Document 0gN902N3V4qkkM6X8gZ7XObgd

AR226-2691 13 AR226-2691 DuPont EMSE Report No. 15-03 Study Title ACCELERATED BlODEGRADATION OF 8-2 TELOMER B ALCOHOL- A PRELIMINARY SCREENING STUDY | Test Guideline This study uses the concept ofOECD Guideline for Testing of Chemicals; Section 3: Inherent Biodegradability (1992). Author Ning Wang, Ph.D. Study Completion Date 20 March 2003 Test Facilities E.I. du Font de Nemours and Company Central Research & Development Corporate Center for Engineering Research Environmental and Microbiological Sciences & Engineering Glasgow Building 300, P.O. Box 6101 Newark, DE 19714-6101, USA and E.I. du Pont de Nemours and Company Haskell Laboratory for Health and Environmental Sciences Newark, DE 19714, USA Submitter E.I. du Pont de Nemours and Company DuPont Chemical Solutions Enterprise Wilmington, DE 19898, USA EMSE Study /Project Number 15-03/4842 Report Number EMSER 15-03 EMSER15-03/4842 Page 1 of 47 DuPont EMSE Report No. 15-03 PAGE RESERVED FOR SPECIFIC COUNTRY REQUIREMENTS EMSER15-03/4842 Page 2 of 47 DuPontEMSE Report No. 15-03 CERTIFICATION OF AUTHENTICITY ACCELERATED BlODEGRADATION 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 of the procedures and results. Report by: ^ Ning Wang, Ph.D. Senior Research Biologist Approved by: V L ^ ^ JRoTiTn, Gannon, Ph.D. gxyx/y^rz^^s.^ (Research Manager 63J^Q/^ Date 03/3 0 103 / Date Study Initiation Date: 14-July-2002 Date Study Completed: 20-March-2003 Submitter: E.I. du Pont de Nemours and Company DuPont Chemical Solutions Enterprise Wilmington, DE 19898, USA EMSER15-03/4842 Page 3 of 47 DuPont EMSE Report No. 15-03 TABLE OF CONTENTS Page Reserved for Specific Country Requirements........................................................................^ Certification of Authenticity............................................................................................................3 Table of Contents............................................................................................................................^ 1.0 Summary .................................................................................................................................6 2.0 General Study Information......................................................................................................? 3.0 Materials and Methods............................................................................................................8 3.1 TestSystem..........................................................................................................................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 Discussion..........................................................................................................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 Concentrationsf offluorinated 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 # 0118247)................................................................................................................................22 Appendix A:...................................................................................................................................23 Table A-l. Analytical results of 8-2 TBA concentration at day 0 (19-Jul-2002), day 14 (2-Aug-2002), and day 28 (16-Aug-2002)............................................................................24 Table A-l (continued)...................................................................................................................^ Table A-l (continued)...................................................................................................................^ 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 coated glass serum bottles) at day 0 (19-Jul-2002), day 14 (2-Aug-2002), and day 28 (16-Aug-2002) .............27 Table A-3. Analytical results offluoride concentration at day 0 (19-Jul-2002), day 14 (2Aug-2002), and day 28 (16-Aug-2002).......................................................................,.........28 Table A-3 (continued)...................................................................................................................^ Table A-4. Analytical results offluorinated acid metabolite 2-perfluorooctyl ethanoic acid (2-PFOEA) at day 0 (19-Jul-2002), and day 28 (16-Aug-2002)...................................30 Table A-5. Analytical results offluorinated acid metabolite 2H-hexadecafluoro-2- decenoic acid ( 2H-HDF-2-DA) at day 0 (19-Jul-2002), and day 28 (16-Aug-2002) .........31 Table A-6. Analytical results offluorinated acid metabolite perfluorooctanoic acid (PFOA) at day 0 (19-Jul-2002), and day 28 (16-Aug-2002) ................................................32 Table A-7. Analytical results offluorinated acid metabolite perfluorohexanoic acid (PFHA) at day 0 (19-Jul-2002), and day 28 (16-Aug-2002) ................................................33 EMSER15-03/4842 Page 4 of 47 DuPontEMSE Report No. 15-03 Table A-8. Components of 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 of samples TA 50-1 to TA 50-26...............................................................................................................................36 Figure A-3 A chromatograph of sample TA50-3 used for 8-2 TBA analysis .............................37 Figure A-4 A calibration curve used for 8-2 TBA quantification of samples TA 51-1 to TA51-17 and TA 52-1 to TA52-17.......................................................................................38 Figure A-5 A chromatograph of sample TA 52-3 used for 8-2 TBA analysis ............................39 Figure A-6 Chromatographs of samples TA52-3 and TA52-6, 30 ug L"1 of standard acid mix made in sample matrix TA50-15, sample matrix TA52-16, and a methanol solvent blank.........................................................................................................................40 Figure A-6 Chromatographs of samples TA52-3 and TA52-6, 30 ug L'1 of standard acid mix 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 of material characterization of 8-2 TBA test substance used in this study ..................................................................................................42 Appendix B:...................................................................................................................................43 Table B-l. Analytical results of spike recovery of2-perfluorooctyl ethanoic acid (2PFOEA) at day 0 (17-Sep-2002), and day 2 (19-Sep-2002)................................................. 44 Table B-2. Analytical results of spike recovery of2H-hexadecafluoro-2-decenoic acid< 2H-HDF-2-DA) at day 0 (17-Sep-2002), and day 2 (19-Sep-2002)...................... ?;;............45 Table B-3. Analytical results of spike recovery ofperfluorooctanoic acid (BFOA) at day 0 (17-Sep-2002), and day 2 (19-Sep-2002).......................................sn..^.).........................46 Table B-4. Analytical results of spike recovery ofperfluorohexanoic acid (PFHA) afcday 0 (17-Sep-2002), and day 2 (19-Sep-2002)................................................................w,.......47 EMSER15-03/4842 .Page 5 of 47 1.0 DuPont EMSE Report No. 15-03 ACCELERATED BlODEGRADATJON OF 8-2 TELOMER B ALCOHOL- A PRELIMINARY SCREENING STUDY Author Ning Wang, Ph.D. SUMMARY Rationale of the Study This test generates environmental fate information potentially relevant to the persistence of the test substance. When a chemical enters the environment, biodegradation i& one of the major routes that determines the environmental fate of the chemical. The biodegradation of 8-2 Telomer B Alcohol (8-2 TBA, CAS# 678-39-7) may be slow, because only 2 hydrocarbons of the molecule may be readily accessible for microbial metabolism, the rest of 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 of the biotransformation potential for a given test chemical. Such a test may provide optimal conditions for potential microbial metabolism of 8-2 TBA. For example, if a chemicaliis.not metabolized undersuch optimized conditions, it is less likely to be metabolized/biodsgraded in the environmeA^he test system is also useful to rapidly identify potentia.lnrneat.yTh'; biotransformation poducts and this will enable use of authentic standards to facilitafeod identification and quantification of transformation products in the subsequent definitive' .! biodegradability studies. Test System: The primary biotransformation potential of the test substance 8-2 TBA in bacterial growth medium plus bacterial culture that has pre-grown in 50-100 mg/L of 8-2 TBA was determined. The bacterial culture originated from an industrial waste water treatment facility. The test system consisted of 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 0,14, and 28), sample bottles were sacrificed for extraction and analysis of the test chemical, fluorinated acid metabolites, and fluoride (F~ion). Findings: The primary biotransformation of 8-2 TBA was rapid (>70% at day 14 and near 100% at day 28) compared with the abiotic control. Significant defluorination of 8-2 TBA was observed during the test. Four of 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, FQ^gCHhCOOH, 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 of 47 i{n-- WiUd iUlt malar' 2.0 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 of the identified metabolites, it accounted for <2% of mass balance. PFOA apparently may be farther metabolized to form PFHA and may not be an ultimate (stable) metabolite under the test conditions. GENERAL STUDY INFORMATION v w'c-N.rf Study Objectives Determine the biotransformation potential of 8-2 TBA by monitoring its concentration changes during a biodegradation test with optimized conditions Determine the degree of defluorination of 8-2 TBA Identify potential metabolites such as fluorinated acids during the test Assess whether perfluorinated acids can be farther metabolized Determine possible biodegradation pathways of 8-2 TBA Confirm expectatioi^femt ass balance will be difficult to achieve in cold studies due. i.iS.' :',; to potential abiotic rIBBval mechanisms - volatility and adsorption. ' ' mov Test System Justification The test system is modified from OECD 302 guidelines and was requested by the submitter. Study Personnel E.I. du Font de Nemours and Company Central Research & Development - Corporate Center for Engineering Research - Environmental and Microbiological Sciences & Engineering Haskell Laboratory for Health and Environmental Sciences : r.ii- 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 Microbiological 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 of 47 3.0 3.1 3.1.1 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 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 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-Jul-2002 19-Sep-2002 20-March-2003 MATERIALS AND METHODS 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 Alcohol, 99% 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10heptadecafluoro464.12gmole'1 678-39-7 EMSER15-03/4842 Page 8 of 47 Structure: DuPont EMSE Report No. 15-03 3.1.2 3.1.3 3.1.4 Lot Number: EMSE Sample Number: Concentration ofa.s., nominal: Concentration ofa.s., analyzed: Major impurity Certificate of Analysis Date: Date Received: Solubility at 25C.: Vapor pressure: Stability: Appearance/Color: Storage Conditions: Safety Precautions: P.00/001 E93386-80 99% 99.2% 0.8% as C7FisCF=CHCH20H (Figure A-7) 13-Sept-2001 26-Mar-2002 -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 Preparation of Bacterial Growth Medium The 20% yeast extract and different mineral stock solutions were prepared and were autoclaved. The bacterial growth medium was prepared by dilution of the different stock solutions (see Table A-8 for details of 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 TEA solution On 15-Jul-2002, Glass serum bottles (120 mL volume) were filled with 100 mL of sterile water and 10 uL of 8-2 TBA stock solution (3 mg/mL in ethanol) was injected into each of the bottles for a final concentration of 300 ug/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 of 47 3.1.5 3.1.6 3.7.7 3.2 DuPont EMSE Report No. 15-03 Adapted bacterial culture preparation Approximately 500 mL of 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 of the sludge was transferred to a 250 mL sterile cell culture flask that contained about 50 mg/L of 8-2 TBA in 100 mL bacterial growth medium. The bacterial culture was maintained at room temperature by periodically transferring about 0.5 mL of the culture into another 50 mL polypropylene tube that contained about 5 mL of the bacterial growth medium plus 50-100 mg/L of 8-2 TBA. The culture was mixed by constant shaking at about 210 RPM. The day before the initiation of the test (18-Jul-2002), 0.5 mL of the bacterial culture was transferred into a 250 mL flask that already contained 100 mL of the bacterial growth medium plus 600 ug/L of 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 will serve as inoculum for the test. Ten mL of 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 will be used for abiotic control treatment. Activated Sludge Collection Approximately 4 liters of activated sludge was collected from the City ofWilmington (DE) Municipal Waste 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 of the sludge was filtered through a nylon net with a pore size of 85 um. 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 of four specific fluorinated acid standards, which were quantified in the study. These spike recoveries are assumed to be also representative of 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 of 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 times. EMSER15-03/4842 Page 10 of 47 3.2.1 3.2.2 3.2.3 3.2.4 3.2.5 3.2.6 DuPontEMSE Report No. 15-03 Treatment I - 600 /us/L of 8-2 TEA in Bacterial Growth Medium plus the Bacterial Culture Inoculum in Coated Bottles For a total of 15 coated glass serum bottles (5 replicates x 3 sampling time points), 29.34 mL of of the growth medium, 0.663 mL of the washed bacterial culture, and 6 uL of 8-2 TBA stock solution (3 mg/mL in ethanol) were added to each of the glass serum bottles. The bottles were crimped with pre-cleaned aluminum foil and PTFE/silicone septa. Treatment 2 - 600 us/L of 8-2 TBA in Bacterial Growth Medium plus Killed Bacterial Culture in Coated Bottles For a total of 9 coated glass serum bottles (3 replicates x 3 sampling time points), 29.04 mL of the growth medium, 0.663 mL of the killed bacterial culture, 0.3 mL of 0.2 M NaCN, and 6 uL of 8-2 TBA stock solution (3 mg/mL in ethanol) were added to each of 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 of 9 coated glass serum bottles (3 replicates x 3 sampling time points), 29.34 mL of the growth medium and 0.663 mL of the washed bacterial culture was added to each of 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 uL of 8-2 TBA stock solution (3 mg^mL in ethanol) for a final concentration of 600 ug/L. The ,: :;,. bottles were shaken at 200-301PRPM for approximately 30 min before sample collection aWW' sample extraction. Treatment 4 - Growth Medium plus Bacterial Culture Inoculum in Coated Bottles For a total of 9 coated glass serum bottles (3 replicates x 3 sampling time points), 29.34 mL of the growth medium and 0.663 mL of the washed bacterial culture was added to each of 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 of the test vessels was available for biotransformation. Treatment 5 - Growth Medium plus Bacterial Culture Inoculum in non-Coated Bottles For a total of 9 non-coated'gBBs serum bottles (3 replicates x 3 sampling time points), 29.34 mL of the growth medium and 0.663 mL of the washed bacterial culture was added to each of the glass serum bottles. The bottles were crimped with pre-cleaned aluminum foil and PTFE/silicone septa. This treatment will serve as sample matrix control for quantification offluorinated acid metabolites by LC/MS/MS. Spike Recovery of Fluorinated Acids from Sample Matrix On 17 September 2002, a separate experiment was conducted to determine the spike recovery of fluorinated acids from bacterial culture sample matrix. Each of the sample bottles was filled with 30 mL of 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 of 47 DuPont EMSE Report No. 15-03 3.2.7 3.2.7.1 3.2.7.2 3.2.7.3 3.3 3.3.1 3.3.1.1 3.3.1.2 -250 RPM at room temperature to allow the bacteria to grow. For day 0 samples, 3 bottles were spiked with fluorinated acid mix solution (made from individual stock solution in ethanol) to a final concentration of 200 ug/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 of 200 ug/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. Test Conditions and Sampling Sample Incubation The crimped glass serum bottles were incubated with 200-300 RPM of shaking at room temperature in the dark. The room temperature of the test lab was monitored and recorded throughout the course of the study. Sampling Interval 5 crimped serum bottles from Treatment 1 and 3 bottles from the rest of treatments were sampled for extraction of 8-2 TBA, fluoride, and fluorinated acids at day 0 (19-Jul-2002), day 14 (2-Aug-2002), and day 28 (16-Aug-2002). Sample Storage Analytical samples were stored at -10 to -20C. Sample Extraction and Analysis Sample Collection and Extraction Sample Collection At days 0,14, and 28, crimped sample bottles were removed from the shaker and the bottles were turned upside down. A total of 10 mL of the test medium from each of the bottles were withdrawn with a 10-mL polypropylene syringe and was injected into a 15 mL polypropylene tube that contained 100uLof5N 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 of shaking. Then 83.3 uL of6N HzS04 was added to each of the tubes to neutralize the test medium. The sample tubes were stored at approximately -20C for fluoride analysis. 8-2 TEA and fluorinated acid extraction: After withdrawing a 10 mL of test medium for fluoride analysis, 0.34 mLof6N H2SC>4 and 30 mL of chilled MTBE were injected into the sample bottles. After MTBE and H2S04 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 of 47 DuPont EMSE Report No. 15-03 3.3.1.3 the crimped sample bottles were decapped and the MTBE phase from each of 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 of the MTBE phase from each of 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 of the glass scintillation vials, 2 mL aliquot of 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 of 8-2 TBA. From each of the glass scintillation vials, 6 mL aliquot of the MTBE phase was sequentially dried in a GC vial under N2 and was redissolved in 1.5 mL ofmethanol for fluorinated acid analysis. Temperature Measurements The temperature of the test system was monitored and recorded throughout the course of the study. 3.3.2 3.3.2.1 Analytical Methods for Test Substance and Products Analysis of 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, West 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) of 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 of the freshly made 50-mg/L stocks in methanol. Typically, the calibration standards were made in the range of 25-1000 ug/L of 8-2 TBA. Constant level of internal standard was used: approximately 300 ug/L of 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 of 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 of the concentrations of 8-2 TBA and internal standard. An example of a calibration curve was given in Figure A-2 for quantification of samples TA50-1 to TA 50-26. A chromatograph of 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 of 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 of the concentrations of 8-2 TBA and the internal standard. An example of a calibration curve was given in Figure A-4 for quantification of samples TA51 -1 to TA 51 -17 and TA52-1 to TA 52-17. A chromatograph of sample separation (Sample TA52-3) was given in Fugure A-5. Quantification of 8-2 TBA: A 0.5 mL aliquot of 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 uL of 50 ug/mL of 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 of 47 DuPont EMSE Report No. 15-03 GC7MS system: HP 6890 Plus GC (Agilent), HP 5973 Mass Selective Detector (Agilent), MPS2-MultiPurposeSampler (Gerstal) Column: Temp. ramp: Initial: DB-5MS, 30 m x 0.25 mm, 1 u,m film (Agilent) 80C for 2 min 200C/mmtol200C 50C/min to 3000C and hold for 3 min Flow rate: Split: 1.0 mL/min; He; constant flow mode 5:1 Inlet temp.: Injection volume: MSD transfer line temp.: lonization: SIM ions monitored: 250C 2uL 280C El, 70 eV m/z: 31, 95, 131; dwell time: 50 ms for each ion (day 0 samples) m/z: 31,33,95,98; dwell time: 25 ms for each ion (days 14 and 28 samples) Retention time: 8-2 Telomer B Alcohol (C8-2A): Internal standard (D-8-2 TBA): Internal Standard (C-l 1 Iso) 4.97 min 4.93 - 4.95 min 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 offluorinated acids in test samples. The analytical standard stock solutions for each of the above acids were prepared at approx. 200 u,g/mL concentration by dissolving appropriate amount of the acids in methanol. A single, mixed analytical stock solution at approx. 200 ug/L for each acid was prepared by dilution of each of the above primary stock solutions with methanol in a single volumetric flask. An aliquot of the mixed acid stock solution (~200 u.g/L) and an aliquot of a negative control sample matrix (Treatment 5) were diluted together in a single vial to produce approx. 30 ug/L concentration of each acid in lOx diluted sample matrix in methanol. A separate 30 ug/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 ug/L standards and a pure methanol was designated as a 0 ug/L standard. For fluorinated acid spike recovery experiment (Section 3.2.6), a calibration standard curve was constructed in the range of 10 - 50 u.g/L for quantification of individual fluorinated acid in the acid-spiked samples. EMSER15-03/4842 Page 14 of 47 DuPont EMSE Report No. 15-03 Sample Analysis: Aliquots of all samples were diluted lOx with methanol before the analysis. In addition, aliquots of all samples were transferred to HPLC vials for direct analysis without dilution. All lOx diluted and all undiluted samples were analyzed by liquid chromatography-tandem mass spectrometry (LC/MS/MS). A set of standards (see above) was run at the beginning and at the end of 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 of the analysis sequence. For maximal accuracy of 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 ofanalytes in samples were as close as possible to those of 30 ug/L standards. For fluorinated acid spike recovery experiment (Section 3.2.6), a calibration standard curve was constructed in the range of 10 - 50 ug/L for quantification of individual fluorinated acid in the acid-spiked samples. Chromatographs of samples TA52-3 and TA52-6,, 30 ng/L of 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 twice according to the following conditions: HPLC instrument: Waters 2795 HPLC MS instrument: HPLC Column: Micromass Quattro Micro Phenomenex Luna C18 (2), 20 mm x 2.0 mm, 3 urn particle size Mobile Phase: Mobile Phase Gradient: A: 2 mM ammonium acetate in deionized water (18.2M2cm) C: methanol purge solvent: deionized water (18.2 MSl cm) needle wash solvent: isopropanol Time (min) Flow Rate %A %C (mL/min) 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: 0 to 1.0 min - flow to waste to 4.5 min - flow to MS Column Temperature: 4.5 to 6.5 min 35C flow to waste Injection Volume: 10u,L lonization mode: Capillary voltage: electrospray ionization in negative mode 3.50 kV EMSER15-03/4842 Page 15 of 47 DuPontEMSE 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-HDF-2-DA 2-PFOEA 140C 350C N2,500 War N2, 100 1/hr Ql - 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) Collision 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 of 100 mg/L offluoride in water (Thermo Orion ) was used for standard calibration. The calibration standards normally were made in the range of 5 - 100 ug/L by dilution of the 100 mg/L offluoride standard with TISABII (total ionic strength adjustment buffer II; Thermo Orion) and deionized water from Bamstead E-Pure system (Megohm-cm = 16 - 18). Half strength TISABII (one part of TISABII plus one part of deionized water) was used for the dilution. A 10 mL aliquot of each of the standard solution was used for fluoride analysis to construct a standard curve for quantification offluoride in samples. Quantification of Fluoride: The 10 mL test medium that was treated with NaOH and HzS04 (Section 3.3.1.2) from each of the 15 mL polypropylene tubes was thawed at room temperature and was centrifiiged. Five milliliter from each of the tubes was transferred to a 50 mL polypropylene tube that contained 5 mL of TISABII solution. After mixing, the medium was analyzed for fluoride using a 710 A Plus pH/ISE meter (Thermo Orion, serial # 066814) and a lonplus 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 of the sample medium and fluoride standard solution and the conductivity in millivolts was recorded after 5 min of incubation. Each sample was measured 1 - 2 times. A standard curve was generated by plotting'the standard fluoride concentration in LOG scale versus millivolts (Figure A-l) for quantification offluoride. EMSER15-03/4842 Page 16 of 47 4.0 DuPont EMSE Report No. 15-03 RESULTS AND DISCUSSION 4.1 Under the test conditions, 8-2 TBA was rapidly transformed (Figure 1 and Table A-l). Spike recovery of 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 recovery of 8-2 TBA from the test medium. Spike recovery of fluorinated acids for day 0 samples was moderate, ranged 62-76% (Table B-l, B-2, B-3, and B-4). Spike recovery of2-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 ofPFOA 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 of2-PFOEA and 2H-HDF-2-DA in day 14 and day 28 samples as well. At day 14, >70% of 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 of parent chemical was due to a combination ofbiotransformation and abiotic removal. In the abiotic controls, the concentration of 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 of the test vessels and/or the sludge inoculum. The 8-2 TBA that adsorbed to the walls of the test vessels during the coating was fully accessible for metabolism, with more than 90% ofbiotransformation 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 ug/L at day 0 to 64 ng/L at day 14, and 70 ug/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)?CF=CHCOOH (2H-HDF-2-DA) is the most abundant metabolite and accounted for at least 10% of total mass present. F(CF2)gCH2COOH (2-PFOEA) is the second abundant metabolite, accounted for ~2% of total mass present. F(CF2)7COOH (PFOA) accounted for <2% of total mass present. F(CF2)5COOH (PFHA) accounted for -0.4% of total mass present. The formation of PFHA is not due to impurity of the test chemical, since 6-2 Telomer B Alcohol [F(CF2)6CH2CH20H, CAS# 647-42-7], which could lead to PFHA formation, was not detected in the test chemical (Figure A-7). The concentration of EMSER15-03/4842 Page 17 of 47 5.0 6.0 DuPontEMSE Report No. 15-03 PFHA increased significantly from day 0 to day 28 in test vessels, while only background level of PFHA was present in the abiotic controls during the test. Defluorination ofPFOA may lead to the formation of PFHA during the test. 4.4 Based on the metabolites identified, the following is a potential pathway for 8-2 TBA biotransformation (a simplified pathway which does not include all potential biotransfonnation products): F(CF2)8CH2CH20H F(CF2)8CHzCOOH F(CFz)7CF=CHCOOH + F- F(CFz)7COOH +F- F(CF2)5COOH + 4 F- A notable feature of this pathway is that PFOA may be defluorinated (metabolized) to form PFHA. CONCLUSIONS -WC. -jStON? Under the test conditions, 8-2 TBA is being rapidly transformed to fluorinated acids and other unidentified transformation products. Although PFOA is one of the identified metabolites, it accounted for <2% of mass balance. PFOA apparently mayAe farther metabolized to form PFHA and may not be an ultimate (stable) metabolite under the test conditions. REFERENCE 6.1 OECD Guideline for Testing of Chemicals, 302B: Zahn-Wellens/EMPA Test (1992); 302C: Inherent Biodegradability: Modified MITI Test (II) (1981). EMSER15-03/4842 Page 18 of 47 DuPont EMSE Report No. 15-03 FIGURE 1 8-2 TELOMER B ALCOHOL (8-2 TBA) CONCENTRATION DURING THE 28-DAY TEST* 5 10 15 20 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 of Table A-l. EMSER15-03/4842 Page 19 of 47 DuPont EMSE Report No. 15-03 FIGURE 2 FLUORIDE CONCENTRATION DURING THE 28-DAY TEST* Adapted bacterial culture + 8-2 TBA Killed bacterial culture + 2 mM NaCN + 8-2 TBA 80 U) ^ fl) 40 a *zz 0 J3 U- 20 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 of Table A-3. EMSER15-03/4842 Page 20 of 47 DuPontEMSE Report No. 15-03 TABLE 1: ESTIMATED CONCENTRATIONS! OF FLUORINATED ACID METABOLITES AT DAY 0 (19-JUL-2002) AND DAY 28 (16-AUG-2002). Type of Treatments Treatment 1 - Adapted bacterial culture in growth medium plus 600 ug/L of 8-2 TBA in coated serum bottle Treatment 2 - Killed bacterial culture in growth medium plus 2mM KCN and 600 tig/l ot 8-2 TBA in coated serum bottle Day 0 Dw!2Q Day 0 Day 28 2-PFOEA ND^I 20 ND ND 2H-HDF-2-DA pgL 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 of Table A-4, A-5, A-6, A-7, B-l, B-2, B-3, and B-4. f Estimated concentration, Ce = Cf/Sr where: Cf= final concentration of individual fluorinated acid metabolites at day 0 or 28 Sr = % Spike recovery of the individual fluorinated acids. Due to the low spike recovery of 2-PFOEA and 2H-HDF-2-DA and moderate spike recovery of PFOA and PFHA, the % spike recovery value was used for correcting possible underestimation of fluorinated acids in test samples. ^ ND: Not detected above sample matrix background level EMSER15-03/4842 Page 21 of 47 DuPont EMSE Report No. 15-03 TABLE 2: DAILY TEMPERATURE READINGS IN FHE LAB WH!ERE THE TEST WAS CONDUCTED. THE TEMPERATURE WAS RECORDED> BY A CALIB RATED DIQKS0N 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-Jul-2002 22-Jul-2002 23-Jul-2002 24-Jul-2002 25-Jul-2002 26-Jul-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 IO-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 24.2S 24.2S 24.25 24.5 25.0 24.1 0.5 EMSER15-03/4842 Page 22 of 47 ____ ________DuPontEMSE Report No. 15-03 APPENDIX A: EMSER15-03/4842 . Page 23 of 47 DuPont EMSE Report No. 15-03 TABLE A-1. ANALYTICAL RESULTS OF 8-2 TBA C()NCESNTRATION fIT DAY 0 (19-JUL-2002), DAY 14 (2-AUG-2002), AND DAY 28 ( 16-/kUG-2002) Type of Rep 8-2 TBA 8-2TBA 8-2 TBA final Treatment Analytical Analytical concsntrationt No Time concentration Average TA50-1 TA50-2 day Treatment 1 - Adapted 0 1 bacterial culture in growth 1 medium plus 600 ug/L of 8- 2 TBA in coated serum o 2 bottle 2 ML-' 637 652 694 693 WL-' 645 694 pgL-' 968 1041 TA50-3 0 3 699 690 1035 3 681 TA50-4 0 4 769 781 1172 4 792 TA50-5 0 5 710 Average Standard Deviation f (n =5) 5 724 TA50-6 Treatment 2-Killed 0 1 757 bacterial culture in growth 1 740 medium plus 2mM KCN and TA 50-7 600 ug/L of 8-2 TBA in 0 2 801 coated serum bottle 2 793 717 1076 1058 . 75 749 797 1124 1196 ;; OWit; <CN an: ' .-' TA50-8 0 3 592 599 899 3 606 Average Standard Deviation (n =3) 1073 155 TA50-12 Treatment 4-Adapted 0 1 139 147 221 bacterial culture in Growth medium in coated serum 1 154 TA50-13 bottle o 2 194 189 284 2 184 TA50-14 0 3 125 124 186 Average Standard Deviation (n = 3) TA51-1 TA51 -2 Treatments-Adapted bacterial culture in growth medium plus 600 ug/L of 8-2 TBA in coated serum bottle TA51-3 3 14 1 1 14 2 2 14 3 3 , 123 13.0 14.6 12.6 13.3 83.2 79.4 13.8 13.0 81.3 23050 20.7 19.5 122 TA51-4 14 4 3.65 3.37 5.1 TA51-5 4 3.08 14 5 79.5 75.9 114 5 72.3 Average Standard Deviation (n = 5) 56.3 57.8 EMSER15-03/4842 Page 24 of 47 DuPont EMSE Report No. 15-03 TABLE A-1 (CONTINUED) No TA51 -6 TA51-7 TA51-8 Type of Treatment Rep Time day Treatment 2 - Killed 14 1 bacterial culture in growth 1 medium plus 2mM KCN and 14 2 600 tig/I-of 8-2 TBA in coated serum bottle 2 14 3 3 Average Standard Deviation (n = 3) 8-2 TBA Analytical CIancentratlon pgL-' 499 502 607 615 521 531 TA51-12 Treatment 4 -Adapted 14 1 bacterial culture in Growth 1 medium in coated serum TA51-13 bottle 14 2 2 TA51-14 14 3 3 Average Standard Deviation (n = 3) 8.14 4.53 12.5 9.92 7.05 9.03 TA52-1 Treatment 1 - Adapted 28 1 0 bacterial culture in growth medium plus 600 ug/L of 1 0 TA52-2 8-2 TEA in coated serum 28 2 0 bottle 2 0 TA52-3 28 3 0 3 0 TA52-4 28 4 0 4 0 TA52-5 28 5 0 5 0 Average Standard Deviation (n =5) TA52-6 Treatment 2 - Kilted 28 1 388 bacterial culture in growth 1 388 TA52-7 medium plus 2mM KCN and 28 2 321 600 ug/L of 8-2 TBA in 2 322 TA52-8 coated serum bottle 28 3 500 3 523 Average Standard Deviation (n =5) S-2 TBA Analytical Average ML-' 501 8-2 TBA final concentrationf WL-' 752 611 917 526 789 81987 6.34 11.2 9.51 16.8 8.04 0 12.1 12.8 3.7 0 0 0 0 0 0 0 0 0 0 388 582 322 483 512 768 611 145 EMSER15-03/4842 Page 25 of 47 DuPont EMSE Report No. 15-03 TABLE A-1 (CONTINUED) Type of Treatment No TA52-12 TA52-13 TA52-14 Treatment 4 - Adapted bacterial culture in Growth medium in coated serum bottle Average Standard Deviation Rep Time day 28 1 1 28 2 2 28 3 3 8-2 TBA Analytical concentration ML-' 0 0 6.36 3.96 0.65 1.37 8-2 TBA Analytical Average WL-' 6.34 8-2 TBA final concentration}: WL'' 0 5.16 7.7 1.01 1.5 3.1 4.1 t Standard Deviation was calculated using the Final concentration of 8-2 TBA. f Final concentration of 8-2 TBA, Cf = Ca x (Vi^iBE/Vt,) where: Ca= 8-2 TBA analytical average value, VMTBE = 30 tnL (MTBE used to extract the test medium), V( = 20 mL (Test medium used fro extraction). EMSER15-03/4842 Page 26 of 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 COATED GLASS SERUM BOTTLES) AT DAY 0 (19-JUL-2002), DAY 14 (2-AUG-2002), AND DAY 28 (16-AUG-2002) Time Rep 8-2 TBA 8-2 TBA Final 8-2 TBA % of Spike Analytical Analytical Concentration-1 Recovery^ No Ciancentration Average day WL-' WL-' WL-' TA50-9 0 1 638 628 942 119 1 617 TA50-10 0 2 624 626 939 118 2 627 TA50-11 0 3 678 683 1025 133 3 688 Average Standard Deviation 123 8 TA51-9 14 1 459 460 1 461 TA51-10 14 2 502 506 2 509 TA51-11 14 3 502 496 3 489 Average Standard Deviation 690 113 759 124 744 122 120 6 " TA52-9 28 1 586 568 852 1 549 TA52-10 28 2 572 580 870 2 588 TA52-11 28 3 594 578 867 145 144.0 3 562 Average Standard Deviation 1442 f Final 8-2 TBA concentration, Cf = Ca (VMroE/Vt,) where: Ca = 8-2 TBA analytical average VMTBE = 30 mL (MTBE used to extract the test medium) V( = 20 mL (Test medium used fro extraction) % % of spike recovery, R = [(Cf-Cc)/Cy x 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 of 8-2 TBA spiked into the sample bottles (600 ug/L; Treatment 3) EMSER15-03/4842 Page 27 of 47 DuPont EMSE Report No. 15-03 TABLE A-3. ANALYTICAL RESULTS OF FLUORIt)EC ONCENITRATION.^T DAY O (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 C<incentration Average day WL-' ML-' WL'' TA50-1 Treatment 1 - Adapted 0 1 5.3 bacterial culture in growth medium plus 600 ug/L of 8- 1 5.4 TA50-2 2 TBA in coated serum 0 2 4.5 bottle 2 4.8 TA50-3 0 3 4.4 3 4.2 TA50-4 0 4 4.4 4 4.1 TA50-5 0 5 4.1 Average Standard Deviation (n = 5)f 5 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 Treatment 2 - Killed 0 1 bacterial culture in growth 1 medium plus 2mM KCN and TA 50-7 600 ug/L of 8-2 TBA in 0 2 coated serum bottle 2 TA50-8 Average Standard Deviation (n = 3) 0 3 3 TA51 -1 Treatment 1 - Adapted 14 1 bacterial culture in growth 1 medium plus 600 ug/L of 8- TA51-2 2 TBA in coated serum 14 2 bottle 2 TA51-3 14 3 3 TA51-4 14 4 4 TA51-5 Average Standard Deviation (n = 5) 14 5 5 TA51 -6 Treatment 2 - Killed 14 1 bacterial culture in growth 1 medium plus 2mM KCN and TA 51 -7 600 ug/L of 8-2 TBA in 14 2 coated serum bottle 2 TA51-8 Average Standard Deviation (n = 3) 14 3 7.3 7.8 7.6 34.4 33.5 31.6 26.8 32.8 6.4 6.3 6.9 7.3 7.8 7.6 34.4 33.5 31.6 26.8 32.8 6.4 6.3 6.9 fMi& / tW! W:'!-!- r : i-i:r5.: 8-& ^b- ?-" ^r-i^ ^nMSt 15.2 15.1 0.5 68.8 67.0 63.2 53.6 65.6 63.6 6.0 12.8 12.6 13.8 13.1 0.6 EMSER15-03/4842 Page 28 of 47 DuPont EMSE Report No. 15-03 TABLE A-3 (c;ONTINUED) Type of Treatment No TA52-1 TA52-2 TA52-3 Treatment 1 - Adapted bacterial culture in growth medium plus 600 ug/L of 82 TBA in coated serum bottle TA52-4 TA52-5 Average Standard Deviation (n = 5) Rep Time day 28 1 1 28 2 2 28 3 3 28 4 4 28 5 5 Fluoride Analytical 1Concentration WL-' 36.0 37.6 34.3 34.3 34.3 34.0 36.1 37.4 33.5 34.0 TA52-6 Treatment 2-Killed 28 1 6.5 bacterial culture in growth 1 medium plus 2mM KCN and TA 52-7 600 ug/L of 8-2 TBA in 28 2 5.4 coated serum bottle 2 TA52-8 28 3 5.0 3 Average Standard Deviation (n = 3) Fluoride Analytical Average WL-' 36.8 34.3 34.2 36.8 33.7 6.5 5.4 5.0 Final Fluoride concentration:): WL-1 73.6 68.6 68.4 73.6 67.4 70.3 3.0 13.0 10.8 .tAtf. ,CN an:! ' 10.0 11.3 1.6 t Standard Deviation was calculated using the final fluoride concentration. % Final fluoride concentration, Cf = Cax [(Vi+Vt,)/Vt] where: Ca^ Fluoride analytical average value, V; = 5 mL (TISABII buffer added to the test medium for fluoride measurement), Vt = 5 mL (Test medium used for fluoride measurement). EMSER15-03/4842 Page 29 of 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 TA50-2 Treatment 1 - Adapted bacterial 0 1 culture in growth medium plus 1 600 (Jg/L of 8-2 TBA in coated TA50-3 serum bottle 0 2 ML-' ND ND ND ML-' ND ND WL-' NDf ND 2 ND Average (n =2) ND TA50-6 Treatment 2 - Killed bacterial 0 1 ND culture in growth medium plus 2mM KCN and 600 ug/L of 8-2 1 ND TA 50-7 TBA in coated serum bottle 0 2 ND 0 2 ND Average (n=2) ND ND ND ND ND TA52-2 Treatment 1 - Adapted bacterial 28 1 culture in growth medium plus 1 600 pg/L of 8-2 TBA in coated TA52-3 serum bottle 28 2 2 Average {n =2) 15.4 15.3 22.1 22.6 15.4 22.4 5.8 8.4 7.1 TA52-6 Treatment 2 - Killed bacterial 28 1 ND culture in growth medium plus 2mM KCN and 600 ug/L of 8-2 1 ND TA 52-7 TBA in coated serum bottle 28 2 ND 2 ND Average (n = 2) ND ND ND ND ND f ND: Not detected above sample matrix background level. $ Final 2-PFOEA concentration, Cf = Ca x [CvNrrBE/Vt,)/(Vs/Vni)] where: Ca= 2-PFOEA analytical average value, VMTBE = 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 30 of 47 DuPont EMSE Report No. 15-03 TABLE A-5. ANALYTICAL RESULTS OF FLUORINATED ACID METABOLITE 2HHEXADECAFLUORO-2-DECENOIC ACID ( 2H-HDF-2-DA) AT DAY 0 2002), AND DAY 28 (16-AUG-2002) (19-JUL- Type of Treatment No TA50-2 TA50-3 Treatment 1 - Adapted bacterial culture in growth medium plus 600 ug/L of 8-2 TBA in coated serum bottle Average (n=2) Time Rep 2H-HDF-2-DA Analytical 1soncentration 2H-HDF-2-IDA Analyticsil Average Final 2H-HDF-2-DA ( ;oncentrationt day 0 1 1 WL-' ND ND WL-' ND ML-' NDf 0 2 ND ND ND 2 ND ND TA50-6 Treatment 2- Killed bacterial 0 1 ND culture in growth medium plus 2mM KCN and 600 ug/L of 8-2 TBA in 1 ND TA 50-7 coated serum bottle 0 2 ND 0 2 ND Average (n=2) ND ND ND ND ND TA52-2 Treatment 1 - Adapted bacterial 28 1 117 culture in growth medium plus 600 ug/L of 8-2 TBA in coated serum 1 111 TA52-3 bottle 28 2 156 2 147 Average (n =2) 114 sti; ."<: 42.8 152 57.0 50 TA52-6 Treatment 2 - Killed bacterial 28 1 ND culture in growth medium plus 2mM KCN and 600 ug/L of 8-2 TBA in 1 ND TA 52-7 coated serum bottle 28 2 ND 2 ND Average (n = 2) ND ND ND ND ND ND: Not detected above sample matrix background level Final 2H-HDF-2-DA concentration, Cf = Ca x [(VMTBE/Vt,)/(Vs/Vn)] where: Ca= 2H-HDF-2-DA analytical average value, VMTBE = 30 mL (MTBE used to extract the test medium), V( = 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 of 47 DuPont EMSE Report No. 15-03 TABLE A-6. ANALYTICAL RESULTS OF FLUORINATED ACID METABOLITE PERFLUOROOCTANOIC ACID (PFOA) AT DAY 0 (19-JUL-2002), AND DAY 28 (16-AUG-2002) Type of Treatment No TA50-2 TA50-3 Treatment 1 - Adapted bacterial culture in growth medium plus 600 ug/L of 8-2 TBA in coated serum bottle Time day 0 0 Rep 1 1 2 2 PFOA Analytical concentration WL-' ND ND ND ND PFOA Analytical Average ML-' ND Final PFOA concentration!: Wi-' NDf ND ND Average (n=2) ND TA50-6 Treatment 2 -Killed bacterial 0 culture in growth medium plus 2mM KCN and 600 pg/L of 8-2 TBA in TA 50-7 coated serum bottle 0 Average (n=2) ND ND ND ND ND ND ND ND ND TA52-2 Treatment 1 - Adapted bacterial 28 culture in growth medium plus 600 ug/L of 8-2 TBA in coated serum TA52-3 bottle 28 Average (n =2) 27,5 27.5 25.1 25.8 27.5 10.3 wwo- 25.5 9.6 10.0 TA52-6 Treatment 2-Killed bacterial 28 culture in growth medium plus 2mM KCN and 600 pg/L of 8-2 TBA In TA52-7 coated serum bottle 28 Average (n = 2) ND ND ND ND ND ND ND ND ND f ND: Not detected above sample matrix background level. t Final PFOA concentration, Cf = Ca x [(VMTBE/Vt,)/(V/Vni)] where: Ca^ PFOA analytical average value, VMTBB = 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 of 47 DuPont EMSE Report No. 15-03 TABLE A-7. ANALYTICAL RESULTS OF FLUORINATED ACID METABOLITE PERFLUOROHEXANOIC ACID (PFHA) AT DAY 0 (19-JUL-2002), AND DAY 28 (16-AUG-2002) Type of Treatment No TA50-2 TA50-3 Treatment 1 - Adapted bacterial culture in growth medium plus 600 ug/L of 8-2 TBA in coated serum bottle Time day 0 0 Rep 1 1 2 2 PFHA Analytical concentration mL-' 0.6 0.4 ND ND PFHA Analytical Average WL-1 0.5 Final PFHA concentration:): WL-' 0.2f NO ND Average (n=2) 0.1 TA50-6 Treatment 2 -Killed bacterial 0 culture in growth medium plus 2mM KCN and 600 ug/L of 8-2 TBA in TA 50-7 coated serum bottle o Average (n=2) 0.2 ND 0.09 0.03 0.1 0.06 0.04 0.02 0.03 TA52-2 Treatment 1 - Adapted bacterial 28 1 culture in growth medium plus 600 - ug/L of 8-2 TBA in coated serum TA52-3 bottle 28 2 Average (n =2) 4.6 5.0 9.4 10.8 4.8 10.1 1.8 UW w-ic.w.r":w. p;>A <;' 3.8 2.8 TA52-6 Treatment 2 -Killed bacterial 28 culture in growth medium plus 2mM KCN and 600 ug/L of 8-2 TBA in TA 52-7 coated serum bottle 28 Average (n = 2) ND 0.2 0.08 0.4 0.1 0.05 0.02 ND 0.05 f ND: Not detected above sample matrix background level. $ Final PFHA concentration, Cf = Ca x [(VMTBE/Vt,)/(V./V,n)] where: Ca = PFHA analytical average value VMTBE = 30 mL (MTBE used to extract the test medium) t 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 the dried MTBE phase for metabolite analysis). EMSER15-03/4842 Page 33 of 47 DuPont EMSE Report No. 15-03 TABLE A-8. COMPONENTS OF BACTERIAL GROWTH MEDIUM USED FOR THE TEST. KH2P04 NaH2P04*H20 KC1 MgS04*7H20 CaCL2*2H20 NaCI FeS04*7H20 CuS04*5H20 CoCl2*6H20 MnCl2*4H20 Na2Mo02*2H20 CfiH5Na307*2H20 (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 1.0 g/1 0.5mg/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 of 47 DuPontEMSE Report No. 15-03 FIGURE A-1 A FLUORIDE STANDARD CALIBRATION CURVE F~ Standard Curve (for TA51, 8/2/02) 180 200 Millivolts EMSER15-03/4842 Page 35 of 47 DuPont EMSE Report No. 15-03 FIGURE A-2 A CALIBRATION CURVE USED FOR 8-2 TBA QUANTIFICATION OF SAMPLES TA 50-1 TO TA 50-26 Response Ratio C8-Z& fit; afctp Ktlteio 1.04~c000 Ant - .<7e-00i Cool! at Wt (r*a} 0.99S> Curve wird/a? Calibration curve used for samples TA 50 - 1:26. The concentration of the internal standard: 300 pg/L ofCl 1-iso; the range of concentrations of 8-2 TBA: 25.1- 4020 ug/L. EMSER15-03/4842 Page 36 of 47 DuPont EMSE Report No. 15-03 FIGURE A-3 A CHROMATOGRAPH OF SAMPLE TA50-3 USED FOR 8-2 TBA ANALYSIS Abundance 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000 800 600 400 200 Tlme-> 4.97 Ion 95.00 (94.70 to 95.70): 08140227.D 5.42 ^ ^ j wf \^f\^-^/ Ak Wj^^'^ JM^^ 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00 Sample TA50-3. Quantification was done using peak area of ion m/z 95; retention time 4.97 min = 8-2 TBA; retention time 5.42 min = Cl 1-iso. EMSER15-03/4842 Page 37 of 47 DuPont EMSE Report No. 15-03 FIGURE A-4 A CALIBRATION CURVE USED FOR 8-2 TBA QUANTIFICATION OF SAMPLES TA 51-1 TO TA51-17 AND TA 52-1 TO TA52-17 Response .Ratio CB-2& lasawt Batio Heap Ratio S.BSe-OOl * tab * l.SSe-OOS Coet of Bt tK'^!t) 9.999 CtH-w Pits wl(l/a) Calibration curve used for samples TA 51-1:17 and TA 52-1:17. The concentration of the internal standard: 514 ug/L ofD-8-2 TBA; the range of concentrations of 8-2 TBA: 25.1 -1005 ug/L. EMSER15-03/4842 Page 38 of 47 DuPont EMSE Report No. 15-03 FIGURE A-5 A CHROMATOGRAPH OF SAMPLE TA 52-3 USED FOR 8-2 TEA ANALYSIS Abundance Ion 31.00 (30.70 to 31.70): 09160259.D 12000 10000 8000 6000 4000 2000 0 |Time-> 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 Abundance 4.93 Ion 33.00 (32.70 to 33.70): 09160259.D 12000 10000 8000 6000 4000 2000 0 n"ime~> 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 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 3 9 of 47 DuPont EMSE Report No. 15-03 FIGURE A-6 CHROMATOGRAPHS OF SAMPLES TA52-3 AND TA52-6, 30 |JG L'1 OF STANDARD ACID MIX MADE IN SAMPLE MATRIX TA50-15, SAMPLE MATRIX TA52-16, AND A METHANOL SOLVENT BLANK. TA 52-6 (Abiotic control, Treatment 2) TA 52-3 (Test sample, Treatment 1) 1D1502VC61 Smoolh(Mn.2}<2) 100-, I PFHA 3.26 1 35.10 MRMof4channBls,ES313>269 s.9718+002 101502cS8 SmoDthfMn^ia) ^r-l 1 PFHA_ 3.25 167.45 1 | MRMof4 channels .ES313 > 268 2.338e+003 101502W61 Smoolh(Mn,22) 100-1 . nin MRMtH4channels.ES- PFOA "] 3.43 144.06 f 413>369 2.023e+003 0-t; . 101502\ic58 ^''-j Smi)Olh(Mn,2ia) ~rf min MRMof4 channels ,ES- PFOA "j 3.43 828.02 | 413 > 369 1.110B+004 101502W6I Srnoolh(Mn,2>a) MRMof4channels.ES- l01502ic58 Snioolh(Mn,22) 3.52 457 > 393 1.07le*oo2 '""'I MRMof4channels,ES- 2H-HDF-2-DA 1 3.53 | 6121.70 457 > 393 7.970e*004 (''-'(' MRMof4channels,ES- 3.96 477 > 383 1.193E+002 101502wSS Smoolh(Mn^i<2) ^^ ."rl'nTOi'i'r'i4fHw|-m^ min MRMof4 channels,ES- | 2-PFOEA_ 3.52 1 63.79 477 > 393 8.900e*002 min 0-t l.^ili.lili.lpi i >^l.'^pnll^lr.l.^.u.^u.ll^lJl^l.T^'j'^l<.lii^l.AI^-'AP"P"^l"^"i min 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 Standard: TA50-15 (Sample matrix. Treatment 5) + 30 ng L'1 acids PFHA_ 3.24 | 485.33 TA 52-16 (Sample matrix, Treatment-5) t00i y.-| PFOA'_] 3.41 996.53 2H-HDF-2-DA_ 3.51 1519.72 100-| y. 1.13 1.63 100,0.05 "" 1-06 PFOA_ 3.43 | 142.99 j 100-| % 2-PFOEA_ ' 3.51 96.56 ' ' 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 EMSER15-03/4842 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 Page 40 of 47 DuPont EMSE Report No. 15-03 FIGURE A-6 CHROMATOGRAPHS OF SAMPLES TA52-3 AND TA52-6, 30 |JG L'1 OF STANDARD ACID MIX MADE IN SAMPLE MATRIX TA50-15, SAMPLE MATRIX TA52-16. AND A METHANOL SOLVENT BLANK - CONTINUED FROM PAGE 42. Methanol solvent blank 1B15B2iieB8 Smoolh(Mn,2Q) tSO-i PFHA_ | 3.25 23.58 MRMof4channels,ES313>269 3.52SfH102 101502vc68 Smaoth(Mn,2i2) iW-1 1015Q2vc6a Smootl(l>*l^x2) 1A1 MRMl)f4chBliels,ES- PFOA 413 > 369 S 3 43 I 125.73 1.731e+003 3.05 MRMof4cliannels^S- EMSER15-03/4842 Page 41 of 47 DuPont EMSE Report No. 15-03 FIGURE A-7 A GC/MS CHROMATOGRAPH OF MATERIAL CHARACTERIZATION OF 8-2 TBA TEST SUBSTANCE USED IN THIS STUDY '1 9wstats ?SOOS90 361166011 ^ asoao StWW " WWW itBOian iaeoe t.'3 & -t- Mf- ,'<Mi' 1 '*& ?i4it.to , """ " "N"1-"-- ' i . ,, ,.--. ^ t -1 ; ; * ^^ u " ^- 8-2 TBA (99.2%) ' Z;B l ^ C7FisCF=CHCH20H ( 0.8%) : t*:(a , at:fla , . ..'.....aii^ BC'IH. ,,,, 4.^ EMSER15-03/4842 Page 42 of 47 APPENDIX B: DuPont EMSE Report No. 15-03 EMSER15-03/4842 Page 43 of 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-SSP-t002). No TA86-4 TA86-5 TA86-6 Tinm day 0 0 0 Rep 1 1 2 2 3 3 2i-PFOEA A inalytlcal coricentration ML-' 113 95.7 99.0 98.7 94.7 108 2-PFOEA Analytical Average ML-' 104 98.9 101 Final 2-PFC>EA concentrati "* ML-' 156 148 152 % of Spike Recovery 78 74 76 Average Standard Deviation"^ (n = 3) .. 76 2 TA87-4 2 1 1 TA87-5 2 2 2 TA87-6 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 <-.-. i awa.'36 i 5 f Standard Deviation was calculated using the final 2-PFOEA concentration. % Final 2-PFOEA concentration, Cf = Ca x CvMi-BE/Vt,) where: Ca= 2-PFOEA analytical average value, VMTBE = 30 mL (MTBE used to extract the test medium), Vt = 20 mL (Test medium used for extraction). % of spike recovery, R = (Cf/Cs) x 100, where: Cf = Final 2-PFOEA concentration in the spiked bottles, Cs = Concentration of 2-PFOEA spiked into the sample bottles (200 u.g/L). EMSER15-03/4842 Page 44 of 47 DuPont EMSE Report No. 15-03 TABLE B-2. ANALYTICAL RESULTS OF SPIKE RECOVERY OF 2H-HEXADECAFLUORO-2- DECENOIC 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 concentrationt Recovery No Time Rep concentration Average day WL-' P9L-' ML-' TA86-4 0 1 91.2 90.4 136 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) 'aBii'ttteSBsSMattOR'i TA87-4 TA87-5 TA 87-6 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 61.5 82.7 30 31 41 w;tSMf.fc6t""n f Standard Deviation was calculated using the final 2H-HDF-2-DA concentration. % Final 2H-HDF-2-DA concentration, Cf = Ca x (VMTBE/VI,) where: Ca= 2H-HDF-2-DA analytical average value, VMTBE = 30 mL (MTBE used to extract the test medium), V( = 20 mL (Test medium used for extraction). % of spike recovery, R = (Cf/Cs) x 100, where: Cf= Final 2H-HDF-2-DA concentration in the spiked bottles, Cs = Concentration of 2H-HDF-2-DA spiked into the sample bottles (200 ug/L). EMSER15-03/4842 Page 45 of 47 DuPont EMSE Report No. 15-03 TABLE B-3. ANALYTICAL RESULTS OF SPIKE RECOVERY OF PERFLUOROOCTAN01C ACID (PFOA) AT DAY 0 (17-SEP-2002), AND DAY 2 (19-SEP-2002) No TA86-4 TA86-5 Timia day 0 0 PFOA PFOA Final PFOA % of Spike Ainalytical Analytical concentration:]' Recovery Rep coricentration Average ML-' ML-' pgL-' 1 88.2 88.5 133 67 1 88.7 2 89.4 90 135 68 2 90.6 TA86-6 0 3 94.3 94.8 142 71 3 95.3 Average Standard Deviation'1' (n = 3) 692 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 10 "oi ', f Standard Deviation was calculated using the final 2-PFOA concentration. 3: Final PFOA concentration, Cf = Ca x (VM-TBE/VI,) where: Ca= PFOA analytical average value, VMTBE = 30 mL (MTBE used to extract the test medium), V( = 20 mL (Test medium used for extraction). % of spike recovery, R = (Cf/Cs) x 100, where: Cf= Final PFOA concentration in the spiked bottles, Cs = Concentration of PFOA spiked into the sample bottles (200 u.g/L). EMSER15-03/4842 Page 46 of 47 DuPont EMSE Report No. 15-03 TABLE B-4. ANALYTICAL RESULTS OF SPIKE RECOVERY OF PERFLUOROHEXAN01C ACID (PFHA) AT DAY 0 (17-SEP-2002), AND DAY 2 (19-SEP-2002) No TA86-4 TA86-5 TA86-6 Timie day 0 0 0 Rep 1 1 2 2 3 3 PFHA A.nalyticai coricentration ML-' 78.8 79.5 82.1 80.7 82.7 89.7 PFHA Analytical Average WL-' 79.2 81.4 86.2 Final PFHA concentrationt WL-' 119 % of Spike Recovery 60 122 61 129 65 Average Standard Deviation'!" (n = 3) 623 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 t Standard Deviation was calculated using the final 2-PFHA concentration. f Final PFHA concentration, Cf = Ca x (VMTBE/V(,) where: Ca= PFHA analytical average value, VMTBE = 30 mL (MTBE used to extract the test medium), V( = 20 mL (Test medium used for extraction). % of spike recovery, R = (Cf/Cs) x 100, where: Cf= Final PFHA concentration in the spiked bottles, Cs = Concentration of PFHA spiked into the sample bottles (200 |ig/L). EMSER15-03/4842 Page 47 of 47