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AR226-2693 15 AR226-2693 DuPont EMSE Report No. 92-02 Study Title AQUEOUS STABILITY OF 8-2 TELOMER B ALCOHOL AS A FUNCTION OF PH Test Guideline OECD (1981). Hydrolysis as a Function ofpH. OECD Guideline for the Testing of Chemicals Method 111, adopted 12-May-1981. Author William R. Berti, Ph.D. Study Completion Date 27-March-2003 Test Facilities E.I. du Pont de Nemours and Company Central Research & Development Corporate Center for Engineering Research Environmental and Microbiological Sciences & Glasgow Building 300, P.O. Box 6101 Newark, DE 19714-6101 USA Engineering and E.I. du Pont de Nemours and Company Haskell Laboratory for Health and Environmental Sciences Newark, DE 19714 USA Sponsor Telomers Research Program The Rand Corporation 1200 South Hayes Street Arlington, VA 22202 USA EMSE Study /Project Number TO 106/4842 Report Number EMSER 92-02 TO 106/4842 Page lof31 DuPont EMSE Report No. 92-02 PAGE RESERVED FOR SPECIFIC COUNTRY REQUIREMENTS T0106/4842 - Page 2 of 31 DuPont EMSE Report No. 92-02 CERTIFICATION OF AUTHENTICITY AQUEOUS STABILITY OF 8-2 TELOMER B ALCOHOL AS A FUNCTION OF PH We, the undersigned, declare that the work described in this report was performed under our supervision, and that this report provides an accurate record of the procedures and results. Report by: " N ^ . ^ William R. Berti, Ph.D. Senior Research Biologist Approved by: C??-A^-(te).3 Date o^-^MA-^^ Date Study Initiation Date: 25-April-2002 Date Study Completed: 27-March-2003 Submitter: Telomers Research Program The Rand Corporation 1200 South Hayes Street Arlington, VA 22202 USA TO 106/4842 Page 3 of 31 DuPont EMSE Report No. 92-02 TABLE OF CONTENTS Page Reserved for Specific Country Requirements........................................................................^ Certification of Authenticity............................................................................................................3 Table of Contents.............................................................................................................................4 1.0 Summary .................................................................................................................................6 2.0 General Study Information.....................................................................................................^ 3.0 Materials and Methods............................................................................................................8 3.1 Test Guidelines....................................................................................................................8 3.2 Test System.........................................................................................................................^ 3.2.1 3.2.2 Chemical System........................................................................................................8 Test System ..............................................................................................................10 3.3 Test Conduct......................................................................................................................10 3.3.1 Buffer Solutions........................................................................................................10 3.3.2 3.3.3 Preliminary Test .......................................................................................................11 Aqueous Stability of an Unstable Substance............................................................! 1 3.4 Parameters Observed .........................................................................................................11 3.4.1 3.4.2 Analysis of Test Systems..........................................................................................11 Analytical Methods for Test Substance and Products..............................................! 1 3.5 Statistical Analysis .........................................................................................v..................13 3.6 Protocol Deviations ...........................................................................................................13 4.0 Results and Discussion.......................................................................................................... 13 5.0 Conclusions...........................................................................................................................15 6.0 Retention of Records.............................................................................................................15 7.0 Disposal of Test Substance ...................................................................................................15 8.0 References.............................................................................................................................15 Tables Table 1 Concentration of 8-2 TBA at pH 1.2 at 37C and at pH 4, 7, and 9 at 50C after 0 and 5 Days.............................................................................................................................16 Table 2 pH of Buffer Solutions Containing 8-2 TBA at Days 0 and 5..........................................17 Table 3 Daily Temperature Readings of Shaking Water Bath for Aqueous Stability of 8-2 TBA Conducted at pH 1.2 ..............................................................................................18 Table 4 Daily Temperature Readings of Shaking Water Bath for Aqueous Stability of 8-2 TBA Conducted at pH 4 .................................................................................................19 Table 5 Daily Temperature Readings of Shaking Water Bath for Aqueous Stability of 8-2 TBA Conducted at pH 7 and 9.............'...........................................................................20 Table 6 Sterility of Test Solutions at Day 5 in Sterile Trypticase Soy Broth (TSB) Medium.................................................................................................................................21 T0106/4842 Page 4 of 31 DuPont EMSE Report No. 92-02 Appendix A Table A-l Analytical Results of the Aqueous Stability of 8-2 TBA, pH 1.2, 37C, Days 0 (21-Aug-2002) and 5 (26-Aug-2002)....................................................................................22 Table A-2 Analytical Results of the Aqueous Stability of 8-2 TBA, pH 4.0, 50C, Days 0 (24-Jul-2002) and 5 (29-Jul-2002) ........................................................................................23 Table A-3 Analytical Results of the Aqueous Stability of 8-2 TBA, pH 7.0, 50C, Days 0 (21-Aug-2002) and 5 (26-Aug-2002)....................................................................................24 Table A-4 Analytical Results of the Aqueous Stability of 8-2 TBA, pH 9.0, 50C, Days 0 (21-Aug-2002) and 5 (26-Aug-2002)....................................................................................25 Appendix B Table B-l Analytical Results of the Aqueous Stability of 8-2 TBA for Pilot Experiment Conducted at pH 4 and Room Temperature, Day 0 only (16-Jul-2002)f ......;..;;.;................26 Table B-2 Analytical Results of the Aqueous Stability of 8-2 TBA, pH 1.2, 50,G, Days 0 (31-Jul-2002) and 5 (5-Aug-2002)f......................................................................................28 Table B-3 Analytical Results of the Aqueous Stability of 8-2 TBA, pH 7.0, 50C, Days 0 (31-Jul-2002) and 5 (5-Aug-2002)t......................................................................................29 Table B-4 Analytical Results of the Aqueous Stability of 8-2 TBA, pH 9.0, 50C, Days 0 (31-Jul-2002) and 5 (5-Aug-2002)t......................................................................................30 Appendix C Appendix C Certificate of Test Substance Analysis............................................. ;au..',s;.....^s.^.^fe' " T0106/4842 Page 5 of 31 1.0 2.0 DuPont EMSE Report No. 92-02 AQUEOUS STABILITY OF 8-2 TELOMER B ALCOHOL AS A FUNCTION OF PH Author William R. Berti, Ph.D. SUMMARY Test System: The aqueous stability of the test substance 8-2 Telomer B Alcohol (i.e., 8-2 TBA) in sterile aqueous solutions buffered at pH 1.2, 4.0, 7.0, and 9.0 was determined. Test systems consisted of the test substance in aqueous buffered solutions in sterilized containers. The test system was incubated in the dark at 50C at pH 4, 7, and 9 and at 37C at pH 1.2. An unstable test substance is indicated by a 10% or greater loss of test substance within 5 days. Findings: The test substance, 8-2 TBA, is stable under the conditions of the test at pH 4,7, and 9 at 50C andpH1.2at37C. Conclusions: The time in which 50% of the test substance will transform is estimated as greater than one year: t% >1 year. GENERAL STUDY INFORMATION Study Objectives This test generates essential environmental fate information relevant to the persistence of the test substance. The aqueous stability of a substance (e.g., hydrolytic stability) is one of the most common reactions controlling abiotic degradation and is therefore one of the main potential degradation paths of substances in the environment. A procedure to determine aqueous stability rates also is important in indicating whether other testing should be performed on a parent substance or on its aqueous stability products. Aqueous stability behavior needs to be examined at pH values normally found in the environment (pH 4 to 9) at environmentally relevant temperatures (10 to 60C). It is also sometimes desirable to determine aqueous stability of a test substance at physiologically relevant pH and temperatures, such as pH 1.2 and 37C, respectively. Specific objectives include: 1. To determine if a compound is stable in an aqueous environment, 2. To provide a material mass balance that accounts for greater then 90% of the initially applied test substance. 3. For unstable compounds: a. To determine the aqueous stability rate of the test substance in sterile buffer solutions at pH 4, 7, and 9 between 10 to 60C and at pH 1.2 and 37C, T0106/4842 Page 6 of 31 DuPont EMSE Report No. 92-02 b. To identify and follow the formation and decline of the aqueous stability produces) of the test substance if formed, c. To determine a first-order aqueous stability rate constant and half-life of the test substance (if rate constant and half-life of major aqueous stability products cannot be determined, then a aqueous stability study with the aqueous stability produces) will be conducted exactly as the parent study). Test System Justification The test system is outlined by OECD Guideline 111 and was requested by the sponsor. Study Personnel E.I. du Pont de Nemours and Company Central Research and Development - Corporate Center for Engineering Research Environmental and Microbiological Sciences & Engineering Haskell Laboratory for Health and Environmental Sciences Management: Study Director: Analytical Chemist: Technical Personnel: John T. Gannon, Ph.D. E.I. du Pont de Nemours and Company Central Research and Development Corporate Center for Engineering Research Environmental and Microbiological Sciences & Engineering Glasgow Building 300, P.O. Box 6101 Newark, DE 19714-6101 USA and S. Mark Kennedy, Ph.D. E.I. du Pont de Nemours and Company Haskell Laboratory for Health and Environmental Sciences Newark, DE 19714 USA William R. Berti, Ph.D. E.I. du Pont de Nemours and Company Central Research and Development Corporate Center for Engineering Research Environmental and Microbiological Sciences & Glasgow Building 300, P.O. Box 6101 Newark, DE 19714-6101 USA Engineering Bogdan Szostek, Ph.D. E.I. du Pont de Nemours and Company Haskell Laboratory for Health and Environmental Sciences Newark, DE 19714 USA Lisa M. Sulecki, DuPont Central Research & Development Mark G. Starr, DuPont Central Research & Development Keith B. Prickett, DuPont Haskell Laboratory T0106 / 4842 Page 7 of 31 DuPont EMSE Report No. 92-02 Study Execution Dates Study Initiation Date: Experimental Start Date: Experimental Completion Date: Study Completion Date: 25-April-2002 15-July-2002 9-September-2002 27-March-2003 3.0 MATERIALS AND METHODS 3.1 Test Guidelines The aqueous stability of the test substance 8-2 TBA in sterile aqueous solutions buffered at pH 1.2, 4.0, 7.0, and 9.0 was determined. Test systems consisted of the test substance in aqueous buffered solutions in sterilized containers made of non-adsorbing materials. The test systems were incubated in the dark at 50C at pH 4, 7, and 9 and 37C at pH 1.2. Day 0 and Day 5 samples were analyzed for the concentration of the test substance. If less than 10% of the test substance degrades in 5 days (t./, is greater than 1 year at 25C), the test substance is considered stable and no additional testing for aqueous stability is performed. If degradation of the test substance had occurred at pH 4, 7, and/or 9 and 50C, aqueous stability would also be performed at two additional temperatures separated by at least 15C (e.g., 20C and 35C) at the pH values at which the test substance was shown to be unstable. To test for first-order behavior, each test vessel would have been analyzed in time intervals that provided a minimum of six-spaced data points normally between 20% and 70% of aqueous stability of the test substance. Test solution would have been analyzed for the concentration of the test substance and its transformation product(s). The test substance concentration would be plotted against the sampling intervals to determine its degradation rate constant and half-lives at each pH. 3.2 3.2.1 3.2.1.1 Test System Chemical System Test Substance Name: Synonym: Active substance(s): Molecular Weight: CAS Name: CAS Number(s): 8-2 Telomer B Alcohol (Perfluorooctyl)ethanol, 8-2 TBA 8-2 Telomer B Alcohol, 99% 464.12 g mole-' 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10heptadecafluoro- 678-39-7 TO 106/4842 Page 8 of 31 Structure: DuPont EMSE Report No. 92-02 3.2.1.2 3.2.1.3 H Number: Lot Number: EMSE Sample Number: Concentration ofa.s., nominal: Concentration ofa.s., analyzed: Certificate of Analysis Date: Date Received: Supplier: Solubility at 25C: Vapor pressure: Stability: Appearance/Color: Storage Conditions: Safety Precautions: H-24691 P.00/001 E93386-63 99% 99.2% 13-Sept-2001 26-Mar-2002 Clariant Co. -140 ug L-1 0.023 mm Hg Stable at ambient room temperature White solid Room temperature; keep tightly closed Wear lab coat, protective gloves, and safety glasses Reference Substance None Test Vehicle Solutions buffered at pH 1.2,4,7, and 9 were added to glass test vessels containing 8-2 TBA L"1 methanol to attain an initial concentration of 150 ug 8-2 TBA L"'. For test solutions with a volume of 4.5 mL, 33.8 uL of 20 mg 8-2 TBA L"1 methanol was used to attain a concentration of 150 ug 8-2 TBA L"1. For test solutions with a volume of 3.0 mL, 22.5 U.L of 20 mg 8-2 TBA L"' methanol was used to attain a concentration of 150ug8-2TBAL-1. T0106/4842 Page 9 of 31 DuPont EMSE Report No. 92-02 3.2.1.4 Application Information The test substance in methanol was mixed with pH 1.2, 4, 7, or 9 buffer solutions to attain a concentration of 150 ug 8-2 TBA L'1 buffer solution. The amount of methanol in the final test solution was less than 1% by volume. 3.2.2 3.2.2.1 3.2.2.2 Test System Test Units Different types of test vessels were used to reduce or eliminate the amount of test substance lost during the test period. Test vessels used to conduct this study were either 10 mL borosilicate glass vials with aluminum-lined crimp caps (pH 1.2, 7, and 9 samples) or 7 mL borosilicate glass vials with aluminum-lined screw caps (pH 4). All test vessels were gravity sterilized by autoclaving for 20 minutes at 121C. Test Conditions Test solutions buffered at pH 4,7, and 9 were held at 50 1C for 5 days in the dark by covering the test vessels using the lid of the shaking water bath. The test conditions were replicated 4 times at each pH and time. Test solutions buffered at pH 1.2 were held at 37 1C for 5 days in the dark by covering the test vessels using the lid of the shaking water bath. This test condition was also replicated 4 times. 3.3 Test Conduct Sterile buffer concentrations of 0.05 M were used. After preparation, each solution was filter: sterilized by passing through a 0.2-mm filter. 3.3.1 3.3.1.1 3.3.7.2 3.3.7.3 3.3.7.^ Buffer Solutions pH 1.2 Buffer: 250 mL of 0.200 M potassium chloride (KC1) and 475 mL of 0.200 N HC1 were diluted to 1.000 L with water. Final pH adjustments were made with 1.0 N HC1 or NaOH as necessary. pH4 Buffer: 500 mL of 0.100 M potassium hydrogen phthalate [KH^fiHA)] and 4.00 mL of 0.100 N sodium hydroxide (NaOH) were diluted to 1.000 L with water. No final pH adjustment was necessary. pH 7 Buffer: 500 mL of 0.100 M potassium dihydrogen phosphate (KHzP04) and 296 mL of 0.100 M sodium hydroxide were diluted to 1.000 L with water. Final pH adjustments were made with 5N HC1. pH9 Buffer: 500 mL 0.050 M boric acid (HzBC^) and 213 mL of 0.100 M NaOH were diluted to 1.000 L with water. Final pH adjustments were made with 5N HC1. T0106/4842 Page 10 of 31 DuPont EMSE Report No. 92-02 3.3.2 Preliminary Test A preliminary test was performed on the test substance at 50C at pH 4.0, 7.0, and 9.0 and at 37CandpH1.2. 3.3.3 Aqueous Stability of an Unstable Substance The test substance is stable; therefore, testing for the aqueous stability of an unstable substance was not performed. 3.3.4 3.3.4.1 3.3.4.2 Sample Collection and Storage Sampling Intervals Day 0 samples were frozen immediately after the addition of the test substance until extracted and analyzed. Day 5 samples were shaken at 37C (buffer pH 1.2) or 50C buffer pH 4, 7, and 9) for 5 days, at which time they were frozen until extracted and analyzed. Test Solution Storage Test solutions were stored at approximately -20C. ...uia-.i;., .,, ipfcs wen; s! 3.4 3.4.1 3.4.1.1 3.4.1.2 3.4.1.3 3.4.2 3.4.2.1 Parameters Observed Analysis of Test Systems Sterility Measurements Sterility was determined on Day 5 samples of each of the test systems. Sterile trypticase soy broth (TSB) medium was used to check the sterility of each sample. At sampling, 0.5 mL of the test solution was aseptically added to a culture tube containing 4.5 mL of TSB medium. The TSB medium was incubated 3 to 4 days in darkness at 30C. The presence or absence of cloudiness as compared to sterile TSB medium controls determined sterility. pH Measurement Test solution pH was measured and documented at Day 0 and at the end of the experiment. Temperature Measurements The temperature of the test system was monitored and recorded daily throughout the course of the study. Analytical Methods for Test Substance and Products Analysis of Test Substance Sample preparation: The GC/MS analysis of 8-2 TBA samples involved use of an internal standard and surrogate spiked into each sample to trace the extraction recovery. The 1-Octanol, 3,3,4,4,5,5,6,6,7,8,8,8-dodecafluoro-7-(trifluoromethyl)- (CAS# 20015-46-7, 98%, Oakwood Products, West Columbia, SC), referred to as C9-iso, was used as the surrogate. The 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,10,10,10-hexadecafluoro-9-(trifluoromethyl)- (CAS# 31200-98-3, 98%, Oakwood Products, West Columbia, SC), referred to as Cl 1-iso, was used TO106/4842Page 11 of31 DuPont EMSE Report No. 92-02 as the internal standard. The 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluoro(CAS# 678-39-7, 97.6%, Oakwood Products, West Columbia, SC), referred to as 8-2 TBA, was used as the analytical standard of 8-2 TBA. Stock solutions at approximately 1000 ug mL'1 of internal standard and surrogate were prepared in methanol and stored refrigerated. The stocks were diluted to appropriate concentration with methanol before use for each set of samples analyzed. The pH 1.2,7, and 9 samples (4.5-mL test solution volume) in crimp-capped 10-mL glass vials were spiked with 13.5 uL of 50 ug C9-iso mL'1 methanol by injecting the solution into the closed vial through the vial septum using a syringe. Then, 2.25-mL propane, 2-methoxy2-methyl- [CAS# 1634-04-4 (methyl t-butyl ether, referred to as MTBE] was introduced into the vial using a syringe. The contents of the vial were vortexed for 15 min using a multi-tube vortexer. The vial was opened and 1 mL of the MTBE layer was sampled to a glass GC vial (1.7 mL), spiked with 6 uL of a 50 u,g Cl 1-iso mL"1 methanol, crimped, and subjected to GC/MS analysis. The calibration standards were prepared by spiking appropriate volumes of 50 ug mL'1 methanol of C9-iso, Cl 1-iso, 8-2 TBA to 1-mL MTBE. The calibration curves were constructed using peak area of signal obtained for ion m/z: 95. The pH 4 samples (3-mL test solution volume) required a different preparation procedure because the presence ofphthalates in the sample buffer interfered with the analysis of the test substance. The pH 4 samples in 7 mL glass vials with screw caps were opened and 9 uL of 50-ug C9-iso mL"1 methanol was added to the vial using a syringe, followed by 1.5 mL of hexane (CAS# 110-54-3). The contents of the vial were vortexed for 15 min using a multi-tube vortexer. The SPE column (Isolute Si, 100 mg /10mL, Jones Chromatography) was conditioned by cleaning it with 2 mL ofisopropanol (IPA) and conditioning with 2 mL hexane. The hexane extract was loaded onto the conditioned SPE column. After loading the hexane extract, the column was washed with 2 mL of hexane and the analytes were eluted with 1 mL IPA. No drying of the column was applied after the hexane wash. A 0.5 mL aliquot of the IPA extract was sampled to a glass GC vial (1.7 mL), spiked with 3 uL of 50 ug Cl 1-iso mL'1 methanol, crimped, and subjected to GC/MS analysis. The calibration standards for the pH 4 samples were prepared by spiking appropriate volumes of 50 ug mL'1 methanol of C9-iso, Cl 1-iso, 8-2 TBA to 1 mL of matrix matched IPA. The matrix matched IPA was prepared by subjecting the blank pH 4 buffer to the SPE procedure, the same way as the samples. The IPA eluate was used to prepare the matrix matched calibration standards. The calibration curves were constructed using peak area of signal obtained for ion m/z: 95. Instrumentation and conditions: GC/MS system: HP 6890 Plus GC (Agilent, Wilmington, DE USA), HP 5973 Mass Selective Detector (Agilent), MPS2-MultiPurposeSampler (Gerstal, Baltimore, MD USA) Column: DB-5MS, 30 m x 0.25 mm, 1 urn film (Agilent, Wilmington, DE USA) Temp. ramp: Initial: Flow rate: Split: Inlet temp.: Injection volume: MSD transfer line temp.: SIM ions monitored: 80C for 2 min 200C/mintol200C 50G/min to 300C and hold for 3 min 1.0 mL/min; He; constant flow mode 5:1 250C 2uL 280C m/z: 31,95, 131 T0106/4842 Page 12 of 31 3.5 3.6 4.0 DuPont EMSE Report No. 92-02 Retention time: 8-2 TBA: C9-iso: Cll-iso: 4.93 min 4.60 min 5.38mm Statistical Analysis Paired t-test tests were used to determine if the mean test substance concentrations in paired Day 0 and Day 5 buffer solutions are the same (JMP, 1995). Protocol Deviations None RESULTS AND DISCUSSION Under the conditions of the test at pH 1.2 and 37C and at pH 4,7, and 9 and 50C, 8-2 TBA is stable (Table 1). Tables in Appendix A (Tables A-l, A-2, A-3, and A-4) contain the analytical results of the aqueous stability study of the test substance that are summarized in Table 1. Stability of the test substance, 8-2 TBA: At pH 1.2, 8-2 TBA concentration after five days at 37C was 195 8.00 ug L"1, compared to 183 10.9 ug L''at the start of the test (Day 0). The difference between the Day 5 and Day 0 concentrations of the test substance was +6.7%, indicating that 8-2 TBA is stable at this pH. The difference between Day 5 and Day 0 was not statistically significant (p = 0.05). At pH 4, 8-2 TBA concentration after five days at 50C was 143 10.0 ug L"1, compared to 156 4.22 ug L''at the start of the test (Day 0). The difference between the Day 5 and Day 0 concentrations of the test substance was -8.7%, indicating that 8-2 TBA is stable at this pH. The difference between Day 5 and Day 0 was not statistically significant (p=0.05). At pH 7, 8-2 TBA concentration after five days at 50C was 181 12.6 u,g L"1, compared to 199 9.12 ug L'' at the start of the test (Day 0). The difference between the Day 5 and 0 concentrations of the test substance was -9.6%, indicating that 8-2 TBA is stable at this pH. The difference between Day 5 and Day 0 was not statistically significant (p = 0.05). At pH 9, 8-2 TBA concentration after five days at 50C was 175 10.5 ug L'1, compared to 169 5.99 u.g L'' at the start of the test (Day 0). The difference between the Day 5 and Day 0 concentrations of the test substance was +3.8%, indicating that 8-2 TBA is stable at this pH. The difference between Day 5 and Day 0 was not statistically significant (p=0.05). Experimental conditions -pH: The pH 1.2 test solutions were pH 1.11 and 1.54 at days 0 and 5, respectively, a variation of more 0.1 units (Table 2). This had no significant effect on the outcome of the test because the compound was stable. The pH 4 test solutions were pH 4.02 and 4.12 at days 0 and 5, respectively; a variation of more 0.1 units (Table 2). This had no significant effect on the outcome of the test because the compound was stable. T0106/4842 Page 13 of 31 DuPont EMSE Report No. 92-02 The pH 7 test solutions were pH 7.03 and 7.04 at days 0 and 5, respectively; and the pH 9 test solutions were pH 9.02 and 8.97 at days 0 and 5, respectively (Table 2). The pH of the test solutions at these 2-pH levels did not change significantly (pH change less than 0.1 unit) during the test. Experimental conditions - temperature: The temperature of the shaking water bath used to maintain the pH 1.2 test solution at a constant temperature did not fluctuate by a total of more than 0.2C at 37C (Table 3). The average temperature of the shaking water bath was 37.1C and the standard deviation was0.1C(n=6). The temperature of the shaking water bath used to maintain the pH 4 test solution at a constant temperature did not fluctuate by more than 0.1C at 50C during the 5 days of the test (Table 4). The average temperature of the shaking water bath was 50.0C and the standard deviation was 0.05C (n = 6). The temperature of the shaking water bath used to maintain the pH 7 and 9 test solutions at a constant temperature did not fluctuate by more than 0.1C at 50C during the 5 days of the test (Table 5). The average temperature of the shaking water bath was 50.0C and the standard deviation was 0.1 C (n = 6). Experimental conditions - sterility: The sterility check solutions for the control, pH 1.2 at 37C, and pH 7 and 9 at 50C were sterile at the end of the test, as indicated by the lack of bacteria growth in the Trypticase Soy Broth (TSB) medium (Table 6). The sterility check solution for the pH 4 at 50C was not sterile as indicated by a slightly cloudiness of the TSB medium after the incubation period. (Table 6). This did not affect the outcome of the study because the test substance was stable at this pH and temperature (Table 1). Trial studies: Prior to the definitive studies, several trial studies were performed. The results of these trial studies are found in Appendix B (Tables B-l, B-2, B-3, and B-4). The information below summarizes these results. Table B-l lists the results of a pilot experiment that was conducted to demonstrate that the experimental design, implementation, and subsequent analysis of the test substance was adequate to determine its aqueous stability. This study was conducted at pH 4 and room temperature for Day 0 only. Table B-2 lists the results of the aqueous stability of the test substance at pH 1.2 and 50C. The test substance was shown to be stable at this pH and temperature after 5 days. The study was repeated because it should have been conducted at a pH of 1.2 and 37C. Both demonstrate that the test substance is stable (Tables 1 and A-l). Table B-3 lists the results of the. aqueous stability of the test substance at pH 7 and 50C on Day 0 and Day 5. This test was repeated using different test vessels because of the large standard deviation of the average test substance concentration at Day 5 (standard deviation of 39.8 ug L'1 for Day 5 compared to 6.25 ug L"1 for Day 0), which appears to be due to the loss of the test substance in two or more of the Day 5 replications. The repeated study demonstrated that the test substance is stable at pH 7 (Tables 1 and A-3). T0106/4842 Page 14 of 31 5.0 6.0 7.0 8.0 DuPont EMSE Report No. 92-02 Table B-4 lists the results of the aqueous stability of the test substance at pH 9 and 50C on Day 0 and Day 5. This test was repeated using different test vessels because of the large standard deviation of the average test substance concentration at Day 5 (standard deviation of 42.9 ug L'1 for Day 5 compared to 11.5 ug L'' for Day 0), which appears to be due to the loss of the test substance in two of the Day 5 replications. The repeated study demonstrated that the test substance is stable at pH 9 (Tables 1 and A-3). CONCLUSIONS The time in which 50% of the test substance will transform in water at environmentally relevant pH ranges and temperatures is greater than one year (ii/, >1 year), because >90% of the test substance added at Day 0 was recovered after 5 days at pH 1.2 and 37C and pH 4.0, 7.0, and 9.0 and 50C. RETENTION OF RECORDS For the periods demanded by GLP guidelines and specific country requirements, study documents and materials will be stored in the archives of the test facility, including but not limited to: study protocol; any protocol and/or report amendments or addenda or SOP deviations; all raw data; one original signed copy of the final report; laboratory-specific or site-specific raw data such as personnel files, instrument, equipment, refrigerator, and/or freezer raw data. Documents and materials are archived according to the principles of Good Laboratory Practice in the organization of the testing facility. DISPOSAL OF TEST SUBSTANCE After issuance of the final report, the remaining test substance will be stored at the DuPont EMSE laboratory until its expiration date and then destroyed by incineration, unless other arrangements are made between the submitter and the Test Facility. REFERENCES 8.1 OECD Guidelines for the Testing of Chemicals, Section 1, Method 111, Hydrolysis as a Function ofpH. 8.2 Environmental Protection Agency. 1989. Good Laboratory Practice Standards, 40 CFR, Part 160, Final Rule. EPA, Washington, DC. 8.3 JMP. 1995. JMP Statistical and Graphics Guide. Version 3 ofJMP. SAS Institute Inc. Cary, NC USA. T0106/4842 Page 15 of 31 DuPont EMSE Report No. 92-02 TABLE 1 CONCENTRATION OF 8-2 TBA AT PH 1.2 AT 37C AND AT PH 4, 7, AND 9 AT 50C AFTER 0 AND 5 DAYS 8-2 TBA Average Difference between pH Time Date Concentration Day 5 and Day Of Day ugL-1 % 1.2 0 21-Aug-2002 183 10.9:): 1.2 5 26-Aug-2002 195 8.00 +6.4 4 0 24-Jul-2002 156+4.22 4 5 29-Jul-2002 143 10.0 -8.7 7 0 21-Aug-2002 199 9.12 7 5 26-Aug-2002 181 12.6 -9.5 9 0 21-Aug-2002 169 5.99 9 5 26-Aug-2002 175 10.5 +3.5 t Difference between Day 5 and Day 0, % = Co = 8-2 TBA concentration at Day 0 C5 = 8-2 TBA concentration at Day 5 3: Mean standard deviation; n = 4. (Cs - Co)/((C5 + Co)/2) x 100, where: -ai; T0106/4842 Page 16 of 31 DuPont EMSE Report No. 92-02 TABLE 2 PH OF BUFFER SOLUTIONS CONTAINING 8-2 TBA AT DAYS 0 AND 5 Time Day 0 5 Date 21-Aug-2002 26-Aug-2002 Nominal PH S.U.f 1.2 1.2 Measured PH s.a 1.11 1.54 Nominal temperature C Room temperature 37 Temperature of pH measurement C 22.5 37.1 24-Jul-2002 4 29-Jul-2002 4 4.02 4.12 Room temperature 50 23.0 50.0 21-Aug-2002 7 26-Aug-2002 7 7.03 7.04 Room temperature 50 22.5 50.0 21-Aug-2002 26-Aug-2002 t S.U. = Standard Units 9.02 8.97 Room temperature 50 22.5 50.0 TO 106/4842 Page 17 of 31 DuPont EMSE Report No. 92-02 TABLE 3 DAILY TEMPERATURE READINGS OF SHAKING WATER BATH FOR AQUEOUS STABILITY OF 8-2 TBA CONDUCTED AT pH 1.2 Time Day 0 1 2 3 4 5 Average, n = 6 Standard Deviation Date 21-Aug-2002 22-Aug-2002 23-Aug-2002 24-Aug-2002 25-Aug-2002 26-Aug-2002 Temperature C 37.1 37.0 37.2 37.0 37.1 37.1 37.1 0.1 T0106/4842 Page 18 of 31 DuPont EMSE Report No. 92-02 TABLE 4 DAILY TEMPERATURE READINGS OF SHAKING WATER BATH FOR AQUEOUS STABILITY OF 8-2 TBA CONDUCTED AT PH 4 Time Day 0 1 2 3 4 5 Average, n = 6 Standard Deviation Date 24-Jul-2002 25-Jul-2002 26-Jul-2002 27-Jul-2002 28-Jul-2002 29-JUI-2002 Temperature C 49.9 50.0 49.9 49.9 50.0 50.0 50.0 0.05 T0106/4842 Page 19 of 31 DuPont EMSE Report No. 92-02 TABLE 5 DAILY TEMPERATURE READINGS OF SHAKING WATER BATH FOR AQUEOUS STABILITY OF 8-2 TBA CONDUCTED AT PH 7 AND 9 Time Day 0 1 2 3 4 5 Average, n = 6 Standard Deviation Date 21-Aug-2002 22-Aug-2002 23-Aug-2002 24-Aug-2002 25-Aug-2002 26-Aug-2002 Temperature C 49.9 50.0 50.1 49.9 50.0 50.0 50.0 0.1 T0106/4842 Page 20 of 31 DuPont EMSE Report No. 92-02 TABLE 6 STERILITY OF TEST SOLUTIONS AT DAY 5 IN STERILE TRYPTICASE SOY BROTH (TSB) MEDIUM Treatment Result Conclusion Control Buffer1.2pH,37C,Day5 Buffer4pH,50C,Day5 Buffer7pH,50C,Day5 Buffer9pH,50C,Day5 Clear Clear Cloudy Clear Clear Sterile Sterile Non-sterile Sterile Sterile Sterile indicates that no bacteria were observed in 4.5 mL of TSB containing 0.5 mL test solution after 3 to 4 days of incubation in the dark at 30C. T0106/4842 Page 21 of 31 DuPont EMSE Report No. 92-02 TABLE A-1 ANALYTICAL RESULTS OF THE AQUEOUS STABILITY OF 8-2 TBA, PH 1.2, 37C, DAYS 0 (21 -AUG-2002) AND 5 (26-AUG-2002) No pH Time day 1 1.2 0 2 1.2 0 3 1.2 0 4 1.2 0 5 1.2 0 6 1.2 0 7 1.2 0 8 1.2 0 Average Standard Deviation Rep 1 1 2 2 3 3 4 4 Test Substance ugL-' 171 178 201 192 190 183 171 176 Test Substance Analytical Replication Average ugL-' 175 197 186 174 183 10.9 Difference Between Analytical Replications! % 4.0 4.6 4.0 2.9 9 1.2 5 1 200 200 0.0 10 1.2 5 1 200 11 1.2 5 2 186 189 4.0 12 1.2 5 2 193 13 1.2 5 3 204 204 0.2 14 1.2 5 3 205 15 1.2 5 4 203 188 15 16 1.2 5 4 174 Average 195 Standard Deviation 8.00 t Calculated by dividing the absolute difference of the two analytical replications by their average value, times 100. TO 106 / 4842 Page 22 of 31 DuPont EMSE Report No. 92-02 TABUEA-2 ANALYTICAL RlESUU"SOF THE AQIJEOUS STABILIT^ rOF8-2TBA,pH4.0,500C, DAYS 0 (24-JlJL-201)2)A>ND 5 (23I-JUL-2002) No pH 1 4 2 4 3 4 4 4 5 4 6 4 7 4 8 4 Average Standard Deviation Time day 0 0 0 0 0 0 0 0 Rep 1 1 2 2 3 3 4 4 Test Substance ugL-' Test Substance Analytical Replication Average ugL-' 162 162 162 156 157 157 150 153 156 155 153 151 156 4.22 Difference Between Analytical Replications! % 0.2 0.4 3.7 3.1 9 4 5 1 128 130 2.6 10 4 5 1 132 11 4 5 2 155 154 1.3 12 4 5 2 153 13 4 5 3 143 143 0.2 14 4 5 3 143 15 4 5 4 145 145 0.0 16 4 5 4 145 Average 143 Standard Deviation 10.0 f Calculated by dividing the absolute difference of the two analytical replications by their average value, times 100. T0106/4842 Page 23 of 31 DuPont EMSE Report No. 92-02 TABLE A-3 ANALYTICAL RESULTS OF THE AQUEOUS STABILITY OF 8-2 TBA, PH 7.0, 50C, DAYS 0 (21-AUG-2002) AND 5 (26-AUG-2002) No PH 1 7 2 7 3 7 4 7 5 7 6 7 7 7 8 7 Average Standard Deviation Time day 0 0 0 0 0 0 0 0 Rep 1 1 2 2 3 3 4 4 Test Substance ugL-' Test Substance A nalytical Replication Average ugL-1 Difference Between Analytical Replications! % 202 199 2.3 197 195 195 0.3 196 201 190 12 179 211 211 0.7 212 199 9.12 9 7 5 1 195 192 3.1 10 7 5 1 189 11 7 5 2 169 166 3.6 12 7 5 2 163 13 7 5 3 178 175 3.4 14 7 5 3 172 15 7 5 4 202 191 12 16 7 5 4 180 Average 181 Standard Deviation 12.6 t Calculated by dividing the absolute difference of the two analytical replications by their average value, times 100. TO 1067 4842 Page 24 of 31 DuPont EMSE Report No. 92-02 TABLE ^k-4 ANALYT ICAL FIESULTSOIF THE AQIJE;OUS STABIUIY OF8-2TBA,PH9.0,50C. DAYSO (21 -ALUG-2(D02) AND 5 (2 6-Auo-2002) Test Substance Test Analytical Replication Difference Between No pH Time Rep Substance Average Analytical Replications! day ugL-' ugL-' % 1 9 0 1 185 176 10 2 9 0 1 167 3 9 0 2 168 170 2.6 4 9 0 2 173 5 9 0 3 159 162 3.7 6 9 0 3 165 7 9 0 4 163 167 4.5 8 9 0 4 171 Average Standard Deviation 169 5.99 9 9 5 1 172 182 12 10 9 5 1 193 11 9 5 2 173 179 6.7 12 9 5 2 185 13 9 5 3 160 160 0.6 14 9 5 3 159 15 9 5 4 169 180 13 16 9 5 4 191 Average 175 Standard Deviation 10.5 t Calculated by dividing the absolute difference of the two analytical replications by their average value, times 100. T0106/4842 Page 25 of 31 DuPont EMSE Report No. 92-02 TABLE B-1 ANALYTICAL RESULTS OF THE AQUEOUS STABILITY OF 8-2 TBA FOR PILOT EXPERIMENT CONDUCTED AT PH 4 AND ROOM TEMPERATURE, DAY 0 ONLY (16-JUL-2002)f Test Substance Test Analytical Replication Difference Between No Rep Substance Average Analytical Replications:}: ^p^-p^_ 1 1 106 112 10 2 1 117 3 2 104 108 8.2 4 2 113 5 3 109 110 2.4 6 3 111 7 4 102 113 19 8 4 123 9 5 114 110 7.3 10 5 106 11 6 125 117 13 12 6 109 13 7 103 106 6.9 14 7 110 15 8 118 115 4.9 16 8 112 17 9 114 115 3.2 18 9 117 19 10 100 94.1 12 20 10 88.4 t Experiment performed to assess the adequacy of the test methods and analytical procedure.; It was performed using pH 4 buffer at room temperature. Day 0 samples only. Test vessels were .7 mL glass scintillation vials with aluminum-lined screw caps. 22.5 uL of a 20 mg 8-2 TBA L"1 methanol solution was added to each test vessel with 3 mL of the pH 4 buffer solution. After capping, the samples were vortex-mixed for 10s. Samples were frozen until analyzed. ^ Calculated by dividing the absolute difference of the two analytical replications by their average value, times 100. TO 106/4842 Page 26 of 31 DuPont EMSE Report No. 92-02 TABLE B-1 (CONT'D) Test Substance Test Analytical Replication Difference Between No Rep Substance Average Analytical Replications]: ugC' ugL-' % 21 11 108 106 3.6 22 11 104 23 12 120 103 35 24 12 84.9 25 13 117 119 4.4 26 13 122 27 14 101 108 12 28 14 115 29 15 112 108 7.4 30 15 104 31 16 106 114 13 32 16 121 Average 110 Standard Deviation 6.09 f Experiment performed to assess the adequacy of the test methods and anaallyyttiiccal procedure. It-was performed using pH 4 buffer at room temperature. Day 0 samples only. Test vessels wei?e'7.mL . glass scintillation vials with aluminum-lined screw caps. 22.5 uL of a 20 mg 8-2 TBA L'tttt. K. >'"- methanol solution was added to each test vessel with 3 mL of the pH 4 buffer solution. After capping, the samples were vortex-mixed for 10s. Samples were frozen until analyzed. Calculated by dividing the absolute difference of the two analytical replications by their average value, times 100. T0106/4842 Page 27 of 31 DuPont EMSE Report No. 92-02 TABUEB-2 ANAL YTICAL R ESULT)S OF THE.AQUEOUS SSTABILITY OF t?-2TBA,PH1.2,50C, DAYS 0 (31-JlJL-200 2) AND 5 (5-AUG-20I02)+ ,r' Test Substance Test Analytical Difference Between No pH Time Repf Substance Rreplication Average Analytical Replications^ day UgL-' ugL-' % 1 1.2 0 1 136 137 1.2 2 1.2 0 1 138 3 1.2 0 2 117 119 2.0 4 1.2 0 2 120 5 1.2 0 3 157 150 8.7 6 1.2 0 3 144 7 1.2 0 3 152 1.6 8 1.2 0 3 149 1.2 4 114 110 8.8 1.2 4 105 Average 129 Standard Deviation 18.4 9 1.2 5 1 143 141 3.8 10 1.2 5 1 138 11 1.2 5 2 140 134 9.0 12 1.2 5 2 128 13 1.2 5 3 102 102 NA 14 1.2 5 4 165 164 0.8 15 1.2 5 4 163 Average 135 Standard Deviation 25.6 f Test was performed at 50C instead of37C. Experiment was performed using pH 1.2 buffer at 50C. Test vessels were 7 mL glass scintillation vials with aluminum-lined screw caps. 22.5 uL of a 20 mg 8-2 TBA L"' methanol solution was added to each test vessel with 3 mL of the pH 1.2 buffer solution. After capping, the samples were vortex-mixed for 10 s. Samples were frozen until analyzed. $ Day 0, replicate 3 measured four times; Day 5 replicate 3 measured once. ^ Calculated by dividing the absolute difference of the two analytical replications by their average value, times 100. NA = Not applicable; only one analytical measurement of sample. T0106/4842 Page 28 of 31 DuPont EMSE Report No. 92-02 TABLE B-3 ANALYTICAL RESULTS OF THE AQUEOUS STABILITY OF 8-2 TBA, PH 7.0, 50C, DAYS 0 (31-JUL-2002) AND 5 (5-AUG-2002)+ Test Substance Test Analytical Replication Difference Between No PH Time Rep Substance Average Analytical Replications:): day ugL-' ugL-' % 1 7 0 1 103 104 1.6 2 7 0 1 104 3 7 0 2 114 111 5.7 4 7 0 2 108 5 7 0 3 98.0 95.7 4.9 6 7 0 3 93.3 7 7 0 4 102 102 1.3 8 7 0 4 101 Average Standard Deviation 103 6.25 58.7 57.2 5.3 10 55.7 11 24.6 24.6 0.3 12 7 5 13 7 5 24.7 NDH 14 7 5 15 7 5 96.7 91.7 11 16 7 5 86.7 Average 43.4 Standard Deviation 39.8 t Test was repeated because of the variation in replicate results of test substance concentration at Day 5. Experiment was performed using pH 7 buffer at 50C. Test vessels were 7 mL glass scintillation vials with aluminum-lined screw caps. 22.5 uL of a 20 mg 8-2 TBA L'1 methanol solution was added to each test vessel with 3 mL of the pH 1.2 buffer solution. After capping, the samples were vortex-mixed for 10s. Samples were frozen until analyzed. f Calculated by dividing the absolute difference of the two analytical replications by their average value, times 100. If ND = Not determined; samples were lost prior to analysis T0106/4842 Page 29 of 31 ^WSil^'SSSSlSSSlWiSSlSiS DuPont EMSE Report No. 92-02 TABLE B-4 ANALYTICAL RESULTS OF THE AQUEOUS STABILITY OF 8-2 TBA, PH 9.0, 50C, DAYS 0 (31-JUL-2002) AND 5 (5-AUG-2002)t Test Substance Test Analytical Replication Difference Between No PH Time Rep Substance Average Analytical Replications^ day ugL-' ugL-' % 1 9 0 1 162 162 0.6 2 9 0 1 163 3 9 0 2 149 142 9.6 4 9 0 2 135 5 9 0 3 153 150 4.4 6 9 0 3 147 7 9 0 4 132 136 5.2 8 9 0 4 139 Average 147 Standard Deviation 11.5 9 9 5 1 150 143 9.8 10 9 5 1 136 11 9 5 2 71.3 80.8 24 12 9 5 2 90.3 13 9 5 3 59.7 59.0 2.3 14 9 5 3 58.3 15 9 5 4 131 142 15 16 9 5 4 152 Average 106 Standard Deviation 42.9 t Test was repeated because of the variation in replicate results of test substance concentration at Day 5. Experiment was performed using pH 9 buffer at 50C. Test vessels were 7 mL glass scintillation vials with aluminum-lined screw caps. 22.5 uL of a 20 mg 8-2 TBA L'1 methanol solution was added to each test vessel with 3 mL of the pH 1.2 buffer solution. After capping, the samples were vortex-mixed for 10 s. Samples were frozen until analyzed. { Calculated by dividing the absolute difference of the two analytical replications by their average value, times 100. TO 106/4842 Page 30 of 31 DuPont EMSE Report No. 92-02 APPENDIX C CERTIFICATE OF TEST SUBSTANCE ANALYSIS Olfjani SrnbH. Wctt Gcndorf CQG / Qualitiuniuiagenleiu M50 Bilftkitdien Fu: <-1-49 867S/2676 X^ dl ^ <S^ (V"-1I (,I,yH- -TitTtL- aIcncosrdpingec;otiEoN1n0204-c3.1eB rtificate Oate: 13.11.2001 Page: 1 / I 3^r .onsi.9."i'ient Material "aterial-no. Sdt;.i No. : 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10, IQ-heptddfcaflaoro : 104705 : ?Ou/n'Jl Ci; ^-. bd*:-:.', or whi:" ;-.^ rona i a";renc i.5 a pact, the following values ^{st.arir.ir.eu. ri-.ey conx^x.-n ':.'j -.-.^ aqeeed product specification. ;"s?"cti^". c;.3r3"":.--i;:.=t.i';/ -methcrtSpeCLSicaEiori Rejult ;3-?er;*^--a-:<,l.a--;-.ar.cl ^api-i-aLy ^C S-a Parf-uocoalkyiethanol ^splli.acy GC Su.-n Pacf--ocallcylbutanoi CaCL'L.ar'/ 3C S^n ?er:-uor5^itylethene Sapiliary GC Mater ccr.te-it Xsri Fi.s:her 3:N 51777 M-y.ef-.y-Dyrtoi.laci-.e content CapiLlary 3C >- 97,S >- 98,0 <-1,0 <"0,5 <-0,2 <-0,Z 99,Jl!a/a] 99,9ta/al < 0,lla/a] < 0,l%(a/ai < 0,1* < 0,11 ^?:e ascve particuLars co not release the custonier froiti the obLigation to sarry SL;': ar. --.spectio'"- or ^cods received. y.s. sa'-T.c-irmar - Woricsinspector T0106/4842 Page 31 of 31