Document 9940pM0jXv0wQBz03ojB6jvnD

BACK TO MAIN Study Title Microbial Metabolism (Biodegradation) Studies of PerfluorooctaneSulfonate (PFOS) IV. Pure Culture Study 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, Massachusetts02571-1075 Laboratory Project ID Springborn Study No.: 290.6120 Page 1 of 21 PFOS - Pure Culture SUBMITTED BY: SIGNATURES AND APPROVAL Springborn Laboratories Inc. 790 Main Street Wareham, Massachusetts02571-1075 William E. Gledhill, Ph.D. Director, Environmental Fate and Microbiological Programs Date BACK TO MAIN Page 2 Principal investigator Senior Research Chemist Senior Research Chemist BACK TO MAIN PFOS .Pure Culture Page 3 TABLE OF CONTENTS Page SIGNATURES AND APPROVAL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 LISTOFTABLES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 LIST OF FIGURES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 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 2.2.2PFOS Stock Solution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 2.2.3Tetrabutylammonium Sulfate Solution. . . . . . . . . . . . . . . . . . . . . . . . . . . 10 2.2.4Sodium Hydroxide Solution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IO 2.2.5Ammonium Acetate Solution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 3.0 ANALYTICAL METHODS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 3.1 Pure Culture Study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 . 3.1.1 Preparation of the Broth Test Samples . . . . . . . . . . . . . . . . . . . . . . . . . 10 3.1.2 Preparation of the Cell Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1 3.1.3 Quality Control Sample Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1 3.2Closed Vial (Headspace) Study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 3.2.1Preparation of Broth Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 3.2.2Preparation of the Cell Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 3.2.3Quality Control Sample Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 3.3 Instrumental Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 4.0 TEST PROCEDURES. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 4.1 Pure Culture Study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 4.1.1 Test Procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 4.1.2 Results and Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 4.2 Closed Vial Headspace . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 4.2.1Test Procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 4.2.2Results and Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 5.0 CONCLUSIONS AND FUTURE STUDIES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 BACK TO MAIN PFOS - Pure Culture Page 4 LIST OF TABLES Page Table 1. Results from the microbial transformation study, Day 0 and Day 7 broth and cell samples , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 Table 2. Results from the closed vial microbial transformation study, Day 3 broth and cell samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 BACK TO MAIN PFOS - Pure Culture Page 5 LIST OF FIGURES Page Figure 1. Flow chart of extraction procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 BACK TO MAIN PFOS - Pure Culture 1.O 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 for the mixed culture systems (soil, activated and anaerobic sludge) 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 P450 monooxygenase). 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 from natural environments and pure cultures 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 replenishmentof natural nutrientswithout 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. BACK TO MAIN PFOS - Pure Culture Page 7 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 of fluorochemicals. This report summarizes the results of exposure of PFOS to several pure culture of organism. 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 off- white 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 I-pL and IO-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 assumptionthat the signal ratio for each is 1:1with PFOS. BACK TO MAIN PFOS - Pure Culture Page 8 PerfluorinatedAlkyl Sulfonates Observed Analyte Peak Response (Area) Perfluorononanesulfonate (C9) response 0.990 Perfluorooctane sulfonate (C8) response 223.245 Perfluoroheptane sulfonate (C7) response 5.786 Perfluorohexane sulfonate (C6) response 4.262 Perfluoropentanesulfonate (C5) response 5.326 Perfluorobutane sulfonate (C4) response 5.190 Perfluoropropane sulfonate (C3) response 2.622 Total 247.421 Total (Yo) 0.400 90.23 2.34 1.72 2.15 2.10 1.06 100 The internal standard, perfluorooctanoicacid (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 1OO-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 1OO-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 solutionwere placed in a 1OO-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/I 5.0mg/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 50mL 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 BACK TO MAIN PFOS - Pure Culture Page 9 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 IOO-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 IOO-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 l-mL aliquot of an aqueous PFOS stock solution that 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.OO mg/mL PFOS primary stock solution (SLI No. 70-93C) was prepared by placing 0.1004 g of PFOS in a IOO-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 IOO-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 Fortification Volume (mL) Final Volume (mL) Diluent Standard Concentration (mglL) 1.22 mg/mL 1.22 mg/mL 1.22 mg/mL 122 mg/L 12.2 mg/L 1.22 ma/L 0.100 0.0500 0.0250 0.0500 0.125 0.410 50.0 Methanol 50.0 Methanol 50.0 Methanol 50.0 Methanol 50.0 Met h a n o I 50.0 Methanol 2.44 1.22 0.610 0.122 0.0305 0.0100 BACK TO MAIN PFOS - Pure Culture Page 10 The calibration standards were stored in amber bottles with Teflon@-linedcrimp 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 (Le., 25 pL of 25 mg/L PFOA/15 mg/L THPFOS to 2.00 mL of standard). 2.2.3 Tetrabutylammonium Sulfate Solution. Tetrabutylammoniumsulfate (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 HydroxideSolution. Sodium hydroxidesolutions (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 Solution. Ammonium acetate solut'ions (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 ANALYTICAL METHODS 3.1 Pure Culture Study On day zero, one-half of the flask cultures were removed and centrifuged at 1200 x g with a Beckman Model 65-6R centrifuge (rotating radius of 12.5 cm, from RCF Nomagraph, IEC (International Equipment Company),300 SecondAve., Needham Heights, Massachusetts, 02194) for 30 minutes at 4C to separate cells from medium. The methods used for extraction and analysis of the broth and cell samples are summarized in the flow chart presented in Figure 1. Supernatant and the cell samples were then extracted and analyzed as described below. 3.1.1 Preparation of the Broth Test Samples A 1.0-mL aliquot of the 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-vm filter (Titan, nylon BACK TO MAIN PFOS - Pure Culture Page 11 membrane) prior to analysis. A 2-mL aliquot was removed from each sample and 25 pL of the 25.0 mg/L PFOAl15.0 mg/L THPFOS internal standard solution was added to the sample aliquot. 3.1.2 Preparation of the Cell Samples Each solid sample was extracted once with 40 mL of methanol, vortexed for 30 seconds, sonicated for 15 minutes, and shaken on a shaker table at 200 rpm for 30 minutes. 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 then diluted as appropriate in methanol. A 2.0-mL aliquot was removed from each sample extract and 25 pL of the 25.0 mg/L PFOAl15.0 mg/L THPFOS internal standard solution was added. 3.1.3 Quality Control Sample Preparation Preparation of the aqueous and solid quality control (QC) samples is summarized in the following table. When necessary and prior to QC fortification, the pure culture samples were centrifuged at 1200 x g for 30 minutes to separate the broth and cell portions. All QC samples were prepared by fortifying the appropriate control matrix (broth or cells) at the concentrations listed below. The QC samples were then treated following the same procedures as the test samples (described above). QC Sample TY Pe PFOS Stock Concentration (mglL) Davs 0 & 7 Broth 1000 1000 1000 Cells 1000 1000 1000 1 = Mixed both matrix 2 = Solids from 10.0mL broth Volume of Stock Solution Used (mL) 0.0500 0.100 0.100 0.0250 0.0250 0.0500 Control Matrix Volume of Control Matrix (mL) 1 5.00 1 5.00 1 5.00 2 10.0 2 10.0 2 10.0 QC Sample Concentration 10.0mglL 20.0 mg/L 20.0 mg/L 25.0 pg 25.0 pg 50.0pg 3.2 Closed Vial (Headspace) Study On day three all of the flask cultures were removed and centrifuged at 1200 x g with a Beckman Model 65-6R centrifuge (rotating radius of 12.5 cm, from RCF Nomagraph, IEC (International EquipmentCompany), 300 SecondAve., Needham Heights, Massachusetts, 02194) for 30 minutes at 4C to separate cells from medium. The methods used for extraction and analysis of the broth BACK TO MAIN BFOS - Pure Culture Page 12 and cell samples are summarized in the flow chart presented in Figure 1. Supernatant and cell samples were then extracted and analyzed as described below. 3.2.1 Preparation of Broth Samples The entire volume (4.00 mL) of the centrifuged supernatant from each sample was transferred to a separatory funnel. An aliquot (10.0 mL) of 0.5M pH10 TBA-S solution and an aliquot (50.0 mL) of purified reagent water was added to each separatory funnel. The samples were extracted three times with 300 ml of a 5050 ethyl acetate/hexane solution. When emulsions formed an additional 1 to 2 mL of methanol was added to the separatory funnel to dissipate them. The extracts were reduced in volume by rotary evaporation at 35 "Cto approximately2 mL (sampleswere not allowed to go dry). When water was present in the round bottom flask methanol was added to remove it. The concentrated extracts were transferred to glass tubes and the round bottom flasks were rinsed methanol which was also added to the tubes. The volume of the extracts was further reduced under a gentle stream of nitrogen at room temperature to an approximate final volume of 100 pL. These were then reconstituted to the appropriate final volume (4.00 mL) with 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 PFOAll5.0 mg/L THPFOS internal standard solution was added. 3.2.2 Preparation of the Cell Samples Each solid sample was extracted once with 20 mL of methanol, vortexed for 30 seconds, sonicated for 15 minutes, and shaken on a shaker table at 200 rpm for 30 minutes. The samples were then centrifuged at 1200 x g for 30 minutes using a Beckman Model 656R 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 then diluted as appropriate in methanol. A 2.0-mL aliquot was removed from each sample extract and 25 pL of the 25.0 mg/L PFOAll 5.0mg/L THPFOS internal standard solution was added. 3.2.3 Quality Control Sample Preparation Preparation of the aqueous and solid quality control (QC) samples is summarized in the following table. When necessary and prior to QC fortification, the pure culture samples were centrifuged at 1200 x g for 30 minutes to separate the broth and cell portions. All QC samples were prepared by fortifying the appropriate control matrix (broth or cells) at the concentrations listed below. The QC samples were then treated following the same procedures as the test samples (described above). PFOS - Pure Culture BACK TO MAIN Page 13 QC Sample TY Pe PFOS Stock Concentration (mglL) Volume of Stock Solution Used (mL) Control Matrix Volume of Control Matrix (rnL) QC Sample Concentration Broth Cells 10.0 10.0 10.0 10.0 1 = 1:lOO broth:purified reagent water 2 = 1 : l O broth:purified reagent water 3 = Solids cells. 0.0800 0.0800 0.0750 0.0750 1 4.00 0.200 mg/L 2 4.00 0.200 mglL 3 0.1078 0.750 pg 3 0.1158 0.750 pg 3.3 Instrumental Conditions The following instrumental conditions were used during the analysis of the test samples. Instrumental System Column: Mobile phases: Flow Rate: Gradient program: Run time: Equilibration delay: Injection volume: Hewlett-Packard Model 1050 quaternary pump, membrane degasser, autosampler, PE Sciex APl 100 LC/MS, PE Sciex TurbolonSpray@(electrospray) Keystone Betasil C18, 5 pm, 100 A, 150 x 2 mm column with a Betasil C18 guard column A: 2 mM ammonium acetate in purified reagent water B: 100% Methanol 0.3 mUmin Time (min) - %A - %B 0 60 40 8.5 10 90 I1 10 90 13 0 100 17 0 100 20 60 40 20 min 10 min 10 pL LC/MS Darameters Experiment information: Scan type: Scan time: Peak Hopping: Mass defect: Pause time: Q1, SIM 2.01 sec Disabled 0 mmu/100 amu 2 msec BACK TO MAIN PFOS - Pure Culture Page 14 Dwell time: 400 msec Masses scanned (amu)*: 413 (PFOA),427 (THPFOS),499 (PFOS), 616,630 (N-EtFOSE-OH) * Based on 3M analytical method No. ETS-8-11.O 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 DISCUSSIONS 4.1 Pure Culture Study 4.1.ITest Procedures Microbial transformations of PFOS were conducted using four species of microorganisms representative of soil microflora microflora using the two-stage incubation procedure of Betts et a/. (1974). These species, originally obtained from the American Type Culture Collection (ATCC), were selected on the basis of their performance during previous biotransformation studies conducted at Springborn, and their documented history in metabolizing xenobiotics. The fungi Cunninghamella echinulafa var. echinulafa (ATCC No. 9244), Mucor circinelloides f. griseocyanus (ATCC No.1207a), and Phanerochaete chrysosporium (ATCC No. 24725), are cultured on Potato Dextrose Agar slants, and the actinomycete Streptomyces griseus (ATCC No. 13273) is cultured on Yeast Malt Extract Agar (ATCC No. 196) according to ATCC recommendations. The stock slants for each species were maintained under refrigeration at approximately 4 "C. The microorganisms were inoculated onto agar slants directly from the preserved in-house slants, and the freshly prepared slants were incubated at a temperature range of 22 to 26 "C. Once sufficient growth was observed (4 days), these slants were used directly to inoculate the Stage I flasks. All work performed with the pure cultures utilized strict aseptic technique, up until the time of harvest of the dosed systems. For all microorganisms, Stage I cultures were initiated with actively growing microorganisms. Cells were inoculated into 250-mL Erlenmeyer flasks with polyurethane foam plugs and stainless steel BACK TO MAIN PFOS - Pure Culture Page 15 caps. Each flask received 60 mL of a soybean grits-glucose (SGG)medium and was incubated for 3 days in an incubatorkhaker set at 250 rpm at 24 to 26 "C. After 3 days, the Stage II flasks were initiated by transferring 6 mL of each culture to 60 mL of freshly prepared soybean gritsglucose medium. After approximately 24 hours, one Stage II culture flask per species was dosed with 20.9 mg/L of PFOS. This concentration was achieved by addition of 1.3 mL of a 1.06 mg/mL aqueous PFOS stock solution that had been vortexed and vigorously shaken. Corresponding Stage II culture flasks for each species were left undosed to provide controls. After allowing the Stage II cultures to shake for approximately 20 minutes, Day 0 samples were removed (25-mL aliquots), centrifuged at approximately 1400 x g (3500 rpm) for 30 minutes (Phanerochaete chrysosporiurn for 45 minutes) at 4 "C to separate broth and medium and refrigerated prior to analysis. Purified reagent water (2 to 3 mL) was added to the medium of some species to aid in the transfer. The Stage II cultures were returned to the incubator at approximately 26 "C and 250 rpm for 7 days. After 7 days, the cultures were harvested by centrifugation at approximately 1400 x g (3500rpm) for 30 minutes at 4 "Cto separate broth and medium. The same procedure was also followed to provide undosed matrix samples which were used to prepare for quality control samples. The broth and cells were refrigerated prior to analysis. 4.1.2 Results and Discussion Table 1 summarizes the fate of PFOS in the microbiological media inoculated with four species of microorganisms. Mass balances for the fortified cultures ranged from 90% to 125%. No evidence of significant metabolism of PFOS by any culture was noted. 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. 4.2 Closed Vial Headspace 4.2.1 Test Procedures Additional sealed vessel test systems were also set up with lower PFOS concentrations in an attempt to obtain better mass balance measurements. These systems were used to culture Phanerochaete and contained 1/ I 0 and 1/100 strength soybean grits-glucose medium and resazurin. The test systems were prepared by centrifuging approximately 50 mL of SGG media BACK TO MAIN PFOS - Pure Culture Page 16 at 1200x g (3000 rpm) for 30 minutes. Simultaneously,two IO-mL aliquots of Phanerochaefe were centrifuged at 1200x g (3000 rpm) for 30 minutes. The SGG supernatant was filtered using a 0.22pm filter and serial ten-fold dilutionswere made to obtain SGG concentrations of IO-' and 1O-'. The medium from the Phanerochaefe tubes was decanted, sterile reagent grade water was added to a volume of 10 mL, and the medium was vortexed and centrifuged again. The medium was again decanted from the tubes and SGG was added to each tube, IO-' to one tube and IO-* to the other, until each tube contained an approximate volume of 10 mL. A 4-mL aliquot of each SGG/culture mixture was added to a 22-mL sterile vial. A 90-pL aliquot of resazurin (0.445 mg/mL) was added to each vial resulting in a resazurin concentration of 10 mg/L. The 10 mg/mL PFOS stock solution (SLI No. 70-938) was diluted 1 : l O O in methanol to produce a secondary stock solution with a concentration of 0.1 mg/mL. An 8-pL aliquot of this stock solution was then added to each sterile vial for a final PFOS concentration of 0.0002 mg/mL. Abiotic control vials, which contained no inoculum, and matrix control vials, which contained no PFOS, were prepared in the same manner. The headspace of each vial was purged with 0, and the caps were crimped immediately. Each vial was incubated at 26 "Cand 250 rpm. Daily thereafter, color was observed and growth was recorded. 4.2.2 Results and Discussion Preliminary analytical results of the broth (Section 4.1.2) suggested possible biotransformation of PFOS by Phanerochaefe. Based on these results, several closed bottle experiments were conducted with Phanerochaefe and PFOS, however, difficulty was experienced keeping the systems aerobic. Since aerobicity was lost, bottles were analyzed on Day 3 even though it was not known if the culture had been exposed for a sufficient period of time to allow for biotransformation of PFOS. Abiotic controls were also run to provide controls for potential hydrolysis or adsorption versus biotransformation. Data, summarized in Table 2, however, indicates no significant biotransformationof PFOS by Phanerochaefe. 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. PFOS - Pure Culture 5.0 CONCLUSIONS AND FUTURE STUDIES BACK TO MAIN Page 17 It does not appear that the four species studied are capable of metabolizing PFOS. 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 in an attempt to enrich for PFOS degradates. 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 - Pure Culture Paae 18 REFERENCES 3M. Technical Report Summary, Fate of Fluorochemicals, 11/15/77. 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 4546-1 07, OECD Guideline for the Testing of Chemicals, 105 Water Solubility, May 3, 1999. Betts, R.E., Walters, D.E. and Rosazza, J.P. 1974. Microbial Transformations of Antitumor Compounds. 1. Conversion of Acronycine to 9-hydroxyacronycine by Cunninghamella echinulata. Journal ofMedical Chemistry. 17:599-602. BACK TO MAIN PFOS - Pure Culture Table 1. Resultsfrom the microbialtransformationstudy, Day 0 and Day 7 broth and cell samples. PFOS Concentration Measured (mglL) Measured (%) Page 19 Sample NO./ Species Day 0 Nominal (mglL) Biomass Medium Total 499-09/9244a 20.9 499-1019244 0.00 499-11I1207ad 20.9 499-1211207a 0.00 499-1312472Y 20.9 499-14124725 0.00 499-15113273' 20.9 499-16113273 0.00 Day 7 499-09/9244a 20.9 499-1019244 0.00 499-11I 1207ad 20.9 499-1211207a 0.00 499-13124725" 20.9 499-14124725 0.00 499-15113273' 20.9 499-16113273 0.00 14.5 0.0100 15.3 c 0.0100 7.38 < 0.0100 15.1 0.01 00 16.9 0.0100 11.2 0.0100 22.3 0.0100 15.1 0.0100 7.98 0.01 00 7.57 0.01 00 11.5 0.01 00 11.1 0.0100 8.18 0.0100 11.1 0.0100 1.20 0.0100 6.88 0.0100 22.5 0.0100 22.9 0.0100 18.9 0.0100 26.2 0.0100 25.1 0.01 00 22.3 0.01 00 23.5 0.01 00 22.0 0.0100 QC brothQ c599-33 10.0 NA 7.89 NA 099-34 20.0 NA 11.8 NA c599-35 20.0 NA 18.7 NA QC cellsh C599-152 25.0 26.2 NA NA C599-153 25.0 22.3 NA NA C599-154 50.0 48.8 NA NA 9244 = Cunninghamella echinulata var. echinulata Values expressed as 0.0100 were below the limit of quantitation. NA = not applicable 1207a = Mucor circinelloides f. griseocyanus 24725 = Phanemchaete chrysosporium 13273 = Streptomyces griseus QC = quality control sample QC cells nominal and measured concentrations are in pg Biomass 69.6 NAC 73.3 NA 35.3 NA 72.1 NA 80.8 NA 53.6 NA 107 NA 72.3 NA NA NA NA 105 89.0 97.6 Medium 38.2 NA 36.2 NA 55.0 NA 53.1 NA 39.1 NA 53.1 NA 5.76 NA 32.9 NA 78.9 89.6 93.7 NA NA NA Total (Mass Balance) 108 NA 110 NA 90.3 NA 125 NA 120 NA 107 NA 112 NA 105 NA NA NA NA NA NA NA BACK TO MAIN PFOS - Pure Culture Page 20 Table 2. Results from the closed vial microbial transformation study, Day 3 broth and cell samples. Measured (mg/L) Measured (%) Sample No./ Nominal Species Sample Concentration ID (mg/L) Biomass Medium Total Biomass Medium 799-01/24725 0.209 0.0720 0.127 0.199 34.4 60.8 1O(-1) SGG" 799-02/Abiotic 0.209 NA" 0.197 0.197 NA 94.3 ctrl, IO(-I) SGGb 799-031Matrix 0.000 < 0.0100" < 0.0100 0.0100 NA NA ctrl, IO(-I) SGGd 799-04124725 0.209 0.0280 0.145 0.173 13.4 69.4 IO(-2) SGG 799-05/Abiotic 0.209 NA 0.185 0.185 NA 88.5 ctrl, lo(-2) SGG 799-06/Matrix 0.000 0.0100 0.0100 < 0.0100 NA NA ctrl, IO(-2) SGG Total (Mass Balance) 95.2 94.3 NA 82.8 88.5 NA QC broth' C799-16 C799-17 0.200 0.200 NA 0.214 NA NA 107 NA NA- 0.193 NA NA 96.5 NA QC cellsg C799-18 C799-19 0.750 0.768 NA NA 102 NA NA 0.750 0.784 NA NA 105 NA NA a 24725 = Phanerochaete chrysosporium Abiotic controls do not contain biomass " NA = not applicable Matrix controls contain biomass but no PFOS e Values 0.0100 were below LOQ QC = quality control sample QC cell concentration values are in pg PFOS - Pure Culture Figure I. Flow chart of extraction procedures. BACK TO MAIN Page 21 SAMPLES I All samples: Centrifuge @ 1200 x g for 30 minutes PORTION 0.209 mg/L Add 1 volume 0.5 M TBA-HSO, 20.9 mg/L Extract3 x with 300 mL EtOAc/hexane PORTION 37 IN MEOH 4 Filter ANALYSIS - I I A I , NITROGEN (low volume) I W/ MEOH I 4 Filter m J.I (Discard) 1 W/ MEOH Filter 1LC/MS r l ANALYSIS