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AR226-2692 \ AR226-2692 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: 301 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 E M S E R 13-03 ' EMSER13-03/4842 Page 1 of 28 DuPont EMSE Report No. 13-03 Page Reserved for Specific Country Requirements EMSERl 3-03/4842 Page 2 of 28 DuPont EMSE Report No. 13-03 Certification of Authenticity Ready Biodegradation of 8-2 Telomer B Alcohol (Modiei O E C D 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: jpi T. Gannon, Ph.D. ssearch 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 Date 03 EMSER13-03/4842 Page 3 of 28 DuPont EMSE Report No. 13-03 Table of Contents Page Reserved for Specific Country Requirements....................................................................................2 Certification o f Authenticity............................................................................................................................ 3 Table o f Contents................................................................................................................................................ 4 1.0 Summary................................................................................................ 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................................................................................................... --lO 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 o f 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 o f 8-2 TBA at day 0 (31-Dec-2002), day 7 (7-Jan-2003), day 14 (14-Jan-20Q3), and day 28 (28-Jan-2003)*............................................................................. .^....16 Table 2 Daily temperature readings with a calibrated digital thermometer inside a ...r 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 (7- Jan-2003), day 14 (14-Jan-2003), and day 28 (28-Jan-2003)......................................................19 Table A-2. Analytical results of Spike recovery o f 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 o f fluoride concentration at day 0 (31-Dec-2002), day 7 (7Jan-2003), day 14 (14-Jan-2003), and day 28 (28-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 o f samples E93384EJ-1 to E93384EJ-64..............................................................................................26 Figure A-2 A calibration curve used for 8-2 TBA quantification of samples E93384EJ-1 to E93384EJ-28............................................................................................ Figure A-3 A chromatograph o f sample E93384EJ-29 used for 8-2 TBA analysis........................ 28 EMSER13-03/4842 Page 4 of 28 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 fthe Study This test generates environmental fate information relevant to assess the potential for environmental persistence o f a test substance. When a chemical enters the environment, biodegradation is one of the major routes that determines the environmental fate o f 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 o f the 301 D test guidelines. Because the duration o f 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 v-.,ou-?< primary biodegradation potential of 8-2 TBA, through measurement o f parent loss and a ui ; formation o f fluoride, not the ultimate environmental fate of the test chemical. To assess foil umme ?* biodegradation potential o f fluorinated chemicals such as 8-2 TBA, a test system with ./maiee 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 o f 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 o f individually crimped test vessels (glass serum bottles) and the test was conducted at room temperature (~22C). The saturated solution o f 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 o f different experimental treatments were sacrificed for extraction and analysis. Potential degradation o f 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 and 13% more conditions, the loss of 8-2 TBA from the test vessels at day 14 compared with the abiotic control vessels. wAatsd~a8y%28m, tohree at day 7 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 DuPont EMSE Report No. 13-03 Conclusion: Under the test conditions, 8-2 TEA 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 o f 8-2 TBA by monitoring its concentration during the test Determine the degree o f defluorination o f 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 30ID 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 EMSER13-03/4842 Page 6 of 28 DuPont EMSE Report No. 13-03 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 3.0 Materialsand Methods 3.1 Test System 3.1.1 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, h3,e3p,t4a,d4e,5c,a5f,l6u,o6r,o7-,7,8,8,9,9,10,10,10464.12 g mole'1 678-39-7 F OH Lot Number: EMSE Sample Number: Concentration o f a.s., nominal: Concentration of a.s., analyzed: Major impurity P.00/001 E93386-80 99% 99.2% 0.8% as C7F15CFCHCH2OH EMSER13-03/4842 Page 7 of 28 DuPont EMSE Report No. 13-03 Certificate o f 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 3.1.2 Reference Substance None 3.1.3 Preparation o fMineral Medium One day before the initiation o f the test, 2 mL each o f 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. 3.1.4 Container Coating with 8-2 TBA Solution Several polypropylene gallon jugs with lids (Pretium packaging, Case ED# YZ2Q07) were sterilized under UV light overnight in a Biohood. Five days before the initiation o f 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 o f 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. 3.1.5 Preparation o fSaturated 8-2 TBA Solution Five days before the initiation o f the test, anotherjug 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 o f 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 The supernatant was transferred to the apnred-cwoaastecdenjutrgifpurgeepdartoedreinmSoevcetitohne p3a.1r.t4icaunladrwmaastter. referred as saturated 8-2 TBA solution. 3.1.6 Preparation o f8-2 TBA Saturated Mineral Medium Three milliliter each o f 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. 3.1.7 Activated Sludge Collection Approximately 4 liters of activated sludge was collected from the City o f 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 o f the EMSER13-03/4842 Page 8 o f28 DuPont EMSE Report No. 13-03 sludge was filtered through a Nylon net with a pore size o f 85 pm. This is referred to as the activated sludge filtrate. A 20 mL aliquot o f 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. 3.1.8 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. 3.2 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. 3.2.1 Treatment 1 - 8-2 TBA Saturated Mineral Medium plus Activated Sludee 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 o f 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. 3.2.2 Treatment 2 - 8-2 TBA Saturated Mineral Medium plus Killed Sludee Filtrate For a total o f 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 o f 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. 3.2.3 T R reatmen ecovery t 3 - Mineral Medium plus Activated Sludee Filtrate for 8-2 TBA Spike For a total o f 16 glass serum bottles (4 replicates x 4 sampling time points), 34.82 mL of the mineral medium and 0.175 mL o f activated sludge filtrate was added to each o f 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 ,1 4 , and 28), 4 bottles were spiked (dosed) with 140 pL o f 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. 3.2.4 Treatment 4 - Mineral Medium plus Activated Sludee Filtrate For a total of 8 glass serum bottles (2 replicates x 4 sampling time points), 34.82 mL of mineral medium and 0.175 mL o f 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 DuPont EMSE Report No. 13-03 3.2.5 3.2.5.1 3.2.5.2 3.2.5.3 Test Conditions and Sampling Sample Incubation The crimped glass serum bottles were incubated with 200-300 RPM o f 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 o f the test system was monitored and recorded throughout the course of the study. Sampling Interval Four crimped serum bottles for Treatment 1-3 and 2 crimped serum bottles for Treatment 4 were sampled for extraction o f 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). Sample Storage Analytical samples were stored at approximately -20C. 3.3 Sample Extraction andAnalysis 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 o f the test medium from each o f 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 pfroeleyzperrofpoyrlfeunrethtuerbeanthaalytscios.ntAainnoetdhe0r.055mmLLloefft5inNthseodsiyurmingheydwraosxiindjeefcoterdfliunotroidae1e5xmtraLction. 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 sample tubes 6wNerHe2sSto0r4edwains added to a -20C each o freezer f the tubes to neutralize the for later fluoride analysis. test medium. The tMa8ed-s2Tdt iBmTnEBgeA-d5Hi.eu52xmS6tr0mwa4cLawtsiaoowsnf it6:thNhBedneHrfaio2nwSrjnee0ce4ftrxeiotndrmai5nc0ttth0oioemenba,LocMthtolToef sBcf.htEhiA-leHlfect2derSriMm0th4pTeeBsdMoElsvT.aemBAnEtp3-lse0yHsbm2toeSLtmt0lae4wlswiqaaasufstopetirrneotjphefacetrhtee1edd0c,bmhtyhiLleleodf sample bottles were incubated at room temperature for approximately 250 - 300 RPM shaking. After settling the MTBE phase, the crimped 2sahmwpliethbottles 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 o f 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 DuPont EMSE Report No. 13-03 3.3.1.3 3.3.2 3.3.2.1 2 mL aliquot o f 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. Temperature Measurements The temperature o f the test system was monitored and recorded throughout the course of the study. Analytical Methods for Test Substance and Products Analysis o f Test Substance 8-2 TBA Analysis: Analytical standards: The 1H,1H, 2H, 2H-perfluorodecan-l-ol (8-2 TBA, CAS# 678-39-7, 97.6%, Oakwood Products, West Columbia, SC) was used as the analytical standard. The ID, ID, 2D, 2D, 3-13C-heptadecafluoro decanol (D-8-2 TBA, DuPont) was used as the internal standard. Stock solutions (1000 mg/L) o f 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 o f25-1000 pg/L 8-2 TBA. Constant level o f internal standard was used: ~300 pg/L o f 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 o f a calibration curve was given in Figure A-2 and an example o f a chromatograph of sample (E93384EJ-29) separation was given in Figure A-3. tAwgolaa0tssh.s5aeGnmvaCiLalylvzauielasidqlintu(wg1o.tia7coGemfbCtLhysevayoMrGliunTCmgB/eMeE,)t,Sph3heinpavssLitearolufrmwfo5aem0ns ttcphaagecp/cmpGoeLCrddoavinnifagDdlts-so8u(St-bh2ejeceTctfiBtooelAndlot3iwon.3itane.n1rgna.2clay)olsnwsitdsaa.instiEdopaanlarcsdch:ewsdaaimsnpaadleded 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: Flow rate: Split: Inlet temp.: Injection volume: MSD transfer line temp.: Ionization: SIM ions monitored: 80C for 2 min 20C/min to 120C 50C/min to 300C and hold for 3 min 1.0 mL/min; He; constant flow mode 5:1 250C 2 pL 280C El, 70 eV m/z: 31,33,95,98; dwell time: 25 ms for each ion EMSER13-03/4842 Page 11 of 28 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 o f 100 mg/L o f fluoride in water (Thermo Orion ) was used for standard calibration. The calibration standards were made in the range o f 5 - 100 pg/L by dilution of the 100 mg/L o f fluoride standard with TISABII (total ionic strength adjustment buffer II; Thermo Orion) and deionized water from Bamstead E-Pure system (Megohm-cm = 1 6 -1 8 ). Half strength TISABII (one part o f TISABII plus one part of deionized water) was used afonratlhyseisditloutcioonns.trAuc8t mL aliquot o f each o f the standard a standard curve for quantification solution was used for fluoride of fluoride in samples. Quantification o f Fluoride: pTohleyp5rmopLylteenstemtuebdeisumwatshatht awwaesdtraetarteodomwittehmNpaeOraHturaendanHd2wSa0s4cfreonmtriefaucghedo. f the 15 mL Four milliliter aliquot from each o f 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 Ionplus fluoride selective electrode (Thermo Orion, model 96-09, lot # GX1). After filling the reference chamber with reference electrode filling solution (Thermo Orion), the electrode was inserted into each 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 -l) for quantification o f 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 compared with abiotic control in test vessels (Treatment vessels (Treatment 2). 1) was ~ 8% more (p <0.05) A(pt<d0a.y011)4,cothmeplaorsesdowf 8it-h2aTbBioAticincotensttrovlevsseeslsse(lsT{rTearetmatemnet n1t)2w).as -13% more At day 28, the concentrations o f 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 o f 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 DuPont EMSE Report No. 13-03 extraction due to adsorption to the glass walls o f 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 pmeorlfalurobraosoisc,tafonroiocnaecmido, ltewoofm8-o2leTs BoAf fltuoobreidceownvoeurltdedbetoreolneeasmedo.leNoof 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) o f 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 o f Chemicals, Section 3: Ready Biodegradability: 301 D Closed Bottle Test. (1992). EMSER13-03/4842 Page 13 of 28 DuPont EMSE Report No. 13-03 I 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 --o-- Killed sludge filtrate + 8-2 TBA saturated mineral medium 250 OJ 200 oOO 150 <m 100 IN004E--01) 50 0 0 5 10 15 20 25 30 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 o f Table A -l. ill EMSER13-03/4842 Page 14 of 28 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 o f Table A-3. EMSER13-03/4842 Page 15 of 28 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-AN-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 209 5 f 185 10 153 11 144 + 13 Test Substance remaining At day 7, day 14, and day 28 Compared to Day Of % 100 89 73 69 Treatment 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 o f Table A -l. tJ TMeesatnsubssttaanndcaerrdemdeaviinaitniogna;tnD=ay4s. 7, 14, and 28 compared to Day 0, % = (Cx'/:Co)-x 100, where: Co = 8-2 TBA concentration at Day 0, Cx = 8-2 TBA concentration at Days 7,14, and 28. EMSER13-03/4842 Page 16 of 28 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 Date Temperature Day C 0 31-Dec-2002 21.6 1 1-Jan-2003 22.8f 2 2-Jan-2003 21.8 3 3-Jan-2003 21.1 4 4-Jan-2003 23.5f 5 5-Jan-2003 23.5f 6 6-Jan-2003 22.4 7 7-Jan-2003 22.3 8 8-Jan-2003 21.9 9 9-Jan-2003 22.1 10 10-Jan-2003 22.0 11 11-Jan-2003 23.8f 12 12-Jan-2003 23.8f 13 13-Jan-2003 22.0 14 14-Jan-2003 22.2 15 15-Jan-2003 22.0 16 16-Jan-2003 22.3 17 17-Jan-2003 21.9 18 18-Jan-2003 23.0t 19 19-Jan-2003 23.0f 20 20-Jan-2003 21.9 21 21-Jan-2003 22.1 22 22-Jan-2003 22.1 23 23-Jan-2003 22.3 24 24-Jan-2003 21.9 25 25-Jan-2003 25.0f 26 26-Jan-2003 25.3T 27 27-Jan-2003 21.7 28 28-Jan-2003 22.0 Average, n = 20 22.0 Standard Deviation 0.3 f 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 DuPont EMSE Report No. 13-03 Appendix A: EMSERl 3-03/4842 Page 18 of 28 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 E93384 EJ-3 0 E93384 EJ-4 Average Standard Deviation:): 0 1 1 2 2 3 3 4 4 8-2 TBA Analytical concentration vgV 107 104 101 103 102 101 107 106 8-2 TBA Analytical Average mV 106 102 102 107 8-2 TBA final concentrationf mV 212 204 204 214 209 5 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 Standard Deviation 0 1 1 2 2 3 3 4 4 102 99.4 97.4 98.2 106 97.2 107 104 101 202 97.8 196 102 204 106 212 204 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 85.4 171 185 10 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 ^ j-2003 EMSER13-03/4842 Page 19 of 28 DuPont EMSE Report No. 13-03 Table A-1 (continued) Type of Rep Treatment No Time day E93384 EJ-29 Treatment 1 - Activated 14 sludge filtrate plus 8-2 TBA 1 1 E93384 EJ-30 saturated mineral medium 14 2 2 E93384 EJ-31 14 3 3 E93384 EJ-32 14 4 Average Standard Deviation 4 E93384 EJ-33 Treatment 2 - Killed sludge 14 filtrate plus 8-2 TBA 1 1 E93384 EJ-34 saturated mineral medium 14 2 2 E93384 EJ-35 14 3 3 E93384 EJ-36 14 4 Average Standard Deviation 4 8-2 TBA Analytical concentration 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 ugL" 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 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 28 1 66.2 64.2 filtrate plus 8-2 TBA 1 62.1 E93384 EJ-62 saturated mineral medium 28 2 91.9 86.8 2 81.7 E93384 EJ-63 28 3 58.1 53.9 3 49.6 E93384 EJ-64 28 4 63.4 66.9 4 70.4 Average Standard Deviation t Final concentration of 8-2 TBA, Cf = Cax (Vmxbe/V^) where: QVVtm==tb82e-52=mT5L0BAm(TLaesn(taMmlyTetiBdciaEulmuavsueedsreatdgoefervxoatreluaxectrt,athcteiotnes)t. medium), $ Standard Deviation was calculated using the final concentration of 8-2 TBA; n = 4. 8-2 TBA final concentrationf ggL'1 162 156 137 157 153 11 181 163 176 182 159 150 131 134 144 13 128 174 108 134 136 28 EMSER13-03/4842 Page 20 of 28 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 (3 1 -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 Concentrationf Recovery^ No Concentration Average day v a t1 ggL-' 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 Standard Deviation 238 106 5 E93384 EJ-23 7 1 205 204 408 91 1 202 E93384 EJ-24 7 2 202 202 404 90 2 202 E93384 EJ-25 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 of 28 DuPont EMSE Report No. 13-03 Table A-2 (continued) Time Rep 8-2 TBA 8-2 TBA Final 8-2 TBA Analytical Analytical Concentrationf No Concentration Average day w '' M L '1 jgL-` E93384 EJ-65 28 1 163 162 324 1 160 E93384 EJ-66 28 2 168 168 336 2 168 E93384 EJ-67 28 3 164 171 342 3 177 E93384 EJ-68 28 4 161 163 326 4 Average Standard Deviation 164 t Final 8-2 TBA concentration, Cf = Ca x (Vmtbe/VJ where: Ca= 8-2 TBA analytical average value, Vmtbe = 50 mL (MTBE used to extract the test medium), Vt= 25 mL (Test medium used fro extraction). $ %of spike recovery, R = (Cf/Cs) x 100, where: Cf= final 8-2 TBA concentration, Cs= Concentration of 8-2 TBA spiked into the sample bottles (448 |ig/L). % of Spike Recovery^ 72 75 76 73 74 2 EMSER13-03/4842 Page 22 of 28 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 0 1 1 E93384 EJ-2 saturated mineral medium 0 2 2 E93384 EJ-3 03 3 E93384 EJ-4 04 4 Average Standard Deviation^ Fluoride Analytical concentration jgL-` 10.7 10.4 9.3 9.4 7.4 8.3 7.6 8.1 Fluoride Analytical Average vgL'' 10.6 9.4 7.9 7.9 Fluoride final concentrationf vgL'1 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 saturated mineral medium E93384 EJ-16 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 DuPont EMSE Report No. 13-03 Table A-3 (continued) Type of Rep Fluoride Fluoride Treatment Analytical Analytical No Time concentration Average day m l -' FSfi'' E93384 EJ-29 Treatment 1 - Activated 14 sludge filtrate plus 8-2 TBA 1 1 8.9 7.9 8.4 E93384 EJ-30 saturated mineral medium 14 2 8.3 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 14 4 1 1 8.3 8.3 8.6 8.5 E93384 EJ-34 saturated mineral medium 14 2 7.8 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 4 8.0 Average Standard Deviation E93384 EJ-57 Treatment 1 - Activated sludge filtrate plus 8-2 TBA 28 1 1 9.3 9.3 9.3 E93384 EJ-58 saturated mineral medium 28 2 8.6 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 28 filtrate plus 8-2 TBA 1 1 8.5 9.4 9.0 E93384 EJ-62 saturated mineral medium 28 2 8.8 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 | FiAnvaelrfalgueoridSetacnodnacrdenDtreavtiaiotino,nCf = Cax [(Vj+VJ/VJ where: CVai == F4lmuoLri(dTeIaSnAaBlyItIicalbauvffeerragaeddveadluteo, the test medium for fluoride measurement), V{Stt=an4damrLd D(Teevsitatmioendiwumasucsaelcdufloartefdluuosriindgetmheeafsiunraelmfluenotr)i.de concentration; n = 4. Fluoride final concentrationf M 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 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 PO4 8.50 g Dipotassium hydrogen orthophosphate, K2 HPO4 21.75 g Disodium hydrogen orthophosphate, Ammonium chloride, NH4C1 N a2HP04 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, FeCl3.6H20 Dissolve in water and make up to 1 liter. 22.50 g ',i: 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 o f concentrated HC1 per liter. If a precipitate forms in a stock solution, replace it with a fresh-made solution. EMSER13-03/4842 II Page 25 of 28 DuPont EMSE Report No. 13-03 F ig u r e A-1 A FLUORIDE STANDARD CALIBRATION CURVE USED FOR FLUORIDE QUANTIFICATION OF SAMPLES E93384EJ-1 TO E93384EJ-64 EMSER13-03/4842 Page 26 of 28 DuPont EMSE Report No. 13-03 F ig u r e A-2 A CALIBRATION CURVE USED FOR 8-2 TBA QUANTIFICATION OF SAMPLES E93384EJ-1 TO E93384EJ-28 internal standard: 308 pg/L o f D-8-2 TBA. The range o f concentrations of 8-2 TBA: 25.5 - 1020 pg/L. EMSER13-03/4842 Page 27 of 28 DuPont EMSE Report No. 13-03 F ig u r e A-3 A CHROMATOGRAPH OF SAMPLE E93384EJ-29 USED FOR 8-2 TBA ANALYSIS 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). EMSER13-03/4842 Page 28 of 28