Document jBJRbpLDN37wGy4ejZejxEYdQ

AR226-2691 13 AR226-2691 DuPont EMSE Report No. 15-03 Study Title Accelerated Biodegradation of 8-2 Telomer B Alcohol- A preliminary Screening Study j I Test Guideline This study uses the concept o f OECD Guideline for Testing o f Chepiicals; Section 3: Inherent Biodegradability (1992). Author Ning Wang, Ph.D. Study Completion Date 20 March 2003 Test Facilities E.I. du Pont de Nemours and Company Central Research & Development Corporate Center for Engineering Research Environmental and M icrobiological Sciences & Engineering Glasgow Building 300, P.O. Box 6101 Newark, DE 19714-6101, USA and E.I. du Pont de Nemours and Company Haskell Laboratoiy for Health and Environmental Sciences Newark, DE 19714, USA Submitter E.I. du Pont de Nemours and Company DuPont Chemical Solutions Enterprise W ilmington, DE 19898, USA EMSE Study /Project Number 15-03/4842 Report Number EM SER15-03 EMSER15-03/4842 Page 1 o f 47 DuPont EMSE Report No. 15-03 Page Reserved for Specific Country Requirements EMSERl 5-03/4842 Page 2 o f 47 DuPont EMSE Report No. 15-03 Certification of Authenticity Accelerated Biodegradation of 8-2 Telomer B Alcohol - A PRELIMINARY SCREENING STUDY * We, the undersigned, declare that the work described in this report was performed under our supervision, and that this report provides an accurate record o f the procedures and results. Report by: Ning Wang, Ph.D. Senior Research Biologist Approved by: Date Study Initiation Date: 14-July-2002 Date Study Completed: 20-M arch-2003 Submitter: E.I. du Pont de Nemours and Company DuPont Chemical Solutions Enterprise Wilmington, DE 19898, USA EMSER15-03/4842 Page 3 o f 47 DuPont EMSE Report No. 15-03 Table of Contents Page Reserved for Specific Country Requirements.................................................................................2 Certification o f Authenticity.......................................................................................................................... 3 Table o f Contents..............................................................................................................................................4 1.0 Summary.................................................................................................................................................. 6 2.0 General Study Information................................................................................................................... 7 3.0 Materials and M ethods.........................................................................................................................8 3.1 Test System.......................................................................................................................................... 8 3.2 Test Conduct......................................................................................................................................10 3.3 Sample Extraction and Analysis.................................................................................................... 12 3.3.1 Sample Collection and Extraction...................................................................................... 12 3.3.2 Analytical Methods for Test Substance and Products................................................... 13 4.0 Results and D iscussion........................................................................................................................ 17 5.0 Conclusions...........................................................................................................................................18 6.0 Reference................................................................................ 18 Figure 1 8-2 Telomer B Alcohol (8-2 TBA) concentration during the 28-day test*.....................19 Figure 2 Fluoride concentration during the 28-day test*.....................................................................20 Table 1: Estimated Concentrations! o f fluorinated acid metabolites at day 0 (19-Jul- 2002) and day 28 (16-Aug-2002).....................................................................................................21 Table 2: Daily temperature readings in the lab where the test was conducted. The temperature was recorded by a calibrated Dickson Recorder (Model THDx, serial # 0 1 1 8 2 4 7 ) .................................................................................................................................................22 Appendix A :.................................................................................................................................................... 23 Table A -l. Analytical results o f 8-2 TBA concentration at day 0 (19-M -2002), day 14 (2-Aug-2002), and day 28 (16-Aug-20Q2)..................................................................................... 24 Table A -l (continued)....... ......................... 25 Table A -l (continued)................................................................................................................................... 26 Table A -2. Analytical results o f Spike recovery o f 8-2 TBA from the sample matrix (Treatment 3, adapted bacterial culture plus growth medium in coated glass serum bottles) at day 0 (19-Jul-2002), day 14 (2-Aug-2002), and day 28 (16-A ug-2002)..............27 Table A -3. Analytical results o f fluoride concentration at day 0 (19-Jul-2002), day 14 (2Aug-2002), and day 28 (16-Aug-2002).......................... 28 Table A-3 (continued)...................................................................................................................................29 Table A-4. Analytical results o f fluorinated acid metabolite 2-perfluorooctyl ethanoic acid (2-PFOEA) at day 0 (19-Jul-2002), and day 28 (16-A ug-2002)....................................... 30 Table A-5. Analytical results o f fluorinated acid metabolite 2H-hexadecafluoro-2decenoic acid ( 2H-HDF-2-DA) at day 0 (19-Jul-2002), and day 28 (16-A ug-2002)......... 31 Table A-6. Analytical results o f fluorinated acid metabolite perfluorooctanoic acid (PFOA) at day 0 (19-M-2Q2), and day 28 (16-A ug-2002)......................................................32 Table A -7. Analytical results o f fluorinated acid metabolite perfluorohexanoic acid (PFHA) at day 0 (19-Jul-2002), and day 28 (16-A ug-2002)...................................................... 33 EMSER15-03/4842 Page 4 o f 47 DuPont EMSE Report No. 15-03 Table A -8. Components o f bacterial growth medium used for the test............................................. 34 Figure A -l A fluoride standard calibration curve................................................................................. 35 Figure A -2 A calibration curve used for 8-2 TBA quantification o f samples TA 50-1 to TA 5 0 -2 6 ...................... 36 Figure A -3 A chromatograph o f sample TA50-3 used for 8-2 TBA analysis................................ 37 Figure A -4 A calibration curve used for 8-2 TBA quantification o f samples TA 51-1 to T A 51-17 and T A 52-1 to T A 52-17.................................................................................................. 38 Figure A-5 A chromatograph o f sample TA 52-3 used for 8-2 TBA analysis...............................39 Figure A -6 Chromatographs o f samples TA52-3 and TA52-6, 30 pg L'1o f standard acid m ix made in sample matrix TA50-15, sample matrix TA52-16, and a methanol solvent blank.......................................................................................................................................... 40 Figure A -6 Chromatographs o f samples TA52-3 and TA52-6, 30 pg L'1o f standard acid m ix made in sample matrix TA50-15, sample matrix TA52-16, and a methanol solvent blank - Continued from page 42.........................................................................................41 Figure A -7 A GC/MS chromatograph o f material characterization o f 8-2 TBA test substance used in this study...............................................................................................................42 Appendix B :.................................................................................................................................................... 43 Table B - l . Analytical results o f spike recovery o f 2-perfluorooctyl ethanoic acid (2PFOEA) at day 0 (17-Sep-2002), and day 2 (19-Sep-2002)..................................... ..................44 Table B-2. Analytical results o f spike recovery o f 2H-hexadecafluoro-2-decenoic acid ( 2H-HDF-2-DA) at day 0 (17-Sep-2002), and day 2 (19-Sep-2002)........................ a:............. 45 Table B-3. Analytical results o f spike recovery o f perfluorooctanoic acid (BFOA) at day 0 (17-Sep-20O2), and day 2 (19-Sep-2Q02)........................................... .in.-../"..:..........................46 Table B-4. Analytical results o f spike recovery o f perfluorohexanoic acid (PFHA) at-day 0 (17-Sep-2002), and day 2 (19-Sep-2002).......................................................................jshx....... 47 EMSER15-03/4842 Page 5 o f 47 DuPont EMSE Report No. 15-03 Accelerated Biodegradation of 8-2 Telomer B Alcohol- A PRELIMINARY SCREENING STUDY Author Ning Wang, Ph.D. 1.0 Summary Rationale o f the Study This test generates environmental fate information potentially relevant to the persistence o f the test substance. When a chemical enters the environment, biodegradation is one o f the major routes that determines the environmental fate o f the chemical. The biodegradation o f 8-2 Telomer B Alcohol (8-2 TBA, CAS# 678-39-7) may be slow, because only 2 hydrocarbons o f the molecule may be readily accessible for microbial metabolism, the rest o f the 8 perfluorinated carbons are expected to be difficult to be metabolized by microorganisms. Hypothetically, microorganisms that have been pre-exposed to the test chemical may adapt to have a higher metabolic capacity to transform and potentially defluorinate 8-2 TBA under favorable growth conditions. A test with pre-adapted bacterial culture under favorable growth conditions may give a clearer indication o f the biotransformation potential for a given test chemical. Such a test may provide optimal conditions for potential microbial metabolism o f 8-2 TBA. For example, if a chemicahis not metabolized under such optimized conditions, it is less likely to be metabolized/biodegraded in the environm e|i| J.The test system is also useful to rapidly identify p o te n tia l -itl. 'i he biotransformation products and this w ill enable use o f authentic standards to facilitateocl identification and quantification o f transformation products in the subsequent definitive' ! biodegradability studies. Test System: The primary biotransformation potential o f the test substance 8-2 TBA in bacterial growth medium plus bacterial culture that has pre-grown in 50-100 mg/L o f 8-2 TBA was determined. The bacterial culture originated from an industrial waste water treatment facility. The test system consisted o f individually crimped test vessels (glass serum bottles). The test was conducted at room temperature (~24C). The 8-2 TBA stock solution (made in ethanol) was introduced into the bacterial growth medium (Table A -8) with the bacterial inoculum and was kept in closed bottles in the dark at room temperature. Periodically (days 0 ,1 4 , and 28), sample bottles were sacrificed for extraction and analysis o f the test chemical, fluorinated acid metabolites, and fluoride (F- ion). Findings: The primary biotransformation o f 8-2 TBA was rapid (>70% at day 14 and near 100% at day 28) compared with the abiotic control. Significant defluorination o f 8-2 TBA was observed during the test. Four o f the potential biotransformation products were monitored for and were identified through tandem mass spectrometry analysis and comparison with analytical standards: 1) 2-perfluorooctyl ethanoic acid (2-PFOEA, F(CF2 )8CH2COOH, CAS# 27854-31-5); 2) 2H-hexadecafluoro-2-decenoic acid (2H-HDF-2-DA, F(CF2)7CF=CHCOOH, CAS# 161094-76-4); 3) perfluorooctanoic acid (PFOA, EMSER15-03/4842 Page 6 o f 47 ai h no: mmHtA' . '\ ) {K-*! mum am Maft' DuPont EMSE Report No. 15-03 F(CF2)7COOH, CAS# 335-67-1); and 4) perfluorohexanoic acid (PFHA, F(CF2)5COOH, CAS# 307-24-4). Other potential transformation products that formed during the test were not determined. Conclusions: Under the test conditions, 8-2 TBA is being rapidly transformed to fluorinated acids and other unidentified transformation products. Although PFOA is one o f the identified metabolites, it accounted for <2% o f mass balance. PFOA apparently may be further metabolized to form PFHA and may not be an ultimate (stable) metabolite under the test conditions. 2.0 General Study Information w Study Objectives Determine the biotransformation potential o f 8-2 TBA by monitoring its concentration changes during a biodegradation test with optimized conditions Determine the degree o f defluorination o f 8-2 TBA Identify potential metabolites such as fluorinated acids during the test A ssess whether perfluorinated acids can be further metabolized Determine possible biodegradation pathways o f 8-2 TBA Confirm expectation th |t mass balance w ill be difficult to achieve in cold studies due to potential abiotic remdval mechanisms - volatility and adsorption. ' Test System Justification The test system is modified from OECD 302 guidelines and was requested by the submitter. Study Personnel E.I. du Pont de Nemours and Company Central Research & Development - Corporate Center for Engineering Research Environmental and M icrobiological Sciences & Engineering Haskell Laboratory for Health and Environmental Sciences Management: John T. Gannon, Ph.D. E.I. du Pont de Nemours and Company Central Research and Development Corporate Center for Engineering Research Environmental and M icrobiological Sciences & Engineering Glasgow Building 300, P.O. Box 6101 Newark, DE 19714-6101, USA and S. Mark Kennedy, Ph.D. Haskell Laboratory for Health and Environmental Sciences Newark, DE 19714, USA EMSER15-03/4842 Page 7 o f 47 DuPont EMSE Report No. 15-03 Study Director: Analytical Chemists: Technical Personnel: Ning Wang, Ph.D. E.I. du Pont de Nemours and Company Central Research and Development Corporate Center for Engineering Research Environmental and M icrobiological Sciences & Engineering Glasgow Building 300, P.O. Box 6101 Newark, DE 19714-6101, USA Bogdan Szostek, Ph.D. E.I. du Pont de Nemours and Company Haskell Laboratory for Health and Environmental Sciences Newark, DE USA 19714, USA Vladimir Capka, Ph.D. E.I. du Pont de Nemours and Company Haskell Laboratory for Health and Environmental Sciences Newark, DE USA 19714, USA Patrick W. Folsom, DuPont Central Research & Development Keith B. Prickett, DuPont Haskell Laboratory Richard F. Rossi, DuPont Haskell Laboratory Study Execution Dates Experimental Start Date: Experimental Completion Date: Study Completion Date: 19-M -2002 19-Sep-2002 20-M arch-2003 3.0 Materials and Methods 3.1 3.1.1 Test System Test Substance Name: Synonym: Active substance(s) CAS Name: Molecular weight CAS Number(s): 8-2 Telomer B Alcohol (Perfluorooctyl)ethanol, 8-2 TBA 8-2 Telomer B A lcohol, 99% 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10heptadecafluoro464.12 g mole"1 678-39-7 EMSER15-03/4842 Page 8 o f 47 Structure: DuPont EMSE Report No. 15-03 F OH 3.1.2 3.1.3 3.1.4 Lot Number: EMSE Sample Number: Concentration o f a.s., nominal: Concentration o f a.s., analyzed: Major impurity Certificate o f Analysis Date: Date Received: Solubility at 25C.: Vapor pressure: Stability: Appearance/Color: Storage Conditions: Safety Precautions: P.00/001 E 9 3 386-80 99% 99.2% 0.8% as C7F15CF=CHCH2OH (Figure A -7) 13-Sept-2001 26-M ar-2002 -1 4 0 pg 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 Preparation o fBacterial Growth Medium The 20% yeast extract and different mineral stock solutions were prepared and were autoclaved. The bacterial growth medium was prepared by dilution o f the different stock solutions (see Table A-8 for details o f the medium components). The growth medium was sterile filtered into Nalgene 1 liter filter units and was stored at room temperature. Test vessel Coating with 8-2 TBA solution On 15-Jul-2002, Glass serum bottles (120 mL volume) were filled with 100 mL o f sterile water and 10 pL o f 8-2 TBA stock solution (3 mg/mL in ethanol) was injected into each o f the bottles for a final concentration o f 300 pg/L. The bottles were sealed and incubated for 3 days at room temperature with about 250 RPM shaking. The bottles were rinsed with sterile deionized water and capped for the test the next day. EMSER15-03/4842 Page 9 o f 47 DuPont EMSE Report No. 15-03 3.1.5 3.1.6 3.1.7 3.2 Adapted bacterial culture preparation Approximately 500 mL o f industrial waste sludge from an industrial waste water treatment facility was collected from aeriation tanks on 13-June-2002 and were sent to the test lab. Upon arriving at the test lab the same day, the sludge was assigned an ID number E93386-87. The sludge was mixed by briefly shaking the jug to suspend the microorganisms. After settling the sludge for approximately 15 min to remove coarse matters, about 2 mL aliquot o f the sludge was transferred to a 250 mL sterile cell culture flask that contained about 50 mg/L o f 8-2 TBA in 100 mL bacterial growth medium. The bacterial culture was maintained at room temperature by periodically transferring about 0.5 mL o f the culture into another 50 mL polypropylene tube that contained about 5 mL o f the bacterial growth medium plus 50-100 mg/L o f 8-2 TBA. The culture was mixed by constant shaking at about 210 RPM. The day before the initiation o f the test (18-M -2002), 0.5 mL o f the bacterial culture was transferred into a 250 mL flask that already contained 100 mL o f the bacterial growth medium plus 600 pg/L o f 8-2 TBA and incubated overnight at room temperature. The cell culture was centrifuged and the pellet was redissolved in the growth medium after discarding the supernatant. This wash step was repeated once. The washed bacterial culture w ill serve as inoculum for the test. Ten mL o f the washed bacterial culture was transferred with a plastic pipette to a 20 mL scintillation vila and was autoclaved. The autoclaved culture was referred to as killed bacterial culture and w ill be used for abiotic control treatment. Activated Sludge Collection Approximately 4 liters o f activated sludge was collected from the City o f Wilmington (DE) Municipal W aste Treatment Facility (POTW) - Aeriation Basin # 2 on 16 September 2002. Upon arriving at the test lab, the sludge was assigned an ID number E93386-100. The sludge was mixed by briefly shaking the jug to suspend the microorganisms. After settling the sludge for approximately 15 min to remove coarse matters, the upper aqueous phase o f the sludge was filtered through a nylon net with a pore size o f 85 pm. The filtrate was inoculated into the bacterial growth medium and incubated overnight. This bacterial culture was used as inoculum in a separate experiment to conduct spike recovery o f four specific fluorinated acid standards, which were quantified in the study. These spike recoveries are assumed to be also representative o f fluorinated acids recovery from the adapted baterial culture. Test Units Test vessels were 120 mL borosilicate glass serum bottles with pre-cleaned aluminum-lined crimp caps. The pre-cleaning was done by rinsing the aluminum foil and septa with methanol once and then with sterile deionized water three times. Test Conduct Five types o f experimental treatments were conducted. The pre-cleaning was done by rinsing the aluminum foil and septa with methanol once and then with sterile deionized water three tim es. EMSER15-03/4842 Page 10 o f 47 DuPont EMSE Report No. 15-03 3.2.1 3.2.2 3.2.3 3.2.4 3.2.5 3.2.6 Treatment 1 - 600 uz/L o f8-2 TBA in Bacterial Growth Medium plus the Bacterial Culture Inoculum in Coated Bottles For a total o f 15 coated glass serum bottles (5 replicates * 3 sampling time points), 29.34 mL o f o f the growth medium, 0.663 mL o f the washed bacterial culture, and 6 pL o f 8-2 TBA stock solution (3 mg/mL in ethanol) were added to each o f the glass serum bottles. The bottles were crimped with pre-cleaned aluminum foil and PTFE/silicone septa. Treatment 2 - 600 us/L o f8-2 TBA in Bacterial Growth Medium plus Killed Bacterial Culture in Coated Bottles For a total o f 9 coated glass serum bottles (3 replicates x 3 sampling time points), 29.04 mL o f the growth medium, 0.663 mL o f the killed bacterial culture, 0.3 mL o f 0.2 M NaCN, and 6 pL o f 8-2 TBA stock solution (3 mg/mL in ethanol) were added to each o f the glass serum bottles. The bottles were crimped with pre-cleaned aluminum foil and PTFE/silicone septa. This treatment served as an abiotic control. Treatment 3 - Growth Medium plus Bacterial Culture Inoculum in Coated Bottles for 8-2 TBA Spike Recovery ^ For a total o f 9 coated glass serum bottles (3 replicates * 3 sampling time points), 29.34 mL o f the growth medium and 0.663 mL o f the washed bacterial culture was added to each o f the glass serum bottles. The bottles were crimped with pre-cleaned aluminum foil and PTFE/silicone septa. At each sampling time points (Day 0, 14, and 28), 3 bottles were spiked (dosed) with 6 pL o f 8-2 TBA stock solution (3 mg/mL in ethanol) for a final concentration o f 600 pg/L. The ; bottles were shaken at 200-300 RPM for approximately 30 min before sample collection 5tM*f* sample extraction. Treatment 4 --Growth Medium plus Bacterial Culture Inoculum in Coated Bottles For a total o f 9 coated glass serum bottles (3 replicates x 3 sampling time points), 29.34 mL o f the growth medium and 0.663 mL o f the washed bacterial culture was added to each o f the glass serum bottles. The bottles were crimped with pre-cleaned aluminum foil and PTFE/silicone septa. This treatment was used to provide an indication whether 8-2 TBA that has been adsorbed to the glass wall o f the test vessels was available for biotransformation. Treatment 5 - Growth Medium plus Bacterial Culture Inoculum in non-Coated Bottles For a total o f 9 non-coated p lf s serum bottles (3 replicates x 3 sampling time points), 29.34 mL o f the growth medium and 0.663 mL o f the washed bacterial culture was added to each o f the glass serum bottles. The bottles were crimped with pre-cleaned aluminum foil and PTFE/silicone septa. This treatment w ill serve as sample matrix control for quantification o f fluorinated acid metabolites by LC/MS/MS. Spike Recovery o fFluorinated Acids from Sample Matrix On 17 September 2002, a separate experiment was conducted to determine the spike recovery o f fluorinated acids from bacterial culture sample matrix. Each o f the sample bottles was filled with 30 mL o f bacterial growth medium plus the bacterial inoculum that had been prepared the day before (Section 3.1.6). For day 2 samples, the bottles were crimped with pre-cleaned aluminum foil and PTFE/silicone septa and were kept shaken at EMSER15-03/4842 Page 11 o f 47 DuPont EMSE Report No. 15-03 -2 5 0 RPM at room temperature to allow the bacteria to grow. For day 0 samples, 3 bottles were spiked with fluorinated acid m ix solution (made from individual stock solution in ethanol) to a final concentration o f 200 pg/L for each acid. The four acids used are: 1) 2perfluorooctyl ethanoic acid (2-PFOEA; CAS# 27854-31-5, DuPont,); 2) 2H-hexadecafluoro2-decenoic acid (2H-HDF-2-DA; CAS# 161094-76-4, DuPont,); 3) perfluorooctanoic acid (PFOA; CAS# 335-67-1,97% , Oakwood Products); and 4) perfluorohexanoic acid (PFHA; CAS # 307-24-4, +98%, TCI America). Another 3 bottles were not spiked to serve as sample matrix control. The bottles were shaken at 200-300 RPM for approximately 30 min before sample collection and sample extraction. At day 2 sampling time point (19 September 2002), 3 crimped bottles were spiked with fluorinated acid mix solution (made from individual stock solution in ethanol) to a final concentration o f 200 pg/L for each acid. Another 3 bottles were not spiked to serve as sample matrix control. The bottles were shaken at 200-300 RPM for approximately 30 min before sample collection and sample extraction. 3.2.7 3.2.7.1 Test Conditions and Sampling Sample Incubation The crimped glass serum bottles were incubated with 200-300 RPM o f shaking at room temperature in the dark. The room temperature o f the test lab was monitored and recorded throughout the course o f the study. 3.2.7.2 Sampling Interval 5 crimped serum bottles from Treatment 1 and 3 bottles from the rest o f treatments were sampled for extraction o f 8-2 TBA, fluoride, and fluorinated acids at day 0 (19-Jul-2002), day 14 (2-A ug-2002), and day 28 (16-Aug-2002). 3.2.7.3 Sample Storage Analytical samples were stored at -1 0 to -20C . 3.3 3.3.1 3.3.1.1 3.3.1.2 Sample Extraction andAnalysis Sample Collection and Extraction Sample Collection At days 0 ,1 4 , and 28, crimped sample bottles were removed from the shaker and the bottles were turned upside down. A total o f 10 mL o f the test medium from each o f the bottles were withdrawn with a 10-mL polypropylene syringe and was injected into a 15 mL polypropylene tube that contained 100 pL o f 5 N sodium hydroxide for fluoride extraction. Sample Extraction Fluoride extraction: The 15 mL polypropylene tubes containing the 10 mL test medium plus NaOH were incubated at room temperature for 3 - 4 h with 250 - 300 RPM o f shaking. Then 83.3 pL o f 6N H2S 0 4 was added to each o f the tubes to neutralize the test medium. The sample tubes were stored at approximately -20C for fluoride analysis. 8-2 TBA andfluorinated acid extraction: After withdrawing a 10 mL o f test medium for fluoride analysis, 0.34 mL o f 6 N H2S 0 4 and 30 mL o f chilled MTBE were injected into the sample bottles. After MTBE and H2S 0 4 were injected, the sample bottles were shaken (250 - 300 RPM) at room temperature for approximately 2 h. After settling the MTBE phase, EMSER15-03/4842 Page 12 o f 47 ifl i e 1111 DuPont EMSE Report No. 15-03 3.3.1.3 the crimped sample bottles were decapped and the MTBE phase from each o f the sample bottles was transferred to 50 mL polypropylene centrifuge tubes with a glass transfer pipette. The MTBE phase was centrifuged at approximately 2000 RPM for 10 min. A 20 mL aliquot o f the MTBE phase from each o f the centrifuge tubes was transferred with a glass pipette to a 20 mL glass scintillation vial with a foil-lined cap and the scintillation vials were stored at -- 20C. From each o f the glass scintillation vials, 2 mL aliquot o f the MTBE phase was transferred with a glass pipette to a GC vial and was sealed with aluminum-lined crimp cap for GC/MS quantification o f 8-2 TBA. From each o f the glass scintillation vials, 6 mL aliquot o f the MTBE phase was sequentially dried in a GC vial under N2 and was redissolved in 1.5 mL o f methanol for fluorinated acid analysis. Temperature Measurements The temperature o f the test system was monitored and recorded throughout the course o f the study. 3.3.2 3.3.2.1 Analvtical Methods for Test Substance and Products Analysis o f Test Substance 8-2 TBA Analysis: Analytical standards: The 1H,1H, 2H, 2H-perfluorodecan-l-ol (8-2 TBA, CAS# 678-39-7, 97.6%, Oakwood Products, W est Columbia, SC) was used as the analytical standard. The 1H, 1H, 2H, 2H-perfluoro-9-methyldecan-l-ol (C -l 1 Iso; CAS# 31200-98-3,98% , Oakwood Products,) was used as an internal standard for day 0 samples and the ID, ID , 2D, 2D, 3 -13Cheptadecafluoro decanol (M +5) (D -8-2 TBA, DuPont,) was used as the internal standard for day 14 and day 28 sample analysis. Stock solutions (1000 mg/L) o f the analytical standard and the internal standards were prepared in methanol and refrigerated. The calibration standards were freshly prepared for each calibration in MTBE by dilution o f the freshly made 50-mg/L stocks in methanol. Typically, the calibration standards were made in the range o f 25-1000 pg/L o f 8-2 TBA. Constant level o f internal standard was used: approximately 300 pg/L o f C -l 1 Iso for day 0 samples and D-8-2 TBA for day 14 and day 28 samples. For day 0 samples, the calibration curves were constructed using the ratio o f the peak area for ion m/z 95 (8-2 TBA) and peak area for ion m /z 95 (C -l 1 Iso) and the ratio o f the concentrations o f 8-2 TBA and internal standard. An example o f a calibration curve was given in Figure A-2 for quantification o f samples TA50-1 to TA 50-26. A chromatograph o f sample separation (Sample TA50-3) was given in Fugure A -3. For day 14 and day 28 samples, the calibration curves were constructed using the ratio o f the peak area for ion m /z 31 (8-2 TBA) and peak area for ion m /z 33 (D-8-2 TBA) and the ratio o f the concentrations o f 8-2 TBA and the internal standard. An example o f a calibration curve was given in Figure A -4 for quantification o f samples TA51-1 to TA 51-17 and TA52-1 to TA 52-17. A chromatograph o f sample separation (Sample TA52-3) was given in Fugure A-5. Quantification o f 8-2 TBA: A 0.5 mL aliquot o f the MTBE phase from the GC vials {Section 3.3.1.2) was placed in a glass GC vial (1.7 mL volume), 3 - 6 pL o f 50 pg/mL o f D-8-2 TBA internal standard was added to the vial using a GC syringe, the vial was capped and subjected to analysis. Each sample was analyzed twice by a GC/MS instrument according to the following conditions: EMSER15-03/4842 Page 13 o f 47 DuPont EMSE Report No. 15-03 GC/MS system: HP 6890 Plus GC (Agilent), HP 5973 Mass Selective Detector (A gilent), MPS2-MultiPurposeSampler (Gerstal) Column: DB-5M S, 30 m x 0.25 mm, 1 pm film (Agilent) Temp, ramp: Initial: 80C for 2 min 20C/min to 120C 50C/min to 300C and hold for 3 min Flow rate: 1.0 mL/min; He; constant flow mode Split: 5:1 Inlet temp.: 250C Injection volume: 2 pL MSD transfer line temp.: 280C Ionization: El, 70 eV SIM ions monitored: m/z: 31, 95, 131; dwell time: 50 ms for each ion (day 0 samples) m/z: 3 1 ,3 3 ,9 5 ,9 8 ; dwell time: 25 ms for each ion (days 14 and 28 samples) Retention time: 8-2 Telomer B Alcohol (C8-2A): 4.97 min Internal standard (D -8-2 TBA): 4.93 - 4.95 min Internal Standard (C -l 1 Iso) 5.42 min Fluorinated acid Analysis: Analytical standards: The perfluorohexanoic acid (PFHA; CAS # 307-24-4, +98%, TCI America), perfluorooctanoic acid (PFOA; CAS# 335-67-1,97% , Oakwood Products), 2Hhexadecafluoro-2-decenoic acid (2H-HDF-2-DA; CAS# 161094-76-4, DuPont), and 2perfluorooctyl ethanoic acid (2-PFOEA; CAS# 27854-31-5, DuPont) were used as standards for analysis o f fluorinated acids in test samples. The analytical standard stock solutions for each o f the above acids were prepared at approx. 200 pg/mL concentration by dissolving appropriate amount o f the acids in methanol. A single, mixed analytical stock solution at approx. 200 pg/L for each acid was prepared by dilution o f each o f the above primary stock solutions w ith methanol in a single volumetric flask. An aliquot o f the mixed acid stock solution (-2 0 0 pg/L) and an aliquot o f a negative control sample matrix (Treatment 5) were diluted together in a single vial to produce approx. 30 pg/L concentration o f each acid in lOx diluted sample matrix in methanol. A separate 30 pg/L solution was prepared with a day 0 negative control (Sample TA 50-15) and a day 28 negative control (Sample TA 52-15). These two solutions were designated as 30 pg/L standards and a pure methanol was designated as a 0 pg/L standard. For fluorinated acid spike recovery experiment (Section 3.2.6), a calibration standard curve was constructed in the range o f 10 - 50 pg/L for quantification o f individual fluorinated acid in the acid-spiked samples. EMSER15-03/4842 Page 14 o f 47 DuPont EMSE Report No. 15-03 Sample Analysis: Aliquots o f all samples were diluted lOx with methanol before the analysis. In addition, aliquots o f all samples were transferred to HPLC vials for direct analysis without dilution. A ll lOx diluted and all undiluted samples were analyzed by liquid chromatography-tandem mass spectrometry (LC/MS/MS). A set o f standards (see above) was run at the beginning and at the end o f the analysis sequence. Quantitation was done using a single two-point calibration curve for each acid constructed using standards from the beginning and the end o f the analysis sequence. For maximal accuracy o f results, the results for undiluted samples for PFHA, PFOA, and 2-PFOEA were considered final. For 2H-HDF-2-DA, the results from lOx diluted samples were considered final. This was done to ensure that the peak area data values o f analytes in samples were as close as possible to those o f 30 pg/L standards. For fluorinated acid spike recovery experiment (Section 3.2.6), a calibration standard curve was constructed in the range o f 10 - 50 pg/L for quantification o f individual fluorinated acid in the acid-spiked samples. Chromatographs o f samples TA52-3 and T A 52-6,, 30 pg/L o f standard acid mix made in sample matrix TA50-15, sample matrix TA52-16, and a methanol solvent blank were given in Figure A -6. Each sample was analyzed tw ice according to the following conditions: HPLC instrument: Waters 2795 HPLC MS instrument: Micromass Quattro Micro HPLC Column: Phenomenex Luna C l8 (2), 20 mm x 2.0 mm, 3 pm particle size Mobile Phase: Mobile Phase Gradient: A: 2 mM ammonium acetate in deionized water (18.2 MG cm) C: methanol purge solvent: deionized water (18.2 M il cm) needle wash solvent: isopropanol Time (min) Flow Rate % A %C (mL/rr 0 90 10 0.3 0.5 90 10 0.3 1.5 0 100 0.3 4.5 0 100 0.3 4.6 90 10 0.4 6.5 90 10 0.3 Flow Direction Switching: O tol.O m in - flow to waste to 4.5 min flow to MS 4.5 to 6.5 min - flow to waste Column Temperature: 35C Injection Volume: 10 pL Ionization mode: electrospray ionization in negative mode Capillary voltage: 3.50 kV EMSER15-03/4842 Page 15 o f 47 DuPont EMSE Report No. 15-03 Ion source temperature: Desolvation temperature: Desolvation Gas Flow: Cone Gas Flow: Quadrupole Resolution: Collision Gas, Pressure: Data acquisition mode: PFHA PFOA 2H -H D F -2-D A 2-PFOEA 140C 350C N 2, 500 1/hr N2, 1 0 0 1/hr Q1 - low and high mass resolution 10 Q2 - low and high mass resolution 15 argon, 3.5 x 10'3 mbar multiple reaction monitoring (0-4.5 min), inter-channel delay 0.05 sec Reaction Dwell Time (sec) Cone Voltage (V ) C ollision Energy (eV) 313 > 269 0.1 15 15 413 > 369 0.1 15 15 457 > 393 0.1 15 15 477 > 393 0.1 20 20 Fluoride Analysis: Analytical standard: Certified standard o f 100 mg/L o f fluoride in water (Thermo Orion ) was used for standard calibration. The calibration standards normally were made in the range o f 5 - 100 pg/L by dilution o f the 100 mg/L o f fluoride standard with TISABII (total ionic strength adjustment buffer II; Thermo Orion) and deionized water from Bamstead E-Pure system (Megohm-em = 1 6 -1 8 ). H alf strength TISABII (one part o f TISABII plus one part o f deionized water) was used for the dilution. A 10 mL aliquot o f each o f the standard solution was used for fluoride analysis to construct a standard curve for quantification o f fluoride in samples. Quantification o f Fluoride: The 10 mL test medium that was treated with NaOH and H2S 0 4{Section 3.3.1.2) from each o f the 15 mL polypropylene tubes was thawed at room temperature and was centrifuged. Five m illiliter from each o f the tubes was transferred to a 50 mL polypropylene tube that contained 5 mL o f TISABII solution. After mixing, the medium was analyzed for fluoride using a 710 A Plus pH/ISE meter (Thermo Orion, serial # 066814) and a Ionplus fluoride selective electrode (Thermo Orion, model 96-09, lot # GX1). After filling the reference chamber with reference electrode filling solution (Thermo Orion), the electrode was inserted into each o f the sample medium and fluoride standard solution and the conductivity in m illivolts was recorded after 5 min o f incubation. Each sample was measured 1 - 2 times. A standard curve was generated by plotting the standard fluoride concentration in LOG scale versus m illivolts (Figure A -l) for quantification o f fluoride. EMSER15-03/4842 Page 16 o f 47 DuPont EMSE Report No. 15-03 4.0 Results and Discussion 4.1 Under the test conditions, 8-2 TBA was rapidly transformed (Figure 1 and Table A-l). Spike recovery o f 8-2 TBA from the test medium averaged 123 8 % at day 0,120 6.0% at day 14, and 144 2% at day 28 (Table A -2), indicating that MTBE extraction method obtained a good recoveiy o f 8-2 TBA from the test medium. Spike recovery o f fluorinated acids for day 0 samples was moderate, ranged 62-76% (Table B -l, B-2, B-3, and B-4). Spike recoveiy o f 2-PFOEA and 2H-HDF-2-DA was poor for day 2 samples (Section 3.2.6, full-grown bacterial culture), averaged about 36% and 34%, respectively; spike recovery o f PFOA and PFHA was moderate, averaged about 81% and 66%, respectively (Table B -l, B-2, B -3, and B-4). This indicated that full grown bacterial culture matrix may cause significant underestimation o f 2-PFOEA and 2H-HDF-2-DA in day 14 and day 28 samples as w ell. At day 14, >70% o f 8-2 TBA has been biotransformed in test vessels ( Treatment 1) compared with abiotic control vessels (Treatment 2). At day 28, no 8-2 TBA was detected in test vessels. The loss o f parent chemical was due to a combination o f biotransformation and abiotic removal. In the abiotic controls, the concentration o f 8-2 TBA decreased continuously. The loss is possibly caused by volatilization during the incubation or by incomplete extraction due to adsorption to the glass walls o f the test vessels and/or the sludge inoculum . The 8-2 TBA that adsorbed to the walls o f the test vessels during the coating was fully accessible for metabolism, with more than 90% o f biotransformation at day 14 and 99% at day 28 (Table A-l). 4.2 Under the test conditions, significant defluorination occurred during the test, suggesting that the adapted microorganisms were able to metabolize 8-2 TBA to fluorinated acids and other metabolites. The fluoride concentration in test vessels increased from ~10 pg/L at day 0 to 64 pg/L at day 14, and 70 pg/L at day 28, while there was no increase for the abiotic controls (Table A-3). 4.3 Four fluorinated acid metabolites (biotransformation products) have been identified from the test system and their mass balance contributions at day 28 are roughly estimated based in Table 1 and Table A - l : The F(CF2)7CF=CHCOC)H (2H-HDF-2-DA) is the most abundant metabolite and accounted for at least 10% o f total mass present. F(CF2)gCH2COOH (2-PFOEA) is the second abundant metabolite, accounted for ~2% o f total mass present. F(CF2)7COOH (PFOA) accounted for <2% o f total mass present. F(CF2)5COOH (PFHA) accounted for ~0.4% o f total mass present. The formation o f PFHA is not due to impurity o f the test chemical, since 6-2 Telomer B A lcohol [F(CF2)6CH2CH2OH, CAS# 647-42-7], which could lead to PFHA formation, was not detected in the test chemical (Figure A-7). The concentration o f EMSER15-03/4842 Page 17 o f 47 DuPont EMSE Report No. 15-03 PFHA increased significantly from day 0 to day 28 in test vessels, while only background level o f PFHA was present in the abiotic controls during the test. Defluorination o f PFOA may lead to the formation o f PFHA during the test. 4.4 Based on the metabolites identified, the following is a potential pathway for 8-2 TBA biotransformation (a sim plified pathway which does not include all potential biotransformation products): F(CF2)8CH2CH2OH F(CF2)8CH2COOH F(CF2)7CF=CHCOOH + F- F(CF2)7COOH + F- F(CF2)5COOH + 4 F - A notable feature o f this pathway is that PFOA may be defluorinated (metabolized) to form PFHA. 5.0 Conclusions 'we. Under the test conditions, 8-2 TBA is being rapidly transformed to fluorinated acids and other unidentified transformation products. Although PFOA is one o f the identified metabolites, it accounted for <2% o f mass balance. PFOA apparently may be further metabolized to form PFHA and may not be an ultimate (stable) metabolite under the test conditions. 6.0 Reference 'eferemce 6.1 OECD Guideline for Testing o f Chemicals, 302B: Zahn-Wellens/EMPA Test (1992); 302C: Inherent Biodegradability: Modified MITI Test (II) (1981). EMSER15-03/4842 Page 18 o f 47 DuPont EMSE Report No. 15-03 Figure 1 8-2 Telomer B Alcohol (8-2 TBA) concentration during the 28-day TEST* * The test was initiated on 19 July 2002 and finished on 16 August 2002. The graph is derived from the original data o f Table A - l . EMSER15-03/4842 Page 19 o f 47 DuPont EMSE Report No. 15-03 Figure 2 Fluoride concentration during the 28-day test* 0 5 10 15 20 25 30 Days after the initiation * The test was initiated on 19 July 2002 and finished on 16 August 2002. The graph is derived from the original data o f Table A-3. EMSER15-03/4842 Page 20 o f 47 DuPont EMSE Report No. 15-03 Table 1: Estimated CoNCENTRATiONSf of fluorinated acid metabolites at day 0 (19-JUL-2002) and day 28 (16-AUG-2002). Type of Treatments 2-PFOEA T reatm ent 1 - Adapted bacterial culture in growth medium plus 600 pg/L of 8-2 TBA in coated serum bottle T reatm ent 2 - Killed bacterial culture in growth medium plus 2mM KCN and 600 pg/L of 8-2 TBA in coated serum bottle Day 0 Day 28 Day 0 Day 28 NDf 20 ND ND 2H-HDF-2-DA w L -1 ND PFOA ND 147 12 ND ND ND ND PFHA 0.2 4.2 0.05 0.08 * This table is derived from the original data o f Table A -4, A -5, A -6, A -7, B -l, B-2, B -3, and B-4. f Estimated concentration, Ce = Cf/Srwhere: Cf = final concentration o f individual fluorinated acid metabolites at day 0 or 28 Sr= % Spike recovery o f the individual fluorinated acids. Due to the low spike recovery o f 2-PFOEA and 2H-HDF-2-DA and moderate spike recovery o f PFOA and PFFLA, the % spike recovery value was used for correcting possible underestimation o f fluorinated acids in test samples. f ND: Not detected above sample matrix background level EMSER15-03/4842 Page 21 o f 47 DuPont EMSE Report No. 15-03 Table 2: Daily temperature readings in the lab where the test was conducted. The temperature was recorded by a calibrated Dickson Recorder (Model THDx, serial # 0118247) Time Day 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 Average, n = 20 Standard Deviation Date 19-Jul-2002 20-Jul-2002 21-JUI-2002 22-Jul-2002 23-Jul-2002 24-Jul-2002 25-Jul-2002 26-JUI-2002 27-Jul-2002 28-Jul-2002 29-Jul-2002 30-Jul-2002 31-Jul-2002 1-Aug-2002 2-Aug-2002 3-Aug-2002 4-Aug-2002 5-Aug-2002 6-Aug-2002 7-Aug-2002 8-Aug-2002 9-Aug-2002 10-Aug-2002 11-Aug-2002 12-Aug-2002 13-Aug-2002 14-Aug-2002 15-Aug-2002 16-Aug-2002 Temperature C 24.0 23.5 23.5 23.75 24.0 23.75 25.50 23.75 23.5 24.0 24.50 24.0 24.5 24.5 24.0 24.0 23.75 24.25 24.0 23.75 24.0 24.0 23.5 23.75 24.25 24.25 24.25 24.5 25.0 24.1 0.5 EMSERl 5-03/4842 Page 22 o f 47 DuPont EMSE Report No. 15-03 Appendix A: EMSERl 5-03/4842 .Page 23 o f 47 DuPont EMSE Report No. 15-03 Table A-1. Analytical results of 8-2 TBA concentration at day 0 (19-Jul-2002), day 14 (2-AUG-2002), AND DAY28 (16-AUG-2002) Type of Rep 8-2 TBA 8-2 TBA 8-2 TBA final Treatment Analytical Analytical concentration^: No Time concentration Average TA50-1 TA50-2 TA50-3 T reatm ent 1 - Adapted bacterial culture in growth medium plus 600 pg/L of 8- 2 TBA in coated serum bottle day 0 0 0 TA50-4 0 TA50-5 0 Average Standard Deviation f (n =5) TA50-6 TA 50-7 T reatm ent 2 - Killed bacterial culture in growth medium plus 2mM KCN and 600 pg/L of 8-2 TBA in coated serum bottle 0 0 TA50-8 0 Average Standard Deviation (n =3) 1 1 2 2 3 3 4 4 5 5 1 1 2 2 3 3 U 9 -'' 637 652 694 693 699 681 769 792 710 724 757 740 801 793 592 606 M l'' 645 694 690 781 717 749 797 599 fjg L '1 968 1041 1035 1172 1076 1058 75 1124 1196 899 1073 155 <CH arc TA50-12 Treatm ent 4 - Adapted 01 bacterial culture in Growth medium in coated serum 1 TA50-13 bottle 02 2 TA50-14 03 3 Average Standard Deviation (n = 3) TA51-1 TA51-2 Treatm ent 1 - Adapted bacterial culture in growth medium plus 600 pg/L of 8-2 TBA in coated serum bottle 14 1 1 14 2 2 TA51-3 14 3 3. TA51-4 14 4 4 TA51-5 14 5 5 139 154 194 184 125 123 13.0 14.6 12.6 13.3 83.2 79.4 3.65 3.08 79.5 72.3 147 221 189 284 124 186 230 50 13.8 20.7 13.0 19.5 81.3 122 3.37 5.1 75.9 114 Average Standard Deviation (n = 5) EMSER15-03/4842 Page 24 o f 47 DuPont EMSE Report No. 15-03 Table A-1 (continued) No TA51-6 TA51-7 TA51-8 Type of Treatment Rep Time day T reatm ent 2 - Killed bacterial culture in growth medium plus 2mM KCN and 600 pg/L of 8-2 TBA in coated serum bottle 14 14 14 1 1 2 2 3 3 8-2 TBA Analytical concentration M L -1 499 502 607 615 521 531 Average Standard Deviation (n = 3) 8-2 TBA Analytical Average M L '1 501 611 526 8-2 TBAfinal concentration} 752 917 789 819 87 TA51-12 T reatm ent 4 - Adapted 14 1 bacterial culture in Growth medium in coated serum TA51-13 bottle 14 2 2 TA51-14 14 3 3 Average Standard Deviation (n = 3) 8.14 12.5 9.92 7.05 9.03 6.34 9.51 11.2 16.8 8.04 12.1 12.8 3.7 TA52-1 TA52-2 TA52-3 Treatm ent 1 - Adapted bacterial culture in growth medium plus 600 pg/L of 8-2 TBA in coated serum bottle TA52-4 TA52-5 Average Standard Deviation (n =5) 28 1 28 2 2 28 3 3 28 4 4 28 5 5 0 0 0 0 0 0 0 0 0 00 00 00 00 00 0 TA52-6 TA52-7 TA52-8 Treatm ent 2 - Killed bacterial culture in growth medium plus 2mM KCN and 600 pg/L of 8-2 TBA in coated serum bottle 28 28 28 Average Standard Deviation (n =5) 1 1 2 2 3 3' 388 388 321 322 500 523 388 582 322 483 512 768 611 145 EMSER15-03/4842 Page 25 o f 47 DuPont EMSE Report No. 15-03 Table A-1 (continued) Type of Treatment No TA52-12 TA52-13 TA52-14 Treatm ent 4 - Adapted bacterial culture in Growth medium in coated serum bottle Average Standard Deviation Rep Time day 28 28 28 1 1 2 2 3 3 8-2 TBA Analytical concentration W 0 0 6.36 3.96 0.65 1.37 8-2 TBA Analytical Average 4- li'' 6.34 8-2 TBAfinal concentration^ v g L '' 0 5.16 7.7 1.01 1.5 3.1 4.1 f Standard Deviation was calculated using the Final concentration o f 8-2 TBA. J Final concentration o f 8-2 TBA, Cf = Ca x (VmtbeA^J where: Ca= 8-2 TBA analytical average value, Vmtbe = 30 mL (MTBE used to extract the test medium), Vt = 20 mL (Test medium used fro extraction). EMSER15-03/4842 Page 26 o f 47 DuPont EMSE Report No. 15-03 Table A-2. Analytical results of Spike recovery of 8-2 TBA from the sample matrix (Treatment 3, adapted bacterial culture plus growth medium in COATEDGLASSSERUM BOTTLES) AT DAY0 (19-JUL-2002), DAY 14 (2-AUG-2002), and DAY28 (16-AUG-2002) Time Rep 8-2 TBA Analytical 8-2 TBA Final 8-2 TBA Analytical Concentrationf % of Spike Recovery^ No Concentration Average day TA50-9 01 638 628 942 119 1 617 TA50-10 02 624 626 939 118 2 627 TA50-11 03 678 683 1025 133 3 688 Average Standard Deviation 123 8 TA51-9 14 1 1 TA51-10 14 2 2 TA51-11 14 3 3 Average Standard Deviation 459 461 502 509 502 489 460 506 496 690 113 759 124 744 122 120 6 TA52-9 TA52-10 TA52-11 28 1 1 28 2 2 28 3 586 549 572 588 594 568 580 578 852 870 867 3 562 AverageStandard Deviation ________________________________ 142 145 144.0 1 4 4 2 ______ f Final 8-2 TBA concentration, Cf = Ca (V mtbe/ V J where: Ca= 8-2 TBA analytical average V mtbe = 30 mL (MTBE used to extract the test medium) Vt = 20 mL (Test medium used fro extraction) J % o f spike recovery, R = [(Cf-Cc)/Cs] * 100, where: Cf = Final 8-2 TBA concentration in the spiked bottles Cc = Final 8-2 TBA concentration in the coated bottles without adding or spiking with 8-2 TBA stock solution (Treatment 4) Cs = Concentration o f 8-2 TBA spiked into the sample bottles (600 pg/L; Treatment 3) EMSER15-03/4842 Page 27 o f 47 DuPont EMSE Report No. 15-03 Table A-3. Analytical results of fluoride concentration at day 0 (19-Jul-2002), day 14 (2-AUG-2002), AND DAY 28 (16-AUG-2002) Type of Rep Fluoride Fluoride Final Fluoride Treatment Analytical Analytical concentration): No Time Concentration Average day ygL-' /ug'' W L-` TA50-1 TA50-2 TA50-3 Treatm ent 1 - Adapted bacterial culture in growth medium plus 600 pg/L of 8- 2 TBA in coated serum bottle 0 0 0 TA50-4 0 TA50-5 Average Standard Deviation (n = 5)f 0 1 1 2 2 3 3 4 4 5 5 5.3 5.4 4.5 4.8 4.4 4.2 4.4 4.1 4.1 5.9 5.4 10.8 4.7 9.4 4.3 8.6 4.3 8.6 5.0 10.0 9.5 0.9 TA50-6 TA 50-7 Treatm ent 2 - Killed bacterial culture in growth medium plus 2mM KCN and 600 pg/L of 8-2 TBA in coated serum bottle 0 0 TA50-8 0 Average Standard Deviation (n = 3) 1 1 2 2 3 3 7.3 7.8 7.6 7.3 / ci,?fi>.?" ' 7.8 7.6 15.2 15.1 0.5 TA51 -1 TA51-2 TA51-3 T reatm ent 1 - Adapted bacterial culture in growth medium plus 600 pg/L of 8- 2 TBA in coated serum bottle 14 14 14 TA51-4 14 TA51-5 14 Average Standard Deviation (n = 5) TA51-6 TA 51-7 T reatm ent 2 - Killed bacterial culture in growth medium plus 2mM KCN and 600 pg/L of 8-2 TBA in coated serum bottle 14 14 TA51-8 Average Standard Deviation (n = 3) 14 1 1 2 2 3 5 4 4 5 5 1 1 2 2 3 34.4 33.5 31.6 26.8 32.8 6.4 6.3 6.9 34.4 68.8 33.5 67.0 31.6 63.2 26.8 53.6 32.8 65.6 63.6 6.0 6.4 12.8 6.3 12.6 6.9 13.8 13.1 0.6 EMSER15-03/4842 Page 28 o f 47 DuPont EMSE Report No. 15-03 Table A-3 (continued) Type of Rep Fluoride Treatment Analytical No Time Concentration day TA52-1 TA52-2 TA52-3 Treatm ent 1 - Adapted bacterial culture in growth medium plus 600 pg/L of 82 TBA in coated serum bottle 28 28 28 TA52-4 28 TA52-5 28 Average Standard Deviation (n = 5) 1 1 2 2 3 3 4 4 5 5 36.0 37 34.3 34.3 34.3 34.0 36.1 37.4 33.5 34.0 Fluoride Analytical Average m i'' 36.8 34.3 34.2 36.8 33.7 Final Fluoride concentration! p g i'' 73.6 68.6 68.4 73.6 67.4 70.3 3.0 TA52-6 TA 52-7 Treatm ent 2 - Killed bacterial culture in growth medium plus 2mM KCN and 600 pg/L of 8-2 TBA in coated serum bottle 28 28 TA52-8 28 Average Standard Deviation (n = 3) 1 2 2 3 3 6.5 5.4 5.0 6.5 13.0 Ji&'i.Ci \C M g nti 5.4 10.8 5.0 10.0 11.3 1.6 f Standard Deviation was calculated using the final fluoride concentration. % Final fluoride concentration, Cf = Ca x [(Vi+VJ/VJ where: Ca= Fluoride analytical average value, Vi = 5 mL (TISABII buffer added to the test medium for fluoride measurement), Vt = 5 mL (Test medium used for fluoride measurement). EMSER15-03/4842 Page29 o f 47 DuPont EMSE Report No. 15-03 Table A-4. Analytical results of fluorinated acid metabolite 2-perfluorooctyl ETHANOIC ACID (2-PFOEA) AT DAY 0 (19-JUL-2002), AND DAY 28 (16-AUG-2002) Type of Rep 2-PFOEA 2-PFOEA Final 2-PFOEA Treatment Analytical Analytical concentration]: No Time concentration Average day ijg L -' jugf' V 9 L '1 TA50-2 TA50-3 Treatm ent 1 - Adapted bacterial culture in growth medium plus 600 pg/L of 8-2 TBA in coated serum bottle 0 0 1 1 2 ND ND ND ND NDf ND ND 2 ND Average (n =2) ND TA50-6 TA 50-7 T reatm ent 2 - Killed bacterial culture in growth medium plus 2mM KCN and 600 pg/L of 8-2 TBA in coated serum bottle Average (n = 2) 0 0 0 1 1 2 2 ND ND ND ND ND ND ND ND ND.-V TA52-2 TA52-3 T reatm ent 1 - Adapted bacterial culture in growth medium plus 600 pg/L of 8-2 TBA in coated serum bottle 28 28 Average (n =2) 1 1 2 2 15.4 15.3 22.1 22.6 15.4 5.8 22.4 8.4 7.1 TA52-6 TA 52-7 T reatm ent 2 - Killed bacterial culture in growth medium plus 2mM KCN and 600 pg/L of 8-2 TBA in coated serum bottle 28 28 Average (n = 2) 1 1 2 2 ND ND ND ND ND ND ND ND ND t ND: N ot detected above sample matrix background level, f Final 2-PFOEA concentration, Cf = Ca * [(Vmtbe /Vt_)/(VJVm)] where: Ca= 2-PFOEA analytical average value, V mtbe = 30 mL (MTBE used to extract the test medium), V, = 20 mL (Test medium used for extraction), Vs = 6 mL (MTBE phase dried for metabolite analysis), Vm= 1.5 mL (Methanol used to redissolve th dried MTBE phase for metabolite analysis). EMSER15-03/4842 Page 30 o f 47 DuPont EMSE Report No. 15-03 Table A-5. Analytical results o f fluorinated acid metabolite 2Hhexadecafluoro-2-decenoic acid ( 2H-HDF-2-DA) at day 0 (19-Jul2002), AND DAY 28 (16-AUG-2002) Type of Rep 2H-HDF-2-DA 2H-HDF-2-DA Final Treatment Analytical Analytical 2H-HDF-2-DA No Time concentration Average concentration! day TA50-2 Treatm ent 1 - Adapted bacterial culture in growth medium plus 600 pg/L of 8-2 TBA in coated serum TA50-3 bottle 0 0 1 1 2 2 Average (n =2) ND ND ND ND ugL-' ND NDf ND ND ND TA50-6 Treatm ent 2 - Killed bacterial culture in growth medium plus 2mM KCN and 600 pg/L of 8-2 TBA in TA 50-7 coated serum bottle Average (n = 2) 0 0 0 1 2 2 ND ND ND ND ND ND ND ND ND TA52-2 Treatm ent 1 - Adapted bacterial 28 1 culture in growth medium plus 600 pg/L of 8-2 TBA in coated serum 1 TA52-3 bottle 28 2 2 Average (n =2) 117 111 156 147 114 aor.eni 42.8 iff .* - 152 57.0 50 TA52-6 Treatm ent 2 - Killed bacterial culture in growth medium plus 2mM KCN and 600 pg/L of 8-2 TBA in TA 52-7 coated serum bottle 28 28 Average (n = 2) 1 2 2 ND ND ND ND ND ND ND ND ND f ND: N ot detected above sample matrix background level X Final 2H-HDF-2-DA concentration, Cf = Ca x [(V mtbe /Vt,)/(V s/V m)] where: Ca= 2H-HDF-2-DA analytical average value, V mtbe = 30 mL (MTBE used to extract the test medium), Vt= 20 mL (Test medium used for extraction), Vs = 6 mL (MTBE phase dried for the metabolite analysis), Vm= 1.5 mL (Methanol used to redissolve the dried MTBE phase for metabolite analysis). EMSER15-03/4842 Page 31 o f 47 DuPont EMSE Report No. 15-03 Table A-6. Analytical results of fluorinated acid metabolite perfluorooctanoic ACID (PFOA) AT DAY0 (19-JUL-2002), AND DAY28 (16-AUG-2002) Type of Rep PFOA Treatment Analytical No Time concentration day jgL-` TA50-2 Treatm ent 1 - Adapted bacterial culture in growth medium plus 600 pg/L of 8-2 TBA in coated serum TA50-3 bottle 0 0 1 J 2 2 ND NO ND ND Average (n =2) PFOA Analytical Average V9L-' ND Final PFOA concentration^ pgL'' NDf ND ND ND TA50-6 Treatm ent 2 - Killed bacterial culture in growth medium plus 2mM KCN and 600 pg/L of 8-2 TBA in TA 50-7 coated serum bottle Average (n = 2) 0 0 0 1 1 2 2 ND NO ND ND ND ND ND ND ND TA52-2 Treatm ent 1 - Adapted bacterial culture in growth medium plus 600 pg/L of 8-2 TBA in coated serum TA52-3 bottle 28 28 Average (n =2) 1 2 2 27.5 ?7 25.1 25.8 27.5 10.3 whs* 25.5 9.6 10.0 TA52-6 Treatm ent 2 - Killed bacterial culture in growth medium plus 2mM KCN and 600 pg/L of 8-2 TBA in TA 52-7 coated serum bottle 28 28 Average (n = 2) 1 2 2 ND ND ND ND ND ND ND ND ND t ND: Not detected above sample matrix background level. $ Final PFOA concentration, Cf = Ca * [(VMtbe /Vt,)/(V /V m)] where: Ca= PFOA analytical average value, Vmtoe = 30 mL (MTBE used to extract the test medium), Vt = 20 mL (Test medium used for extraction), Vs = 6 mL (MTBE phase dried for metabolite analysis), Vm= 1.5 mL (Methanol used to redissolve the dried MTBE phase for metabolite analysis). EMSER15-03/4842 Page 32 o f 47 DuPont EMSE Report No. 15-03 Table A-7. Analytical results of fluorinated acid metabolite perfluorohexanoic ACID (PFHA) AT DAY0 (19-JUL-2002), ANDDAY 28 (16-AUG-2002) Type of Treatment No TA50-2 Treatm ent 1 - Adapted bacterial culture in growth medium plus 600 (jg/L of 8-2 TBA in coated serum TA50-3 bottle Average (n =2) Rep Time day 0 0 1 1 2 2 PFHA Analytical concentration /jg i-~' 0.6 0.4 ND ND PFHA Analytical Average P 0.5 Final PFHA concentration^ Ijg L -' 0 .2 f ND ND 0.1 TA50-6 Treatm ent 2 - Killed bacterial culture in growth medium plus 2mM KCN and 600 pg/L of 8-2 TBA in TA 50-7 coated serum bottle Average (n = 2) 0 0 0 1 1 2 2 0.2 ND 0.09 0.03 0.1 0.04 0.06 0.02 0.03 TA52-2 Treatm ent 1 - Adapted bacterial 28 1 culture in growth medium plus 600 pg/L of 8-2 TBA in coated serum TA52-3 bottle 28 2 2 Average (n =2) 4.6 5.0 9.4 10.8 4.8 1.8 I 10.1 3.8 2.8 TA52-6 Treatm ent 2 - Killed bacterial culture in growth medium plus 2mM KCN and 600 pg/L of 8-2 TBA in TA 52-7 coated serum bottle 28 28 Average (n = 2) 1 1 2 2 ND 0.4 0.1 ND 0.2 0.08 0.05 0.02 0.05 f ND: Not detected above sample matrix background level. $ Final PFHA concentration, Cf = Ca x [(Vmtbe/%,)/(VS/Vm)] where: Ca= PFHA analytical average value V mtbe = 30 mL (MTBE used to extract the test medium) # Vt = 20 mL (Test medium used for extraction) Vs = 6 mL (MTBE phase dried for metabolite .analysis) Vm= 1.5 mL (Methanol used to redissolve the dried MTBE phase for metabolite analysis). EMSER15-03/4842 Page 33 o f 47 DuPont EMSE Report No. 15-03 Table A-8. Components of bacterial growth medium used for the test. KH2 PO4 NaH2 P 0 4 *H20 KC1 M gS04 *7H20 CaCL2 *2H20 NaCl FeS04 *7H20 CuS 0 4 *5H20 CoC12 *6H20 MnCl2 *4H20 Na2 M o02 *2H20 C6 H5 Na3 O7 *2 H2 0 (Sodium citrate dihydrate tribasic) NiCl2 *6H20 ZnCl2 Yeast Extract Final Concentration 0.70 g/1 0.395g/l 0.5 g/1 0.5 g/1 0.025 g/1 g1 . 0 / 1 0.5 mg/L 0.05 mg/L 0.1 mg/L 0.01 mg/L 0.025 mg/L 1 mg/L 0.1 mg/L 0.005 mg/L 2g/L PH = 7.2 EMSER15-03/4842 Page 34 o f 47 DuPont EMSE Report No. 15-03 Figure A-1 A FLUORIDE STANDARDCALIBRATION CURVE F Standard Curve (for TA51, 8/2/02) EMSER15-03/4842 Page 35 o f 47 DuPont EMSE Report No. 15-03 Figure A-2 A CALIBRATION CURVE USED FOR8-2 TBA QUANTIFICATION OFSAMPLES TA 50-1 TOTA 50-26 Calibration curve used for samples TA 5 0 --1:26. The concentration o f the internal standard: 300 pg/L o f C l 1-iso; the range o f concentrations o f 8-2 TBA: 2 5 .1 -4 0 2 0 pg/L. EMSER15-03/4842 Page 36 o f 47 DuPont EMSE Report No. 15-03 Figure A-3 A CHROMATOGRAPH OFSAMPLETA50-3 USED FOR8-2 TBA ANALYSIS Sample TA50-3. Quantification was done using peak area o f ion m/z 95; retention time 4.97 min = 8-2 TBA; retention time 5.42 min = C l 1-iso. EMSER15-03/4842 Page 37 o f 47 DuPont EMSE Report No. 15-03 Figure A-4 A CALIBRATION CURVE USEDFOR8-2 TBA QUANTIFICATION OFSAMPLES TA 51-1 TOTA51-17 ANDTA 52-1 TOTA52-17 * Calibration curve used for samples TA 5 1 - 1 : 1 7 and TA 52-1:17. The concentration o f the internal standard: 514 pg/L o f D-8-2 TBA; the range o f concentrations o f 8-2 TBA: 25.1 -1005 pg/L. EMSER15-03/4842 Page 38 o f 47 DuPont EMSE Report No. 15-03 Figure A-5 A CHROMATOGRAPH OFSAMPLETA 52-3 USED FOR8-2 TBA ANALYSIS Abundance Ion 31.00 (30.70 to 31.70): 09160259.D Time--> Abundance 4.93 Ion 33.00 (32.70 to 33.70): 09160259.D Time--> Sample TA52-3; Ion 31, retention time 4.95 min = 8-2 TBA; Ion 33, RT 4.93 min = D-8-2 TBA (internal standard). EMSER15-03/4842 Page 39 o f 47 DuPont EMSE Report No. 15-03 Figure A-6 Chromatographs of samples TA52-3 and TA52-6,30 |jg L'1 OF STANDARDACID MIX MADEINSAMPLE MATRIXTA50-15, SAMPLE MATRIX TA52-16, ANDA METHANOLSOLVENT BLANK. TA 52-6 (Abiotic control, Treatment 2) TA 52-3 (Test sample, Treatment 1) 1D15Q2vc61 Smooth(Mn,2>2) M R M of4 channels,ES- 101S02vc58 Smooth(Mn,2x2) M R M of4 channels.ES- Standard: TA50-15 (Sample matrix, Treatment 5) + 30 pg L"1acids TA 52-16 (Sample matrix, T reatm ents) EMSER15-03/4842 Page 40 o f 47 DuPont EMSE Report No. 15-03 Figure A-6 Chromatographs of samples TA52-3 and TA52-6,30 |jg L'1 OF STANDARDACID MIX MADE INSAMPLE MATRIXTA50-15, SAMPLE MATRIX TA52-16, ANDA METHANOLSOLVENT BLANK- CONTINUED FROM PAGE42. Methanol solvent blank EMSER15-03/4842 Page 41 o f 47 DuPont EMSE Report No. 15-03 Figure A-7 A GC/MS chromatograph of material characterization of 8-2 TBA TEST SUBSTANCE USEDINTHIS STUDY EMSER15-03/4842 Page 42 o f 47 DuPont EMSE Report No. 15-03 Appendix B: EMSERl 5-03/4842 Page 43 o f 47 DuPont EMSE Report No. 15-03 Table B-1. Analytical results of spike recovery of 2-perfluorooctyl ethanoic ACID (2-PFOEA) AT DAY 0 (17-SEP-2002), AND DAY 2 (19-SE^002). No TA86-4 TA86-5 TA86-6 Time day 0 0 0 2-PFOEA Analytical Rep concentration 1 113 1 95.7 2 99.0 2 98.7 3 94.7 3 108 2-PFOEA Analytical Average v g L '` 104 98.9 101 Final 2-PFOEA concentration! U9L-' 156 % of Spike Recovery 78 148 74 152 76 Average Standard Deviationf (n = 3) - 76 2 TA87-4 2 1 1 TA87-5 2 2 2 TA 87-6 2 3 3 Average Standard Deviation (n = 3) 45.0 43.5 44.8 44.0 54.6 57.6 44.3 44.4 56.1 66.5 33 66.6 33 84.2 42 x itwKiwSS 5 f Standard Deviation was calculated using the final 2-PFOEA concentration. J Final 2-PFOEA concentration, Cf = Ca x (VmtbeA^J where: Ca= 2-PFOEA analytical average value, V Mtbe = 30 mL (MTBE used to extract the test medium), V, = 20 mL (Test medium used for extraction). % o f spike recovery, R = (Cf/Cs) x 100, where: Cf = Final 2-PFOEA concentration in the spiked bottles, Cs = Concentration o f 2-PFOEA spiked into the sample bottles (200 pg/L). EMSER15-03/4842 Page 44 o f 47 DuPont EMSE Report No. 15-03 Table B-2. Analytical results of spike recovery of 2H-hexadecafluoro-2decenoic ACID ( 2H-HDF-2-DA) at day 0 (17-Sep-2002), and day 2 (19-SEP-2002) 2H-HDF-DA 2H-HDF-DA Final 2H-HDF-DA % of Spike Analytical Analytical concentration^ Recovery No Time Rep concentration Average TA86-4 day 0 1 Pfifi-' 91.2 PSfi'' 90.4 u g i'' 136 68 1 89.5 TA86-5 0 2 88.0 88.1 132 66 2 88.1 TA86-6 0 3 93.5 94.3 141 71 3 95.0 Average Standard Deviation^ (n = 3) t. 's#i680a3tetlii TA87-4 2 1 1 TA87-5 2 2 2 TA 87-6 2 3 3 Average Standard Deviation (n = 3) 38.1 41.1 41.3 40.6 54.6 55.5 39.6 41.0 55.1 59.4 30 61.5 31 82.7 41 iii34f,6tMin f Standard Deviation was calculated using the final 2H-HDF-2-DA concentration. % Final 2H-HDF-2-DA concentration, Cf = Ca x (Vmtbe/ Vt) where: Ca= 2H-HDF-2-DA analytical average value, V mtbe 30 mL (MTBE used to extract the test medium), Vt = 20 mL (Test medium used for extraction). % o f spike recovery, R = (Cf/Cs) x 100, where: Cf = Final 2H-HDF-2-DA concentration in the spiked bottles, Cs = Concentration o f 2H-HDF-2-DA spiked into the sample bottles (200 pg/L). EMSER15-03/4842 Page 45 o f 47 DuPont EMSE Report No. 15-03 Table B-3. Analytical results of spike recovery of perfluorooctanoic acid (PFOA) at DAY0 (17-SEP-2002), AND DAY2 (19-SEP-20Q2) PFOA PFOA Final PFOA % of Spike Analytical Analytical concentration^ Recovery No Time Rep concentration Average TA86-4 day 0 1 vgL~l 88.2 ijg L '1 88.5 M L' 133 67 1 88.7 TA86-5 0 2 89.4 90 135 68 2 90.6 TA8 6 - 6 0 3 94.3 94.8 142 71 3 95.3 Average Standard Deviationf (n = 3) 69 2 TA87-4 2 1 1 TA87-5 2 2 2 TA 87-6 2 3 3 Average Standard Deviation (n = 3) 98.0 102 95.9 103 122 124 100 99.5 123 150 75 149 75 185 93 81 1 0 enfin f Standard Deviation was calculated using the final 2-PFOA concentration. % Final PFOA concentration, Cf = Ca x (VmtbeA^J where: Ca= PFOA analytical average value, V mtbe = 30 mL (MTBE used to extract the test medium), V, = 20 mL (Test medium used for extraction). % o f spike recovery, R = (Cf/Cs) x 100, where: Cf = Final PFOA concentration in the spiked bottles, Cs = Concentration o f PFOA spiked into the sample bottles (200 pg/L). EMSER15-03/4842 Page 46 o f 47 DuPont EMSE Report No. 15-03 Table B-4. Analytical results of spike recovery of perfluorohexanoic acid (PFHA) AT DAY0 (17-SEP-2002), and DAY2 (19-SEP-2002) PFHA PFHA Final PFHA % of Spike Analytical Analytical concentration^: Recovery No Time Rep concentration Average day pgi"' TA86-4 0 1 78.8 79.2 119 60 TA86-5 0 1 2 79.5 82.1 81.4 122 61 2 80.7 TA86-6 0 3 82.7 86.2 129 65 3 89.7 Average Standard Deviation'!" (n = 3) 62 3 TA87-4 2 1 1 TA87-5 2 2 2 TA 87-6 2 3 3 Average Standard Deviation (n = 3) 80.4 79.3 84.8 84.7 100 97.4 79.9 84.8 98.7 120 60 127 64 148 74 66 7 f Standard Deviation was calculated using the final 2-PFHA concentration. J Final PFHA concentration, Cf = Ca x (Vmtbe/ Vt_) where: Ca= PFHA analytical average value, V mtbe = 30 mL (MTBE used to extract the test medium), Vt = 20 mL (Test medium used for extraction). % o f spike recovery, R = (Cf/Cs) x 100, where: C f= Final PFHA concentration in the spiked bottles, Cs = Concentration o f PFHA spiked into the sample bottles (200 pg/L). EMSER15-03/4842 Page 47 o f 47