Document 5MMV9JKqY2z5Bke7Qe2XEwyN

AR226-1030A034 Study Title Microbial Metabolism (Biodegradation) of Perfluorooctane Sulfonate (PFOS) I. Activated Sludge/Sediment Authors William E. Gledhill, Ph.D. Barbara J. Markley, Ph.D. Study Completed On 3 November 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 36 BACK TO MAIN BACK TO MAIN PFOS - Activated Sludge/Sediment SIGNATURES AND APPROVAL SUBMITTED BY: Springborn Laboratories Inc. 790 Main Street Wareham, Massachusetts 02571-1075 Page 2 ,// II " J1 William E. Gledhill, Ph.D. Director, Environmental Fate and Microbiological Programs Date Barbara J. Markley, Ph.D. Director, Chemistry Date Sean P. McLaughlin Principal Investigator Date S /l ' v \ / < n>L \ Marjorie E. Dix Senior Research Chemist i\:3 ,0 Date Nj H. Mahle, Ph.D Senior Research Chemist Date BACK TO MAIN PFOS - Activated Sludge/Sediment__________________________________________ Page 3 TABLE OF CONTENTS Page SIGNATURES AND A P P R O V A L .................................................................................................... 2 LIST OF T A B L E 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 Standards ...................................................................... 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 Tetrabutylammonium Sulfate Stock Solution ............................................. 10 2.2.4 Sodium Hydroxide Stock Solution .............................................................. 10 2.2.5 Ammonium Acetate Stock Solution ........................................................... 10 3.0 PREPARATION OF SAMPLES AND ANALYTICALMETHODS ........................................ 10 3.1 Preparation of the 20 and 105 mg/L Aqueous Test Samples .................................. 10 3.2 Preparation of the 0.20 mg/L Aqueous Test Sam ples............................................... 11 3.3 Preparation of the Solid S a m p le s............................................................................... 11 3.4 Preparation of Quality Control Samples .....................................................................11 3.5 Instrumental Conditions .............................................................................................. 12 4.0 TEST PROCEDURES, RESULTS, AND DISCUSSION........................................................13 4.1 Toxicity T e s t..................................................................................................................13 4.1.1 Test Procedures............................................................................................ 13 4.1.2 Results and Discussion ............................................................................... 14 4.2 Activated Sludge/Sediment Biodegradation Study - Acclimation P h a s e ................. 14 4.2.1 Test Procedures............................................................................................ 14 4.2.2 Results and Discussion ............................................................................... 16 4.3 Closed Vial (Headspace) Aerobic Biodegradation Test ........................................... 17 4.3.1 Test Procedures............................................................................................ 17 4.3.2 Results and Discussion ............................................................................... 19 5.0 CONCLUSIONS........................................................................................................................20 6.0 FUTURE STUDIES ................................................................................................................. 20 REFERENCES 21 BACK TO MAIN / PFQS - Activated Sludge/Sediment__________________________________________ Page 4 LIST OF TABLES Page Table 1. Composition of OECD inorganic salts m e d iu m ................................................. 22 Table 2. Toxicity assay of PFOS in activated s lu d g e ....................................................... 23 Table 3. Total organic carbon (TOC) analyses during the pre- and post dosing phases of the activated sludge/sediment acclimation study24 ........................................................................................................................ Table 4. PFOS concentration measurements from the activated sludge/sediment acclimation flasks .................................................................... 25 Table 5. PFOS concentration measurements from the analytical control fla s k s .......................................................................................................................... 26 Table 6. Selection of test conditions for the sealed PFOS COz evolution study .......................................................................................................................... 27 Table 7. C 0 2 analysis of the headspace in sample vials during the closed vial (headspace) aerobic biodegradation test with PFOS.3 ..............................28 Table 8. PFOS concentration measurements for the 0.200 mg/L test samples from the sealed vessels (headspace) .................................................................. 29 Table 9. PFOS concentration measurements for the 105 mg/L test samples from the sealed vessels (headspace).....................................................................31 BACK TO MAIN PFOS - Activated Sludge/Sediment_____________________________________________Page 5 LIST OF FIGURES Page Figure 1. Flow chart of extraction procedures......................................................................33 Figure 2. Total measured PFOS concentration duringacclimation in activated sludge/sedim ent......................................................................................34 Figure 3. PFOS concentration in the analytical control f la s k ............................................ 35 Figure 4. Concentration of PFOS versus time during the closed vial (headspace) aerobic biodegradation t e s t ............................................................36 BACK TO MAIN PFOS - Activated Sludge/Sediment 1.0 INTRODUCTION Page 6 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 (sandy soil) 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 P450monooxygenase enzyme system known to metabolize complex molecules. Throughout the experimental program the principles outlined above for enrichment were incorporated. The results of the studies are summarized in four separate reports and provide a BACK TO MAIN PFOS - Activated Sludge/Sediment__________________________________________ Page 7 basis for the future direction of the program to better understand the environmental fate of fluorochemicals. This report summarizes the results of exposure of PFOS to an activated sludge/sediment 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 perfluorooctane 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 - Activated Sludge/Sediment__________________________________________ 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-mL aliquot of the PFOA solution and a 30-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 2.5 mL of the 500 mg/L PFOA solution containing 300 mg/L THPFOS solution in a 50 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 - Activated Sludge/Sedimenl__________________________________________ 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 secondary stock solution was prepared by diluting 0.500 mL of this stock to 50.0 mL final volume to produce a 10.0 mg/L PFOS stock. 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). BACK TO MAIN PFOS - Activated Sludge/Sediment_________________________________________ Page 10 2.2.3 Tetrabutylammonium Sulfate Stock Solution. Tetrabutylammonium sulfate (TBA-S) solutions (0.5 M) were prepared by placing 35.0 g of TBA-S in a 200-mL volumetric flask and bringing to volume with purified reagent water. This solution was then brought to pH 10 with 10 N NaOH. 2.2.4 Sodium Hydroxide Stock Solution. Sodium hydroxide solutions (10 N) were prepared by placing 400 g of sodium hydroxide in a 1000-mL volumetric flask and bringing to volume with purified reagent water. 2.2.5 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 all shake-flask and closed vial test samples, an appropriate volume of test sample (10 mL for the shake-flask samples, and 15 mL for the closed vial samples) was placed into a NalgeneTM centrifuge tube 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 at room temperature with a Beckman Model 65-6R centrifuge. The procedures used for the resultant aqueous (supernatant) and solid samples are detailed below. 3.1 Preparation of the 20 and 105 mg/L Aqueous Test Samples A 1.00-mL aliquot of the supernatant was added to a volumetric flask and diluted to volume (25.0 and 100 mL for the 20.8 and 105 mg/L samples, respectively) with methanol. The samples were then filtered through a 0.2-pm filter (Titan, nylon membrane) prior to analysis. A 2.0-mL aliquot was removed from each filtered sample, placed into an autosampler vial, and 25 pL of the 25.0 mg/L PFOA/15.0 mg/L THPFOS internal standard solution was added. BACK TO MAIN PFOS - Activated Sludge/Sediment_________________________________________ Page 11 3.2 Preparation of the 0.20 mg/L Aqueous Test Samples The entire volume (15 ml.) of supernatant from each sample was transferred to a separatory funnel. Ten milliliters of the 0.5 M pH 10 TBA-S solution was added to each separatory funnel. The samples were then extracted twice with 300 mL of 50:50 ethyl acetate/hexane. When emulsions formed, an additional 1.00 to 2.00 mL of methanol was added to the separatory funnel and gently mixed until the emulsions vanished. All extracts were reduced to approximately 2.00 mL by rotary evaporation at approximately 35 C (samples were not allowed to go dry). When water was present in the round-bottom flask, methanol was added and rotary evaporation was continued until the amount of water was minimized. The sample extract was then quantitatively transferred to a centrifuge tube. The sample extract was reduced to a volume of approximately 100-pL under a gentle stream of nitrogen at room temperature, brought to a volume of 10 mL with methanol, vortexed for 30 seconds, and sonicated for 15 minutes. The sample extracts were then filtered through a 0.2-pm filter (Titan, nylon membrane) prior to analysis. A 2.00-mL aliquot was removed from each sample and 15 pL of the 25.0 mg/L PFOA/15.0 mg/L THPFOS internal standard solution was added. 3.3 Preparation of the Solid Samples Each solid sample was extracted by adding 40 mL of methanol and vortexing for 30 seconds, sonicating for 15 minutes, and shaking on a shaker table at 200 rpm for 30 minutes at room temperature. The samples were then centrifuged at 1200 x g for 30 minutes using a Beckman Model 65-6R centrifuge, 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 diluted in methanol. A 2.0-mL aliquot was removed from each sample extract, placed into an autosampler vial, and 25 pL of the 25.0 mg/L PFOA/15.0 mg/L THPFOS internal standard solution was added. 3.4 Preparation of Quality Control Samples 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, biomass, etc.) at the concentrations listed below. The QC samples were then treated following the same procedures as the test samples (aqueous samples, Sections 3.1 and 3.2; biomass samples, Section 3.3). BACK TO MAIN PFOS - Activated Sludge/Sediment Page 12 Test ID QC Sample Type Shake-Flask Days 7, 14,21, 28, and 35 Aqueous PFOS Stock Concentraron (mg/L) 1000 1000 1000 Volume of Stock Solution Used (mL) Control Matrix 0.100 1 0.100 1 0.200 2 Volume of Control Matrix (mL) 10.0 10.0 10.0 QC Sample Concentration (mg/L) 10.0 10.0 20.0 Solids 1000 0.0250 3 10.0 2.50 1000 0.0250 3 10.0 2.50 1000 0.0500 3 10.0 5.00 Closed Vial Low Cone. Day 0, 16, 30, 61 Aqueous 10.0 10.0 0.300 0.300 1 15.0 0.200 1 15.0 0.200 10.0 0.300 2 15.0 0.200 Solids 10.0 10.0 0.300 0.300 2 15.0 0.200 3 15.0 0.200 10.0 0.300 3 15.0 0.200 High Cone. Day 0, 15, 30, 63 Aqueous 1000 1000 1000 0.250 0.500 1.00 1 10.0 25.0 1 10.0 50.0 2 10.0 100 Solids 1000 1000 1000 1 = OECD mineral media 2 = Aqueous portion from centrifuged inoculum blank 3 = Solid portion from centrifuged inoculum blank 0.500 1.00 1.50 3 15.0 33.3 3 15.0 66.7 3 15.0 100 3.5 Instrumental Conditions The following instrumental conditions were used during the analysis of the test samples. Instrumental System: HPLC Column: Hewlett-Packard Model 1050 quaternary pump, membrane degasser, autosampler, PE Sciex API 100 LC/MS (single quadrupole), PE Sciex TurbolonSpray (electrospray) Keystone Betasil C18, 5 pm, 100 , 150 x 2 mm column with a Betasil C18 guard column BACK TO MAIN PFOS - Activated Sludge/Sediment Page 13 Mobile phases: Flow Rate: Gradient program: Run time: Equilibration delay: Injection volume: A: 2 mM ammonium acetate in purified reagent water B: 100% methanol 0.3 mL/min Time (min) %A %B 0 60 40 8.5 10 90 11 10 90 13 0 100 17 0 100 20 60 40 20 min 10 min 10 pL LC/MS parameters Experiment information: Scan type: Q1, selected ion monitoring 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: Turbolon spray 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 Toxicity Test 4.1.1 Test Procedures. The potential toxicity of PFOS was investigated concurrently with the activated sludge/sediment acclimation biodegradation study. The assay system consisted of a single stoppered 250-mL Erlenmeyer flask to which 100 mL of an OECD mineral salts medium BACK TO MAIN PFOS - Activated Sludge/Sediment_________________________________________ Page 14 (Table 1), containing 20.8 mg/L of PFOS (2.0 mL of SLI No. 70-93A) and 5.00 mg/L [14C]sodium benzoate, was added. The radioactivity was delivered by adding 0.50 mL of a 0.220 mg/L [14C]sodium benzoate stock solution (SLI No. 71-52 RA). The remaining concentration of sodium benzoate was made up by adding 0.080 mL of a non-radiolabeled stock solution containing 6.00 mg/mL of sodium benzoate (SLI No. 61-34V). A 1.0-mL aliquot of activated sludge (approximately 3.0 mg dry weight) collected from the Wareham Wastewater Treatment Plant aeration basin was then added to the flask. A control flask was prepared in the same manner but with the addition of 2 mL of reagent water (no methanol) in place of the PFOS. To the small headspace trap in each flask, 2.0 mL of 1M KOH was added. Flasks were placed on a shaker table set at 150 rpm and incubated at 22 C. A 100-pL sample was removed from each trap approximately 6 hours after test initiation and again at 24 and 48 hours. The aliquots were analyzed by liquid scintillation counting (LSC). Toxicity was assessed by comparing the rate of 14C 0 2 evolved from the test system flask and control system flask. 4.1.2 Results and Discussion. Table 2 presents the results of the toxicity study with PFOS (20.8 mg/L) and activated sludge. Evolution of 14C 0 2from the sodium benzoate control flask was not substantially different in the presence of PFOS based on comparison of the rate of 14C 0 2 evolution from both flasks. Thus, at this concentration, PFOS was not anticipated to inhibit the microbial population in this study. However, after the first week, the PFOS concentration in acclimation flasks began to increase due to weekly feeding. Therefore, conclusions about toxicity at concentrations above 20.8 mg/L can not be substantiated, but it could be assumed that PFOS toxicity w ould not be an issue since all m easured concentrations still in the m edium (see Table 4) were below 100% of the 20.8 mg/L nominal concentration. 4.2 Activated Sludge/Sediment Biodegradation Study - Acclimation Phase 4.2.1 Test Procedures. The acclimation phase of the biodegradation study in activated sludge/sediment was conducted in 300-mL baffled Erlenmeyerflasks. Inoculum was collected from several sites: the aeration basin from the Wareham, Massachusetts activated sludge plant, the secondary return activated sludge line from the New Bedford, Massachusetts wastewater treatment plant, one disk from a rotating biological contacter (RBC) plant (Bridgewater, Massachusetts), sediment from below the Wareham activated sludge plant's outfall, and a sandy soil collected from a site in Wilson County, North Carolina. The sediment was not characterized, but was a typical dark organic type sediment found below most sewage treatment plant outfalls. The sandy soil was BACK TO MAIN PFOS - Activated Sludge/Sediment_________________________________________ Page 15 collected by personnel at American Agricultural Services, Inc., Lucama, North Carolina and was added to provide a surface (protection) for microbial growth and soil microorganisms. The soil was characterized by Agvise Laboratories, Northwood, North Dakota and reported to have the following characteristics: sand, 93%; silt, 4%; clay, 3%; pH 4.4; organic matter, 0.8%; phosphorus, 19 mg/kg; nitrogen, 0.043%; and cation exchange capacity, 3.6 meq/100 g. Aerobic sludge from each wastewater treatment plant was added to the acclimation flasks at a concentration of 1.00 mg/mL (dry weight basis). Sandy soil was added at 10.0 mg/mL medium, and sediment at 1.00 mg/mL medium (dry weight basis) at day 7. Test medium consisted of natural sewage (primary effluent) obtained from the Wareham wastewater treatment plant which had been supplemented with the four OECD mineral salt solutions (at the concentration specified in the OECD ready biodegradation test guidelines, Table 1), trace minerals (Trace Minerals Corp., 0.020 mL/100 mL), yeast extract (Difco, 5.00 mg/100 mL), and activated sludge extract. Activated sludge extract was prepared by adding one part dried RBC sludge to four parts water, autoclaving for 30 minutes, and then centrifuging and filtering through Whatman No.41 paper and Whatman glass fiber filters. The total organic carbon (TOC) of the extract, measured with a Dohrman DC-80 TOC analyzer, was approximately 3500 mg C/L. Sufficient volume (2.0 mL/100 mL of medium) of the extract was added to the natural sewage to bring the TOC level to approximately 100 mg C/L. The extract was refrigerated at 4 C after preparation and use. The same extract was used throughout the study. The PFOS test flask contained 100 mL of the medium described above to which 2.0 mL of a sonicated 1.06 mg/mL aqueous PFOS stock solution (SLI No. 70-93A) was added. This resulted in a PFOS concentration of approximately 20.8 mg/L. In addition to the test flask, blank control flasks with no test substance were also established in larger flasks at day 21 (1-L of medium in a 2-L Erlenmeyer flask) for use in preparation of analytical QC samples. Additional flasks, containing only PFOS in OECD salts medium (no biomass, sandy soil, or organic feed) were also established to serve as analytical controls. All flasks were then incubated at 22 2 C on a rotary shaker (150 rpm) in an environmental chamber. On a weekly basis, a 10-mL sample of the mixed liquor (10% of the biomass and medium) was removed from the PFOS flasks for analysis of parent material and potential metabolites. The remaining medium in each flask (approximately 90 mL) was centrifuged and 60 mL of the supernatant was removed for analysis. The biomass and medium removed for analysis (70 mL total) were replaced with 70 mL of fresh medium (containing freshly collected natural sewage, BACK TO MAIN PFOS - Activated Sludge/Sediment_________________________________________ Page 16 minerals, and carbon nutrients at concentrations originally supplied at day zero), and 1.4 ml. of the 1.06 mg/mL PFOS stock solution (SLI No. 70-93A). An additional amount of solids consisting of activated sludge from the Wareham and New Bedford wastewater treatment plants and sediment equivalent to 10% of the original amount supplied at day zero, were also collected each week and added to the test vessels. The Bridgewater RBC wastewater treatment plant sludge was generally collected on a monthly basis and maintained under aeration in the laboratory at ambient temperature. The RBC sludge was not fed during this time period. Flasks were reinoculated from this aerated sample. The blank control flask containing 1.0 L of medium was also treated in the same manner each week and received no PFOS. Analytical control samples were also maintained, sampled, and refortified weekly in the same manner as the activated sludge flasks, but received no biomass or nutrients. Carbon analysis (TOC) was generally conducted on a weekly basis on the supernatants from the flasks after filtration through a 1.0-pm Gelman filter by injection of 1,0-mL samples into a Dohrman DC-80 TOC analyzer. Parameters such as pH, suspended solids, and microbial plate counts were not conducted for these test systems. Weekly OECD medium replacement (70 mL/flask) should have maintained the pH near neutrality. The acclimation procedure was conducted over a 112-day (16 weeks) period. However, samples for analytical measurement of PFOS and potential metabolites were only collected for the first 14 weeks. All samples from this acclimation phase were saved in 100-mL amber Wheaton bottles with Teflon-lined rubber septa under refrigeration priorto analysis and completion of the PFOS method validation. At week 10 and12, approximately % of the biomass was removed from the PFOS flask to initiate the sealed vial studies described below. The remaining biomass in the original Erlenmeyer flasks continued to be fed for another four weeks after which time all flasks were transferred to the refrigerator for storage. 4.2.2 Results and Discussion. Table 3 presents the TOC analyses conducted on the centrifuged medium for the blank control, PFOS, and analytical control vessels. The blank control and PFOS vessels were acclimated to the nutrient feed for approximately 27 days prior to dosing. Gradual acclimation to the feed was evidenced by the gradual TOC decrease between days -27 and -1. The PFOS and analytical control vessels were dosed on day 0 and TOC measurements were conducted on a weekly basis for the next ten weeks. The TOC of the analytical control vessel remained in the 0.7 to 1.7 mg/L range throughout the study. The PFOS medium TOC elevated to 34 mg/L on day 14 and, subsequently, settled back to the 15 to 20 mg/L range for the remainder BACK TO MAIN PFOS - Activated Sludge/Sediment_________________________________________ Page 17 of the study. TOC values for the blank control ranged from 10 to 26 mg/L from weeks one through ten. Explanations for the TOC fluctuations cannot be provided. Perhaps, the complex nature of the nutrient feeds and their variation at different collection times may have contributed to these results. Table 4 summarizes the analytical results for days 7,14, 21,28 and 35. For these days, analysis was accomplished by combining the biomass and supernatant samples and centrifuging to create a liquid and solid sample. Day 112 was also analyzed, but the biomass sample was not combined with a supernatant sample priorto analysis. Initially, it was assumed that the PFOS might distribute equally between the solids and liquid in the test system. If so, PFOS concentrations in the two matrices should have remained constant throughout the study, if no biodegradation had occurred. However, this was not the case as PFOS was found to initially be distributed primarily in the medium and as the acclimation progressed was found primarily associated with the cells. With the sampling and feeding regime employed, compounds preferentially adsorbing to biomass and not rapidly biodegrading will tend to accumulate in the acclimation test system. However, the accumulation'should have been more gradual than that reflected by the mass balance in Table 4. Based on partitioning observed in sealed vessels (Table 8 indicates an average 18% partitioning to biomass and 82% to medium), it can be calculated that even for a compound partitioning 18:82 between solids and medium, an increase from 100% of nominal at day zero to 118% of nominal at five weeks would be expected. One possible explanation for the mass balances obtained in Table 4 for the activated sludge system may be explained in Table 5 where the results of the analytical control flasks are presented. Mass balance shows good recovery for the first two weeks then a significant decline in recovery from the medium. Concurrent with this decline in mass balance was the observation of a white precipitate forming on the glass at the interface of the medium and headspace of the vessel. A similar precipitate was observed for the flasks containing biomass. While not verified by specific analysis, it appears reasonable that PFOS concentrations were significantly exceeding water solubility and precipitating on the test vessel walls. 4.3 Closed Vial (Headspace) Aerobic Biodegradation Test 4.3.1 Test Procedures. While awaiting validation of the PFOS analytical method, a biodegradation study was initiated to determine if carbon (C 02) could be evolved from PFOS, BACK TO MAIN PFOS - Activated Sludge/Sediment_________________________________________ Page 18 thereby, providing direct evidence for biodegradation. This study was conducted in 20-mL serum vials with a PFOS concentration of 105 mg/L (or 19 mg C/L). Subsequent to the analytical method validation, an additional sealed vessel study was conducted at a much lower PFOS concentration (0.20 mg/L) in 22-mL serum vials. Both studies employed inoculum from the acclimation flasks after approximately 12 weeks into the acclimation. Inoculum concentration selected for these studies was based on a study in which varying concentrations of activated sludge were added to OECD salts medium containing 50 mg/L yeast extract and resazurin, an oxidation/reduction indicator in sealed 20-mL serum vials. Headspace in the vials was flushed with pure oxygen prior to sealing. A one to ten dilution of this medium was also examined and biomass concentrations ranged from 10 to 6666 mg/L (dry wt. basis). It was found that all of the vessels containing the full strength medium went anaerobic within 2 weeks (Table 6). Biomass concentrations exceeding 2000 mg/L also caused the 1/10 strength medium to go anaerobic within 2 weeks and a biomass concentration of 666 mg/L gave variable results. Concentrations of 10, 30 and 100 mg/L biomass in the 1/10 strength medium remained aerobic for greater than 2 weeks. Based on these data, an activated sludge concentration of 200 mg/L was selected for the sealed vessel studies. For the low concentration (0.20 mg/L), 64 vials (22-mL) were prepared as follows; 12 blank vials received 15 mL of mineral medium, 12 inoculum control vials received 14 mL of mineral media and 1 mL of inoculum, 20 analytical control vials received 14 mL of mineral media and 1 mL of a 3 pg/mL PFOS stock solution, and 20 PFOS biodegradation vials received 13 mL of mineral media, 1 mL of inoculum, and 1 mL of a 3 pg/mL PFOS stock solution. The PFOS aerobic sludge acclimation system served as the source of inoculum (final concentration of solids = 200 mg/L). For the high concentration (105 mg/L), 48 vials (with a measured volume of 20-mL) were prepared (16 blank inoculum control vials, 16 analytical control vials, and 16 PFOS biodegradation (fortified inoculum) vials. The blank inoculum control vials contained a mixture prepared by adding 0.5278 g of wet solids to 250 mL of mineral media (200 mg/L solids on a dry weight basis). The analytical control vials contained a mixture prepared by adding 26 mL of the 1.01 mg/mL PFOS stock solution to 224 mL of mineral media. The biodegradation (fortified inoculum) vials contained a mixture prepared by mixing 0.5346 g of wet solids with 224 mL of mineral media and 26 m Lof the 1 mg/mL BACK TO MAIN PFOS - Activated Sludge/Sediment_________________________________________ Page 19 PFOS stock solution. Each vial contained 13.6 mL of the appropriate medium and the inoculum control and biodegradation vials contained 200 ppm of dry weight biomass. Analytical samples were removed from selected blank inoculum control vials, analytical control vials, and PFOS biodegradation vials on Days 0,16, 30, and 61 for the low concentration, and on Days 0, 15, 30, and 63 for the high concentration. For the high PFOS concentration study, half of the samples were analyzed for C 0 2production in the headspace of the vials (after acidification and shaking for at least 1 hour) and the other half of the vials were refrigerated and kept for specific chemical analysis. Analysis for headspace C 0 2 was performed on duplicate samples using a ThermoGlass 1200TM carbon analyzer by injection of 200 pL of headspace gas. 4.3.2 Results and Discussion. Tables 8 and 9 summarize analytical data from the PFOS closed vial systems. For the system containing 0.20 mg/L PFOS (Table 8), an average of 0.362 mg/L PFOS was observed in the inoculum blanks throughout the 61 -day study. This was due to the fact that both the fortified and blank test vessels received inoculum from the PFOS aerobic sludge acclimation vessel. The fact that this concentration of PFOS remained relatively constant during the course of the study points to the recalcitrance of this molecule. The PFOS concentration in the fortified analytical control (no inoculum) appeared to steadily decrease from 0.232 mg/L at Day 0 to approximately 0.15 mg/L at Day 61. Average PFOS concentrations in test vessels fortified with PFOS and inoculum also showed a slight decline in PFOS concentration from Day 0 (0.333 mg/L) to Day 61 (0.214 mg/L). Since vials and stoppers were extracted with methanol, PFOS was apparently not adsorbing to the test vessel surfaces. Recoveries or mass balances from the test system containing 105 mg/L PFOS (19 mg C/L) and no inoculum (analytical controls) ranged from 95% to 123% (Table 9). Recoveries from the test vessels fortified with PFOS and inocula were higher and ranged from 121% to 145% over the 63-day test period. Apparent PFOS concentrations decreased from 145 mg/L on day 0 to 132, 132, and 121 mg/L on days 15, 30, and 63, respectively. This represents an approximate 20% decline in PFOS concentration over the course of the study. Whether this represents biodegradation or some sort of matrix effect is not certain. Samples have been retained for mass spectral analysis. BACK TO MAIN PFOS - Activated Sludge/Sediment_________________________________________ Page 20 Table 7 presents data for C 0 2 analyses of the headspace gas from these systems. Very little difference in headspace C 0 2production was noted between the blank and PFOS containing test vessels. This is also indicative of PFOS recalcitrance. On day 63, C 0 2in the headspace of one PFOS vial was somewhat higher (33.8%) than the inoculum control flasks; however, without more replication of test vessels, conclusions as to the extent of biodegradation can not be made. The hydrolysis (analytical) control showed much lower C 0 2 evolution than the blank and PFOS inoculated vessels over the 63-day test period. 5.0 CONCLUSIONS Overall, PFOS was found to be very recalcitrant in the activated sludge/sediment system. PFOS removal from acclimation medium occurred via partitioning to biomass in the inoculated test systems or to the glass walls of the vessels in analytical control flasks. From the current data, it cannot be concluded whether apparent decline of PFOS concentration was due to biodegradation or some type of matrix effect. 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. 6.0 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 - Activated Sludge/Sediment REFERENCES Page 21 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 - Activated Sludge/Sediment_________________________________________ Page 22 Table 1. Composition of OECD inorganic salts medium.3 Solution A B C D Reagents0 k h 2p o 4 k2h p o 4 Na2HP04 2H20 n h 4ci CaCI2 MgSO,,- 7H20 FeCI3 6H20 HCI Concentration, g/L 8.50 21.75 33.40 0.50 27.50 22.50 0.25 0.05 a The mineral media was prepared by mixing 10 mL of Solution A with 800 mL of water, then adding 1 mL of Solutions B, C, and D, and then bringing to a volume of 1 L with water. b Proportionate amounts of salts with different waters of hydration may be substituted. 0 The pH of this solution should be 7.4. BACK TO MAIN PFOS - Activated Sludge/Sediment_________________________________________ Page 23 Table 2. Toxicity assay of PFOS in activated sludge. Test Vessel Blank Control13 Time (hours) 6 24 48 [14C]Radioactivity in C02Trap DPMa Percent Applied Dose <LOQc 0 34100 31.0 46700 42.4 PFOSd 6 <LOQ 24 33400 48 47000 0 30.4 42.7 a DPM = dissociations per minute b Blank Control vessels were comprised of 100 mL of mineral salts medium which contained 5 mg/L [14C]sodium benzoate 0 <LOQ = less than the Limit of Quantitation (60 dpm) d PFOS vessels were comprised of 100 mL of mineral salts medium which contained 20 mg/L of PFOS and 5 mg/L [14C]sodium benzoate _ BACK TO MAIN PFOS - Activated Sludge/Sediment_________________________________________ Page 24 Table 3. Total organic carbon (TOC) analyses during the pre- and post dosing phases of the activated sludge/sediment acclimation study.3 Test Day Pre Dose -27 -20 -16 -13 -8 -1 Blank Control NAb 42.35 35.67 24.45 21.67 11.98 TOC (mg/L) PFOS Analytical Control NA 45.75 38.16 24.76 17.48 11.39 NA NA NA NA NA NA 0 NA NA______C 7 10.07 10.23 1.699 14 14.93 33.85 1.467 21 11.17 17.44 1.604 28 15.54 14.86 1.293 35 21.22 14.94d 0.999 42 26.04 15.78 0.876 49 16.62 16.91 0.703 56 16.31 18.56 1.297 63 14.10 19.61 1.224 70 12.06 18.25 0.899 a Samples were filtered through a 1.0-pm Gelman filter and duplicate 1.0 mL samples were analyzed on the Dohrman DC-80 carbon analyzer. b Not analyzed. c Initial TOC measurement at the start of the post-dose phase (T0). Remainder of analyses indicated after 7 days of incubation prior to feeding. d One sample was an outlier and that data point was not used. BACK TO MAIN PFOS - Activated Sludge/Sediment_________________________________________ Page 25 Table 4. PFOS concentration measurements from the activated sludge/sediment acclimation flasks. Sample No./Typea Nominal (mg/L) PFOS Concentration Measured (mg/L) Measured (%) Total Biomass Medium Total Biomass Medium (Mass ______________________________________________________ Balance) Test Samples AS399-01-1/Day 7 AS399-01-2/Day 14 AS399-01-3/Day 21 AS399-01-4/Day 28 AS399-01-5/Day 35 AS399-01 /Day 112 23.5 24.4 24.8 25.0 25.0 22.0 10.0 11.5 19.6 33.3 21.2 24.7 16.5 14.3 9.43 11.6 5.47 3.64 26.5 25.8 29.0 44.8 26.7 28.4 42.6 47.1 78.9 133 84.8 112 70.0 58.6 38.0 46.2 21.9 16.6 113 106 117 179 107 129 Blank Samples AS399-06-01/Day 7 AS399-06-02/Day 14 AS399-06-03/Day 21 AS399-06-04/Day 28 AS399-06-05/Day 35 0.00 0.00 0.00 0.00 0.00 <0.0100b <0.0100 <0.0100 <0.0100 <0.0100 < 0.0100 <0.0100 < 0.0100 <0.0100 <0.0100 <0.0100 <0.0100 <0.0100 <0.0100 <0.0100 NAC NA NA NA NA NA NA NA NA NA NA NA NA NA NA QC Medium Sam ples" C599-19/Days 7-28e 10.0 NA 11.3 C599-20/Days 7-28e 20.0 NA ` 22.6 C599-21/Days 7-28e 20.0 NA 22.1 C599-59/Day 35e 10.0 NA 10.5 C599-60/Day 35e 20.0 NA 22.7 C599-61/Day 35e 20.0 NA 20.9 C899-14/Day 112e 5.00 NA 5.02 C899-15/Day 112s 10.0 NA 10.3 C899-16/Day 112e 20.0 NA 22.5 QC Biom ass Samples C599-95/Days 7-35' 2.50 2.56 NA C599-96/Days 7-35' 2.50 2.58 NA C599-97/Days 7-35* 5.00 5.33 NA C899-11/Day 112' 2.50 2.61 NA C899-12/Day 112' 2.50 2.60 NA C899-13/Dav 112* 5.00 5.76 NA a 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. e QC sample fortified in control medium. ' QC sample fortified in blank biomass. NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 113 NA 113 NA 110 NA 105 NA 114 NA 105 NA 100 NA 103 NA 112 102 NA 103 NA 107 NA 104 NA 104 NA 115 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA BACK TO MAIN PFOS - Activated Sludge/Sediment Table 5. PFOS concentration measurements from the analytical control flasks. Page 26 Sample No./Typea Nominal (mg/L) PFOS Concentration Measured (mg/L) Measured (%) Total Biomass Medium Total Biomass Medium (Mass ______________________________________________________ Balance) Test Samples AS399-07-01/Day 7 AS399-07-02/Day 14 AS399-07-03/Day 21 AS399-07-04/Day 28 AS399-07-05/Day 35 AS399-07/Day 112 Blank Samples AS399-09-01/Day 7 AS399-09-02/Day 14 AS399-09-03/Day 21 AS399-09-04/Day 28 AS399-09-05/Day 35 QC Samples" C599-19/Days 7-28 C599-20/Days 7-28 C599-21/Days 7-28 C599-59/Day 35 C599-60/Day 35 C599-61/Day 35 C899-14/Day 112 C899-15/Day 112 C899-16/Day 112 21.2 21.2 21.2 21.2 21.2 21.2 NAb NA NA NA NA NA 21.1 19.8 11.1 17.8 10.2 5.74 21.1 19.8 11.1 17.8 10.2 5.74 0.00 0.00 0.00 0.00 0.00 <0.0100 < 0.0100 <0.0100 <0.0100 <0.0100 <0.0100 <0.0100 <0.0100 <0.0100 <0.0100 <0.0100 <0.0100 <0.0100 <0.0100 <0.0100 10.0 NA 11.3 NA 20.0 NA 22.6 NA 20.0 NA 22.1 NA 10.0 NA 10.5 NA 20.0 NA 22.7 NA 20.0 NA 20.9 NA 5.00 NA 5.02 NA 10.0 NA 10.3 NA 20.0 NA 22.5 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 99.6 93.2 52.4 83.9 48.0 27.1 NA NA NA NA NA 113 113 110 105 114 105 100 103 112 100 93.2 52.4 83.9 48.0 27.1 NA NA NA NA NA NA NA NA NA NA NA NA NA NA a Day 0 samples were not taken. b Values expressed as < 0.0100 were below the limit of quantitation 0 NA = not applicable d QC = quality control sample. e QC sample fortified in control medium. ' QC sample fortified in blank biomass. BACK TO MAIN PFOS - Activated Sludge/Sediment_________________________________________ Page 27 Table 6. Selection of test conditions for the sealed PFOS C 0 2evolution study.3 Day 10 0 +++ 4 +++ 5 +++ 6 +/-+/-+ 7 -+/-+/9 10 12 -- 14 ... 16 ... 17 -- 21 -- Solids Concentration, mg/L (dry weight basis)b Full Strength Medium 1/10 Strength Medium 30 100 666 2000 6666 10 30 100 666 2000 6666 +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ --+ ... +++ +++ 4-++ +++ +++/- ... +++ +++ --+ ... -- +++ +++ +++ ... +++ +++ --+ ... ... +++ +++ +++ +/-- -- +++ ... ... +++ +++ +++ ... ... +/-- ... -- ... +++ +++ +++ ... . . . +/-- +/-- ... ... ... +++ +++ +++ ... ... . . . . . . ... ... ... +++ +++ +++ ... ... ... ... ... ... ... +++ +++ +++ +/-+/-+/- ... ... ... ... ... ... ... +++ +++ +++ ... ... ... ... -- ... ... +++ +++ +++ ... ... ... ... -- +++ +++ +++ ... ... -- ... ... a Test system = 2-mL serum vials, 15 mL medium consisting of OECD salts, Wareham raw sewage, 100 mg C/L sludge extract, resazurin and activated sludge. For 1/10 strength medium, raw sewage and sludge extract were diluted 1 to 10 in the test system. b Aerobicity was judged by observing medium color; + = pink color and presence of 0 2; - = colorless and no 0 2; +/- = faint pink color. BACK TO MAIN PFOS - Activated Sludge/Sediment_________________________________________ Page 28 Table 7. C 0 2 analysis of the headspace in sample vials during the closed vial (headspace) aerobic biodegradation test with PFOS.a Test Vessel Replicate Headspace C, mg/L Day 0 15 30 63 % Theoretical Day 0 15 30 63 Inoculum Control13 A NA 7.61 9.22 11.54 NA NA NA NA B NA 7.55 9.25 10.49 NA NA NA NA Analytical Control0 A NA 2.32 3.14 3.27 NA NA NA NA B NA 2.68 2.78 3.58 NA NA NA NA PFOS Fortified01 A NA 6.53 8.10 9.99 NA -7.70 -8.33 -7.52 B NA 7.19 8.39 15.62 NA -2.86 -6.20 33.78 a Averaged for duplicate test vessels. b Contains "acclimated' activated sludge in mineral salts medium. c Contains 79.3 mg/L (20 mg C/L) PFOS in mineral salts medium. d Contains "acclimated' activated sludge in mineral salts medium and 79.3 mg/L PFOS. e NA = not applicable BACK TO MAIN PFOS - Activated Sludge/Sediment Page 29 Table 8. PFOS concentration measurements for the 0.200 m g/Ltest samples from the sealed vessels (headspace). Sample No./ Sample Day Nominal (mg/L) Inoculum Control HS0699-10/0 HS799-03/16 HS799-04/16 HS799-121/30 HS799-122/30 HS899-69/61 HS899-70/61 0.000 0.000 0.000 0.000 0.000 0.000 0.000 Avg. PFOS + Inoculum b HS0699-15/0 HS799-08/16 HS799-09/16 HS799-10/16 HS799-126/30 HS799-127/30 HS799-128/30 HS899-74/61 HS899-75/61 HS899-76/61 Avg Blank Analytical Control HS0699-08 HS799-01/16 HS799-02/16 HS799-119/30 HS799-120/30 HS899-67/61 HS899-Q8/61 0.000 0.212 0.212 0.212 0.212 0.212 0.212 0.212 0.212 0.212 0.212 0.212 0.000 0.000 0.000 0.000 0.000 0.000 0.000 PFOS Concentration Measured (mg/L)____________________Measured (%) Total Biomass Medium Total Biomass Medium (Mass Balance) 0.0400 0.023 0.020 0.005 0.019 0.0309 0.0222 0.0229 0.300 0.365 0.373 0.341 0.316 0.352 0.310 0.337 0.340 0.388 0.393 0.346 0.335 0.395 0.340 0.362 NA3 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 0.0841 0.0321 0.0131 0.0351 0.0521 0.0251 0.0471 0.0066 0.0748 0.0273 0.0397 0.249 0.175 0.207 0.271 0.188 0.108 0.202 0.101 0.220 0.100 0.180 0.333 0.207 0.220 0.306 0.240 0.133 0.249 0.108 0.295 0.127 0.219 39.7 15.1 6.18 16.6 24.6 11.8 22.2 . 3.11 35.3 12.9 79.9 c 117 82.5 97.6 128 88.7 50.9 95.3 47.6 104 47.2 85.9 c 157 97.7 104 144 113 62.8 118 50.8 139 60.0 105 NA NA NA NA NA <0.0100 < 0.0100 <0.0100d NA <0.0100 NA <0.0100 NA <0.0100 NA <0.0100 NA <0.0100 < 0.0100 < 0.0100 < 0.0100 NA <0.0100 NA <0.0100 NA <0.0100 NA <0.0100 NA <0.0100 NA NA NA NA NA NA NA NA NA NA NA Analytical Control HS0699-12/0 HS799-05/16 HS799-06/16 HS799-07/16 HS799-123/30 HS799-124/30 HS799-125/30 HS899-71/61 HS899-72/61 HS899-73/61 0.212 0.212 0.212 0.212 0.212 0.212 0.212 0.212 0.212 0.212 NA 0.232 0.232 NA 0.197 0.197 NA 0.198 0.198 NA 0.196 0.196 NA 0.179 0.179 NA 0.187 0.187 NA 0.188 0.188 NA 0.159 0.163 NA 0.142 0.144 NA 0.152 0.154 NA 109 NA 92.9 NA 93.4 NA 92.5 NA 84.4 NA 88.2 NA 88.7 NA 75.0 NA 67.0 NA 71.7 109 92.9 93.4 92.5 84.4 88.2 88.7 75.0 67.0 71.7 a NA = not applicable b Net values after subtraction of inoculum control average values. c Ratio equivalent to 18% PFOS on solids and 82% in medium. d Values presented as less than were below the limit of quantitation BACK TO MAIN PFOS - Activated Sludge/Sediment_________________________________________ Page 30 Table 8. Continued. PFOS concentration measurements for the 0.200 mg/L test samples from the sealed vessels (headspace). Sample No./ Sample Day QCa Medium C699-266/0 C699-267/0 C799-40/16 C799-41/16 C799-42/16 C799-43/16 C799-145/30 C799-146/30 C799-147/30 C799-148/30 C899-94/61 C899-95/61 C899-96/61 C899-97/61 Nominal (mg/L) 0.200 0.200 0.200 0.200 0.200 0.200 0.200 0.200 0.200 0.200 0.200 0.200 0.200 0.200 PFOS Concentration Measured (mg/L) Measured (%) Total Biomass Medium Total Biomass Medium (Mass Balance) NA NA NAb NA NA NA NA NA NA NA NA NA NA NA 0.195 0.186 0.195 0.186 0.195 0.186 0.162 0.176 0.170 0.163 0.221 0.207 0.229 0.218 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 97.5 NA 93.0 NA 106 NA 111 NA 105 NA 98.5 NA 81.0 NA 88.0 NA 85.0 NA 81.5 NA 111 NA 104 NA 114 NA 109 NA NA NA NA NA NA NA NA NA NA NA NA NA NA QC solids C699-262/0 C699-263/0 C799-38/16 C799-39/16 C799-149/30 C799-150/30 C899-98/61 C899-99/61 0.200 0.200 0.200 0.200 0.200 0.200 0.200 0.200 0.219 0.213 0.219 0.213 0.211 0.210 0.211 0.205 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 110 NA 107 NA 115 NA 110 NA 106 NA 105 NA 106 NA 103 NA NA NA NA NA NA NA NA NA a QC = quality control sample b NA = not applicable BACK TO MAIN PFOS - Activated Sludge/Sediment_________________________________________ Page 31 Table 9. PFOS concentration measurements for the 105 m g/Ltest samples from the sealed vessels (headspace). Sample No./ Sample Day Blank Inoculum HS799-41/0 HS799-42/0 HS799-135/15 HS799-136/15 HS899-25/30 HS899-26/30 HS999-25/63 HS999-26/63 Nominal (mg/L) ' PFOS Concentration Measured (mg/L)____________________Measured (%) Total Biomass Medium Total Biomass Medium (Mass Balance) 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 <0.0100a <0.0100 <0.0100 < 0.0100 < 0.0100 <0.0100 < 0.0100 < 0.0100 <0.0100 <0.0100 <0.0100 <0.0100 <0.0100 <0.0100 <0.0100 <0.0100 < 0.0100 <0.0100 <0.0100 < 0.0100 <0.0100 < 0.0100 <0.0100 < 0.0100 NAb NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA PFOS + Inoculum HS799-45/0 HS799-46/0 HS799-139/15 HS799-140/15 HS899-29/30 HS899-30/30 HS999-29/63 HS999-30/63 Analytical Control HS799-43/0 HS99-44/0 HS799-137/15 HS799-138/15 HS899-27/30 HS899-28/30 HS999-27/63 HS999-28/63 105 1.31 151 152 1.25 144 105 2.86 134 136 2.72 127 105 0.912 138 139 0.869 131 105 1.10 145 146 1.05 138 105 1.25 142 143 1.19 135 105 2.50 136 139 2.34 130 105 1.42 125 127 1.35 119 105 2.27 130 132 2.17 124 105 NA 120 120 NAa 114 105 NA 115 115 NA 109 105 NA 129 129 NA 123 105 NA 109 109 NA 103 105 NA 123 123 NA 117 105 NA 124 124 NA 118 105 NA 108 108 NA 103 105 NA 99.5 99.5 NA 94.7 145 130 132 139 136 132 121 126 114 109 123 103 117 118 103 94.7 QCCMedium C799-131/0 C799-132/0 C799-133/0 C799-165/15 C799-166/15 C799-167/15 C899-02/30 C899-03/30 C899-04/30 C999-103/63 C999-104/63 C999-105/63 25.0 50.0 100 25.0 50.0 100 25.0 50.0 100 25.0 50.0 100 NA 25.3 NA NA 53.7 NA NA 104 NA NA 29.5 NA NA 59.1 NA NA 115 NA NA 27.5 NA NA 52.6 NA NA 106 NA NA 26.8 NA NA 53.6 NA NA 110 NA NA 101 NA 107 NA 104 NA 118 NA 118 NA 116 NA 110 NA 105 NA 106 NA 107 NA 107 NA 110 NA NA NA NA NA NA NA NA NA NA NA NA a Values presented as less than were below the limit of quantitation. b NA = not applicable 0 QC = quality control sample BACK TO MAIN PFOS - Activated Sludge/Sediment_________________________________________ Page 32 Table 9. Continued. PFOS concentration measurements for the 105 mg/L test samples from the sealed vessels (headspace). Sample No./ Sample Day Nominal (mg/L) PFOS Concentration Measured (mg/L) Measured (%) Total Biomass Medium Total Biomass Medium (Mass Balance) QCa Solids C799-134/0 C799-135/0 C799-136/0 C799-168/15 C799-169/15 C799-170/15 C899-05/30 C899-06/30 C899-07/30 C999-106/63 C999-107/63 C999-108/63 33.3 66.7 100 33.3 66.7 100 33.3 66.7 100 66.7 66.7 100 35.7 69.9 99.0 37.1 68.9 107 37.7 75.4 102 68.1 72.3 103 NAb NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 107 105 99.0 111 103 107 113 113 102 102 108 103 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA a QC = quality control sample b NA = not applicable BACK TO MAIN PFOS - Activated Sludge/Sediment Figure 1. Flow chart of extraction procedures. Page 33 ACTIVATED SLUDGE/SEDIMENT SAMPLES AND ANALYTICAL CONTROL SAMPLES All samples that contain solids: Centrifuge @ 1200 x g for 30 min AQUEOUS (discard) ORGANIC 1 ROTOVAP/N2 LOW VOLUME 1 R E C O N STITU TE IN MEOH ^ filter LC/MS ANALYSIS MEOH EXTRACT 1filte r LC-MS ANALYSIS S O L ID S (discard) BACK TO MAIN PFOS - Activated Sludge/Sediment_________________________________________ Page 34 Figure 2. Total measured PFOS concentration during acclimation in activated sludge/sediment.9 Week Number Nominal values were calculated using the following assumptions: a) 10% of the biomass and 70% of the medium was removed and replaced each week with fresh biomass and 14.84 mg/L PFOS (70% of nominal) and b) 92.0% of PFOS partitions to biomass in the system, 0.3% remains in the medium, and 1.2% binds to glass. BACK TO MAIN PFOS - Activated Sludge/Sediment_________________________________________ Page 35 Figure 3. PFOS concentration in the analytical control flask. Week Number BACK TO MAIN PFOS - Activated Sludge/Sediment_________________________________________ Page 36 Figure 4. Concentration of PFOS versus time during the closed vial (headspace) aerobic biodegradation test. -6--Nominal - Inoculated Analytical Control