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BACK TO MAIN Biodegradation study Report The Aerobic Biodegradation of W-EtFOSE Alcohol by the Microbial Activity Present in Municipal Wastewater Treatment Sludge Thursday, November 02, 2000 Project Number 3M Project ID: LIMS E00-2252 Pace Contract Analytical Project ID: CA058 Pace Proposal ID: 000511.1 Principal Analytical Investigator Cleston C. Lange, Ph.D. CLIENT James K. Lundberg, Ph.D., 3M Environmental Laboratory Bldg 2-3E-09, P.O. Box 33331, St.Paul, MN 55133-3331 Contract Laboratory Pace Analytical Services, Inc. Science Solutions Division 1700 Elm Street, Suite 200 Minneapolis, Minnesota 55414 Project Dates Project Initiation: July 20, 2000 Project Completion: October 1, 2000 Author Cleston C. Lange, Ph.D. Report 2 of 2 for project CA058, Revised Page 1 of 55 Aerobic Biodegradation of ^-EtFOSE alcohol Cleston C. Lange, Ph.D., Pace Analytical Services, Science Solutions Division Wednesday November 1, 2000 BACK TO MAIN Table of Contents Title Page..................................................................................................................................................... 1 Table of Contents......................................................................................................................................... 2 List of Tables & Figures............................................................................................................................... 3 List of Appendices........................................................................................................................................ 3 Executive Summary.....................................................................................................................................4 1.0 Project Personnel.................................................................................................................................. 5 2.0 Data Requirements...............................................................................................................................5 3.0 Project Objective................................................................................................................................5-6 4.0 Test Article............................................................................................................................................6 5.0 Control & Reference Articles............................................................................................................ 7-8 6.0 Receipt/Generation of Samples............................................................................................................9 7.0 Methods................................................................................................................................................ 9 7.1 Sample Preparation ........................................................................................................................9 7.1.1 Collection of Sludge..........................................................................................................9-10 7.1.2 Culture Setup..................................................................................................................10-12 7.1.3 Solid Phase Extraction (SPE)..........................................................................................12-13 7.2 Instrumental Analysis....................................................................................................................14 7.3 Data Transformations and Calculations........................................................................................15 7.3.1 Molar Calculations..............................................................................................................15 7.3.2 Conversion of ng/mL to micromolar and nanomolar...........................................................15 7.3.3 Mass Balance Calculations.................................................................................................16 7.4 Statistics........................................................................................................................................17 7.4.1 Formula for Standard Deviation..........................................................................................17 7.4.2 Formula for Relative Standard Deviation............................................................................17 7.5 Software Versions.........................................................................................................................17 8.0 Results................................................................................................................................................ 18 8.1 Incubator, Refrigerator, and Freezer Temperature Log Data.......................................................18 8.2 Mishaps recorded during the study..........................................................................................19-20 8.3 Sludge Characterization Report....................................................................................................21 8.3.1 Metals.................................................................................................................................. 21 8.3.2 Wet Chemistry..................................................................................................................... 21 8.4 Quality Control/Sample Matrix Spike Results...............................................................................22 8.5 HPLC/MS Calibration and Calibration Verification.................................................................. 23-24 8.6 Blanks ......................................................................................................................................... 24 8.7 Matrix Spikes................................................................................................................................. 24 8.8 Surrogate Spikes........................................................................................................................... 24 8.9 Sample Results............................................................................................................................. 25 8.9.1 Results ofincubations with 47 ng/mL W-EtFOSE alcohol...............................................25-27 8.9.2 Results of incubations with 2,380 ng/mL W-EtFOSE alcohol........................................ 28-34 9.0 Conclusions.................................................................................................................................. 34-36 10.0 Sample and Data Retention..............................................................................................................37 Page 2 of 55 Aerobic Biodegradation of W-EtFOSE alcohol Cleston C. Lange, Ph.D., Pace Analytical Services, Science Solutions Division Wednesday November 1, 2000 BACK TO MAIN List of Tables & Figures Table 1: Solid phase extraction % recovery table for quality control matrix spike samples..................... 22 Figure 1: Degradation curve of W-EtFOSE alcohol at a test concentration of 47 ng/mL (nM vs. time)....25 Figure 2: Production of W-EtFOSAA in cultures containing 47 ng/mL W-EtFOSE alcohol........................26 Figure 3: Production of metabolite M556 in cultures containing 47 ng/mL W-EtFOSE alcohol................ 26 Figure 4: Production of FOSA in cultures containing 47 ng/mL W-EtFOSE alcohol...........................27 Figure 5: Production of PFOS in cultures containing 47 ng/mL W-EtFOSE alcohol...........................27 Figure 6: Degradation curve of W-EtFOSE alcohol at a test concentration of 2,380 ng/mL (|j.Mvs. time) 28 Figure 7: Degradation curve of W-EtFOSE alcohol at a test concentration of 2,380 ng/mL (% vs.time)..29 Figure 8: Second-Order relationship for W-EtFOSE alcohol biodegradation (1/conc vs. tim e).................30 Figure 9: Production of W-EtFOSAA in cultures containing 2,380 ng/mL of W-EtFOSE alcohol.............. 31 Figure 10: Production of metabolite M556 in cultures containing 2,380 ng/mL of W-EtFOSE alcohol.....32 Figure 11: Production of PFOSulfinate in cultures containing 2,380 ng/mL of W-EtFOSE alcohol......... 33 Figure 12: Production of FOSA in cultures containing 2,380 ng/mL of W-EtFOSE alcohol................33 Figure 13: Production of PFOS in cultures containing 2,380 ng/mL of W-EtFOSE alcohol................34 Figure 14: Production of PFOA in cultures containing 2,380 ng/mL of W-EtFOSE alcohol................34 Figure 15: Deduced metabolic pathway for degradation of W-EtFOSE alcohol........................................ 36 List of Appendices Appendix A: Signatures of Project Personnel........................................................................................... 38 Appendix B: Table 1. Sample results presented as ng/mL (ppb)...................................................... 39-47 Appendix B: Table 2. Sample results presented as molar values (|j.M)............................................. 48-50 Appendix C: Table 1. Biodegradation samples finalized data for 2,380ng/mL samples ........................ 51 Table 2. Biodegradation samples finalized data for 47 ng/mL samples............................. 51 Appendix D: Table 1. Sterilized/inhibited sludge controls finalized data for 2,380 ng/mL samples.........52 Table 2. No-sludge controls finalized data for the 2,380 ng/mL samples...........................52 Table 3. Sterilized/inhibited sludge controls finalized data for the 47 ng/mL samples...... 53 Table 4. No-sludge controls finalized data for the 47ng/mL samples.................................53 Appendix E: Table 1. Percent Degradation data for the 2,380 ng/mL biodegradation samples..............54 Appendix F: Table 1. Mass balance data table for all samples and control samples............................. 55 Page 3 of 55 Aerobic Biodegradation of ^-EtFOSE alcohol Cleston C. Lange, Ph.D., Pace Analytical Services, Science Solutions Division Wednesday November 1, 2000 BACK TO MAIN Executive Summary A study was undertaken to determine the biodegradability of W-EtFOSE alcohol [2-(N-ethylperfluorooctane sulfonamido) ethyl alcohol] when exposed to municipal wastewater treatment sludge. W-EtFOSE alcohol is a monomer used in the synthesis of fluorinated surfactant and protective chemical products. The results of this study elucidate the environmental fate of this compound and related compounds upon entry into the environment, either directly or through municipal waste treatment processes. The aerobic biodegradability of W-EtFOSE alcohol was tested during a 5-week, 8 time-point study. The study utilized municipal wastewater treatment sludge as microbial inoculum and employed test concentrations of 0.047 pg/mL (90 nM) and 2.38 pg/mL (4.168 pM) W-EtFOSE alcohol in a mineral salts medium. Analytical results demonstrate that W-EtFOSE alcohol is readily biodegraded, with 100% degradation of the 0.047pg/mL samples in 14 days and 90% of 2.38 pg/mL in 35 days. The major metabolites identified from test cultures were W-EtFOSAA and M556, which together accounted for 85% of the total products observed in the day 35 cultures. Observed minor metabolites included W-EtFOSA, FOSA, PFOSulfinate, PFOS and PFOA. Mass balance between parent material and measured products was achieved, implying that all significant degradation products of W-EtFOSE alcohol were identified and that PFOS and/or PFOA are the endpoints in the biodegradation. This work promises to serve as a model for studying the biotransformation of other perfluorooctanesulfonate and fluorinated telomer-alcohol based chemistries. Page 4 of 55 Aerobic Biodegradation of ^-EtFOSE alcohol Cleston C. Lange, Ph.D., Pace Analytical Services, Science Solutions Division Wednesday November 1, 2000 BACK TO MAIN 1.0 Project Personnel 1.1 Sponsor 1.2 Sponsor Representative 1.3 Contract Facility Personnel: 1.3.1 Study Director 1.3.2 Laboratory Management: 1.3.3 Sample Preparation Analysts 1.3.4 1.3.5 1.3.6 1.3.7 1.3.8 HPLC/MS Analyst Sludge Characterization Report Sample Custodian: Report Author Report Reviewer 3M Company Dr. James K. Lundberg Dr. Cleston C. Lange Mr. Mark T. McCann Ms. Angela L. Schuler Dr. Cleston C. Lange Mr. Anthony E. Scales Mr. Daryl K. Peterson Dr. Cleston C. Lange Dr. Cleston C. Lange Mr. Dirk W. Hoogenboom 2.0 Data Requirements The sponsor representative desired a proposal for a biodegradation study to be conducted using W-EtFOSE Alcohol as the degradant. After brief discussions with Dr. James K. Lundberg and Dr. William K. Reagan of the 3M Environmental Laboratory concerning details of draft proposals, copies of a final proposal (Pace Proposal # 000511.1) were submitted to them on Tuesday, May 18, 2000. The study was to be conducted as a non-GLP study but with the understanding that good data quality objectives be met. Approval was given to proceed with the project via a letter received from Dr. James K. Lundberg and dated May 23, 2000. 3.0 Project Objective The objective of the proposed study was to determine whether W-EtFOSE alcohol will degrade under aerobic conditions using the inherent microbial populations of municipal waste treatment plant mixed liquor suspended solids "sludge" as the inoculum. The proposed study incorporated EPA-published guidelines OPPTS 835.3200, OPPTS Page 5 of 55 Aerobic Biodegradation of W-EtFOSE alcohol Cleston C. Lange, Ph.D., Pace Analytical Services, Science Solutions Division Wednesday November 1, 2000 BACK TO MAIN 835.3210, and OPPTS 835.5045. Degradation rates, identification of product analytes, and quantitation of all suspected products and the parent material for mass balance determinations was proposed. Initially, a 3-day preliminary study was conducted that indicated that W-EtFOSE alcohol was readily biodegraded. This preliminary study showed the production of expected perfluorinated intermediates with the concomitant decrease in W-EtFOSE alcohol concentration. Subsequently, a 2-week multiple sample point screening study was conducted to work out difficulties in the analytical procedures that might prove costly if encountered in a larger study. The 2-week screening study provided direct evidence that W-EtFOSE alcohol is biodegraded at a test concentration well above its suspected water solubility, that the test concentration of 2.5 mg/L is not inhibitory to microbial growth, and that the compound degrades to predictable products in a stoichiometric fashion. Furthermore, that study confirmed that the extraction methods and analytical procedures efficiently recovered products and parent analyte from the culture matrix. A report for the 2-week screening study was provided to 3M on August 11, 2000, and provided the first direct evidence that this compound was biotransformed to smaller molecular weight fluorinated compounds. This 35-day study was conducted in order to elucidate the biodegradation pathway of W- EtFOSE alcohol; to determine the sequence of events leading to the observed products; and to determine whether the perfluorooctanoate (PFOA), which was detected below the MQL near the end of the 2-week screening study, is truly a product of the biodegradation of W-EtFOSE alcohol. 4.0 Test Article The test article used was HPLC purified W-EtFOSE Alcohol (3M Std I.D. # SE-035). The 3M Environmental Laboratory provided approximately 3 grams of W-EtFOSE alcohol for testing on June 8, 2000. The W-EtFOSE alcohol was delivered accompanied by a Material Safety and Data Sheet (MSDS) and a chain of custody (COC) as 3M Environmental Lab. COC # 14681. Upon receipt at Pace, the material was given a test, control and reference (TCR) number CA-TCR02-008. The purity of the material was not provided at the time the study was conducted, but an interim certificate of analysis was provided on October 31, 2000 and the purity was reported at 99.9%. Page 6 of 55 Aerobic Biodegradation of W-EtFOSE alcohol Cleston C. Lange, Ph.D., Pace Analytical Services, Science Solutions Division Wednesday November 1, 2000 BACK TO MAIN 5.0 Control & Reference Articles Reference materials were received from the 3M Environmental Laboratory on June 8, 2000. Approximately 1 gram of each of the following materials was received with accompanying purity information, MSDS, and chain of custody (COC # 14681). Upon receipt at Pace, each was given a Pace test, control, & reference number: Note: M556 was received from the 3M Environmental Laboratory as a methanol solution. An MSDS, and a chain of custody were not provided for this material. NC: purity analysis not completed. All purity information provided for control & reference articles was archived with the raw data for the study. All test, control, and reference materials were stored at -77C + 0.2C. All final data in data tables for this report are calculated assuming 100% purity of test and reference materials because purity information was not complete for all compounds. 5.1. Perfluorooctanoate Ammonium Salt (PFOA) The purity was determined at 97-99% by HPLC/MS as part of this study. 3M#: TCR-99131-37 Pace #: CA-TCR02-001 O' nhJ 5.2. Perfluorooctane sulfinate Potassium Salt (PFOSulfinate) Purity: NC 3M#: SD-007 Pace #: CA-TCR02-002 FFFFFFFF o F FFFFFFF ** 5.3. Perfluorooctane sulfonate Potassium Salt (PFOS) Purity: 86.4% 3M#: SD-009 Pace #: CA-TCR02-003 Page 7 of 55 Aerobic Biodegradation of ^-EtFOSE alcohol Cleston C. Lange, Ph.D., Pace Analytical Services, Science Solutions Division Wednesday November 1, 2000 BACK TO MAIN 5.4. Perfluorooctane sulfonamide (FOSA) Purity: NC 3M#: SD-029 F F F F F F FFO Pace #: CA-TCR02-004 5.5. /V-Ethyl perfluorooctane sulfonamide (W-EtFOSA) Purity: NC 3M#: SD-012 Pace #: CA-TCR02-005 FFFFFFFFO 5.6. [2-(Perfluorooctane sulfonamido) acetic acid] (M556). Purity: NC 3M#: 00001-5-24 Pace#:CA-TCR02-006 FFFFFFFFO 5.7. [2-(W-Ethyl perfluorooctane sulfonamido) acetic acid] (W-EtFOSAA) Purity: NC 3M#: SE-038 Pace #: CA-TCR02-007 F FF FF FF FFF Ff F0 N T FF 1 Oh CH3 5.8. [2-(W-Ethyl perfluorooctane sulfonamido) ethyl alcohol] (W-EtFOSE alcohol) Purity: 99.9% 3M#: SE-035 Pace #: CA-TCR02-008 Page 8 of 55 Aerobic Biodegradation of ^-EtFOSE alcohol Cleston C. Lange, Ph.D., Pace Analytical Services, Science Solutions Division Wednesday November 1, 2000 BACK TO MAIN 6.0 Receipt/Generation of Samples Samples were not received for this project, but were generated as an inherent part of this study. Each of the one hundred and ninety-two experimental cultures were extracted by solid phase extraction (SPE) methodology to generate five hundred and seventy-six analytical samples, and referred to as SPE eluates 1, 2 and 3. Of the three eluates generated for each of the samples, eluate 1 was the aqueous sample wash and eluates 2 and 3 were separately collected 25 mL methanol elutions of the sample washed SPE cartridges. Data obtained for eluates 2 and 3 from the 2-week screening study demonstrated quantitative recovery of target compounds, and thus it was expected that no analytes are lost to the aqueous eluate 1. Because degradation of W-EtFOSE alcohol was observed, the analysis of the positive-control cultures containing sodium dodecyl sulfate (SDS) as a degradant, was deemed unnecessary. 7.0 Methods 7.1 Sample Preparation 7.1.1. Collection of Sludge. To prepare the test cultures, sludge was obtained from the primary municipal waste treatment facility in the Twin Cities area. Arrangements were made for Pace personnel to retrieve fresh mixed liquor suspended solids (MLSS) from the aeration units at the Twin Cites Metro Wastewater Treatment Facility (also referred to as Pig's Eye Sewer Treatment Plant) located in St. Paul, MN. Six liters of MLSS was collected by Pace laboratory personnel on July 31, 2000 and delivered as six 1-liter Nalgene polypropylene bottles containing MLSS, and were accompanied with a corresponding chain of custody (Pace C.O.C. # 465254). Upon receipt, individual bottles were labeled #1 through #6. Bottle #1 was subsequently delivered to Mr. Daryl Peterson, of the Minnesota Environmental Laboratory at Pace Analytical Services, for sludge characterization. The sludge characterization report was received on August 16, 2000 and is summarized in section 8.3. Page 9 of 55 Aerobic Biodegradation of ^-EtFOSE alcohol Cleston C. Lange, Ph.D., Pace Analytical Services, Science Solutions Division Wednesday November 1, 2000 BACK TO MAIN The MLSS in bottles #2 and #3 were allowed to settle overnight and the settled sludge was used to prepare cultures for the 5-week study. At the time that the 5-week study was begun, the settled MLSS had been stored 12 hours at 4C. The settled sludge in each bottle constituted approximately 20% of the volume, or approximately 200 mL in a 1-liter sample, based on visual inspections. 7.1.2. Culture Setup. All cultures were prepared on August 1, 2000 and the final sample collection occurred on September 5, 2000. Slight modifications to the original proposed methods (Pace proposal 05011.1) were deemed necessary based on findings from the 3-day and 2-week screening studies. Changes were made with regard to culture volume (changed from 20 mL to 25 mL) and the number of test concentrations (only two concentrations were tested, not three as originally proposed). Cultures were prepared using a mineral salts medium defined in EPA Guideline OPPTS 835.3200. The mineral salts medium pH was 7.4 and contained per liter, 0.334 g Na2HPO4-2H2O, 0.005 g NH4Cl, 0.2175 g K2HPO4, and 0.085 KH2PO4, 0.0275 g CaCl2-anhydrous, 0.0225 g MgSO4-7H2O, and 0.00025 g FeCl3-6H2O. Per one liter of mineral salts medium in a 2 L Erlenmeyer flask, 50 mL of settled sludge was added. This mineral salts medium plus sludge mixture was then used to prepare the 25 mL sample cultures. A separate 500 mL of mineral salts medium plus sludge was prepared as described above and was sterilized in an autoclave at 121C for 30 minutes and then cooled to ambient room temperature. Upon cooling, this sterile sludge medium was treated with 100 pg/mL of chloramphenicol as a microbial growth inhibitor, and used to prepare the 25 mL sterile/inhibited control cultures. Page 10 of 55 Aerobic Biodegradation of ^-EtFOSE alcohol Cleston C. Lange, Ph.D., Pace Analytical Services, Science Solutions Division Wednesday November 1, 2000 BACK TO MAIN Mineral Salts medium, un-sterilized and without sludge, was used to prepare the 25 mL no sludge controls. Individual cultures and controls were prepared by dispensing 25 mL of appropriate mineral salts medium solution into sterile 125 mL Nalgene polycarbonate culture flasks containing labels with appropriate information. Mineral salts medium containing sludge had to be swirled to homogenize the mixture before dispensing. Samples were spiked with either 5 pL of a 11,900 pg/mL solution of NEtFOSE alcohol in methanol (CA058-SS-001) or 5 pL of a 238 pg/mL solution of N-EtFOSE alcohol prepared in methanol (CA058-SS-012) to prepare the 2.380 pg/ mL (4.168 pM) samples and 0.047 pg/mL (90 nM) samples, respectively. Blank controls received only the appropriate mineral medium and no analyte. The day zero samples were prepared and immediately placed in a freezer that was maintained at -19C + 7C. All other cultures were placed in temperature controlled shaking incubators that were maintained at 28C + 2C. Samples were pulled from incubators at designated time points of days 1, 3, 7, 14, 21, 28 and 35. Following the first week of incubation, the samples to be collected on days 14, 21, 28 and 35, received weekly additions of 1 mL of mineral salts medium plus sludge (stored at 4C) in order to add fresh sludge as a food source to maintain microbial activity. The differences in volume were not a factor in the analysis because the entire sample volumes were extracted using SPE at the end of the incubation period. Upon removal from the incubator, samples were either immediately frozen, or immediately prepared by solid phase extraction. All sample preparation information, including times, analyte additions, etc. were recorded in sample preparation worksheets and signed and dated by the sample preparation analyst. All original data sheets were maintained in project specific binders. Page 11 of 55 Aerobic Biodegradation of ^-EtFOSE alcohol Cleston C. Lange, Ph.D., Pace Analytical Services, Science Solutions Division Wednesday November 1, 2000 BACK TO MAIN The positive control cultures containing sodium dodecyl sulfate (SDS) were prepared by adding 25 |j.L of a stock solution of SDS (50,043 mg/L) to make a final concentration of 50.043 mg/L (173.5 |j.M), as suggested by EPA guideline OPPTS 835.3200. Two sets of QC test control samples were prepared in mineral salts medium containing sludge. Each set consisted of five independently prepared 25 mL cultures containing mineral salts medium plus sludge in a 125 mL flask, to which a multi-component standard containing each of the analytes of interest was added. The final concentrations of the five QC control samples were approximately 10, 50, 200, 500 and 1000 ng/mL (exact amounts are accounted for in data calculations). These QC control samples were allowed to sit for 15 minutes at room temperature and were then extracted by the SPE methodology described for this study. The data obtained from HPLC/MS analysis of eluate 2 and eluate 3 from the QC control samples was used to determine the efficiency of analyte recovery from the culture matrix. 7.1.3. Solid Phase Extraction of Cultures A change was made from the original proposed procedure with regard to the solid phase extraction procedure. Different types of SPE cartridges were used. All Sample cultures and control cultures were prepared by solid phase extraction methodology using SEP-VAC C18 6cc SPE cartridges from Waters Corporation (Part No. WAT036905). A sample label was applied to each SPE cartridge prior to use, and each was packed with plug of quartz glass wool to deter plugging of the SPE filter. Each SPE cartridge was washed prior to use by drawing 5 mL of methanol and then 5 mL of aqueous 1% acetic acid solution through the cartridge. These wash solution eluates were discarded to waste. All of the SPE eluates for this study were collected in clear I-chem vials with labels that identified them as eluate 1, 2 or 3, as defined below. Page 12 of 55 Aerobic Biodegradation of ^-EtFOSE alcohol Cleston C. Lange, Ph.D., Pace Analytical Services, Science Solutions Division Wednesday November 1, 2000 BACK TO MAIN Samples that were frozen had to be thawed before extraction. Following thawing, and prior solid phase extraction, 0.25 mL of glacial acetic acid was added to each of the cultures yielding a final concentration of 1% acetic acid. The content of each acidified culture was swirled to mix, and then drawn by vacuum through the appropriately labeled SPE cartridge by carefully pouring the contents of the culture flask into the SPE cartridge. The aqueous eluate was collected in an I-chem vial labeled eluate 1, removed from the vacuum manifold, and capped. Then, 25 mL of methanol was added to the culture flask, the flask sealed, and vigorously shaken. The cap was then removed from the flask, and the methanol content (25 mL) drawn through the SPE cartridge, collected in an I-chem vial labeled eluate 2. Eluate 2 was expected to contain a majority of the analyte that was in the original culture sample. As a precaution that some analyte may be retained in the SPE cartridge, or in the culture flask, a second 25 mL methanol step was conducted in a similar fashion to that used for eluate 2, and collected in a third I-chem vial, labeled eluate 3. Aliquots of eluates 2 and 3 were transferred to autovials, capped, and then quantitatively analyzed by HPLC/MS. The remaining volume of each eluate was stored at 4C. Page 13 of 55 Aerobic Biodegradation of ^-EtFOSE alcohol Cleston C. Lange, Ph.D., Pace Analytical Services, Science Solutions Division Wednesday November 1, 2000 BACK TO MAIN 7.2 Instrumental Analysis All quantitative analysis was conducted on an HP1100 high performance liquid chromatograph with mass spectrometer detector (HPLC/MSD) system. The HPLC/MSD system was set up with the same chromatography configuration as that described in method ETS 8-136.0 which utilizes a dual-column setup followed by a pressure relief valve serving as a flow through splitter. The MSD was operated in electrospray ionization in negative ion mode using selected ion monitoring (SIM). Ions monitored were: m/z 413 (PFOA); m/z 483 (PFOSulfinate); m/z 498 (FOSA); m/z 499 (PFOS); m/z 526 (W-EtFOSA); m/z 556 (M556); m/z 584 (W-EtFOSAA); and m/z 630 (W-EtFOSE alcohol). Other expected products, such as analytes II, V and VI in Figure 15, were not monitored, as reference standards for those compounds were not available. All analysis was quantitative, with 8 calibration standards prepared from 5 to 1000 ng/mL (ppb) and containing each of the analytes being quantified. Typical injection volumes for samples and calibration standards were 50 |j.L, except where otherwise noted for instrumental dilutions in section 8.3 below. The 4.6 x 150 mm Betasil C8 column used for this study had serial number 0501383T, lot number P9G09 and the 4 x 35 mm NG1 column used had serial number 15123. The pressure-regulated splitter had no identifying number to distinguish it. Part numbers for ordering can be found in method ETS 8-136.0 MSD Settings: Ionization mode: API-ES negative Gas Temp: 300C Drying gas: 8.0 L/minute Nebulizer Pressure: 30 psig Vcap:3500V Fragmentor: 70V EMV Gain: 2.0 Actual Dwell for each ion: 146 SIM resolution: high HPLC Settings: Flow: 1 mL/min, splitter used Time (min). %A %B 0.00 97.0 3.0 0.50 97.0 3.0 6.00 5.0 95.0 8.50 5.0 95.0 8.51 97.0 3.0 10.50 97.0 3.0 Page 14 of 55 Aerobic Biodegradation of ^-EtFOSE alcohol Cleston C. Lange, Ph.D., Pace Analytical Services, Science Solutions Division Wednesday November 1, 2000 BACK TO MAIN 7.3 Data Transformations and Calculations 7.3.1 Molar Calculations: Because all data was collected on an ng/mL basis (part per billion, ppb), a transformation from ng/mL to molar concentrations had to be conducted to obtain mass balance information. The mole conversion values used for each of the analytes were as follows: PFOA- NH4+ salt = 431 nanogram (ng) per nanomole (nmole) PFOSulfinate- K+ salt = 522 ng/nmole PFOS- K+ salt = 538 ng/nmole FOSA = 499 ng/nmole W-EtFOSA = 527 ng/nmole M556 acid = 557 ng/nmole W-EtFOSAA = 585 ng/nmole W-EtFOSE alcohol = 571 ng/nmole 7.3.2 Conversion of ng/mL to micromolar (^M) and nanomolar (nM). (Working Examples): 500 ng/mL PFOS = (500 ng/mL)*(1 nmole/522 ng) = 0.958 nmole/mL = 0.958 ^mole/liter = 0.958 |j.M 50 ng/mL PFOS = (50 ng/mL) * (1 nmole/522 ng) = 0.0958 nmole/mL = 0.0958 |imole/L = 0.0958 |iM = 95.8 nM Page 15 of 55 Aerobic Biodegradation of ^-EtFOSE alcohol Cleston C. Lange, Ph.D., Pace Analytical Services, Science Solutions Division Wednesday November 1, 2000 BACK TO MAIN 7.3.3 Mass Balance Calculations: Convert all ng/mL values to their corresponding molar concentrations as pM or nM (see section 7.3.2, above). Then, sum the quantified molar values (pM) for all analytes in eluate 2 and eluate 3. Divide the sum of the analyte concentrations by the known concentration of starting compound and represent as a percentage of the known starting concentration. (Working Example): If, the starting concentration of W-EtFOSE alcohol was at 1800 pM And, after incubation, the following were determined: PFOA was detected at 50 pM PFOS was detected at 100 pM W-EtFOSAA was detected at 500 pM W-EtFOSE alcohol was detected at 1000 pM Then, the mass balance is as follows: Mass balance = [(50pM + 100pM + 500pM + 1000pM)/1800pM] X 100% Mass balance = (1650pM/1800pM) X 100% = 91.7% Page 16 of 55 Aerobic Biodegradation of W-EtFOSE alcohol Cleston C. Lange, Ph.D., Pace Analytical Services, Science Solutions Division Wednesday November 1, 2000 BACK TO MAIN 7.4 Statistics 7.4.1 Formula used for determination of Standard Deviation (SD): SD =(I%2/n-1 )1/2= [ I (X - X )2/(n-1)]1/2 I%2 = the sum of the squared differences of deviations between the values of individual data points and the mean: = I [(Xi - X )2] I = The sum of all values in a population Xi = the value of an individual data point n = the total number of samples, where one sample is represented as (i). X = mean of all X in the sample population = ( I X) / n 7.4.2 Formula used for Relative Standard Deviation (RSD): The RSD is a ratio of the standard deviation (SD) to the arithmetic mean of the replicate analysis and expressed as a percent. RSD = (SD / mean of Xn) * 100% 7.5 Software Versions MicrosoftTM Excel 2000 was used for data processing and producing tables. MicrosoftTM Word 2000 was used for processing the analytical report text. Page 17 of 55 Aerobic Biodegradation of ^-EtFOSE alcohol Cleston C. Lange, Ph.D., Pace Analytical Services, Science Solutions Division Wednesday November 1, 2000 BACK TO MAIN 8.0 Results 8.1 Incubator, Refrigerator, and Freezer Temperature Log Data. During this study, three reciprocal shaker incubators were used. Pace ID numbers for the incubator-shakers were 0267, 0268 and 0269. Shaker Speeds on incubators were set to 200 rpm, but were not regularly calibrated. A -20 C freezer, Norlake freezer, was used during the study to store the cultures until they could be prepared by solid phase extraction. The freezer ID was 0050 and was situated at Pace/3M Tier-2 facility in location SL-R6. One IsoTemp ultra-low freezer (-80C) was used for storage of test, control and reference materials. The freezer ID was 0241 and was situated at Pace in location SL-R4. Two refrigeration units, a Carroll walk-in cooler and a True refrigerator, were used to store SPE-prepared samples and stock and calibration standards. The coolers were identified as ID 0140 (location SL-R1) and ID 0213 (location SLR8), respectively. The average temperatures + the standard deviation for each incubator, refrigerator, and freezer are shown below for the dates in which they were used. The average temperatures and standard deviations are based on readings that were recorded on a daily basis, excluding weekends and holidays. Incubator-shaker, ID 0267, from dates 7/31/00 to 8/8/00: Incubator-shaker, ID 0268, from dates 7/31/00 to 8/21/00: Incubator-shaker, ID 0269, from dates 7/31/00 to 9/5/00: Norlake Freezer, ID 0050, from dates 7/31/00 to 9/5/00: IsoTemp Freezer, ID 0241, from dates 7/31/00 to 9/5/00: Carroll walk-in cooler, ID 0140, from dates 7/31/00 to 9/5/00: True refrigerator, ID 0213, from dates 7/31/00 to 9/5/00: 27C + 1C 28 + 2C 28C + 1C -19C + 7 C -77.0C + 0.2C 4C + 2C 2.5C + 1.3C Page 18 of 55 Aerobic Biodegradation of ^-EtFOSE alcohol Cleston C. Lange, Ph.D., Pace Analytical Services, Science Solutions Division Wednesday November 1, 2000 BACK TO MAIN 8.2 Mishaps recorded during the Study. During the study, two incubator failures occurred as a result of shaker motor failure and overheating. These failures were addressed immediately upon that finding by transferring all cultures contained within the incubator to another functional incubator. On occasion, incubator temperatures rose above 30C as a result of building air conditioning failure. Low incubator temperatures are highly dependent on ambient room temperature since only 1 of 5 incubators used was refrigerated (ID 0269). Specific temperature data can be found in the incubator logs as part of the facility records. The occasional temperature spikes were not expected to have a profound effect on the study. Below is an account of the mishaps recorded. On August 21, 2000, incubator 0267 failed with a high temp weekend reading of 40 C but had returned to 27C and shaking had been maintained. The study was not affected by this since all cultures in that incubator (days 1, 3 and 7) had been collected and prepared by August 8, 2000. On August 21, 2000, incubator 0268 failed, also with a high temp weekend reading of 36C and a shaker motor failure. Shaking had ceased for an unknown time, but less than 3 days. The study may have been affected by this mishap since cultures for days 21, 28 and a subset of day 35 samples were contained in that incubator when it failed. Immediately upon discovery of the failure on August 21, 2000, all affected cultures were transferred to functioning incubators 0274 and 0269 for the remainder of the study. Day 21 cultures (CA058-0801-SA-106 to -126) were transferred to incubator ID 0274, day 28 cultures (CA058-0801-SA127 to -147), and the subset of day 35 cultures (CA058-0801-SA-148 to -156) were transferred to incubator ID 0269. During the study, all of the long-term test cultures (longer than 7-day incubations) were to receive weekly additions of fresh sludge, with the exception of the sterile/inhibited controls and the no-sludge control cultures. On August 8, 2000, two of the sterile/inhibited control cultures mistakenly received fresh sludge. Because active sludge was added to these negative controls, biodegradation of W-EtFOSE alcohol occurred for two weeks. The Identification numbers for those Page 19 of 55 Aerobic Biodegradation of ^-EtFOSE alcohol Cleston C. Lange, Ph.D., Pace Analytical Services, Science Solutions Division Wednesday November 1, 2000 BACK TO MAIN two controls were CA058-0801-SA-113 and CA058-0801-SA-114. The data for those samples was not used when determining mass balance. Oddly, no biodegradation products were observed in those two samples even though a reduced concentration of the W-EtFOSE alcohol was observed. Page 20 of 55 Aerobic Biodegradation of ^-EtFOSE alcohol Cleston C. Lange, Ph.D., Pace Analytical Services, Science Solutions Division Wednesday November 1, 2000 BACK TO MAIN 8.3 Sludge Characterization Report Analysis of the mixed liquor suspended solids used for this study yielded the following. Sludge was collected on August 1, 2000 and was digested on August 3, 2000. The Pace project number for the sludge characterization report was 1035161. 5-Day BOD determinations were not performed, however, for future studies it is recommended. 8.3.1 Metals: Boron: 600 pg/L Cadmium: 12.9 pg/L Calcium: 124,000 pg/L Chromium: 273 pg/L Cobalt: 15.6 pg/L Copper: 1,450 pg/L Iron: 31,000 pg/L Lead: 140 pg/L Magnesium: 29,700 pg/L Manganese: 12,200 pg/L Molybdenum: 60 pg/L Nickel: 123 pg/L Selenium: 28 pg/L Potassium: 30,500 pg/L Zinc: 1,320 pg/L 8.3.2. Wet Chemistry: Sulfate: 57.1 mg/L Orthophosphate: 6.8 mg/L Phosphorous: 56.7 mg/L Ammonia: 1.3 mg/L Kjeldahl Nitrogen: 197 mg/L Chemical Oxygen Demand (COD): Total Organic Carbon (TOC): Total suspended solids: Total solids: Page 21 of 55 Aerobic Biodegradation of ^-EtFOSE alcohol Cleston C. Lange, Ph.D., Pace Analytical Services, Science Solutions Division Wednesday November 1, 2000 3,440 mg/L 120 mg/L 2,280 mg/L 3,070 mg/L BACK TO MAIN 8.4 Quality Control/Sample Matrix Spike Results. Analyte recovery from sample matrices was determined by conducting solid phase extraction (SPE) of spiked sludge matrix samples. The percent recovery was calculated for each analyte based on the known amount spiked into the sample matrix. The recoveries were determined for 5 analyte concentrations ranging from 10 to 1000 ng/mL. SPE quality control samples were prepared in duplicate and labeled CA058-MW-023A to CA058-MW-027A and CA058-MW023B to CA058-MW-027B. The summed quantitative results of the HPLC/MS evaluation of eluate 2 and eluate 3 from each sample are listed below in Table 1. All analytes, except M556, were recovered at near 90 to 100%. M556 was not recovered from the 10 ppb sample. Recovery from the higher concentrations of M556 averaged 73.1%. S a m p le ID D escrip tio n CA058-MW-023A SPE QC SAMPLE, 10 PPB IN SLUDGE FOR RECOVERY CA058-MW-023B SPE QC DUPLICATE SAMPLE, 10 PPB IN SLUDGE FOR RECOVERY CA058-MW-024A SPE QC SAMPLE, 50 PPB IN SLUDGE FOR RECOVERY CA058-MW-024B SPE QC DUPLICATE SAMPLE, 50 PPB IN SLUDGE FOR RECOVERY CA058-MW-025A SPE QC SAMPLE, 200 PPB IN SLUDGE FOR RECOVERY CA058-MW-025B SPE QC DUPLICATE SAMPLE, 200 PPB IN SLUDGE FOR RECOVERY CA058-MW-026A SPE QC SAMPLE, 500 PPB IN SLUDGE FOR RECOVERY CA058-MW-026B SPE QC DUPLICATE SAMPLE, 500 PPB IN SLUDGE FOR RECOVERY CA058-MW-027A SPE QC SAMPLE, 1000 PPB IN SLUDGE FOR RECOVERY CA058-MW-027B SPE QC DUPLICATE SAMPLE, 1000 PPB IN SLUDGE FOR RECOVERY PFOA 91 .2% 1 0 1 .2% 1 0 7 .1% 105 .4% 1 0 0 .2% 107 .0% 101 .0% 102 .6% 98 .1% 9 7 .9% PFOS R ecovery (% ) PFO S ulfinate M 556 N -E tFO S A A FOSA N -EtFO SEa lc o h o l N -EtFO SA 9 7 .4% 9 1 .6% 0 .0% 8 6 .8% 9 1 .9% 96 .7% 8 5 .4% 97 .3% 94 .1% 0 .0% 8 9 .4% 93 .2% 91 .1% 84 .1% 1 10 .9% 107 .9% 9 7 .4% 112 .1% 110 .1% 1 02 .1% 1 00 .8% 1 13 .9% 107 .6% 8 9 .8% 112 .7% 111 .3% 1 08 .3% 1 0 3 .2% 1 06 .9% 1 0 4 .5% 6 9 .2% 104.3% 103.2% 103.3% 98 .3% 1 08 .0% 1 0 5 .3% 7 2 .1% 104 .6% 102 .1% 1 02 .2% 9 6 .4% 9 7 .6% 1 0 0 .3% 6 5 .0% 9 6 .3% 9 7 .4% 98 .2% 93 .2% 9 9 .8% 100 .9% 7 0 .5% 9 9 .8% 98 .7% 9 9 .6% 9 3 .0% 1 00 .0% 9 7 .6% 5 9 .5% 9 8 .7% 96 .5% 9 5 .0% 9 3 .0% 9 8 .0% 97 .5% 6 1 .4% 9 6 .1% 93 .3% 90 .1% 89 .2% Table 1. Solid phase extraction percent recovery data determined by quantitative HPLC/MS analysis of eluate 2 and eluate 3 for each sample. Each matrix spike was prepared in duplicate and are shown. Page 22 of 55 Aerobic Biodegradation of ^-EtFOSE alcohol Cleston C. Lange, Ph.D., Pace Analytical Services, Science Solutions Division Wednesday November 1, 2000 BACK TO MAIN 8.5 HPLC/MS Calibration and Calibration Verification. HPLC/MS multi-component calibration standards contained PFOA, PFOSulfinate, PFOS, FOSA, W-EtFOSA, M556, W-EtFOSAA and W-EtFOSE alcohol. Eight calibration standards were prepared which contained these analytes at approximately 5, 10, 20, 40, 100, 200, 500 and 1000 ng/mL, and labeled CA058-MW-014 to CA058-MW-021. For quantitation, the exact concentration values were used. Calibration standards were prepared in methanol in 10 mL amounts and stored in clear I-chem vials at 4C in refrigerator ID 0213. Aliquots were transferred to autovials and capped for use in HPLC/MS runs. Continuing calibration verification (CCV) was used in sequence runs, and included both CA058-MW-016 (20 ng/mL) and CA058-MW-019 (200 ng/mL) for checking both low and high range curves data as described below. Instrument calibration was performed for each analyte with all eight calibration standards injected. Analytes were quantified by use of either the low 5 calibration points (5 to 100 ng/mL) or the high 5 calibration points (40 to 1000 ng/mL). Analyte concentrations below 100 ng/mL were reported based on the value given for low calibration curve data, and with rare exception, all residuals in that range were 100% + 15%. Values reported between 100 and 1000 ng/mL were determined from high calibration curve data, and again residuals on values in that range were 100% + 15% with rare exception. For samples with concentrations determined to be above 1000 ng/mL, the samples were injected again as 10 |j.L injections and quantified off of calibration curves of which 50 |j.L was injected, for an instrumental dilution factor of 5. Values reported were multiplied by a factor of 5 for calculation of the final concentration. Data for dilutions was obtained using high range curve values, with residuals in the range of 100 to 1000 ng/mL of 100% + 15% with rare exception. Calibration check and verification standards (CCVs) were used after 15 sample injections and included both calibration standards CA058-MW-016 (20 ng/mL) and CA058-MW-019 (200 ng/mL) for verification of low and high range curves. CCVs were flanked by methanol blanks. All quantitation was conducted using Page 23 of 55 Aerobic Biodegradation of ^-EtFOSE alcohol Cleston C. Lange, Ph.D., Pace Analytical Services, Science Solutions Division Wednesday November 1, 2000 BACK TO MAIN averaged calibration curves flanking 30 samples, and one set of CCVs and accompanying methanol blanks. All calibration curves were slightly quadratic and had R-values of at least 0.990. All data was determined to be of good to excellent quality, with residuals on curves and CCVs generally in the range of 100% + 15%. All data was collected as external standard calibration, without the use of an internal standard as suggested by ETS-8-136.0. Instrumental sequence runs were setup as described in method ETS 8-136.0 and contained both calibration curves and CCVs with accompanying methanol blanks. 8.6 Blanks Methanol blanks were injected onto the HPLC/MS column and quantitatively analyzed to determine the background analyte concentration in the methanol used for sample preparation. Sample blanks, consisting of either mineral salts medium or mineral salts medium containing sludge, and no analyte, were prepared and incubated in an identical manner to real samples. Sample blank results were used to determine whether the sample matrix contained any of the analytes of interest. No fluorochemical analytes were detected in the sample blanks. 8.7 Matrix Spikes Matrix sample spikes were not employed for this study. Matrix spike recovery information was determined using the QC matrix spiked sample results described in section 8.1. 8.8 Surrogate Spikes Surrogate spikes were not employed during this study. Page 24 of 55 Aerobic Biodegradation of ^-EtFOSE alcohol Cleston C. Lange, Ph.D., Pace Analytical Services, Science Solutions Division Wednesday November 1, 2000 BACK TO MAIN 8.9 Sample Results 8.9.1 Results of incubations with 47 ng/mL of W-EtFOSE alcohol. All data for this report are calculated assuming 100% purity of test and reference materials since purity information was not available for all compounds. All reported concentrations for samples and controls described in this section can be found in Table 1 of Appendix B. Summarized data for the 47 ng/mL samples can be found in Table 2 of Appendix C and summarized data for controls can be found in Table 3 and Table 4 of Appendix D. All graphs presented in this section are not "mathematically fit" curves, but are hand drawn to accent the trend of the data. From the graph in Figure 1, it is obvious that biodegradation occurred rapidly and was complete by day 14. Based on Figure 1, the biodegradation half-life estimate at this test concentration was < 2 days. 100.00 90.00 80.00 70.00 60.00 50.00 40.00 30.00 20.00 10.00 0.00 0 5 10 15 20 25 30 35 40 Incubation time (days) Figure 1. The 35-day biodegradation of 47 ng/mL (90 nM) W-EtFOSE alcohol by aerobic microorganisms present in municipal sludge, versus the no-sludge and sterile/inhibited controls, demonstrated significant biodegradation. Values from triplicate samples are shown. The two lowest values at day 21 for the no-sludge controls are from samples CA058-0801-SA-113 and CA058-0801-SA-114 that suffered a sample preparation mishap where active sludge was added inadvertently at day 7. Quantitative results from triplicate samples are shown for each time point. Page 25 of 55 Aerobic Biodegradation of ^-EtFOSE alcohol Cleston C. Lange, Ph.D., Pace Analytical Services, Science Solutions Division Wednesday November 1, 2000 BACK TO MAIN The data demonstrate that W-EtFOSE alcohol is readily biotransformed to W-EtFOSAA (Figure 2), the expected 4-electron oxidation product of W-EtFOSE alcohol. During the 35 days of incubation, the W-EtFOSAA that formed also underwent biodegradation via a subsequent Wdealkylation reaction to yield M556 as shown in Figure 3. The M556 formed then underwent further W-dealkylation and oxidation reactions to yield FOSA (Figure 4) and PFOS (Figure 5). Other expected metabolites W-EtFOSA, PFOSulfinate, and PFOA were not detected above the analytical MQL for any of the 47 ng/mL samples. 0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 Incubation time (days) Figure 2. The production, and subsequent degradation, of W-EtFOSAA observed during the biodegradation of 47 ng/mL (90 nM) of W-EtFOSE alcohol. Quantitative results from triplicate samples are shown for each time point. Figure 3. The production of metabolite M556 observed from the biodegradation of 47 ng/mL (90 nM) of W-EtFOSE alcohol. Quantitative results from triplicate samples are shown for each time point. Page 26 of 55 Aerobic Biodegradation of ^-EtFOSE alcohol Cleston C. Lange, Ph.D., Pace Analytical Services, Science Solutions Division Wednesday November 1, 2000 BACK TO MAIN 0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 Incubation time (days) Figure 4. The production of FOSA observed from the biodegradation of 47 ng/mL (90 nM) of W-EtFOSE alcohol. Quantitative results from triplicate samples are shown for each time point. 0 5 10 15 20 25 30 35 40 Incubation time (days) Figure5. The production of PFOS observed from the biodegradation of 47 ng/mL (90 nM) of W-EtFOSE alcohol. Quantitative results from triplicate samples are shown for each time point. Page 27 of 55 Aerobic Biodegradation of ^-EtFOSE alcohol Cleston C. Lange, Ph.D., Pace Analytical Services, Science Solutions Division Wednesday November 1, 2000 BACK TO MAIN 8.9.2 The Results of incubation with 2,380 ng/mL of M-EtFOSE alcohol. All data for this report are calculated assuming 100% purity of test and reference materials since purity information was not available for all compounds. All raw data collected for samples and controls described in this section can be found in Table 1 of Appendix B. Summarized data for samples can be found in Table 1 of Appendix C and summarized data for controls can be found in Table 1 and Table 2 of Appendix D. All graphs, in this section, except for Figure 14, are not "mathematically fit" curves, but are hand drawn to accent the trend of the data. The 35-day incubation of 2,380 ng/mL (4.168 pM) W-EtFOSE alcohol with aerated sludge provided further insight into the metabolic pathway of biodegradation. As shown in Figure 6, the biodegradation of W-EtFOSE alcohol is rapid, with a majority of the degradation occurring in 21 days. By visual inspection of Figure 6, the half-life of W-EtFOSE alcohol in this test system was approximately 2 to 3 days. Interestingly, after 21 days of incubation, the degradation of W-EtFOSE alcohol nearly ceased with approximately 90% of the parent material degraded, shown in Figure 7 and Appendix E, Table 1. However, production of several products continued after the biodegradation of W-EtFOSE alcohol had ceased. Figure 6. The biodegradation of 2,380 ng/mL W-EtFOSE alcohol by aerobic microorganisms present in municipal sludge over 35 days (triangles). No sludge controls (squares), and sterile/inhibited controls (circles), are also shown. Quantitative results from triplicate samples are shown for each time point. Page 28 of 55 Aerobic Biodegradation of ^-EtFOSE alcohol Cleston C. Lange, Ph.D., Pace Analytical Services, Science Solutions Division Wednesday November 1, 2000 BACK TO MAIN 100.0% 90.0% 80.0% 70.0% 60.0% 50.0% (n 40.0% LU 30.0% 20.0% 10.0% 0.0% 0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 Incubation time (days) Figure 7. The percent biodegradation of W-EtFOSE alcohol during the 35 day incubation. The percentage value is based on the average analytically determined molar value from the day 0 triplicate samples (4.921 pM + 0.176 pM). By day 35, only 11.4% + 1.9% of the starting W-EtFOSE alcohol remained. Quantitative results from triplicate samples are shown for each time point. In this 35-day study, heat-sterilized/chloramphenicol-inhibited sludge showed very little to no biodegradation activity of W-EtFOSE alcohol (Figure 1). This observation conclusively demonstrates that degradation was due to microbiological activity present in the active sludge. The no-sludge control samples, which contained unsterilized mineral salts medium and W-EtFOSE alcohol, began to produce a small amount of WEtFOSAA near the end of the study, indicating microbial contamination. This may be expected since these controls were not heat sterilized, and no steps were taken with these samples to control microbial growth. Turbidity measurements commonly used to monitor for microbial growth in solution, was not part of the study protocol. Because the sample preparation analyst was unaware of making turbidity observations, such information was not recorded. The biodegradation of W-EtFOSE alcohol appeared to be second-order, as evidenced by the plot of 1/(concentration) versus time resulting in a linear relationship (Figure 8). Page 29 of 55 Aerobic Biodegradation of W-EtFOSE alcohol Cleston C. Lange, Ph.D., Pace Analytical Services, Science Solutions Division Wednesday November 1, 2000 BACK TO MAIN Figure 8. The determination of the second order degradation of W-EtFOSE alcohol as evidenced by the linear plot of [1/ (average concentration)] (pMT1) versus time (days) for the first 21 days of the biodegradation of 4.168 pM W-EtFOSE alcohol. The Degradation of W-EtFOSE alcohol at that test concentration ceased after 21 days of incubation. Mass balance calculations throughout the study at the test concentration of 2,380 ng/mL averaged 100.6% + 16.3% for all biodegradation samples, 118.6% + 7.9% for all sterile/inhibited controls, and 110.9% + 8.3% for all of the no-sludge controls (Table 1 of Appendix F). The major metabolites identified from test cultures were W-EtFOSAA and M556, which together accounted for nearly 83.8% of the total products observed at day 35, with W-EtFOSAA accounting for 34.9% of the total products and M556 accounting for 48.9%. The biodegradation dynamics for W-EtFOSAA, Figure 9, was irregular and probably represents the simultaneous production and degradation of W-EtFOSAA by a mixed microbial population. Page 30 of 55 Aerobic Biodegradation of W-EtFOSE alcohol Cleston C. Lange, Ph.D., Pace Analytical Services, Science Solutions Division Wednesday November 1, 2000 BACK TO MAIN Figure 9. The production of the metabolite W-EtFOSAA observed during the biodegradation of 2,380 ng/mL (4.168 pM) W-EtFOSE Alcohol. Quantitative results from triplicate samples are shown for each time point. The concentration increase of M556, Figure 10, and was postulated to be the net result of an W-dealkylation reaction which removed the acetate moiety to produce W-EtFOSA. However, W-EtFOSA was not detected at significantly high levels in degradation samples, and may have formed as a rapidly degraded transient species. The W-EtFOSA likely underwent monooxygenation of the alkyl group to yield an alcohol, which could then undergo subsequent oxidations to yield an aldehyde and then the acid M556. An alternative explanation may be that W-EtFOSAA was monooxygenated prior to dealkylation, which undergoes oxidation to produce a dicarboxylic acid. A dicarboxylic species could undergo one or two W-dealkylations to yield M556 or FOSA, respectively. While Figure 15 suggests the former reaction route, the alternative mechanism is equally likely. Page 31 of 55 Aerobic Biodegradation of W-EtFOSE alcohol Cleston C. Lange, Ph.D., Pace Analytical Services, Science Solutions Division Wednesday November 1, 2000 BACK TO MAIN 3.000 2.500 5 2.000 c 0 1.500 g 1 1.000 co O 0.500 0.000 - 0 .5 0 0 Incubation time (days) Figure 10. The production of the metabolite M556 during the biodegradation of 2,380 ng/mL (4.168 pM) W-EtFOSE Alcohol. Quantitative results from triplicate samples are shown for each time point. FOSA, of which the production is shown in Figure 11, may have been formed from M556 as its direct precursor, as previously discussed. The FOSA intermediate accounted for 5.0% of the total measured product at day 35. Interestingly, PFOSulfinate production (Figure 12) occurred in an analogous manner to the irregular formation of W-EtFOSAA. This may suggest that PFOSulfinate was formed directly from W-EtFOSAA as a precursor or that the same microbial populations were responsible for degrading both compounds. This was not confirmed in the present study design and thus this possible route is not shown in the pathway presented in Figure 15. PFOSulfinate accounted for 3.5% of the total fluorochemical products detected at day 35. Page 32 of 55 Aerobic Biodegradation of W-EtFOSE alcohol Cleston C. Lange, Ph.D., Pace Analytical Services, Science Solutions Division Wednesday November 1, 2000 BACK TO MAIN Figure 11. The production of FOSA observed during the biodegradation of 2,380 ng/mL (4.168 pM) W-EtFOSE Alcohol. Quantitative results from triplicate samples are shown for each time point. Figure 12. The production of PFOSulfinate observed during the biodegradation of 2,380 ng/mL (4.168 pM) W-EtFOSE alcohol. Quantitative results from triplicate samples are shown for each time point. Finally, the formation of PFOS (Figure 13) and PFOA (Figure 14) was observed. PFOS and PFOA appeared to be the ultimate degradation products. PFOS was probably formed from a microbial mediated sulfur oxidation mechanism that converted PFOSulfinate to PFOS. PFOA may have been formed from PFOSulfinate by an abiotic mechanism, as previously described in the literature (Hu et.al. 1990, J. Fluorine Chemistry). PFOS accounted for 7.0% of the final product measured at day 35 and PFOA accounted for 0.6% of the final product measured at day 35. Page 33 of 55 Aerobic Biodegradation of ^-EtFOSE alcohol Cleston C. Lange, Ph.D., Pace Analytical Services, Science Solutions Division Wednesday November 1, 2000 BACK TO MAIN Figure 13. The production of PFOS observed during the biodegradation of 2,380 ng/mL (4.168 pM) W-EtFOSE alcohol. Quantitative results from triplicate samples are shown for each time point. Figure 14. The production of PFOA observed after a 3-day lag period during the biodegradation of 2,380 ng/mL (4.168 pM) W-EtFOSE alcohol. Quantitative results from triplicate samples are shown for each time point. 9.0 Conclusions This study demonstrated that W-EtFOSE alcohol is rapidly biodegraded by the inherent microbial populations of municipal waste treatment facilities sludge, with 100% degradation of a 47 ng/mL (90 nM) aqueous solution of W-EtFOSE alcohol occurring within 14 days. Tests performed at a concentration of 2.38 pg/mL (4.168 pM) of W-EtFOSE alcohol showed that nearly 90% of the original starting material had degraded in 35 days. The data indicated that one of the primary metabolites of W-EtFOSE alcohol is W-EtFOSAA. An alcohol dehydrogenase enzyme likely catalyzes this transformation, Page 34 of 55 Aerobic Biodegradation of W-EtFOSE alcohol Cleston C. Lange, Ph.D., Pace Analytical Services, Science Solutions Division Wednesday November 1, 2000 BACK TO MAIN anticipated to be the first step in a series of enzymatic reactions affecting the hydrocarbon tail of the molecule. Indeed, the subsequent disappearance of W-EtFOSAA and the continued generation of other products support this hypothesis. The second major metabolite, and most predominant product formed in 35 days, was M556. Together, W-EtFOSAA and M556 accounted for 83.8% of the total products at day 35 for the 2,380 ng/mL cultures (48.9% M556, 34.9% W-EtFOSAA). Other degradation products observed at the culmination of this study, and presented as a percentage of the total products formed at day 35, included: W-EtFOSA (0.1%), FOSA (5.0%), PFOSulfinate (3.5%), PFOS (7.0%) and PFOA (0.6%) (Table 1, Appendix E). Further elucidation of the sequence of events yielding these products is the subject of a subsequent study. A possible pathway, based on observed products in this study, is presented in Figure 15. The fact that the study was conducted at concentrations near and above the suspected solubility of W-EtFOSE alcohol did not seem to confer any major observable differences on the ability of microorganisms to degrade the starting material. One notable difference was the complete biodegradation of the W-EtFOSAA formed at the low concentration, while W-EtFOSAA degraded slowly and accumulated significantly during incubations with a high concentration of W-EtFOSE alcohol. The pathway in Figure 15 implies that PFOS and PFOA are the ultimate end-products. This is not substantiated since smaller molecular weight products were not targeted for analysis. However, mass balance was achieved between the parent material and products implying that all significant degradation products were identified and that PFOS and/or PFOA are endpoints in the biodegradation. Page 35 of 55 Aerobic Biodegradation of W-EtFOSE alcohol Cleston C. Lange, Ph.D., Pace Analytical Services, Science Solutions Division Wednesday November 1, 2000 BACK TO MAIN FF FFFFFFFFO OH CH3 W-EtFOSE Alcohol FFFFF II W-EtFOSE Aldehyde FFFFFO CH3 FFFFFFFF FFFFFO OH W-EtFOSAA CH3 FFFFFFFFO IV. F FFF F F F F O W-EtFOSA CH3 V. VI. VII. VIII. IX. FFFFFFF FFFFFFFFO FO FFFFFFFFO FFFF FFFFFFFFO M556 FOSA PFOSulfinate Abiotic mechanism X. PFOSF FF FF FF FF F F F F F F F FO r H XI. Figure 15. The metabolism of W-EtFOSE alcohol as mediated by the microbial activity of aerobic sludge. The pathway shows PFOS and PFOA as end-products. Page 36 of 55 Aerobic Biodegradation of ^-EtFOSE alcohol Cleston C. Lange, Ph.D., Pace Analytical Services, Science Solutions Division Wednesday November 1, 2000 BACK TO MAIN 10.0 Sample and Data Retention Original facility records and original data and a copy of the signed final report will be retained in the Pace Analytical-Tier 2 data archives for a period of 2 years after completion of the project. At the end of 2 years, or upon request before that time, the data will be sent to the 3M Environmental Laboratory for ultimate long-term archiving. The following will be provided to 3M personnel: The original signed analytical report and one copy of the signed original All original Data, correspondence, chromatograms, sample & standards prep sheets, etc., upon request. Upon request of original data and records, copies of all original raw data in the form of chromatograms, reduced data, and written records concerning this project will be prepared and retained in the Pace Analytical-Tier 2 data archives.. All electronic copies of the instrumental raw data will be archived onto CD disks (read-only) and a copy provided to 3M upon request ,or after 2 years, which ever is first. Facility data will be retained for a period of 10 years. Facility data is available for inspection and includes the following records: Training records Controlled storage temperature logs Standard preparation logs Calibration and maintenance logs Chemical and solvent traceability logs Standard Operating Procedures Methods pertaining to the conduct of this project The remaining sample extracts will be retained at the Pace Analytical facility for a period of 1 year after completion of the project at 4C in the Carroll walk-in cooler (Pace ID 0140) located in the Pace Analytical-Tier 2 facility. Page 37 of 55 Aerobic Biodegradation of ^-EtFOSE alcohol Cleston C. Lange, Ph.D., Pace Analytical Services, Science Solutions Division Wednesday November 1, 2000 BACK TO MAIN Appendix A Signatures of Project Personnel Project Title: 5-Week W-EtFOSE Alcohol Aerobic Biodegradation Study Client Project ID: LIMS E00-2252 Contract Analytical Project Number: CA058 The following individuals participated in the conduct of this project: Study Director: Cleston C. Lange ________________ Signature Laboratory Management: Mark T. McCann Report Author: Cleston C. Lange Signature Signature Sample Preparation Analyst: Angela L. Schuler ________________ Signature HPLC/MS Analysis Conducted by: Anthony E. Scales ________________ Signature Final Report Reviewed by: Dirk W. Hoogenboom _________________ Signature Page 38 of 55 Aerobic Biodegradation of ^-EtFOSE alcohol Cleston C. Lange, Ph.D., Pace Analytical Services, Science Solutions Division Wednesday November 1, 2000 Date Date Date Date Date Date BACK TO MAIN Appendix B TABLE 1. The quantitative results of sample analysis, presented as nanogram per milliliter, which is the same as parts per billion (ng/mL; ppb). S a m p le ID C A 0 5 8 -0 8 0 1 -S A -0 0 1 CA058-0801-SA-002 CA058-0801-SA-003 CA058-0801-SA-004 CA058-0801-SA-005 C A 0 58 -0 80 1 -S A -00 6 CA058-0801-SA-007 CA058-0801-SA-008 CA058-0801-SA-009 CA058-0801 -SA-010 C A 0 5 8 -0 8 0 1 -S A -0 1 1 CA058-0801-SA-012 CA058-0801-SA-013 CA058-0801-SA-014 CA058-0801-SA-015 C A 0 58 -0 80 1 -S A -01 6 CA058-0801-SA-017 CA058-0801-SA-018 CA058-0801-SA-019 C A 0 58 -0 80 1 -S A -02 0 C A 0 5 8 -0 8 0 1 -S A -0 2 1 C A 0 5 8 -0 8 0 1 -S A -0 0 1 CA058-0801-SA-002 CA058-0801-SA-003 CA058-0801-SA-004 CA058-0801-SA-005 C A 0 58 -0 80 1 -S A -00 6 CA058-0801-SA-007 CA058-0801-SA-008 CA058-0801-SA-009 C A 0 5 8 -0 8 0 1 -S A -0 1 0 C A 0 5 8 -0 8 0 1 -S A -0 1 1 CA058-0801-SA-012 CA058-0801-SA-013 CA058-0801-SA-014 CA058-0801-SA-015 C A 0 58 -0 80 1 -S A -01 6 CA058-0801-SA-017 CA058-0801-SA-018 CA058-0801-SA-019 C A 0 58 -0 80 1 -S A -02 0 C A 0 5 8 -0 8 0 1 -S A -0 2 1 D escription Day 0 Biodeg Sam ple (50 ppb EtFOSE A lcohol) Day 0 Biodeg Sam ple D uplicate (50 ppb EtFOSE A lcohol) Day 0 Biodeg Sam ple Triplicate (50 ppb EtFOSE A lcohol) Day 0 S terile Sludge Control Sam ple (50 ppb EtFOSE A lcohol) Day 0 S terile Sludge Control D uplicate (50 ppb EtFOSE A lcohol) Day 0 S terile Sludge Control Triplicate (50 ppb EtFOSE A lcohol) Day 0 No Sludge Control Sam ple (50 ppb EtFOSE A lcohol) Day 0 No Sludge Control D uplicate (50 ppb EtFOSE A lcohol) Day 0 No Sludge Control Triplicate (50 ppb EtFOSE A lcohol) Day 0 Sludge Blank, NO EtFOSE A lcohol Day 0 Sludge Blank D uplicate, NO EtFOSE A lcohol Day 0 Sludge Blank Triplicate, No EtFOSE A lcohol Day 0 Biodeg Sam ple (2380 ppb EtFOSE A lcohol) Day 0 Biodeg Sam ple D uplicate (2380 ppb EtFOSE A lcohol) Day 0 Biodeg Sam ple Triplicate (2380 ppb EtFOSE A lcohol) Day 0 S terile Sludge Control (2380 ppb EtFOSE A lcohol) Day 0 S terile Sludge Control D uplicate (2380 ppb EtFOSE A lcohol) Day 0 S terile Sludge Control Triplicate (2380 ppb EtFOSE A lcohol) Day 0 No Sludge Control (2380 ppb EtFOSE A lcohol) Day 0 No Sludge Control D uplicate (2380 ppb EtFOSE A lcohol) Day 0 No Sludge Control Triplicate (2380 ppb EtFOSE A lcohol) Day 0 Biodeg Sam ple (50 ppb EtFOSE A lcohol) Day 0 Biodeg Sam ple D uplicate (50 ppb EtFOSE A lcohol) Day 0 Biodeg Sam ple Triplicate (50 ppb EtFOSE A lcohol) Day 0 S terile Sludge Control Sam ple (50 ppb EtFOSE A lcohol) Day 0 S terile Sludge Control D uplicate (50 ppb EtFOSE A lcohol) Day 0 S terile Sludge Control Triplicate (50 ppb EtFOSE A lcohol) Day 0 No Sludge Control Sam ple (50 ppb EtFOSE A lcohol) Day 0 No Sludge Control D uplicate (50 ppb EtFOSE A lcohol) Day 0 No Sludge Control Triplicate (50 ppb EtFOSE A lcohol) Day 0 Sludge Blank, NO EtFOSE A lcohol Day 0 Sludge Blank D uplicate, NO EtFOSE A lcohol Day 0 Sludge Blank Triplicate, No EtFOSE A lcohol Day 0 Biodeg Sam ple (2380 ppb EtFOSE A lcohol) Day 0 Biodeg Sam ple D uplicate (2380 ppb EtFOSE A lcohol) Day 0 Biodeg Sam ple Triplicate (2380 ppb EtFOSE A lcohol) Day 0 S terile Sludge Control (2380 ppb EtFOSE A lcohol) Day 0 S terile Sludge Control D uplicate (2380 ppb EtFOSE A lcohol) Day 0 S terile Sludge Control Triplicate (2380 ppb EtFOSE A lcohol) Day 0 No Sludge Control (2380 ppb EtFOSE A lcohol) Day 0 No Sludge Control D uplicate (2380 ppb EtFOSE A lcohol) Day 0 No Sludge Control Triplicate (2380 ppb EtFOSE A lcohol) Elu ate PFOA 2 0.0 2 0.0 2 0.0 2 0.0 2 0.0 2 0.0 2 0.0 2 0.0 2 0.0 2 0.0 2 0.0 2 0.0 2 0.0 2 0.0 2 0.0 2 0.0 2 0.0 2 0.0 2 0.0 2 0.0 2 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 PFOS PFOS su lfina te 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 NM556 EtFO S A A FOSA 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 N- EtFOSE a lcoh o l N- E tF O S A 43.3 0.0 39.5 0.0 37.1 0.0 45.9 0.0 44.6 0.0 44.0 0.0 43.3 0.0 45.6 0.0 44.4 0.0 0.0 0.0 0.0 0.0 0.0 0.0 2835.8 0.0 2742.8 0.0 2668.8 0.0 2897.0 0.0 2752.9 0.0 2753.0 0.0 2684.0 0.0 2712.2 0.0 2788.4 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 81.3 0.0 51.7 0.0 49.7 0.0 48.7 0.0 49.8 0.0 32.7 0.0 104.9 0.0 113.4 0.0 84.1 0.0 Page 39 of 55 Aerobic Biodegradation of ^-EtFOSE alcohol Cleston C. Lange, Ph.D., Pace Analytical Services, Science Solutions Division Wednesday November 1, 2000 BACK TO MAIN Appendix B, Table 1 continued. S a m p le ID CA058-0801-SA-022 CA058-0801-SA-023 CA058-0801-SA-024 CA058-0801-SA-025 C A 0 58 -0 80 1 -S A -02 6 CA058-0801-SA-027 CA058-0801-SA-028 CA058-0801-SA-029 C A 0 58 -0 80 1 -S A -03 0 CA058-0801 -SA-031 CA058-0801-SA-032 CA058-0801-SA-033 CA058-0801-SA-034 CA058-0801-SA-035 C A 0 58 -0 80 1 -S A -03 6 CA058-0801-SA-037 CA058-0801-SA-038 CA058-0801-SA-039 C A 0 58 -0 80 1 -S A -04 0 C A 0 5 8 -0 8 0 1 -S A -0 4 1 CA058-0801-SA-042 CA058-0801-SA-022 CA058-0801-SA-023 CA058-0801-SA-024 CA058-0801-SA-025 C A 0 58 -0 80 1 -S A -02 6 CA058-0801-SA-027 CA058-0801-SA-028 CA058-0801-SA-029 C A 0 58 -0 80 1 -S A -03 0 C A 0 5 8 -0 8 0 1 -S A -0 3 1 CA058-0801-SA-032 CA058-0801-SA-033 CA058-0801-SA-034 CA058-0801-SA-035 C A 0 58 -0 80 1 -S A -03 6 CA058-0801-SA-037 CA058-0801-SA-038 CA058-0801-SA-039 C A 0 58 -0 80 1 -S A -04 0 C A 0 5 8 -0 8 0 1 -S A -0 4 1 CA058-0801-SA-042 S am ple D e scrip tio n Day 1 Biodeq Sam ple (50 ppb EtFOSE A lcohol) Day 1 Biodeq Sam ple D uplicate (50 ppb EtFOSE A lcohol) Day 1 Biodeq Sam ple Triplicate (50 ppb EtFOSE A lcohol) Day 1 S terile Sludqe Control Sam ple (50 ppb EtFOSE A lcohol) Day 1 S terile Sludqe Control D uplicate (50 ppb EtFOSE A lcohol) Day 1 S terile Sludqe Control Triplicate (50 ppb EtFOSE A lcohol) Day 1 No Sludqe Control Sam ple (50 ppb EtFOSE A lcohol) Day 1 No Sludqe Control D uplicate (50 ppb EtFOSE A lcohol) Day 1 No Sludqe Control Triplicate (50 ppb EtFOSE A lcohol) Day 1 Sludqe Blank, NO EtFOSE A lcohol Day 1 Sludqe Blank D uplicate, NO EtFOSE A lcohol Day 1 Sludqe Blank Triplicate, No EtFOSE A lcohol Day 1 Biodeq Sam ple (2380 ppb EtFOSE A lcohol) Day 1 Biodeq Sam ple D uplicate (2380 ppb EtFOSE A lcohol) Day 1 Biodeq Sam ple Triplicate (2380 ppb EtFOSE A lcohol) Day 1 S terile Sludqe Control (2380 ppb EtFOSE A lcohol) Day 1 S terile Sludqe Control D uplicate (2380 ppb EtFOSE A lcohol) Day 1 S terile Sludqe Control Triplicate (2380 ppb EtFOSE A lcohol) Day 1 No Sludqe Control (2380 ppb EtFOSE A lcohol) Day 1 No Sludqe Control D uplicate (2380 ppb EtFOSE A lcohol) Day 1 No Sludqe Control Triplicate (2380 ppb EtFOSE A lcohol) Day 1 Biodeq Sam ple (50 ppb EtFOSE A lcohol) Day 1 Biodeq Sam ple D uplicate (50 ppb EtFOSE A lcohol) Day 1 Biodeq Sam ple Triplicate (50 ppb EtFOSE A lcohol) Day 1 S terile Sludqe Control Sam ple (50 ppb EtFOSE A lcohol) Day 1 S terile Sludqe Control D uplicate (50 ppb EtFOSE A lcohol) Day 1 S terile Sludqe Control Triplicate (50 ppb EtFOSE A lcohol) Day 1 No Sludqe Control Sam ple (50 ppb EtFOSE A lcohol) Day 1 No Sludqe Control D uplicate (50 ppb EtFOSE A lcohol) Day 1 No Sludqe Control Triplicate (50 ppb EtFOSE A lcohol) Day 1 Sludqe Blank, NO EtFOSE A lcohol Day 1 Sludqe Blank D uplicate, NO EtFOSE A lcohol Day 1 Sludqe Blank Triplicate, No EtFOSE A lcohol Day 1 Biodeq Sam ple (2380 ppb EtFOSE A lcohol) Day 1 Biodeq Sam ple D uplicate (2380 ppb EtFOSE A lcohol) Day 1 Biodeq Sam ple Triplicate (2380 ppb EtFOSE A lcohol) Day 1 S terile Sludqe Control (2380 ppb EtFOSE A lcohol) Day 1 S terile Sludqe Control D uplicate (2380 ppb EtFOSE A lcohol) Day 1 S terile Sludqe Control Triplicate (2380 ppb EtFOSE A lcohol) Day 1 No Sludqe Control (2380 ppb EtFOSE A lcohol) Day 1 No Sludqe Control D uplicate (2380 ppb EtFOSE A lcohol) Day 1 No Sludqe Control Triplicate (2380 ppb EtFOSE A lcohol) Elu ate PFOA 2 0.0 2 0.0 2 0.0 2 0.0 2 0.0 2 0.0 2 0.0 2 0.0 2 0.0 2 0.0 2 0.0 2 0.0 2 0.0 2 0.0 2 0.0 2 0.0 2 0.0 2 0.0 2 0.0 2 0.0 2 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 PFOS PFOS su lfina te 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 4.8 44.5 4.2 43.7 4.6 48.1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 4.4 0.0 5.6 0.0 3.3 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 NM556 EtFO S A A FOSA 0.0 15.2 0.0 0.0 14.2 0.0 0.0 12.7 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 27.6 329.1 8.2 26.7 324.4 7.8 29.7 350.2 8.5 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 51.9 0.0 0.0 48.4 0.0 0.0 35.9 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 N- EtFOSE a lcoh o l N- E tF O S A 17.6 0.0 17.6 0.0 18.4 0.0 43.9 0.0 44.4 0.0 45.9 0.0 40.7 0.0 41.0 0.0 41.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1604.9 11.2 1681.3 10.7 1704.9 11.6 2820.3 0.0 2711.9 0.0 2605.4 0.0 2719.8 0.0 2673.8 0.0 2593.2 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 165.5 0.0 122.9 0.0 76.1 0.0 80.5 0.0 95.9 0.0 203.2 0.0 131.8 0.0 128.1 0.0 124.4 0.0 Page 40 of 55 Aerobic Biodegradation of ^-EtFOSE alcohol Cleston C. Lange, Ph.D., Pace Analytical Services, Science Solutions Division Wednesday November 1, 2000 BACK TO MAIN Appendix B, Table 1 continued. S a m p le ID CA058-0801-SA-043 CA058-0801-SA-044 CA058-0801-SA-045 C A 0 5 8 -0 80 1 -S A -04 6 CA058-0801-SA-047 CA058-0801-SA-048 CA058-0801-SA-049 C A 0 5 8 -0 80 1 -S A -05 0 C A 0 5 8 -0 8 0 1 -S A -0 5 1 CA058-0801-SA-052 CA058-0801-SA-053 CA058-0801-SA-054 CA058-0801-SA-055 C A 0 5 8 -0 80 1 -S A -05 6 CA058-0801-SA-057 CA058-0801-SA-058 CA058-0801-SA-059 C A 0 5 8 -0 80 1 -S A -06 0 C A 0 5 8 -0 8 0 1 -S A -0 6 1 CA058-0801-SA-062 CA058-0801-SA-063 CA058-0801-SA-043 CA058-0801-SA-044 CA058-0801-SA-045 C A 0 5 8 -0 80 1 -S A -04 6 CA058-0801-SA-047 CA058-0801-SA-048 CA058-0801-SA-049 C A 0 5 8 -0 80 1 -S A -05 0 C A 0 5 8 -0 8 0 1 -S A -0 5 1 CA058-0801-SA-052 CA058-0801-SA-053 CA058-0801-SA-054 CA058-0801-SA-055 C A 0 5 8 -0 80 1 -S A -05 6 CA058-0801-SA-057 CA058-0801-SA-058 CA058-0801-SA-059 C A 0 5 8 -0 80 1 -S A -06 0 C A 0 5 8 -0 8 0 1 -S A -0 6 1 CA058-0801-SA-062 CA058-0801-SA-063 D escription Day 3 Biodeq Sam ple (50 ppb EtFOSE A lcohol) Day 3 Biodeq Sam ple D uplicate (50 ppb EtFOSE A lcohol) Day 3 Biodeq Sam ple Triplicate (50 ppb EtFOSE A lcohol) Day 3 S terile Sludqe Control Sam ple (50 ppb EtFOSE A lcohol) Day 3 S terile Sludqe Control D uplicate (50 ppb EtFOSE A lcohol) Day 3 S terile Sludqe Control Triplicate (50 ppb EtFOSE A lcohol) Day 3 No Sludqe Control Sam ple (50 ppb EtFOSE A lcohol) Day 3 No Sludqe Control D uplicate (50 ppb EtFOSE A lcohol) Day 3 No Sludqe Control Triplicate (50 ppb EtFOSE A lcohol) Day 3 Sludqe Blank, NO EtFOSE A lcohol Day 3 Sludqe Blank D uplicate, NO EtFOSE A lcohol Day 3 Sludqe Blank Triplicate, No EtFOSE A lcohol Day 3 Biodeq Sam ple (2380 ppb EtFOSE A lcohol) Day 3 Biodeq Sam ple D uplicate (2380 ppb EtFOSE A lcohol) Day 3 Biodeq Sam ple Triplicate (2380 ppb EtFOSE A lcohol) Day 3 S terile Sludqe Control (2380 ppb EtFOSE A lcohol) Day 3 S terile Sludqe Control D uplicate (2380 ppb EtFOSE A lcohol) Day 3 S terile Sludqe Control Triplicate (2380 ppb EtFOSE A lcohol) Day 3 No Sludqe Control (2380 ppb EtFOSE A lcohol) Day 3 No Sludqe Control D uplicate (2380 ppb EtFOSE A lcohol) Day 3 No Sludqe Control Triplicate (2380 ppb EtFOSE A lcohol) Day 3 Biodeq Sam ple (50 ppb EtFOSE A lcohol) Day 3 Biodeq Sam ple D uplicate (50 ppb EtFOSE A lcohol) Day 3 Biodeq Sam ple Triplicate (50 ppb EtFOSE A lcohol) Day 3 S terile Sludqe Control Sam ple (50 ppb EtFOSE A lcohol) Day 3 S terile Sludqe Control D uplicate (50 ppb EtFOSE A lcohol) Day 3 S terile Sludqe Control Triplicate (50 ppb EtFOSE A lcohol) Day 3 No Sludqe Control Sam ple (50 ppb EtFOSE A lcohol) Day 3 No Sludqe Control D uplicate (50 ppb EtFOSE A lcohol) Day 3 No Sludqe Control Triplicate (50 ppb EtFOSE A lcohol) Day 3 Sludqe Blank, NO EtFOSE A lcohol Day 3 Sludqe Blank D uplicate, NO EtFOSE A lcohol Day 3 Sludqe Blank Triplicate, No EtFOSE A lcohol Day 3 Biodeq Sam ple (2380 ppb EtFOSE A lcohol) Day 3 Biodeq Sam ple D uplicate (2380 ppb EtFOSE A lcohol) Day 3 Biodeq Sam ple Triplicate (2380 ppb EtFOSE A lcohol) Day 3 S terile Sludqe Control (2380 ppb EtFOSE A lcohol) Day 3 S terile Sludqe Control D uplicate (2380 ppb EtFOSE A lcohol) Day 3 S terile Sludqe Control Triplicate (2380 ppb EtFOSE A lcohol) Day 3 No Sludqe Control (2380 ppb EtFOSE A lcohol) Day 3 No Sludqe Control D uplicate (2380 ppb EtFOSE A lcohol) Day 3 No Sludqe Control Triplicate (2380 ppb EtFOSE A lcohol) E luate PFOA 2 0.0 2 0.0 2 0.0 2 0.0 2 0.0 2 0.0 2 0.0 2 0.0 2 0.0 2 0.0 2 0.0 2 0.0 2 0.0 2 0.0 2 0.0 2 0.0 2 0.0 2 0.0 2 0.0 2 0.0 2 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 PFOS Concentration (ng/mL)____________ N- PFOS N- EtFO SE su lfin a te M556 E tFO S A A FOSA a lco h o l N- E tF O S A 0.0 0.0 6.9 13.7 0.0 7.2 0.0 0.0 0.0 5.1 11.8 0.0 9.8 0.0 0.0 0.9 4.8 11.1 0.0 9.0 0.0 0.0 0.8 0.0 0.0 0.0 40.0 0.0 0.0 0.8 0.0 0.0 0.0 38.6 0.0 0.0 0.0 0.0 0.0 0.0 41.0 0.0 0.0 0.0 0.0 0.0 0.0 36.6 0.0 0.0 0.0 0.0 0.0 0.0 38.2 0.0 0.0 0.0 0.0 0.0 0.0 37.1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 16.3 60.2 112.6 556.4 23.4 1180.2 15.6 13.9 60.7 104.1 529.1 21.1 1227.6 14.9 17.5 66.1 113.3 595.1 25.3 1084.7 16.2 0.0 0.0 0.0 0.0 0.0 2721.4 0.0 0.0 0.0 0.0 0.0 0.0 2644.5 0.0 0.0 0.0 0.0 0.0 0.0 2622.4 0.0 0.0 0.0 0.0 0.0 0.0 2483.8 0.0 0.0 0.0 0.0 0.0 0.0 2523.4 0.0 0.0 0.0 0.0 0.0 0.0 2549.3 0.0 0.0 0.0 0.0 6.3 0.0 0.0 0.0 0.0 0.0 0.0 4.9 0.0 0.0 0.0 0.0 0.9 0.0 5.2 0.0 0.0 0.0 0.0 0.8 0.0 0.0 0.0 0.0 0.0 0.0 0.8 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 6.5 11.4 70.1 0.0 79.0 0.0 0.0 7.1 12.8 89.3 0.0 104.1 0.0 0.0 6.7 12.4 81.7 0.0 75.5 0.0 0.0 0.0 0.0 0.0 0.0 37.4 0.0 0.0 0.0 0.0 0.0 0.0 59.0 0.0 0.0 0.0 0.0 0.0 0.0 59.5 0.0 0.0 0.0 0.0 0.0 0.0 113.6 0.0 0.0 0.0 0.0 0.0 0.0 145.2 0.0 0.0 0.0 0.0 0.0 0.0 115.6 0.0 Page 41 of 55 Aerobic Biodegradation of ^-EtFOSE alcohol Cleston C. Lange, Ph.D., Pace Analytical Services, Science Solutions Division Wednesday November 1, 2000 BACK TO MAIN Appendix B, Table 1 continued. S a m p le ID CA058-0801-SA-064 CA058-0801-SA-065 C A 0 5 8 -0 80 1 -S A -06 6 CA058-0801-SA-067 CA058-0801-SA-068 CA058-0801-SA-069 C A 0 5 8 -0 80 1 -S A -07 0 C A 0 5 8 -0 8 0 1 -S A -0 7 1 CA058-0801-SA-072 CA058-0801-SA-073 CA058-0801-SA-074 CA058-0801-SA-075 C A 0 5 8 -0 80 1 -S A -07 6 CA058-0801-SA-077 CA058-0801-SA-078 CA058-0801-SA-079 C A 0 5 8 -0 80 1 -S A -08 0 C A 0 5 8 -0 8 0 1 -S A -0 8 1 CA058-0801-SA-082 CA058-0801-SA-083 CA058-0801-SA-084 CA058-0801-SA-064 CA058-0801-SA-065 C A 0 5 8 -0 80 1 -S A -06 6 CA058-0801-SA-067 CA058-0801-SA-068 CA058-0801-SA-069 C A 0 5 8 -0 80 1 -S A -07 0 C A 0 5 8 -0 8 0 1 -S A -0 7 1 CA058-0801-SA-072 CA058-0801-SA-073 CA058-0801-SA-074 CA058-0801-SA-075 C A 0 5 8 -0 80 1 -S A -07 6 CA058-0801-SA-077 CA058-0801-SA-078 CA058-0801-SA-079 C A 0 5 8 -0 80 1 -S A -08 0 C A 0 5 8 -0 8 0 1 -S A -0 8 1 CA058-0801-SA-082 CA058-0801-SA-083 CA058-0801-SA-084 D escription Day 7 Biodeq Sam ple (50 ppb EtFOSE A lcohol) Day 7 Biodeq Sam ple D uplicate (50 ppb EtFOSE A lcohol) Day 7 Biodeq Sam ple Triplicate (50 ppb EtFOSE A lcohol) Day 7 S terile Sludqe Control Sam ple (50 ppb EtFOSE A lcohol) Day 7 S terile Sludqe Control D uplicate (50 ppb EtFOSE A lcohol) Day 7 S terile Sludqe Control Triplicate (50 ppb EtFOSE A lcohol) Day 7 No Sludqe Control Sam ple (50 ppb EtFOSE A lcohol) Day 7 No Sludqe Control D uplicate (50 ppb EtFOSE A lcohol) Day 7 No Sludqe Control Triplicate (50 ppb EtFOSE A lcohol) Day 7 Sludqe Blank, NO EtFOSE A lcohol Day 7 Sludqe Blank D uplicate, NO EtFOSE A lcohol Day 7 Sludqe Blank Triplicate, No EtFOSE A lcohol Day 7 Biodeq Sam ple (2380 ppb EtFOSE A lcohol) Day 7 Biodeq Sam ple D uplicate (2380 ppb EtFOSE A lcohol) Day 7 Biodeq Sam ple Triplicate (2380 ppb EtFOSE A lcohol) Day 7 S terile Sludqe Control (2380 ppb EtFOSE A lcohol) Day 7 S terile Sludqe Control D uplicate (2380 ppb EtFOSE A lcohol) Day 7 S terile Sludqe Control Triplicate (2380 ppb EtFOSE A lcohol) Day 7 No Sludqe Control (2380 ppb EtFOSE A lcohol) Day 7 No Sludqe Control D uplicate (2380 ppb EtFOSE A lcohol) Day 7 No Sludqe Control Triplicate (2380 ppb EtFOSE A lcohol) Day 7 Biodeq Sam ple (50 ppb EtFOSE A lcohol) Day 7 Biodeq Sam ple D uplicate (50 ppb EtFOSE A lcohol) Day 7 Biodeq Sam ple Triplicate (50 ppb EtFOSE A lcohol) Day 7 S terile Sludqe Control Sam ple (50 ppb EtFOSE A lcohol) Day 7 S terile Sludqe Control D uplicate (50 ppb EtFOSE A lcohol) Day 7 S terile Sludqe Control Triplicate (50 ppb EtFOSE A lcohol) Day 7 No Sludqe Control Sam ple (50 ppb EtFOSE A lcohol) Day 7 No Sludqe Control D uplicate (50 ppb EtFOSE A lcohol) Day 7 No Sludqe Control Triplicate (50 ppb EtFOSE A lcohol) Day 7 Sludqe Blank, NO EtFOSE A lcohol Day 7 Sludqe Blank D uplicate, NO EtFOSE A lcohol Day 7 Sludqe Blank Triplicate, No EtFOSE A lcohol Day 7 Biodeq Sam ple (2380 ppb EtFOSE A lcohol) Day 7 Biodeq Sam ple D uplicate (2380 ppb EtFOSE A lcohol) Day 7 Biodeq Sam ple Triplicate (2380 ppb EtFOSE A lcohol) Day 7 S terile Sludqe Control (2380 ppb EtFOSE A lcohol) Day 7 S terile Sludqe Control D uplicate (2380 ppb EtFOSE A lcohol) Day 7 S terile Sludqe Control Triplicate (2380 ppb EtFOSE A lcohol) Day 7 No Sludqe Control (2380 ppb EtFOSE A lcohol) Day 7 No Sludqe Control D uplicate (2380 ppb EtFOSE A lcohol) Day 7 No Sludqe Control Triplicate (2380 ppb EtFOSE A lcohol) Elu ate PFOA 2 0.0 2 0.0 2 0.0 2 0.0 2 0.0 2 0.0 2 0.0 2 0.0 2 0.0 2 0.0 2 0.0 2 0.0 2 4.1 2 3.8 2 4.0 2 0.0 2 0.0 2 0.0 2 0.0 2 0.0 2 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 PFOS Concentration (ng/mL)____________ N- PFOS N- EtFO SE su lfin a te M556 E tFO S A A FOSA a lco h o l N- E tF O S A 2.9 0.0 11.5 13.0 4.8 3.0 0.0 2.9 0.0 11.2 11.9 3.2 0.0 0.0 2.8 0.0 12.4 12.0 3.5 0.0 0.0 0.0 0.0 0.0 0.0 0.0 40.4 0.0 0.0 0.0 0.0 0.0 0.0 40.3 0.0 0.0 0.0 0.0 0.0 0.0 39.4 0.0 0.0 0.0 0.0 0.0 0.0 34.7 0.0 0.0 0.0 0.0 0.0 0.0 34.8 0.0 0.0 0.0 0.0 0.0 0.0 36.7 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 44.1 57.0 366.0 559.2 58.1 756.7 16.7 40.9 58.3 363.4 531.1 54.2 771.9 16.3 40.0 59.6 340.4 566.2 54.4 757.0 15.2 0.0 0.0 0.0 0.0 0.0 2667.8 0.0 0.0 0.0 0.0 0.0 0.0 2811.4 0.0 0.0 0.0 0.0 0.0 0.0 2728.0 0.0 0.0 0.0 0.0 0.0 0.0 2449.1 0.0 0.0 0.0 0.0 0.0 0.0 2562.5 0.0 0.0 0.0 0.0 0.0 0.0 2556.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 2.9 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 5.6 7.2 67.0 107.0 4.2 62.2 0.0 4.7 6.9 59.4 93.3 3.6 64.8 0.0 5.9 8.8 70.8 126.2 5.1 87.8 0.0 0.0 0.0 0.0 0.0 0.0 58.3 0.0 0.0 0.0 0.0 0.0 0.0 49.3 0.0 0.0 0.0 0.0 0.0 0.0 49.6 0.0 0.0 0.0 0.0 0.0 0.0 143.0 0.0 0.0 0.0 0.0 0.0 0.0 117.7 0.0 0.0 0.0 0.0 0.0 0.0 137.9 0.0 Page 42 of 55 Aerobic Biodegradation of ^-EtFOSE alcohol Cleston C. Lange, Ph.D., Pace Analytical Services, Science Solutions Division Wednesday November 1, 2000 BACK TO MAIN Appendix B, Table 1 continued. S a m p le ID CA058-0801-SA-085 C A 0 5 8 -0 80 1 -S A -08 6 CA058-0801-SA-087 CA058-0801-SA-088 CA058-0801-SA-089 C A 0 5 8 -0 80 1 -S A -09 0 C A 0 5 8 -0 8 0 1 -S A -0 9 1 CA058-0801-SA-092 CA058-0801-SA-093 CA058-0801-SA-094 CA058-0801-SA-095 C A 0 5 8 -0 80 1 -S A -09 6 CA058-0801-SA-097 CA058-0801-SA-098 CA058-0801-SA-099 C A 0 5 8 -0 80 1 -S A -10 0 C A 0 5 8 -0 8 0 1 -S A -1 0 1 CA058-0801-SA-102 CA058-0801-SA-103 CA058-0801-SA-104 CA058-0801-SA-105 CA058-0801-SA-085 C A 0 5 8 -0 80 1 -S A -08 6 CA058-0801-SA-087 CA058-0801-SA-088 CA058-0801-SA-089 C A 0 5 8 -0 80 1 -S A -09 0 C A 0 5 8 -0 8 0 1 -S A -0 9 1 CA058-0801-SA-092 CA058-0801-SA-093 CA058-0801-SA-094 CA058-0801-SA-095 C A 0 5 8 -0 80 1 -S A -09 6 CA058-0801-SA-097 CA058-0801-SA-098 CA058-0801-SA-099 C A 0 5 8 -0 80 1 -S A -10 0 C A 0 5 8 -0 8 0 1 -S A -1 0 1 CA058-0801-SA-102 CA058-0801-SA-103 CA058-0801-SA-104 CA058-0801-SA-105 D escri Dtion E luate PFOA Day 14 B iodeq Sam ple (50 ppb EtFOSE A lcohol) 2 0.0 Day 14 B iodeq Sam ple Duplicate (50 ppb EtFOSE A lcohol) 2 0.0 Day 14 B iodeq Sam ple Triplicate (50 ppb EtFOSE A lcohol) 2 0.0 Day 14 S terile S ludqe Control Sam ple (50 ppb EtFOSE A lcohol) 2 0.0 Day 14 S terile S ludqe Control D uplicate (50 ppb EtFOSE A lcohol) 2 0.0 Day 14 S terile S ludqe Control Triplicate (50 ppb EtFOSE A lcohol) 2 0.0 Day 14 No S ludqe Control Sam ple (50 ppb EtFOSE A lcohol) 2 0.0 Day 14 No S ludqe Control D uplicate (50 ppb EtFOSE A lcohol) 2 0.0 Day 14 No S ludqe Control Triplicate (50 ppb EtFOSE A lcohol) 2 0.0 Day 14 S ludqe Blank, NO EtFOSE A lcohol 2 0.0 Day 14 S ludqe Blank Duplicate, NO EtFOSE A lcohol 2 0.0 Day 14 S ludqe Blank Triplicate, No EtFOSE A lcohol 2 0.0 Day 14 B iodeq Sam ple (2380 ppb EtFOSE A lcohol) 2 8.9 Day 14 B iodeq Sam ple Duplicate (2380 ppb EtFOSE A lcohol) 2 8.7 Day 14 B iodeq Sam ple Triplicate (2380 ppb EtFOSE A lcohol) 2 8.7 Day 14 S terile S ludqe Control (2380 ppb EtFOSE A lcohol) 2 0.0 Day 14 S terile S ludqe Control D uplicate (2380 ppb EtFOSE A lcohol) 2 0.0 Day 14 S terile S ludqe Control Triplicate (2380 ppb EtFOSE A lcohol) 2 0.0 Day 14 No S ludqe Control (2380 ppb EtFOSE A lcohol) 2 0.0 Day 14 No S ludqe Control D uplicate (2380 ppb EtFOSE A lcohol) 2 0.0 Day 14 No S ludqe Control Triplicate (2380 ppb EtFOSE A lcohol) 2 0.0 Day 14 B iodeq Sam ple (50 ppb EtFOSE A lcohol) 3 0.0 Day 14 B iodeq Sam ple Duplicate (50 ppb EtFOSE A lcohol) 3 0.0 Day 14 B iodeq Sam ple Triplicate (50 ppb EtFOSE A lcohol) 3 0.0 Day 14 S terile S ludqe Control Sam ple (50 ppb EtFOSE A lcohol) 3 0.0 Day 14 S terile S ludqe Control D uplicate (50 ppb EtFOSE A lcohol) 3 0.0 Day 14 S terile S ludqe Control Triplicate (50 ppb EtFOSE A lcohol) 3 0.0 Day 14 No S ludqe Control Sam ple (50 ppb EtFOSE A lcohol) 3 0.0 Day 14 No S ludqe Control D uplicate (50 ppb EtFOSE A lcohol) 3 0.0 Day 14 No S ludqe Control Triplicate (50 ppb EtFOSE A lcohol) 3 0.0 Day 14 S ludqe Blank, NO EtFOSE A lcohol 3 0.0 Day 14 S ludqe Blank Duplicate, NO EtFOSE A lcohol 3 0.0 Day 14 S ludqe Blank Triplicate, No EtFOSE A lcohol 3 0.0 Day 14 B iodeq Sam ple (2380 ppb EtFOSE A lcohol) 3 0.0 Day 14 B iodeq Sam ple Duplicate (2380 ppb EtFOSE A lcohol) 3 0.0 Day 14 B iodeq Sam ple Triplicate (2380 ppb EtFOSE A lcohol) 3 0.0 Day 14 S terile S ludqe Control (2380 ppb EtFOSE A lcohol) 3 0.0 Day 14 S terile S ludqe Control D uplicate (2380 ppb EtFOSE A lcohol) 3 0.0 Day 14 S terile S ludqe Control Triplicate (2380 ppb EtFOSE A lcohol) 3 0.0 Day 14 No S ludqe Control (2380 ppb EtFOSE A lcohol) 3 0.0 Day 14 No S ludqe Control D uplicate (2380 ppb EtFOSE A lcohol) 3 0.0 Day 14 No S ludqe Control Triplicate (2380 ppb EtFOSE A lcohol) 3 0.0 PFOS Concentration (ng/mL)____________ N- PFOS N- EtFO SE su lfin a te M556 E tFO S A A FOSA a lco h o l N- E tF O S A 5.4 0.0 16.2 4.4 7.8 0.0 0.0 4.9 0.0 16.0 4.1 7.3 0.0 0.0 5.0 0.0 15.7 5.0 7.5 0.0 0.0 0.0 0.0 0.0 0.0 0.0 43.9 0.0 0.0 0.0 0.0 0.0 0.0 39.1 0.0 0.0 0.0 0.0 0.0 0.0 43.2 0.0 0.0 0.0 0.0 0.0 0.0 32.2 0.0 0.0 0.0 0.0 0.0 0.0 32.5 0.0 0.0 0.0 0.0 0.0 0.0 32.2 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 85.2 57.1 632.1 488.4 76.6 417.2 11.4 87.8 56.6 705.1 429.1 79.3 402.7 11.2 81.9 51.1 668.6 395.9 78.3 386.4 10.6 0.0 0.0 0.0 3.2 0.0 2702.3 0.0 0.0 0.0 0.0 0.0 0.0 2686.7 0.0 0.0 0.0 0.0 0.0 0.0 2602.5 0.0 0.0 0.0 0.0 5.9 0.0 2331.5 0.0 0.0 0.0 0.0 17.0 0.0 2436.4 0.0 0.0 0.0 0.0 4.2 0.0 2454.1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 7.1 5.1 84.3 80.3 4.8 20.8 0.0 4.9 3.4 68.0 53.3 3.6 15.3 0.0 10.7 7.1 138.3 91.9 8.2 36.8 0.0 0.0 0.0 0.0 0.0 0.0 33.6 0.0 0.0 0.0 0.0 0.0 0.0 42.4 0.0 0.0 0.0 0.0 0.0 0.0 77.2 0.0 0.0 0.0 0.0 0.0 0.0 130.4 0.0 0.0 0.0 0.0 0.0 0.0 122.6 0.0 0.0 0.0 0.0 0.0 0.0 93.8 0.0 Page 43 of 55 Aerobic Biodegradation of ^-EtFOSE alcohol Cleston C. Lange, Ph.D., Pace Analytical Services, Science Solutions Division Wednesday November 1, 2000 BACK TO MAIN Appendix B, Table 1 continued. S a m p le ID C A 0 5 8 -0 80 1 -S A -10 6 CA058-0801-SA-107 CA058-0801-SA-108 CA058-0801-SA-109 C A 0 5 8 -0 80 1 -S A -11 0 C A 0 5 8 -0 8 0 1 -S A -1 1 1 CA058-0801-SA-112 CA058-0801-SA-113 CA058-0801-SA-114 CA058-0801-SA-115 C A 0 5 8 -0 80 1 -S A -11 6 CA058-0801-SA-117 CA058-0801-SA-118 CA058-0801-SA-119 C A 0 5 8 -0 80 1 -S A -12 0 C A 0 5 8 -0 8 0 1 -S A -1 2 1 CA058-0801-SA-122 CA058-0801-SA-123 CA058-0801-SA-124 CA058-0801-SA-125 C A 0 5 8 -0 80 1 -S A -12 6 C A 0 5 8 -0 80 1 -S A -10 6 CA058-0801-SA-107 CA058-0801-SA-108 CA058-0801-SA-109 C A 0 5 8 -0 80 1 -S A -11 0 C A 0 5 8 -0 8 0 1 -S A -1 1 1 CA058-0801-SA-112 CA058-0801-SA-113 CA058-0801-SA-114 CA058-0801-SA-115 C A 0 5 8 -0 80 1 -S A -11 6 CA058-0801-SA-117 CA058-0801-SA-118 CA058-0801-SA-119 C A 0 5 8 -0 80 1 -S A -12 0 C A 0 5 8 -0 8 0 1 -S A -1 2 1 CA058-0801-SA-122 CA058-0801-SA-123 CA058-0801-SA-124 CA058-0801-SA-125 C A 0 5 8 -0 80 1 -S A -12 6 D escri Dtion E luate PFOA D ay 21 B io de q S am ple (50 p pb E tFO S E A lco ho l) 2 0.0 D ay 21 B io de q S am ple D u p lica te (50 ppb E tFO S E A lco ho l) 2 0.0 D ay 21 B io de q S am ple T rip lic a te (50 p pb E tFO S E A lco ho l) 2 0.0 D ay 21 S te rile S lu dq e C ontrol S am ple (50 ppb E tFO S E A lco h o l) 2 0.0 D ay 21 S te rile S lu dq e C ontrol D u p lica te (50 p pb E tFO S E A lco ho l) 2 0.0 D ay 21 S te rile S lu dq e C ontrol T rip lic a te (50 p pb E tFO S E A lco ho l) 2 0.0 D ay 21 No S lu dq e C ontrol S am ple (50 ppb E tFO S E A lco ho l) 2 0.0 D ay 21 No S lu dq e C ontrol D u p lica te (50 p pb E tFO S E A lco ho l) 2 0.0 D ay 21 No S lu dq e C ontrol T rip lic a te (50 p pb E tFO S E A lco ho l) 2 0.0 D ay 21 S lu dq e B lank, NO E tFO S E A lco ho l 2 0.0 D ay 21 S lu dq e B la nk D uplicate , NO E tFO S E A lco ho l 2 0.0 D ay 21 S lu dq e B la nk T rip lica te , No E tFO S E A lco ho l 2 0.0 D ay 21 B io de q S am ple (2 3 80 p pb E tFO S E A lco ho l) 2 10.5 D ay 21 B io de q S am ple D u p lica te (2380 p pb E tFO S E A lco ho l) 2 10.2 D ay 21 B io de q S am ple T rip lic a te (2380 p pb E tFO S E A lco ho l) 2 9.7 D ay 21 S te rile S lu dq e C ontrol (2 3 80 p pb E tFO S E A lco ho l) 2 0.0 D ay 21 S te rile S lu dq e C ontrol D u p lica te (2380 p pb E tFO S E A lco ho l) 2 0.0 D ay 21 S te rile S lu dq e C ontrol T rip lic a te (2380 p pb E tFO S E A lco ho l) 2 0.0 D ay 21 No S lu dq e C ontrol (2 3 80 p pb E tFO S E A lco ho l) 2 0.0 D ay 21 No S lu dq e C ontrol D u p lica te (2380 p pb E tFO S E A lco ho l) 2 0.0 D ay 21 No S lu dq e C ontrol T rip lic a te (2380 p pb E tFO S E A lco ho l) 2 0.0 D ay 21 B io de q S am ple (50 p pb E tFO S E A lco ho l) 3 0.0 D ay 21 B io de q S am ple (50 p pb E tFO S E A lco ho l) 3 0.0 D ay 21 B io de q S am ple D u p lica te (50 ppb E tFO S E A lco ho l) 3 0.0 D ay 21 B io de q S am ple T rip lic a te (50 p pb E tFO S E A lco ho l) 3 0.0 D ay 21 S te rile S lu dq e C ontrol S am ple (50 ppb E tFO S E A lco h o l) 3 0.0 D ay 21 S te rile S lu dq e C ontrol D u p lica te (50 p pb E tFO S E A lco ho l) 3 0.0 D ay 21 S te rile S lu dq e C ontrol T rip lic a te (50 p pb E tFO S E A lco ho l) 3 0.0 D ay 21 No S lu dq e C ontrol S am ple (50 ppb E tFO S E A lco ho l) 3 0.0 D ay 21 No S lu dq e C ontrol D u p lica te (50 p pb E tFO S E A lco ho l) 3 0.0 D ay 21 No S lu dq e C ontrol T rip lic a te (50 p pb E tFO S E A lco ho l) 3 0.0 D ay 21 S lu dq e B lank, NO E tFO S E A lco ho l 3 0.0 D ay 21 S lu dq e B la nk D uplicate , NO E tFO S E A lco ho l 3 0.0 D ay 21 S lu dq e B la nk T rip lica te , No E tFO S E A lco ho l 3 0.0 D ay 21 B io de q S am ple (2 3 80 p pb E tFO S E A lco ho l) 3 0.0 D ay 21 B io de q S am ple D u p lica te (2380 p pb E tFO S E A lco ho l) 3 0.0 D ay 21 B io de q S am ple T rip lic a te (2380 p pb E tFO S E A lco ho l) 3 0.0 D ay 21 S te rile S lu dq e C ontrol (2 3 80 p pb E tFO S E A lco ho l) 3 0.0 D ay 21 S te rile S lu dq e C ontrol D u p lica te (2380 p pb E tFO S E A lco ho l) 3 0.0 D ay 21 S te rile S lu dq e C ontrol T rip lic a te (2380 p pb E tFO S E A lco ho l) 3 0.0 D ay 21 No S lu dq e C ontrol (2 3 80 p pb E tFO S E A lco ho l) 3 0.0 D ay 21 No S lu dq e C ontrol D u p lica te (2380 p pb E tFO S E A lco ho l) 3 0.0 PFOS Concentration (ng/mL)____________ N- PFOS N- EtFO SE su lfin a te M556 E tFO S A A FOSA a lco h o l N- E tF O S A 6.0 0.0 15.1 0.0 7.2 0.0 0.0 5.8 0.0 14.7 0.0 7.4 0.0 0.0 5.3 0.0 15.1 0.0 6.8 0.0 0.0 0.0 0.0 0.0 0.0 0.0 39.9 0.0 0.0 0.0 0.0 0.0 0.0 39.2 0.0 0.0 0.0 0.0 0.0 0.0 40.7 0.0 0.0 0.0 0.0 0.0 0.0 28.6 0.0 0.0 0.0 0.0 0.0 0.0 3.1 0.0 0.0 0.0 0.0 0.0 0.0 22.5 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 116.1 46.2 610.4 679.8 92.3 324.1 6.8 105.6 51.9 811.0 560.6 88.4 221.4 6.3 111.3 57.0 888.5 429.8 86.8 247.2 6.1 0.0 0.0 0.0 2.9 0.0 2658.7 0.0 0.0 0.0 0.0 0.0 0.0 2548.1 0.0 0.0 0.0 0.0 0.0 0.0 2455.2 0.0 0.0 0.0 0.0 16.3 0.0 2119.0 0.0 0.0 6.8 6.8 12.2 0.0 2148.2 0.0 0.0 0.0 0.0 5.7 0.0 2347.1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 3.5 0.0 39.3 45.6 0.0 4.9 0.0 5.3 3.0 77.1 59.3 3.0 5.6 0.0 4.9 2.9 77.9 45.4 3.9 8.2 0.0 0.0 0.0 0.0 0.0 0.0 50.4 0.0 0.0 0.0 0.0 0.0 0.0 45.8 0.0 0.0 0.0 0.0 0.0 0.0 59.2 0.0 0.0 0.0 0.0 0.0 0.0 115.2 0.0 0.0 0.0 0.0 0.0 0.0 144.0 0.0 0.0 0.0 0.0 0.0 0.0 136.3 0.0 Page 44 of 55 Aerobic Biodegradation of ^-EtFOSE alcohol Cleston C. Lange, Ph.D., Pace Analytical Services, Science Solutions Division Wednesday November 1, 2000 BACK TO MAIN Appendix B, Table 1 continued. S a m p le ID CA058-0801-SA-127 CA058-0801-SA-128 CA058-0801-SA-129 C A 0 5 8 -0 80 1 -S A -13 0 C A 0 5 8 -0 8 0 1 -S A -1 3 1 CA058-0801-SA-132 CA058-0801-SA-133 CA058-0801-SA-134 CA058-0801-SA-135 C A 0 5 8 -0 80 1 -S A -13 6 CA058-0801-SA-137 CA058-0801-SA-138 CA058-0801-SA-139 C A 0 5 8 -0 80 1 -S A -14 0 C A 0 5 8 -0 8 0 1 -S A -1 4 1 CA058-0801-SA-142 CA058-0801-SA-143 CA058-0801-SA-144 CA058-0801-SA-145 C A 0 5 8 -0 80 1 -S A -14 6 CA058-0801-SA-147 CA058-0801-SA-127 CA058-0801-SA-128 CA058-0801-SA-129 C A 0 5 8 -0 80 1 -S A -13 0 C A 0 5 8 -0 8 0 1 -S A -1 3 1 CA058-0801-SA-132 CA058-0801-SA-133 CA058-0801-SA-134 CA058-0801-SA-135 C A 0 5 8 -0 80 1 -S A -13 6 CA058-0801-SA-137 CA058-0801-SA-138 CA058-0801-SA-139 C A 0 5 8 -0 80 1 -S A -14 0 C A 0 5 8 -0 8 0 1 -S A -1 4 1 CA058-0801-SA-142 CA058-0801-SA-143 CA058-0801-SA-144 CA058-0801-SA-145 C A 0 5 8 -0 80 1 -S A -14 6 CA058-0801-SA-147 D escri Dtion Day 28 B iodeq Sam ple (50 ppb EtFOSE A lcohol) Day 28 B iodeq Sam ple Duplicate (50 ppb EtFOSE A lcohol) Day 28 B iodeq Sam ple Triplicate (50 ppb EtFOSE A lcohol) Day 28 S terile S ludqe Control Sam ple (50 ppb EtFOSE A lcohol) Day 28 S terile S ludqe Control Duplicate (50 ppb EtFOSE A lcohol) Day 28 S terile S ludqe Control Triplicate (50 ppb EtFOSE A lcohol) Day 28 No S ludqe Control Sam ple (50 ppb EtFOSE A lcohol) Day 28 No S ludqe Control Duplicate (50 ppb EtFOSE A lcohol) Day 28 No S ludqe Control Triplicate (50 ppb EtFOSE A lcohol) Day 28 S ludqe Blank, NO EtFOSE A lcohol Day 28 S ludqe Blank Duplicate, NO EtFOSE A lcohol Day 28 S ludqe Blank Triplicate, No EtFOSE A lcohol Day 28 B iodeq Sam ple (2380 ppb EtFOSE A lcohol) Day 28 B iodeq Sam ple Duplicate (2380 ppb EtFOSE A lcohol) Day 28 B iodeq Sam ple Triplicate (2380 ppb EtFOSE A lcohol) Day 28 S terile S ludqe Control (2380 ppb EtFOSE A lcohol) Day 28 S terile S ludqe Control Duplicate (2380 ppb EtFOSE A lcohol) Day 28 S terile S ludqe Control Triplicate (2380 ppb EtFOSE A lcohol) Day 28 No S ludqe Control (2380 ppb EtFOSE A lcohol) Day 28 No S ludqe Control Duplicate (2380 ppb EtFOSE A lcohol) Day 28 No S ludqe Control Triplicate (2380 ppb EtFOSE A lcohol) Day 28 B iodeq Sam ple (50 ppb EtFOSE A lcohol) Day 28 B iodeq Sam ple Duplicate (50 ppb EtFOSE A lcohol) Day 28 B iodeq Sam ple Triplicate (50 ppb EtFOSE A lcohol) Day 28 S terile S ludqe Control Sam ple (50 ppb EtFOSE A lcohol) Day 28 S terile S ludqe Control Duplicate (50 ppb EtFOSE A lcohol) Day 28 S terile S ludqe Control Triplicate (50 ppb EtFOSE A lcohol) Day 28 No S ludqe Control Sam ple (50 ppb EtFOSE A lcohol) Day 28 No S ludqe Control Duplicate (50 ppb EtFOSE A lcohol) Day 28 No S ludqe Control Triplicate (50 ppb EtFOSE A lcohol) Day 28 S ludqe Blank, NO EtFOSE A lcohol Day 28 S ludqe Blank Duplicate, NO EtFOSE A lcohol Day 28 S ludqe Blank Triplicate, No EtFOSE A lcohol Day 28 B iodeq Sam ple (2380 ppb EtFOSE A lcohol) Day 28 B iodeq Sam ple Duplicate (2380 ppb EtFOSE A lcohol) Day 28 B iodeq Sam ple Triplicate (2380 ppb EtFOSE A lcohol) Day 28 S terile S ludqe Control (2380 ppb EtFOSE A lcohol) Day 28 S terile S ludqe Control Duplicate (2380 ppb EtFOSE A lcohol) Day 28 S terile S ludqe Control Triplicate (2380 ppb EtFOSE A lcohol) Day 28 No S ludqe Control (2380 ppb EtFOSE A lcohol) Day 28 No S ludqe Control Duplicate (2380 ppb EtFOSE A lcohol) Day 28 No S ludqe Control Triplicate (2380 ppb EtFOSE A lcohol) El u ate PFOA 2 0.0 2 0.0 2 0.0 2 0.0 2 0.0 2 0.0 2 0.0 2 0.0 2 0.0 2 0.0 2 0.0 2 0.0 2 11.4 2 10.2 2 10.4 2 0.0 2 0.0 2 0.0 2 0.0 2 0.0 2 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 PFOS Concentration (ng/mL)______ _____ N- PFOS N- EtFO SE su lfin a te M556 EtFOSAA FOSA a lco h o l N- E tF O S A 6.9 0.0 10.1 0.0 7.4 0.0 0.0 6.7 0.0 9.4 0.0 8.0 0.0 0.0 7.0 0.0 11.9 0.0 7.1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 37.5 0.0 0.0 0.0 0.0 0.0 0.0 36.6 0.0 0.0 0.0 0.0 0.0 0.0 37.9 0.0 0.0 0.0 0.0 0.0 0.0 26.7 0.0 0.0 0.0 0.0 0.0 0.0 26.4 0.0 0.0 0.0 0.0 0.0 0.0 23.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 125.1 57.7 786.0 581.7 100.4 324.0 3.2 133.1 49.7 768.0 559.5 108.0 373.0 4.2 138.1 59.7 893.3 470.2 102.3 281.5 3.9 0.0 3.0 0.0 0.0 0.0 2369.0 0.0 0.0 2.9 0.0 0.0 0.0 2343.4 0.0 0.0 4.8 0.0 5.2 0.0 1923.6 0.0 0.0 5.9 0.0 26.2 0.0 1636.3 0.0 0.0 3.6 0.0 16.6 0.0 1736.7 0.0 0.0 7.1 0.0 49.0 0.0 1950.3 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 7.0 3.6 84.2 52.5 2.9 9.6 0.0 6.9 3.1 75.7 52.1 2.6 9.2 0.0 6.0 3.4 89.8 41.3 2.6 7.5 0.0 0.0 0.0 0.0 0.0 0.0 39.3 0.0 0.0 0.0 0.0 0.0 0.0 57.1 0.0 0.0 0.0 0.0 0.0 0.0 66.5 0.0 0.0 0.0 0.0 0.0 0.0 61.0 0.0 0.0 0.0 0.0 0.0 0.0 100.4 0.0 0.0 0.0 0.0 0.0 0.0 108.4 0.0 Page 45 of 55 Aerobic Biodegradation of ^-EtFOSE alcohol Cleston C. Lange, Ph.D., Pace Analytical Services, Science Solutions Division Wednesday November 1, 2000 BACK TO MAIN Appendix B, Table 1 continued. S a m p le ID CA058-0801-SA-148 CA058-0801-SA-149 C A 0 58 -0 80 1 -S A -15 0 C A 0 5 8 -0 8 0 1 -S A -1 5 1 CA058-0801-SA-152 CA058-0801-SA-153 CA058-0801-SA-154 CA058-0801-SA-155 C A 0 58 -0 80 1 -S A -15 6 CA058-0801 -S A -157 CA058-0801-SA-158 CA058-0801-SA-159 C A 0 58 -0 80 1 -S A -16 0 C A 0 5 8 -0 8 0 1 -S A -1 6 1 CA058-0801-SA-162 CA058-0801-SA-163 CA058-0801-SA-164 CA058-0801-SA-165 C A 0 58 -0 80 1 -S A -16 6 CA058-0801-SA-167 CA058-0801-SA-168 CA058-0801-SA-148 CA058-0801-SA-149 C A 0 58 -0 80 1 -S A -15 0 C A 0 5 8 -0 8 0 1 -S A -1 5 1 CA058-0801-SA-152 CA058-0801-SA-153 CA058-0801-SA-154 CA058-0801-SA-155 C A 0 58 -0 80 1 -S A -15 6 CA058-0801 -S A -157 CA058-0801-SA-158 CA058-0801-SA-159 C A 0 58 -0 80 1 -S A -16 0 C A 0 5 8 -0 8 0 1 -S A -1 6 1 CA058-0801-SA-162 CA058-0801-SA-163 CA058-0801-SA-164 CA058-0801-SA-165 C A 0 58 -0 80 1 -S A -16 6 CA058-0801-SA-167 CA058-0801-SA-168 D escriD tion Day 35 Biodeq Sam ple (50 ppb EtFOSE A lcohol) Day 35 Biodeq Sam ple D uplicate (50 ppb EtFOSE A lcohol) Day 35 Biodeq Sam ple Triplicate (50 ppb EtFOSE A lcohol) Day 35 S terile Sludqe Control Sam ple (50 ppb EtFOSE A lcohol) Day 35 S terile Sludqe Control D uplicate (50 ppb EtFOSE A lcohol) Day 35 S terile Sludqe Control Triplicate (50 ppb EtFOSE A lcohol) Day 35 No Sludqe Control Sam ple (50 ppb EtFOSE A lcohol) Day 35 No Sludqe Control D uplicate (50 ppb EtFOSE A lcohol) Day 35 No Sludqe Control Triplicate (50 ppb EtFOSE A lcohol) Day 35 Sludqe Blank, NO EtFOSE A lcohol Day 35 Sludqe Blank D uplicate, NO EtFOSE A lcohol Day 35 Sludqe Blank Triplicate, No EtFOSE A lcohol Day 35 Biodeq Sam ple (2380 ppb EtFOSE A lcohol) Day 35 Biodeq Sam ple D uplicate (2380 ppb EtFOSE A lcohol) Day 35 Biodeq Sam ple Triplicate (2380 ppb EtFOSE A lcohol) Day 35 S terile Sludqe Control (2380 ppb EtFOSE A lcohol) Day 35 S terile Sludqe Control D uplicate (2380 ppb EtFOSE A lcohol) Day 35 S terile Sludqe Control Triplicate (2380 ppb EtFOSE A lcohol) Day 35 No Sludqe Control (2380 ppb EtFOSE A lcohol) Day 35 No Sludqe Control D uplicate (2380 ppb EtFOSE A lcohol) Day 35 No Sludqe Control Triplicate (2380 ppb EtFOSE A lcohol) Day 35 Biodeq Sam ple (50 ppb EtFOSE A lcohol) Day 35 Biodeq Sam ple D uplicate (50 ppb EtFOSE A lcohol) Day 35 Biodeq Sam ple Triplicate (50 ppb EtFOSE A lcohol) Day 35 S terile Sludqe Control Sam ple (50 ppb EtFOSE A lcohol) Day 35 S terile Sludqe Control D uplicate (50 ppb EtFOSE A lcohol) Day 35 S terile Sludqe Control Triplicate (50 ppb EtFOSE A lcohol) Day 35 No Sludqe Control Sam ple (50 ppb EtFOSE A lcohol) Day 35 No Sludqe Control D uplicate (50 ppb EtFOSE A lcohol) Day 35 No Sludqe Control Triplicate (50 ppb EtFOSE A lcohol) Day 35 Sludqe Blank, NO EtFOSE A lcohol Day 35 Sludqe Blank D uplicate, NO EtFOSE A lcohol Day 35 Sludqe Blank Triplicate, No EtFOSE A lcohol Day 35 Biodeq Sam ple (2300 EtFOSE A lcohol) Day 35 Biodeq Sam ple D uplicate (2300 EtFOSE A lcohol) Day 35 Biodeq Sam ple Triplicate (2300 EtFOSE A lcohol) Day 35 S terile Sludqe Control (2300 EtFOSE A lcohol) Day 35 S terile Sludqe Control D uplicate (2300 EtFOSE A lcohol) Day 35 S terile Sludqe Control Triplicate (2300 EtFOSE A lcohol) Day 35 No Sludqe Control (2300 EtFOSE A lcohol) Day 35 No Sludqe Control D uplicate (2300 EtFOSE A lcohol) Day 35 No Sludqe Control Triplicate (2300 EtFOSE A lcohol) Eluate PFOA 2 0.0 2 0.0 2 0.0 2 0.0 2 0.0 2 0.0 2 0.0 2 0.0 2 0.0 2 0.0 2 0.0 2 0.0 2 12.7 2 11.6 2 13.4 2 0.0 2 0.0 2 0.0 2 0.0 2 0.0 2 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 3 0.0 PFOS PFOS su lfina te NM556 EtFO S A A FOSA 9.0 0.0 14.2 0.0 7.2 9.9 0.0 14.1 0.0 5.7 8.1 0.0 12.6 3.2 6.7 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 127.1 62.8 651.1 872.1 96.1 137.6 75.6 1091.4 506.9 94.1 140.3 58.6 977.2 555.1 96.7 0.0 3.3 0.0 0.0 0.0 0.0 3.5 0.0 0.0 0.0 0.0 11.7 0.0 0.0 0.0 0.0 19.0 0.0 321.4 0.0 0.0 18.1 0.0 89.2 0.0 0.0 16.4 0.0 329.6 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 5.4 3.4 57.4 70.6 3.8 5.6 3.4 77.5 40.8 3.4 5.9 3.0 72.0 44.9 3.5 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 16.5 0.0 0.0 0.0 0.0 8.2 0.0 0.0 0.0 0.0 17.8 0.0 N- EtFOSE a lcoh o l N- E tF O S A 0.0 0.0 0.0 0.0 0.0 0.0 36.8 0.0 35.3 0.0 35.1 0.0 29.1 0.0 27.4 0.0 27.4 0.0 0.0 0.0 0.0 0.0 0.0 0.0 274.4 3.2 198.1 2.8 224.1 2.3 2330.8 0.0 2332.4 0.0 2049.8 2.6 1729.5 0.0 1676.3 0.0 1777.8 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 12.0 0.0 9.1 0.0 8.9 0.0 42.9 0.0 63.4 0.0 102.1 0.0 108.8 0.0 96.7 0.0 95.8 0.0 Page 46 of 55 Aerobic Biodegradation of ^-EtFOSE alcohol Cleston C. Lange, Ph.D., Pace Analytical Services, Science Solutions Division Wednesday November 1, 2000 BACK TO MAIN Appendix B, Table 1 continued. S a m p le ID CA058-MW -023A CA058-MW -023B CA058-MW -024A CA058-MW -024B CA058-MW -025A CA058-MW -025B CA058-MW -026A CA058-MW -026B CA058-MW -027A CA058-MW -027B CA058-MW -023A CA058-MW -023B CA058-MW -024A CA058-MW -024B CA058-MW -025A CA058-MW -025B CA058-MW -026A CA058-MW -026B CA058-MW -027A CA058-MW -027B D escription Eluate PFOA SPE Q C S AM PLE , 10 PPB IN S LU D G E FO R R E C O V ER Y 2 10.0 SPE QC D UPLICATE SAM PLE, 10 PPB IN SLU DG E FOR RECOVERY 2 11.1 SPE Q C S AM PLE , 50 PPB IN S LU D G E FO R R E C O V ER Y 2 58.9 SPE QC D UPLICATE SAM PLE, 50 PPB IN SLU DG E FOR RECOVERY 2 58.0 SPE QC SAM PLE, 200 PPB IN SLU DG E FOR RECOVERY 2 220.5 SPE Q C D U P L IC A T E S AM P LE , 200 PPB IN S LU D G E FO R R E C O V ER Y 2 235.3 SPE QC SAM PLE, 500 PPB IN SLU DG E FOR RECOVERY 2 482.2 SPE Q C D U P L IC A T E S AM P LE , 500 PPB IN S LU D G E FO R R E C O V ER Y 2 491.9 SPE QC SAM PLE, 1000 PPB IN SLU DG E FOR RECOVERY 2 943.5 SPE Q C D U P L IC A T E S AM P LE , 1000 PPB IN S LU D G E FO R R E C O V ER Y 2 948.6 SPE Q C S AM PLE , 10 PPB IN S LU D G E FO R R E C O V ER Y 3 0.0 SPE QC D UPLICATE SAM PLE, 10 PPB IN SLU DG E FOR RECOVERY 3 0.0 SPE Q C S AM PLE , 50 PPB IN S LU D G E FO R R E C O V ER Y 3 0.0 SPE QC D UPLICATE SAM PLE, 50 PPB IN SLU DG E FOR RECOVERY 3 0.0 SPE QC SAM PLE, 200 PPB IN SLU DG E FOR RECOVERY 3 0.0 SPE Q C D U P L IC A T E S AM P LE , 200 PPB IN S LU D G E FO R R E C O V ER Y 3 0.0 SPE QC SAM PLE, 500 PPB IN SLU DG E FOR RECOVERY 3 18.7 SPE Q C D U P L IC A T E S AM P LE , 500 PPB IN S LU D G E FO R R E C O V ER Y 3 16.8 SPE QC SAM PLE, 1000 PPB IN SLU DG E FOR RECOVERY 3 30.1 SPE Q C D U P L IC A T E S AM P LE , 1000 PPB IN S LU D G E FO R R E C O V ER Y 3 22.2 PFOS PFOS su lfina te 9.7 10.1 9.7 10.3 53.7 56.0 52.5 53.2 198.8 210.7 205.5 216.5 412.5 461.5 429.1 468.8 866.7 911.5 864.9 928.5 0.0 0.0 0.0 0.0 2.9 3.3 5.6 5.9 20.4 20.3 15.9 16.3 36.3 35.9 29.9 31.6 54.4 57.6 37.4 40.1 NM556 EtFO S A A FOSA 0.0 9.6 10.1 0.0 9.8 10.2 4.9 54.5 55.9 4.5 53.0 55.1 13.8 199.0 206.3 14.4 200.6 207.4 29.3 419.9 444.3 31.7 439.9 454.2 53.5 889.1 895.1 55.3 874.0 874.6 0.0 0.0 0.0 0.0 0.0 0.0 0.0 4.9 2.5 0.0 6.7 3.9 0.0 22.0 14.7 0.0 21.2 11.1 0.0 38.6 24.3 0.0 35.1 20.7 0.0 51.6 33.5 0.0 41.6 23.1 N- EtFOSE a lcoh o l N- E tF O S A 11.6 10.3 10.9 10.1 61.3 56.6 60.1 56.7 230.5 216.6 232.0 215.8 503.5 472.6 514.4 474.5 989.6 958.4 944.2 923.7 0.0 0.0 0.0 0.0 0.0 2.9 4.8 4.2 15.4 14.4 11.3 10.7 22.9 21.6 19.2 18.4 28.3 26.8 21.1 20.6 Page 47 of 55 Aerobic Biodegradation of ^-EtFOSE alcohol Cleston C. Lange, Ph.D., Pace Analytical Services, Science Solutions Division Wednesday November 1, 2000 BACK TO MAIN Appendix B, continued Table 2. Biodegradation tables presented as micromolar concentrations. Results of the analysis of eluate 2 and eluate 3 for each sample are combined to create the final values presented. D escription Day 0 Biodeg Sample (50 ppb EtFOSE Alcohol) Day 0 Biodeg Sample Duplicate (50 ppb EtFOSE Alcohol) Day 0 Biodeg Sample Triplicate (50 ppb EtFOSE Alcohol) Day 0 Sterile Sludge Control Sample (50 ppb EtFOSE Alcohol) Day 0 Sterile Sludge Control Duplicate (50 ppb EtFOSE Alcohol) Day 0 Sterile Sludge Control Triplicate (50 ppb EtFOSE Alcohol) Day 0 No Sludge Control Sample (50 ppb EtFOSE Alcohol) Day 0 No Sludge Control Duplicate (50 ppb EtFOSE Alcohol) Day 0 No Sludge Control Triplicate (50 ppb EtFOSE Alcohol) Day 0 Sludge Blank, NO EtFOSE Alcohol Day 0 Sludge Blank Duplicate, NO EtFOSE Alcohol Day 0 Sludge Blank Triplicate, No EtFOSE Alcohol Day 0 Biodeg Sample (2380 ppb EtFOSE Alcohol) Day 0 Biodeg Sample Duplicate (2380 ppb EtFOSE Alcohol) Day 0 Biodeg Sample Triplicate (2380 ppb EtFOSE Alcohol) Day 0 Sterile Sludge Control (2380 ppb EtFOSE Alcohol) Day 0 Sterile Sludge Control Duplicate (2380 ppb EtFOSE Alcohol) Day 0 Sterile Sludge Control Triplicate (2380 ppb EtFOSE Alcohol) Day 0 No Sludge Control (2380 ppb EtFOSE Alcohol) Day 0 No Sludge Control Duplicate (2380 ppb EtFOSE Alcohol) Day 0 No Sludge Control Triplicate (2380 ppb EtFOSE Alcohol) D escription Day 1 Biodeg Sample (50 ppb EtFOSE Alcohol) Day 1 Biodeg Sample Duplicate (50 ppb EtFOSE Alcohol) Day 1 Biodeg Sample Triplicate (50 ppb EtFOSE Alcohol) Day 1 Sterile Sludge Control Sample (50 ppb EtFOSE Alcohol) Day 1 Sterile Sludge Control Duplicate (50 ppb EtFOSE Alcohol) Day 1 Sterile Sludge Control Triplicate (50 ppb EtFOSE Alcohol) Day 1 No Sludge Control Sample (50 ppb EtFOSE Alcohol) Day 1 No Sludge Control Duplicate (50 ppb EtFOSE Alcohol) Day 1 No Sludge Control Triplicate (50 ppb EtFOSE Alcohol) Day 1 Sludge Blank, NO EtFOSE Alcohol Day 1 Sludge Blank Duplicate, NO EtFOSE Alcohol Day 1 Sludge Blank Triplicate, No EtFOSE Alcohol Day 1 Biodeg Sample (2380 ppb EtFOSE Alcohol) Day 1 Biodeg Sample Duplicate (2380 ppb EtFOSE Alcohol) Day 1 Biodeg Sample Triplicate (2380 ppb EtFOSE Alcohol) Day 1 Sterile Sludge Control (2380 ppb EtFOSE Alcohol) Day 1 Sterile Sludge Control Duplicate (2380 ppb EtFOSE Alcohol) Day 1 Sterile Sludge Control Triplicate (2380 ppb EtFOSE Alcohol) Day 1 No Sludge Control (2380 ppb EtFOSE Alcohol) Day 1 No Sludge Control Duplicate (2380 ppb EtFOSE Alcohol) Day 1 No Sludge Control Triplicate (2380 ppb EtFOSE Alcohol) D escription Day 3 Biodeg Sample (50 ppb EtFOSE Alcohol) Day 3 Biodeg Sample Duplicate (50 ppb EtFOSE Alcohol) Day 3 Biodeg Sample Triplicate (50 ppb EtFOSE Alcohol) Day 3 Sterile Sludge Control Sample (50 ppb EtFOSE Alcohol) Day 3 Sterile Sludge Control Duplicate (50 ppb EtFOSE Alcohol) Day 3 Sterile Sludge Control Triplicate (50 ppb EtFOSE Alcohol) Day 3 No Sludge Control Sample (50 ppb EtFOSE Alcohol) Day 3 No Sludge Control Duplicate (50 ppb EtFOSE Alcohol) Day 3 No Sludge Control Triplicate (50 ppb EtFOSE Alcohol) Day 3 Sludge Blank, NO EtFOSE Alcohol Day 3 Sludge Blank Duplicate, NO EtFOSE Alcohol Day 3 Sludge Blank Triplicate, No EtFOSE Alcohol Day 3 Biodeg Sample (2380 ppb EtFOSE Alcohol) Day 3 Biodeg Sample Duplicate (2380 ppb EtFOSE Alcohol) Day 3 Biodeg Sample Triplicate (2380 ppb EtFOSE Alcohol) Day 3 Sterile Sludge Control (2380 ppb EtFOSE Alcohol) Day 3 Sterile Sludge Control Duplicate (2380 ppb EtFOSE Alcohol) Day 3 Sterile Sludge Control Triplicate (2380 ppb EtFOSE Alcohol) Day 3 No Sludge Control (2380 ppb EtFOSE Alcohol) Day 3 No Sludge Control Duplicate (2380 ppb EtFOSE Alcohol) Day 3 No Sludge Control Triplicate (2380 ppb EtFOSE Alcohol) PFOA 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 PFOA 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 PFOA 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 PFOS 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 PFOS 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.009 0.008 0.009 0.000 0.000 0.000 0.000 0.000 0.000 PFOS 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.030 0.026 0.033 0.000 0.000 0.000 0.000 0.000 0.000 PFOS su lfina te 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 PFOS su lfina te 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.094 0.094 0.098 0.000 0.000 0.000 0.000 0.000 0.000 PFOS su lfina te 0.000 0.000 0.004 0.003 0.003 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.128 0.130 0.139 0.000 0.000 0.000 0.000 0.000 0.000 Page 48 of 55 Aerobic Biodegradation of ^-EtFOSE alcohol Cleston C. Lange, Ph.D., Pace Analytical Services, Science Solutions Division Wednesday November 1, 2000 C o n c e n tr a tio n (u,M) M556 N-EtFO SAA 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 C o n c e n tr a tio n (u.M) M556 N-EtFO SAA 0.000 0.026 0.000 0.024 0.000 0.022 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.049 0.651 0.048 0.637 0.053 0.660 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 C o n c e n tr a tio n (u,M) M556 0.012 0.009 0.009 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.223 0.210 0.226 0.000 0.000 0.000 0.000 0.000 0.000 N-EtFO SAA 0.034 0.029 0.028 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.071 1.057 1.157 0.000 0.000 0.000 0.000 0.000 0.000 FOSA 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 FOSA 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.016 0.016 0.017 0.000 0.000 0.000 0.000 0.000 0.000 FOSA 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.047 0.042 0.051 0.000 0.000 0.000 0.000 0.000 0.000 N-EtFOSE a lcoh o l 0.076 0.069 0.065 0.080 0.078 0.077 0.076 0.080 0.078 0.000 0.000 0.000 5.109 4.894 4.761 5.159 4.908 4.879 4.884 4.948 5.031 N-EtFOSA 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 N-EtFOSE a lcoh o l 0.031 0.031 0.032 0.077 0.078 0.080 0.071 0.072 0.072 0.000 0.000 0.000 3.100 3.160 3.119 5.080 4.917 4.919 4.994 4.907 4.759 N-EtFOSA 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.021 0.020 0.022 0.000 0.000 0.000 0.000 0.000 0.000 N-EtFOSE a lcoh o l 0.013 0.017 0.016 0.070 0.068 0.072 0.064 0.067 0.065 0.000 0.000 0.000 2.205 2.332 2.032 4.831 4.735 4.697 4.549 4.674 4.667 N-EtFOSA 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.030 0.028 0.031 0.000 0.000 0.000 0.000 0.000 0.000 BACK TO MAIN Appendix B, Table 2 continued. D e scrip tio n Dav 7 Biodea Sample (50 DDb EtFOSE Alcohol) Day 7 Biodea Sample Duplicate (50 ppb EtFOSE Alcohol) Dav 7 Biodea Sample Triplicate (50 ppb EtFOSE Alcohol) Dav 7 Sterile Sludae Control Sample (50 ppb EtFOSE Alcohol) Dav 7 Sterile Sludae Control Duplicate (50 ppb EtFOSE Alcohol) Dav 7 Sterile Sludae Control Triplicate (50 ppb EtFOSE Alcohol) Dav 7 No Sludae Control Sample (50 ppb EtFOSE Alcohol) Dav 7 No Sludae Control Duplicate (50 ppb EtFOSE Alcohol) Dav 7 No Sludae Control Triplicate (50 ppb EtFOSE Alcohol) Dav 7 Sludae Blank, NO EtFOSE Alcohol Dav 7 Sludae Blank Duplicate, NO EtFOSE Alcohol Dav 7 Sludae Blank Triplicate, No EtFOSE Alcohol Dav 7 Biodea Sample (2380 ppb EtFOSE Alcohol) Dav 7 Biodea Sample Duplicate (2380 ppb EtFOSE Alcohol) Dav 7 Biodea Sample Triplicate (2380 ppb EtFOSE Alcohol) Dav 7 Sterile Sludae Control (2380 ppb EtFOSE Alcohol) Dav 7 Sterile Sludae Control Duplicate (2380 ppb EtFOSE Alcohol) Dav 7 Sterile Sludae Control Triplicate (2380 ppb EtFOSE Alcohol) Dav 7 No Sludae Control (2380 ppb EtFOSE Alcohol) Dav 7 No Sludae Control Duplicate (2380 ppb EtFOSE Alcohol) Dav 7 No Sludae Control Triplicate (2380 ppb EtFOSE Alcohol) D escriD tion Dav 14 Biodea Sample (50 ppb EtFOSE Alcohol) Dav 14 Biodea Sample Duplicate (50 ppb EtFOSE Alcohol) Dav 14 Biodea Sample Triplicate (50 ppb EtFOSE Alcohol) Dav 14 Sterile Sludae Control Sample (50 ppb EtFOSE Alcohol) Dav 14 Sterile Sludae Control Duplicate (50 ppb EtFOSE Alcohol) Dav 14 Sterile Sludae Control Triplicate (50 ppb EtFOSE Alcohol) Dav 14 No Sludae Control Sample (50 ppb EtFOSE Alcohol) Dav 14 No Sludae Control Duplicate (50 ppb EtFOSE Alcohol) Dav 14 No Sludae Control Triplicate (50 ppb EtFOSE Alcohol) Dav 14 Sludae Blank, NO EtFOSE Alcohol Dav 14 Sludae Blank Duplicate, NO EtFOSE Alcohol Dav 14 Sludae Blank Triplicate, No EtFOSE Alcohol Dav 14 Biodea Sample (2380 ppb EtFOSE Alcohol) Dav 14 Biodea Sample Duplicate (2380 ppb EtFOSE Alcohol) Dav 14 Biodea Sample Triplicate (2380 ppb EtFOSE Alcohol) Dav 14 Sterile Sludae Control (2380 ppb EtFOSE Alcohol) Dav 14 Sterile Sludae Control Duplicate (2380 ppb EtFOSE Alcohol) Dav 14 Sterile Sludae Control Triplicate (2380 ppb EtFOSE Alcohol) Dav 14 No Sludae Control (2380 ppb EtFOSE Alcohol) Dav 14 No Sludae Control Duplicate (2380 ppb EtFOSE Alcohol) Dav 14 No Sludae Control Triplicate (2380 ppb EtFOSE Alcohol) D e scrip tio n Dav 21 Biodea Sample (50 ppb EtFOSE Alcohol) Dav 21 Biodea Sample (50 ppb EtFOSE Alcohol) Dav 21 Biodea Sample Duplicate (50 ppb EtFOSE Alcohol) Dav 21 Biodea Sample Triplicate (50 ppb EtFOSE Alcohol) Dav 21 Sterile Sludae Control Sample (50 ppb EtFOSE Alcohol) Dav 21 Sterile Sludae Control Duplicate (50 ppb EtFOSE Alcohol) Dav 21 Sterile Sludae Control Triplicate (50 ppb EtFOSE Alcohol) Dav 21 No Sludae Control Sample (50 ppb EtFOSE Alcohol) Dav 21 No Sludae Control Duplicate (50 ppb EtFOSE Alcohol) Dav 21 No Sludae Control Triplicate (50 ppb EtFOSE Alcohol) Dav 21 Sludae Blank, NO EtFOSE Alcohol Dav 21 Sludae Blank Duplicate, NO EtFOSE Alcohol Dav 21 Sludae Blank Triplicate, No EtFOSE Alcohol Dav 21 Biodea Sample (2380 ppb EtFOSE Alcohol) Dav 21 Biodea Sample Duplicate (2380 ppb EtFOSE Alcohol) Dav 21 Biodea Sample Triplicate (2380 ppb EtFOSE Alcohol) Dav 21 Sterile Sludae Control (2380 ppb EtFOSE Alcohol) Dav 21 Sterile Sludae Control Duplicate (2380 ppb EtFOSE Alcohol) Dav 21 Sterile Sludae Control Triplicate (2380 ppb EtFOSE Alcohol) Dav 21 No Sludae Control (2380 ppb EtFOSE Alcohol) Dav 21 No Sludae Control Duplicate (2380 ppb EtFOSE Alcohol) PFOA 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0.009 0.009 0.009 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 PFOA 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0.021 0 .0 2 0 0 .0 2 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 PFOA 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0.024 0.024 0.022 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 PFOS 0.005 0.005 0.005 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0.092 0.085 0.085 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 PFOS 0 .0 1 0 0.009 0.009 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0.172 0.172 0.172 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 PFOS 0.011 0.011 0 .0 1 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0.222 0 .2 0 6 0 .2 1 6 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 PFOS su lfina te 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0.123 0.125 0.131 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 PFOS su lfina te 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0.119 0.115 0.111 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 PFOS su lfina te 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0.089 0.105 0.115 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0.013 0 .0 0 0 C o n c e n tra tio n (uM) M556 N-EtFO SAA 0.021 0.022 0 .0 2 0 0.025 0.022 0.021 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0.777 1.139 0.759 1.067 0.738 1.184 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 C o n c e n tra tio n (uM) M556 N-EtFO SAA 0.029 0.008 0.029 0.007 0.028 0.009 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 1.286 0.972 1.388 0.825 1.449 0.834 0 .0 0 0 0.005 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 1 0 0 .0 0 0 0.029 0 .0 0 0 0.007 C o n c e n tra tio n (uM) M556 0.027 0 .0 2 6 0.027 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 1.166 1.594 1.735 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0.012 0 .0 0 0 N-EtFOSAA 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 1.240 1.060 0.812 0.005 0 .0 0 0 0 .0 0 0 0.028 0.021 0 .0 1 0 FOSA 0 .0 1 0 0 .0 0 6 0.007 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0.125 0 .1 1 6 0.119 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 FOSA 0 .0 1 6 0.015 0.015 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0.163 0 .1 6 6 0.173 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 FOSA 0.014 0.015 0.014 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0.185 0.183 0.182 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 N-EtFOSE alcoh o l 0.005 0 .0 0 0 0 .0 0 0 0.071 0.071 0.069 0.061 0.061 0.064 0 .0 0 0 0 .0 0 0 0 .0 0 0 1.434 1.465 1.479 4.774 5.010 4.864 4 .5 4 0 4.694 4.718 N-EtFOSA 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0.032 0.031 0.029 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 N-EtFOSE alcoh o l 0 .0 0 0 0 .0 0 0 0 .0 0 0 0.077 0.068 0 .0 7 6 0 .0 5 6 0.057 0 .0 5 6 0 .0 0 0 0 .0 0 0 0 .0 0 0 0.767 0.732 0.741 4.791 4 .7 8 0 4.693 4.312 4.482 4.462 N-EtFOSA 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0.022 0.021 0 .0 2 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 N-EtFOSE alcoh o l 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 7 0 0.069 0.071 0 .0 5 0 0 .0 0 6 0.039 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .5 7 6 0.398 0.447 4.745 4.543 4.404 3.913 4.014 4.349 N-EtFOSA 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0.013 0.012 0.011 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 Page 49 of 55 Aerobic Biodegradation of ^-EtFOSE alcohol Cleston C. Lange, Ph.D., Pace Analytical Services, Science Solutions Division Wednesday November 1, 2000 BACK TO MAIN Appendix B, Table 2 continued. S am ple D e scrip tio n Dav 28 Biodea Sample (50 DDb EtFOSE Alcohol) Day 28 Biodea Sample Duplicate (50 ppb EtFOSE Alcohol) Dav 28 Biodea Sample Triplicate (50 ppb EtFOSE Alcohol) Dav 28 Sterile Sludae Control Sample (50 ppb EtFOSE Alcohol) Dav 28 Sterile Sludae Control Duplicate (50 ppb EtFOSE Alcohol) Dav 28 Sterile Sludae Control Triplicate (50 ppb EtFOSE Alcohol) Dav 28 No Sludae Control Sample (50 ppb EtFOSE Alcohol) Dav 28 No Sludae Control Duplicate (50 ppb EtFOSE Alcohol) Dav 28 No Sludae Control Triplicate (50 ppb EtFOSE Alcohol) Dav 28 Sludae Blank, NO EtFOSE Alcohol Dav 28 Sludae Blank Duplicate, NO EtFOSE Alcohol Dav 28 Sludae Blank Triplicate, No EtFOSE Alcohol Dav 28 Biodea Sample (2380 ppb EtFOSE Alcohol) Dav 28 Biodea Sample Duplicate (2380 ppb EtFOSE Alcohol) Dav 28 Biodea Sample Triplicate (2380 ppb EtFOSE Alcohol) Dav 28 Sterile Sludae Control (2380 ppb EtFOSE Alcohol) Dav 28 Sterile Sludae Control Duplicate (2380 ppb EtFOSE Alcohol) Dav 28 Sterile Sludae Control Triplicate (2380 ppb EtFOSE Alcohol) Dav 28 No Sludae Control (2380 ppb EtFOSE Alcohol) Dav 28 No Sludae Control Duplicate (2380 ppb EtFOSE Alcohol) Dav 28 No Sludae Control Triplicate (2380 ppb EtFOSE Alcohol) PFOA 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.026 0.024 0.024 0.000 0.000 0.000 0.000 0.000 0.000 S am ple D e scrip tio n Dav 35 Biodea Sample (50 ppb EtFOSE Alcohol) Dav 35 Biodea Sample Duplicate (50 ppb EtFOSE Alcohol) Dav 35 Biodea Sample Triplicate (50 ppb EtFOSE Alcohol) Dav 35 Sterile Sludae Control Sample (50 ppb EtFOSE Alcohol) Dav 35 Sterile Sludae Control Duplicate (50 ppb EtFOSE Alcohol) Dav 35 Sterile Sludae Control Triplicate (50 ppb EtFOSE Alcohol) Dav 35 No Sludae Control Sample (50 ppb EtFOSE Alcohol) Dav 35 No Sludae Control Duplicate (50 ppb EtFOSE Alcohol) Dav 35 No Sludae Control Triplicate (50 ppb EtFOSE Alcohol) Dav 35 Sludae Blank, NO EtFOSE Alcohol Dav 35 Sludae Blank Duplicate. NO EtFOSE Alcohol Dav 35 Sludae Blank Triplicate, No EtFOSE Alcohol Dav 35 Biodea Sample (2380 ppb EtFOSE Alcohol) Dav 35 Biodea Sample Duplicate (2380 ppb EtFOSE Alcohol) Dav 35 Biodea Sample Triplicate (2380 ppb EtFOSE Alcohol) Dav 35 Sterile Sludae Control (2380 ppb EtFOSE Alcohol) Dav 35 Sterile Sludae Control Duplicate (2380 ppb EtFOSE Alcohol) Dav 35 Sterile Sludae Control Triplicate (2380 ppb EtFOSE Alcohol) Dav 35 No Sludae Control (2380 ppb EtFOSE Alcohol) Dav 35 No Sludae Control Duplicate (2380 ppb EtFOSE Alcohol) Dav 35 No Sludae Control Triplicate (2380 ppb EtFOSE Alcohol) PFOA 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.030 0.027 0.031 0.000 0.000 0.000 0.000 0.000 0.000 PFOS 0.016 0.015 0.016 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.306 0.325 0.334 0.000 0.000 0.000 0.000 0.000 0.000 PFOS 0.021 0.023 0.019 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.307 0.332 0.339 0.000 0.000 0.000 0.000 0.000 0.000 PFOS su lfina te 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.142 0.123 0.146 0.007 0.007 0.011 0.014 0.008 0.016 PFOS su lfina te 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.154 0.183 0.143 0.008 0.008 0.027 0.044 0.042 0.038 C o n c e n tr a tio n (u,M) M556 N-EtFOSAA 0.023 0.000 0.022 0.000 0.028 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 2.019 1.472 1.958 1.419 2.281 1.187 0.000 0.000 0.000 0.000 0.000 0.012 0.000 0.061 0.000 0.039 0.000 0.114 C o n c e n tra tio n (uM) M556 0.033 0.033 0.029 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 1.644 2.712 2.434 0.000 0.000 0.000 0.000 0.000 0.000 N-EtFOSAA 0.000 0.000 0.007 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 2.187 1.271 1.392 0.000 0.000 0.000 0.784 0.226 0.806 FOSA 0.017 0.018 0.017 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.240 0.256 0.243 0.000 0.000 0.000 0.000 0.000 0.000 FOSA 0.017 0.013 0.015 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.232 0.226 0.232 0.000 0.000 0.000 0.000 0.000 0.000 N-EtFOSE alcoh o l 0.000 0.000 0.000 0.087 0.085 0.088 0.062 0.061 0.053 0.000 0.000 0.000 0.774 0.887 0.671 5.588 5.570 4.617 3.938 4.262 4.777 N-EtFOSA 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.007 0.010 0.009 0.000 0.000 0.000 0.000 0.000 0.000 N-EtFOSE alcoh o l 0.000 0.000 0.000 0.085 0.082 0.082 0.067 0.064 0.063 0.000 0.000 0.000 0.665 0.481 0.541 5.507 5.559 4.993 4.265 4.114 4.347 N-EtFOSA 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.007 0.007 0.005 0.000 0.000 0.006 0.000 0.000 0.000 Page 50 of 55 Aerobic Biodegradation of ^-EtFOSE alcohol Cleston C. Lange, Ph.D., Pace Analytical Services, Science Solutions Division Wednesday November 1, 2000 BACK TO MAIN Appendix C Biodegradation Samples Final Data TABLE 1. The compiled data results from the biodegradation of 2,380 ng/ml (4.168 pM) NEtFOSE alcohol over 35 days of incubation. Approximately 90% biodegradation was observed after 35 days. All values are presented as micromolar (pM) concentrations. Sample Description Day 0 Biodeq Sam ple (2300 EtFOSE A lcohol) In c u b a tio n tim e (d a y s) 0.0 Day 0 Biodeq Sam ple Duplicate (2300 EtFOSE A lcohol) 0.0 Day 0 Biodeq Sam ple T riplicate (2300 EtFOSE A lcohol) 0.0 Day 1 Biodeq Sam ple (2300 EtFOSE A lcohol) 1.0 Day 1 Biodeq Sam ple Duplicate (2300 EtFOSE A lcohol) 1.0 Day 1 Biodeq Sam ple T riplicate (2300 EtFOSE A lcohol) 1.0 Day 3 Biodeq Sam ple (2300 EtFOSE A lcohol) 2.7 Day 3 Biodeq Sam ple Duplicate (2300 EtFOSE A lcohol) 2.7 Day 3 Biodeq Sam ple T riplicate (2300 EtFOSE A lcohol) 2.7 Day 7 Biodeq Sam ple (2300 EtFOSE A lcohol) 6.7 Day 7 Biodeq Sam ple Duplicate (2300 EtFOSE A lcohol) 6.7 Day 7 Biodeq Sam ple T riplicate (2300 EtFOSE A lcohol) 6.7 Day 14 Biodeq Sam ple (2300 EtFOSE A lcohol) 13.8 Day 14 Biodeq Sam ple D uplicate (2300 EtFOSE A lcohol) 13.8 Day 14 Biodeq Sam ple T riplicate (2300 EtFOSE A lcohol) 13.8 D ay 21 B io de q S am ple (2 3 00 E tFO S E A lco ho l) 20.8 D ay 21 B iodeq S am ple D u p licate (2300 E tFO S E A lco h o l' 20.8 D ay 21 B io de q S am ple T rip lic a te (2 3 00 E tFO S E A lcohol) 20.8 Day 28 Biodeq Sam ple (2300 EtFOSE A lcohol) 27.8 Day 28 Biodeq Sam ple D uplicate (2300 EtFOSE Alcohol) 27.8 Day 28 Biodeq Sam ple T riplicate (2300 EtFOSE Alcohol) 27.8 Day 35 Biodeq Sam ple (2300 EtFOSE A lcohol) 34.8 Day 35 Biodeq Sam ple D uplicate (2300 EtFOSE Alcohol) 34.8 Day 35 Biodeq Sam ple T riplicate (2300 EtFOSE Alcohol) 34.8 PFOA 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0 .0 0 0 0.009 0.009 0.009 0.021 0 .0 2 0 0 .0 2 0 0.024 0.024 0.022 0 .0 2 6 0.024 0.024 0 .0 3 0 0.027 0.031 PFOS 0 .0 0 0 0 .0 0 0 0 .0 0 0 0.009 0.008 0.009 0 .0 3 0 0 .0 2 6 0.033 0.092 0.085 0.085 0.172 0.172 0.172 0.222 0 .2 0 6 0 .2 1 6 0 .3 0 6 0.325 0.334 0.307 0.332 0.339 PFOS su lfina te 0.000 0.000 0.000 0.094 0.094 0.098 0.128 0.130 0.139 0.123 0.125 0.131 0.119 0.115 0.111 0.089 0.105 0.115 0.142 0.123 0.146 0.154 0.183 0.143 M556 0.000 0.000 0.000 0.049 0.048 0.053 0.223 0.210 0.226 0.777 0.759 0.738 1.286 1.388 1.449 1.166 1.594 1.735 2.019 1.958 2.281 1.644 2.712 2.434 N-EtFO SAA 0.000 0.000 0.000 0.651 0.637 0.660 1.071 1.057 1.157 1.139 1.067 1.184 0.972 0.825 0.834 1.240 1.060 0.812 1.472 1.419 1.187 2.187 1.271 1.392 FOSA 0.000 0.000 0.000 0.016 0.016 0.017 0.047 0.042 0.051 0.125 0.116 0.119 0.163 0.166 0.173 0.185 0.183 0.182 0.240 0.256 0.243 0.232 0.226 0.232 N-EtFOSE alcoh o l 5.109 4.894 4.761 3.100 3.160 3.119 2.205 2.332 2.032 1.434 1.465 1.479 0.767 0.732 0.741 0.576 0.398 0.447 0.774 0.887 0.671 0.665 0.481 0.541 N-EtFOSA 0.000 0.000 0.000 0.021 0.020 0.022 0.030 0.028 0.031 0.032 0.031 0.029 0.022 0.021 0.020 0.013 0.012 0.011 0.007 0.010 0.009 0.007 0.007 0.005 TABLE 2. The compiled results for the biodegradation samples tested at 47 ng/ml (90 nM) NEtFOSE alcohol over 35 days of incubation. Complete degradation was observed by day 7. All analytical concentrations are presented as nanomolar (nM). Sample Description Incu b atio r tim e (days) Day 0 Bide Sam ple (50 ppb EtFOSE A lcohol) 0.0 Day 0 B iodea Sam ple Duplicate (50 ppb EtFOSE A lcohol) 0.0 Day 0 B iodea Sam ple T riplicate (50 ppb EtFOSE A lcohol) 0.0 Day 1 B iodea Sam ple (50 ppb EtFOSE A lcohol) 1.0 Day 1 B iodea Sam ple Duplicate (50 ppb EtFOSE A lcohol) 1.0 Day 1 B iodea Sam ple T riplicate (50 ppb EtFOSE A lcohol) 1.0 Day 3 B iodea Sam ple (50 ppb EtFOSE A lcohol) 2.7 Day 3 B iodea Sam ple Duplicate (50 ppb EtFOSE A lcohol) 2.7 Day 3 B iodea Sam ple T riplicate (50 ppb EtFOSE A lcohol) 2.7 Day 7 B iodea Sam ple (50 ppb EtFOSE A lcohol) 6.7 Day 7 B iodea Sam ple Duplicate (50 ppb EtFOSE A lcohol) 6.7 Day 7 B iodea Sam ple T riplicate (50 ppb EtFOSE A lcohol) 6.7 Day 14 B iodea Sam ple (50 ppb EtFOSE A lcohol) 13.8 Day 14 B iodea Sam ple Duplicate (50 ppb EtFOSE A lcohol) 13.8 Day 14 B iodea Sam ple T riplicate (50 ppb EtFOSE A lcohol) 13.8 Day 21 B io de a S am ple (50 p pb E tFO S E A lco ho l) 20.8 Day 21 B io de a S am ple D u p lica te (50 p pb E tFO S E A lcohol) 20.8 Day 21 B io de a S am ple T rip lic a te (50 p pb E tFO S E A lco ho l) 20.8 Day 28 B iodea Sam ple (50 ppb EtFOSE A lcohol) 27.8 Day 28 B iodea Sam ple Duplicate (50 ppb EtFOSE Alcohol) 27.8 Day 28 B iodea Sam ple T riplicate (50 ppb EtFOSE A lcohol) 27.8 Day 35 B iodea Sam ple (50 ppb EtFOSE A lcohol) 34.8 Day 35 B iodea Sam ple Duplicate (50 ppb EtFOSE Alcohol) 34.8 Day 35 B iodea Sam ple T riplicate (50 ppb EtFOSE A lcohol) 34.8 PFOA 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 PFOS 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 5.4 5.3 5.1 10.0 9.2 9.3 11.2 10.7 9.9 15.9 15.4 16.3 20.9 22.9 18.7 PFOS su lfina te 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 3.6 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 C o n c e n tra tio n (nM) M556 0.0 0.0 0.0 0.0 0.0 0.0 12.4 9.1 8.6 20.7 20.1 22.3 29.1 28.7 28.3 27.1 26.4 27.1 23.4 21.9 27.7 32.9 32.7 29.2 N-EtFOSAA 0.0 0.0 0.0 26.0 24.2 21.7 34.2 28.5 27.9 22.2 25.2 20.5 7.5 7.0 8.6 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 7.3 FOSA 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 9.6 6.4 7.0 15.7 14.7 14.9 14.5 14.8 13.7 17.2 18.5 16.5 16.8 13.2 15.5 N-EtFOSE a lcoh o l 75.8 69.2 65.0 30.9 30.9 32.2 12.6 17.2 15.8 5.2 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 N-EtFO SA 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 Page 51 of 55 Aerobic Biodegradation of ^-EtFOSE alcohol Cleston C. Lange, Ph.D., Pace Analytical Services, Science Solutions Division Wednesday November 1, 2000 BACK TO MAIN Appendix D Control Samples Final Data TABLE 1. The compiled results of the sterile/inhibited control samples at 2,380 ng/ml (4.168 mM) N-EtFOSE alcohol over 35 days of incubation. No significant abiotic degradation was observed. All concentrations are presented as micromolar (pM). S am ple D e scrip tio n Day 0 Sterile S ludqe Control (2300 EtFOSE A lcohol) In c u b a tio n tim e (day s) 0.0 Day 0 Sterile S ludqe Control Duplicate (2300 EtFOSE A lcohol) 0.0 Day 0 Sterile S ludqe Control T riplicate (2300 EtFOSE A lcohol) 0.0 Day 1 Sterile S ludqe Control (2300 EtFOSE A lcohol) 1.0 Day 1 Sterile S ludqe Control Duplicate (2300 EtFOSE A lcohol) 1.0 Day 1 Sterile S ludqe Control T riplicate (2300 EtFOSE A lcohol) 1.0 Day 3 Sterile S ludqe Control (2300 EtFOSE A lcohol) 2.7 Day 3 Sterile S ludqe Control Duplicate (2300 EtFOSE A lcohol) 2.7 Day 3 Sterile S ludqe Control T riplicate (2300 EtFOSE A lcohol) 2.7 Day 7 Sterile S ludqe Control (2300 EtFOSE A lcohol) 6.7 Day 7 Sterile S ludqe Control Duplicate (2300 EtFOSE A lcohol) 6.7 Day 7 Sterile S ludqe Control T riplicate (2300 EtFOSE A lcohol) 6.7 Day 14 Sterile S ludqe Control (2300 EtFOSE A lcohol) 13.8 Day 14 Sterile Sludqe Control Duplicate (2300 EtFOSE A lcohol) 13.8 Day 14 Sterile Sludqe Control Triplicate (2300 EtFOSE A lcohol) 13.8 D ay 21 S terile S lu dq e C ontrol (2 3 00 E tFO S E A lco ho l) 20.8 D ay 21 S terile S lu dq e C ontrol D u p lica te (2300 E tFO S E A lco ho l) 20.8 D ay 21 S terile S lu dq e C ontrol T rip lica te (2300 E tFO S E A lcohol) 20.8 Day 28 Sterile S ludqe Control (2300 EtFOSE A lcohol) 27.8 Day 28 Sterile Sludqe Control Duplicate (2300 EtFOSE Alcohol) 27.8 Day 28 Sterile Sludqe Control Triplicate (2300 EtFOSE Alcohol) 27.8 Day 35 Sterile S ludqe Control (2300 EtFOSE A lcohol) 34.8 Day 35 Sterile Sludqe Control Duplicate (2300 EtFOSE Alcohol) 34.8 Day 35 Sterile Sludqe Control Triplicate (2300 EtFOSE Alcohol) 34.8 PFOA 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 PFOS 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 PFOS su lfina te 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.01 0.01 0.01 0.01 0.01 0.03 C o n c e n tr a tio n (u,M) M556 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 N-EtFO SAA 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.01 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.01 0.00 0.00 0.00 FOSA 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 N-EtFOSE alcoh o l 5.16 4.91 4.88 5.08 4.92 4.92 4.83 4.73 4.70 4.77 5.01 4.86 4.79 4.78 4.69 4.74 4.54 4.40 5.59 5.57 4.62 5.51 5.56 4.99 N-EtFOSA 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.01 TABLE 2. The compiled results of the no-sludge control samples at 2,380 ng/ml (4.168 mM) NEtFOSE alcohol over 35 days of incubation. Some degradation due to suspected microbial contamination was observed due to un-sterilized mineral media being used to set up those controls. This suggests that the first reaction is a ubiquitous alcohol dehydrogenase reaction. All concentrations are presented as micromolar (pM). S am ple D e scrip tio n Day 0 No Sludqe Control (2300 EtFOSE A lcohol) Day 0 No Sludqe Control Duplicate (2300 EtFOSE A lcohol) Day 0 No Sludqe Control T riplicate (2300 EtFOSE A lcohol) Day 1 No Sludqe Control (2300 EtFOSE A lcohol) Day 1 No Sludqe Control Duplicate (2300 EtFOSE A lcohol) Day 1 No Sludqe Control T riplicate (2300 EtFOSE A lcohol) Day 3 No Sludqe Control (2300 EtFOSE A lcohol) Day 3 No Sludqe Control Duplicate (2300 EtFOSE A lcohol) Day 3 No Sludqe Control T riplicate (2300 EtFOSE A lcohol) Day 7 No Sludqe Control (2300 EtFOSE A lcohol) Day 7 No Sludqe Control Duplicate (2300 EtFOSE A lcohol) Day 7 No Sludqe Control T riplicate (2300 EtFOSE A lcohol) Day 14 No Sludqe Control (2300 EtFOSE A lcohol) Day 14 No S ludqe C ontrol Duplicate (2300 EtFOSE Alcohol) Day 14 No S ludqe Control T riplicate (2300 EtFOSE Alcohol) D ay 21 No S lu dq e C ontrol (2 3 00 E tFO S E A lco ho l) D ay 21 No S lu dq e C o ntrol D u p lica te (2300 E tFO S E A lcohol) D ay 21 No S lu dq e C ontrol T rip lica te (2300 E tFO S E A lcohol) Day 28 No Sludqe Control (2300 EtFOSE A lcohol) Day 28 No S ludqe C ontrol Duplicate (2300 EtFOSE Alcohol) Day 28 No S ludqe Control Triplicate (2300 EtFOSE A lcohol) Day 35 No Sludqe Control (2300 EtFOSE A lcohol) Day 35 No S ludqe Control Duplicate (2300 EtFOSE A lcohol) Day 35 No S ludqe Control T riplicate (2300 EtFOSE Alcohol) Incu b atio n tim e (days) 0.0 0.0 0.0 1.0 1.0 1.0 2.7 2.7 2.7 6.7 6.7 6.7 13.8 13.8 13.8 20.8 20.8 20.8 27.8 27.8 27.8 34.8 34.8 34.8 PFOA 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 PFOS 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 PFOS su lfina te 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.01 0.00 0.01 0.01 0.02 0.04 0.04 0.04 Page 52 of 55 Aerobic Biodegradation of ^-EtFOSE alcohol Cleston C. Lange, Ph.D., Pace Analytical Services, Science Solutions Division Wednesday November 1, 2000 M556 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.01 0.00 0.00 0.00 0.00 0.00 0.00 0.00 N-EtFO SAA 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.01 0.03 0.01 0.03 0.02 0.01 0.06 0.04 0.11 0.78 0.23 0.81 FOSA 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 N-EtFOSE alcoh o l 4.88 4.95 5.03 4.99 4.91 4.76 4.55 4.67 4.67 4.54 4.69 4.72 4.31 4.48 4.46 3.91 4.01 4.35 3.94 4.26 4.78 4.27 4.11 4.35 N-EtFOSA 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 BACK TO MAIN Appendix D, continued TABLE 3. The compiled results of the sterile/inhibited sludge control samples at 47 ng/ml (90 nM) W-EtFOSE alcohol over 35 days of incubation. Degradation was observed in day 21 sterile sludge controls because on day 7 they received fresh sludge by mistake. All concentrations are presented as nanomolar (nM). _____ _____________________________________________ Sam ple Description Day 0 Sterile S ludge Control Sam ple (50 ppb EtFOSE A lcohol) Day 0 Sterile S ludge Control Duplicate (50 ppb EtFOSE A lcohol) Day 0 Sterile S ludge Control T riplicate (50 ppb EtFOSE A lcohol) Day 1 Sterile S ludge Control Sam ple (50 ppb EtFOSE A lcohol) Day 1 Sterile S ludge Control Duplicate (50 ppb EtFOSE A lcohol) Day 1 Sterile S ludge Control T riplicate (50 ppb EtFOSE A lcohol) Day 3 Sterile S ludge Control Sam ple (50 ppb EtFOSE A lcohol) Incu b atio r tim e (d a VS) 0.0 0.0 0.0 1.0 1.0 1.0 2.7 PFOA 0.0 0.0 0.0 0.0 0.0 0.0 0.0 PFOS 0.0 0.0 0.0 0.0 0.0 0.0 0.0 PFOS su lfina te 0.0 0.0 0.0 0.0 0.0 0.0 2.9 C o n c e n tra tio n (nM) M556 0.0 0.0 0.0 0.0 0.0 0.0 0.0 N-EtFOSAA 0.0 0.0 0.0 0.0 0.0 0.0 0.0 FOSA 0.0 0.0 0.0 0.0 0.0 0.0 0.0 N-EtFOSE a lcoh o l 80.4 78.2 77.0 76.9 77.8 80.4 70.1 Day 3 Sterile S ludge Control Duplicate (50 ppb EtFOSE A lcohol) 2.7 0.0 0.0 2.9 0.0 0.0 0.0 67.6 Day 3 Sterile S ludge Control T riplicate (50 ppb EtFOSE A lcohol) 2.7 0.0 0.0 0.0 0.0 0.0 0.0 71.9 Day 7 Sterile S ludge Control Sam ple (50 ppb EtFOSE A lcohol) 6.7 0.0 0.0 0.0 0.0 0.0 0.0 70.8 Day 7 Sterile S ludge Control Duplicate (50 ppb EtFOSE A lcohol) 6.7 0.0 0.0 0.0 0.0 0.0 0.0 70.6 Day 7 Sterile S ludge Control T riplicate (50 ppb EtFOSE A lcohol) 6.7 0.0 0.0 0.0 0.0 0.0 0.0 68.9 Day 14 Sterile S ludge Control Sam ple (50 ppb EtFOSE A lcohol) 13.8 0.0 0.0 0.0 0.0 0.0 0.0 76.9 Day 14 Sterile S ludge Control Duplicate (50 ppb EtFOSE A lcohol) 13.8 0.0 0.0 0.0 0.0 0.0 0.0 68.4 Day 14 Sterile S ludge Control T riplicate (50 ppb EtFOSE A lcohol) 13.8 0.0 0.0 0.0 0.0 0.0 0.0 75.6 Day 21 S terile S lu dg e C ontrol S am ple (50 p pb E tFO S E A lco ho l) 20.8 0.0 0.0 0.0 0.0 0.0 0.0 69.9 Day 21 S terile S lu dg e C ontrol D u p lica te (50 p pb E tFO S E A lco ho l) 20.8 0.0 0.0 0.0 0.0 0.0 0.0 68.7 Day 21 S terile S lu dg e C ontrol T rip lic a te (50 ppb E tFO S E A lco ho l) 20.8 0.0 0.0 0.0 0.0 0.0 0.0 71.2 Day 28 Sterile S ludge Control Sam ple (50 ppb EtFOSE A lcohol) 27.8 0.0 0.0 0.0 0.0 0.0 0.0 86.9 Day 28 S te rile S lu dg e C o ntrol D u p lica te (50 ppb EtF OS E A lco ho l) 27.8 0.0 0.0 0.0 0.0 0.0 0.0 84.9 Day 28 Sterile Sludge Control Triplicate (50 ppb EtFOSE A lcohol) 27.8 0.0 0.0 0.0 0.0 0.0 0.0 88.0 Day 35 Sterile S ludge Control Sam ple (50 ppb EtFOSE A lcohol) 34.8 0.0 0.0 0.0 0.0 0.0 0.0 85.4 Day 35 Sterile S ludge Control Duplicate (50 ppb EtFOSE A lcohol) 34.8 0.0 0.0 0.0 0.0 0.0 0.0 81.8 Day 35 Sterile S ludge Control T riplicate (50 ppb EtFOSE A lcohol) 34.8 0.0 0.0 0.0 0.0 0.0 0.0 81.5 N-EtFOSA 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 TABLE 4. The compiled results of the no-sludge control samples at 47 ng/ml (90 nM) W-EtFOSE alcohol over 35 days of incubation. Some degradation due to suspected microbial contamination was observed due to un-sterilized mineral media being used to set up those controls (shown shaded). This suggests that the first reaction may be catalyzed by a ubiquitous alcohol dehydrogenase reaction. All concentrations are presented as nanomolar (nM). Sam ple D e scrip tio n Day 0 No S ludge Control Sam ple (50 ppb EtFOSE A lcohol) In c u b a tio n tim e (days) 0.0 Day 0 No S ludge Control D uplicate (50 ppb EtFOSE A lcohol) 0.0 Day 0 No S ludge Control Triplicate (50 ppb EtFOSE A lcohol) 0.0 Day 1 No S ludge Control Sam ple (50 ppb EtFOSE A lcohol) 1.0 Day 1 No S ludge Control D uplicate (50 ppb EtFOSE A lcohol) 1.0 Day 1 No S ludge Control Triplicate (50 ppb EtFOSE A lcohol) 1.0 Day 3 No S ludge Control Sam ple (50 ppb EtFOSE A lcohol) 2.7 Day 3 No S ludge Control D uplicate (50 ppb EtFOSE A lcohol) 2.7 Day 3 No S ludge Control Triplicate (50 ppb EtFOSE A lcohol) 2.7 Day 7 No S ludge Control Sam ple (50 ppb EtFOSE A lcohol) 6.7 Day 7 No S ludge Control D uplicate (50 ppb EtFOSE A lcohol) 6.7 Day 7 No S ludge Control Triplicate (50 ppb EtFOSE A lcohol) 6.7 Day 14 No S lu dg e C ontrol S am ple (50 p pb EtF OS E A lco ho l) 13.8 Day 14 No S lu dg e C ontrol D u plicate (50 ppb EtF OS E A lco ho l) 13.8 Day 14 No S ludge Control Triplicate (50 ppb EtFOSE A lcohol) 13.8 Day 21 No S lu dg e C ontrol S am ple (50 p pb E tFO S E A lco ho l) 20.8 Day 21 No S lu dg e C ontrol D u plicate (50 ppb E tFO S E A lco ho l) 20.8 Day 21 No S lu dg e C ontrol T rip lica te (50 p pb E tFO S E A lco ho l) 20.8 Day 28 No Sludge Control Sam ple (50 ppb EtFOSE A lcohol) 27.8 Day 28 No Sludge Control D uplicate (50 ppb EtFOSE A lcohol) 27.8 Day 28 No Sludge Control Triplicate (50 ppb EtFOSE A lcohol) 27.8 Day 35 No Sludge Control Sam ple (50 ppb EtFOSE A lcohol) 34.8 Day 35 No Sludge Control D uplicate (50 ppb EtFOSE A lcohol) 34.8 Day 35 No Sludge Control Triplicate (50 ppb EtFOSE A lcohol) 34.8 PFOA 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 PFOS 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 PFOS su lfina te 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 Page 53 of 55 C o nce n tratio n (nM) M556 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 N-EtFOSAA 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 FOSA 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 N -E tF O S E a lcoh o l 75.797 79.884 77.736 71.322 71.869 71.725 64.028 66.874 64.970 60.718 61.016 64.331 56.389 56.944 57.000 50.068 5.501 39.405 61.838 61.144 53.334 67.445 63.636 63.476 N-EtFO SA 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 Aerobic Biodegradation of W-EtFOSE alcohol Cleston C. Lange, Ph.D., Pace Analytical Services, Science Solutions Division Wednesday November 1, 2000 BACK TO MAIN Appendix E Table 1. The percent degradation of W-EtFOSE alcohol and percent of products based on molar equivalents of each analyte detected through the study for the 2,380 ng/mL samples. A value of 100% is based on the average value from triplicate samples for the day 0 W-EtFOSE alcohol samples. The average value at day 0 was analytically determined to be 4.921 pM. The averaged percentage of each analyte at day 35 is shown at the bottom. The sum of the analyte percentages at day 35 was equal to 105.6 Days Incubation 0.0 0.0 0.0 1.1 1.1 1.1 2.7 2.7 2.7 6.7 6.7 6.7 13.7 13.7 13.7 20.7 20.7 20.7 27.8 27.8 27.8 34.8 34.8 34.8 PFOA 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.2% 0.2% 0.2% 0.4% 0.4% 0.4% 0.5% 0.5% 0.5% 0.5% 0.5% 0.5% 0.6% 0.5% 0.6% PFOS 0.0% 0.0% 0.0% 0.2% 0.2% 0.2% 0.6% 0.5% 0.7% 1.9% 1.7% 1.7% 3.5% 3.5% 3.5% 4.5% 4.2% 4.4% 6.2% 6.6% 6.8% 6.2% 6.8% 6.9% % of total fluorochemicals Measured PFOS sulfinate M556 N-EtFOSAA FOSA 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 1.9% 1.0% 13.2% 0.3% 1.9% 1.0% 12.9% 0.3% 2.0% 1.1% 13.4% 0.3% 2.6% 4.5% 21.8% 1.0% 2.6% 4.3% 21.5% 0.9% 2.8% 4.6% 23.5% 1.0% 2.5% 15.8% 23.1% 2.5% 2.5% 15.4% 21.7% 2.4% 2.7% 15.0% 24.1% 2.4% 2.4% 26.1% 19.8% 3.3% 2.3% 28.2% 16.8% 3.4% 2.3% 29.4% 16.9% 3.5% 1.8% 23.7% 25.2% 3.8% 2.1% 32.4% 21.5% 3.7% 2.3% 35.3% 16.5% 3.7% 2.9% 41.0% 29.9% 4.9% 2.5% 39.8% 28.8% 5.2% 3.0% 46.4% 24.1% 4.9% 3.1% 33.4% 44.4% 4.7% 3.7% 55.1% 25.8% 4.6% 2.9% 49.5% 28.3% 4.7% N-EtFOSE alcohol 103.8% 99.5% 96.7% 63.0% 64.2% 63.4% 44.8% 47.4% 41.3% 29.1% 29.8% 30.1% 15.6% 14.9% 15.1% 11.7% 8.1% 9.1% 15.7% 18.0% 13.6% 13.5% 9.8% 11.0% N-EtFOSA 0.0% 0.0% 0.0% 0.4% 0.4% 0.4% 0.6% 0.6% 0.6% 0.6% 0.6% 0.6% 0.4% 0.4% 0.4% 0.3% 0.2% 0.2% 0.2% 0.2% 0.2% 0.2% 0.1% 0.1% Average value at day 35 0.6% Standard Deviation 0.0% RSD (%)____________________ 7.5% 6.6% 0.3% 5.1% 3.3% 0.4% 13.1% 46.0% 11.3% 24.5% 32.9% 10.1% 30.8% 4.7% 0.1% 1.4% 11.4% 1.9% 16.7% 0.1% 0.0% 16.6% Page 54 of 55 Aerobic Biodegradation of ^-EtFOSE alcohol Cleston C. Lange, Ph.D., Pace Analytical Services, Science Solutions Division Wednesday November 1, 2000 BACK TO MAIN Appendix F Table 1. Mass Balance data for all biodeg samples, sterile control sample, and no-sludge control samples. Note that for the day 21 no-sludge controls there was only one good value due to a sample preparation error. The % mass balance shown was calculated using expected concentration of 4.168 pM. 90 nM m ass balance SamDlinc B iodea sam ple Sterile co n tro ls no -slu d a e controls Day 0 90.9% Day 0 83.0% Day 0 78.0% Day 0 96.4% Day 0 93.7% Day 0 92.3% Day 0 90.9% Day 0 95.8% Day 0 93.2% Day 1 68.2% Day 1 66.1% Day 1 64.6% Day 1 92.2% Day 1 93.3% Day 1 96.4% Day 1 85.5% Day 1 86.2% Day 1 86.0% Day 3 71.0% Day 3 65.8% Day 3 67.0% Day 3 87.5% Day 3 84.5% Day 3 86.2% Day 3 76.8% Day 3 80.2% Day 7 77.9% Day 7 75.6% Day 7 68.4% Day 7 65.9% Day 7 84.9% Day 7 84.6% Day 7 82.7% Day 7 72.8% Day 7 73.2% Day 7 77.1% Day 14 74.8% Day 14 71.4% Day 14 73.2% Day 14 92.2% Day 14 82.0% Day 14 90.7% Day 14 67.6% Day 14 68.3% Day 14 67.5% Day 21 63.3% Day 21 62.2% Day 21 60.8% Day 21 83.8% Day 21 82.4% Day 21 85.4% Day 21 60.0% Day 28 67.7% Day 28 66.9% Day 28 72.5% Day 28 Day 28 Day 28 Day 28 Day 28 Day 28 Day 35 84.7% Day 35 82.5% Day 35 84.8% Day 35 102.4% Day 35 98.1% Day 35 97.7% Day 35 80.9% Day 35 76.3% Day 35 76.1% Day 35 Day 35 Average= 72.0% 90.0% 78.5% S td Dev= 8.2% 6.1% 9.4% RSD = 11.3% 6.8% 12.0% 4.168 uM m ass balance A ll sam ples SamDlinc B iodea sample Sterile con tro ls no-sludae controls A ll samples 90.9% Day 0 122.6% 122.6% 83.0% Day 0 117.4% 117.4% 78.0% Day 0 114.2% 114.2% 96.4% Day 0 123.8% 123.8% 93.7% Day 0 117.8% 117.8% 92.3% Day 0 117.0% 117.0% 90.9% Day 0 117.2% 117.2% 95.8% Day 0 118.7% 118.7% 93.2% Day 0 120.7% 120.7% 68.2% Day 1 94.6% 94.6% 66.1% Day 1 95.6% 95.6% 64.6% Day 1 95.4% 95.4% 92.2% Day 1 121.9% 121.9% 93.3% Day 1 118.0% 118.0% 96.4% Day 1 118.0% 118.0% 85.5% Day 1 119.8% 119.8% 86.2% Day 1 117.7% 117.7% 86.0% Day 1 114.2% 114.2% 71.0% Day 3 89.6% 89.6% 65.8% Day 3 91.8% 91.8% 67.0% Day 3 88.0% 88.0% 87.5% Day 3 115.9% 115.9% 84.5% Day 3 113.6% 113.6% 86.2% Day 3 112.7% 112.7% 76.8% Day 3 109.1% 109.1% 80.2% Day 3 112.1% 112.1% 77.9% Day 7 112.0% 112.0% 75.6% Day 7 89.5% 89.5% 68.4% Day 7 87.7% 87.7% 65.9% Day 7 90.6% 90.6% 84.9% Day 7 114.5% 114.5% 84.6% Day 7 120.2% 120.2% 82.7% Day 7 116.7% 116.7% 72.8% Day 7 108.9% 108.9% 73.2% Day 7 112.6% 112.6% 77.1% Day 7 113.2% 113.2% 74.8% Day 14 84.5% 84.5% 71.4% Day 14 82.5% 82.5% 73.2% Day 14 84.5% 84.5% 92.2% Day 14 115.1% 115.1% 82.0% Day 14 114.7% 114.7% 90.7% Day 14 112.6% 112.6% 67.6% Day 14 103.7% 103.7% 68.3% Day 14 108.2% 108.2% 67.5% Day 14 107.2% 107.2% 63.3% Day 21 84.3% 84.3% 62.2% Day 21 85.9% 85.9% 60.8% Day 21 85.0% 85.0% 83.8% Day 21 113.9% 113.9% 82.4% Day 21 109.0% 109.0% 85.4% Day 21 105.6% 105.6% 60.0% Day 21 94.5% 94.5% 67.7% Day 28 97.4% 97.4% 66.9% Day 28 104.6% 104.6% 72.5% Day 28 119.6% 119.6% 104.2% Day 28 120.0% 120.0% 101.8% Day 28 117.4% 117.4% 105.5% Day 28 134.2% 134.2% 74.1% Day 28 133.8% 133.8% 73.3% Day 28 111.3% 111.3% 63.9% Day 28 96.3% 96.3% 84.7% Day 35 103.4% 103.4% 82.5% Day 35 117.7% 117.7% 84.8% Day 35 125.4% 125.4% 102.4% Day 35 125.7% 125.7% 98.1% Day 35 122.8% 122.8% 97.7% Day 35 132.3% 132.3% 80.9% Day 35 133.6% 133.6% 76.3% Day 35 120.6% 120.6% 76.1% Day 35 122.2% 122.2% Day 35 105.1% 105.1% Day 35 124.5% 124.5% 80.5% Averages 100.6% 118.6% 110.9% 110.0% 11.8% Std Devs 16.3% 7.9% 8.3% 13.5% 14.6% RSD s 16.2% 6.6% 7.5% 12.3% Page 55 of 55 Aerobic Biodegradation of ^-EtFOSE alcohol Cleston C. Lange, Ph.D., Pace Analytical Services, Science Solutions Division Wednesday November 1, 2000 BACK TO MAIN Appendix A Signatures of Project Personnel Project Title: 5-Week N-EtFOSE Alcohol Aerobic Biodegradation Study Client Project ID: LIMS E00-2252 Contract Analytical Project Number: CA058 The following individuals participated in the conduct of this project: Study Director: Cleston C. Lange Signature Laboratory Management: Mark T. McCann Report Author: Cleston C. Lange /T /-- - Signature ^nature Sample Preparation Analyst: Angela L. Schuler Date "M * Date HPLC/MS Analysis Conducted by: Anthony E. Scales Final Report Reviewed by: Dirk W. Hooaenboom Date Oi 2 Date Page 38 of 55 Aerobic Biodegradation of A'-EtFOSE alcohol Cleston C. Lange, Ph.D., Pace Analytical Services, Science Solutions Division Wednesday November 1,2000 Original