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BACK TO MAIN Study Title Microbial Metabolism (Biodegradation) Studies of Perfluorooctane Sulfonate (PFOS) 11!. Anaerobic Sludge Biodegradation Authors William E. Gledhill, Ph.D. Barbara J. Markley, Ph.D. Study Completed On 31 October 2000 Submitted To 3M Environmental Laboratory 935 Bush Avenue, BLDG 2-3E-09 St. Paul, Minnesota 55133-3331 Performing Laboratory Springborn Laboratories, Inc. 790 Main Street Wareham, Massachusetts 02571-1075 Laboratory Project ID Springborn Study No.: 290.6120 Page 1 of 16 PFQS - Anaerobic Sludge Biodegradation SIGNATURES AND APPROVAL SUBMITTED BY: Springborn Laboratories Inc. 790 Main Street Wareham, Massachusetts 02571-1075 BACK TO MAIN Page 2 William E. Giedhill, Ph.D. Director, Environmental Fate and Microbiological Programs Date ZX / * 1 Sean P. McLaughlin y Principal Investigator h h>!eDate Barbaija' J. Markley, P h| ). Director, Chemistry lk iU Date / 1'vj Cry^, \OvyC 1 [1! Mrjorie E. Dix Date Senior Research Chemist BACK TO MAIN PFOS - Anaerobic Sludge Biodegradation_________________________________ Page 3 TABLE OF CONTENTS Page SIGNATURES AND A PPR O VA L....................................................................................................2 LIST OF TA B LE S ............................................................................................................................. 4 LIST OF F IG U R E S ........................................................................................................................... 5 1.0 INTRODUCTION ....................................................................................................................... 6 2.0 TEST SUBSTANCE, INTERNAL STANDARDS, AND SOLUTION PREPARATION ----- 7 2.1 Test Substance and internal S ta n d a rds...................................................................... 7 2.2 Preparation of Stock Solutions and Reagents ..........................................................8 2.2.1 Internal Standard Stock Solutions ................................................................ 8 2.2.2 PFOS Stock S o lu tio n ..................................................................................... 8 2.2.3 Ammonium Acetate Stock Solution ............................................................10 3.0 PREPARATION OF SAMPLES AND ANALYTICAL METHODS .................................... 10 3.1 Preparation of the Aqueous Test S a m p le s.............................................................. 10 3.2 Preparation of the Biomass S a m p le s .........................................................................10 3.3 Quality Control Sample P reparation...........................................................................10 3.4 Instrumental Conditions . . .......................................................................................... 11 4.0 TEST PROCEDURES, RESULTS, AND DISCUSSION........................................................12 4.1 Test Procedures...........................................................................................................12 4.2 Results and Discussion .............................................................................................. 13 5.0 CONCLUSIONS AND FUTURE STUDIES ...........................................................................13 REFERENCES 14 BACK TO MAIN PFOS - Anaerobic Sludge Biodegradation LIST OF TABLES Table 1. Results for test samples from the anaerobic biodegradation test Page 4 Page 15 PFOS - Anaerobic Sludge Biodegradation LIST OF FIGURES Figure 1. Flow chart of extraction procedures BACK TO MAIN Page 5 Page 16 PFOS - Anaerobic Sludge Biodegradation Page 6 1.0 INTRODUCTION The biodegradation program for perfluorooctane sulfonate (PFOS) was designed to offer a wide range of conditions to maximize the chance for selection and enrichment of microbial populations that could metabolize this chemical as well as other unique fluorochemicals. In addition to enrichment, the program was also designed to optimize for co-metabolism of fluorochemicals. This was done by continual addition of fresh inoculum and complex natural nutrients. The overall goal of these studies, therefore, was to observe loss of parent material and formation of degradation products as quantitatively as possible within the limits of the study designs. Three aerobic systems were examined: a sewage treatment based system to select for faster growing species (Zymogenous), a soil based system to select for slower growing species (Autochthonous), and a pure culture system for examining specific metabolic capabilities (Cytochrome P450monooxygenase). One anaerobic system was studied: 10% anaerobic digester sludge. The overall screening program was based on key factors to maximize the chance for enrichment of those organisms capable of metabolizing unique chemicals. Among these factors were: - testing and enrichment under non-toxic conditions - the use of natural ecosystems as the basis for enrichment - the use of natural nutrients from those ecosystems with supplemental trace minerals, co-factors and vitamins - the continual introduction of new microbes from different natural sources - the periodic replenishment of natural nutrients without diluting out the species being enriched - the provision for a realistic time frame for enrichment and acclimation - the protection of microorganisms from toxic products or metabolites by use of low substrate concentrations, replenishment of nutrients, balanced medium (C:N:P, etc.), proper pH and provision of a protective surface for growth (vermiculite, sand, soil, activated C, diatomaceous earth, etc.) - the enrichment in more concentrated (higher biomass and test substance concentration) systems and examination of biodegradation in more dilute systems - the separation of systems selective for fast growing (zymogenous) and slow growing (autochthonous) species - use of pure cultures containing the cytochrome P450 monooxygenase enzyme system known to metabolize complex molecules. Throughout the experimental program the principles outlined above for enrichment were incorporated. The results of the studies for PFOS are summarized in four separate reports and provide a basis for the future direction of the program to better understand the environmental fate BACK TO MAIN PFOS - Anaerobic Sludge Biodegradation_______________________________________ Page 7 of fluorochemicals. This report summarizes the results of exposure of PFOS to an anaerobic sludge biodegradation system. 2.0 TEST SUBSTANCE, INTERNAL STANDARDS, AND SOLUTION PREPARATION 2.1 Test Substance and Internal Standards The test substance, perfluorooctane sulfonate potassium salt (PFOS, lot no. TN-A-2130), an offwhite powder, was received on 25 January 1999 (SLI No. 70-93) from 3M Environmental, St. Paul, Minnesota. Prior to study completion, analytical characterization of the PFOS test substance was not conducted. Therefore, all calculations in the report are based on PFOS purity of 100%. After study completion, a sample of a 1.06 mg/mL PFOS stock solution (SLI No. 70-93A), see Section 2.2.2) was sent to 3M Environmental Laboratory for evaluation of impurities using LC/MS-TOF. Compounds looked for were the C2 to C10 PFOS analogous materials, and many of them were observed in both the 1-pL and 10-pL injections (e.g., masses 249, 299, 349, 399, 449, 499, and 549 were observed as peaks). The C2 to C10 carboxylates related to PFOA were looked for, and some were found. For example, the masses 213, 263, 313, 363, 413, and 463 which correspond to the C4, C5, C6, C7, C8, and C9 perfluorinated carboxylates, respectively, were observed; however, perfluorinated carboxylates were estimated to be present at < 0.2% of the total material. FOSA was looked for but not observed. The perfluorooctance sulfinate was also looked for at mass 483, but not found. The percentage of each component, based on signal intensity, is presented in the following table and is based on an assumption that the signal ratio for each is 1:1 with PFOS. Perfluorinated Alkyl Sulfonates Observed Analyte Peak Response (Area) Perfluorononane sulfonate (C9) response 0.990 Perfluorooctane sulfonate (C8) response 223.245 Perfluoroheptane sulfonate (C7) response 5.786 Perfluorohexane sulfonate (C6) response 4.262 Perfluoropentane sulfonate (C5) response 5.326 Perfluorobutane sulfonate (C4) response 5.190 Perfluoropropane sulfonate (C3) response 2.622 Total 247.421 Total (%) 0.400 90.23 2.34 1.72 2.15 2.10 1.06 100 BACK TO MAIN PFOS - Anaerobic Sludge Biodegradation_______________________________________ Page 8 The internal standard, perfluorooctanoic acid (PFOA, lot no. 07216AS), an off-white solid wax, was received on 1 April 1999 (SLI No. 71-94) from Aldrich, Milwaukee, Wisconsin. An additional internal standard, 1,1,2,2-tetrahydroperfluorooctane sulfonate (THPFOS), a brown crystal was received on 18 January 1999 (SLI No. 70-83) from 3M Environmental, St. Paul, Minnesota. Upon receipt at Springborn, the samples of test substance and internal standards were stored in their original containers at room temperature in a dark, ventilated cabinet. 2.2 Preparation of Stock Solutions and Reagents 2.2.1 Internal Standard Stock Solutions. A 25.0 mg/L PFOA stock solution containing 15.0 mg/L THPFOS internal standard was prepared in the following manner. A 1000 pg/mL PFOA solution was prepared by placing 0.1002 g of PFOA in a 100-mL volumetric flask and bringing to volume with methanol. A 1000 pg/mL THPFOS solution was prepared by placing 0.1002 g of THPFOS in a 100-mL volumetric flask and bringing to volume with methanol. A 50.0-mL aliquot of the PFOA solution and a 30.0-mL aliquot of the THPFOS solution were placed in a 100-mL volumetric flask and brought to volume with methanol which resulted in a 500 mg/L PFOA solution containing 300 mg/L THPFOS. The 25.0 mg/L PFOA/15.0 mg/L THPFOS internal standard solution was then prepared by placing 2.50 mL of the 500 mg/L PFOA solution containing 300 mg/L THPFOS solution in a 50.0-mL volumetric flask and bringing to volume with methanol. No visible signs of undissolved substances were observed in any of the methanol solutions. 2.2.2 PFOS Stock Solutions. Two PFOS stock solutions were prepared and used for dosing the activated sludge/sediment, closed vial and toxicity test systems. At the time of test initiation, the actual purity of the PFOS sample had not been determined and was assumed to be 100% for preparation of stock solutions. A 1.06 mg/mL PFOS stock solution (SLI No. 70-93A) was prepared by placing 0.1061 g of PFOS in a 100-mL volumetric flask and bringing to volume with purified reagent water. This stock solution was used to dose the toxicity assay and activated sludge/sediment acclimation flasks. A 1.01 mg/mL PFOS stock solution (SLI No. 70-93E) was prepared by placing 0.1011 g of PFOS in a 100-mL volumetric flask and bringing to volume with purified reagent water. This stock solution was used to dose the closed vial (headspace) aerobic biodegradation test system. These stock solutions were suspensions and were used after vigorous shaking and sonication to deliver homogeneous suspensions to the test systems. Homogeneity was confirmed by the LC/MS analysis of a 1-mL aliquot of an aqueous PFOS stock solution that BACK TO MAIN PFOS - Anaerobic Sludge Biodegradation__________________________ Page 9 resulted in a 100.2% recovery (SLI No. F499-59). Note that the solubility of PFOS in purified reagent grade water is 0.567 mg/mL (SD = 52.8, CV = 9.31 %, n = 6) (VanHoven and Nixon, 1999). A 1.00 mg/mL PFOS primary stock solution (SLI No. 70-93C) was prepared by placing 0.1004 g of PFOS in a 100-mL volumetric flask and bringing to volume with methanol. This stock solution was used in the preparation of quality control samples. A final PFOS primary stock solution with a concentration of 1.22 mg/mL (SLI No. 70-93D) was prepared by placing 0.1223 g of PFOS in a 100-mL volumetric flask and bringing to volume with methanol. Secondary stock solutions with concentrations of 1.22, 12.2, and 122 mg/L were prepared by placing the appropriate volume of the 1.22 mg/mL primary stock solution in a 50.0-mL volumetric flask and bringing to volume with methanol. The primary and secondary stock solutions were used to prepare calibration standards. Preparation of the calibration standards is detailed in the following table. Concentration of Stock Solution 1.22 mg/mL 1.22 mg/mL 1.22 mg/mL 122 mg/L 12.2 mg/L 1.22 mg/L Fortification Volume (mL) 0.100 0.0500 0.0250 0.0500 0.125 0.410 Final Volume (mL) 50.0 50.0 50.0 50.0 50.0 50.0 Diluent - Standard Concentration (mg/L) Methanol Methanol Methanol Methanol Methanol Methanol 2.44 1.22 0.610 0.122 0.0305 0.0100 The calibration standards were stored in amber bottles with Teflon-lined crimp caps. Aliquots were removed as needed for each LC/MS analysis. The internal standards were mixed in the same proportions and added (prior to LC/MS analysis)to the calibration standards in the same manner they were added to the test samples (i.e., 25 pL of 25 mg/L PFOA/15 mg/L THPFOS to 2.00 mL of standard). Only the 1.06 mg/mL PFOS aqueous stock solution was used to dose the PFOS anaerobic sludge system. BACK TO MAIN PFOS - Anaerobic Sludge Biodegradation______________________________________ Page 10 2.2.3 Ammonium Acetate Stock Solution. Ammonium acetate solutions (2 mM) were prepared by adding 0.151 g of ammonium acetate to a 1000-mL volumetric flask and bringing to volume with purified reagent water. 3.0 PREPARATION OF SAMPLES AND ANALYTICAL METHODS The methods used in the analysis of the aqueous and solid samples are summarized in the flow chart presented in Figure 1. For each anaerobic biodegradation test sample a 10.0-mL aliquot was removed from the vessel containing the sample and centrifuged at 1200 x g (rotating radius of 12.5 cm, from RCF Nomagraph, IEC (International Equipment Company), 300 Second Ave., Needham Heights, Massachusetts, 02194) for 30 minutes with a Beckman Model 65-6R centrifuge. The procedures used for the resultant aqueous and biomass samples are detailed below. 3.1 Preparation of the Aqueous Test Samples A T.O-mL aliquot of the centrifuged supernatant was added to a volumetric flask and diluted to a volume of 25 mL with methanol. The samples were then filtered through a 0.2-pm filter (Titan, nylon membrane) prior to analysis. A 2-mL aliquot was removed from each sample and 25 pL of the 25.0 mg/L PFOA/15.0 mg/L THPFOS internal standard solution was added to the sample aliquot. 3.2 Preparation of the Biomass Samples Each solid sample was extracted once with 40 mL of methanol by vortexing for 30 seconds, sonicating for 15 minutes, and shaking on a shaker table at 200 rpm for 30 minutes. The samples were then centrifuged at 1200 x g (rotating radius of 12.5 cm, from RCF Nomagraph, IEC (International Equipment Company), 300 Second Ave., Needham Heights, Massachusetts, 02194) for 30 minutes and the supernatant was decanted. The supernatant was then filtered through a 0.2-pm filter (Titan, nylon membrane) prior to analysis. If necessary, the sample extract (supernatant) was then diluted as appropriate in methanol. A 2-mL aliquot was removed from each sample extract and 25 pL of the 25 mg/L PFOA/15 mg/L THPFOS internal standard solution was added. 3.3 Quality Control Sample Preparation PFOS - Anaerobic Sludge Biodegradation Page 11 Preparation of the aqueous and solid quality control (QC) samples is summarized in the following table. When necessary and prior to QC fortification, the media were centrifuged at 1200 x g for 30 minutes to separate the solid and aqueous portions. All QC samples were prepared by fortifying the appropriate control matrix (aqueous medium, solid medium) at the concentrations listed below. The QC samples were then treated following the same procedures as the test samples (as described above). QC Sam ple Type Aqueous S o lid s PFOS Stock C o n c e n tra tio n (m g/m L) 1.00 1.00 1.00 1.00 1.00 1.00 Volum e of Stock Solution Used (mL) 0.0250 0.0500 0.100 0.0250 0.0250 0.0500 Control Matrix 1 1 1 2 2 2 Volum e of Control Matrix (mL) 5.00 5.00 5.00 10.0 10.0 10.0 QC Sam ple C o n c e n tra tio n (m g/L) 5.00 10.0 20.0 2.50 2.50 5.00 1 = Aqueous portion from centrifuged anaerobic blank 2 = Solid portion from centrifuged anaerobic blank (10 mL) 3.4 Instrumental Conditions The following instrumental conditions were used during the analysis of the test samples. Instrumental System: Hewlett-Packard Model 1050 quaternary pump, membrane degasser, autosampler, PE Sciex API 100 LC/MS, PE Sciex Column: TurbolonSpray (electrospray) Keystone Betasil C18, 5 urn, 100 A, 150 x 2 mm column with a Betasil C18 guard column Mobile phases: A: 2 mM ammonium acetate in purified reagent water B: 100% Methanol Flow Rate: 0.3 mL/min Gradient program: Time (min) %A %B 0 60 40 8.5 10 90 11 10 90 13 0 100 17 0 100 20 60 40 Run time: 20 min Equilibration delay: 10 min Injection volume: 10 pL PFOS - Anaerobic Sludge Biodegradation Page 12 LC/MS parameters Experiment information: Scan type: Q1, SIM Scan time: 2.01 sec Peak Hopping: Disabled Mass defect: 0 mmu/100 amu Pause time: 2 msec Dwell time: 400 msec Masses scanned (amu)* 413 (PFOA), 427 (THPFOS), 499 (PFOS), 616, 630 (N-EtFOSE- alcohol) Based on 3M analytical method No. ETS-8-11.0 State file information: Source parameters: Polarity: TurbolonSpray voltage: Temperature: Orifice Potential: Nebulizer gas: Auxiliary gas: Negative -5000 volts 400 C -20 volts air (high purity) nitrogen 4.0 TEST PROCEDURES, RESULTS, AND DISCUSSION 4.1 Test Procedures Twenty 160-mL serum bottles with crimped butyl rubber tops were established for this test, ten PFOS bottles and ten blank bottles. On test day 0, two 3.0-L batches of OECD mineral media were prepared and purged with nitrogen. The batches of mineral media were enriched and inoculated as follows: A 323-m l aliquot was removed from each mineral media batch to make space for the following, which were added to each batch while stirring; 6.00 mL of a 0.500 mg/mL resazurin stock solution to produce a 1.00 mg/L concentration in each mineral media batch, 16.5 mL of dried sludge extract to produce approximately 100 mg C/L concentration in each batch, and 300 mL of anaerobic digester sludge. The dried sludge extract was prepared on 4/29/99 as follows: dry sludge was collected from the rotating biological contacter (RBC) wastewater treatment, Bridgewater, Massachusetts. A 200-g aliquot was added to 800 mL of purified reagent grade water, and the sludge mixture was autoclaved for 30 minutes at 121 C. After autoclaving, the sludge mixture was centrifuged and then filtered twice; the first time through a Whatman #41 paper filter and the second time through a Whatman glass fiber filter. The resultant sludge extract was BACK TO MAIN PFOS - Anaerobic Sludge Biodegradation_________________________ ____________ Page 13 refrigerated at 4 C until used and was determined to contain approximately 18,400 mg C/L via analysis on a Dohrmann DC-80 carbon analyzer. The anaerobic sludge was collected from an anaerobic digestor at the Rockland, Massachusetts wastewater treatment plant, and was maintained so as to minimize exposure of sludge to oxygen. A 100-mL aliquot of the enriched and inoculated mineral media solution was added to each serum bottle. The PFOS bottles were then dosed with 2.0 ml. of a 1.06 mg/mL PFOS stock solution to produce a PFOS concentration of 20.8 mg/L. Nitrogen was used to purge the headspace in all the PFOS and blank bottles and the bottles were crimped closed. The bottles were maintained in the dark at 35 C and were not fed with additional nutrients during the study. On test Days 7, 14, 21, 28, 35, 42, 49, and 56, one inoculum blank bottle and one PFOS bottle were removed from incubation and stored at 4 C prior to analysis. Day zero samples were not obtained. One blank tube was analyzed on days 7 and 56. 4.2 Results and Discussion - Table 1 presents the anaerobic sludge analytical data. Mass balances remained in the 101% to 108% range and indicated no apparent PFOS biodegradation over the 56-day period. Analysis of the QC samples with each set of test system samples resulted in measured concentrations which were consistent with the recovery range determined during the method validation study. Based on these results, it was established that the appropriate quality control was maintained during the analyses of the test samples. 5.0 CONCLUSIONS AND FUTURE STUDIES Acclimation to PFOS degradation, if it occurs, may take substantial time. It has been noted that anoxic settling ponds at the 3M plant in Decatur, Alabama contain materials tentatively identified as perfluorooctane sulfinate (reduced PFOS). Thus, it may be beneficial to sample both aerobic and anaerobic sites known to have been exposed to PFOS for a prolonged time period. Microbes in such a system may be able to use PFOS as an electron acceptor or possibly a source of sulfur. Time decay studies with these inocula may provide the best chance to demonstrate PFOS metabolism. BACK TO MAIN PFOS - Anaerobic Sludge Biodegradation ___________________________________ Page 14 REFERENCES VanHoven, Raymond L. and Willard B. Nixon. 1999. Determination of the Water Solubility of PFOS by The Shake Flask Method, Wildlife International LTD. Project Number 454C-107, OECD Guideline for the Testing of Chemicals, 105 Water Solubility, May 3, 1999. BACK TO MAIN PFOS - Anaerobic Sludge Biodegradation______________________________________ Page 15 Table 1. Results for test samples from the anaerobic biodegradation test. Sample No./Typea Nomina! (mg/L) PFOS Concentration Measured (mg/L) Measured {%) Biomass Medium Total Biomass Medium Total (Mass Balance) Test Samples AN599-01/ Day 7 20.8 7.36 AN599-02/ Day 14 20.8 7.13 AN599-03/ Day 21 20.8 7.23 AN599-04/ Day 28 20.8 6.09 AN599-05/ Day 35 20.8 6.28 AN599-06/ Day 42 20.8 6.93 AN599-07/ Day 49 20.8 6.09 AN599-08/ Day 56 20.8 . 6.41 13.6 21.0 35.4 15.3 22.5 34.3 15.1 22.3 34.8 15.8 21.9 29.3 14.8 21.1 30.2 15.2 22.2 33.3 15.3 21.4 29.3 15.9 22.3 30.8 65.5 73.7 72.5 75.9 71.3 73.3 73.4 76.4 101 108 107 105 102 107 103 107 Blank Samples AN599-11 0.000 AN599-18 0.000 < 0.0100b <0.0100 < 0.0100 < 0.0100 <0.0100 <0.0100 NAC NA NA NA NA NA QC Samplesd C799-1918 5.00 NA 5.05 NA NA C799-1928 10.0 NA 10.2 NA NA C799-1938 20.0 NA 19.4 NA NA C799-194f 2.50 2.27 NA NA 90.9 C799-195f 2.50 2.62 NA NA 105 C799-196f 5.00 5.00 NA NA 100 3 Day 0 samples were not taken. b Values expressed as < 0.0100 were below the limit of quantitation c NA = not applicable d QC = quality control sample. 6 QC sample fortified in control medium. ' QC sample fortified in blank biomass. 101 102 96.9 NA NA NA NA NA NA NA NA NA PFOS - Anaerobic Sludge Biodegradation Figure 1. Flow chart of extraction procedures. BACK TO MAIN Page 16 ANAEROBIC SLUDGE BIODEGRADATION SAMPLES i AQUEOUS PORTION "DILUTE IN MEOH ^ Filter " LC/MS ANALYSIS All samples: Centrifuge @ 1200 x g for 30 minutes Y Y SOLIDS PORTION Y EXTRACT W/ MEOH l MEOH EXTRACT ^ Filter LC/MS ANALYSIS i 1 SOLIDS (Discard)