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BACK TO MAIN Study Title Microbial Metabolism (Biodegradation) Studies of Perfluorooctane Sulfonate (PFOS) I I . Aerobic Soil Biodegradation Authors William E.Gledhill, Ph.D. Barbara J. Markley, Ph.D. Study Completed On 31 October 2000 Submitted To 3M Environmental Laboratory 935 Bush Avenue, BLDG 2-3E-09 St. Paul, Minnesota 55133-3331 Performing Laboratory Springborn Laboratories, Inc. 790 Main Street Wareham, Massachusetts 02571-1075 Laboratory Project ID Springborn Study No.: 290.6120 Page 1 of 17 PFOS - Aerobic Soil Biodegradation SIGNATURES AND APPROVAL SUBMITTED BY: Springborn Laboratories Inc. 790 Main Street Wareharn, Massachusetts 02571-1075 BACK TO MAIN Page 2 Director, Environmental Fate and Microbiological Programs Principal Investigator Senior Research Chemist Dirgctor, Chemistry `J Senior Research Chemist BACK TO MAIN PFOS .Aerobic Soil Biodegradation 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 2.2.2 PFOS Stock Solution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 2.2.3 Ammonium Acetate Stock Solution . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 3.0 PREPARATION OF SAMPLES AND ANALYTICAL METHODS . . . . . . . . . . . . . . . . . . 10 3.3 Dionex ASET" Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 3.2 Quality Control Sample Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 3.3 Instrumental Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 4.0 TEST PROCEDURES, RESULTS, AND DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . . . . 12 4.1 Test Procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 4.2 Results and Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 5.0 CONCLUSIONS AND FUTURE STUDIES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. BACK TO MAIN PFOS - Aerobic Soil Biodegradation Page 4 LIST OF TABLES Page Table 1. Results for the test samples from the aerobic soil biodegradation study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 BACK TO MAIN PFOS - Aerobic Soil Biodegradation Page 5 LIST OF FIGURES Page Figure 1. Flow chart of extraction procedures . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . 17 BACK TO MAIN PFOS - Aerobic Soil Biodegradation 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 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 P,,, 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 capable of metabolizing unique chemicals. Among these factors were: - testing and enrichment under non-toxic conditions - the use of natural ecosystems as the basis for enrichment - the use of natural nutrients from those ecosystems with supplemental trace minerals, co-factors and vitamins - the continual introduction of new microbes from different natural sources - the periodic replenishment of natural nutrients without diluting out the species being enriched - the provision for a realistic time frame for enrichment and acclimation - the protection of microorganisms from toxic products or metabolites by use of low substrate concentrations, replenishment of nutrients, balanced medium (C:N:P, etc.), proper pH and provision of a protective surface for growth (vermiculite, sand, soil, activated C, diatomaceous earth, etc.) - the enrichment in more concentrated (higher biomass and test substance concentration) systems and examination of biodegradation in more dilute Systems - the separation of systems selective for fast growing (zymogenous) and slow growing (autochthonous) species - use of pure cultures containing the cytochrome P,50 monooxygenase enzyme system known to metabolize complex molecules. Throughout the experimental program the principles outlined above for enrichment were incorporated. The results of the studies are summarized in four separate reports and provide a BACK TO MAIN PFOS - Aerobic Soil Biodearadation Paae 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 aerobic soil biodegradation system. 2.0 TEST SUBSTANCE, INTERNAL STANDARDS, AND SOLUTION PREPARATION 2.1 Test Substance and Internal Standards The test substance, perfluorooctane sulfonate potassium salt (PFOS, lot no. TN-A-2130), an offwhite powder, was received on 25 January 1999 (SLI No. 70-93) from 3M Environmental, St. Paul, Minnesota. Prior to study completion, analytical characterization of the PFOS test substance was not conducted. Therefore, all calculations in the report are based on PFOS purity of 100%. After study completion, a sample of a 1.06 mg/mL PFOS stock solution (SLI No. 70-93A), see Section 2.2.2) was sent to 3M Environmental Laboratory for evaluation of impurities using LCIMS-TOF. Compounds looked for were the C2 to C10 PFOS analogous materials, and many of them were observed in both the l-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 : l with PFOS. Perfluorinated Alkyl Sulfonates Observed Analyte Peak Response (Area) Perfluorononane sulfonate (C9) response 0.990 Perfluorooctanesulfonate (C8) response 223.245 Perfluoroheptanesulfonate (C7) response 5.786 Perfluorohexanesulfonate (C6) response 4.262 Perfluoropentanesulfonate (C5) response 5.326 Perfluorobutanesulfonate (C4) response 5.190 Perfluoropropanesulfonate (C3) response 2.622 Total 247.421 Total (YO) 0.400 90.23 2.34 1.72 2.15 2.10 1.06 100 BACK TO MAIN PFQS - Aerobic Soil Biodegradation Page 8 The internal standard, perfluorooctanoic acid (PFOA, lot no. 07216AS), an off-white solid wax, was received on 1 April 1999 (SLI No. 71-94) from Aldrich, Milwaukee, Wisconsin. An additional internal standard, 1,1,2,2-tetrahydroperfluorooctane sulfonate (THPFOS), a brown crystal was received on 18 January I 9 9 9 (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 pglmL 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 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 mgiL THPFOS. The 25.0 mg/L PFOAl15.0 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 50me 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 sludgekediment, 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 PFQS 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 PFQS stock solution (SLINo. 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 BACK TO MAIN PFOS - Aerobic Soil Biodegradation Page 9 resulted in a 100.2% recovery (SLI No. F499-59). Note that the solubility of PFOS in purified reagent grade water is 0.567 mg/mL (SD= 52.8, CV = 9.31%, n = 6) (VanHoven and Nixon, 1999). A 1.OO mg/mL PFOS primary stock solution (SLI No. 70-93C) was prepared by placing 0.1004 g of PFOS in a 100-mL volumetric flask and bringing to volume with methanol. This stock solution was used in the preparation of quality control samples. A final PFOS primary stock solution with a concentration of 1.22 mg/mL (SLI No. 70-93D) was prepared by placing 0.1223 g of PFOS in a 100-mL volumetric flask and bringing to volume with methanol. Secondary stock solutions with concentrations of 1.22, 12.2, and 122 mg/L were prepared by placing the appropriate volume of the 1.22 mg/mL primary stock solution in a 50.0-mL volumetric flask and bringing to volume with methanol. The primary and secondary stock solutions were used to prepare calibration standards. Preparation of the calibration standards is detailed in the following table. Concentration of Stock Solution 1.22 mg/mL 1.22 mg/mL 1.22 mg/mL 122 mg/L 12.2 mg/L 1.22 mg/L Fortification Volume (mL) 0.100 0.0500 0.0250 0.0500 0.125 0.41 0 Final Volume (mL) 50.0 50.0 50.0 50.0 50.0 50.0 Diluent Standard Concentration (mglL) 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 LCIMS ana1ysis)to the calibration standards in the same manner they were added to the test samples ( k . , 25 pL of 25 mg/L P F O N I5 mg/L THPFOS to 2.00 mL of standard). Only the 1.06 mg/mL PFOS (70-93A) stock was used to dose the soil for the soil biodegradation test. BACK TO MAIN PFOS - Aerobic Soil Biodegradation Page 10 2.2.3 Ammonium Acetate Stock Solution. Ammonium acetate solutions (2 mM) were prepared by adding 0.151 g of ammonium acetate to a 1000-rnLvolumetricflask and bringing to volume with purified reagent water. 3.0 PREPARATION OF SAMPLES AND ANALYTICAL METHODS The methods used in the analysis of the aqueous and solid samples are summarized in the flow chart presented in Figure 1. For each test sample, the entire test sample was transferred to a 22-mL accelerated solvent extractor (ASE) cell. Any space remaining in the ASE cell was filled with Ottawa sand. The cells were capped, shaken, and then placed on the DionexASETMfor extraction. The extraction was performed following the program presented below. The program was performed twice; the extracts collected separately and analyzed. 3.d Dionex ASE" Method Pressure: Tem perat ure: Preheat Time: Purge During Preheat : Heat Time: Static Time: Flush Volume: Purge time: Static Cycles: Solvent : 1500 psi 100 "C 0 minutes Off 5 minutes 5 minutes 60% 60 seconds 1 100% methanol Prior to analysis, the sample extracts were filtered through a 0.2-p-17filter (Titan, nylon membrane) and diluted as appropriate in methanol. A 2-mL aliquot was removed from each sample extract and 25 pL of the 25.0 mg/L PFON15.0 mg/L THPFOS internal standard solution was added. 3.2 Quality Control Sample Preparation Preparation of the quality control (QC) samples is summarized in the following table. The IO-g (dry weight) aliquots were removed from each QC sample, supplemented with PFOS, mixed using a spatula and placed into 22-mL ASE cells for extraction on the Dionex ASETM. ~~ ~~~ BACK TO MAIN PFOS - Aerobic Soil Biodegradation Page 11 PFOS Stock Concentration (mglmL) 1.oo 1.QO 4 .oo a Soil from t h e study Dry weight ~~ Volume of Stock Solution Used (mL) 0.250 0.250 0.250 Amount of Control Matrixa (sib 10.0 10.0 10.0 QC Sample Concentration (mglkg) 25.0 25.0 25.0 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 Seiex API 100 LC/MS, PE Sciex Turboionspray@(electrospray) Keystone Betasil 618, 5 vm, 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 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: QI, SIM Scan time: 2.01 sec Peak Hopping: Disabled Mass defect: 0 mmu/l00 amu Pause time: 2 msec Dwell time: 400 msec Masses scanned (amu)*: 413 (PFOA),427 (THPFOS), 499 * Based on 3M analytical method No. ETS-8-11.O (PFOS), 616,630 (N-EtFOSE-OH) State file information: BACK TO MAIN PFQS - Aerobic Soil Biodegradation 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 Page 12 4.1 Test Procedures Best vessels used during the aerobic soil study were 40-mL l-Chem glass vials with silicone/Teflon@-linedseptum screw caps. Three soils and two sediments from diverse sources in Massachusetts were collected, air dried briefly by spreading out on aluminum foil on a laboratory bench to remove excess water, and sieved through a 2-mm screen. Soils were collected from a hardwood forest in Hanson, MA, a pine forest in Onset, MA and a river bank in Bridgewater, MA. Sediments were from brackish sites below the Wareham wastewater treatment plant outfall and from the Narrows area in Wareham, MA. The percent moisture was then determined for each soil and sediment and a 200-9 (dry wt.) aliquot of each soil and sediment were thoroughly mixed using a spatula. The water holding capacity (WHC) of the soikediment mixture was determined by adding 10 g of the mixture to a glass column, pumping water up through the soil column until breakthrough was observed and recordingthe amount of water added. The volume of water added was divided by the amount of dry soil present and resulted in a measured value of 39.5% WHC. Ten gram aliquots (based on dry weight) of the soil sediment/mixture were added to each of twenty test vessels (ten for the test substance, ten blanks). The soils were initially adjusted to 75% of the WHC by adding water and the nutrients described below. During the study, soil moisture was monitored each week by weighing individual vessels and adding reagent water, if required. Soil biomass was not measured prior to initiation of the study, but was determined on day 83 to confirm that a viable microbial population still existed. Potting soil extract was prepared by mixing 400-g soil potting soil (from a local nursery) with one liter of water and autoclaving for 45 minutes. After cooling and settling, the potting soil extract was filtered and the TOC was determined to be 16,360 mg/L via analysis on a Dohrmann Model DC80 BACK TO MAIN PFOS - Aerobic Soil Biodegradation Page 13 total organic carbon analyzer. Sample injections for each replicate were performed in duplicate to demonstrate reproducibility. Soils in each of the twenty test vessels were dosed with, 10 pL of a 1/1OOth strength trace mineral solution (Trace Minerals Research, Roy, Utah), 100 pL of yeast extract (5 mg/mL stock, Acumedia, Baltimore, MD), 30 pL of potting soil extract and 100 pL reagent water. These additions brought the soil rnoistures to approximately 75% of WHC. A 200-vL aliquot of the 1.06 mg/mL PFOS stock solution (SLI No. 70-93A)was then added to the test substance test vials, resulting in an PFOS concentration of approximately 21.2 mglkg soil. Blank control soils received 20-pL of methanol. Following dosing, the vials were incubated in the dark at 22 -I 3 "C for twenty weeks in an environmental chamber. Soil microbial biomass was determined at day 83 of the study by both fumigation/extraction and standard plate counts to evaluate the microbial population. On test Days 7, 14, 21, 28, 35, 42, 49, 56, and 63, one blank tube and one PFOS tube were removed from incubation and stored at 4 "Cprior to analysis. 4.2 Results and Discussion The fumigation/extraction technique (SLI SOP No. 2.4.17) is a measure of the carbon from microbial sources and is determined as the difference in TOC between soils fumigated with chloroform and non-fumigated (Brookes, et. ai., 1987). The biomass of the control soil at day 83 of the study was determined to be 17.4mg M O O g soil. Standard plate counts (SLI SOP No. 7.26) on nutrient agar determined the microbial population to be 6 x 1O5cells/g. The biomass values are typical for an active microbial community. Table 1 summarizes the fate of PFOS in the soil system over the 63-day test period. Mass balances ranged from 102% to 121% and indicated that essentially no PFOS metabolism occurred during the study. 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. BACK TO MAIN PFOS - Aerobic Soil Biodegradation 5.0 CONCLUSIONS AND FUTURE STUDIES Page 14 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 PFBS - Aerobic Soil Biodegradation REFERENCES Page 15 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. Brooks, P.C, D.S. Jenkinson, and E.D. Vance. 1987. An extraction method for measurement of soil microbial biomass carbon. Soil Biol. Biochem. =:703-707. BACK TO MAIN PFOS - Aerobic Soil Biodegradation Table 1. Results for the test samples from the aerobic soil biodegradation study. Page 16 Sample N0.V Type Test Samples S0499-01/Day 7 S0499-02/Day 14 S0499-03/Day 21 S0499-04/Day 28 S0499-05/Day 35 S0499-06/Day 42 S0499-07/Day 49 S0499-08/Day 56 S0499-09/Day 63 Nominal (mg/kg) 21.2 21.2 21.2 21.2 21.2 21.2 21.2 21.2 21.2 PFOS Concentration Measured (mglkg) Measured (%) Total Total (Mass Balance) 22.6 107 21.3 101 20.4 96.1 23.4 111 22.7 107 22.8 108 21.8 103 21.9 103 24.1 114 Blank Samples S0499-11/Day 7 0.00 < O.O1OOb NAc S0499-12IDay 14 0.00 < 0.0100 NA S0499-19/Day 63 0.00 < 0.0100 NA QCdSamples c999-75 25.0 26.2 105 C999-76 25.0 27.0 108 c999-77 25.0 27.1 108 a Day 0 samples were not taken. Values represented by 0.0100 were below the limit of quantitation. NA = not applicable QC = quality control sample PFOS - Aerobic Soil Biodegradation Figure 1. Flow chart of extraction procedures. BIODEGRADATION SAMPLES BACK TO MAIN Page 17 EXTRACT W/ MEOH F 4 lFilter (Discard)