Document ba3OJyOnDN0deZ2rN1MKg3O33

3 Final Report Study Title Anaerobic Biodegradability Testing of the Perfluoroalkyl Substances PFOS, PFBS, PFOA, PFBA, MeFBSE, EtFOSE, 6:2-FTS, 6:2 FTOH and 8:2 FTOH, and also 246-Trichlorophenol and Pentachlorophenol Data Requirement Exploratory/Method Development Author Cleston C. Lange, Ph.D. Study Completion Date Date of Final Report Signature Performing Laboratory 3M Environment, Health, Safety and Sustainability Environmental Laboratory 3M Center, Bldg. 260-05-N-17 St. Paul, MN 55144 Project Identification E05-0544 Number of Pages 101 3M ENVIRONMENTAL LABORATORY PROJECT NO. E05-0544 SUMMARY This report summarizes the results of anaerobic biodegradation experiments conducted from August 2005 to August 2006 under 3M Project E05-0544. The goal of the project was to develop effective and safe equipment procedures and test methods which would enable researchers of the 3M Environmental Laboratory to conduct anaerobic biodegradation testing of fluorinated surfactants and fluorinated polymers. This testing ability was needed to meet conditional requirements of U.S. EPA consent order agreements for the manufacture and sale of fluorinated materials in the U.S. To accomplish this goal, municipal anaerobic digester sludge was acquired from a local Twin Cities (MN) municipal anaerobic digester (Empire, MN) and the sludge was used to establish several sets of anaerobic test cultures. Those cultures were then dosed with a variety of perfluoroalkylated substances (PFAS). It is noteworthy that since the time that this work was performed, a number of research laboratories have published results which have confirmed many of the findings from this study (e.g. Zhang et al. 2013). Those findings and their relevance to this study's results are discussed herein. In total nine PFAS test substances were chosen for the evaluation. They were chosen for a variety of reasons, including the following: First, several of them are synthetic building blocks for the manufacture of specialty fluorinated surfactants and fluorinated polymer protectant products and are expected environmental degradants of those fluorinated polymers (Schultz et al. 2003). Secondly, some of them are perfluorooctyl based (i.e. C8) synthons historically used in the manufacture of legacy fluorinated polymers and fluorinated surfactants which have been phased out in 2000-2001. Thirdly, some of the PFAS test substances are perfluorohexyl (C6) or perfluorobutyl (C4) based and were planned to be C8 replacements because of their anticipated more favorable environmental and toxicological profiles. Fourthly, some of the PFAS test substances were identified as the penultimate environmental degradation endpoints of PFASderived fluorinated polymers and fluorinated surfactants. Finally, some of the PFAS test substances were derived from electrochemical fluorination (ECF) process and others were derived from a telomerization (TM) process, and represent the two primary industrial processes used for making PFAS materials. The differences between ECF and TM have been discussed previously (Saez et al. 2008), amongst them one important difference is that ECF results in PFAS that are a mixture of linear and branched isomers, and also odd and even carbon perfluoroalkyl chain length, because of isomerization reactions that occur during the ECF process (Ignat'ev et al. 2003). The TM process results in predominantly even number perfluoroalkyl carbon chains and only linear isomer PFAS because of sequential step wise tetrafluoroethylene additions at the end of an elongating perfluoroalkyl chain during that process. Most of the PFAS test substances were evaluated at a nominal concentration 1 g/mL (1 ppm). The ECF-derived chemistries were perfluorooctanesulfonate (PFOS), perfluorobutanesulfonate (PFBS), perfluorooctanoate (PFOA), perfluorobutanoate (PFBA), 2-[(Nethyl)perfluorooctanesulfonamido]ethanol (EtFOSE), and 2-[(N-methyl)perfluorobutanesulfonamido]ethanol (MeFBSE). The TM-derived PFAS were 1,1,2,2-tetrahydro- Study Report: E05-0544 Page 2 of 101 3M ENVIRONMENTAL LABORATORY PROJECT NO. E05-0544 perfluorooctanesulfonate (a.k.a. 6:2 fluorotelomer sulfonate; 6:2 FTS), 1,1,2,2-tetrahydroperfluorooctanol (a.k.a. 6:2 fluorotelomer alcohol; 6:2 FTOH) and 1,1,2,2-tetrahydroperfluorodecanol (a.k.a. 8:2 fluorotelomer alcohol; 8:2 FTOH). In addition to the nine PFASs tested, 2,4,6-trichlorophenol (246-TCP) and pentachlorophenol (PCP) were also evaluated as potential positive controls for anaerobic biodegradation testing because they had previously been shown to undergo reductive dehalogenation to lesser halogenated phenol end products under methanogenic conditions (Mohn & Kennedy, 1992). The 246-TCP and PCP were dosed at nominal 1 g/mL and 2 g/mL, respectively. Some exceptions to the dosed concentrations listed above are noted in the individual test substance results sections. Cultures were prepared as sets of replicate cultures, some contained sterilized sludge and acted as killed controls and others contained bioactive sludge with live microorganisms. Each culture consisted of 5 mL of anaerobic sludge culture in sealed nominal 42.5 mL amber-glass test vessels and were prepared in a nitrogen filled glove box devoid of oxygen (O2). Low level addition of yeast extract solution to each provided necessary nutrients to sustain microorganisms in the sludge. The incubations were carried out in a glove box for up to 108 days. Many of the sets also were prepared in parallel with blank water cultures, blank sterile cultures and blank bioactive cultures for evaluating background from sludge and nutrients which were added to the cultures; blanks however were not dosed with equivalent 5-10 L of methanol carrier solvent that the test cultures received. Each culture set was prepared to allow for at least duplicate or triplicate culture collections at each time point, with some exceptions as noted in the report. At select time points, cultures were collected and evaluated for biogas production. Select culture headspace gases were monitored by gas-phase FTIR analysis and by semi-quantitative full scan GC/MS analysis for gas composition and for potential volatile fluorocarbon products. The cultures were then extracted with acetone and analyzed for the test substance by LC/MS/MS or direct-inject GC/MS and for anticipated soluble biotransformation products by LC/MS/MS. Formation of biogas occurred in all bioactive cultures and was determined by a volumetric displacement method. The composition of the biogas was verified as methane (CH4) and carbon dioxide (CO2) by gas-phase FTIR. Biogas did not form in sterilized control cultures and demonstrated that methanogenic conditions were successfully established during the study. The reductive dehalogenation of 246-TCP and PCP were observed in bioactive cultures based on measured loss of test substance and concomitant formation of lesser-chlorinated phenols in the culture medium. The incubation with 246-TCP resulted in stoichiometric formation of 4chlorophenol (4-CP) and occurred without a significant lag period for its degradation to occur. PCP biodegradation occurred with an apparent 10 day lag phase, but ultimately formed several tetrachlorinated and trichlorinated phenols, but which was not consistent amongst replicates by the last time point. The degradation of 246-TCP and PCP did not occur in the sterile control cultures. The reductive dehalogenation of 246-TCP and PCP confirmed the establishment of healthy anaerobic methanogenic consortia inclusive of halorespiring microorganisms (i.e. microbes which use halogenated organics as terminal electron acceptor for anaerobic respiration). While 246-TCP was found to be a reliable positive control substance for future anaerobic biodegradation testing, PCP was determined to be a poor positive control substance Study Report: E05-0544 Page 3 of 101 3M ENVIRONMENTAL LABORATORY PROJECT NO. E05-0544 due to inconsistent results; inconsistency was likely due to toxicity of PCP and the tetrachlorophenol biotransformation product(s). Of the nine PFAS that were tested, five were concluded to be recalcitrant to anaerobic biodegradation during the evaluations. This was not surprising given that all five are commonly detected in environmental matrices and four of the five are considered benign to normal environmental degradation mechanisms; 6:2 FTS has been shown to biodegrade aerobically. The five anaerobically stable substances were PFOS, PFBS, PFOA, PFBA and 6:2 FTS. Four of the nine PFAS that were tested did undergo biodegradation anaerobically and all were PFAS alcohol substances. Two PFAS alcohols were derived from ECF synthesis, a C8 PFAS as EtFOSE and a C4 PFAS as MeFBSE. The other PFAS alcohols were TM-derived and were a C8 PFAS as 8:2 FTOH and a C6 PFAS as 6:2 FTOH. Each of the PFAS alcohols underwent biotransformation reactions at the non-fluorinated portion of the molecule to generate a carboxylic or sulfinic acid end product. EtFOSE was biodegraded with the slowest rate and with only approximately 2.66 mole percent (mole%) biodegraded. The anaerobic biodegradation of EtFOSE resulted in the formation of 2[(N-ethyl) perfluorooctanesulfonamido]acetate (EtFOSAA) and perfluorooctane sulfinate (PFOSi). EtFOSE anaerobic biodegradation did not form (N-ethyl)perfluorooctane sulfonamide (EtFOSA), perfluorooctane sulfonamide (FOSA), PFOS or PFOA, each of which were previously observed as transient and/or terminal biotransformation products in aerobic municipal sludge cultures (Lange 2000, 2001a, Rhoads 2005). The low level of EtFOSE biodegraded and the slow degradation rate could not be determined by direct measure of EtFOSE and was only determined via the measurement of the accumulated biotransformation products in the culture medium. MeFBSE biodegraded with the fastest apparent rate and approximately 75 mole% of MeFBSE was biodegraded to 2[(N-methyl)-perfluorobutanesulfonamido]acetate (MeFBSAA) and perfluorobutane sulfinate (PFBSi). Other anticipated PFAS products such as (N-methyl)perfluorobutane sulfonamide (MeFBSA), perfluorobutane sulfonamide (FBSA), PFBA and PFBS were not formed. Both of the fluorotelomer alcohol (FTOH) test substances biodegraded anaerobically. The FTOHs were removed from the bioactive and sterile cultures, however, biotransformation products were only observed in bioactive cultures. Approximately 8 mole% of the dosed 8:2 FTOH was biotransformed to 8:2 fluorotelomer carboxylate (8:2-FTCA) and 8:2 fluorotelomer unsaturated carboxylate (8:2 FTUCA). Similarly, 11 mole% of the dosed 6:2 FTOH biotransformed in analogous fashion to give 6:2 FTCA and 6:2 FTUCA. Neither FTOH test substance yielded perfluorinated carboxylates (PFCAs) which have been observed in other studies using aerobic sludge (Lange 2001b, Dinglasen et al. 2004, Wang et al. 2005). The reason for loss of FTOHs in sterile sludge controls was not determined, but could be due to irreversible binding of FTOHs to the sludge or the PTFE liner on the test vessel cap. The loss of FTOHS in sterile sludge controls has been observed in other studies (Saez et al., 2008, Wang et Study Report: E05-0544 Page 4 of 101 3M ENVIRONMENTAL LABORATORY PROJECT NO. E05-0544 al. 2005). Additionally, anaerobic biotransformation products of FTOHs discovered since 20052006 were not measured for (e.g. 5:3 acid from 6:2 FTOH and 7:3 acid from 8:2 FTOH, Zhang et al. 2013), therefore it is likely that the estimated biodegradation rates for the FTOHs are biased low and this is discussed later in the report. Overall, several conclusions were drawn from the results of this study. First, PFAS with a nonfluorinated alcohol moiety biotransform anaerobically in a predictable fashion and the biotransformation products were identified and quantified. Secondly, the rates of biodegradation appeared to be dependent on molecular weight, with the lower molecular weight substances biotransformed faster than the higher molecular weight substances (i.e. MeFBSE > 6:2 FTOH > 8:2 FTOH > EtFOSE); the faster and more abundant degradation of lower MW substances is likely related to an increased water solubility which in turn increases bioavailability. Thirdly, anaerobic biotransformation of the PFAS alcohols does not result in penultimate environmental endpoint perfluorinated sulfonates (PFSAs) or perfluorinated carboxylates (PFCAs). Accumulation of PFAS sulfinate end products from EtFOSE and MeFBSE suggests there is a requirement for O2 and/or O2-requiring enzymes/microbes for the next stage biotransformation reactions to generate perfluorinated sulfonates. Fourthly, the results of this study showed that 6:2 FTS, PFBS, PFOS, PFBA and PFOA are stable and do not undergo biological attack under anaerobic conditions. Fifth, degradation products identified in this study can be expected to sequester in low oxygen environments such as groundwater, sediment and wastewater treatment effluents and those are likely environmental sinks for precursors to perfluorinated sulfonates and carboxylates. Finally, this study resulted in development of a reliable analytical method for quantifying the target analyte PFASs in sludge matrices and established general method criteria results of batch QC elements. In general, it can be expected that matrix-matched calibration can be achieved with accuracy of within 100 + 25% (+30% LLOQ) using the sample preparation and LC/MS/MS methods described, and QC results can be expected to be within 100 + 30%. Calibration can be achieved over the range of approximately 5 ng/mL to 1000 ng/mL with quadratic fit and 1/x weighting to aid in low end quantitative accuracy, but LLOQs as low as 1.00 ng/mL were achieved for some analytes. Both internal standard and external standard methods of quantitation suffice, and it is analyte and IS dependent in terms of which quantitation method performs best. While ultimately this work was performed to establish methods suitable for testing anaerobic biodegradability of fluorinated polymer and fluorinated surfactants for studies conducted under good laboratory practice (GLP) standards, two additional non-fluorinated substances 246-TCP and PCP were evaluate for their potential use as positive controls in such testing. The 246-TCP biodegraded reproducibly to a single end product and without any lag phase or toxicity effects. Degradation of 246-TCP was not observed in sterile controls. These observations indicate 246TCP is a good positive control candidate. In contrast, pentachlorophenol (PCP) showed a long lag period and results on day-74 for 2 of 3 culture replicates showed loss of PCP with concomitant formation 2345- and 2346-tetrachlorophenol, and some 345-trichlorophenol, while the third replicate did not. Due to the observed lag, the inconsistent results and possible toxicity effects, PCP was not a good positive control substance candidate. Study Report: E05-0544 Page 5 of 101 TABLE OF CONTENTS 3M ENVIRONMENTAL LABORATORY PROJECT NO. E05-0544 SUMMARY ......................................................................................................................2 TABLE OF CONTENTS ..................................................................................................6 1 INTRODUCTION......................................................................................8 2 TEST, CONTROL AND REFERENCE (TCR) SUBSTANCES ........10 3 METHODS ...............................................................................................14 3.1 Collection, Storage and Handling of Anaerobic Digester Sludge .............15 3.2 Culture Preparations...................................................................................18 3.3 Anaerobic Glove Box "Incubation" Conditions ........................................20 3.4 Culture Collections ....................................................................................21 3.5 Analytical Determinations .........................................................................22 3.5.1 Gas Production Measurement ....................................................................22 3.5.2 Gas Phase FTIR Analysis ..........................................................................23 3.5.3 Headspace-GC/MS Analysis .....................................................................23 3.5.4 Analytical Sample Preparation ..................................................................23 3.5.5 Direct Inject GC/MS Analysis ...................................................................24 3.5.6 LC/MS/MS Analysis..................................................................................25 4 RESULTS & DISCUSSION....................................................................35 4.1 Anaerobic Culture Test Parameters ...........................................................35 4.2 Anaerobic Biodegradation Testing Results ...............................................38 Study Report: E05-0544 Page 6 of 101 4.2.1 4.2.2 4.2.3 4.2.4 4.2.5 4.2.6 4.2.7 4.2.8 4.2.9 4.2.10 4.2.11 5 3M ENVIRONMENTAL LABORATORY PROJECT NO. E05-0544 EtFOSE Results .........................................................................................38 MeFBSE Results ........................................................................................43 PFOS Results .............................................................................................48 PFBS Results .............................................................................................51 PFOA Results.............................................................................................54 PFBA Results.............................................................................................57 6:2 FTS Results..........................................................................................60 6:2 FTOH Results ......................................................................................64 8:2 FTOH Results ......................................................................................69 246-TCP Results ........................................................................................74 PCP Results................................................................................................77 RESULTS SYNOPSIS.............................................................................81 6 CONCLUSION/DISCUSSION ...............................................................89 7 LITERATURE CITED ...........................................................................93 8 REPORT APPROVAL............................................................................98 APPENDIX A: Data Calculations .................................................................................99 Study Report: E05-0544 Page 7 of 101 3M ENVIRONMENTAL LABORATORY PROJECT NO. E05-0544 1 INTRODUCTION Anaerobic biodegradation is defined as the microbiologically catalyzed degradation of a substance in the absence of molecular oxygen (O2). While typically under aerobic conditions O2 serves as a final electron acceptor for metabolic processes, but in the absence of O2 microorganisms find alternative electron acceptors to complete the biochemical redox reactions needed for cell growth and maintenance. Anaerobic microorganisms capable of such reactions are found in natural environments such as bogs, hot springs, subsurface water (i.e. groundwater), marine and freshwater sediments and submerged soils (i.e. paddy soil), and in manmade environments such anaerobic digesters, septic tanks and landfills, and in the gastrointestinal tract (colon) of animals and humans and specialized gut of ruminants. During anaerobic digestion the microbiological breakdown of organic polymers involves four stages as shown in Figure 1. These stages are: 1) hydrolysis stage, in which organic polymers are broken down to form simple sugars, amino acids and hydroxylated fatty acids; 2) acidogenesis, or fermentation stage, is where further breakdown of products of the hydrolysis stage forms small organic acids; 3) acetogenesis stage is where the organic acids from acidogenesis are further broken down to form acetic acid, carbon dioxide and hydrogen; and 4) the methanogenesis stage, where conversion of acetate to methane by acetotrophic methanogens and/or conversion of hydrogen and carbon dioxide into methane by hydrogenotrophic methanogens occurs. In traversing from stages 1 to 4 of the anaerobic process, the redox potential decreases, with methanogenesis occurring under the more negative redox conditions. Some anaerobic environments can attain redox values as low as -530 mV (Mah & Sussman, 1967). For biogenic methane production to occur from a complex organic substrates such as a polymer, one can assume that the necessary microbial consortia for stages 1 through 4 are functioning and that redox is below 0 mV. Figure 1. The four step anaerobic digestion Process (Source: BEEMS Module B7) Study Report: E05-0544 Page 8 of 101 3M ENVIRONMENTAL LABORATORY PROJECT NO. E05-0544 The work described in this report was initiated to develop test methods which could be used for reproducible establishment of anaerobic cultures for conducting GLP anaerobic biodegradability testing fluoropolymers; and conducted in support of consent order agreements between 3M Company and the U.S. EPA. While a draft protocol was written with the intent of conducting a more defined anaerobic test initially, it was determined early on that there was not enough information available regarding anaerobic culture preparations, proper dosing of fluorinated test substances into anaerobic cultures, identity of degradation products, nor recovery of test substance and degradation products from digester sludge matrix. Therefore the draft protocol was mostly ignored during the method development process. While the unsigned, draft protocol is included in the raw data package, it was not approved and was not a method development protocol and is only included to offer some insight to the mindset of the author during the refinement of the anaerobic biodegradation test method developed. During this method development project, the establishment of methanogenic anaerobic cultures was accomplished using municipal anaerobic digester sludge as microbial inoculum. Cultures were maintained as biogas-producing cultures, with analytical confirmation of methane and carbon dioxide formation in the bioactive culture headspaces. The anaerobic cultures were dosed with several perfluoroalkyl substances (PFAS) and were incubated for up to 108 days. The test substances evaluated were inclusive of electrochemical fluorination (ECF) derived and telomerization (TM) derived PFAS. Several of the tested substances had been previously evaluated under aerobic biodegradation conditions during 3M Environmental Laboratory testing and the same biotransformation products identified in those studies served as the basis for the target biotransformation products during this study. Those study IDs were E00-2252 (EtFOSE), E01-0415 (EtFOSE, EtFOSAA, EtFOSA, FOSA, PFOSi , PFOS and PFOA), E01-0444 (PFOS), E01-0684 (6:2 FTOH and 8:2 FTOH) and E02-1325 (MeFBSE). In addition to the PFASs, 2,4,6-trichlorophenol (246-TCP) and pentachlorophenol (PCP) were evaluated as potential positive control substances for future anaerobic biodegradation testing studies. Both biotransform via halorespiring microorganism under anaerobic conditions (i.e. reductive dehalogenation) and form stable, predictable end products (Woods et al. 1989, Miksell & Boyd 1985, and Mohn & Kennedy 1992). From the results obtained during this study, 246-TCP was determined to be a good candidate as a positive control substance for anaerobic biodegradation testing, while PCP was determined to not be a good positive control. The description of the anaerobic test system developed and the results of incubations with the aforementioned test substances and control substances are described herein. The report is divided into sections based on each test substance evaluated during this study. Several of the PFAS test substances did undergo anaerobic biodegradation and biotransformation products were identified and quantified. Based on rates of formation for total products formed, estimated rates of biodegradation were determined. The implications of these findings to potential anaerobic environmental fate of those tested PFASs are also discussed. Study Report: E05-0544 Page 9 of 101 3M ENVIRONMENTAL LABORATORY PROJECT NO. E05-0544 2 TEST, CONTROL AND REFERENCE (TCR) SUBSTANCES Table 1. Test Substances Evaluated During this Study (E05-0544) Test Substances Molecular Formula 3M TCR No. Purity (%) Stock Sol. ID Stock Conc. in Methanol (g/mL; ppm) ECF-derived MeFBSE C4F9SO2N[(CH3)(CH2CH2OH)] TCR-631 95.55 05007-92 976 PFBS C4F9SO3K TCR-282 97.3 05007-102 901 EtFOSE C8F17SO2N[(CH2CH3)(CH2CH2OH)] SE-031 97.7 05007-91 1010 PFOS C8F17SO3K TCR-741 99.9 05007-101 1060 PFOA C7F15CO2H TCR-617 99.51 05007-99 1010 PFBA C3F7CO2H TCR-757 99.2 05007-145 1030 Fluorotelomerization-Derived 6:2 FTS C6F13CH2CH2SO3K TCR-343 93.5 05007-100 954 6:2 FTOH C6F13CH2CH2OH TCR-1056 97 05017-66 1230 8:2 FTOH C8F17CH2CH2OH TCR-1058 97 05017-68 1300 Chlorinated Phenols PCP C6HCl5O TCR-1053 98.2 05017-62 1040 246-TCP C6H3Cl3O TCR-937 99.6 05007-146 1220 A figures are shown to 3 significant figures, however more significant figure values were used in data calculations All substances were soluble in methanol at the stock solution concentrations described in Table 1. References substances used for quantitation were chosen based on predicted biodegradation products as shown in Table 2. The identity of the reference substances, molecular formula, abbreviations and purities are given in Table 3. Study Report: E05-0544 Page 10 of 101 3M ENVIRONMENTAL LABORATORY PROJECT NO. E05-0544 Table 2. Selection of Target Analytes Based on Test Substance Dosed Target Analytes to Measure Test Substance Dosed (i.e. Anticipated Biodegradation Products) EtFOSE PFOS PFOA MeFBSE PFBS EtFOSE * EtFOSAA * EtFOSA * FOSA * * PFOSi * * PFOS * * MeFBSE * MeFBSAA * MeFBSA * FBSA * * PFBSi * * PFBS * * 6:2 FTS PFBA 6:2 FTOH 6:2 FTCA 6:2 FTUCA 8:2 FTOH 6:2 FTS 6:2 FTOH * * [a] * * * * * 8:2 FTCA 8:2 FTUCA PFBA PFHxA PFHpA PFOA PFNA * * * * * * * * * [a] 6:2 FTOH was intended to be measured in the 6:2 FTS cultures, but was not. However the 6:2 FTS culture did not demonstrate any loss of test substance based on direct measure of 6:2 FTS. 8:2 FTOH * * * * * * Study Report: E05-0544 Page 11 of 101 Table 3. Reference Substance (Study E05-0544) Analyte Abbreviation Perfluorobutanesulfonyl-Related Analytes 2[(N-methyl)perfluorobutanesulfonamido]ethanol [a] 2[(N-methyl)perfluorobutanesulfonamido]acetate N-Methyl Perfluorobutane sulfonamide Perfluorobutane sulfonamide Perfluorobutane sulfinate Perfluorobutane sulfonate [a] Perfluorooctanesulfonyl-Related Analytes 2[(N-ethyl)perfluorooctanesulfonamido]ethanol [a] 2[(N-ethyl)perfluorooctanesulfonamido]acetate N-Ethyl Perfluorooctane sulfonamide Perfluorooctane sulfonamide Perfluorooctane sulfinate Perfluorooctane sulfonate [a] Fluorotelomer-Related Analyte 6:2 Fluorotelomer Sulfonate [a] 6:2 Fluorotelomer Alcohol [a] 8:2 Fluorotelomer Alcohol [a] 6:2 Fluorotelomer acid 6:2 Fluorotelomer unsaturated carboxylic acid 8:2 Fluorotelomer carboxylic 8:2 Fluorotelomer unsaturated carboxylic acid Perfluorinated Carboxylic Acid Analytes Perfluorononanoate Perfluorooctanoate [a] Perfluoroheptanoate MeFBSE MeFBSAA MeFBSA FBSA PFBSi PFBS EtFOSE EtFOSAA EtFOSA FOSA PFOSi PFOS 6:2 FTS (aka THPFOS) 6:2 FTOH 8:2 FTOH 6:2 FTCA 6:2 FTUCA 8:2 FTCA 8:2 FTUCA PFNA PFOA PFHpA Study Report: E05-0544 Page 12 of 101 Molecular Formula C4F9SO2N[(CH3)(CH2CH2OH)] C4F9SO2N [(CH3)(CH2CO2 -] C4F9SO2NH(CH3) C4F9SO2NH2 C4F9SO2 C4F9SO3 - C8F17SO2N[(CH2CH3)(CH2CH2OH)] C8F17SO2N [(CH2CH3)(CH2CO2 -] C8F17SO2NH(CH2CH3) C8F17SO2NH2 C8F17SO2 C8F17SO3 - C6F13CH2CH2SO3 C6F13CH2CH2OH C8F17CH2CH2OH C6F13CH2CO2 C5F11CFCHCO2 C8F17CH2CO2 C7F15CFCHCO2 - C8F17CO2 C7F15CO2 C6F13CO2 - 3M ENVIRONMENTAL LABORATORY PROJECT NO. E05-0544 Reference Substance ID Purity TCR-631 TCR-707 TCR-632 TCR-314 TCR-723 TCR-282 95.55% 50.1% 97.25% 99.3% 51.2% [b] 97.3% TCR-078, SE-031 TCR-881 TCR-871 TCR-280 TCR-7 (SD-007) TCR-741 97.7% 98.63% 100% [c] 98.94% 97.2% 99.9% TCR-343 TCR-1056 TCR-1058 TCR-679 TCR-681 TCR-1156 TCR-1089 93.5% 97% 97% 97% 97% 95% 98% TCR-618 TCR-617 TCR-267 98.02% 99.51% 98.2% 3M ENVIRONMENTAL LABORATORY PROJECT NO. E05-0544 Table 3. Reference Substance (Study E05-0544) Analyte Abbreviation Molecular Formula Reference Substance ID Purity Perfluorohexanoate PFHxA C5F11CO2 - TCR-673 97.7% Perfluorobutanoate [a] PFBA C3F7CO2 - TCR-757 99.2% Chlorinated Phenol Analytes Pentachlorophenol [a] PCP C6HCl5O TCR-1053 98.2% 2,3,5,6-Tetrachlorophenol 2356-TeCP C6H2Cl4O TCR-1050 99.96% 2,4,6-Trichlorophenol [a] 246-TCP C6H3Cl3O TCR-937 99.6% 2,6-Dichlorophenol 2,6-DCP C6H4Cl2O TCR-1051 99.9% 2,4-Dichlorophenol 24-DCP C6H4Cl2O TCR-1052 99.2% 2-Chlorophenol 2-CP C6H5ClO TCR 1055 99% 4-Chlorophenol 4-CP C6H5ClO TCR-1054 99.01% Internal Standards Trimethylsilylpropane sulfonate (IS) TMSPS Si(CH3)3(C3H6SO3 -) TCR-938 100% Perfluorohexane sulfonate PFHS C6F13SO3 - TCR-890 99.98% [a] indicates a reference which was also a test substance in this study [b] Purity was determined for the same lot of PFBSi material (3M lot 41-2601-2249-8) as TCR-1099 at 51.2%, but was determined after use of the substance in this study as TCR-723 (3M lot 41-2601-2249-8) with purity designated at 50% based on average of MSDS 45-55% value and was only value available at time of use. [c] Purity not determined, assumed 100% Study Report: E05-0544 Page 13 of 101 3M ENVIRONMENTAL LABORATORY PROJECT NO. E05-0544 3 METHODS The biodegradation of each test substance was inferred by loss of test substance, and/or formation of predicted polyfluorinated degradation products (see Table 3), and all determinations were compared to the results of killed (sterile sludge) and abiotic (no-sludge) controls which were prepared in parallel with the bioactive (live sludge) test cultures. None of the cultures were pre-acclimated to the test substances and as such the estimated initial rates are reflective of native ability of the digester sludge microorganisms to biodegrade the test substances and not reflective of adapted ability which could possibly have occurred if given more time to acclimate to the test substances. Most of the culture preparation and collection data and calibration and QC preparations for this project were documented in 3M Technical Notebooks No. 139716 (Cleston Lange) and No. 140568 (Peter Radford). This work was method development/exploratory in nature and therefore culture and standard preparations and extractions were not always recorded in accordance with the laboratory SOPs for documenting sample and calibration standard preparations. Also, there were no analytical criteria or analytical procedures specified by any signature-approved protocol or general project outline (GPO), albeit a draft protocol was written apriori and which did aid in defining the scope and general goals for this method development work. All of the reported results were evaluated for their merit based on the acquired analytical results with matrix-matched calibration standards and continuing calibration verification (CCV) checks, and select quality control (QC) samples prepared for select culture sets, and all results from bioactive cultures were compared results from sterile control cultures as confirmation of biodegradation when test substance loss or product formation was observed. The operation and parameters of the test equipment, as described herein, were in many instances developed during this study in 2005-2006 with the intention of defining Standard Operating Procedures (SOPs) to support planned GLP studies at that time. Additional references that aided in this method development project were Tiedje and Shelton (1984), the U.S.EPA Fate, Transport and Transformation Test Guidance OPPTS 835.3400 and the OECD Guideline for the testing of Chemicals No. 308 and No. 311. However, many aspects of those guidance's were not followed or were considered irrelevant to this work and several changes were adopted during this development project to allow for ease of culture preparations in the glove box, as well as to allow for replicate cultures to be prepared, and to accommodate the sensitive analyte-specific analyses by liquid chromatography with tandem mass spectrometric detection (LC/MS/MS) and gas chromatography with mass spectrometric detection (GC/MS). As a result of the method development work described herein, and previously conducted aerobic biodegradation studies up to that time, a final equipment procedure for operation and maintenance of the anaerobic glove Study Report: E05-0544 Page 14 of 101 3M ENVIRONMENTAL LABORATORY PROJECT NO. E05-0544 box (ETS-9-028) and an analytical method for the extraction of biodegradation cultures for LC/MS analysis (ETS-8-001) were finalized in 2006 and added to the 3M Environmental Laboratory quality system. However, it is noted that not all results reported herein were consistent with the final parameters or criteria established in those SOPs since most of this work preceded those SOPs, nor did the results of this study always meet the acceptance criteria established in the final analytical method ETS-8-001. The SOPs developed were more importantly put in place during later stages of this study with the intended purpose to support the GLP studies for fluoropolymer anaerobic biodegradation conducted in 2006 (i.e. 3M anaerobic biodegradation studies E05-0627 and E05-0630). In some instances the reanalysis of all time points for a test substance was conducted in a single analytical batch after completion of the incubations, and any previous intermittent analysis results were disregarded for determining the final biodegradation conclusions (i.e. MeFBSE). Calibration curves were typically constructed from analyzing sterilized sludge matrix extracted calibration standards. In some instances, QCs were prepared by fortifying target analytes into the equivalent of sterilized sludge cultures and were extracted the same as samples and analyzed. Analyses were typically, but not always, performed by internal standard (IS) method using trimethylsilylpropane sulfonate (TMSPS) as IS in extraction solutions and used to aid in baselining analytical results for test substance loss and/or product formation. In some instances, one or more anchor points [i.e. standards below the batch-defined lower level of quantitation (LLOQ)], were used to improve low end calibration accuracy. Also, in some instances, the LLOQ peak area response was not always 2-times the response of equivalent prepared method blanks. However, in all instances those data were evaluated more closely for merit and potential effects on final results and conclusions were considered, and in some instances were not used for final reporting. The use of laboratory control spikes (LCSs) and laboratory matrix spikes (LMSs) was not included during this study and some test, control and reference substances were not fully characterized at the time of use. However, as a result of findings during this study several were later more thoroughly characterized for use in GLP biodegradation studies once it was established they were anticipated degradation products of later tested fluoropolymers. 3.1 Collection, Storage and Handling of Anaerobic Digester Sludge The anaerobic digester sludge for this study was obtained from the municipal anaerobic digester located in Empire, Minnesota and was collected and delivered to the 3M Environmental laboratory by Pace Analytical Services personnel. At the time of sludge collection the Empire wastewater treatment plant (WWTP) constituted one component of the larger Twin Cities metropolitan wastewater treatment system comprised of eight large interconnected WWTPs servicing over 300 million gallons of wastewater daily at that time. The Empire plant was the only Twin Cities Metro WWTP that had an on-sight anaerobic digester. For this study, the anaerobic digester sludge was delivered to 3M Environmental Laboratory on four occasions for testing purposes, as listed in Table 4. Study Report: E05-0544 Page 15 of 101 3M ENVIRONMENTAL LABORATORY PROJECT NO. E05-0544 Anaerobic sludge was typically received at ambient temperature in polypropylene 1-L bottles sealed tightly with white non-transparent lids. Once received at 3M each was labeled with unique IDs, described in Table 4, and then stored refrigerated until use. The bottles of anaerobic sludge used for the earlier phases of this study were originally logged into the 3M Environmental Laboratories LIM system under project E05-0544 as samples and were given unique sample IDs starting with E05-0544-xxx. Later, these bottles were reassigned with traceability (TN-A-) numbers and treated thereafter as reagents for preparation of cultures. Sludges were always stored with the lids tightly sealed and refrigerated prior to use. The anaerobic sludge was always used within 4 weeks of collection for preparation of test cultures. Anaerobic sludge has been reported to be viable for up to four weeks for anaerobic biodegradation evaluations (Shelton & Tiedje, 1984) and this storage period is more than the two weeks refrigerated storage suggested by U.S. EPA and OECD guidance. Sludge was typically transferred from the refrigerator to the glove box the day of or the day before culture preparations. The sludge was always received as a dark-black semi-solid liquid with a texture of fine mud that maintained itself as a non-partitioning single phase. Pressure within sealed containers increased when stored at ambient temperature and was easily recognizable by bulging lids on the container. This was presumed to be due to very rapid anaerobic microbial gas production. The sludge had a distinct sulfide smell. Storage of the sludge at 4C alleviated excessive pressure during storage, presumably due to the anticipated decrease in microbial activity at cold temperatures. Evaluation of the content or chemical makeup of the sludge was not typically performed, but some evaluations were performed on certain sludge collections and used to generalize the makeup of the sludge used throughout the study since it was anticipated that the anaerobic digester is maintained as a semi-continuous yet generally homeostatic system over time. Sterilized sludge was prepared by autoclaving 500 mL of the anaerobic digester sludge for 30-45 minutes in a tempered-glass NalgeneTM bottle with autoclavable orange polypropylene lid and This study report and data were partially audited by the 3M Environmental Laboratory Quality Assurance Unit (QAU). Study Report: E05-0544 Page 16 of 101 Table 4. Digester Sludge Collection Information 3M ENVIRONMENTAL LABORATORY PROJECT NO. E05-0544 Sludge Source Date Collected 3M Env. Lab LIMS ID Volume (L) Total Suspended Solids (g/L)* 3M Env. Lab Chain of Custody No. Empire WWTP 10-August-2005 E05-0544-87193 2 ND (MN) 08429 Empire WWTP 29-August-2005 E05-0544-87854/87855 2 14.3 (MN) 09251 Empire WWTP 18-October- E05-0544-90220 2 14.3 (MN) 2005 09599 Empire WWTP (MN) 14-February2006** TNA-07526** 2 11.1; 18.1*** 09435 *; Total suspended solids (TSS) determined by a modification of EPA method 160.2 and determined by weight of filtered sludge that was dried at between 85-110 C. ND; not determined. **; sludge was not used in this study, but was characterized for content and reported to provide general characterization data for sludge obtained from the Empire (MN) WWTP anaerobic digester. ***; TSS was determined at both the 3M Environmental Laboratory; and Pace Analytical Services and different values reported (1st value is 3M result). Sludge from the Empire, MN wastewater treatment plant anaerobic digester was collected on 2/14/2006 for another study, and was characterized by Pace Analytical Services (Pace project no.1027866, Report dated March 2006). The general sludge characterization results are shown below in Table 5 to provide some insight to the sludge used in 2005 from the same Empire digester; however, the sludge evaluated was not used in this study. Table 5. Empire Plant Digester Sludge Characterization Data Parameter Result Units Analysis Method Total Suspended Solids (dry) Total Solids (dry) Nitrogen (Ammonia) 18,100 20,600 722 mg/L mg/L mg/L EPA 160.2 EPA 160.3 EPA 350.1 Nitrogen (Total Kjeldahl) 984 mg/L EPA 351.2 Phosphorous (Total) 454 mg/L EPA 365.2 Ortho-Phosphate 47.8 mg/L EPA 365.2 Sulfate < 2.5 mg/L EPA 375.4 Chemical Oxygen Demand (COD) 47,800 mg/L EPA 410.4 Non-Purged Organic Carbon (TOC-NPOC) 1,100 mg/L EPA 415.2 [a] these data determined for digester sludge that was not used in this study, but is included to provide general characterization data for digester sludge obtained from the Empire (MN) WWTP anaerobic digester around the time frame that sludge was collected for this study. These results are for Empire plant digester sludge obtained on February 2, 2006. Study Report: E05-0544 Page 17 of 101 3M ENVIRONMENTAL LABORATORY PROJECT NO. E05-0544 3.2 Culture Preparations Anaerobic test cultures were prepared as one of five independently prepared sets of cultures over a period of approximately three months. The first set of cultures was prepared as a very small exploratory set of test cultures and blank cultures and was primarily used for optimizing the methods of culture preparation and analysis and for some preliminary evaluation of potential anaerobic product formation from EtFOSE and MeFBSE. This first set of cultures (IDs E05-0544-001 to E05-0544-024) were prepared on 8/17/2005 using anaerobic sludge provided from the digester on 8/10/2005. This first set of cultures was dosed with and without EtFOSE or MeFBSE. Preparation of that set of cultures is referenced in 3M Technical notebook # 139716, pages 10-12 and most of the data associated with this set is excluded from this report due to the exploratory nature of that set. The second preparation of culture sets (IDs E05-0544-025 thru E05-0544-129) occurred on 8/29/2005 using anaerobic sludge provided from the digester on 8/29/2005. Those cultures were dosed with either EtFOSE or MeFBSE at nominal 1 g/mL (1 ppm) of each, and set up for time dependent collection and analysis. The preparation of that set of cultures is referenced in 3M technical notebook # 139716, pages 25-26. The third preparation of culture sets (IDs E05-0544-130 thru E05-0544-219) occurred on 9/1/2005 using anaerobic sludge provided from the digester on 8/29/2005. That set of cultures was prepared with either 6:2 FTS, PFOS or PFBS for time dependent sample collection and analysis. Preparation of the third set of cultures is referenced in 3M technical notebook # 139716, pages 31-32. The fourth preparation of culture sets (IDs E05-0544-220 thru E05-0544-240) occurred on 9/22/2005 using sludge collected on 8/29/2005. Those cultures were prepared with 246-TCP at nominal 1 ppm of each for time dependent collection and analysis. The preparation of those cultures is referenced in 3M Technical notebook (TNB) 139716, page 59. Finally, a fifth preparation of cultures (IDs E05-0544-500 thru E05-0544-694) occurred on 11/11/2005 using sludge collected from the digester on 10/18/2005. Those sets of cultures were prepared for time dependent collection and analysis, and were dosed with PCP, PFOA, PFBA, 6:2 FTOH or 8:2 FTOH. Preparation of those cultures is referenced in 3M technical notebook # 139716, pages 79-84. With the exception of the first set of cultures and the fourth set of cultures, all of the culture sets were prepared with ample cultures to harvest cultures five times in triplicate per time point per culture type. Study Report: E05-0544 Page 18 of 101 3M ENVIRONMENTAL LABORATORY PROJECT NO. E05-0544 Note that for the apparent missing cultures with IDs E05-0544-241 to E05-0544-499 there were no culture prepared and those IDs were skipped. The culture sets 2 through 5 included the following: active-sludge (live) cultures, sterilizedsludge (killed) control cultures, water control (abiotic) blanks, active-sludge (live) blanks, and sterilized-sludge (killed) blanks. The final prepared active cultures contained 4 mL of activesludge plus 1 mL of sterile 10 mg/ml yeast extract (YE solution) and test substance. Sterile control cultures contained 4 mL of autoclave-sterilized sludge plus 1 mL of sterile 10 mg/mL YE solution and test substance. Active and sterile blank cultures were prepared identically, except they were not dosed with test substance. Abiotic control blanks received 4 mL of autoclavesterilized Milli-QTM water and 1 mL of sterile YE solution, and no test substance. All cultures were prepared in nominal 42.5 mL amber-glass I-ChemTM vials with screw-caps containing PTFE-lined silicon-rubber septa. To each test vessel vial was added 1 mL of sterile 10 mg/mL YE solution under a normal atmosphere of air and then followed by the transfer of those vials to the glove box while keeping caps loose during transfer in the ante-chamber to displace air with N2 gas. Once equilibrated to a low-oxygen environment in the glove box, 4 mL of anaerobic digester sludge was added. After adding sludge, the caps were sealed and test substance was then added to the appropriate cultures through the septa using a 25 L or 50 L Hamilton gas-tight syringe and methanol as carrier solvent. All test substances were added as stock solutions prepared in methanol and are listed in Table 1 along with the other reference substances used in this study listed in Table 3. All fluorochemical (FC) test substances were dosed into cultures to nominal concentration of 1 g/mL (1 ppm) by adding 5 L of a nominal 1000 g/mL stock solution into a 5 mL culture using a glass Hamilton gas-tight syringe. The 246-TCP cultures were tested at approximately 1.2 ppm of 246-TCP by adding 5 L of a 1,220 ppm stock solution to a 5 mL cultures, except the last time point cultures which were dosed 2X at 2.44 ppm to evaluate higher level 246-TCP effects on the cultures. The pentachlorophenol (PCP) cultures were tested at approximately 2.0 ppm of PCP by adding 10 L of a nominal 1000 ppm stock solution to a 5 mL culture. In many instances, blank cultures were not fortified with equivalent doses of blank methanol. This could have influenced the levels of methane/CO2 gas productions in test cultures versus blanks by providing the additional methanol which would be expected to biodegrade, and therefore is acknowledged as a potential positive effect on rates and gas production in those cultures dosed with methanolic solutions. However, sterile controls and abiotic controls did receive the same quantity of test substance as bioactive test cultures and no significant difference was noted in gas production of blanks for culture sets prepared. Once the test substance was added to the appropriate cultures, the cultures were mixed gently and incubated static and inverted (cap down) in the glove box to minimize losses of volatiles through the cap. Study Report: E05-0544 Page 19 of 101 3M ENVIRONMENTAL LABORATORY PROJECT NO. E05-0544 Note: Resazurin is a recommended colorimetric indicator of O2 by some guidance documents to ensure anaerobic conditions, but was excluded from these study cultures. The conditions of the glove box (i.e. O2 levels) during preparation and incubation from the oxygen sensor in the glove box served as the indicator for anaerobic conditions, as did positive formation of methane in headspace of bioactive cultures which is inhibited by O2 and also was not formed in sterile cultures. Cultures were prepared and stored under strictly anaerobic conditions in the glove box which was maintained as described. Cultures were typically incubated static, but were periodically (approximately once every week or two) gently mixed to maintain culture homogeneity during the incubation period. 3.3 Anaerobic Glove Box "Incubation" Conditions The anaerobic glove box (instrument name: Durga) used for this study was a Vacuum Atmospheres Inc. Nexus Model 100043 (S/N # 00420) controlled atmosphere glove box. The glove box was maintained at between 20-26C during the incubation periods. The glovebox atmosphere was maintained with ultra-pure nitrogen (N2) provided from a pressurized tank (Oxygen Service Company, MN). Trace oxygen and water in the glove box atmosphere were continuously removed using a built in circulating blower and regenerative O2 and water removal system. Transfer of materials into and out of the glove box was performed using one of two vacuum antechambers. The levels of O2 (in ppm or percent) within the chamber were monitored using a calibrated solid state oxygen sensor (Advanced Micro Instruments, Inc.) installed on the glove box. Moisture content was monitored by a Panametrix AMX 1 dew point meter model 705-844 installed on the glove box. . All anaerobic cultures were prepared, sealed tightly, and then incubated within the glove box to ensure maintenance of anaerobic conditions. During culture incubation periods, the glove box was maintained with an atmosphere of ultra-pure nitrogen and was typically < 1 ppm of O2. On occasion, the O2 levels spiked to levels greater than 10 ppm due to transfer of materials in/out of the glove box. The transient oxygen spikes returned to 1 ppm, or less, soon thereafter. The recorded oxygen and temperature readings during the incubation time frames are shown in Figure 2 and original records can be found in 3M Technical Notebook No. 139716 (Cleston Lange) or in the instrument logbook which was put into place for the glove box Study Report: E05-0544 Page 20 of 101 3M ENVIRONMENTAL LABORATORY PROJECT NO. E05-0544 Figure 2. Glovebox temperature () and oxygen levels () during study E05-0544. 3.4 Culture Collections At each time point, select culture vessels were collected (harvested) from the glove box and were evaluated for production of gases (based on volumetric displacement method of a syringe barrel in a syringe). The gases were pushed back into the culture and were then frozen until such time that they could be extracted for analysis by LC/MS/MS and direct inject GC/MS. Cultures were typically harvested in duplicate or triplicate for each culture type at each collection time, except for the single culture collected for days 66 (MeFBSE) & 72 (EtFOSE0 for culture set 2, as described below. Five sets of cultures were prepared during the course of this project. Due to the preliminary and highly developmental nature of culture set #1 prepared on 8/17/2005, and the lack of sufficient replicates and splitting the cultures out for several different analytical techniques, discussion about the 1st set of cultures is largely excluded from this report. The preparation dates, test substances dosed and incubation dates for the other culture sets were as follows: Set # 2 cultures were prepared on 8/29/2005 with MeFBSE or EtFOSE. The MeFBSE cultures were harvested on days 0, 10, 21, 29, 66 (70) and 108 and those dosed with EtFOSE were harvested on days 0, 10, 21, 29, 72 and 108. Cultures for day 70 (MeFBSE) sat on the bench for ~4 days after headspace analysis but prior to being frozen and extracted for analysis by LC/MS/MS, hence, the parenthetic value of 70 days which indicate LC/MS/MS results were for ~day 70. Headspace result for those MeFBSE cultures were for day 66. Study Report: E05-0544 Page 21 of 101 3M ENVIRONMENTAL LABORATORY PROJECT NO. E05-0544 Set # 3 cultures were dosed with PFOS, PFBS and 6:2 FTS (a.k.a. THPFOS) on 9/1/2005 and harvested on days 0, 7, 15, 26, 69 and 105. Set # 4 cultures were dosed with 246-TCP on 9/22/2005 and harvested on days 0, 5, 7, 15 and 47. Set # 5 cultures were prepared on 11/11/2005 and dosed with PFOA, PFBA, 6:2 FTOH, 8:2 FTOH or PCP and those cultures harvested on days 0, 10, 20, 74 and 94; the PCP cultures were harvested on different days 0, 3, 10, 20 and 74. Special collection time points other than those listed above were as follows: on day 66 for culture set # 2 (MeFBSE), one active and one sterile culture for MeFBSE were removed from the glove box to perform headspace GC/MS analysis for potential volatile products, and those two samples sat on the lab bench until day 70 when they were frozen for later extraction and LC/MS/MS analysis (IDs E05-0544-112 and -129). Likewise, on day 72 for set # 2, one active and one sterile culture dosed with EtFOSE (IDs E05-0544-083 and -099) were removed from the glove box for headspace GC/MS analysis, along with cultures representing a day-72 active blank culture (E05-0544-054), a day-72 sterile blank culture (E05-0544-066) and a day-72 abiotic blank culture (E05-0544-033). All of those day-72 cultures were frozen immediately after headspace GC/MS analysis on that same day. On day 69 for culture set # 3, and active and sterile culture representing each for PFOS (E05-0544-204 and -219), PFBS (E05-0544-174 and 189) and 6:2 FTS (E05-0544-144 and -159) were removed from the glove box for headspace GC/MS analysis and then frozen immediately following the analysis on the same day. 3.5 Analytical Determinations 3.5.1 Gas Production Measurement General gas production in the headspace of cultures was determined using a syringe barrel displacement method, which briefly was a 10 mL lure-lock polypropylene syringe with rubber tipped plunger coupled to a 21 Gauge 1 inch beveled needle. The syringe plunger was pushed all the way into the syringe barrel to the zero mark and then the needle pierced thru the septa of a culture into the headspace, which promptly resulted in the plunger traveling up the barrel of the syringe, if gas pressure was present. Gas production was measured as the change in gas volume by reading the graduations on the syringe barrel after the plunger travel had ceased (measured to + 0.1 mL). The same plunger was used for all cultures at the same sample collection day with replacement of the needle between samples and purging the syringe with air between different sets to avoid cross contamination of gases in the headspace. Gas volumes were recorded in 3M Technical notebook TNB 139716. All syringes and needles were used as provided by the manufacturer (Becton-Dickinson) and were not treated with silicon grease or other lubricants. Study Report: E05-0544 Page 22 of 101 3.5.2 Gas Phase FTIR Analysis 3M ENVIRONMENTAL LABORATORY PROJECT NO. E05-0544 Gas phase Fourier transform infrared (FTIR) spectroscopy analysis was conducted using a Midac Model I2001 FTIR with a nominal 10 cm (~ 100 mL) gas cell and using a 2001 ppm ethylene calibration standard (balanced with N2) For sampling, 1 mL of static headspace from a culture was taken by gas tight syringe and introduced to the FTIR cell and analyzed. Nitrogen was used as a makeup gas for all measurements. The gas phase FTIR analysis was conducted primarily to quantify methane and carbon dioxide in culture headspaces following 3M Environmental Laboratory method ETS-8-031 and to qualitatively evaluate the gases for any potential high level volatile FC products. Only methane, carbon dioxide and water were observed by gas phase FTIR of culture headspaces; volatile fluorocarbon (C-F stretch) absorbance was not observed. 3.5.3 Headspace-GC/MS Analysis Semi-quantitative/qualitative analysis of culture headspace gases for volatile fluorocarbon analytes was performed by gas chromatography with mass spectrometric detection (GC/MS) and was performed on cultures collected on day 66 (MeFBSE cultures only) and day 72 for cultures sets #2 and #3. The method used was slightly modified from that described in method ETS-8016 and was modified for means of sample introduction via a syringe (ETS-8-016 is essentially EPA method TO15). A volatile surrogate standard was added to the culture headspace prior to sampling and then approximately 20 mL of headspace was withdrawn from each culture into a glass 50 mL Hamilton syringe fitted with a gas tight-valve. This was then injected onto a cryogenically cooled solid-phase TenaxTM trap to focus the volatiles for heated injection onto the GC/MS column. See data for analytical runs conducted on instrument ID "Zeke" in OctoberNovember 2005. Analytical batch Z051031.s was analysis of calibration standards. Batch Z051103.s included headspace analysis for cultures E05-0544-112 (bioactive MeFBSE, day 66) and -129 (Sterile MeFBSE, day 66). Batch Z051109.s included headspace analysis for cultures E05-0544-054 (bioactive blank, day 72), -144 (bioactive 6:2 FTS, day 69), -083 (bioactive EtFOSE, day 72), -174 (bioactive PFBS, day 69) and -204 (bioactive PFOS, day 69). Volatile fluorocarbon analytes were not detected above background for any of the analyzed culture headspaces, indicating they were not formed for EtFOSE, MeFBSE, 6:2 FTS, PFBS and PFOS. Culture sets #4 and #5 which were dosed with PFOA, PFBA, 6:2 FTOH, 8:2 FTOH, 246-TCP and PCP were not analyzed by headspace GC/MS. 3.5.4 Analytical Sample Preparation Following headspace gas sampling/analysis, cultures were either frozen until later extraction with acetone, or were extracted immediately with acetone. Study Report: E05-0544 Page 23 of 101 3M ENVIRONMENTAL LABORATORY PROJECT NO. E05-0544 All 5 mL cultures were extracted by addition of 15 mL of acetone containing nominal 250 ng/mL of trimethylsilylpropane sulfonate (TMSPS) and 250 ng/mL of perfluorohexane sulfonate (PFHS) as internal standards (ISs). After addition of acetone extraction solution, each was briefly shaken to mix and then centrifuged at 1500 rpm to pellet the solids. Aliquots of supernatant were transferred to autovials for analyses. Analyses were performed against matrix matched calibration standards which were prepared by fortifying known quantities of target analytes into 4 mL of sterilized sludge plus 1 mL yeast extract solution followed up with extraction similar manner as samples using acetone extraction solution. All preparations associated with analytical samples were documented in 3M Technical notebook #139716 (Cleston Lange) and #140568 (Peter Radford). 3.5.5 Direct Inject GC/MS Analysis The GC/MS analysis of acetone extracts for test substances 6:2 FTOH and 8:2 FTOH was performed using a Hewlett Packard 6890A gas chromatograph equipped with a 5973 massselective detector (MSD); shown as analytical run #24 (R060517a.S) in Table 8. The associated blank cultures prepared with the FTOH cultures were accidentally excluded from the analysis by the analyst, hence no background levels were established for the blank sludge cultures. The GC parameters included a split-less injection of 5.0 L of acetone sample extract onto a 30 m x 0.250 mm ID x 0.001 mm film DB-1MS column was performed. A 15.5 minute temperature program was used with the following parameters: initial temp at 50C for 1.00 minute; 20C/minute to 300C; hold 2 minutes at 300C. The mass spectrometer was operated using electron impact (EI) source at 69.9 eV and 230C, the MS quadrapole was at 150C, GC to MS transfer line was at 250C. Selected ion monitoring (SIM) mode was used to collect data for mass-to-charge ratio (m/z) positively charged ions with m/z 131, 169, 395, 405, 414 and 444. Calibration standards for 8:2 FTOH were standards 140568-039D1 to -039D8 and for 6:2 FTOH were 140568-039C1 to -039C8. For GC/MS analysis of the 8:2 FTOH cultures, two QCs were analyzed in the analytical batch R060517.S approximately every 15 samples as CCVs and were identified as 140568-039D9 and 140568-039D10, described on page 41 of 3M TNB 140568. For GC/MS analysis of the 6:2 FTOH cultures, two QCs were analyzed in the analytical batch R060517a.S approximately every 15 samples as CCVs and were identified as 140568-039C9 and 140568-039C10. Those QC were prepared at 40 ng/mL and 400 ng/mL, respectively. Samples were analyzed for 6:2 FTOH and 8:2 FTOH with calibration standards identified as 140568039C1 to -039C8 and described on page 41 also. Quantitation was performed by external standard method using Analyst software version 1.6.2 after conversion of Chemstation *.d files to common data format *.cdf file extension using Vx CaptureTM and then to Analyst readable *.wiff extension using the Analyst Translat.exe program. Integration of the respective FTOH peaks was performed from the total ion chromatogram (TIC) Study Report: E05-0544 Page 24 of 101 3M ENVIRONMENTAL LABORATORY PROJECT NO. E05-0544 created by summing SIM ions m/z 169, 295, 305 and 314 for 6:2 FTOH (Peak Rt. 4.68 min.) and SIM ions m/z 169, 395, 405, and 414 for 8:2 FTOH (Peak Rt. 5.70 min.). Specific instrument parameters can be found with the raw data and the analytic run log associated with analytical run R060517a.S. 3.5.6 LC/MS/MS Analysis After establishing the presence of and identities of biodegradation products by qualitative analysis (data not shown, see TNB 139716, pages 17-23), the quantitative LC/MS/MS analyses for degradation products was conducted for all test culture extracts. The LC/MS/MS method utilized an Agilent 1100 series high-pressure liquid chromatograph (HPLC: degasser, binary pump, autosampler and thermostat controlled column compartment) coupled to a triple quadrapole mass spectrometer with electrospray interface. Ions monitored for the target analytes are listed in Table 6. Chromatographic separations of fluorochemical analytes was performed at a flow rate of 1 mL/minute by 2-D chromatography which used an NG1 anion exchange (4 x 35 mm, 5; Dionex) guard column in-line with a Betasil C8 reverse-phase (4.6 x 150 mm, 5; Keystone) analytical column. Injection of sample extracts varied for different batches and ranged between 5 L and 75 L. The HPLC gradient was as described in Table 7. Quantitative LC/MS/MS analysis of chlorinated phenols was performed using the C8 column only (non NG1 column), but similar mobile phase conditions and gradient as described in Table 7. Analysis of the 6:2 FTS culture extracts was performed either by the NG1-C8 configuration above or using a different column configuration [Betasil C8 (4.6 x 150mm, 5) and Rocket C18 (3 x 37 mm, 3)] in tandem, with slightly modified aqueous/methanol gradient profile (see raw data). The specific HPLC and mass spectrometer operating parameters used for each analytical run are recorded within the raw data. Quantitative analysis was typically performed using an 8 point calibration curve, with some exceptions as noted for EtFOSE which had 11 calibration standards analyzed. Curves were constructed using results from analysis of matrix-matched calibration standards typically ranging at nominal concentrations of 10.0, 25.0, 100, 250, 500, 750, 1000 and 2000 ng/mL of target analytes (5.00, 12.5, 50.0, 125, 250, 375 and 1000 ng/mL for PFBSi). For quantitation of the EtFOSE cultures at analyte concentrations below 10.0 ng/mL, three additional standards (total of 11 standards) were included with the additional three prepared at 1.00, 4.00 and 7.50 ng/mL. Quality control samples (QCs) were prepared at 40 ng/mL and 400 ng/mL and analyzed as continuing calibration verification (CCVs) in all LC/MS/MS analytical batches used to analyze culture extracts for dosed substances MeFBSE, EtFOSE, PFOS, PFBS, PFOA and PFBA, except run #26 in which the CCVs were reinjection of the 250 ng/mL standard. Similarly, QCs were prepared at 40 ng/mL and 400 ng/mL and analyzed in direct inject GC/MS analytical batch for analysis of 6:2 FTOH and 8:2 FTOH. Study Report: E05-0544 Page 25 of 101 3M ENVIRONMENTAL LABORATORY PROJECT NO. E05-0544 The cultures dosed with TM derived substances (i.e. 6:2 FTOH, 8:2 FTOH and 6:2 FTS) were analyzed by LC/MS/MS for PFCAs, FTCAs, 6:2 FTS and FTUCAs. The CCVs in those analytical batches were the re-injection of a low-level (25.0 ng/mL) and mid-level (750 ng/mL) calibration standard throughout the run. CCVs were injected approximately every 10-15 samples and at the end of the run and were within 100 + 30% accuracy. The cultures dosed with 246-TCP were analyzed by LC/MS/MS in analytical run #12 and the CCVs in that run were the 800 ng/mL calibration standard. The PCP cultures were analyzed by LC/MS/MS in analytical run #27 and the CCVs in that run were reinjection of the 600 ng/mL calibration standard. The lower limit of quantitation was analyte and batch specific and was defined as the lowest standard that gave good accuracy with the fitted calibration curve (100+ 30%) and that gave a peak area response at least 2-times the response of the associated blank analyzed just prior to the 1st calibration standard injection. LLOQs were < 25.0 ng/mL for all analytes reported, and commonly were < 10.0 ng/mL. Exceptions to the normal rules for assigning LLOQs were in analytical run #21 where the LLOQ for PFOA was assigned as 4.00 ng/mL, but the curve included the 1.00 ng/mL standard (as an anchor point); but the 1.00 ng/mL standard was not 2-times the blank response. Also, the LLOQ in run #21 for PFOS was assigned as 7.5 ng/mL, however that calibration standard was not included in the calibration curve, but was 109% accurate and was more than 2-times the response of the blanks. The actual analyte LLOQs are shown in the individual results sections for each test substance. All calibration standards included in calibration curves were within 100 + 25% of theoretical and CCVs/QC results were within 100 +30%, with the following exceptions: 1) one QC analyzed as a CCV for PFBSi in analytical run #5 recovered at 145% but did not affect the reported PFBSi results since all samples flanked by that QC were below the LLOQ (BLOQ); 2) five CCVs in the latter half of the run #8 for 6:2 FTS were at 152%, 131%, 144%, 154% and 151%, data was reported based on most results flanked by those CCVs were BLOQ and the 6:2 FTS cultures analyzed gave results consistent with the dose levels; 3) two CCVs in the latter half of analytical run #8 for 6:2 FTCA were 147% and 143%, but flanked samples were all BLOQ; 4) two CCVs for 6:2 FTS in analytical run #8 were 152% and 131% and data flanked by those CCVs was reported and those data are flagged in the appropriate tables, and three other CCVs in that run for 6:2 FTS were 144%, 154% and 151% but flanked samples which were all < LLOQ and did not affect the reported results; 5) one calibration standard for 24-DCP in analytical run #12 was 126%; 6) one CCV in analytical run #26 was 69% for MeFBSE. Study Report: E05-0544 Page 26 of 101 3M ENVIRONMENTAL LABORATORY PROJECT NO. E05-0544 Table 6. Analytes and MS ions for LC/MS/MS and Direct Inject GC/MS Analyses (Study E05-0544) LC/MS/MS Analiysis Reference Material Abbreviation [a] Molecular Formula (anion shown) 2[(N-Methyl)perfluorobutanesulfonamido]ethanol; acetate adduct MeFBSE C4F9SO2N[(CH3)(CH2CH2OH)] CH3CO2 - 2[(N-methyl)perfluorobutanesulfonamido]acetate MeFBSAA C4F9SO2N[(CH3)(CH2CO2 -)] N-Methyl perfluorobutanesulfonamide MeFBSA C4F9SO2N(CH3) - Perfluorobutanesulfonamide FBSA C4F9SO2NH - ECF-Derived Perfluorobutane sulfinate Perfluorobutane sulfonate 2[(N-Ethyl)perfluorooctanesulfonamido]ethanol; acetate adduct 2[(N-Ethyl)perfluorooctanesulfonamido]acetate N-Ethyl perfluorooctanesulfonamide Perfluorooctanesulfonamide Perfluorooctane sulfinate PFBSi PFBS EtFOSE EtFOSAA EtFOSA FOSA PFOSi C4F9SO2 C4F9SO3 - C8F17SO2N[(CH2CH3)(CH2CH2OH)]CH3CO2 C8F17SO2N[(CH2CH3)(CH2CO2 -)] C8F17SO2N(CH2CH3) C8F17SO2NH C8F17SO2 - Perfluorinated Carboxylates (can be ECF derived or TM derived) Perfluorooctane sulfonate Perfluorobutanoate Perfluorohexanoate Perfluoroheptanoate Perfluorooctanoate PFOS PFBA PFHxA PFHpA PFOA C8F17SO3 C3F7CO2 C5F11CO2 C6F13CO2 C7F15CO2 - Internal Standards (ISs) Perfluorononanoate Trimethylsilylpropane sulfonate (IS) Perfluorohexanesulfonate (IS) PFNA TMSPS PFHS C8F17CO2 Si(CH3)3(C3H6SO3 -) C6F13SO3 - Parent Ion (m/z) 416 370 312 298 283 299 630 584 526 498 483 499 213 313 363 413 463 195 399 Daughter Ion (m/z) 59 219 69 78 219 80 59 169 65 78 219 80 169 or 119 269 319 369, 319 or 169 419 80 80 Study Report: E05-0544 Page 27 of 101 3M ENVIRONMENTAL LABORATORY PROJECT NO. E05-0544 Table 6. Analytes and MS ions for LC/MS/MS and Direct Inject GC/MS Analyses (Study E05-0544) LC/MS/MS Analiysis Reference Material Abbreviation [a] Molecular Formula (anion shown) Pentachlorophenol PCP C6Cl5O - Chlorinated Phenols 2356-Tetrachlorophenol 246-Trichlorophenol 24-Dichlorophenol 4-Chlorophenol 6:2 Fluorotelomer sulfonate 2356-TeCP 246-TCP 24-DCP 4-CP 6:2 FTS (a.k.a THPFOS) C6HCl4O C6H2Cl3O C6H3Cl2O C6H4ClO C6F13CH2CH2SO3- TM Derived 6:2-fluorotelomer carboxylate Unsaturated 6:2 Fluorotelomer carboxylate 6:2-FTCA 6:2 FTUCA C6F13CH2CO2 C5F11CFCHCO2 - Direct inject GC/MS Analysis 8:2-fluorotelomer carboxylate Unsaturated 8:2 Fluorotelomer carboxylate Analyte 8:2-FTCA 8:2 FTUCA Abbreviation C8F17CH2CO2 C7F15CFCHCO2 - Molecular Formula (anion) TM Derived 6:2 Fluorotelomer Alcohol 6:2 FTOH C6F13C2H4OH 8:2 Fluorotelomer Alcohol 8:2 FTOH C8F17C2H4OH [a] Naming of fluorinated compounds in this report is consistent with the terminology and classification set forth by Buck et al. 2011. Parent Ion (m/z) 263, 261 229,227 195, 197 161, 163 127, 129 427 377 357 Daughter Ion (m/z) 35 35 35 35 35 80 293 293 477 393 457 393 Quant Ion (m/z) 169, 295, 305, 314 (sum) 169, 395, 405, 414 (sum) Study Report: E05-0544 Page 28 of 101 3M ENVIRONMENTAL LABORATORY PROJECT NO. E05-0544 Table 7. Typical HPLC Mobile Phase Gradient for NG1-C8 method (E05-0544) Time (minutes) % A % B 0.00 3.0 97.0 0.50 3.0 97.0 5.00 95.0 5.0 9.00 95.0 5.0 9.01 3.0 97.0 12.00 3.0 97.0 Mobile Phase A equals 2 mM aqueous Ammonium Acetate Solution Mobile Phase B equals HPLC grade Methanol Flow rate was 1.00 mL/ minute Note: Holding times between 5-9 minutes and 9.01 and 12.00 minutes may have varied to accommodate later eluting peaks during analysis of some analytes on alternative column setups, but gradients always remained the same. Study Report: E05-0544 Page 29 of 101 3M ENVIRONMENTAL LABORATORY PROJECT NO. E05-0544 The analytical batches used to report or confirm results for this study are described in Table 8. Table 8. Study Analytical Batch Summary (Study E05-0544) R#un Purpose for Analysis Date of Analysis Analytical Run/Batch ID Instrument Name/Type Data Used for Reporting (Y/N) Analytes Reported Stan Data files\Projects\Anae robic Qualitative LC/MS & Biodeg\Anaerobic Stan (API 1 LC/MS/MS of cultures 25-Aug-2005 Screening 08-24- 4000) NO from set # 1 2005\Data None Anaerobic Set 08- 25-2005 Set #1.wiff Rufus Qualitative direct inject 2\MSDChem\1\Dat Rufus 2 2 GC/MS of cultures from 06-Sept-2005 a\Anaerobic (Agilent NO None set #1 Headspace06-sept- GC- MSD) 2005 Stan Data files\Projects\Anae robic Qualitative LC/MS/MS of Biodeg\Anaerobic Stan (API 3 cultures from set #1 08-Sept-2005 Screening 08-24- 4000) NO 2005\08-Sept- None 200508-Sept-2005 (C4s MRM) and (C8s MRM) Ollie Quantitative. LC/MS/MS data\Projects\E05- Ollie (API 4 EtFOSE cultures (set 2) 06-Oct-2005 0544\2005_10_06\ 4000 Q- NO days 0 to 29 E05-0544 EtFOSE Trap) Cultures & Blanks- None -06-OCT-2005 Ollie Quant LC/MS/MS data\Projects\E05- Ollie (API 5 MeFBSE cultures (set 2) 11-Oct-2005 0544\2005_10_06\ 4000 Q- days 0 to 29 E05-0544 MeFBSE Trap) Cultures & Blanks- -11-OCT-2005 MeFBSE, MeFBSAA, YES MeFBSA, FBSA, PFBSi, PFBS & PFBA Study Report: E05-0544 Page 30 of 101 3M ENVIRONMENTAL LABORATORY PROJECT NO. E05-0544 Table 8. Study Analytical Batch Summary (Study E05-0544) R#un Purpose for Analysis Date of Analysis Analytical Run/Batch ID Instrument Name/Type Data Used for Reporting (Y/N) Analytes Reported 6 Quant LC/MS/MS PFBS 19-Oct-2005 a051019a.spl Amelia YES cultures days 0, 7, 18 & 26 (Quattro II) PFBS & PFBA 7 Quant LC/MS/MS, PFOS 20-Oct-2005 a051020b.spl Amelia YES cultures days 0, 7, 18 & 26 (Quattro II) PFOS & PFOA Quant LC/MS/MS, 6:2 Amelia 8 FTS cultures days 0,7,18 & 25-Oct-2005 a051025a.spl (Quattro II) YES 26 9 Quant LC/MS/MS, PFBS 28-Oct-2005 a051028a.spl Amelia NO cultures day 26 (Quattro II) 6:2 FTS, 6:2 FTUCA & PFHxA None, confirmatory results only 10 Quant LC/MS/MS, PFOS 31-Oct-2005 a051031a.spl Amelia YES FOSA & PFOSi cultures day 26 (Quattro II) 11 Quant LC/MS/MS, PFOS 01-Nov-2005 a051101a.spl Amelia YES FOSA & PFOSi cultures day 0 (Quattro II) Quant LC/MS/MS, TCP Amelia 246-TCP, 12 cultures days 0, 5, 7, 15 & 08-Nov-2005 a051108a.spl (Quattro II) YES 24-DCP & 4-CP 47 Quant LC/MS/MS for PFOS, PFBS and 6:2 FTS Amelia 13 cultures (day 69), MeFBSE 22-Nov-2005 a051122a.spl (Quattro II) NO (day 70), and EtFOSE (day 72) None, data not used to simplify reporting Quant LC/MS/MS, Amelia 14 MeFBSE cultures (set 2) 23-Nov-2005 a051123a.spl (Quattro II) NO day 70 None, confirmatory results only Study Report: E05-0544 Page 31 of 101 3M ENVIRONMENTAL LABORATORY PROJECT NO. E05-0544 Table 8. Study Analytical Batch Summary (Study E05-0544) R#un Purpose for Analysis Date of Analysis Analytical Run/Batch ID Instrument Name/Type Data Used for Reporting (Y/N) Analytes Reported 15 Quant LC/MS/MS, PFBS 30-Nov-2005 culture day 69 Quant LC/MS/MS, PCP 16 cultures day 0, 3, 10 & 20 06-Dec-2005 and TCP cultures day 0, 5, 7, 15 and 47 Quant LC/MS/MS, 6:2 17 FTOH and 8:2 FTOH 14-Dec-2005 cultures days 0, 10 & 20 Quant LC/MS/MS, 6:2 18 FTOH and 8:2 FTOH 15-Dec-2005 cultures days 0, 10 & 20 Quant LC/MS/MS, PFOA 19 and PFBA cultures days 0, 19-Dec-2005 10 & 20 Quant LC/MS/MS, PFBS cultures (day 105) and 20 MeFBSE cultures (days 70 27-Dec-2005 and 108); and EtFOSE cultures (days 0,10,21,29,72 and 108) a051130a.spl a051206a.spl a051214a.spl 051215a.spl a051219a.spl a051227a.spl Amelia (Quattro II) Amelia (Quattro II) Amelia (Quattro II) Amelia (Quattro II) Amelia (Quattro II) Amelia (Quattro II) Quant LC/MS/MS, PFOS 21 cultures (day 69) and 6:2 29-Dec-2005 FTS cultures (days 69 and 105) a051229a.spl Amelia (Quattro II) YES PFBS, PFBSi, FBSA, PFBA None, NO confirmatory results only None; Data was accidentally NO deleted and lost- results reported from a051215a PFHxA, PFHpA, YES 6:2 FTCA & 6:2 FTUCA PFBA, PFHpA, YES PFOA, PFNA MeFBSE, MeFBSAA, MeFBSA, FBSA, PFBSi, PFBS & YES PFBA; EtFOSE, EtFOSAA, EtFOSA, FOSA, PFOSi, PFOS & PFOA PFOS, PFOSi, FOSA, PFOA; 6:2 YES FTS, 6:2 FTUCA, 6:2 FTCA, PFHxA Study Report: E05-0544 Page 32 of 101 3M ENVIRONMENTAL LABORATORY PROJECT NO. E05-0544 Table 8. Study Analytical Batch Summary (Study E05-0544) R#un Purpose for Analysis Date of Analysis Quant LC/MS/MS, 6:2 07-March- 22 FTOH cultures for days 74 2006 and 94 Quant LC/MS/MS, 8:2 08-March- 23 FTOH and PFOA cultures 2006 for days 74 and 94 Quantitative direct inject 24 GC/MS for 6:2 FTOH and 17-May-2006 8:2 FTOH for days, 0, 10, 20, 74 and 94 25 Quant LC/MS/MS, PFOS 14-July-2006 cultures for day 105 26 Quant LC/MS/MS, PFBA 01-August- cultures for day 74 and 94 2006 Quant LC/MS/MS of PCP 16-August- 27 cultures for days 0, 3, 10, 2006 20 & 74 Quantitative LC/MS/MS of 8:2 FTOH cultures (day 0, 28 10, 20, 74 & 94) and 18-August- Qualitative analysis of 6:2 2006 FTOH Cultures (day 74 and 94) Semi 29 quantitative/qualitative 03-Nov.-2005 Headspace GC/MS of day 66 MeFBSE Cultures Study Report: E05-0544 Page 33 of 101 Analytical Run/Batch ID A060307a.spl A060308a.spl R060517a.S A060714a.spl A060801b.spl A060816b.spl A060818a.spl Z051103.s Instrument Name/Type Data Used for Reporting (Y/N) Amelia YES (Quattro II) Analytes Reported 6:2 FTCA, 6:2 FTUCA, PFHxA and PFHpa Amelia YES (Quattro II) PFOA & PFNA Rufus (HP GC/MS) Amelia (Quattro II) Amelia (Quattro II) Amelia (Quattro II) Amelia (Quattro II) Zeke (Agilent GC- MSD) YES 6:2 FTOH & 8:2 FTOH PFOS, PFOSi, FOSA, PFOA, YES EtFOSE, EtFOSAA, EtFOSA PFBA, PFBSi, PFBS and FBSA [Also MeFBSE, YES MeFBSAA, MeFBSA were analyzed, data not used] PCP, 2356-TeCP, YES 246-TCP, 24-DCP and 4-CP PFHxA, PFHpA, YES PFOA, PFNA, 8:2 FTCA, 8:2 FTUCA YES Volatiles 3M ENVIRONMENTAL LABORATORY PROJECT NO. E05-0544 Table 8. Study Analytical Batch Summary (Study E05-0544) R#un Purpose for Analysis Date of Analysis Analytical Run/Batch ID Instrument Name/Type Data Used for Reporting (Y/N) Analytes Reported Semi quantitative/qualitative Headspace GC/MS of day Zeke 30 69 PFBS, PFOS and 6:2 03-Nov.-2005 Z051109.s (Agilent YES FTS cultures; and day 72 GC- MSD) bioactive blank and EtFOSE culture Volatiles Note: This table is a comprehensive list of all analyses conducted for this study. However, not all data was used because of either its qualitative nature or redundancy with other runs for the same samples where better data quality was achieved. Data quality objectives were not defined for this study, hence data was never rejected because of failure to attain specific data quality objectives and the quality of the data reported are specified in each analytical data set. Study Report: E05-0544 Page 34 of 101 3M ENVIRONMENTAL LABORATORY PROJECT NO. E05-0544 4 RESULTS & DISCUSSION The results reported herein were compiled from laboratory work conducted as part of a method development project for anaerobic methods and procedures needed to conduct GLP anaerobic biodegradability testing studies. The results of this discovery study provided early insight to the potential anaerobic biodegradability of several fluorochemical substrates (test substances), or the lack thereof for some. The cultures that were prepared utilized a rich anaerobic microbial inoculum from a municipal anaerobic digester sludge (Empire MN). The intent of this study was neither for regulatory submission nor to extrapolate potential rates of degradation in natural anaerobic environments, nor anaerobic wastewater treatment systems. However, these data do provide some insight into the potential fate of these substances in anaerobic environments in general. Degradation curve fits were performed manually using ACD Labs Chemsketch 2012 software (freeware version 14.01). Chemical structures shown in degradation pathway figures were also generated using ACD Labs ChemsketchTM 2012. 4.1 Anaerobic Culture Test Parameters Increases in culture gas volumes were determined after removal of the culture from the glove box on collection days and equilibration to ambient temperature. Changes in gas volume (V) in the headspace was determined by a syringe barrel displacement method described in section 3.5.1. The change in gas volume are plotted in Figures 3 and 4. It was apparent within the first week of incubation that gas volume had increased in all of the bioactivesludge cultures resulting in syringe barrel displacement values of approximately 5 mL to 7 mL, while no significant increase was realized in the sterile-sludge controls (not shown). Changes in gas volumes continued to rise throughout the experiment for the first three to four weeks in the bioactive cultures, after which the gas pressures appeared to plateau and then decreased during the remainder of the incubations. Increased gas volume was observed for bioactive cultures dosed with test substance versus bioactive blanks, and was assumed to be formed predominantly from the 5 L of methanol solvent used to deliver test substances and which would have been converted to additional methane and carbon dioxide; methanol was not added to bioactive blanks and therefore only reflect methane and carbon dioxide formed from organic carbon in sludge and dilute yeast extract solution. Semi-quantitative analysis by extractive gas-phase FTIR of headspace gases from day-26 and day-29 bioactive-sludge and sterile-sludge cultures for culture sets #3 and #2 (see section Study Report: E05-0544 Page 35 of 101 3M ENVIRONMENTAL LABORATORY PROJECT NO. E05-0544 3.2), respectively, showed methane and carbon dioxide were the primary gases in the headspaces of those bioactive cultures. Methane and carbon dioxide were not appreciably produced in sterile-sludge cultures. Additionally, the ratio of methane-to-carbon dioxide (approximately 4:1) was in good agreement with the ratio of methane and carbon dioxide expected for biogas (https://en.wikipedia.org). The gas-phase FTIR results for methane and carbon dioxide concentrations are presented in Table 9. No volatile fluorochemicals were observed in headspaces by gas-phase FTIR analysis. Additional analyses for volatile fluorochemicals at for day 66, 69 and day 72 for cultures from culture sets #2 and #3 by a sensitive headspace GC/MS method showed that no volatile FCs were formed from any of the fluorochemical test substances. However, the GC/MS data did show the presence of volatile hydrogen sulfide and alkylated sulfides in the headspace of active cultures, but not in sterile cultures (data not shown), and suggests the cultures likely contained sulfate-reducing microorganisms. V ( m L) Average Gas Volume Produced 8 7 6 L) 5 (m4 V 3 2 1 0 0 20 40 D 6a 0y s 80 100 120 Days Figure 3. The average change in gas volumes measured for all bioactive cultures (blank cultures and dosed cultures) from culture sets #2, #3, #4, and #5. (Data manually fit for appearance) Study Report: E05-0544 Page 36 of 101 3M ENVIRONMENTAL LABORATORY PROJECT NO. E05-0544 Table 9. Methane & Carbon Dioxide Concentrations by Gas-Phase FTIR Sample ID E05-0544-0028 E05-0544-0061 Description Water Blank, Day 29 Sterile-sludge Blank, Day 29 Methane (ppm) < MDL < MDL Carbon Dioxide (ppm) 1240 542 E05-0544-0049 Active-sludge Blank, Day 29 99,600 28,300 Gas Volume (V; mL) 0.0 0.0 5.0 E05-0544-0079 E05-0544-0094 EtFOSE, Day 29, Active-sludge EtFOSE, Day 29, Sterile-sludge 131,00 34,700 7.7 1,860 681 0 E05-0544-0109 E05-0544-0124 MeFBSE, Day 29, Active-sludge MeFBSE, Day 29, Sterile-sludge 131,000 1,830 25,100 < MDL 7.2 < MDL E05-0544-0139 E05-0544-0154 6:2 FTS, Day 26; Active-sludge 6:2 FTS, Day 26; Sterile-sludge 118,000 < MDL 36,300 1660 7.5 < MDL E05-0544-0199 E05-0544-0214 PFOS, Day 26; Active-sludge PFOS, Day 26; Sterile-sludge 119,000 1,810 35,300 2,720 6.9 < MDL E05-0544-0169 PFBS, Day 26; Active-sludge 124,000 41,100 7.4 E05-0544-0184 PFBS, Day 26; Sterile-sludge 786 1,660 < MDL Note: The minimum detection limit (MDL) for CO2 was 418 ppm, and the MDL for CH4 was 780 ppm by extractive gas phase FTIR. All values in the table are corrected for dilution factors applied during analysis and are shown to 3 significant figures only. Study Report: E05-0544 Page 37 of 101 3M ENVIRONMENTAL LABORATORY PROJECT NO. E05-0544 4.2 Anaerobic Biodegradation Testing Results 4.2.1 EtFOSE Results The test substance [2-(N-ethyl)-perfluorooctanesulfonamido]ethyl alcohol (EtFOSE) was dosed into anaerobic cultures at 1010 ng/mL (1.01 ppm) and incubated anaerobically for up to 108 days. Quantitative LC/MS/MS analysis of processed culture extracts was conducted for EtFOSE and the anticipated biodegradation products EtFOSAA, EtFOSA, FOSA, PFOSi, PFOS and PFOA. Data was collected from analytical run #20 (A051227a-2.spl) as described in Table 10. While internal standard PFHS peaks were integrated and included in raw data results, quantitation was performed by external standard method for all target analyte and the IS was not used for quantitation. Calibration curves contained six or more calibration standards. Curve fits were quadratic with either 1/x or 1/x^2 weighting. The coefficient of determination (R) values for all calibration curves were > 0.990. The lower limit of quantitation (LLOQ) for each analyte were as follows: PFOA (7.50 ng/mL), PFOSi (1.00 ng/mL), PFOS (7.50 ng/mL), FOSA (7.50 ng/mL), EtFOSA (7.50 ng/mL), EtFOSAA (4.00 ng/mL) and EtFOSE (1.00 ng/mL). Quantitative accuracy for all analytes was within 100+ 25% for calibration standards and QCs (used as analytical run CCVs). While data from analytical run #4 for analysis of EtFOSE day 0 to day 29 cultures was technically usable, it was not used for reporting and only served as confirmation of results for the entire culture set day 0 to day 108 determined in the single analytical run #20. The LC/MS/MS analysis showed that EtFOSAA and PFOSi were produced at measurable levels in bioactive cultures, but not in sterile cultures. Both products steadily increased in concentration over the course of the 108 days of incubation. The maximum concentration of EtFOSAA reached 21.9 ng/mL at day 72, and PFOSi maximum value was reached at 4.58 ng/mL on day 72. The EtFOSE concentration in active cultures was not significantly reduced, suggesting the observed biotransformation was very slow and was likely the only reactions taking place. Furthermore, analysis of active and sterile culture headspaces at day72 of incubations by a sensitive GC/MS assay showed that volatile fluorocarbon products were not generated. At day-72, the time point with the maximal measured total product, there was 235 pmole of total product (EtFOSAA+ PFOSi), from a theoretical 8,844 pmole of EtFOSE dosed on day 0. This accounts for a maximum biotransformation of 2.66 mole% of the dosed EtFOSE. The average concentration of products at day-108 appeared to be less than at Study Report: E05-0544 Page 38 of 101 3M ENVIRONMENTAL LABORATORY PROJECT NO. E05-0544 day 72 and was likely an artifact of the analysis. Anticipated products EtFOSA, FOSA, PFOS and PFOA were not formed from anaerobic biodegradation of EtFOSE. The semi-quantitative analysis of culture headspace for the day 72 EtFOSE-dosed bioactive culture E05-0544-083 was performed using a sensitive GC/MS assay in analytical run Z051109.s and showed that volatile fluorocarbons were not formed from the anaerobic biodegradation of EtFOSE. Plots showing the measured concentrations for EtFOSE and the anaerobic transformation products N-EtFOSAA and PFOSi during 108 days of incubation are shown in Figure 4. Analysis of sterile culture extracts showed that EtFOSAA and PFOSi were not formed in sterile cultures (not shown). The measured concentrations of EtFOSE and anticipated products for active and sterile cultures are presented in Table 10. C o n c e n tr a ti o n ( n g / m L ) 1600 1400 1200 itle 1000 isT 800 Ax 600 400 200 0 0 A EtFOE St EF O-SS tE e- S rt ieler i l Ce uC lu tl ut u rr ee 20 40 60 80 AxiD sa Ty its le 100 120 C o n c e n tr a ti o n ( n g / m L ) 1600 1400 1200 itle 1000 isT 800 Ax 600 400 200 0 0 B E t F O S EE - tBFi Oo a Sc Et i v e C u l t u r e 20 40 60 80 100 120 AxiD sa Ty its le C o n c e n tr a ti o n ( n g / m L ) 25 L) 20 g/m (n15 tion tra 10 cen on C 5 0 0 C E t F O SEA tFA O- B Si o Aa Ac t i v e C u l t u r e 5 4.5 C o n c e n tr a ti o n ( n g / m L) 4 3.5 itle 3 isT 2.5 Ax 2 1.5 1 0.5 0 D 20 40 60 80 100 120 0 DD aa yy ss P F O S i P- BFi Oo a Sc it i v e C u l t u r e 20 40 60 80 100 120 D ays Axis Title Figure 4. Measured EtFOSE, EtFOSAA and PFOSi concentrations in sterile cultures (A) and bioactive cultures (B, C &D), each dosed with EtFOSE. Data manually fit for appearance of trend. Study Report: E05-0544 Page 39 of 101 3M ENVIRONMENTAL LABORATORY PROJECT NO. E05-0544 EtFOSE biotransformed anaerobically at a very slow rate and could not be discerned by measurement of EtFOSE concentrations. However, it was discernible from formation of measureable levels of anticipated products EtFOSAA and PFOSi. The rate of product formation (summed for both products) appeared to be linear over the first 29 days and based on assumed zero-order rate gave an initial degradation rate of 4.11 pmole/day (Figure 5). An initial sludge-dependent biotransformation rate was calculated at 0.0718 pmole/day/mg sludge (dw). Based on the determined initial zero-order degradation rate, a zero-order halflife (t1/2) of 1077 days was determined for EtFOSE; the t1/2 does not take into consideration the fact that degradation was pseudo-first order over the length of the incubation, thus the half-life is likely underestimated. Values used for converting ng/mL quantities of analytes to pmole quantities and determination of rates and mass balance were as describe in Appendix A. Figure 5. Linear plot of total products formed (pmole) from EtFOSE for the first 29 days. Study Report: E05-0544 Page 40 of 101 3M ENVIRONMENTAL LABORATORY PROJECT NO. E05-0544 Table 10. Analyte concentrations measured in bioactive and sterile anaerobic cultures incubated with EtFOSE. Culture ID E05-0544-070 E05-0544-071 E05-0544-072 E05-0544-073 E05-0544-074 E05-0544-075 E05-0544-076 E05-0544-077 E05-0544-078 E05-0544-079 E05-0544-080 E05-0544-081 E05-0544-083 E05-0544-082 E05-0544-084 E05-0544-085 E05-0544-086 E05-0544-087 E05-0544-088 E05-0544-089 E05-0544-090 Culture Type Bioactive Sludge w/ EtFOSE Bioactive Sludge w/ EtFOSE Bioactive Sludge w/ EtFOSE Bioactive Sludge w/ EtFOSE Bioactive Sludge w/ EtFOSE Bioactive Sludge w/ EtFOSE Bioactive Sludge w/ EtFOSE Bioactive Sludge w/ EtFOSE Bioactive Sludge w/ EtFOSE Bioactive Sludge w/ EtFOSE Bioactive Sludge w/ EtFOSE Bioactive Sludge w/ EtFOSE Bioactive Sludge w/ EtFOSE Bioactive Sludge w/ EtFOSE Bioactive Sludge w/ EtFOSE Sterile Sludge w/ EtFOSE Sterile Sludge w/ EtFOSE Sterile Sludge w/ EtFOSE Sterile Sludge w/ EtFOSE Sterile Sludge w/ EtFOSE Sterile Sludge w/ EtFOSE Incubation Time (days) 0 0 0 10 10 10 21 21 21 29 29 29 72 108 108 0 0 0 10 10 10 EtFOSE 977 865 879 881 1130 999 1040 1350 925 1220 1130 1100 1250 1140 681 1100 849 851 1040 645 883 EtFOSAA <4.00 <4.00 <4.00 3.40 [a] 4.08 4.17 7.98 7.19 5.92 11.7 11.4 10.5 21.9 22.1 13.3 <4.00 <4.00 <4.00 <4.00 <4.00 <4.00 Concentration (ng/mL) EtFOSA <7.50 <7.50 <7.50 <7.50 <7.50 <7.50 <7.50 <7.50 <7.50 <7.50 <7.50 <7.50 <7.50 <7.50 <7.50 <7.50 <7.50 <7.50 <7.50 <7.50 <7.50 FOSA <7.50 <7.50 <7.50 <7.50 <7.50 <7.50 <7.50 <7.50 <7.50 <7.50 <7.50 <7.50 <7.50 <7.50 <7.50 <7.50 <7.50 <7.50 <7.50 <7.50 <7.50 PFOSi <1.00 <1.00 <1.00 <1.00 <1.00 <1.00 <1.00 1.84 2.16 3.06 2.13 2.25 4.58 3.35 1.40 <1.00 <1.00 <1.00 <1.00 <1.00 <1.00 PFOS <7.50 <7.50 <7.50 <7.50 <7.50 <7.50 <7.50 <7.50 <7.50 <7.50 <7.50 <7.50 <7.50 <7.50 <7.50 <7.50 <7.50 <7.50 <7.50 <7.50 <7.50 PFOA <7.50 <7.50 <7.50 <7.50 <7.50 <7.50 <7.50 <7.50 <7.50 <7.50 <7.50 <7.50 <7.50 <7.50 <7.50 <7.50 <7.50 <7.50 <7.50 <7.50 <7.50 Study Report: E05-0544 Page 41 of 101 3M ENVIRONMENTAL LABORATORY PROJECT NO. E05-0544 Table 10. Analyte concentrations measured in bioactive and sterile anaerobic cultures incubated with EtFOSE. Culture ID Culture Type Incubation Time (days) EtFOSE EtFOSAA Concentration (ng/mL) EtFOSA FOSA PFOSi PFOS PFOA E05-0544-091 Sterile Sludge w/ EtFOSE 21 950 <4.00 <7.50 <7.50 <1.00 <7.50 <7.50 E05-0544-092 Sterile Sludge w/ EtFOSE 21 1240 <4.00 <7.50 <7.50 <1.00 <7.50 <7.50 E05-0544-093 Sterile Sludge w/ EtFOSE 21 852 <4.00 <7.50 <7.50 <1.00 <7.50 <7.50 E05-0544-094 Sterile Sludge w/ EtFOSE 29 939 <4.00 <7.50 <7.50 <1.00 <7.50 <7.50 E05-0544-095 Sterile Sludge w/ EtFOSE 29 877 <4.00 <7.50 <7.50 <1.00 <7.50 <7.50 E05-0544-096 Sterile Sludge w/ EtFOSE 29 905 <4.00 <7.50 <7.50 <1.00 <7.50 <7.50 E05-0544-099 Sterile Sludge w/ EtFOSE 72 924 <4.00 <7.50 <7.50 <1.00 <7.50 <7.50 E05-0544-097 Sterile Sludge w/ EtFOSE 108 752 <4.00 <7.50 <7.50 <1.00 <7.50 <7.50 E05-0544-098 Sterile Sludge w/ EtFOSE 108 1390 <4.00 <7.50 <7.50 <1.00 <7.50 <7.50 Notes: Results for the EtFOSE-dosed samples were reported from analytical run #20 of Table 8. Analysis included a set of matrix matched calibration standards (140568-045A01 to -045A11). Quantitation was by external standard method. Curve fits were quadratic with 1/x weighting, except for PFOS which was weighted 1/x2. R2 values for all curves were > 0.99. A low-level and mid-level QC at 40.0 ng/mL and 400 ng/mL (140568-45A12 and -45A13), respectively, were injected approximately every 15 samples as CCVs during the analysis. Quantitative accuracy for all analytes was within 100+ 25% for standards (+ 30% at LLOQ) and within 100 + 30% for QCs (treated as CCVs throughout the run. All values in Table 10 are reported to three significant figures but higher precision data used in calculations. Associated bioactive and sterile sludge blanks analyzed with the samples were all below the LLOQ for all analytes [a] Value was determined by extrapolation of the curve slightly below the LLOQ of 4.00 ng/mL but within 75% of LLOQ acceptance range. Study Report: E05-0544 Page 42 of 101 3M ENVIRONMENTAL LABORATORY PROJECT NO. E05-0544 4.2.2 MeFBSE Results The test substance [2-(N-methyl)-perfluorobutanesulfonamido]ethyl alcohol (MeFBSE) was dosed into anaerobic cultures at 976 ng/mL (2.73 M) and then incubated anaerobically for up to 108 days. Cultures were extracted for days 0, 10, 21, 29, 70)and 108. Quantitative LC/MS/MS analysis of processed culture extracts was conducted for MeFBSE, MeFBSAA, MeFBSA, FBSA, PFBSi, PFBS and PFBA. Data was collected from two analytical runs #5 and #20 as described in Table 8. Quantitation was by internal standard method for all analytes. Calibration curves contained six or more calibration standards, except PFBA in run #20 which had only 5 active points in the curve as footnoted in Table 8. The coefficient of determination (R) values for all calibration curves were > 0.990. The lower limit of quantitation (LLOQ) for each analyte were always < 25.0 ng/mL. Calibration standards included in the calibration curve all were within 100 + 25% and QCs/CCVs were all within 100 + 30%, except one QC analyzed as a CCV for PFBSi in analytical run #5 at 145%--but did not affect the reported PFBSi results since all samples flanked by that QC were below the LLOQ. The LC/MS/MS results showed that MeFBSE was biodegraded with approximately 75% removed by day-72 (Figure 6) with concomitant formation of MeFBSAA and PFBSi as shown in Figure 7. Other anticipated products as MeFBSA, FBSA, PFBS and PFBA were not detected above the LLOQ during the 108 days of incubation (Table 11). MeFBSE biodegradation occurred with near equimolar production of MeFBSAA and PFBSi (summed), with mass balance calculated at 103%, 91.3%, 93.7%, 99.0%, 91.4% and 120% for cultures collected on days 0, 10, 21, 29, 70 and 108, respectively. The MeFBSE was not degraded in sterile controls, nor were any products formed. The semi-quantitative analysis of the headspace gas for the MeFBSE-dosed bioactive and sterile cultures (E05-0544-112 and -129, respectively) occurred on day 66, and was performed using a sensitive GC/MS assay in analytical run Z051103.s. Results of headspace analysis showed that volatile fluorocarbons were not formed from the anaerobic biodegradation of MeFBSE. After the headspace analysis, those cultures sat on the bench top for ~3 days prior to being extracted for LC/MS/MS analysis and likely continued to degrade while stored on the lab bench, and is the reason why LC/MS/MS results indicate those culture times as day 70 rather than day 66. Study Report: E05-0544 Page 43 of 101 3M ENVIRONMENTAL LABORATORY PROJECT NO. E05-0544 MeFBM Se EF BBS iEoBai co ta icvt iev e 1200 MeFBSM Ee F SB tS eE rSilt ee r i l e 1200 C o n c e n tra tio n (n g / m L ) C o n c e n tra tio n (n g / m L ) 1000 1000 itle 800 itle 800 isT 600 isT 600 Ax Ax 400 400 200 200 0 0 20 40 60 80 100 120 D ays Axis Title 0 0 20 40 60 80 100 120 D ays Axis Title Figure 6. The MeFBSE (a.k.a. N-MeFBSE Alcohol) concentrations in active cultures (left) and sterile cultures (right). Data manually fit for appearance. C o n c e n tra tio n (n g / m L ) MM ee FF BBS SA AA AB i o a c t i v e P F BPS Fi BB iSo ia c t i v e 700 400 C o n c e n tra tio n (n g / m L ) 600 350 500 300 itle400 itle250 isT isT200 Ax 300 Ax 150 200 100 100 50 0 0 20 40 60 80 100 120 AxDisa Ty its le 0 0 20 40 60 80 100 120 AxD isa yTsitle Figure 7. The MeFBSAA and PFBSi concentrations measured in active cultures dosed with MeFBSE (red circles). Data manually fit for appearance. The formation of total products in pmole over the first 29 days (days 0, 10, 21 and 29) of incubation was linear and the slope of the fitted line was used to estimate a zero-order rate of degradation at 186 pmole/day (Figure 8). Based on that rate, a sludge-dependent Study Report: E05-0544 Page 44 of 101 3M ENVIRONMENTAL LABORATORY PROJECT NO. E05-0544 biodegradation rate for MeFBSE was determined at 3.26 pmole/day/mg sludge (dw) for MeFBSE. Based on the determined initial zero-order degradation rate, a zero-order half-life (t1/2) of 36.7 days was determined for EtFOSE; the t1/2 does not take into consideration the fact that degradation was pseudo-first order over the length of the incubation, thus the halflife value is likely underestimated. Values used for converting ng/mL quantities of analytes to pmole quantities and determination of rates and mass balance were as describe in Appendix A. Figure 8. Plot of MeFBSE total products formation over first 29 days. Note (MeFBSE Results): measured concentrations of PFBSi were determined using standards prepared from a reference substance which was assumed at 50% (w/v) in water at the time of preparation, but later this was shown by NMR analysis to be actually 51.2% (w/v) in water; however, measured values were not corrected for the 1.2% difference. Therefore, PFBSi values could be biased slightly low as a result, hence rates may be biased slightly low also. Study Report: E05-0544 Page 45 of 101 3M ENVIRONMENTAL LABORATORY PROJECT NO. E05-0544 Table 11. Analyte concentrations measured in active and sterile cultures incubated with MeFBSE Culture ID Culture Type Incubation Time (days) MeFBSE MeFBSAA Concentration (ng/mL) MeFBSA FBSA [s] PFBSi [b] E05-0544-100 Active Sludge w/ MeFBSE 0 926 <10.0 <10.0 <10.0 16.3 E05-0544-101 Active Sludge w/ MeFBSE 0 982 <10.0 <10.0 <10.0 16.3 E05-0544-102 Active Sludge w/ MeFBSE 0 1050 <10.0 <10.0 <10.0 18.0 E05-0544-103 Active Sludge w/ MeFBSE 10 738 140 <10.0 <10.0 20.6 E05-0544-104 Active Sludge w/ MeFBSE 10 672 126 <10.0 <10.0 13.8 E05-0544-105 Active Sludge w/ MeFBSE 10 803 147 <10.0 <10.0 25.2 E05-0544-106 Active Sludge w/ MeFBSE 21 593 266 <10.0 <10.0 31.8 E05-0544-107 Active Sludge w/ MeFBSE 21 588 259 <10.0 <10.0 24.7 E05-0544-108 Active Sludge w/ MeFBSE 21 629 305 <10.0 <10.0 63.0 E05-0544-109 Active Sludge w/ MeFBSE 29 606 374 <10.0 <10.0 37.5 E05-0544-110 Active Sludge w/ MeFBSE 29 569 353 <10.0 <10.0 38.4 E05-0544-111 Active Sludge w/ MeFBSE 29 559 370 <10.0 <10.0 39.3 E05-0544-112 Active Sludge w/ MeFBSE 70 [e] 241 423 <25.0 <25.0 205 E05-0544-113 Active Sludge w/ MeFBSE 108 251 555 <25.0 <25.0 291 E05-0544-114 Active Sludge w/ MeFBSE 108 237 598 <25.0 <25.0 335 E05-0544-115 Sterile Sludge w/ MeFBSE 0 975 <10.0 <10.0 <10.0 <5.00 E05-0544-116 Sterile Sludge w/ MeFBSE 0 1070 <10.0 <10.0 <10.0 <5.00 E05-0544-117 Sterile Sludge w/ MeFBSE 0 1000 <10.0 <10.0 <10.0 <5.00 E05-0544-118 Sterile Sludge w/ MeFBSE 10 990 <10.0 <10.0 <10.0 <5.00 E05-0544-119 Sterile Sludge w/ MeFBSE 10 979 <10.0 <10.0 <10.0 <5.00 E05-0544-120 Sterile Sludge w/ MeFBSE 10 910 <10.0 <10.0 <10.0 <5.00 E05-0544-121 Sterile Sludge w/ MeFBSE 21 766 <10.0 <10.0 <10.0 <5.00 E05-0544-122 Sterile Sludge w/ MeFBSE 21 848 <10.0 <10.0 <10.0 <5.00 E05-0544-123 Sterile Sludge w/ MeFBSE 21 863 <10.0 <10.0 <10.0 <5.00 E05-0544-124 Sterile Sludge w/ MeFBSE 29 934 <10.0 <10.0 <10.0 <5.00 PFBS <10.0 <10.0 <10.0 <10.0 <10.0 <10.0 <10.0 <10.0 <10.0 <10.0 <10.0 <10.0 <25.0 <25.0 <25.0 <10.0 <10.0 <10.0 <10.0 <10.0 <10.0 <10.0 <10.0 <10.0 <10.0 PFBA [c] <10.0 <10.0 <10.0 <10.0 <10.0 <10.0 <10.0 <10.0 <10.0 <10.0 <10.0 <10.0 <25.0 <25.0 <25.0 <10.0 <10.0 <10.0 <10.0 <10.0 <10.0 <10.0 <10.0 <10.0 <10.0 Study Report: E05-0544 Page 46 of 101 3M ENVIRONMENTAL LABORATORY PROJECT NO. E05-0544 Table 11. Analyte concentrations measured in active and sterile cultures incubated with MeFBSE Culture ID Culture Type Incubation Time (days) MeFBSE MeFBSAA Concentration (ng/mL) MeFBSA FBSA [s] PFBSi [b] PFBS PFBA [c] E05-0544-125 Sterile Sludge w/ MeFBSE 29 863 <10.0 <10.0 <10.0 <5.00 <10.0 <10.0 E05-0544-126 Sterile Sludge w/ MeFBSE 29 947 <10.0 <10.0 <10.0 <5.00 11.9 [d] <10.0 E05-0544-129 Sterile Sludge w/ MeFBSE 70 [e] 837 <25.0 <25.0 <25.0 <12.5 <25.0 <25.0 E05-0544-127 Sterile Sludge w/ MeFBSE 108 944 <25.0 <25.0 <25.0 <12.5 <25.0 <25.0 E05-0544-128 Sterile Sludge w/ MeFBSE 108 968 <25.0 <25.0 <25.0 <12.5 <25.0 <25.0 Notes: Results for the MeFBSE-dosed samples were reported from analytical runs #5 and #20 in Table 8. Culture extracts were analyzed against a set of matrix matched calibration standards. All curves contained minimally 6 data points and were quadratic fit with 1/x weighting, except for PFBS in run #20 which was weighted 1/x2. Quantitation was by internal standard method. All calibration standards in calibration curves were within 100+25% (+ 30% at LLOQ) of their theoretical value and CCVs (low and mid-level QCs at 40.0 ng/mL and 400 ng/mL) were injected every 10 to 15 samples and were within 100+30% of their theoretical values for all analytes, except one QC analyzed as a CCV for PFBSi in analytical run #5 which recovered at 145% but did not affect the reported PFBSi results since all samples flanked by that QC were below the LLOQ. All values in table are reported to 3 significant figures while more precise data was used in data calculations. Associated bioactive and sterile sludge blanks analyzed with the samples were all below the LLOQ for all analytes [a] Purity of FBSA was assumed 100% at the time of use, but determined later at 97.3% and was not corrected for. [b] Purity of PFBSi was assumed 50% at the time of use, but later determined to by 51.2% and was not corrected for. [c] PFBA quantification in analytical run #20 included only 5 calibration standards in the curve with mid-points dropped. Data not affected since all results were < LLOQ. [d] An aberrant PFBS result in the day 70 sterile culture was slightly above the LLOQ of 10 ng/mL but ignored for interpretation of results since PFBS was not observed in bioactive cultures; possibly derived from the sludge as background. [e] Culture was collected on day 66 for headspace analysis, but sat out on bench for additional ~4 days prior to LC/MS/MS analysis, hence was designated as a day 70 culture. Study Report: E05-0544 Page 47 of 101 3M ENVIRONMENTAL LABORATORY PROJECT NO. E05-0544 4.2.3 PFOS Results The test substance perfluorooctanesulfonate (PFOS) was dosed into anaerobic cultures at 1060 ng/mL (2.12 M) and cultures incubated anaerobically for up to 105 days. Cultures were collected on days 0, 7, 18, 26, 69 and 105. Quantitative LC/MS/MS analysis of culture extracts was conducted for PFOS and the anticipated products PFOSi, FOSA and PFOA. Quantitative LC/MS/MS analysis of processed culture extracts was conducted for PFOS, FOSA, PFOSi and PFOA. Data for reporting results of PFOS cultures were collected in analytical runs #7, #10, #11, #21 and #25 in Table 8. Results are shown in Table 12. Cultures for day-7 and day-18 were not determined for PFOSi or FOSA. The lower limits of quantitation (LLOQ) varied by batch for each analyte, but were always < 25.0 ng/mL; LLOQs are indicated in Table 12. Calibration standards included in the calibration curve were determined at within 100 + 25%, QCs analyzed as CCVs in the run were recovered within 100 + 30%. The results showed that PFOS did not biodegrade under anaerobic methanogenic sludge conditions for up to 105 days. This was demonstrated by the lack of any appreciable loss of PFOS (Figure 9) and the lack of formation for any fluorinated products PFOA, PFOSi or FOSA. The semi-quantitative analysis of culture headspace for the day 69 PFOS-dosed bioactive culture E05-0544-204 was performed using a sensitive GC/MS assay in analytical run #30 of Table 8 and showed that volatile fluorocarbons were not formed from anaerobic sludge incubations with PFOS. In addition to not degrading, PFOS did not appear to inhibit methanogenisis since gas production seemed comparable in bioactive blanks as in bioactive PFOS cultures (data not shown). The results of this study showed PFOS does not biodegrade under methanogenic conditions. These results corroborate the findings of Hollingsworth et al. (2005) who similarly found that anaerobic biodegradation of PFOS did not occur under methanogenic conditions during a 45 day trial. Those authors also concluded that inhibition of methanogenesis did not occur at up to 600 g/mL of PFOS. Study Report: E05-0544 Page 48 of 101 C o n c e n tra tio n (n g / m L ) C o n c e n tra tio n (n g / m L ) 3M ENVIRONMENTAL LABORATORY PROJECT NO. E05-0544 ChP aF rO tS TB ii to laec t i v e 1400 ChP aF OrtS TS tite lr ei l e 1400 1200 1200 1000 1000 itle 800 T itle 800 T xis 600 xis 600 A A 400 400 200 200 0 0 20 40 6D 0a y s 80 100 120 Axis Title 0 0 20 40 60 80 100 120 D ays Axis Title Figure 9. The PFOS concentrations measured in active cultures (left) and sterile cultures (right) dosed with PFOS. One apparent double spiked sterile culture was omitted (see Table 12). The line represents the dosed concentration of 1060 ng/mL Study Report: E05-0544 Page 49 of 101 3M ENVIRONMENTAL LABORATORY PROJECT NO. E05-0544 Table 12. Analyte concentrations measured in active and sterile anaerobic cultures dosed with PFOS. Culture ID E05-0544-190 Culture Type Active Sludge with PFOS Incubation Time (days) 0 FOSA < 10.0 Concentration (ng/mL) PFOS PFOSi 1140 < 10.0 PFOA < 25.0 E05-0544-191 Active Sludge with PFOS 0 < 10.0 1020 < 10.0 < 25.0 E05-0544-192 Active Sludge with PFOS 0 < 10.0 1060 < 10.0 < 25.0 E05-0544-193 Active Sludge with PFOS 7 Not Determined 1120 Not Determined < 25.0 E05-0544-194 Active Sludge with PFOS 7 Not Determined 1070 Not Determined < 25.0 E05-0544-195 Active Sludge with PFOS 7 Not Determined 1100 Not Determined < 25.0 E05-0544-196 Active Sludge with PFOS 18 Not Determined 1160 Not Determined < 25.0 E05-0544-197 Active Sludge with PFOS 18 Not Determined 1200 Not Determined < 25.0 E05-0544-198 Active Sludge with PFOS 18 Not Determined 1160 Not Determined < 25.0 E05-0544-199 Active Sludge with PFOS 26 < 10.0 1220 < 10.0 < 25.0 E05-0544-200 Active Sludge with PFOS 26 < 10.0 1110 < 10.0 < 25.0 E05-0544-201 Active Sludge with PFOS 26 < 10.0 1120 < 10.0 < 25.0 E05-0544-202 Active Sludge with PFOS [a] 105 < 10.0 1160 < 10.0 < 10.0 E05-0544-203 Active Sludge with PFOS [a] 105 < 10.0 966 < 10.0 < 10.0 E05-0544-204 Active Sludge with PFOS 69 < 4.00 1050 < 10.0 < 4.00 E05-0544-205 Sterile Sludge with PFOS 0 < 10.0 802 < 10.0 < 25.0 E05-0544-206 Sterile Sludge with PFOS 0 E05-0544-207 Sterile Sludge with PFOS 0 < 10.0 < 10.0 1770 [double spiked] 836 < 10.0 < 10.0 < 25.0 < 25.0 E05-0544-208 Sterile Sludge with PFOS 7 Not Determined 1120 Not Determined < 25.0 E05-0544-209 Sterile Sludge with PFOS 7 Not Determined 1030 Not Determined < 25.0 E05-0544-210 Sterile Sludge with PFOS 7 Not Determined 1010 Not Determined < 25.0 E05-0544-211 Sterile Sludge with PFOS 18 Not Determined 1030 Not Determined < 25.0 E05-0544-212 Sterile Sludge with PFOS 18 Not Determined 1010 Not Determined < 25.0 E05-0544-213 Sterile Sludge with PFOS 18 Not Determined 1020 Not Determined < 25.0 E05-0544-214 Sterile Sludge with PFOS 26 < 10.0 1010 < 10.0 < 25.0 E05-0544-215 Sterile Sludge with PFOS 26 < 10.0 987 < 10.0 < 25.0 E05-0544-216 Sterile Sludge with PFOS 26 < 10.0 962 < 10.0 < 25.0 E05-0544-219 Sterile Sludge with PFOS 69 < 4.00 1390 < 1.00 < 4.00 E05-0544-217 Sterile Sludge with PFOS [a] 105 < 10.0 973 < 10.0 < 10.0 E05-0544-218 Sterile Sludge with PFOS [a] 105 < 10.0 887 < 10.0 < 10.0 Note: Results for the PFOS-dosed samples were reported from analytical runs # 7, #10, #11, #21 and #25 in Table 8. Culture extracts were analyzed against a set of matrix matched calibration standards. Quantitation was by external standard method, except PFOA and PFOS in run # 7 which were quantified by internal standard method. Calibration curves contained minimally six data points with quadratic fit and 1/x weighting, except PFOS in run #21 and #25 which was weighted 1/x2. Calibration accuracy for standards included in the curve fit were within 100 + 25% (+ 30% at LLOQ). CCVs (low and mid-level QCs at 40.0 ng/mL and 400 ng/mL, respectively) were injected approximately every 15 samples and were within 100+30% of their theoretical values for all analytes. LLOQs were always < 25.0 ng/mL. All values in the table are reported to 3 significant figures while more precise data was used in data calculations. Associated bioactive and sterile sludge blanks analyzed with the samples were all below the LLOQ for all analytes [a] Culture extracts were also analyzed in run #25 for EtFOSE, EtFOSAA and EtFOSA, but results excluded from table for brevity since those analytes were not expected products of PFOS degradation and were < 10.0 ng/mL in all samples. Study Report: E05-0544 Page 50 of 101 3M ENVIRONMENTAL LABORATORY PROJECT NO. E05-0544 4.2.4 PFBS Results The test substance perfluorobutanesulfonate (PFBS) was dosed into cultures at 990 ng/mL and incubated anaerobically for up to 105 days. Quantitative LC/MS/MS analysis of processed culture extracts was conducted for PFBS and anticipated biodegradation products FBSA, PFBSi and PFBA. Cultures analyzed were collected on days 0, 7, 18, 26, 69 and 105. Cultures for day-0, day-7, day-18 and day-26 were not measured for PFBSi nor FBSA. The LLOQ for all analytes were always < 25.0 ng/mL. Data is reported from three analytical runs as described at the bottom of Table 13. PFBS concentrations measured in active cultures and sterile cultures are plotted in Figure 10. Concentrations of PFBS for day-0 sterile control cultures appeared low, but returned to near expected levels by day 7. The semi-quantitative analysis of culture headspace for the day 69 PFBS-dosed bioactive culture E05-0544-174 in analytical run #30 of Table 8 and showed that volatile fluorocarbons were not significantly formed from the anaerobic biodegradation of PFBS. Overall, the results showed that PFBS did not biodegrade anaerobically over 105 days under methanogenic conditions. No anticipated products were measured above the LLOQ in any cultures and PFBS was recovered at near 100% at all time points. Furthermore, analysis of active and sterile culture headspaces at day 69 of incubations by a sensitive GC/MS assay showed no generation of volatile fluorocarbons, but production of sulfide compounds was observed in active cultures. The PFBS concentrations determined by LC/MS/MS in active and sterile cultures are plotted in Figure 10. Study Report: E05-0544 Page 51 of 101 3M ENVIRONMENTAL LABORATORY PROJECT NO. E05-0544 P F CB ShaB i ro ta Tc t ii tv leeC u l t u r e s 1200 P F B S S t e rile C u lt u r e s PFBS Sterile 1200 C o n c e n tra tio n (n g / m L ) C o n c e n tra tio n (n g / m L ) 1000 1000 itle 800 itle 800 isT 600 isT 600 Ax Ax 400 400 200 200 0 0 20 40 D a60y s 80 100 120 Axis Title 0 0 20 40 D 60a y s 80 100 120 Axis Title Figure 10. The PFBS concentrations measured in PFBS-dosed bioactive cultures (left) and sterile cultures (right). The line represents the dosed concentration of 990 ng/mL. Table 13. Analyte concentrations measured in active and sterile anaerobic cultures dosed with PFBS. Culture ID Culture Type Incubation Time (days) FBSA [a] Concentration (ng/mL) PFBS PFBSi [b] PFBA E05-0544-160 Active Sludge with PFBS 0 Not Determined 908 Not Determined < 10.0 E05-0544-161 Active Sludge with PFBS 0 Not Determined 823 Not Determined < 10.0 E05-0544-162 Active Sludge with PFBS 0 Not Determined 868 Not Determined < 10.0 E05-0544-163 Active Sludge with PFBS 7 Not Determined 927 Not Determined < 10.0 E05-0544-164 Active Sludge with PFBS 7 Not Determined 977 Not Determined < 10.0 E05-0544-165 Active Sludge with PFBS 7 Not Determined 979 Not Determined < 10.0 E05-0544-166 Active Sludge with PFBS 18 Not Determined 966 Not Determined < 10.0 E05-0544-167 Active Sludge with PFBS 18 Not Determined 928 Not Determined < 10.0 E05-0544-168 Active Sludge with PFBS 18 Not Determined 995 Not Determined < 10.0 E05-0544-169 Active Sludge with PFBS 26 Not Determined 969 Not Determined < 10.0 E05-0544-170 Active Sludge with PFBS 26 Not Determined 998 Not Determined < 10.0 E05-0544-171 Active Sludge with PFBS 26 Not Determined 1000 Not Determined < 10.0 Study Report: E05-0544 Page 52 of 101 3M ENVIRONMENTAL LABORATORY PROJECT NO. E05-0544 Table 13. Analyte concentrations measured in active and sterile anaerobic cultures dosed with PFBS. Culture ID Culture Type Incubation Time (days) FBSA [a] Concentration (ng/mL) PFBS PFBSi [b] PFBA E05-0544-174 Active Sludge with PFBS 69 < 10.0 850 < 5.00 < 10.0 E05-0544-172 Active Sludge with PFBS 105 < 25.0 969 < 12.5 < 25.0 E05-0544-173 Active Sludge with PFBS 105 < 25.0 1020 < 12.5 < 25.0 E05-0544-175 Sterile Sludge with PFBS 0 Not Determined 585 Not Determined < 10.0 E05-0544-176 Sterile Sludge with PFBS 0 Not Determined 438 Not Determined < 10.0 E05-0544-177 Sterile Sludge with PFBS 0 Not Determined 551 Not Determined < 10.0 E05-0544-178 Sterile Sludge with PFBS 7 Not Determined 882 Not Determined < 10.0 E05-0544-179 Sterile Sludge with PFBS 7 Not Determined 868 Not Determined < 10.0 E05-0544-180 Sterile Sludge with PFBS 7 Not Determined 894 Not Determined < 10.0 E05-0544-181 Sterile Sludge with PFBS 18 Not Determined 803 Not Determined < 10.0 E05-0544-182 Sterile Sludge with PFBS 18 Not Determined 731 Not Determined < 10.0 E05-0544-183 Sterile Sludge with PFBS 18 Not Determined 684 Not Determined < 10.0 E05-0544-184 Sterile Sludge with PFBS 26 < 10.0 910 < 10.0 < 10.0 E05-0544-185 Sterile Sludge with PFBS 26 < 10.0 937 < 10.0 < 10.0 E05-0544-186 Sterile Sludge with PFBS 26 < 10.0 896 < 10.0 < 10.0 E05-0544-189 Sterile Sludge with PFBS 69 < 10.0 850 < 5.00 < 10.0 E05-0544-187 Sterile Sludge with PFBS 105 < 25.0 946 < 12.5 < 25.0 E05-0544-188 Sterile Sludge with PFBS 105 < 25.0 864 < 12.5 < 25.0 Note: Results for the PFBS-dosed samples were reported from analytical runs # 6, #15 and #20 in Table 8. Culture extracts were analyzed against a set of matrix matched calibration standards. Quantitation was by internal standard method for analytical runs #6 and #20, and external standard method for analytical run #15. Calibration curves contained minimally six data points. Curves in analytical run #6 were quadratic fit and weighted 1/x2, curves in analytical runs #6 and #20 were quadratic fit with 1/x weighting, except FBSA in run #15 which was 1/x2. Calibration accuracy for standards included in the curve fit were within 100 + 25% (+ 30% at LLOQ). CCVs (low and mid-level QCs at 40.0 ng/mL and 400 ng/mL, respectively) were injected approximately every 15 samples and were within 100+30% of their theoretical values for all analytes. LLOQs were always < 25.0 ng/mL. All values in the table are reported to 3 significant figures while more precise data was used in data calculations. Associated bioactive and sterile sludge blanks analyzed with the samples were all below the LLOQ for all analytes. [a] Purity of FBSA was assumed 100% at the time of use, but determined later at 97.3% and was not corrected for. [b] Purity of PFBSi was assumed 50% at the time of use, but later determined to by 51.2% and was not corrected for. Study Report: E05-0544 Page 53 of 101 3M ENVIRONMENTAL LABORATORY PROJECT NO. E05-0544 4.2.5 PFOA Results The test substance perfluorooctanoate (PFOA) was dosed into cultures at 1010 ng/mL and incubated anaerobically for up to 94 days. Cultures analyzed were collected on days 0, 10, 20, 74 and 94. Quantitative LC/MS/MS analysis of processed culture extracts was conducted for PFOA, perfluoroheptanoate (PFHpA), perfluorononanoate (PFNA) and PFBA on days 0, 10 and 20; and only PFNA and PFOA were measured at all other time points. Additionally, qualitative analysis for 8:2 fluorotelomer acid (8:2 FTCA) and 8:2 unsaturated fluorotelomer acid (8:2 FTUCA) was also conducted at all time points. Data was collected from two analytical runs #19 and #23 of Table 8. The PFOA, PFNA, PFHpA and PFBA concentrations measured in active and sterile cultures are shown in Table 14 and plotted in Figure 11. The analysis results showed that PFOA did not biodegrade anaerobically over 94 days under methanogenic conditions. There was no discernible difference between PFOA in bioactive cultures versus sterile cultures. Variation in the results can be attributed to the analytical method precision and accuracy. Additionally, lower molecular weight PFCAs as PFBA and PFHpA did not form in the first ~3 weeks of incubation, and the higher molecular weight PFNA did not form over the incubation period. Qualitative analysis also showed no anticipated fatty acid synthesis products 8:2 FTCA and 8:2 FTUCA (data not shown). Note: Smaller perfluorinated acids could be anticipated degradation products of PFOA, but only PFBA and PFHpA were measured at days 0, 10 and 20. While the PFOA did not measurably biodegrade in active cultures versus the levels measured in sterile cultures, future studies might include analysis at later time points for low molecular weight perfluorinated acids in addition to those measured here, such as trifluoroacetate (TFA), perfluoropropanoate (PFPA), PFPeA and PFHxA, to ensure that none of those potential fluorinated acid products formed at low levels. Study Report: E05-0544 Page 54 of 101 3M ENVIRONMENTAL LABORATORY PROJECT NO. E05-0544 BBiioo aa cc tti ivve eC u l t u r e s 1200 SSttee rr iilleeC u l t u r e s 1200 C o n c e n tra tio n (n g / m L ) C o n c e n tra tio n (n g / m L ) 1000 1000 800 itle itle 800 isT 600 x isT 600 x A 400 A 400 200 200 0 0 0 20 40 D a y s 60 80 100 Axis Title 0 20 40 60 80 100 ADxD iasa yTy sis tle Figure 11. The PFOA concentrations measured in PFOA-dosed bioactive cultures (left) and sterile cultures (right). The line represents the dosed concentration of 1010 ng/mL Table 14. Analyte concentrations measured in active and sterile anaerobic cultures dosed with PFOA. Culture ID E05-0544-635 E05-0544-636 E05-0544-637 E05-0544-638 E05-0544-639 E05-0544-640 E05-0544-641 E05-0544-642 E05-0544-643 E05-0544-644 E05-0544-645 E05-0544-646 E05-0544-647 E05-0544-648 E05-0544-649 Culture Type Active Sludge with PFOA Active Sludge with PFOA Active Sludge with PFOA Active Sludge with PFOA Active Sludge with PFOA Active Sludge with PFOA Active Sludge with PFOA Active Sludge with PFOA Active Sludge with PFOA Active Sludge with PFOA Active Sludge with PFOA Active Sludge with PFOA Active Sludge with PFOA Active Sludge with PFOA Active Sludge with PFOA Incubation Time (days) 0 0 0 10 10 10 20 20 20 74 74 74 94 94 94 PFOA 880 762 815 887 790 758 866 753 881 1080 981 988 944 983 982 Concentration (ng/mL) PFNA PFHpA < 10.0 < 10.0 < 10.0 < 10.0 < 10.0 < 10.0 < 10.0 < 10.0 < 10.0 < 10.0 < 10.0 < 10.0 < 10.0 < 10.0 < 10.0 < 10.0 < 10.0 < 10.0 < 10.0 [b] < 10.0 [b] < 10.0 [b] < 10.0 [b] < 10.0 [b] < 10.0 [b] PFBA [a] < 10.0 < 10.0 < 10.0 < 10.0 < 10.0 < 10.0 < 10.0 < 10.0 < 10.0 [c] [c] [c] [c] [c] [c] Study Report: E05-0544 Page 55 of 101 3M ENVIRONMENTAL LABORATORY PROJECT NO. E05-0544 Table 14. Analyte concentrations measured in active and sterile anaerobic cultures dosed with PFOA. Culture ID Culture Type Incubation Time (days) PFOA Concentration (ng/mL) PFNA PFHpA PFBA [a] E05-0544-650 Sterile Sludge with PFOA 0 925 < 10.0 < 10.0 < 10.0 E05-0544-651 Sterile Sludge with PFOA 0 905 < 10.0 < 10.0 < 10.0 E05-0544-652 Sterile Sludge with PFOA 0 931 < 10.0 < 10.0 < 10.0 E05-0544-653 Sterile Sludge with PFOA 10 1000 < 10.0 < 10.0 < 10.0 E05-0544-654 Sterile Sludge with PFOA 10 1000 < 10.0 < 10.0 < 10.0 E05-0544-655 Sterile Sludge with PFOA 10 1090 < 10.0 < 10.0 < 10.0 E05-0544-656 Sterile Sludge with PFOA 20 1160 < 10.0 < 10.0 < 10.0 E05-0544-657 Sterile Sludge with PFOA 20 1050 < 10.0 < 10.0 < 10.0 E05-0544-658 Sterile Sludge with PFOA 20 987 < 10.0 < 10.0 < 10.0 E05-0544-659 Sterile Sludge with PFOA 74 927 < 10.0 [b] [c] E05-0544-660 Sterile Sludge with PFOA 74 1050 < 10.0 [b] [c] E05-0544-661 Sterile Sludge with PFOA 74 891 < 10.0 [b] [c] E05-0544-662 Sterile Sludge with PFOA 94 943 < 10.0 [b] [c] E05-0544-663 Sterile Sludge with PFOA 94 947 < 10.0 [b] [c] E05-0544-664 Sterile Sludge with PFOA 94 688 < 10.0 [b] [c] Note: Data for PFOA-dosed samples were reported from analytical runs # 19 and #23 in Table 8. Culture extracts were analyzed against a set of matrix matched calibration standards. Quantitation was by internal standard method. Calibration curves contained minimally six data points. Curves in analytical run #19 and #23 were quadratic fit and weighted 1/x. Calibration accuracy for standards included in the curve fit were within 100 + 25% (+ 30% at LLOQ). CCVs (low and mid-level QCs at 40.0 ng/mL and 400 ng/mL, respectively) were injected approximately every 15 samples in analytical runs #19 and #23, and all CCVs/QCs were within 100+30% of their theoretical values for all analytes. LLOQs for all data in Table 14 were 10.0 ng/mL All values in the table are reported to 3 significant figures while more precise data was used in data calculations. Associated bioactive and sterile sludge blanks analyzed with the samples were all below the LLOQ for all analytes. [a] Results for PFBA were reported from analysis of PFOA cultures in run #19, however, CCVs for PFBA were not included as bracketing QC for those samples. [b] PFHpA data was collected for day74 and day 94 samples, but PFHpA is not reported. The PFHpA data was errantly not worked up at the time of analysis in 2006. Electronic data for run #23 was not retrievable in 2016 and only possession of original printed chromatograms and results, therefore PFHpA could not be determined. [c] PFBA was not a target analyte during analysis of day 74 and 94 samples. Study Report: E05-0544 Page 56 of 101 3M ENVIRONMENTAL LABORATORY PROJECT NO. E05-0544 4.2.6 PFBA Results The test substance perfluorobutanoate (PFBA) was dosed into cultures at 1030 ng/mL and incubated anaerobically for up to 94 days. Cultures analyzed were collected on days 0, 10, 20, 74 and 94. Quantitative LC/MS/MS analysis results are reported for PFBA only. Data was collected from two analytical runs as described at the bottom of Table 15. PFBA concentrations measured in active and sterile cultures are plotted in Figure 12 and showed no discernible loss of PFBA in either culture type. These results showed that PFBA did not biodegrade anaerobically over 94 days under methanogenic conditions in the active cultures, nor in the sterile cultures. C o n c e n tra tio n (n g / m L ) C o n c e n tra tio n (n g / m L ) P F B A Bio a ctiv e C ult u re s PFBA Bioactive P PF BFABSAt e Sr itl ee rC ilu el t u r e s 1400 1400 1200 1200 1000 1000 itle 800 itle 800 isT isT Ax 600 Ax 600 400 400 200 200 0 0 0 20 40 D a y s 60 80 100 Axis Title 0 20 40 D a y s 60 80 100 Axis Title Figure 12. The PFBA concentrations measured in PFBA-dose bioactive cultures (left) and sterile cultures (right) dosed with PFBA. The line represents the dosed concentration of 1030 ng/mL. Note: It may be expected that lower molecular weight perfluorinated acids could be generated as biodegradation products of PFBA, but none were analytically determined as part of this study. Future studies might include analysis for perfluorinated acids that are lower molecular weight than PFBA such as perfluoroacetate (i.e. trifluoroacetic acid, TFA) or perfluoropropanoate (PFPA). Study Report: E05-0544 Page 57 of 101 3M ENVIRONMENTAL LABORATORY PROJECT NO. E05-0544 Table 15. Culture ID E05-0544-605 E05-0544-606 E05-0544-607 E05-0544-608 E05-0544-609 E05-0544-610 E05-0544-611 E05-0544-612 E05-0544-613 E05-0544-614 E05-0544-615 E05-0544-616 E05-0544-617 E05-0544-618 E05-0544-619 E05-0544-620 E05-0544-621 E05-0544-622 E05-0544-623 E05-0544-624 E05-0544-625 E05-0544-626 E05-0544-627 E05-0544-628 Analyte concentrations measured in active and sterile anaerobic cultures dosed with PFBA. Culture Type Active Sludge with PFBA Active Sludge with PFBA Active Sludge with PFBA Active Sludge with PFBA Active Sludge with PFBA Active Sludge with PFBA Active Sludge with PFBA Active Sludge with PFBA Active Sludge with PFBA Active Sludge with PFBA Active Sludge with PFBA Active Sludge with PFBA Active Sludge with PFBA Active Sludge with PFBA Active Sludge with PFBA Sterile Sludge with PFBA Sterile Sludge with PFBA Sterile Sludge with PFBA Sterile Sludge with PFBA Sterile Sludge with PFBA Sterile Sludge with PFBA Sterile Sludge with PFBA Sterile Sludge with PFBA Sterile Sludge with PFBA Incubation Time (days) 0 0 0 10 10 10 20 20 20 74 74 74 94 94 94 0 0 0 10 10 10 20 20 20 Concentration (ng/mL) PFBA 1030 818 1030 1170 1120 1250 1240 1080 1290 1180 960 1130 861 1340 1070 848 936 832 988 1060 1000 999 979 991 Study Report: E05-0544 Page 58 of 101 3M ENVIRONMENTAL LABORATORY PROJECT NO. E05-0544 Table 15. Analyte concentrations measured in active and sterile anaerobic cultures dosed with PFBA. Culture ID Culture Type Incubation Time (days) Concentration (ng/mL) PFBA E05-0544-629 Sterile Sludge with PFBA 74 725 E05-0544-630 Sterile Sludge with PFBA 74 1090 E05-0544-631 Sterile Sludge with PFBA 74 713 E05-0544-632 Sterile Sludge with PFBA 94 953 E05-0544-633 Sterile Sludge with PFBA 94 1040 E05-0544-634 Sterile Sludge with PFBA 94 936 Note: Results for the PFBA-dosed samples were reported from analytical runs # 19 and #26 in Table 8. Culture extracts were analyzed against a set of matrix matched calibration standards. Quantitation was by internal standard method for both runs. Calibration curves contained minimally six data points. Curves in analytical runs #19 and #26 were quadratic fit with 1/x weighting for PFBA. Calibration accuracy for standards included in the curve fit were within 100 + 25% (+ 30% at LLOQ). CCVs (low and mid-level QCs at 40.0 ng/mL and 400 ng/mL, respectively) were injected approximately every 15 samples in analytical run #19, and mid-level calibration standard (250 ng/mL) was re-injected as CCVs in analytical run #26, and all CCVs/QCs were within 100+30% of their theoretical values for all analytes. LLOQs for PFBA were 10 ng/mL in both runs. All values in the table are reported to 3 significant figures while more precise data was used in data calculations. Associated bioactive and sterile sludge blanks analyzed with the samples were all below the LLOQ for all analytes. [a] PFBA cultures for days 74 and 94 were additionally analyzed for PFBS, PFBSi, FBSA, MeFBSA, MeFBSAA and MeFBSE in analytical run #26, but those results excluded from the table because they are not anticipated degradation products of PFBA. Study Report: E05-0544 Page 59 of 101 3M ENVIRONMENTAL LABORATORY PROJECT NO. E05-0544 4.2.7 6:2 FTS Results The test substance 1,1,2,2-tetrahydro-tetradecafluorooctane sulfonate (6:2 fluorotelomer sulfonate, 6:2 FTS, a.k.a. THPFOS in data) was dosed into anaerobic cultures at a nominal concentration of 954 ng/mL and incubated anaerobically for up to 105 days. The results of the quantitative LC/MS/MS analysis for 6:2 FTS in the bioactive culture extracts versus sterile control culture extracts showed no discernible loss of 6:2 FTS over 105 days (Figure 13). In addition, the anticipated products perfluorohexanoate (PFHxA) and 6:2 unsaturated fluorotelomer acid (6:2 FTUCA) were not observed as products. The 6:2 FTS concentrations determined by LC/MS/MS in active and sterile cultures are shown in Table 16. Matrix-interference appeared to cause signal enhancement for 6:2 FTS which was observed for mid-study time points, possibly due to transformation of sludge-related compounds in the cultures which facilitated signal enhancement for 6:2 FTS. In the day 69 and day-105 culture extracts a peak corresponding to the 6:2 unsaturated fluorotelomer carboxylate (6:2 FTUCA) was detected below the LLOQ in the sterile cultures, but was not present in the active cultures. However, coincidentally a peak was detected for 6:2 fluorotelomer carboxylate (6:2 FTCA) in the day-69 and day-105 active cultures that was not present in the equivalent sterile cultures. This could imply that a low concentration of 6:2 FTUCA may have been a contaminant of the 6:2 FTS and transformed anaerobically to 6:2 FTCA. Semi-quantitative analysis of bioactive culture E05-0544-144 headspace at day-69 by a sensitive GC/MS assay showed no detectable volatile fluorocarbons (analytical run # 30 of Table 8). The net result established from this data was that 6:2 FTS does not appear to biodegrade anaerobically at any appreciable rate over 105 days under methanogenic conditions. In the future, the anaerobic biodegradability of 6:2 FTS might be further evaluated by including the analysis of 6:2 fluorotelomer alcohol, a likely biotransformation product of 6:2 FTS that was not measured for the 6:2 FTS cultures. Additionally, analysis for smaller molecular weight perfluorinated acids such as TFA, PFPA, PFBA and PFPeA should also be included in future analyses of 6:2 FTS anaerobic cultures. Study Report: E05-0544 Page 60 of 101 3M ENVIRONMENTAL LABORATORY PROJECT NO. E05-0544 C o n c e n tra tio n (n g / m L ) C o n c e n tra tio n (n g / m L ) BB i iooa ac tci tv ieveC u l t u r e s S tSe tr ei l eriCleu l t u r e s 1400 1400 1200 1200 1000 1000 itle 800 itle 800 isT isT Ax 600 Ax 600 400 400 200 200 0 0 20 40 60 80 100 120 D ays Axis Title 0 0 20 40 60 80 100 120 D ays Axis Title Figure 13. The 6:2 FTS concentrations measured in active cultures (left) and sterile cultures (right) dosed with 6:2 FTS. The line represents the dosed concentration of 954 ng/mL. Study Report: E05-0544 Page 61 of 101 3M ENVIRONMENTAL LABORATORY PROJECT NO. E05-0544 Table 16. Analyte concentrations measured in active and sterile anaerobic cultures dosed with 6:2 FTS Culture ID E05-0544-130 Culture Type Active Sludge with 6:2 FTS Incubation Time (days) 0 6:2 FTS 920 Concentration (ng/mL) 6:2 FTCA 6:2 FTUCA [b] Not Determined < 10.0 E05-0544-131 Active Sludge with 6:2 FTS 0 E05-0544-132 Active Sludge with 6:2 FTS 0 E05-0544-133 Active Sludge with 6:2 FTS 7 E05-0544-134 Active Sludge with 6:2 FTS 7 E05-0544-135 Active Sludge with 6:2 FTS 7 E05-0544-136 Active Sludge with 6:2 FTS 18 E05-0544-137 Active Sludge with 6:2 FTS 18 E05-0544-138 Active Sludge with 6:2 FTS 18 E05-0544-139 Active Sludge with 6:2 FTS 26 E05-0544-140 Active Sludge with 6:2 FTS 26 E05-0544-141 Active Sludge with 6:2 FTS 26 E05-0544-144 Active Sludge with 6:2 FTS 69 E05-0544-142 Active Sludge with 6:2 FTS 105 E05-0544-143 Active Sludge with 6:2 FTS 105 E05-0544-145 Sterile Sludge with 6:2 FTS 0 E05-0544-146 Sterile Sludge with 6:2 FTS 0 E05-0544-147 Sterile Sludge with 6:2 FTS 0 E05-0544-148 Sterile Sludge with 6:2 FTS 7 E05-0544-149 Sterile Sludge with 6:2 FTS 7 E05-0544-150 Sterile Sludge with 6:2 FTS 7 E05-0544-151 Sterile Sludge with 6:2 FTS 18 E05-0544-152 Sterile Sludge with 6:2 FTS 18 E05-0544-153 Sterile Sludge with 6:2 FTS 18 930 1010 907 915 805 1030 1000 1010 1080 1200 1140 1100 837 834 804 [a] 730 [a] 1150 [a] 903 [a] 862 [a] 938 [a] 1190 [a] 1040 [a] 1180 [a] Not Determined Not Determined Not Determined Not Determined Not Determined Not Determined Not Determined Not Determined Not Determined Not Determined Not Determined < 10.0 < 10.0 < 10.0 Not Determined Not Determined Not Determined Not Determined Not Determined Not Determined Not Determined Not Determined Not Determined < 10.0 < 10.0 < 10.0 < 10.0 < 10.0 < 10.0 < 10.0 < 10.0 < 10.0 < 10.0 < 10.0 < 10.0 < 10.0 < 10.0 < 10.0 < 10.0 < 10.0 < 10.0 < 10.0 < 10.0 < 10.0 < 10.0 < 10.0 Study Report: E05-0544 Page 62 of 101 PFHxA < 10.0 < 10.0 < 10.0 < 10.0 < 10.0 < 10.0 < 10.0 < 10.0 < 10.0 < 10.0 < 10.0 < 10.0 < 10.0 < 10.0 < 10.0 < 10.0 < 10.0 < 10.0 < 10.0 < 10.0 < 10.0 < 10.0 < 10.0 < 10.0 3M ENVIRONMENTAL LABORATORY PROJECT NO. E05-0544 Table 16. Analyte concentrations measured in active and sterile anaerobic cultures dosed with 6:2 FTS Culture ID E05-0544-154 Culture Type Sterile Sludge with 6:2 FTS Incubation Time (days) 26 6:2 FTS 1180 [a] Concentration (ng/mL) 6:2 FTCA 6:2 FTUCA [b] Not Determined < 10.0 PFHxA < 10.0 E05-0544-155 Sterile Sludge with 6:2 FTS 26 1370 [a] Not Determined < 10.0 < 10.0 E05-0544-156 Sterile Sludge with 6:2 FTS 26 1230 [a] Not Determined < 10.0 < 10.0 E05-0544-159 Sterile Sludge with 6:2 FTS 69 939 < 10.0 < 10.0 < 10.0 E05-0544-157 Sterile Sludge with 6:2 FTS 105 805 < 10.0 < 10.0 < 10.0 E05-0544-158 Sterile Sludge with 6:2 FTS 105 773 < 10.0 < 10.0 < 10.0 Note: Results for the 6:2 FTS-dosed samples were reported from analytical runs # 8 and #21 in Table 8. Culture extracts were analyzed against a set of matrix matched calibration standards. Quantitation was by internal standard method for both runs. Calibration curves contained minimally six data points. Quantitation was by internal standard method and calibration curves were quadratic fit with 1/x weighting, except 6:2 FTS in run #21 which was quantified by external standard method. Calibration accuracy for standards included in the curve fit were within 100 + 25% (+ 30% at LLOQ). CCVs were re-injection of the 25.0 and 750 ng/mL calibration standard, and were injected approximately every 15 sample. CCVs were within 100+30% of their theoretical values for all analytes, with exceptions of 6:2 FTS in run #8 which had five CCVs between 131% and 154% and 6:2 FTUCA in run #8 which had two CCVs at 143% and 147%. LLOQs were 25.0 ng/mL for 6:2 FTS (however 10 ng/mL standard included in curve fit and 10 ng/mL for PFHxA and 6:2 FTUCA in run #8. LLOQs were 10.0 ng/mL for 6:2 FTS, PFHxA, 6:2 FTCA and 6:2 FTUCA in analytical run #21. Results shown are rounded to 3 significant figures while more precise values were used in data calculations. Associated bioactive and sterile sludge blanks analyzed with the samples were all below the LLOQ for all analytes. [a] Results were reported from analytical run #8 and the CCVs analyzed immediately after the 6:2-FTS dosed culture extracts were 152% and 131%, CCVs prior to those samples were 121% and 113%. Data was reported despite the high CCV results since there were not prerequisite pass/fail criteria. [b] 6:2 FTUCA in run #8 had CCV results of 147% and 143% but those CCVs did not flank results for 6:2 FTS-dosed culture extracts and only flanked blank controls. Study Report: E05-0544 Page 63 of 101 3M ENVIRONMENTAL LABORATORY PROJECT NO. E05-0544 4.2.8 6:2 FTOH Results The test substance 1,1,2,2-tetrahydro-tridecafluorooctan-1-ol (6:2 FTOH) was dosed into cultures at a nominal concentration of 1,230 ng/mL and were incubated anaerobically for up to 94 days. Extracted samples were analyzed by direct inject GC/MS for 6:2 FTOH and by LC/MS/MS for potential soluble degradation products 6:2 FTCA, 6:2 FTUCA, PFHpA and PFHxA. The results for 6:2 FTOH showed that there was considerable loss of 6:2 FTOH over the 94 days in bioactive cultures and in sterile cultures (Figure 14). The loss of 6:2 FTOH in sterile cultures was possibly due to irreversible adsorption to sludge or to the test vessel cap (i.e. PTFE liner). Irreversible adsorptive loss of FTOHs has been previously observed to PTFE liners of test vessels (Wang et al. 2005). The loss of 6:2 FTOH was more pronounced in bioactive cultures and was likely the result of the additive effect of 6:2 FTOH being biodegraded and lost to adsorption. The biotransformation product 6:2 FTCA was formed in bioactive cultures at levels exceeding the LLOQ (Figure 15). Based on the average measured value at day 94, 10.9 mole% of the initial dosed 6:2 FTOH was accounted for as 6:2 FTCA (Table 17). The 6:2 FTCA product was not observed in sterile cultures, confirming its formation was due to microbiological activity. In addition, the anticipated product 6:2 FTUCA was observed qualitatively as a low level peak in chromatograms, and that peak response increased over time, but remained below the LLOQ of 10.0 ng/mL and thus was not quantifiable; the 6:2 FTUCA peak was not observed in sterile culture extracts. The anticipated products PFHxA and PFHpA were measured for but not observed as anaerobic degradation products. During this study the 6:2 FTOH biotransformation product 5:3 acid and an aldehyde product 6:2 FTAL (Wang et al. 2005) were not measured. The 5:3 acid was recently identified as an anaerobic biodegradation product by Zhang et al. (2013a). However, the 6:2 FTAL is considered minor and short lived and is not a concern that it was not measured. However 5:3 acid is expected at significant levels anaerobically. The quantitative GC/MS results showed that approximately 300 to 500 ng/mL of additional 6:2 FTOH was lost in bioactive cultures versus the levels lost in sterile cultures, the maximum concentration of 6:2 FTCA was only 150 ng/mL and gave poor mass balance of approximately 40 mole% relative to the lost 6:2 FTOH. Therefore it is speculated that approximately 60 mole% of the lost 6:2 FTOH may have been biotransformed to 5:3 acid. The relevance of this is discussed later in this report. Study Report: E05-0544 Page 64 of 101 3M ENVIRONMENTAL LABORATORY PROJECT NO. E05-0544 M e asu re d C o n c. (n g / m L) M e asu re d C o n c. (n g / m L) 6 : B2 iFoT aO cH tiBvi eo a Cc tui vlte uC ruel tsu r e s 6:2 F6T: O2 FHT OinH SS tt ee r ri lieleC Cu l ut ultr ue res 2000 2000 1800 1800 1600 1600 1400 1400 itle1200 T itle1200 T1000 xis 1000 A 800 xis A 800 600 600 400 400 200 200 0 0 0 20 40 60 80 100 Days Days Axis Title 0 20 40 D a y s 60 80 100 D a Ay xsis Title Figure 14. The measured 6:2 FTOH concentrations by GC/MS in bioactive cultures (left) and sterile cultures (right); dosed with nominal 1230 ng/mL 6:2 FTOH. Data manually fitted for appearance. M e a s u re d C o n c. ( n g / m L) 6 : 2 F T C A f r o Cm h6 a: 2 rtF T TO itH lei n B i o a c t i v e C u l t u r e 250 200 itle 150 isT Ax 100 50 0 0 20 40 D a y s 60 80 100 Axis Title Figure 15. The formation of 6:2-FTCA from 6:2 FTOH in bioactive cultures. Data manually fitted for appearance. The formation of 6:2 FTCA in the active cultures over the first 3 time points (i.e. first 20 days of incubation) was linear and those data were used to estimate an initial Study Report: E05-0544 Page 65 of 101 3M ENVIRONMENTAL LABORATORY PROJECT NO. E05-0544 biotransformation reaction rate, based on the 6:2 FTOH conversion to 6:2 FTCA only (Figure 16). The initial rate was calculated to be 102 pmole of 6:2 FTOH biotransformed to 6:2 FTCA per day under these test conditions. However, 5:3 acid was not measured and possibly accounted for ~60% of the degraded FTOH, therefore the rate is likely an underestimate and may be ~2 times faster than estimated. The sludge-dependent estimated biotransformation rate of 6:2 FTOH to 6:2 FTCA was calculated at 1.76 pmole per day per mg sludge (dry weight). An estimated t1/2 was estimated at 83.8 days based on the initial rate and dose level (see Table 21). Rate calculations were performed as described in Appendix A. Figure 16. The linear curve fit of 6:2-FTCA (pmole) formed in cultures from 6:2 FTOH. Study Report: E05-0544 Page 66 of 101 3M ENVIRONMENTAL LABORATORY PROJECT NO. E05-0544 Table 17. Analyte concentrations measured in active and sterile anaerobic cultures dosed with 6:2 FTOH Culture ID Culture Type Incubation Time (days) 6:2 FTOH (GC/MS) 6:2 FTCA Concentration (ng/mL) 6:2 FTUCA PFHpA E05-0544-575 Active Sludge with 6:2 FTOH 0 1030 0 < 10.0 < 10.0 E05-0544-576 Active Sludge with 6:2 FTOH 0 1930 0 E05-0544-577 Active Sludge with 6:2 FTOH 0 1310 0 E05-0544-578 Active Sludge with 6:2 FTOH 10 983 82.1 < 10.0 < 10.0 < 10.0 < 10.0 < 10.0 < 10.0 E05-0544-579 Active Sludge with 6:2 FTOH 10 1140 90.3 < 10.0 < 10.0 E05-0544-580 Active Sludge with 6:2 FTOH 10 640 67.8 < 10.0 < 10.0 E05-0544-581 Active Sludge with 6:2 FTOH 20 674 164 < 10.0 < 10.0 E05-0544-582 Active Sludge with 6:2 FTOH 20 380 154 < 10.0 < 10.0 E05-0544-583 Active Sludge with 6:2 FTOH 20 419 139 < 10.0 < 10.0 E05-0544-584 Active Sludge with 6:2 FTOH 74 319 183 < 10.0 < 10.0 E05-0544-585 Active Sludge with 6:2 FTOH 74 106 167 < 10.0 < 10.0 E05-0544-586 Active Sludge with 6:2 FTOH 74 44.7 62.7 < 10.0 < 10.0 E05-0544-587 Active Sludge with 6:2 FTOH 94 48.4 131 < 10.0 < 10.0 E05-0544-588 Active Sludge with 6:2 FTOH 94 368 220 < 10.0 < 10.0 E05-0544-589 Active Sludge with 6:2 FTOH 94 56.4 64.8 < 10.0 < 10.0 E05-0544-590 Sterile Sludge with 6:2 FTOH 0 1290 < 10.0 < 10.0 < 10.0 E05-0544-591 Sterile Sludge with 6:2 FTOH 0 1340 < 10.0 < 10.0 < 10.0 E05-0544-592 Sterile Sludge with 6:2 FTOH 0 1370 < 10.0 < 10.0 < 10.0 E05-0544-593 Sterile Sludge with 6:2 FTOH 10 950 < 10.0 < 10.0 < 10.0 E05-0544-594 Sterile Sludge with 6:2 FTOH 10 1090 < 10.0 < 10.0 < 10.0 E05-0544-595 Sterile Sludge with 6:2 FTOH 10 1040 < 10.0 < 10.0 < 10.0 E05-0544-596 Sterile Sludge with 6:2 FTOH 20 1040 < 10.0 < 10.0 < 10.0 E05-0544-597 Sterile Sludge with 6:2 FTOH 20 803 < 10.0 < 10.0 < 10.0 E05-0544-598 Sterile Sludge with 6:2 FTOH 20 793 < 10.0 < 10.0 < 10.0 E05-0544-599 Sterile Sludge with 6:2 FTOH 74 429 < 10.0 < 10.0 < 10.0 PFHxA < 10.0 < 10.0 < 10.0 < 10.0 < 10.0 < 10.0 < 10.0 < 10.0 < 10.0 < 10.0 < 10.0 < 10.0 < 10.0 < 10.0 < 10.0 < 10.0 < 10.0 < 10.0 < 10.0 < 10.0 < 10.0 < 10.0 < 10.0 < 10.0 < 10.0 Study Report: E05-0544 Page 67 of 101 3M ENVIRONMENTAL LABORATORY PROJECT NO. E05-0544 Table 17. Analyte concentrations measured in active and sterile anaerobic cultures dosed with 6:2 FTOH Culture ID Culture Type Incubation Time (days) 6:2 FTOH (GC/MS) 6:2 FTCA Concentration (ng/mL) 6:2 FTUCA PFHpA PFHxA E05-0544-600 Sterile Sludge with 6:2 FTOH 74 445 < 10.0 < 10.0 < 10.0 < 10.0 E05-0544-601 Sterile Sludge with 6:2 FTOH 74 551 < 10.0 < 10.0 < 10.0 < 10.0 E05-0544-602 Sterile Sludge with 6:2 FTOH 94 553 < 10.0 < 10.0 < 10.0 < 10.0 E05-0544-603 Sterile Sludge with 6:2 FTOH 94 524 < 10.0 < 10.0 < 10.0 < 10.0 E05-0544-604 Sterile Sludge with 6:2 FTOH 94 326 < 10.0 < 10.0 < 10.0 < 10.0 Note: Concentrations of 6:2 FTOH in dosed cultures were determined in GC/MS analytical run #24 in Table 8. Soluble biotransformation products were quantified in LC/MS/MS analytical runs #18 and #22 in Table 8. Culture extracts were analyzed against a set of matrix matched calibration standards. Calibration curves contained minimally six data points. Quantitation was by external standard method and calibration curve fit was quadratic with 1/x weighting for 6:2 FTOH. Calibration accuracy for standards included in the curve fit were within 100 + 25% (+ 30% at LLOQ). The analytes 6:2 FTCA, 6:2 FTUCA, PFHpA and PFHxA were quantified by internal standard method and calibration curves were quadratic fit with 1/x weighting. Calibration accuracy for standards included in the curve fit were within 100 + 25% (+ 30% at LLOQ). CCVs were a low and mid-level QCs (40.0 ng/mL and 400 ng/mL) and were injected approximately every 15 samples in analytical run #24 and results were within 100 + 30%. Results shown are rounded to 3 significant figures while more precise values were used in data calculations. Associated bioactive and sterile sludge blanks analyzed with the samples were all below the LLOQ for all analytes. LLOQs were 100 ng/mL for 6:2 FTOH, and 10 ng/mL for 6:2 FTCA, 6:2 FTUCA, PFHpA and PFHxA. Study Report: E05-0544 Page 68 of 101 3M ENVIRONMENTAL LABORATORY PROJECT NO. E05-0544 4.2.9 8:2 FTOH Results The test substance 8:2 fluorotelomer alcohol (8:2 FTOH) was dosed into anaerobic cultures at a nominal concentration of 1210 ng/mL and incubated for up to 94 days. Culture extracts were analyzed by GC/MS and LC/MS/MS for 8:2 FTOH and anticipated PFAS products, respectively. The results showed that 8:2 FTOH was lost in both bioactive and sterile cultures (Figure 17). Loss of 8:2 FTOH in sterile cultures was presumed due to adsorptive loss to the sludge and/or test vessel, while loss in the bioactive cultures was due to an additive effect of adsorption and biodegradation. The LC/MS/MS results showed that 8:2 FTOH biotransformed to give 8:2-fluorotelomer acid (8:2 FTCA) and 8:2-fluorotelomer unsaturated acid (8:2 FTUCA), plotted in Figure 18. Anaerobic biodegradation of 8:2 FTOH did not result in formation of anticipated perfluorinated carboxylate products PFHxA, PFHpA, PFOA or PFNA. Based on the average measured concentrations for 8:2-FTCA and 8:2 FTUCA (Table 18), roughly 8.8 mole% of the 8:2 FTOH biotransformed to those two products over 94 days of incubation, with 8:2 FTCA being produced at about 5-times the level of 8:2 FTUCA. Associated water blanks (no sludge) and sterile sludge and bioactive sludge blanks were less than LLOQ for all analytes. The formation of 8:2 FTCA and 8:2 FTUCA in the active cultures over the first 3 time points (i.e. first 20 days of incubation) was linear and those data were used to estimate an initial biotransformation reaction rate for 8:2 FTOH (Figure 19). The initial rate was calculated to be 23.6 pmole of 8:2 FTOH biotransformed to 8:2 FTCA and 8:2 FTUCA per day under these test conditions. The sludge-dependent estimated biotransformation rate of 8:2 FTOH to 6:2 FTCA was calculated at 0.413 pmole per day per mg sludge (dry weight). A t1/2 was estimated at 275 days based on the initial rate of product formation and dose level of 8:2 FTOH (see Table 21). Rate calculations were performed as described in Appendix A. Note: The 7:3 acid was recently identified as an anaerobic biodegradation product of 8:2 FTOH under anaerobic conditions (Zhang et al. 2013a). However, the 7:3 acid product of 8:2 FTOH was not measured during this study and possibly accounted for a significant portion of the degraded FTOH, therefore the rate is likely an underestimate and may be significantly higher than estimated here. Study Report: E05-0544 Page 69 of 101 3M ENVIRONMENTAL LABORATORY PROJECT NO. E05-0544 C o n c e n tra tio n (n g / m L ) 1600 L) 1400 g/m1200 .(n1000 nc Co 800 red 600 asu 400 e M 200 0 0 8 : A2 cF tT iOveH B8i :o 2a c Ft itvOe HC u l t u r e s 20 40 D a y s 60 80 Days 1600 1400 C o n c e n tra tio n (n g / m L ) 1200 itle1000 isT 800 Ax 600 400 200 0 100 0 8 : 2 CF hT aO rHt STt ietrliel e C u l t u r e s 20 40 D a y s 60 80 100 Axis Title Figure 17. The 8:2 FTOH concentrations measured by GC/MS in bioactive cultures (left) and sterile cultures (right). Data manually fitted to demonstrated data trend. M e asu re d C o n c. (n g / m L) 8 : 2 F T C A f r o mCh8 : a2 rF tT TO iHtleB i o a c t i v e C u l t u r e s 8 : 2 F T U C A f r o Cm h8 a: 2rtF TT iO tHleB i o a c t i v e C u l t u r e s 140 18 M e asu re d C o n c. (n g / m L) 120 16 14 100 itle 80 itle1102 isT x 60 isT x 8 A A6 40 4 20 2 0 0 0 20 40 60 80 100 0 20 40 60 80 100 D ays Axis Title ADDxiaasyy Tss itle Figure 18. The 8:2 FTCA and 8:2 FTUCA formed in bioactive cultures dosed with 8:2 FTOH . Data manually fitted for appearance. Study Report: E05-0544 Page 70 of 101 3M ENVIRONMENTAL LABORATORY PROJECT NO. E05-0544 The initial biotransformation rate for 8:2 FTOH was calculated based on the cumulative formation of 8:2 FTCA and 8:2 FTUCA which only formed in the bioactive cultures. The summed values (pmole) were plotted versus incubation time (days) and fit with a line over the first 20 days (Figure 19). The slope of the fitted line was used to assign the rate of degradation. That initial rate was calculated at 23.6 pmole per day and equated to 0.413 pmole/day per mg sludge (dw). A t1/2 value was estimated at 275 days based on initial rate. Calculations were performed as shown in Appendix A. Figure 19. The linear curve fit of total product formed as 8:2 FTCA plus 8:2 FTUCA (pmole) formed in cultures from 8:2 FTOH. Study Report: E05-0544 Page 71 of 101 3M ENVIRONMENTAL LABORATORY PROJECT NO. E05-0544 Table 18. Analyte concentrations measured in active and sterile cultures dosed with 8:2 FTOH Culture ID E05-0544-545 E05-0544-546 E05-0544-547 E05-0544-548 E05-0544-549 E05-0544-550 E05-0544-551 E05-0544-552 E05-0544-553 E05-0544-554 E05-0544-555 E05-0544-556 E05-0544-557 E05-0544-558 E05-0544-559 E05-0544-560 E05-0544-561 E05-0544-562 E05-0544-563 E05-0544-564 E05-0544-565 E05-0544-566 E05-0544-567 E05-0544-568 Culture Type Active Sludge with 8:2 FTOH Active Sludge with 8:2 FTOH Active Sludge with 8:2 FTOH Active Sludge with 8:2 FTOH Active Sludge with 8:2 FTOH Active Sludge with 8:2 FTOH Active Sludge with 8:2 FTOH Active Sludge with 8:2 FTOH Active Sludge with 8:2 FTOH Active Sludge with 8:2 FTOH Active Sludge with 8:2 FTOH Active Sludge with 8:2 FTOH Active Sludge with 8:2 FTOH Active Sludge with 8:2 FTOH Active Sludge with 8:2 FTOH Sterile Sludge with 8:2 FTOH Sterile Sludge with 8:2 FTOH Sterile Sludge with 8:2 FTOH Sterile Sludge with 8:2 FTOH Sterile Sludge with 8:2 FTOH Sterile Sludge with 8:2 FTOH Sterile Sludge with 8:2 FTOH Sterile Sludge with 8:2 FTOH Sterile Sludge with 8:2 FTOH Incubation Time (days) 0 0 0 10 10 10 20 20 20 74 74 74 94 94 94 0 0 0 10 10 10 20 20 20 8:2 FTOH (GC/MS) 1050 873 1100 838 785 674 743 769 547 519 345 385 439 443 342 1110 1200 1170 1090 984 1030 1550 819 855 8:2 FTOH (LC/MS) 1030 [b] 857 1010 [b] 820 760 551 711 734 572 549 305 442 532 586 308 1040 [b] 1340 [b] 1290 [b] 1090 [b] 1030 [b] 1110 [b] 1240 [b] 1100 [b] 1020 [b] 8:2 FTCA < 5.00 < 5.00 < 5.00 16.9 18.1 17.1 39.2 37.8 32.9 78.0 53.9 59.6 93.5 117 54.0 < 5.00 < 5.00 < 5.00 < 5.00 < 5.00 < 5.00 < 5.00 < 5.00 < 5.00 Concentration (ng/mL) 8:2 FTUCA PFHxA < 1.00 < 1.00 < 1.00 3.86 4.19 2.76 7.39 7.10 4.59 10.6 7.02 7.75 11.7 15.6 7.76 < 1.00 < 1.00 < 1.00 < 1.00 < 1.00 < 1.00 < 1.00 < 1.00 < 1.00 < 1.00 < 1.00 < 1.00 < 1.00 < 1.00 < 1.00 < 1.00 < 1.00 < 1.00 < 1.00 < 1.00 < 1.00 < 1.00 < 1.00 < 1.00 < 1.00 < 1.00 < 1.00 < 1.00 < 1.00 < 1.00 < 1.00 < 1.00 < 1.00 PFHpA < 1.00 1.76 [a] 1.20 [a] < 1.00 < 1.00 1.18 < 1.00 < 1.00 < 1.00 < 1.00 < 1.00 < 1.00 < 1.00 < 1.00 < 1.00 < 1.00 < 1.00 < 1.00 < 1.00 < 1.00 < 1.00 < 1.00 < 1.00 < 1.00 PFOA < 25.0 < 25.0 < 25.0 < 25.0 < 25.0 < 25.0 < 25.0 < 25.0 < 25.0 < 25.0 < 25.0 < 25.0 < 25.0 < 25.0 < 25.0 < 25.0 < 25.0 < 25.0 < 25.0 < 25.0 < 25.0 < 25.0 < 25.0 < 25.0 PFNA < 1.00 < 1.00 < 1.00 < 1.00 < 1.00 < 1.00 < 1.00 < 1.00 < 1.00 < 1.00 < 1.00 < 1.00 < 1.00 < 1.00 < 1.00 < 1.00 < 1.00 < 1.00 < 1.00 < 1.00 < 1.00 < 1.00 < 1.00 < 1.00 Study Report: E05-0544 Page 72 of 101 3M ENVIRONMENTAL LABORATORY PROJECT NO. E05-0544 Table 18. Analyte concentrations measured in active and sterile cultures dosed with 8:2 FTOH Culture ID Culture Type Incubation Time (days) 8:2 FTOH (GC/MS) 8:2 FTOH (LC/MS) 8:2 FTCA Concentration (ng/mL) 8:2 FTUCA PFHxA PFHpA PFOA PFNA E05-0544-569 Sterile Sludge with 8:2 FTOH 74 639 848 < 5.00 < 1.00 < 1.00 < 1.00 < 25.0 < 1.00 E05-0544-570 Sterile Sludge with 8:2 FTOH 74 595 866 < 5.00 < 1.00 < 1.00 < 1.00 < 25.0 < 1.00 E05-0544-571 Sterile Sludge with 8:2 FTOH 74 382 455 < 5.00 < 1.00 < 1.00 < 1.00 < 25.0 < 1.00 E05-0544-572 Sterile Sludge with 8:2 FTOH 94 627 875 < 5.00 < 1.00 < 1.00 < 1.00 < 25.0 < 1.00 E05-0544-573 Sterile Sludge with 8:2 FTOH 94 588 863 < 5.00 < 1.00 < 1.00 < 1.00 < 25.0 < 1.00 E05-0544-574 Sterile Sludge with 8:2 FTOH 94 605 798 < 5.00 < 1.00 < 1.00 < 1.00 < 25.0 < 1.00 Note: Concentrations of 8:2 FTOH in dosed cultures were determined in GC/MS analytical run #24 in Table 8. Later, a LC/MS/MS method was developed for 8:2 FTOH and was determined in LC/MS/MS analytical run #28 of Table 8, but that data only used as confirmation of GC/MS results. Anticipated soluble biotransformation products were quantified and reported from LC/MS/MS analytical runs #18 and #28 of Table 8. Culture extracts were analyzed against a set of matrix matched calibration standards. Calibration curves contained minimally six data points. Quantitation of 8:2 FTOH by GC/MS was done by external standard method and calibration curve fit was quadratic with 1/x weighting. Soluble analytes determined by LC/MS/MS were quantified by internal standard method and curve fits were quadratic with 1/x weighting. Calibration accuracy for standards included in the curve fit were within 100 + 25% (+ 30% at LLOQ). For analytical runs #18 and #24, CCVs were a low and mid-level QCs (40.0 ng/mL and 400 ng/mL) and were injected approximately every 15 samples, and results were within 100 + 30%. For analytical run #28, CCVs were the re-injection of the 250 ng/mL calibration standards approximately every 15 samples, and result were within 100 + 30%. Results shown are rounded to 3 significant figures while more precise values were used in data calculations. Associated bioactive and sterile sludge blanks analyzed with the samples were all below the LLOQ for all analytes. LLOQs were 100 ng/mL for 8:2 FTOH, and 5.00 ng/mL for 8:2 FTCA, 1.00 ng/mL for 8:2 FTUCA, 1.00 ng/mL for PFNA, PFHxA and PFHpA, and 25.0 ng/mL for PFOA. 8:2 FTOH culture extracts for day 0, 10 and 20 were additionally analyzed for 6:2 FTCA and 6:2 FTUCA in analytical run #18, but those data not reported in the table for brevity, those results were < 10 ng/mL. [a] PFHpA was detected slightly above the LLOQ of 1.00 ng/mL on day 0. [b] Values were determined by extrapolation of the curve beyond the upper limit of quantitation (ULOQ) of 1000 ng/mL (used only as confirmatory results). Study Report: E05-0544 Page 73 of 101 3M ENVIRONMENTAL LABORATORY PROJECT NO. E05-0544 4.2.10 246-TCP Results The 2,4,6-trichlorophenol (246-TCP) was tested because chlorophenols are amenable to LC/MS/MS quantitation by electrospray ionization and negative ion mode, similar to PFASs, and the anaerobic biotransformation of 246-TCP had been described as only occurring under methanogenic conditions using municipal anaerobic digester sludge granules (Mohn & Kennedy 1992.). Also, the biotransformation results in formation of 4-chlorophenol (4-CP) as a stable end product which can also be quantified by LC/MS/MS. It was expected that 246-TCP would undergo sequential reductive dehalogenations in the ortho positions of the molecule to form 4-chlorophenol if the cultures were methanogenic. In this study, the 246-TCP was dosed into active-sludge and sterilized-sludge cultures at 1220 ng/mL and were then incubated anaerobically for up to 15 days. Duplicate cultures were established per culture type and were then incubated and collected on days 0, 5, 7, and 15. Additionally, two active and two sterile cultures were prepared with a dose of 246-TCP that was at 2X-concentrated as the other cultures (2440 ng/mL) and those were incubated for 47-days. Quantitative LC/MS/MS analysis for 246-TCP, and the anticipated ortho-dechlorination products 2,4-dichlorophenol (24-DCP) and 4-CP, showed complete loss of the 246-TCP from active cultures by day-5, the first time point, transient formation of 24-DCP, and ultimately stoichiometric formation of 4-CP (Table 19). The 246-TCP did not degrade in the sterilesludge controls and 4-CP was not formed. The anticipated intermediate product 24-DCP was transiently observed in active cultures, but was below the LLOQ of 100 ng/mL. The 2Xconcentrated (2440 ng/mL) cultures collected on day-47 showed loss of 246-TCP in bioactive cultures and resulted in formation of 2X levels of 4-CP (avg. 2310 ng/mL). The 246-TCP was not biodegraded in sterile control cultures. The 4-CP product accumulated in the anaerobic bioactive cultures dosed with 246-TCP and corroborates the findings of Mohn & Kennedy who showed that 4-CP was the primary product from 246-TCP incubation with acclimated sludge granules and that the 4-CP product was recalcitrant to further anaerobic biotransformation. However, in contrast to their study, the anaerobic digester sludge used here did not appear to require acclimation to 246-TCP to be able to degrade it. The lack of an acclimation period may be due to the additional inclusion of yeast extract nutrients to the cultures or may reflect the different sources of anaerobic sludge used or could be due to the lower, possibly less toxic dose evaluated here Study Report: E05-0544 Page 74 of 101 3M ENVIRONMENTAL LABORATORY PROJECT NO. E05-0544 versus the study by Mohn & Kennedy. The 246-TCP is less toxic than PCP (Vallecillo et al., 1999), and has a determined IC50 of 7900 and 16000 ng/mL for anaerobic Nitrosomonas sp. and methanogens, respectively (Blum and Speece, 1991). This likely explains why 246-TCP was a better positive control substance in this study than PCP. Mass balance determined for 4-CP on days 5, 7 and 15 ranged from 130 mole% to 165 mole% (relative to dose level of 1220 ng/mL 246-TCP). These high mass balance values could be due to enhanced signal response for 4-CP due to matrix effects in bioactive sludge versus calibration standards which were prepared in sterilized sludge. 2 4 6 - T C P B iTo Ca Pc t i v e C u l t u r e s 4 - C4P-CB Pi o Fa oc tri mv e eCdu l t u r e s 1400 1400 1200 1200 1000 1000 itle 800 itle 800 isT isT Ax 600 Ax 600 400 400 200 200 0 0 2 4 6 8 10 12 14 16 AxD isa Ty its le 0 0 2 4 6 8 10 12 14 16 AxD isa Ty istle Figure 20. The loss of 246-TCP in bioactive cultures (left) and formation of 4-CP (right) for cultures dosed at 1220 ng/mL 246-TCP. Data manually fitted for appearance. C o n c e n tr a tio n (n g / m L ) C o n c e n tr a tio n (n g / m L ) Study Report: E05-0544 Page 75 of 101 3M ENVIRONMENTAL LABORATORY PROJECT NO. E05-0544 Table 19. Analyte concentrations measured in active and sterile cultures dosed with 246-TCP Culture ID E05-0544-221 Culture Type Active Sludge with 246-TCP Incubation Time (days) 0 Concentration (ng/mL) 246-TCP 24-DCP 4-CP 1280 < 100 < 100 E05-0544-222 Active Sludge with 246-TCP 0 975 < 100 < 100 E05-0544-223 Active Sludge with 246-TCP 5 < 100 < 100 1070 E05-0544-224 Active Sludge with 246-TCP 5 < 100 < 100 1120 E05-0544-225 Active Sludge with 246-TCP 7 < 100 < 100 1040 E05-0544-226 Active Sludge with 246-TCP 7 < 100 < 100 1320 E05-0544-227 Active Sludge with 246-TCP 15 < 100 < 100 1140 E05-0544-228 Active Sludge with 246-TCP 15 < 100 < 100 1180 E05-0544-229 Active Sludge with 246-TCP 47 [a] < 100 < 100 2250 E05-0544-230 Active Sludge with 246-TCP 47 [a] < 100 < 100 2370 E05-0544-233 Sterile Sludge with 246-TCP 0 1170 < 100 < 100 E05-0544-234 Sterile Sludge with 246-TCP 5 1220 < 100 < 100 E05-0544-235 Sterile Sludge with 246-TCP 5 1140 < 100 < 100 E05-0544-236 Sterile Sludge with 246-TCP 7 1190 < 100 < 100 E05-0544-237 Sterile Sludge with 246-TCP 7 975 < 100 < 100 E05-0544-238 Sterile Sludge with 246-TCP 15 1120 < 100 < 100 E05-0544-239 Sterile Sludge with 246-TCP 15 1140 < 100 < 100 E05-0544-240 Sterile Sludge with 246-TCP 47 [a] 3060 < 100 < 100 E05-0544-241 Sterile Sludge with 246-TCP 47 [a] 2850 < 100 < 100 Note: Concentrations of 246-TCP, 24-DCP and 4-CP determined by LC/MS/MS in analytical run #12 of Table 8. Culture extracts were analyzed against a set of matrix matched calibration standards. Calibration curves contained minimally six data points. Quantitation was by external standard method and calibration curve fit was quadratic with 1/x weighting. Calibration accuracy for standards included in the curve fit were within 100 + 25% (+ 30% at LLOQ). CCVs were the re-injection of the 800 ng/mL calibration standards approximately every 15 samples, and result were within 100 + 30%. Results shown are rounded to 3 significant figures while more precise values were used in data calculations. Associated bioactive and sterile sludge blanks analyzed with the samples were all below the LLOQ for all analytes. LLOQs were 100 ng/mL for 246-TCP, 24-DCP and 4-CP [a] Indicates the cultures were dosed at 2440 ng/mL (2x the normal dose concentration of the other cultures). Study Report: E05-0544 Page 76 of 101 3M ENVIRONMENTAL LABORATORY PROJECT NO. E05-0544 4.2.11 PCP Results Pentachlorophenol (PCP) was dosed into cultures at 2070 ng/mL and cultures incubated anaerobically for up to 74 days (collected on days 0, 3, 10, 20 and 74). Quantitative LC/MS/MS analysis with chromatographic separation of chlorinated phenol analytes on a C8 reversed-phase column was achieved. Calibration standards were prepared with reference substances PCP, 2,3,5,6-tetrachlorophenol (2356-TeCP), 246-TCP, 24-DCP and 4-CP. PCP appeared to require an acclimation period of approximately 10 days before significant biodegradation occurred. The PCP did not biodegrade in the sterile-sludge controls. As early as day 10, a major TeCP with peak retention times of 7.2 minutes and minor TeCP peak at 6.6 minutes were observed to form. The TeCP peaks were different retention time than the 2356-TeCP reference substance (6.5 min) and were tentatively assigned as 2,3,4,5tetrachlorophenol (2345-TeCP) as major product and 2,3,4,6-tetrachlorophenol (2346-TeCP) as a minor product. In addition, a trichlorophenol peak was observed at 7.2 minutes, but did not share the same retention time with 246-TCP reference (7.0 min). This peak was tentatively identified as 3,4,5-trichlorophenol (345-TCP). Identities of the chlorinated phenols were assigned based on previous identification of PCP metabolites in methanogenic cultures by Mohn & Kennedy (1992) and Woods et al. (1989). The biodegradation of PCP in the bioactive cultures by day 74 was apparent, but was not consistent amongst the replicates. For example, one culture showed near complete loss of PCP with formation of 2345-TeCP and 345-TCP, while another showed about 50% loss of the PCP, and the third replicate showed very little degradation of PCP. The semi-quantitative results for 2345-TeCP and 345-TCP, determined using surrogate calibration with 2356-TeCP and 246-TCP, respectively, gave results higher than the PCP dose concentration (i.e. > 100% mass balance), indicating the 2345-TeCP and 345-TCP give MS/MS signal responses greater than the 2356-TeCP and 246-TCP reference substances, respectively. Study Report: E05-0544 Page 77 of 101 C o n c e n tr a tio n (n g / m L ) 3M ENVIRONMENTAL LABORATORY PROJECT NO. E05-0544 P C P B Ci ho aac rt ti v Te itC leu l t u r e s 3000 P C P CS the ar irl te TC iutllteu r e s 3000 C o n c e n tr a tio n (n g / m L ) 2500 2500 itle2000 itle2000 isT1500 isT1500 Ax Ax 1000 1000 500 500 0 0 10 20 30 40 50 60 70 80 D ays Axis Title 0 0 10 20 30 D a 4y0s 50 60 70 80 Axis Title Figure 21. The measured PCP concentrations in bioactive cultures (left) and sterile cultures (right). Data manually fitted for appearance. 2 3 4 5 - 2T 3e 4C P5-i Tn eB Ci Po a c t i v e C u l t u r e 4000 3500 3000 itle2500 isT2000 Ax 1500 1000 500 0 0 10 20 30 40 50 60 70 80 AxDisa Ty its le C o n c e n tr a tio n (n g / m L ) 3 4 5 - T C P i n34B i5o -aTc Ct i Pv e C u l t u r e 7000 6000 5000 itle4000 isT Ax 3000 2000 1000 0 0 10 20 30 40 50 60 70 80 AxD isa Ty its le Figure 22. The measured 345-TCP (left) and 2345-TeCP (right) concentrations in bioactive PCP cultures; quantified from surrogate calibration using 246-TCP and 2356-TeCP calibration. Data manually fitted for appearance. C o n c e n tr a tio n (n g / m L ) Study Report: E05-0544 Page 78 of 101 3M ENVIRONMENTAL LABORATORY PROJECT NO. E05-0544 Table 20. Analyte concentrations measured in active and sterile cultures incubated with PCP Culture ID E05-0544-665 E05-0544-666 E05-0544-667 E05-0544-668 E05-0544-669 E05-0544-670 E05-0544-671 E05-0544-672 E05-0544-673 E05-0544-674 E05-0544-675 E05-0544-676 E05-0544-677 E05-0544-678 E05-0544-679 E05-0544-680 E05-0544-681 E05-0544-682 E05-0544-683 E05-0544-684 E05-0544-685 E05-0544-686 E05-0544-687 E05-0544-688 E05-0544-689 Culture Type Active Sludge with PCP Active Sludge with PCP Active Sludge with PCP Active Sludge with PCP Active Sludge with PCP Active Sludge with PCP Active Sludge with PCP Active Sludge with PCP Active Sludge with PCP Active Sludge with PCP Active Sludge with PCP Active Sludge with PCP Active Sludge with PCP Active Sludge with PCP Active Sludge with PCP Sterile Sludge with PCP Sterile Sludge with PCP Sterile Sludge with PCP Sterile Sludge with PCP Sterile Sludge with PCP Sterile Sludge with PCP Sterile Sludge with PCP Sterile Sludge with PCP Sterile Sludge with PCP Sterile Sludge with PCP Incubation Time (days) 0 0 0 3 3 3 10 10 10 20 20 20 74 74 74 0 0 0 3 3 3 10 10 10 20 PCP [a] 2460 2200 2310 2120 2050 2150 2020 2100 2310 2140 2070 1670 1940 219 246 1930 1990 1910 1950 1840 2060 1860 1960 1910 1930 2356-TeCP [a] < 100 < 100 < 100 < 100 < 100 < 100 < 100 < 100 < 100 < 100 < 100 < 100 < 100 < 100 < 100 < 100 < 100 < 100 < 100 < 100 < 100 < 100 < 100 < 100 < 100 2345-TeCP [b] < 100 < 100 < 100 < 100 < 100 < 100 168 168 181 231 249 1380 305 3460 2640 < 100 < 100 < 100 < 100 < 100 < 100 < 100 < 100 < 100 < 100 Concentration (ng/mL) 2346-TeCP [b] 246-TCP [a] < 100 < 100 < 100 < 100 < 100 < 100 < 100 < 100 < 100 < 100 < 100 < 100 < 100 < 100 < 100 < 100 < 100 < 100 < 100 < 100 < 100 < 100 < 100 < 100 < 100 <100 <100 <100 <100 <100 <100 <100 <100 <100 <100 <100 <100 <100 <100 <100 <100 <100 <100 <100 <100 <100 <100 <100 <100 <100 345-TCP [c] < 100 < 100 < 100 < 100 < 100 < 100 < 100 < 100 < 100 < 100 114 388 136 4460 6100 < 100 < 100 < 100 < 100 < 100 < 100 < 100 < 100 < 100 < 100 24-DCP [a] <100 <100 <100 <100 <100 <100 <100 <100 <100 <100 <100 <100 <100 <100 <100 <100 <100 <100 <100 <100 <100 <100 <100 <100 <100 4-CP [a] <100 <100 <100 <100 <100 <100 <100 <100 <100 <100 <100 <100 <100 <100 <100 <100 <100 <100 <100 <100 <100 <100 <100 <100 <100 Study Report: E05-0544 Page 79 of 101 3M ENVIRONMENTAL LABORATORY PROJECT NO. E05-0544 Table 20. Analyte concentrations measured in active and sterile cultures incubated with PCP Culture ID Culture Type Incubation Time (days) PCP [a] 2356-TeCP [a] 2345-TeCP [b] Concentration (ng/mL) 2346-TeCP [b] 246-TCP [a] 345-TCP [c] 24-DCP [a] 4-CP [a] E05-0544-690 Sterile Sludge with PCP 20 1940 < 100 < 100 < 100 <100 < 100 <100 <100 E05-0544-691 Sterile Sludge with PCP 20 1870 < 100 < 100 < 100 <100 < 100 <100 <100 E05-0544-692 Sterile Sludge with PCP 74 2000 < 100 < 100 < 100 <100 < 100 <100 <100 E05-0544-693 Sterile Sludge with PCP 74 2220 < 100 < 100 < 100 <100 < 100 <100 <100 E05-0544-694 Sterile Sludge with PCP 74 2140 < 100 < 100 < 100 <100 < 100 <100 <100 Note: Concentrations of phenolic target analyzed were determined by LC/MS/MS in analytical run # 27 of Table 8. Extracts for days 0, 3, 10 and 20 were also analyzed in analytical run #16 but that data not reported due to unusually high PCP results and because the entire set of cultures were analyzed together in run #27 making the data more cohesive. Culture extracts in run #27 were analyzed against a set of matrix matched calibration standards. Calibration curves contained minimally six data points. Quantitation was by internal standard method using PFHS as IS, and calibration curve fit was quadratic with 1/x weighting. Calibration accuracy for standards included in the curve fit were within 100 + 25% (+ 30% at LLOQ). CCVs were the re-injection of the 600 ng/mL calibration standard approximately every 15 samples, and result were within 100 + 30%. Results shown are rounded to 3 significant figures while more precise values were used in data calculations. Associated bioactive and sterile sludge blanks analyzed with the samples were all below the LLOQ for all analytes. [a] indicates the substance was a commercial reference substance included in the calibration standards [b] Indicates the substance was quantified from surrogate calibration using response of 2356-TeCP reference (i.e. semi-quantitative results). [c] Indicates the substance was quantified from surrogate calibration using response of 246-TCP reference (i.e. semi-quantitative results). LLOQs were 100 ng/mL for PCP, 2356-TeCP, 246-TCP, 24-DCP and 4-CP. Study Report: E05-0544 Page 80 of 101 3M ENVIRONMENTAL LABORATORY PROJECT NO. E05-0544 5 RESULTS SYNOPSIS Anaerobic methanogenic test conditions were successfully established as demonstrated by changes in gas pressure (measured by change in gas volume) in sealed cultures over the course of the experiment. Additionally, the composition of the gas was confirmed as primarily methane and CO2 by gas phase FTIR analysis. Gas formation appeared to be stimulated by the presence of methanol, which was coincidentally added as the test substance solvent, but was not similarly stimulated in cultures that did not receive test substance. This increased gas production likely resulted from degradation of the methanol which likely was converted to form methane and/or carbon dioxide. Gas production in cultures did not appear to be inhibited by any of the tested fluorochemical substances at the nominal concentration of 1 mg/L. Cultures appeared to additionally contain a sulfate-reducing microbial community based on the observation of reduced hydrogen sulfide and alkyl-sulfide formation in the headspace of active cultures by GC/MS analysis. The existence of a sulfate-reducing microbial community might explain the loss of gas pressure in later time points as a result of possible methane oxidation by anaerobic methanotrophic bacteria that typically cohabitate with sulfate reducing consortia in anaerobic environments (Hanson and Hanson, 1996). The substances 246-TCP and PCP were tested as potential positive control substrates for anaerobic biodegradation testing. The levels of PCP and 246-TCP, and their anticipated biotransformation products, were monitored by electrospray LC/MS/MS in negative ion mode. The 246-TCP biotransformed rapidly and was completely removed within 5 days, without any need for acclimation. The loss of 246-TCP occurred with transient formation of 24-DCP on day 15, and ultimately formation of only 4-CP on day 47. The identification of 24-DCP (transient) and 4-CP (final product) are consistent with the findings of previous work (Mohn & Kennedy, 1992). However, in that previous work, the authors showed that 246-TCP anaerobic biodegradation required acclimation of the sludge to 246-TCP before degradation occurred, whereas in this study acclimation of the sludge did not appear to be required. This could be due to the fact that in the previous study the authors tested 246-TCP at between 0.050 mM (9800 ng/ml) to 1.75 mM (346,000 ng/mL), levels that exceed the toxicity of 246-TCP to anaerobic bacteria; the IC50 for 246-TCP is 7900 ng/mL for methanogens and is 16000 ng/mL for anaerobic Nitrosomonas sp. The toxicity of the lesser halogenated phenols 24-DCP and 4-CP is significantly less than 246-TCP (Valecillo et al. 1999). The dose levels tested in this study were well below the IC50 concentrations, therefore the acclimation requirement in that pervious work could have been due to a toxicity effect which did not occur in this study. The incubations with PCP showed that it was successfully biodegraded over 74 days, and did not biodegrade in the sterile-sludge controls. However, there was an apparent lag phase of ~10 days before PCP degradation occurred. Biotransformation reactions appeared to be Study Report: E05-0544 Page 81 of 101 3M ENVIRONMENTAL LABORATORY PROJECT NO. E05-0544 consistent with the reactions for 246-TCP, with reductive dehalogenation occurring at ortho positions to the hydroxyl group to yield 2345-TeCP and 345-TCP as tentatively identified major products; while dehalogenation in the meta and para position did not occur significantly occur. In contrast to 246-TCP results, the biodegradation of PCP in active cultures was not consistent for all culture replicates on day 74. For example, one culture showed near complete loss of PCP with formation of 2345-TeCP and 345-TCP, while another showed about 50% loss of the PCP, and the third replicate showed very little degradation of PCP. These results indicate the need for acclimated sludge to efficiently degrade PCP and that PCP may not serve as a good positive control substance for that reason. While it was speculated that PCP degradation proceeded by sequential ortho-dehalogenation to 2345-TeCP and then 345-TCP, as previously described by Woods et al. (1989) and Miksell & Boyd (1992), the identities of the products were not verified due to the lack of authentic reference substances for those two compounds. The lack of 2345-TeCP and 345TCP references resulted in quantitation using surrogate calibration with 2356-TeCP and 246TCP references, and which resulted in artificially high concentration values for 2345-TeCP and 345-TCP, and shown in Table 20. It had been previously shown that PCP reductive dehalogenation only occurs under highly reducing environments (i.e. low redox conditions) where methanogenic or sulfate-reducing conditions exist (Hendriksen, et al. 1992). This work corroborates the work of other researchers and verifies that methanogenic cultures were successfully established in this study since PCP was reductively dechlorinated as expected. However, based on the results of this study, PCP was a poor positive control substances for these types of tests due to the need for acclimation, the formation of more than one product, and the inconsistent degradation results amongst replicates on day 74. These results were likely due to toxicity effects of PCP at the dosed concentration, as well as the concentrations of biotransformation products which formed in the cultures. Previous studies have shown that PCP is toxic to anaerobic Nitrosomonas sp. and methanogens at relative low levels with IC50 values of 60 ng/mL and 40 ng/mL, respectively (Blum and Speece, 1991). Also, the TeCPs are toxic at relative low levels, with IC50 values for 2356-TeCP at 1300 ng/mL and 130 ng/mL for anaerobic Nitrosomonas sp. and methanogens, respectively. Study Report: E05-0544 Page 82 of 101 3M ENVIRONMENTAL LABORATORY PROJECT NO. E05-0544 Both 246-TCP and PCP were biotransformed during this study via reductive dehalogenation reactions. Based on the tentative identification of the metabolites, the degradation pathways for 246-TCP and PCP are shown in Figure 23. PCP OH Cl Cl Cl Cl Cl OH Cl Cl Cl Cl OH Cl Cl Cl OH OH OH Cl Cl Cl 2,4,6-TCP Cl Cl Cl Figure 23. The anaerobic biotransformation pathways of PCP (top) and 246-TCP (bottom), as observed in this study (E05-0544). Study Report: E05-0544 Page 83 of 101 3M ENVIRONMENTAL LABORATORY PROJECT NO. E05-0544 Four of the nine tested PFASs in this study showed evidence of biodegradation, and all four were PFAS alcohols used as synthetic building blocks for fluorinated surfactants and fluorinated polymer protectant products. These included EtFOSE, MeFBSE, 6:2-FTOH and 8:2 FTOH. The five that did not biodegraded were PFOS, PFBS, PFOA, PFBA and 6:2 FTS. EtFOSE was biotransformed with the slowest rate of the four alcohols which degraded during this study, and approximately 2.66 mole% biotransformed to 2[(Nethyl)perfluorooctane-sulfonamido]acetate (EtFOSAA) and perfluorooctane sulfinate (PFOSi) after 108 days. The anaerobic degradation pathway for EtFOSE, as discerned from this study, is shown in Figure 24. The results are interesting because the EtFOSE terminal biotransformation products derived from anaerobic incubations are considered transient intermediate products during aerobic biodegradation of EtFOSE, and which ultimately degrades to PFOS under aerobic conditions (Lange 2000, 2001a, Rhoads 2008, Boulanger et al. 2005). Rhoads et al. (2008) concluded that EtFOSE does not biodegrade under anaerobic conditions, however, the findings from this study show differently and demonstrated that EtFOSE does biotransform anaerobically, albeit very slowly. The difference in results could be due to the very slow rate EtFOSE degradation, which could have been missed due to analytical limits or possibly differences from using sludge from other sources. The findings of this study do help explain the detection of EtFOSAA and PFOSi in environmental samples expected to have low oxygen such as wastewater treatment effluents (Boulanger et al. 2005, Higgins et al. 2007), marine sediments (Higgins e.t. al.2005, Benskin et al. 2013), biosolids (Sepulvado et al. 2011), and landfill leachates (Allred et al. 2015). Based on chemical similarity, 2-[(N-methyl)perfluorooctanesulfonamido]ethanol (MeFOSE) is expected to biodegrade anaerobically via the same reactions to form PFOSi and the analogous 2-[(Nmethyl)perfluorooctanesulfonamido]acetate (MeFOSAA). Study Report: E05-0544 Page 84 of 101 3M ENVIRONMENTAL LABORATORY PROJECT NO. E05-0544 F F F F F FF FF F F F F F F O F S F O N CH3 EtFOSE OH EtFOSAA (major) F F F F F F F F F F F FF F F O F F F F S F O N CH3 O F F F F F OH F FF FF F F OH F S F O PFOSi (minor) Figure 24. The anaerobic biotransformation pathway of EtFOSE observed (study E05-0544) Study Report: E05-0544 Page 85 of 101 3M ENVIRONMENTAL LABORATORY PROJECT NO. E05-0544 MeFBSE biodegraded with the fastest rate of the four PFAS alcohols that biodegraded during this study. After 108 days of incubation, approximately 75 mole% of the dosed MeFBSE was lost and with concomitant formation of two products, MeFBSAA and PFBSi. The degradation rate, determined as the rate of total product formed (pmole) per day per mg sludge (dw), was approximately 45-times faster than the rate determined for the slowest degraded PFAS alcohol, EtFOSE (Table 21). Anaerobic degradation of MeFBSE did not form any other products such as MeFBSA, FBSA, PFBS or PFBA, several of which do form during aerobic biodegradation of MeFBSE (Ellefson 2002b). The anaerobic degradation pathway of MeFBSE is shown in Figure 25. F FF F FF FO F F S N CH3 O MeFBSE OH MeFBSAA (major) F FF F FF FO F F S N CH3 O O F F F OH F F F F F S OH F O PFBSi (minor) Figure 25. The anaerobic biotransformation pathway for MeFBSE determined in this study (E05-0544). Study Report: E05-0544 Page 86 of 101 3M ENVIRONMENTAL LABORATORY PROJECT NO. E05-0544 The 8:2 FTOH biodegraded with the second slowest rate, but was approximately 6-times the rate determined for EtFOSE (Table 21). The anaerobic biotransformation products identified from 8:2 FTOH in this study were 8:2 FTCA (major) and 8:2 FTUCA (minor), as shown in Figure 26. Those two products, in addition to a new product 7:3 acid, were verified in 2013 by Zhang et al., also shown in Figure 26 for comparison. The lack of formation of perfluorinated carboxylates under anaerobic conditions, but previously identified as significant products from aerobic biodegradation (Lange. 2001 and Dinglasan et al. 2004), suggests that the formation of perfluorinated carboxylates from FTOHs may be the strictly confined to aerobic environments and likely catalyzed by microbial O2-requiring enzymes. F F F F F F F F OH F F F F F F F F F 8:2 F T O H F F F F F F F F OH F F F F F F F F F 8:2 F T O H HO F F F F F F F F O F F F F F F F F F 8:2 F T C A ( m aj o r) H O F F F F F F F F O F F F F F F F F F 8 :2 F T C A ( m aj o r) F F F F F F F F F OH F F F F F F F O 8:2 F T U C A ( m in o r) F F F F F F F F F OH F F F F F F F O 8:2 F T U C A ( m in o r) F F F F F F F H H O H F F F F F F F F H H O 7:3 a ci d ( m aj o r) Figure 26. The anaerobic biotransformation pathway for 8:2 FTOH determined in this study shown on the left (E05-0544), and primary products determined by Zhang et al. (2013) shown on right. Study Report: E05-0544 Page 87 of 101 3M ENVIRONMENTAL LABORATORY PROJECT NO. E05-0544 The 6:2 FTOH degraded at about 25-times the rate determined for EtFOSE (Table 21), but approximately the rate of MeFBSE in this study. Anaerobic biodegradation of 6:2 FTOH formed products analogous to those observed for 8:2 FTOH (Figure 27), however, while a 6:2 FTUCA product peak was observed it was not measurable above the LLOQ of 10 ng/mL. The only measurable product was the 6:2 FTCA. These products, in addition to 5:3 acid product, were verified in 2013 by Zhang et al., also shown in Figure 27 for comparison. As with 8:2 FTOH, perfluorinated carboxylates did not form from 6:2 FTOH incubations under anaerobic conditions. F F F F F F OH F F F F F F F 6:2 F T O H F F F F F F OH F F F F F F F 6:2 F T O H HO F F F F F F O F F F F F F F 6:2 F T C A ( m aj o r) HO F F F F F F O F F F F F F F 6:2 F T C A ( m aj o r) F F F F F F F OH F F F F F O 6 :2 F T U C A ( m in o r) F F F F F F F OH F F F F F O F F F F F H H O H F F F F F F H H O 5:3 A ci d ( m aj o r) Figure 27. The anaerobic biotransformation pathway for 6:2 FTOH determined in this study shown on the left (E05-0544), and primary products determined by Zhang et al. (2013) shown on right. Study Report: E05-0544 Page 88 of 101 3M ENVIRONMENTAL LABORATORY PROJECT NO. E05-0544 6 CONCLUSION/DISCUSSION Nine PFAS test substances were evaluated in this study, most of which had not been thoroughly evaluated under anaerobic conditions prior to this study in 2005-2006. Six were derived from ECF synthesis and were EtFOSE, MeFBSE, PFOS, PFBS, PFOA and PFBA and three were derived from TM synthesis (8:2 FTOH, 6:2 FTOH and 6:2 FTS). The four PFAS alcohols are common precursors for synthesis of fluorinated polymers used as protectant products and specialty fluorinated surfactants. All four of the PFAS alcohols biodegraded under anaerobic sludge conditions, but with different rates of degradation that appeared to be related to water solubility and/or molecular weight (see Table 21). In contrast, the PFAS sulfonates (PFOS, PFBS and 6:2 FTS) and PFAS carboxylates (PFOA and PFBA) were all recalcitrant to anaerobic biodegradation during this study. The formation of PFOSi and EtFOSAA anaerobically from EtFOSE, and likewise PFBSi and MeFBSAA from MeFBSE, is of importance because it shows that the sulfonamido alcohol precursors do not biotransform further to perfluorinated sulfonates (PFSAs) anaerobically, as has been observed under aerobic conditions (Lange 2001a, Ellefson 2002b). Therefore, analysis of environmental samples obtained from low oxygen environments (i.e. sediment, groundwater, sludge, etc.) may contain more sulfinate and sulfonamido acetate products than perfluorinated sulfonates and perfluorinated carboxylates. The formation and sequestration of the perfluoroalkyl sulfinates under anaerobic conditions also suggests that the biological mechanism underlying oxidation of perfluorinated sulfinates likely involves an oxidative step via O2-requiring microbes/enzymes, and may not be the result of a hydrolysis reaction. Perhalofluoroalkyl sulfinates undergo oxidation to perfluorinated carboxylates with oneelectron oxidants or by photooxidation reactions, but they can also undergo H2SO4 catalyzed acid hydrolysis to a mixture PFCAs and PFSAs (Hu et al. 1990). Fluorotelomer carboxylates (FTCAs) and unsaturated carboxylates (FTUCAs) were identified as biotransformation products of 6:2 FTOH and 8:2 FTOH anaerobic biodegradation. The same products were observed as transient intermediates during aerobic biodegradation of FTOHs (Lange 2001b, Dinglasan et al. 2004, Wang et al. 2005), but not considered end products. The more recent work of Zhang et al. (2013) has confirmed that FTCAs and FTUCAs are products from FTOH anaerobic sludge incubations, but also identified a 7:2 acid and 5:3 acid as additional significant products from 8:2 FTOH and 6:2 FTOH, respectively. In that study the authors showed that 6:2 FTCA, 6:2-FTUCA and 5:3 acid were formed from 6:2 FTOH at a ratio of approximately 8:1:4, and that 8:2 FTCA, 8:2 FTUCA and 7:3 acid formed from 8:2 FTOH with a ratio of approximately 4:1:5. Because 5:3 acid and 7:3 acid were not known at the time this study was performed in 20052006, they were not quantified. However, based on the determinate ratios by Zhang et al. Study Report: E05-0544 Page 89 of 101 3M ENVIRONMENTAL LABORATORY PROJECT NO. E05-0544 (2013), the anaerobic biodegradation rates of 6:2 FTOH and 8:2 FTOH reported herein could be up to ~ 2-times faster than reported in Table 21. Therefore, the calculated half-life for 6:2 FTOH and 8:2 FTOH in this study could be ~ one-half of the value in Table 21. Rates in this study were calculated for domestic anaerobic sludge from Empire, MN and half-lives for 6:2 FTOH and 8:2 FTOH were determined at 83.8 days and 275 days, respectively, and were based on a lack of measurement for 5:3 acid and 7:3 acid. Therefore, the half-lives could be as short as ~42 days and ~140 days, respectively. Those half-life values are in good agreement with the determined half-lives by Zhang et al. for Wilmington, DE domestic anaerobic sludge which were determined at 30 days for 6:2 FTOH and 145 days for 8:2 FTOH. The formation of FTCAs and FTUCAs as anaerobic end products is an important finding because both are known to be more toxic to aquatic organisms than perfluorinated carboxylic acids (Phillips et al. 2007) which are the anticipated penultimate end products of FTOH aerobic biodegradation. The loss of FTOHs in sterile sludge controls was observed in this study and hampered the ability to determine degradation rates based on loss of parent FTOH. However, irreversible binding of 8:2 FTOH to soils has been observed and increases with time and with organic carbon content (Liu & Lee, 2005). This phenomena has also been observed in other sludge biodegradation studies conducted since 2005-2006 (Saez et al., 2008). Wang et al. 2005 suggested loss to PTFE containers as well, and in this study the caps of the culture vessels had a PTFE liner, and therefore that may also have contributed to loss of FTOHs in the controls. Similar types of losses were not observed for any of the other tested substances in this study: EtFOSE, MeFBSE, PFOS, PFBS, PFOA, PFBA or 6:2 FTS. Prior to this study, the stability of 6:2 FTS, PFOS, PFBS, PFBA and PFOA under anaerobic conditions was not entirely clear. Although it had been speculated that they were stable, some laboratory results indicated they might not be. Unlike the PFAS alcohol test substances, the PFAS sulfonates (PFBS, PFOS and 6:2 FTS) and PFAS carboxylates (PFBA, PFOA) did not show evidence of anaerobic biodegradation under methanogenic conditions of this study. These results confirm the conclusions of Remde et al. (1996) who showed that PFOS does not biodegradable under anaerobic conditions in a modified ECETOC test over 28 days. Furthermore, a study by Hollingsworth et al. (2005) showed that PFOS did not biodegrade under methanogenic conditions or sulfate-reducing conditions for up to 45 days and 30 days of incubation, respectively, and that PFOS also did not appear to inhibit methanogenesis at up to 600 mg/L. In contrast it was reported by Schrder (2003) that PFOS was removed anaerobically within two days in anaerobic bioreactors using sewage sludge immobilized onto glass-foam beads. Additionally, they reported that PFOA was removed in 25 days in the same reactors once PFOS was removed. However, the authors did not detect any volatile fluorochemical products in the gases from the reactor by GC-ECD, nor were they able to measure any fluoride production by fluoride-selective electrode or fluorinated metabolites. Hence, biodegradation was not conclusively shown. Based on the relative Study Report: E05-0544 Page 90 of 101 3M ENVIRONMENTAL LABORATORY PROJECT NO. E05-0544 inertness of PFOS and PFOA to degradative attack in general, and lack of degradation in this study and by others, it is more likely that the loss of PFOS and PFOA observed by Schrder was due to adsorption to the test vessel or the sludge immobilized on glass-foam beads, or the glass foam beads themselves, and loss of PFOS and PFOA was likely not due to biodegradation. The anaerobic stability of 6:2 FTS was somewhat surprising given that it is biodegraded under aerobic conditions (Key et al. 1996, Wang et al. 2011). 6:2 FTS is of environmental relevance because it has been identified as a component of some brands of aqueous firefighting foams (AFFF) and is a biodegradation product of 6:2 fluorotelomer thioether amido sulfonate (FtTAoS) which is also present in some AFFF formulations (HardingMarjanovic et al. 2015) . Also, 6:2 FTS has been detected in ground water (Schultz and Barofsky, 2004) and municipal wastewater treatment plant effluents (Schultz et al. 2006). The initial rates of degradation were determined from linear plots of the concentration data for total products formed the first few weeks of incubation (i.e. during the fastest rates of degradation); with the assumption of constant saturation of enzymes/cells with test substance above the Cmax concentration needed to maintain a constant maximal rate during that period. The optimization of test substance concentration was not performed during this study and was outside the original scope, therefore all of the PFAS degradation rates were determined for a nominal test concentration of 1 g/mL. It is noteworthy that this test concentration is higher than the reported water solubility concentrations for EtFOSE and 8:2 FTOH (Flaherty 2000, and Liu & Lee 2005). Nonetheless, the measured degradation rates appeared to increase as molecular weight of the PFAS test substance decreased (Table 21), and this likely is reflective of better bioavailability of the lower molecular weight substances due to greater water solubility. Future studies could be performed to evaluate effects of varying test concentration on rates of biotransformation and biotransformation product yields, as well as to test other PFAS compounds such as perfluoroalkyl sulfonamides. Rates were based on assumption of quantifying all degradation products formed, and was an assumption which might have resulted in biased low rate estimates for 6:2 FTOH and 8:2 FTOH by a factor of ~2. Additionally, the incubation temperature may have been a factor for some data sets since modest variations in ambient temperature were observed. For example, the mean temperature for the first 4 weeks of MeFBSE and EtFOSE incubations was ~ 24C, but for 6:2 FTOH and 8:2 FTOH, the mean temperature for the first four weeks of incubation was ~ 22C due to ambient seasonal temperature fluctuations in the laboratory. The effects of temperature variations could have had an impact on the observed rates. Additionally, adsorption to sludge likely occurred in many cases and could have affected bioavailability and biodegradation rates. This was likely a factor for the 6:2 FTOH and 8:2 FTOH where apparent irreversible binding/loss of test substance in sterile controls was observed. Study Report: E05-0544 Page 91 of 101 3M ENVIRONMENTAL LABORATORY PROJECT NO. E05-0544 Table 21. Comparison of anaerobic biodegradation rates determined Test Substance MW Water Solubility (ng/mL) Sludge Biodeg. Rate (K0) [pmole/day/mg(dw)] t1/2 [a] Rate Normalized to EtFOSE Rate EtFOSE 571 151 [c] 0.0718 1077 days 1.00 (2.95 yrs.) 8:2 FTOH 464 194 [d] 0.413 [b] 275 days 5.75 (0.753 yrs.) [b] 6:2 FTOH 364 18,800 [e] 1.76 [b] 83.8 days 24.5 (.230 yrs.) [b] MeFBSE 357 141,000 [f] 3.26 36.7 days 45.4 (0.101 yrs.) Note: Estimated rates are specific to the conditions of the test described herein and are only presented for comparing the four biotransformed PFAS alcohol test substances to each other. The biodegradation of PFAS is very dependent on the physiological conditions of the test and is influenced by presence of enzyme inducers, microbial strain types present, and level of reducing energy present (Kim et al. 2013). PFOS, PFBS, 6:2 FTS, PFOA and PFBA were stable under anaerobic conditions and are therefore excluded from the table. [a] The half-life estimates were determined based on initial rates and as described in Appendix A. [b] Degradation rates may be biased low by a factor of ~ 2 due to not measuring 5:3 acid and 7:3 acid from 6:2 FTOH and 8:2 FTOH, respectively. That product(s) were described as additional major products of anaerobic degradation of FTOHs by Zhang et al. 2013; this also may result in overestimate of half-life (t1/2) for each by a factor of ~ 2. [c] Flaherty 2000 [d] Liu & Lee, 2005. [e] Liu & Lee, 2007 [f] Ellefson 2002 Based on the results of this work it can be expected that the anaerobic end-products identified in this study may be dominant PFAS substances present in anaerobic environmental samples and that anaerobic environments may be environmental sinks for partially transformed fluorochemical precursors that are potential sources of perfluorinated sulfonates and carboxylates. It is comprehensible that if these environments are disturbed or oxygenated, that these substances may become aerobically degraded to generate perfluorinated carboxylates and perfluorinated sulfonates. Environments one might consider as anaerobic sinks for such precursors may include landfills and their associated leachates, wetlands, freshwater and marine sediments, groundwater and biosolids from WWTPs, and these findings could suggest that the limited analysis for PFOS and PFOA in environmental samples of such types may be skewed in terms of total PFOS/PFOA equivalents that are actually present. These conclusions are supported by the measurement of EtFOSAA and PFOSi as dominant PFAS in marine sediment collected from both the east and west coasts of the U.S. (Higgins et al. 2005) and also their presence in wastewater treatment plant (WWTP) sludge samples (Rhoads et al. 2005). Likewise, MeFBSAA, PFBSi, 8:2 FTCA and 8:2 FTUCA have been shown to be formed in leachate collected from anaerobic landfill Study Report: E05-0544 Page 92 of 101 3M ENVIRONMENTAL LABORATORY PROJECT NO. E05-0544 simulation chambers loaded with domestic waste solids (Allred et al. 2015). It was also shown by Sinclair and Kannan (2006) that 8:2 FTCA and 8:2 FTUCA were significant in the effluent waters of a wastewater treatment plant (WWTP) from New York State that received domestic, commercial and industrial waste. Future environmental monitoring studies testing anaerobic environmental samples should consider monitoring for anaerobic degradation products identified in this study because they may be sequestered as potential sources of PFOA and PFOS if they are transported or oxygenated waters where they will biotransform to perfluorinated carboxylates and perfluorinated sulfonates, and will be a long-term continued source of such compounds into the environment. 7 LITERATURE CITED 3M Technical Notebook ID 139716, Cleston C. Lange, issued 2005. 3M Technical Notebook ID 140568. Peter M. Radford, issued 2005. Allred, B.M., J.R. Lang, M.A Barlz, J.A Field. 2015. "Physical and biological release of polyand perfluoroalkyl substance s(PFASs) from municipal solid waste in anaerobic model landfill reactors' Environ. Sci. Technol. 49:7648-7656. BEEMS Module B7-Anaerobic Digestion; http://www.oardc.ohiostate.edu/beems/images/BEEMS_B7_Anaerobic_digestion.pdf Benskin, J.., M.G. Ikonomou, F.A.P.C. Gobas, T.H. Begley, M.B. Woudneh, J.R. Cosgrove. 2013. "Biodegradation of N-ethyl perfluorooctane sulfonamido ethanol (EtFOSE) and EtFOSE-based diester (SAmPAP Diester) in marine sediments". Environ. Sci. Technol. 47:1381-1389. Blum, D.J.W and R.E. Speece. 1991. "A database of chemical toxicity to environmental bacterial and its use in interspecies comparison and correlations". J. Water Pollut. Control Fed. 63(3):198-207. Boulanger, B. J.D. Vargo, J.L. Schnoor, K.C. Hornbuckle. 2005. "Evaluation of perfluorooctane surfactant in a wastewater treatment system and in a commercial surface protection product". Environ. Sci. Technol. 39:5524-5530/ Buck, R.C., J. Franklin, U. Berger, J.M. Conder, I.T. Cousins, P. deVoogt, A.A. Jensen, K. Kannan, S.A. Mabury, S.P.J. van Leeuwen. 2011. "Perfluoroalkyl and polyfluoroalkyl Study Report: E05-0544 Page 93 of 101 3M ENVIRONMENTAL LABORATORY PROJECT NO. E05-0544 substances in the environment: terminology, classification, and origins." Integrated Environmental Assessment and Management-SETAC. 7(4):513-541. Dinglasan, J.J.A., Y. ye, E.A. Edwards, and S. A. Mabury. 2004. "Fluorotelomer alcohol biodegradation yields poly and perfluorinated acids". Environ. Sci. Technol. 38:2857-2864 Ellefson, M.E. 2002a. "Solubility of N-MeFBSE-OH in water". 3M Environmental Laboratory Report E02-0094. Ellefson, M.E 2002b. "Aerobic biodegradation of MeFBSE-OH". 3M Environmental Laboratory Report E02-1325. Flaherty, J. 2000. "Characterization of EtFOSE-OH. Phase: solubility of EtFOSE-OH in water, methanol and acetone". 3M Environmental Laboratory Report E00-1715 Hanson, R.S. and T.E. Hanson. 1996. "Methanotrophic Bacteria". Microbiol. Rev. 60:439471 Harding-Marjanovic, K.C., E.F. Houtz, S.Yi, J.A. Field, D.L. Sedlak, L. Alvarez-Cohen. 2015. "Aerobic biotransformation of fluorotelomer thioether amido sulfonate (Lodyne) in AFFFamended microcosms". Environ. Sci. Technol. 49(13):766-7674. Hekster, F.M., R.W.P.M. Laane, and P. de Voogt. 2003. "Environmental and toxicity effects of perfluoroalkylated substances". Rev. Environ. Contam. Toxicol. 179:99-121 Higgins, C.P., Field, J.A., Criddle, C.S., and R.G. Luthy. 2005. Quantitative determination of perfluorochemicals in sediments and domestic sludge". Environ. Sci. Technol. 39(11): 39463956. Hollingsworth, J., R. Sierra-Alvarez, M. Zhou, K. L. Ogden and J. A. Field. 2005. "Anaerobic biodegradability and methanogenic toxicity of key constituents in copper chemical mechanical planarization effluents of the semiconductor industry". Chemosphere 59: 1219-1228. Hu, C., Z. Xu, W. Huang. 1990. "Reaction of perhalofluoroalkyl sufinates with one-electron transfer oxidants. A facile method for the synthesis of perhalofluorocarboxylic acids." J. Fluorine Chem. 49:433-437. Ignat'ev, N.V., U. Welz-Biermann, U.Heider., A. Kucheryna, S. vonAhsen, W. Habel, P. Sartori, H. Willner. 2003. "Carbon-chain isomerization during the electrochemical fluorination in anhydrous hydrogen fluoride-a mechanistic study". J. Fluorine Chem. 124:2137. Study Report: E05-0544 Page 94 of 101 3M ENVIRONMENTAL LABORATORY PROJECT NO. E05-0544 Lange, C.C. 2000. "The aerobic biodegradation of N-EtFOSE alcohol by the microbial activity present in municipal wastewater treatment sludge". 3M Environmental Laboratory Report E002252. Lange, C.C. 2001a. "The 18-day aerobic biodegradation study of perfluorooctanesulfonylbased chemistries". 3M Environmental Laboratory Report E01-0415 Lange, C.C. 2001b. "Biodegradation screen study for DuPont Zonyl BA-type telomer alcohol". 3M Environmental Laboratory Report E01-0684 Liu, J and L.S. Lee. 2005. "Solubility and sorption by soils of 8:2 fluorotelomer alcohol in water and cosolvent systems". Environ. Sci. Technol. 39(19):7535-7540 Liu, J and L.S. Lee. 2005. "Effect of fluorotelomer alcohol chain length on aqueous solubility and sorption by soils". Environ. Sci. Technol. 41(15):5357-5362 Liu, J. N. Wang, B. Szostek, R.C. Buck, P.K. Panciroli, P.W Folsom, L.M. Sulecki, C.A. Bellin. 2010. "6-2 Fluorotelomer alcohol aerobic biodegradation in soil and mixed bacterial culture". Chemosphere 78(4):437-444. Mah, R.A. and C. Sussman. 1967. "Microbiology of anaerobic sludge fermentation: enumeration of the non-methanogenic anaerobic bacteria". Appl. Microbiol. 16:358-361 Miksell, M.D. and S.A. Boyd. 1985. "Reductive dechlorination of the pesticides 24-D, 24,5-T and pentachlorophenol in anaerobic Sludges". J. Environ. Qual. 14:337-340 Mohn, W.W. and K.J. Kennedy. 1992. "Limited degradation of chlorophenols by anaerobic sludge granules". Appl. Env. Microbiol. 58: 2131-2136. Kim, H.M., N. Wang, K.H. Chu. 2013. "6:2 Flurotelomer alcohol (6:2 FTOH) biodegradation by multiple microbial species under different physiological conditions". Appl. Microbiol. Biotechnol. 98(4):1831-1840 Organization for Economic Cooperation and Development (OECD). 2002. "OECD Guideline for the Testing of Chemicals: Aerobic and Anaerobic Transformation in Aquatic Sediment Systems". OECD No. 308. Phillips, M.M., M.J.A. Dinglasan-Panlilio, S.A. Mabury, K.R. Solomon, P.K. Sibley. 2007. "Fluorotelomer acids are more toxic than perfluorinated acids". Environ. Sci. Technol. 41:7159-7163. Study Report: E05-0544 Page 95 of 101 3M ENVIRONMENTAL LABORATORY PROJECT NO. E05-0544 Saez, M., P. deVoogt, J.R. Parsons. 2008. "Persistence of perfluoroalkylated substances in closed bottle tests with municipal sewage sludge". Environ. Sci. Pollut. Res. 15:472-477 Schultz, M.M., D.F. Barofsky, J.A. Field. 2003. "Fluorinated Alkyl Surfactants" Env. Eng. Sci. 20:487-501 Schultz, M.M. and D.F. Barofsky. 2004. "Quantitative determination of fluorotelomer sulfonates in groundwater by LC MS/MS". Environ. Sci. Technol. 38(6):1828-1835. Schultz, M.M. and D.F. Barofsky. 2006. "Quantitative determination of fluorinated alkyl substances by large-volume-injection liquid chromatography tandem mass spectrometry characterization of municipal wastewater". Environ. Sci. Technol. 40(1):289-295. Sepulvado, J.G., Blaine, A.C., Hundal, L.S. and C.P. Higgins. 2011. "Occurrence and fate of perfluorochemicals in soil following the land application of municipal Biosolids". Environ. Sci. Technol. 45(19):8106-8112. Schrder, H.F. 2003. "Determination of fluorinated surfactants and their metabolites in sewage sludge samples by liquid chromatography with mass spectrometry and tandem mass spectrometry after pressurized liquid extraction and separation on fluorine-modified reversedphase sorbents". J. Chromatography A, 1020: 131-151 Shelton, D.R. and J.M. Tiedje. 1984. "General method for determining anaerobic biodegradation potential". Appl. Env. Microbiol. 47: 850-857 Sinclair, E. and K. Kannan. 2006. "Mass loading and fate of perfluoroalkyl surfactants in wastewater treatment plants". Environ. Sci. Technol. 40:1408-1414. United States Environmental Protection Agency (U.S.E.P.A.) 1998. Fate, Transport and Transformation Test Guidelines. OPPTS 835.3400. Anaerobic Biodegradability of Organic Chemicals. EPA 712-C-98-090 Vallecillo A. , P.A. Garcia-Encina, M. Pena. 1999. "Anaerobic biodegradability and toxicity of chlorophenols". Wat. Sci. Tech. 49(8):161-168. Wang, N., B. Szostek, P. W. Folsom, L.M. Sulecki, V. Capka, R.C. Buck, W. R. Berti, and J.T. Gannon. 2005. Aerobic Biotransformation of 14C-Labeled 8-2 Telomer B Alcohol by Activated Sludge from a Domestic Sewage Treatment Plant. Environ. Sci. Technol 39:531-538 Wang, N., J. Liu, R.C. Buck, S. H. Korzeniowski, B.W. Wolstenholme, P.W. Fosom, L.M. Sulecki. 2011. "6:2 Fluorotelomer sulfonate aerobic biotransformation in activated sludge of waste water treatment plants". Chemosphere 82(6):853-858. Study Report: E05-0544 Page 96 of 101 3M ENVIRONMENTAL LABORATORY PROJECT NO. E05-0544 Wang, Z., I.T. Cousins, M. Scheringer, K. Hungerbuhler. 2013. "Fluorinated alternatives to long-chain perfluoroalkyl carboxylic acids (PFCAs), perfluoroalkane sulfonic acids (PFSA) and their potential precursors". Environ. Int. J. 60:242-248. Woods, S. L., J.F. Ferguson and M.M. Benjamin. 1989. "Characterization of chlorophenol and chloromethoxybenzene biodegradation during anaerobic treatment". Environ. Sci. Technol. 23:62-68 Zhang, S. B. Szostek, P.K. McCausland, B.W. Wolstenhom, X. Lu, N. Wang, R.C. Buck. 2013a. "6:2 and 8:2 Fluorotelomer alcohol anaerobic biotransformation in digester sludge from a WWTP under methanogenic conditions". Env. Sci. Technol. 47:4227-4235 Zhang,S. P. W. Folsom, B.W Wolstenhome, H. Sun, N. Wang, R.C. Buck. 2013b "6:2 Fluorotelomer alcohol biotransformation in an aerobic river sediment system". Chemosphere 90(2):203-209 Study Report: E05-0544 Page 97 of 101 3M ENVIRONMENTAL LABORATORY PROJECT NO. E05-0544 8 REPORT APPROVAL Digitally signed by Cleston C. Lange DN: c=US, st=MN, l=St. Paul, ou=3M Environmental Laboratory - authenticated by LRA, email=clange@mmm.com, o=3M, cn=Cleston C. Lange Reason: I am the author of this document Date: 2016.05.12 10:05:36 -05'00' Cleston C. Lange, Ph.D., Project Lead and Report Author Date Digitally signed by William K. Reagen DN: c=US, st=MN, l=St. Paul, ou=Laboratory Director, ou=3M Environmental Laboratory authenticated by LRA, email=wkreagen@mmm.com, o=3M, cn=William K. Reagen Reason: I am approving this document Date: 2016.05.12 12:26:39 -05'00' William K. Reagen, Ph.D., Technical Director Date This study report and data were audited by the 3M Environmental Laboratory Quality Assurance Unit (QAU). QAU Representative Digitally signed by Kent R. Lindstrom DN: c=US, st=MN, l=St. Paul, ou=3M Environmental Laboratory - authenticated by LRA, email=krlindstrom@mmm.com, o=3M, cn=Kent R. Lindstrom Reason: I have reviewed this document Date: 2016.05.12 10:17:46 -05'00' Date Study Report: E05-0544 Page 98 of 101 APPENDIX A: Data Calculations 3M ENVIRONMENTAL LABORATORY PROJECT NO. E05-0544 Recovery Calculations: Recovery = [measured concentration known concentration] x 100% Calculation of picomole (pmole): Example: If MeFBSE was dosed at 976 ng/mL in a 5 mL total volume culture, and MeFBSE is 357 ng/nmole, then the picomole amount dosed is calculated as follows: [976 ng/mL] x [1 nmole/571 ng] x [5 mL] = 13.7 nmole 13.7 nmole X (1000 pmole/nmole) = 13700 pmole Hence, there is nominal 13700 pmole of MeFBSE dosed into the 5 mL culture. Study Report: E05-0544 Page 99 of 101 APPENDIX A (Continued): Data Calculations Molecular weight values used for pmole calculations were as follows: EtFOSE (571 ng/nmole) EtFOSAA (584 ng/nmole); anion MW used PFOSi (483 ng/nmole); anion MW used MeFBSE (357 ng/nmole) MeFBSAA (371 ng/nmole) PFBSi (283 ng/nmole); anion MW used 6:2 FTOH (364 ng/nmole) 6:2 FTCA (378 ng/nmole); anion MW used 6:2 FTUCA (358 ng/nmole); anion MW used 8:2 FTOH (464 ng/nmole) 8:2 FTCA (478 ng/nmole); anion MW used 8:2 FTUCA (458 ng/nmole); anion MW used PFOS (499 ng/nmole); anion MW used PFBS (299 ng/nmole); anion MW used PFOA (413 ng/nmole); anion MW used PFBA (213 ng/nmole); anion MW used 6:2 FTS (425 ng/nmole) anion MW used PCP (266 ng/nmole) 246-TCP (197 ng/nmole) 24-DCP (163 ng/nmole) 4-CP (129 ng/nmole) 3M ENVIRONMENTAL LABORATORY PROJECT NO. E05-0544 Study Report: E05-0544 Page 100 of 101 3M ENVIRONMENTAL LABORATORY PROJECT NO. E05-0544 APPENDIX A (Continued): Data Calculations Calculation of Mass Balance and Percent Degradation: Example: If there were 13700 pmole of MeFBSE dosed into a 5 mL total liquid volume culture and on day-72 there was 3380 pmole of MeFBSE remaining and 5510 pmole of MeFBSAA and 3610 pmole of PFBSi had formed, and no other analytes were detected, then the sum of total substances measured on day-72 would equal nominal 12500 pmole. The sum of total products formed would be 9120 pmole. Overall mass balance would be [12500/13700] x 100% = 91.4 mole% Percent degradation of dose would be [(9120)/13700] x 100% = 66.7 mole% Calculation of Biodegradation/Biotransformation Rates and Half-lives:. Using Excel 2007TM, plot the measured pmole amount of test substance degraded, or pmole of total products formed, versus incubation time (days). The pmole should be on the y-axis and incubation time on x-axis. Only plot the first 3 or 4 time points to create a best fit with linear equation, and exclude later time points because of quadratic response as degradation slows down. Do not force the data through the origin, use the actual day-0 values measured. In the plot, include the linear equation of the fitted line and the R2 value. The equation should be in the general format of y = mx + b. From the equation of the line, the slope of the line is the x-value in the equation and is calculated in pmole/day. Divide that rate value by the mass of sludge (dry wt.) initially added in each culture to get a sludge-dependent degradation rate in (pmole/day/mg sludge (dw). Rates were normalized to sludge content to account for the different mass of sludge (dry weight) in different culture sets and enabling comparison of rates for the different test substances that degraded. All calculations in this study were based on nominal 5 mL culture volumes containing 4 mL of sludge; the sludge total suspended solids (TSS) value used for all experiments was 14.3 mg sludge (dw) per milliliter of digester sludge. This calculates to 57.2 mg sludge (dw) added per culture. For half-life estimation (days): divide the dose at time zero (pmole) by the determined rate pmole/day, then divide that value by 2. Study Report: E05-0544 Page 101 of 101