt Synonyms/Codes: H-24215 Haskell Number: Perfluorooctanoate, ammonium salt PFOA & 24215 CAS Registry Number: 3825-26-1 Composition: Known Impurities: |iB| \ 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 Font de Nemours and Company Wilmington, Delaware 19898 U.S.A. Study Initiated/Completed: November 5,1999-/ (see report cover page) In-Life Initiated/Completed: November 8, 1999 / November 12, 1999 -7- Company Sanitised. Docs not contain TSCA CJ^ STUDY PERSONNEL Study Director: Management: Primary Technician: Animal Culturist: Carl F. Muska, Ph.D. Robert W. Rickard, Ph.D. Laurie D. Bouchelle Erica Flanders Analytical Chemist: Management: <".^. ' Bogdan Sz'ostek, Ph.D. S. Mark Kennedy, Ph.D. " ' " ' *' ' Toxicology Report Preparation: Bonnie J. Bieber, B.S. ;-... '- 'fr ^39t '...^ fi' ^ "^~ '9' oo----too^"^0^81 Company Sand. ed, . 00- SUMMARY The acute toxicity ofH-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.I; and the United States Environmental Protection Agency, Pesticide Assessment Guidelines Subdivision E, 72-1. The study was conducted with 5 concentrations ofH-24215 and a dilution water control at a mean temperature of 11.8C (range of 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 of rainbow trout to mean, measured H-24215 concentrations of 554,1090,2280,4560, and 9360 mg/L resulted in 0,0,0,75, 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 ofLCso values. Since H-24215 rsa 20% solution ofQIHBlin water, results are also expressed in terms of mean measuredy|M||foncentrations. The results are summarized as follows: Nominal concentrations of H-24215, mg/L8' Mean, measured concentrations ofH-24215, mg/L 96-hour LCso (95% confidence interval) for H-24215, based on mean, measured concentrations^mg/L Mean, measured concentrations oi^l----K"1^ 96-hour LC5o (95% confidence interval) foiM----M 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- Company Ssaann^'.di-eDvd"^,. Dnosos ^nTSCACB/ INTRODUCTION The objective of this study was to assess the acute toxicity ofH-24215 to unfed fingerling rainbow trout, Oncorhynchus mykiss, during an unaerated, static, 96-hour test. MATERIALS AND METHODS A. Test Substance The test substance, H-24215, was supplied as a light coloredjiqu^ The test substance contains 99.4% H-24215 by analysis. H-24215 is a 20% solution ofJ|BB|^Ammonium perfluorooctanoate) in water. B. Test Solution Preparation Test substance solutions were prepared by direct addition of 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 ;;*iyA Dilution water'^riginatedfrom the Haskell Laboratory well which is 378-feet deep and is cased and sealed into bedrock to prevent contamination. The hardness of the well water is adjusted to approximately 110-140 mg/L as CaCOa by the flow-proportioned addition ofCaCh. The well water is then aerated, passed through a green sand filter to remove iron, and filtered through 50-, 10-, and 5-fJi.m 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 OECD05 and ASTM^ criteria and specifications. ^aiS." '^tSJftse. 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, 49cm 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 12.1C). The fish were fed freshly-hatched, live brine shrimp (Prime Artemia me., 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- conta-snTSCACBI San..t.-.zeHd. nDocees.nncot Company 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^'4^ Five nominal concentrations, and a dilution water control were used in this study. Nominal concentrations ofH-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 of 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 offish to the test solutions was initiated approximately 41 minutes after mixing of 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 ofopercular movement and lack of 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 of transitional light (11-157 Lux) preceding and following the 16-hour light interval. Dissolved oxygen, pH, and temperature were measured in all replicate chambers of 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 of 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- Como-snv Sanm;:"!-. c"^"^'"" ^w^" TSCA C&7 back-up sample for A and B replicates was also received on Day 1 because of 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 ofH-24215 were measured directly by high performance liquid chromatography/tandem mass spectrometry (LC/MS/MS). All scheduled samples were analyzed on the day of 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 n 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 /zm particle size 25% 2 mM ammonium acetate in water 75% methanol 0.2 mL/min 25C 50/zL 1.70mm MS parameters: lonization mode: Capillary voltage: Source Temperature: Desolvation Temp: Collision Energy: Scan Function: electrospray (ESI), negative ions -2.8 kV 150C 300C 25V 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 of 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 of the calibration solutions. A typical calibration curve is shown in Figure -12- Company Sanitized. Does not contain TSCA CB; 1. Data for test solutions were compared to the working curve to determine concentrations of H-24215. Representative chromatograms of a calibration standard solution, a test solution and a control solution are presented in Figures 2 to 4, respectively. The limit ofquantitation (LOQ) and limit of 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 LCso and 95% confidence interval was calculated by the moving average-angle method^ 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, ^,^n^.T3CACB. Coinp^ San^-00-1- H-24215: Static, Acute, 96-Hour LC5o 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 /Ag/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 of 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 of 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^ and ASTM^ criteria, and no quantifiable concentrations of 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 CaCOa, 122 mg/L CaC03, 240 /xmhos/cm, respectively. During the test, dissolved oxygen concentrations ranged from 7.5 ^ o ^ ^ " -14Coin,p.3^"' san^-wo TSCACO1. to 11.2 mg/L, pH ranged from 7.1 to 7.2, and mean temperature was 11.8C with a range of 11.6 tol2.1C. 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 of 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 of 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 of the LC5o values and 95% confidence interval at specific time intervals is presented in Table 9. The 96-hour LCso, based on mean, measured H-24215 concentrations, was efMBHp 4001 mg/L with 95% confidence interval of 3327 to 4932 mg/L. Since H-24215 isa20%-, solution y^ aSrfl water, results are also expressed in terms^ofmean, measure^d"^-^--^^^--^^--^Uj concentrations. Tne 96-hour LC5O based on mean, measured|------Bboncentratwioasns80, 0 mg/L with 95% confidence interval of 665 to 986 mg/L. CONCLUSION H-24215 was assessed for acute toxicity to unfed fingeriing rainbow trout, Oncorhyfichus mykiss, in an unaerated, static, acute 96-hour test. The 96-hour LC5o, 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. ~ T - . . .^. ^^o o ^ ______________________.^GCAC61 Co^5"1 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 of 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- ^pa"*""'""'1-.1.-^0O-' C^"'"5'*08' TABLES 17,;ompany Sanis^ec:. H-24215: Static, Acute, 96-Hour LCso to Rainbow Trout, Oncorhynchus mykiss TABLE 1 CHEMICAL CHARACTERISTICS OF HASKELL LABORATORY WELL WATER a Parameter MDL" Analytical Value Parameter MDL" BOD, mg/L 0.29 0.34 J1 Manganese, mg/L 0.0016 COD, mg/L DOC, mg/L 1.9 0.30 ND" ND'1 Magnesium, mg/L MBAS/LAS, mg/L 0.040 0.024 TOC, mg/L 0.30 ND Mercury, mg/L 0.000042 Total Kjeldahl N, mg/L Ammonia N, mg/L Turbidity, NTU 0.17 0.030 0.12 0.36 J 0.038 J 0.16 J Nickel, mg/L Nitrite, mg/L Nitrate, mg/L 0.0030 0.40 0.030 Phenolics, mg/L 0.011 ND Ortho-phosphate, mg/L 1.8 Color, apparent Co/Pte 5.0 ND Potassium, mg/L 0.36 Chlorine, Residual 0.040 0.041 J Selenium, mg/L 0.0016 Solids, tot. susp., mg/L 1.3 ND Silver, mg/L Aluminum, mg/L 0.052 ND Sodium, mg/L 0.000066 0.093 Antimony, mg/L 0.025 ND Sulfate, mg/L 1.5 Arsenic, mg/L 0.0010 ND Sulfide, mg/L 0.011 Beryllium, mg/L 0.0017 ND Zinc, mg/L 0.0036 Boron, mg/L Bromide, mg/L 0.019 ND Ca/Mg ratio 2.0 ND NaTK ratio NA8 NA Cadmium, mg/L 0.00011 ND Volatile priority Calcium, mg/L 0.038 44.4 pollutants, fifs/L 1-40 Chloride, mg/L 3.0- 59.4 Acid extractable Chromium, mg/L 0.00056 ND priority pollutants, /ig/L 1-16 Cobalt, mg/L 0.0011 ND Base/neutral Copper, mg/L 0.0011 ND priority pollutants, ^g/L 1-21 Cyanide, mg/L 0.0040 ND Pesticides/PCBs, ^g/L 0.0020-0.20 Iron, mg/L 0.016 ND Organophosphorus Fluoride, mg/L 0.40 ND pesticides, /ig/L 0.064-0.20 Lead, mg/L 0.023 ND ' Sample analyses performed at Lancaster Laboratories, Lancaster, Pennsylvania, date of sample collection 11 February 1999 unless in b MDL = minimum detection limit, c ND indicates not detected at the MDL, ''Blank corrected, Units based on cobalt/platinum referen analytical values which were greater than the MDL but less than the limit ofquantitation, g NA = not applicable. -18- n^n CompaW58"^.Do^100 TABLE 2 MEASURED CONCENTRATIONS OF H-24215 IN TEST SOLUTION SAMPLES Nominal H-24215 Concentration Nnminal Concentration Measured H-24215 Concentration (mg/L) (mg/L) (mg/L)3 DayO Day 4 Mean, Measured H-24215 Concentration (mg/L)" Mean, Percent Recovery Concentration (mg/L) (%)c Water Control A 0.0 ND" ND" Water Control B 0.0 ND'1 ND'1 ---- 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 1000 5150 5300 4040 3760 4560 912 91 10,000 A 10,000 B 2000 2000 10,500 10,400 84706 80706 9360 1872 94 " The test substance (H-24215) is a 20 % solution OQ----IP water. b Mean, measured concentration was calculated as [(Day 0^+Day Oe)/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/tg/L. c 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. Dsss not contain TSCA CBl TABLE 3 WATER CHEMISTRY OF THE DILUTION WATER CONTROL AT TEST START Total Alkalinity (mg/L as CaC03) EDTA Hardness (mg/L as CaC03) Conductivity (^mhos/cm) Dilution 49 Water Control 122 240 -20- i-ompany Sanitized. Dees net contain TSCA CB1 H-24215: Static, Acute, 96-Hour LCso to Rainbow Trout, Oncorhynchus mykiss TABLE 4 DISSOLVED OXYGEN CONCENTRATION (mg/L)1f O, F 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 10.9 11.0 10.5 10.6 11.1 10.8 10.8 10.9 10.7 10.7 24 Hours A+ B+ 10.1 9.3 8.9 8.9 8.8 10.2 9.6 8.8 8.6 8.6 8.6 10.2 48 Hours3 A+ B+ 9.8 9.6 9.1 8.6 8.7 8.6 8.8 8.5 9.2 9.0 - - 72 Hours3 ^ ^ 8.7 8.6 8.3 7.8 8.0 7.8 7.8 7.5 8.2 8.3 - - 'V The theoretical dissolved oxygen concentrations at saturation is approximately 10.8 mg/L at 12.0C. f 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. -21ulTSC ^^iEW?WMIa t H-24215: Static, Acute, 96-Hour LC5p to Rainbow Trout, Oncorhynchus mykiss 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+ B+ 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 ^ B+ 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 A+ 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. .22- ^c^3 H-24215: Static, Acute, 96-Hour LC5o to Rainbow Trout, Oncorhynchus mykiss 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 ^ B+ 11.8 11.8 11.8 11.8 11.8 11.8 11.8 11.8 11.8 11.8 11.7 11.8 24 Hours A+ Bl 11.9 11.9 12.0 12.0 12.0 11.9 12.0 12.0 12.0 12.0 12.0 11.9 48 Hours ^ B+ 11.6 11.6 11.6 11.6 11.6 - 11.6 11.6 11.6 11.6 11.6 - 72 Hours A+ B+ 11.6 11.6 11.6 11.6 11.6 - 11.6 11.6 11.6 11.6 11.6 - f 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- 5^a^anY S- an.,d. e^d. r-^o.-. "-- al co <b H-24215: Static, Acute, 96-Hour LCso to Rainbow Trout, Oncorhynchus mykiss TABLE 7 MORTALITY OF RAINBOW TROUT, ONCORHYNCHUS MYKISS, IN AN UNAERATED, STATIC, ACUT WITH H-24215 Mean, Measured H-24215 Concentration (mg/L) Water Control 554 1090 2280 4560 9360 24 Hours A+ BT Number of Dead Fish / Number of Fish at Study Start 48 Hours A+ Bt 72 Hours A+ B+ 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 t A and B represent replicate test chambers containing 10 fish each (total 20 fish per test concentration) at test start. -24- Company s,^..^^"'''"'" H-24215: Static, Acute, 96-Hour LCso to Rainbow Trout, Oncorhynchus mykiss TABLES SUBLETHAL EFFECTS IN RAINBOW TROUT, ONCORHYNCHUS MYKISS, IN AN UNAERATED, ST 96-HOUR TEST WITH H-24215 Mean, Measured H-24215 Concentration (mg/L) Water Control 554 1090 2280 4560 9360 24 Hours ^ B1 Number of Fish with Effects / Number of Fish Alive 48 Hours A+ B+ 72 Hours Al B+ 0/10 0/10 0/10 0/10 pk^ 1^/3 L 0/10 0/10 0/10 0/10 1^, 2^, 1^ 5"79 L 0/10 0/10 0/10 0/10 1^ 1^/2 L 0/10 0/10 0/10 0/10 l3'^ 1^, 2^ 3ck/7 L 0/10 0/10 0/10 0/10 1^/1 L 0/10 0/10 0/10 0/10 icdj i adi 1^/5 L 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 lethargic J partial loss o k rapid respira L total mortali -25- Company Sanitized. Dcss net contain TABLE 9 MEAN, MEASURED CONCENTRATIONS OF H-24215 CAUSING MORTALITY TO 50% (LC5o) OF RAINBOW TROUT, ONCORHYNCHUS MYKISS. AT SPECIFIC TIME INTERVALS Time LC5o (mg/L)8 95% Confidenc e Intervals (mg/L)" Lower Upper 24 Hours 48 Hours 72 Hours 96 Hours 4802(960)" 4479(896) 4123(825) 4001(800) 4036(807) 3713(743) 3426(685) 3327(665) 5619(1124) 5606(1121) 5100(1020) 4932(986) id y-intercept are not calculated by the moving average-angle method. 'alues reported in parenthesis. Company Sanitized. Doss not contain TSCA CK 26- FIGURES -27. Comply San;^^. ^c ^ cnnlain TSCA ca( FIGURE 1 REPRESENTATIVE ANALYTICAL CALIBRATION STANDARD CURVE FOR H-24215 30000 25000 -{ Pe 20000 ak Ar 15000 ea 10000 5000 d ^ -i----------i----------i----------i 0.0 1.0 2.0 3.0 4.0 5.0 6.0 H-24215 Concentration, mg/L 28- . . ^.-..-.conta^SCACBl Company Sanitized.^-- H-24215: Static, Acute, 96-Hour LC5p to Rainbow Trout, Oncorhynchus mykiss____________DuPont-3381 FIGURE 2 REPRESENTATIVE HPLC CHROMATOGRAM OF A CALIBRATION STANDARD SOLUTION ln.Tcne:lAS-7;l; D>(e 12-Niw-19 Imbuin S^i^t[BTM:liCTraTSinHLWW VuU:5 991112.9 Sm(Mn. 2x3) 10(h "7-. 7623 fl 68486 1 MRM oil Channel ES- <13XIO> 168.80 6.88e4 AlEa.Heigrt I 020 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.60 2.00 220 2.40 2.60 2.60 3.00 120 3.40 3.60 3.0) Calibration standard solution contains H-24215 at concentration of 2.5 mg/L. 29- Company San!t:2cd. Do^ ^ C3.ta.n TSCA CBl FIGURES REPRESENTATIVE HPLC CHROMATOGRAM OF A H-24215 TEST SOLUTION SAMPLE Ini. Time: lft4(k51 Dle 13-N(>r-19g9 InilninieicOUATTI Stn^lnie:(a5ppiaArti/Dy4 VaUft 9 991112-l7Sm(Mn.2x3) MUlUU Hn4215 KXh NB: B-7B236-JM 2.U 6881 64325 MRM of < Channel ES413.00> 16680 6.46e4 Ansa.HelgIt 0.20 0.40 0.60 0.60 1.00 1-20 1.40 1.60 1.80 2.00 220 2.40 2.60 2.80 3.00 3^0 3.40 3.60 3.80 Test solution sample contains H-24215 at nominal concentration of 625 mg/L. Sample was diluted 250 times before analysis. -30- Company San^ari. C-;-;3 net contain TSCA CS, FIGURE 4 REPRESENTATIVE HPLC CHROMATOGRAM OF A WATER CONTROL SOLUTION SAMPLE Inj.Tjm:15;!4: Dte t}-N(w-199 tRllilliiiert:OUATT2 Sun(Jlwn;:CixibalA/UD*y4 Vulf: 3 hWlWU Hn42I5 NB:B-78236-JM 991112-3 Sm (Mil. 2x3) 10&1 U RM 0(1 Channel ES413.00 > 168.80 336 020 0.40 0.60 0.80 1.00 120 1.40 1.60 1.00 2.00 220 2.40 2.60 2.80 3.00 3.20 3.40 3.60 160 H-24215 would elute at retention time of approximately 2.5 minutes. -31 CQn'ipK-iy-'3''^-- ' ' ^nTSCAC