Document 1QBrJ2wXwpnJzgR337KrnmKwX

t /> DuPont EMSE Report No. 13-03 Study Title Ready Biodegradation of 8-2 Telomer B Alcohol (Modified OECD 301D Closed Bottle Test) Test Guideline Organization for Economic and Cooperative Development (OECD) Guideline for Testing of Chemicals; Section 3: Ready Biodegradability: 30l D Closed Bottle Test (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 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 Submitter E.I. du Pont de Nemours and Company DuPont Chemical Solutions Enterprise Wilmington, DE 19898, USA EMSE Study /Project Number 13-03/4842 Report Number EMSER 13-03 E M S E R 13-03/4842 Page 1 of 28 :-iO CBi OOOOOS DuPont EMSE Report No. 13-03 Page Reserved for Specific Country Requirements EMSERl 3-03/4842 Page 2 o f 28 000006 DuPont EMSE Report No. 13-03 Certification of Authenticity Ready Biodegradation of 8-2 Telomer B Alcohol (Modified OECD 301D Closed Bottle Test) 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: jn T. Gannon, Ph.D. ^search Manager Study Initiation Date: 26- December-2002 Date Study Completed: 20-March-2003 Submitter: E.I. du Pont de Nemours and Company DuPont Chemical Solutions Enterprise Wilmington, DE 19898, USA Date 03 20/03 Date EMSER13-03/4842 Page 3 of 28 000007 DuPont EMSE Report No. 13-03 Table of Contents Page Reserved for Specific Country Requirements............................................................................ 2 Certification of Authenticity................................................................................................................. 3 Table of Contents................................................................................................................................... 4 1.0 Summary........................................................................................................................................5 2.0 General Study Information........................................................................................................... 6 3.0 Materials and Methods................................................................................................................. 7 3.1 Test System................................................................................................................................ 7 3.2 Test Conduct.............................................................................................................................. 9 3.3 Sample Extraction and Analysis.............................................................................................10 3.3.1 Sample Collection and Extraction................................................................................ 10 3.3.2 Analytical Methods for Test Substance and Products................................................11 4.0 Results and Discussion................................................................................................................12 5.0 Conclusion....................... ...........................................................................................................13 6.0 Limitations of the test............................................................... ................................................. 13 7.0 Reference...................................................................................................................................... 13 Figure 1 8-2 Telomer B Alcohol (8-2 TBA) concentration during the 28-day test*................... 14 Figure 2 Fluoride concentration during the 28-day test*................................................................15 Table 1 Concentration of 8-2 TBA at day 0 (31-Dec-2002), day 7 (7-Jan-2003), day 14 (14-Jan-2003), and day 28 (28-Jan-2003)*............................................................................... 16 Table 2 Daily temperature readings with a calibrated digital thermometer inside a chemical hood where the test vessels were incubated............................................................. 17 Appendix A :.......................................................................................................................................... 18 Table A -l. Analytical results of 8-2 TBA concentration at day 0 (31-Dec-2002), day 7 (7Jan-2003), day 14 (14-Jan-2003), and day 28 (28-Jan-2003)..................................................19 Table A-2. Analytical results of Spike recovery of 8-2 TBA from the sample matrix (Treatment 3, activated sludge filtrate plus mineral medium) at day 0 (31-Dec2002), day 7 (7-Jan-2003), day 14 (14-Jan-2003), and day 28 (28-Jan-2003)..................... 21 Table A-3. Analytical results of fluoride concentration at day 0 (31-Dec-2002), day 7 (7Jan-2003), day 14 (l 4-Jan-2003), and day 28 (2 8-Jan-2003)................................................ 23 Table A-4. Preparation o f the mineral medium for the test............................................................. 25 Figure A-l A fluoride standard calibration curve used for fluoride quantification of samples E93384EJ-1 toE93384EJ-64....................................................................................... 26 Figure A-2 A calibration curve used for 8-2 TBA quantification of samples E93384EJ-1 to E93384EJ-28........................................................................................................................... 27 Figure A-3 A chromatograph of sample E93384EJ-29 used for 8-2 TBA analysis...................... 28 000008 DuPont EMSE Report No. 13-03 Ready Biodegradation of 8-2 Telomer B Alcohol (Modified OECD 301D Closed Bottle Test) Author Ning Wang, Ph.D. 1.0 Summary Rationale o f the Study This test generates environmental fate information relevant to assess the potential for environmental persistence of a test substance. When a chemical enters the environment, biodegradation is one of the major routes that determines the environmental fate of the chemical. The "OECD 301 D Closed Bottle Test" is a widely accepted test to assess the "ready" biodegradability of a test chemical. Due to the stringent test conditions, if it passes this test, a given chemical is unlikely to be persistent in the environment. Because activated sludge will be used as the inoculum for the test, the outcome of the test will indicate what may occur if this chemical enters a domestic sewage treatment plant (POTW). The low water solubility, volatility, and strong surface adsorption properties of 8-2 Telomer B Alcohol (8-2 TBA, CAS # 678-39-7) to be used in this test requires a modification of the 301 D test guidelines. Because the duration of the test is relatively short (28 days) and 8-2 TBA is not expected to be ultimately metabolized, this specific study will only provide information on primary biodegradation potential of 8-2 TBA, through measurement of parent loss and formation of fluoride, not the ultimate environmental fate of the test chemical. To assess full biodegradation potential of fluorinated chemicals such as 8-2 TBA, a test system with adapted microorganisms to the test chemical and a favorable growth medium (mineral medium plus additional carbon source) may be needed. Test System: The biodegradability and biotransformation potential of the test substance 8-2 Telomer B Alcohol in mineral medium plus activated sludge inoculum (5 mL activated sludge per liter of mineral medium) from a POTW was determined. The test system consisted of individually crimped test vessels (glass serum bottles) and the test was conducted at room temperature (~22C). The saturated solution of 8-2 Telomer B Alcohol in mineral medium was inoculated with activated sludge and kept in closed bottles in the dark at room temperature. Periodically (days 0, 7, 14, and 28), sample bottles of different experimental treatments were sacrificed for extraction and analysis. Potential degradation of the test chemical and formation of potential metabolites (transformation products) was followed by analysis of the concentration of the test chemical and by analysis for fluoride (F- ion) during the 28-d study. Findings: Under the test conditions, the loss of 8-2 TBA from the test vessels was ~8% more at day 7 and 13% more at day 14 compared with the abiotic control vessels. At day 28, the concentrations of 8-2 TBA were not distinguishable between the test system and the abiotic controls. No defluorination of 8-2 TBA was observed during the test. EMSER13-03/4842 Page 5 of 28 oooooa DuPont EMSE Report No. 13-03 Conclusion: Under the test conditions, 8-2 TBA is not readily biodegradable. Confirmed expectations that abiotic removal mechanisms - volatility and/or adsorption - will be factors that need consideration when designing future studies with objective of achieving mass balance. 2.0 General Study Information Study Objectives Determine the biotransformation potential of 8-2 TBA by monitoring its concentration during the test Determine the degree of defluorination of 8-2 TBA Determine if abiotic removal mechanisms - volatility and/or adsorption - will be factors that may affect ability to achieve mass balance. Test System Justification The test system is outlined by the OECD 301D guideline and readily accepted. Study Personnel E.I. du Pont de Nemours and Company Central Research and Development - Corporate Center for Engineering Research Environmental and Microbiological Sciences & Engineering Haskell Laboratory for Health and Environmental Sciences Management: Study Director: John T. Gannon, Ph.D. E.I. du Pont de Nemours and Company Central Research and Development Corporate Center for Engineering Research Environmental and Microbiological Sciences & Engineering Glasgow Building 300, P.O. Box 6101 Newark, DE 19714-6101 USA and S. Mark Kennedy, Ph.D. E.I. du Pont de Nemours and Company Haskell Laboratory for Health and Environmental Sciences Newark, DE 19714 USA 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 & Engineering Glasgow Building 300, P.O. Box 6101 Newark, DE 19714-6101 USA E M S E R 13-03/4842 Page 6 of 28 OOOO10 DuPont EMSE Report No. 13-03 3.0 3.1 3.1.1 Analytical Chemist: Technical Personnel: Bogdan Szostek, Ph.D. E.I. du Pont de Nemours and Company Haskell Laboratory for Health and Environmental Sciences Newark, DE 19714, USA Patrick W. Folsom, DuPont Central Research & Development Keith B. Prickett, DuPont Haskell Laboratory Study Execution Dates Experimental Start Date: Experimental Completion Date: Study Completion Date: 31 -December-2002 28-January-2003 20-March-2003 Materials and Methods Test System Test Substance Name: Synonym: Active substance(s) CAS Name: Molecular weight CAS Number(s): Structure: 8-2 Telomer B Alcohol (Perfluorooctyl)ethanol, 8-2 TBA 8-2 Telomer B Alcohol, 99% 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10heptadecafluoro464.12 g mole'1 678-39-7 OH Lot Number: EMSE Sample Number: Concentration of a.s., nominal: Concentration of a.s., analyzed: Major impurity P.00/001 E93386-80 99% 99.2% 0.8% as C7Fi5CF=CHCH2OH EMSER13-03/4842 Page 7 o f 28 O O O O ll DuPont EMSE Report No. 13-03 3.1.2 3.1.3 3.1.4 3.1.5 3.1.6 3.1.7 Certificate of Analysis Date: Date Received: Solubility at 25C.: Vapor pressure: Stability: Appearance/Color: Storage Conditions: Safety Precautions: 13-Sept-2001 26-Mar-2002 -140 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 f Mineral Medium One day before the initiation of the test, 2 mL each of mineral stock solutions A, B, C, and D (See Table A-4 for mineral medium stock solution preparation) were added to 2 liters of sterile deionized water and the mineral medium was sterile filtered into two Nalgene 1 liter filter units. Container Coating with 8-2 TBA Solution Several polypropylene gallon jugs with lids (Pretium packaging, Case ID# YZ2Q07) were sterilized under UV light overnight in a Biohood. Five days before the initiation of the test, one of the jugs was filled with 4 liters of sterile water from Bamstead E-Pure system (Megohm-cm = 17.5). A 0.4 mL aliquot of 8-2 TBA stock solution (3 mg/mL in ethanol) was added to the jug for a final concentration of 300 pg/L and was stirred for 4 days. After rinsing with sterile water, the jug was capped for later use to make 8-2 TBA saturated mineral medium. Preparation o f Saturated 8-2 TBA Solution Five days before the initiation of the test, another jug was filled with 3 liters of sterile water from Bamstead E-Pure system (Megohm-cm = 17.5) and was autoclaved. After cooling down, 0.6042 g of 8-2 TBA was added to the jug for a final concentration of 201 mg 8-2 TBA/L and the solution was stirred on a stir plate for 4 days. The solution in the jug was transferred to sterile centrifuge bottles and was centrifuged to remove the particular matter. The supernatant was transferred to the pre-coated jug prepared in Section 3.1.4 and was referred as saturated 8-2 TBA solution. Preparation o f 8-2 TBA Saturated Mineral Medium Three milliliter each of Mineral stock solutions A, B, C, and D was added to the saturated 8-2 TBA solution (3 liters) and was mixed by inversely shaking the jug. The mixed solution was referred as 8-2 TBA saturated mineral medium and was used the next day. Activated Sludge Collection Approximately 4 liters of activated sludge was collected from the City of Wilmington (DE) Municipal Waste Treatment Facility (POTW) - Aeriation Basin #2 on December 31, 2002. After arriving at the test lab, the sludge was assigned an ID number E93386-105. 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 E M S E R 13-03/4842 Page 8 of 28 000012 3.1.8 3.2 3.2.1 3.2.2 3.2.3 3.2.4 DuPont EMSE Report No. 13-03 sludge was filtered through a Nylon net with a pore size of 85 pm. This is referred to as the activated sludge filtrate. A 20 mL aliquot of the filtrate was transferred to a glass scintillation vial with a plastic transfer pipette and was autoclaved. The autoclaved sludge filtrate was referred to as killed sludge filtrate. Test Units Test vessels were 60 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 Four types of experimental treatments were conducted with pre-cleaned glassware and septa and aluminum foil. The pre-cleaning was done by rinsing them with methanol once and then with sterile deionized water 3 times. Treatment 1 - 8-2 TBA Saturated Mineral Medium plus Activated Sludse Filtrate For a total of 16 glass serum bottles (4 replicates x 4 sampling time points), 34.82 mL of 8-2 TBA saturated mineral medium and 0.175 mL of activated sludge filtrate was added to each of the glass serum bottles with plastic transfer pipettes. The bottles were crimped with pre-cleaned aluminum foil and PTFE/silicone septa. Treatment 2 - 8-2 TBA Saturated Mineral Medium plus Killed Sludse Filtrate For a total of 16 glass serum bottles (4 replicates * 4 sampling time points), 34.82 mL of 8-2 TBA saturated mineral medium and 0.175 mL of killed sludge filtrate was added to each of the glass serum bottles with plastic transfer pipettes. The bottles were crimped with pre cleaned aluminum foil and PTFE/silicone septa. This treatment served as an abiotic control. Treatment 3 - Mineral Medium plus Activated Sludse Filtrate for 8-2 TBA Spike Recovery For a total of 16 glass serum bottles (4 replicates x 4 sampling time points), 34.82 mL of the mineral medium and 0.175 mL of activated sludge filtrate was added to each of the glass serum bottles with plastic transfer pipettes. The bottles were crimped with pre-cleaned aluminum foil and PTFE/silicone septa. At each sampling time points (Days 0, 7, 14, and 28), 4 bottles were spiked (dosed) with 140 pL of 8-2 TBA stock solution (112.5 mg/L in ethanol) for a final concentration of 448 pg/L. The bottles were incubated for approximately 30 min with 200-300 RPM shaking with an orbitory shaker (New Brunswick Scientific Company, Model G-10, Serial number 880511137) before sample collection and sample extraction. Treatment 4 - Mineral Medium plus Activated Sludge Filtrate For a total of 8 glass serum bottles (2 replicates * 4 sampling time points), 34.82 mL of mineral medium and 0.175 mL of activated sludge filtrate was added to each of the glass serum bottles with plastic transfer pipettes. The bottles were crimped with pre-cleaned aluminum foil and PTFE/silicone septa. These samples served as sample matrix controls during quantification of 8-2 TBA and its possible transformation products. EMSER13-03/4842 Page 9 of 28 000013 DuPont EMSE Report No. 13-03 3.2.5 3.2.5.1 Test Conditions and Sampling Sample Incubation The crimped glass serum bottles were incubated with 200-300 RPM of shaking with an orbitory shaker (New Brunswick Scientific Company, Model G-10, Serial number 880511137) at room temperature in the dark inside a chemical hood. The temperature of the test system was monitored and recorded throughout the course of the study. 3.2.5.2 Sampling Interval Four crimped serum bottles for Treatment 1-3 and 2 crimped serum bottles for Treatment 4 were sampled for extraction of 8-2 TBA, fluoride (F~ion), and other metabolites at day 0 (31Dec-2002), day 7 (7-Jan-2003), day 14 (14-Jan-2003), and day 28 (28-Jan-2003). 3.2.5.3 Sample Storage Analytical samples were stored at approximately -20C. 3.3 Sample Extraction and Analysis 3-3.1 3.3.1.1 3.3.1.2 Sample Collection and Extraction Sample Collection At days 0, 7,14, 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 was withdrawn with a 10-mL polypropylene syringe. Five milliliter was injected into a 30 mL glass serum bottle and covered with pre-cleaned aluminum foil and was stored in a freezer for further analysis. Another 5 mL left in the syringe was injected into a 15 mL polypropylene tube that contained 0.05 mL of 5 N sodium hydroxide for fluoride extraction. Sample Extraction Fluoride extraction: The 15 mL polypropylene tubes containing the 5 mL test medium plus NaOH were incubated at room temperature for 3 - 4 h with 250 - 300 RPM of shaking. Then 0.042 mL of 6N H2SO4 was added to each of the tubes to neutralize the test medium. The sample tubes were stored in a-20C freezer for later fluoride analysis. 8-2 TBA extraction: Before extraction, MTBE-H2S 04 solvent system was prepared by adding 5.56 mL of 6N H2SO4 in 500 mL of chilled MTBE. A 30 mL aliquot of the chilled MTBE-H2SO4was then injected into each of the crimped sample bottles after the 10 mL of test medium was withdrawn from the bottles. After the MTBE- H2SO4 was injected, the sample bottles were incubated at room temperature for approximately 2 h with 250 - 300 RPM shaking. After settling the MTBE phase, the crimped sample bottles were decapped and the MTBE phase from each of the sample bottles was transferred with a glass pipette to 50 mL polypropylene centrifuge tubes and kept in a freezer. A 20 mL aliquot of MTBE was added to the sample bottles after the first MTBE phase was transferred to the polypropylene centrifuge tubes. The sample bottles were recapped with the original aluminum-lined Teflon septa and were incubated at room temperature for approximately 1 h with 250 - 300 RPM shaking. The MTBE phase was then transferred with a glass pipette to the polypropylene centrifuge tubes that contained the first MTBE phase. The combined 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. From each of the glass scintillation vials, EMSER13-03/4842 Page 10 of 28 000014 DuPont EMSE Report No. 13-03 2 mL aliquot of the MTBE phase was transferred to a GC vial and was sealed with pre cleaned aluminum-lined crimp cap for GC/MS quantification of 8-2 TBA. 3.3.1.3 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 o f Test Substance 8-2 TBA Analysis: Analytical standards: The 1H,1H, 2H, 2H-perfluorodecan-1-ol (8-2 TBA, CAS# 678-39-7,97.6%, Oakwood Products, West Columbia, SC) was used as the analytical standard. The ID, ID, 2D, 2D, 3-l3C-heptadecafluoro decanol (D-8-2 TBA, DuPont) was used as the internal standard. Stock solutions (1000 mg/L) of the analytical standard and the internal standard were prepared in methanol and refrigerated. The calibration standards were prepared freshly 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 pg/L 8-2 TBA. Constant level of internal standard was used: -300 pg/L of D-8-2 TBA. The calibration curves were constructed using the ratio of the peak area for ion m/z 31 (8-2 TBA) and 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-2 and an example of a chromatograph of sample (E93384EJ-29) separation was given in Figure A-3. 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 pL of 50 pg/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: GC/MS system: HP 6890 Plus GC (Agilent), HP 5973 Mass Selective Detector (Agilent), MPS2-MultiPurposeSampler (Gerstal) Column: DB-5MS, 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, 33,95, 98; dwell time: 25 ms for each ion EMSER13-03/4842 Page 11 of 28 o o o o iS DuPont EMSE Report No. 13--03 Retention time: 8-2 Telomer B Alcohol (8-2 TBA): 4.97 min Internal standard (D-8-2 TBA): 4.95 min Fluoride Analysis: Analytical standard: Certified standard of 100 mg/L of fluoride in water (Thermo Orion ) was used for standard calibration. The calibration standards were made in the range of 5 - 100 pg/L by dilution of the 100 mg/L of fluoride 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 8 mL aliquot of each of the standard solution was used for fluoride analysis to construct a standard curve for quantification of fluoride in samples. Quantification of Fluoride: The 5 mL test medium that was treated with NaOH and H2SO4from each of the 15 mL polypropylene tubes was thawed at room temperature and was centrifuged. Four milliliter aliquot from each of the tubes was transferred to a 50 mL polypropylene tube that contained 4 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 twice. A standard curve was generated by plotting the standard fluoride concentration in LOG scale versus millivolts (Figure A -1) for quanti fication of fluoride. 4.0 Results and Discussion 4.1 Under the test conditions, 8-2 TBA is not readily biodegradable (Figure 1, Table 1, and Table A-l). Spike recovery of 8-2 TBA from the test medium averaged 106 5% at day 0, 91 3% at day 7, 94 2% at day 14, and 74 2% at day 28 (Table A-2), indicating that MTBE extraction method obtained a good recovery of 8-2 TBA from the test medium, except for day 28 samples. At day 7, the loss of 8-2 TBA in test vessels (Treatment 1) was ~8% more {p <0.05) compared with abiotic control vessels (Treatment 2). At day 14, the loss of 8-2 TBA in test vessels ( Treatment 1) was -13% more (p <0.0 1) compared with abiotic control vessels (Treatment 2). At day 28, the concentrations of 8-2 TBA were not distinguishable between the test system and the abiotic controls. Most likely, the loss of 8-2 TBA during this 28-day test was due to abiotic removal mechanisms - volatility and/or adsorption rather than biotransformation. 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 EMSER13-03/4842 Page 12 of 28 000016 DuPont EMSE Report No. 13-03 extraction due to adsorption to the glass walls of the test vessels and/or the sludge inoculum. 4.2 Under the test conditions, no defluorination of 8-2 TBA occurred during the 28-d test. The fluoride concentration was the same in the test medium during the test period, averaged 17.9 2.6 pg/L at day 0, 17.1 0.8 pg/L at day 7, 17.4 1.4 pg/L at day 14, and 17.5 1.0 pg/L at day 28 (Table A-3). Under the test conditions, perfluorooctanoic acid (CAS# 335-67-1) is unlikely to be a major metabolite because defluorination was not observed during the test. On a molar basis, for one mole of 8-2 TBA to be converted to one mole of perfluorooctanoic acid, two moles of fluoride would be released. No increase in fluoride concentration was observed during the test. As a result, the absence of fluoride concentration increase during the test indicated that perfluorooctanoic acid is unlikely a major metabolite (>10% of total mass present) of 8-2 TBA biotransformation under these test conditions. 5.0 Conclusion Under the test conditions at room temperature (~22C), 8-2 TBA is not readily biodegradable. Confirmed expectations that abiotic removal mechanisms - volatility and/or adsorption will be factors that need consideration when designing future studies with objective of achieving mass balance. 6.0 Limitations of the test Due to the low water solubility, volatility, and strong surface adsorption properties of 8-2 TBA, it is difficult to conduct a test with a good mass balance (90%) for the abiotic control, whereas, the mass balance achieved in this study was only 67%. During the test, the concentration of 8-2 TBA continued to decrease in the abiotic controls, complicating the experimental data interpretation. Taken together, the conclusion drawn from this test is indicative, not conclusive. Further studies will be conducted to determine the potential major biotransformation products of 8-2 TBA. 7.0 Reference 7.1 OECD Guideline for Testing of Chemicals, Section 3: Ready Biodegradability: 301 D Closed Bottle Test. (1992). E M S E R 13-03/4842 Page 13 of 28 000017 DuPont EMSE Report No. 13-03 Figure 1 8-2 Telomer B Alcohol (8-2 TBA) concentration during the 28-day test* Activated sludge filtrate + 8-2 TBA saturated mineral medium Killed sludge filtrate + 8-2 TBA saturated mineral medium 250 0 5 10 15 20 Days after the initiation * The test was initiated on 31 December 2002 and finished on 28 January 2003. The graph is derived from the original data of Table A -l. E M S E R 13-03/4842 Page 14 of 28 000018 DuPont EMSE Report No. 13-03 Figure 2 Fluoride concentration during the 28-day test* -- Activated sludge filtrate + 8-2 TBA saturated mineral medium --o -- Killed sludge filtrate + 8-2 TBA saturated mineral medium * The test was initiated on 31 December 2002 and finished on 28 January 2003. The graph is derived from the original data of Table A-3. EMSER13-03/4842 Page 15 of 28 000019 DuPont EMSE Report No. 13-03 Table 1 Concentration of 8-2 TBA at day 0 (31-Dec-2002), day 7 (7-Jan-2003), day 14 (14-JAN-2003), AND day 28 (28-JAN-2003)* Type of Treatments Time Treatment 1 - Activated sludge filtrate plus 8-2 TBA saturated mineral medium Day 0 7 14 28 Date 31-Dec-2002 7-Jan-2003 14-Jan-2003 28-Jan-2003 8-2 TBA Concentration u g i'' 209 5 f 185 10 153 11 144 13 Test Substance remaining At day 7, day 14, and day 28 Compared to Day 0$ % 100 89 73 69 Tnatm ant 2 - Killed sludge filtrate plus 8-2 TBA saturated mineral medium 0 7 14 28 31-Dec-2002 7-Jan-2003 14-Jan-2003 28-Jan-2003 204 7 197 6 176 9 136 28 100 97 86 67 * This table is derived from the original data of Table A-1. f Mean standard deviation; n = 4. Test substance remaining at Days 7, 14, and 28 compared to Day 0, % = (Cx/C0) * 100, where: C0= 8-2 TBA concentration at Day 0, C* = 8-2 TBA concentration at Days 7, 14, and 28. EMSER13-03/4842 Page 16 o f 28 OOOOk.0 DuPont EMSE Report No. 13-03 Table 2 Daily temperature readings with a calibrated digital thermometer INSIDE A CHEMICAL HOOD WHERE THE TEST VESSELS WERE INCUBATED 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 Date 31-Dec-2002 1-Jan-2003 2-Jan-2003 3-Jan-2003 4-Jan-2003 5-Jan-2003 6-Jan-2003 7-Jan-2003 8-Jan-2003 9-Jan-2003 10-Jan-2003 11-Jan-2003 12-Jan-2003 13-Jan-2003 14-Jan-2003 15-Jan-2003 16-Jan-2003 17-Jan-2003 18-Jan-2003 19-Jan-2003 20-Jan-2003 21-Jan-2003 22-Jan-2003 23-Jan-2003 24-Jan-2003 25-Jan-2003 26-Jan-2003 27-Jan-2003 28-Jan-2003 Temperature C 21.6 22.8t 21.8 21.1 23.5t 23.5f 22.4 22.3 21.9 22.1 22.0 23.8t 23.8t 22.0 22.2 22.0 22.3 21.9 23.0f 23.0f 21.9 22.1 22.1 22.3 21.9 25.0t 25.3f 21.7 22.0 22.0 Standard Deviation 0.3 t Temperature reading in the test lab recorded by a calibrated Dickson recorder (Model THDx, serial number 01118-247). The temperature readings by the recorder were not used for calculation of the daily average temperature. EMSER13-03/4842 Page 17 of 28 000021 DuPont EMSE Report No. 13-03 Appendix A: EMSER13-03/4842 Page 18 o f 28 000022 DuPont EMSE Report No. 13-03 Table A-1. Analytical results of 8-2 TBA concentration at day 0 (31-Dec-2002), DAY 7 (7-JAN-2003), DAY 14 (14-JAN-2003), AND DAY 28 (28-JAN-2003) Type of Rep Treatment No Time day E93384 EJ-1 Treatment 1 - Activated sludge filtrate plus 8-2 TBA saturated mineral medium E93384 EJ-2 0 0 1 1 2 2 E93384 EJ-3 03 3 E93384 EJ-4 04 Average 1 Standard Deviation! 4 8-2 TBA Analytical concentration /# '' 107 104 101 103 102 101 107 106 E93384 EJ-5 Treatment 2 - Killed sludge filtrate plus 8-2 TBA saturated mineral medium E93384 EJ-6 0 0 E93384 EJ-7 0 E93384 EJ-8 Average 1 Standard Deviation 0 1 1 2 2 3 3 4 4 102 99.4 97.4 98.2 106 97.2 107 104 8-2 TBA Analytical Average UBL-' 106 102 102 107 101 97.8 102 106 8-2 TBA final concentration! ugL-' 212 204 204 214 209 5 202 196 204 212 204 7 E93384 EJ-15 Treatment 1 - Activated sludge filtrate plus 8-2 TBA saturated mineral medium E93384 EJ-16 7 7 E93384 EJ-17 7 E93384 EJ-18 Average Standard Deviation 7 1 1 2 2 3 3 4 4 89.9 91.0 92.8 93.8 89.9 91.0 85.6 85.2 90.5 191 93.3 187 90.5 191 85.4 171 188110 E93384 EJ-19 Treatment 2 - Killed sludge filtrate plus 8-2 TBA saturated mineral medium E93384 EJ-20 7 7 E93384 EJ-21 7 E93384 EJ-22 Average Standard Deviation 7 1 2 2 3 3 4 4 98.4 97.9 96.6 97.0 103 103 97.4 95.1 98.2 196 96.8 194 103 206 96.3 193 197 6 EMSERl 3-03/4842 Page 19 of 28 000023 DuPont EMSE Report No. 13-03 Table A-1 (continued) Type of Rep Treatment No Time day E93384 EJ-29 Treatment 1 - Activated sludge filtrate plus 8-2 TBA E93384 EJ-30 saturated mineral medium 14 14 1 1 2 2 E93384 EJ-31 14 3 3 E93384 EJ-32 14 4 Average Standard Deviation 4 E93384 EJ-33 Treatment 2 - Killed sludge filtrate plus 8-2 TBA E93384 EJ-34 saturated mineral medium 14 14 1 1 2 2 E93384 EJ-35 14 3 3 E93384 EJ-36 14 4 Average Standard Deviation 4 8-2 TBA Analytical concentration fjgL' 78.5 83.7 78.3 77.7 68.6 68 77.6 78.9 90.5 90.7 83.1 80.1 88.4 87.6 91.0 91.1 8-2 TBA Analytical Average tjg i' 81.1 78 68.3 78.3 90.6 81.6 88.0 91.1 E93384 EJ-57 Treatment 1 - Activated sludge filtrate plus 8-2 TBA E93384 EJ-58 saturated mineral medium 28 28 E93384 EJ-59 28 E93384 EJ-60 Average 1 Standard Deviation 28 1 1 2 2 3 3 4 4 75 84 74.4 75.1 64.4 66.4 61.6 72.0 79.5 74.8 65.4 66.8 E93384 EJ-61 Treatment 2 - Killed sludge filtrate plus 8-2 TBA E93384 EJ-62 saturated mineral medium 28 28 E93384 EJ-63 28 E93384 EJ-64 Average 1 Standard Deviation 28 1 1 2 2 3 3 4 4 66.2 62.1 91.9 81.7 58.1 49.6 63.4 70.4 64.2 86.8 53.9 66.9 t Final concentration o f 8-2 TBA, Cf = Ca * (V MTBE/V, ) where: Ca= 8-2 TBA analytical average value, V mtbe = 50 mL (MTBE used to extract the test medium), V, = 25 mL (Test medium used fro extraction). J Standard Deviation was calculated using the final concentration o f 8-2 TBA; n = 4. 8-2 TBA final concentrationf vgC 162 156 137 157 153 11 181 163 176 182 176 9 159 150 131 134 144 13 128 174 108 134 136 28 E M S E R 13-03/4842 Page 20 of 28 000024 DuPont EMSE Report No. 13-.03 Table A-2. Analytical results of Spike recovery of 8-2 TBA from the sample matrix (Treatment 3, activated sludge filtrate plus mineral medium) at day 0 (31-Dec-2002), day 7 (7-Jan-2003), day 14 (14-Jan-2003), and day 28 (28-JAN-2003) Time Rep 8-2 TBA 8-2 TBA Final 8-2 TBA % of Spike Analytical Analytical Concentrationt Recovery^ No Concentration Average day M L 1 /#' E93384 EJ-9 0 1 223 227 454 101 1 230 E93384 EJ-10 0 2 249 252 504 113 2 254 E93384 EJ-11 0 3 232 233 466 104 3 234 E93384 EJ-12 0 4 240 239 478 107 4 Average 1 Standard Deviation 238 106 8 E93384 EJ-23 7 1 205 204 408 91 1 202 E93384 EJ-24 7 2 202 202 404 90 2 202 E93384 EJ-2S 7 3 196 196 392 88 3 195 E93384 EJ-26 7 4 213 212 424 95 4 Average Standard Deviation 210 91 3 E93384 EJ-37 14 1 1 E93384 EJ-38 14 2 2 E93384 EJ-39 14 3 3 E93384 EJ-40 14 4 4 Average Standard Deviation 211 212 212 212 201 211 213 211 212 212 206 212 424 95 424 95 412 92 424 95 94 2 EMSERl 3-03/4842 Page 21 o f 28 000025 DuPont EMSE Report No. 13-03 Table A-2 (continued) Time Rep 8-2 TBA Analytical No Concentration day V g V ` E93384 EJ-65 28 1 1 E93384 EJ-66 28 2 2 E93384 EJ-67 28 3 E93384 EJ-6S 28 3 4 4 Average Standard Deviation 163 160 168 168 164 177 161 164 8-2 TBA Analytical Average p g '; 162 168 171 163 Final 8-2 TBA Concentrationt M L ' 324 336 342 326 tFinal 8-2TBA concentration, Cf = Ca * (VMTBE/Vt-)where: Ca= 8-2 TBA analytical average value, V mtbe = 50 mL (MTBE used to extract the test medium), V, = 25 mL (Test medium used fro extraction). $ % of spike recovery, R = (Cf/C,) * 100, where: Cf = final 8-2 TBA concentration, C, = Concentration of 8-2 TBA spiked into the sample bottles (448 pg/L). % of Spike Recovery* 72 75 76 73 74 2 EMSER13-03/4842 Page 22 of 28 000026 DuPont EMSE Report No. 13-03 Table A-3. Analytical results of fluoride concentration at day 0 (31-Dec-2002), DAY 7 (7-Jan-2003), day 14 (14-JAN-2003), and DAY 28 (28-JAN-2003) Type of Rep Treatment No Time day E93384 EJ-1 Treatment 1 - Activated sludge filtrate plus 8-2 TBA E93384 EJ-2 saturated mineral medium 0 0 1 1 2 2 E93384 EJ-3 03 3 E93384 EJ-4 04 Average Standard Deviation} 4 Fluoride Analytical concentration ugL-' 10.7 10.4 9.3 9.4 7.4 8.3 7.6 8.1 Fluoride Analytical Average pgL' 10.6 9.4 7.9 7.9 Fluoride final concentrationf 21.2 18.8 15.8 15.8 17.9 2.6 E93384 EJ-5 Treatment 2 - Killed sludge filtrate plus 8-2 TBA E93384 EJ-6 saturated mineral medium 0 0 E93384 EJ-7 0 E93384 EJ-8 Average Standard Deviation 0 1 1 2 2 3 3 4 4 9.8 8.3 8.9 8.2 8.4 7.7 8.1 7.4 9.1 18.2 8.6 17.2 8.1 16.2 7.8 15.6 16.8 1.1 E93384 EJ-15 Treatment 1 - Activated sludge filtrate plus 8-2 TBA E93384 EJ-16 saturated mineral medium 7 7 E93384 EJ-17 7 E93384 EJ-18 7 Average Standard Deviation 1 1 2 2 3 3 4 4 7.8 9.0 7.6 8.6 7.1 9.8 9.3 8.8 8.4 16.8 8.1 16.2 8.5 17.0 9.1 18.2 17.1 0.8 E93384 EJ-19 Treatment 2 - Killed sludge filtrate plus 8-2 TBA E93384 EJ-20 saturated mineral medium 7 7 E93384 EJ-21 7 E93384 EJ-22 Average Standard Deviation 7 1 1 2 2 3 3 4 4 10.0 7.5 8.8 7.2 8.6 7.5 8.3 7.3 8.8 17.6 8.0 16.0 8.1 16.2 7.8 15.6 16.4 0.9 EMSER13-03/4842 Page 23 of 28 000027 DuPont EMSE Report No. 13-03 Table A-3 (continued) Type of Rep Fluoride Fluoride Treatment Analytical Analytical No Time concentration Average day ugL* E93384 EJ-29 Treatment 1 - Activated sludge filtrate plus 8-2 TBA E93384 EJ-30 saturated mineral medium 14 14 1 1 2 8.9 7.9 8.3 8.4 8.1 2 7.9 E93384 EJ-31 14 3 8.4 8.5 3 8.6 E93384 EJ-32 14 4 11.1 9.7 Average Standard Deviation E93384 EJ-33 Treatment 2 - Killed sludge filtrate plus 8-2 TBA E93384 EJ-34 saturated mineral medium 14 14 4 1 1 2 8.3 8.3 8.6 7.8 8.5 8.0 2 8.2 E93384 EJ-35 14 3 8.0 8.1 3 8.1 E93384 EJ-36 14 4 8.1 8.1 Average * Standard Deviation E93384 EJ-57 Treatment 1 - Activated sludge filtrate plus 8-2 TBA E93384 EJ-58 saturated mineral medium 28 28 4 1 1 2 8.0 9.3 9.3 8.6 9.3 9.0 2 9.3 E93384 EJ-59 28 3 8,0 8.2 3 8.3 E93384 EJ-60 28 4 8.3 8.5 Average Standard Deviation 4 8.6 E93384 EJ-61 Treatment 2 - Killed sludge filtrate plus 8-2 TBA E93384 EJ-62 saturated mineral medium 28 28 1 1 2 8.5 9.4 8.8 9.0 9.4 2 10.0 E93384 EJ-63 28 3 8.5 8.7 3 8.9 E93384 EJ-64 28 4 7.9 8.6 4 9.3 Average * Standard Deviation t Final fluoride concentration, Cf = Ca * [(V,+Vt,)/Vt] where: Ca= Fluoride analytical average value, V, = 4 mL (TISABII buffer added to the test medium for fluoride measurement), V, = 4 mL (Test medium used for fluoride measurement). JStandard Deviation was calculated using the final fluoride concentration; n = 4. Fluoride final concentrationt tjg L 1 16.8 16.2 17.0 19.4 17.4 * 1.4 17.0 16.0 16.2 16.2 16.4 * 0.4 18.6 18.0 16.4 17.0 17.5*1.0 18.0 18.8 17.4 17.2 17.9*0.7 EMSER13-03/4842 Page 24 of 28 000028 DuPont EMSE Report No. 13-03 Table A-4. Preparation of the mineral medium for the test Prepare the following stock solutions, using analytical grade reagents (From OECD 301 D Test Guidelines) (a) Potassium dihydrogen orthophosphate, KH2 P04 8.50 g Dipotassium hydrogen orthophosphate, K2HP04 21.75 g Disodium hydrogen orthophosphate, Na2HP04 Ammonium chloride, NH4C1 28.39g 0.50 g Dissolve in water and make up to 1 liter. The pH of the solution should be 7.4. (b) Calcium chloride, anhydrous, CaCl2 or Calcium chloride dihydrate, CaCl2.2H2 0 Dissolve in water and make up to 1 liter. 27.50 g 36.40 g (c) Magnesium sulfate heptahydrate, MgS04.7H20 Dissolve in water and make up to 1 liter. (d) Iron (III) chloride hexahydrate, FeCl2.6H20 Dissolve in water and make up to 1 liter. 22.50 g 0.25 g (e) Filter each stock solution through a sterilized filtration unit with 0.2 pm size pore. Note: In order to avoid having to prepare iron (III) chloride stock solution immediately before use, add one drop of concentrated HCI per liter. If a precipitate forms in a stock solution, replace it with a fresh-made solution. E M S E R 13-03/4842 Page 25 of 28 000029 DuPont EMSE Report No. 13-03 Figure A-1 A FLUORIDE STANDARD CALIBRATION CURVE USED FOR FLUORIDE QUANTIFICATION OF SAMPLES E93384EJ-1 TO E93384EJ-64 E M S E R 13-03/4842 Page 26 of 28 000030 DuPont EMSE Report No. 13-03 F ig ure A-2 A CALIBRATION CURVE USED FOR 8-2 TBA QUANTIFICATION OF SAMPLES E93384EJ-1 TO E93384EJ-28 A calibration curve used for samples E93384 EJ-1 to E93384 EJ-28. The concentration of the internal standard: 308 pg/L of D-8-2 TBA. The range of concentrations of 8-2 TBA: 25.5- 1020 pg/L. E M S E R 13-03/4842 Page 27 of 28 000031 DuPont EMSE Report No. 13-03 F igure A-3 A CHROMATOGRAPH OF SAMPLE E93384EJ-29 USED FOR 8-2 TBA ANALYSIS Abundance Ion 31.00 (30.70 to 31.70): 01220323.D Tim e- Abundance 8000 4.95 Ion 33.00 (32.70 to 33.70): 01220323.D 6000 4000 2000 0 Time-> I I I | I I I I f I I I / p T T T 'll I I I I | T I I I | r n ' P I I I | I I I I | I'T I ! | 'H "H | )""l I I | I I I I | I' H I [ I 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 E93384 EJ-29; Ion 31, retention time 4.97 min = 8-2 TBA; Ion 33, RT 4.95 min = D-8-2 TBA (internal standard). E M S E R 13-03/4842 Page 28 of 28 00003*