Document KRBbBx0oKnX08RKezBM7XG9Rx
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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
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PFOS - Activated Sludge/Sediment
SIGNATURES AND APPROVAL
SUBMITTED BY:
Springborn Laboratories Inc. 790 Main Street
Wareham, Massachusetts 02571-1075
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Director, Environmental Fate and Microbiological Programs
d~J3isa
Sean P. MdLaughlin'
Date
Principal Investigator
Director, Chemistry
Marjorie E. Dix
Senior Research Chemist
Date
Senior Research Chemist
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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 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 ANALYTICAL METHODS . . . . . . . . . . . . . . . . . . 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 Samples . . . . . . . . . . . . . . . . . . . . . . 11 3.3 Preparation of the Solid Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
3.4 Preparation of Quality Control Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 3.5 Instrumental Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
4.0 TEST PROCEDURES. RESULTS. AND DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . . . . 13
4.1 ToxicityTest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 4.1.1 Test Procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 4.1.2 Results and Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
4.2 Activated Sludge/Sediment Biodegradation Study - Acclimation Phase . . . . . . . . 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 FUTURESTUDIES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
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LIST OF TABLES
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Table 1. Composition of OECD inorganic salts medium . . . . . . . . . . . . . . . . . . . . . . . 22
Table 2. Toxicity assay of PFOS in activated sludge . . . . . . . . . . . . . . . . . . . . . . . . . . 2 3
Table 3. Table 4.
Total organic carbon (TOC) analyses during the pre- and post dosing phases of the activated sludgelsediment acclimation study24 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
PFOS concentration measurements from the activated sludgelsediment acclimation flasks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 5
Table 5.
PFOS concentration measurements from the analytical control flasks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
Table 6. Table 7.
Selection of test conditions for the sealed PFOS CO, evolution study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
CO, analysis of the headspace in sample vials during the closed vial (headspace) aerobic biodegradation test with PFOS." . . . . . . . . . . . . . .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 mglL test samples from the sealed vessels (headspace). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
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LIST OF FIGURES
Page
Figure 1. Flow chart of extraction procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
Figure 2.
Total measured PFOS concentration during acclimation in
activated sludgelsediment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
Figure 3. PFOS concentration in the analytical control flask . . . . . . . . . . . . . . . . . . . . 35
Figure 4. Concentration of PFOS versus time during the closed vial (headspace) aerobic biodegradation test . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
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1.O INTRODUCTION
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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 P450 monooxygenase enzyme system known to metabolize complex molecules.
Throughout the experimental program the principles outlined above for enrichment were
incorporated. The results of the studies are summarized in four separate reports and provide a
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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 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 c 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: I 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 (Yo)
0.400 90.23 2.34 1.72 2.15
2.10
1.06 100
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The internal standard, perfluorooctanoic acid (PFOA, lot no. 07216AS), an off-white solid wax, was received on IApril 1999 (SLI No. 71-94) from Aldrich, Milwaukee, Wisconsin. An additional internal standard, 1, I,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 StandardStock 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 IOO-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/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 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
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 solutionswere suspensionsand 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
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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 1OO-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
1.22 rng/rnL 1.22 mg/rnL 1.22 rng/mL
122 mg/L
12.2 mg/L 1.22 ma/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
Methanol Methanol Methanol
Methanol Methanol
Methanol
Standard
Concentration (mglL)
2.44 1.22 0.610
0.122 0.0305
0.0100
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).
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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-mLvolumetric 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 ( I0 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 65-6Rcentrifuge. The procedures used for the resultant aqueous (supernatant) and solid samples are detailed below.
3.1 Preparation of the 20 and 105 mglL Aqueous Test Samples A 1.OO-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.
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3.2 Preparation of the 0.20 mglL 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 5050 ethyl acetate/hexane. When emulsions formed, an additional 1.OO 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 PFON15.0 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 WLof the 25.0 mg/L PFON15.0 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).
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Test
QC
ID
Sample
TY Pe
Shake-Flask
Days 7, 14, 21, 28. and 35
Aqueous
PFOS Stock Concentration
(mglL)
1000 1000 1000
Volume of
Stock Solution Used
(mL)
0.100
0.100
0.200
Control Matrix
1 1 2
Volume of Control Matrix (mu
10.0
10.0
10.0
QC Sample Concentration
(mglL)
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 Conc. Day Aqueous
10.0
0.300
1
15.0
0.200
0, 16,30,61
10.0
0.300
1
15.0
0.200
10.0
0.300
2
15.0
0.200
10.0
0.300
2
15.0
0.200
Solids
10.0
0.300
3
15.0
0.200
10.0
0.300
3
15.0
0.200
High Conc. Day 0, 15, 30,
63
Aqueous
1000 1000
0.250 0.500
1
10.0
25.0
1
10.0
50.0
1000
1.oo
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.oo
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 vm, 100 8, 150 x 2 mm column with a
Betasil C18 guard column
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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
IO
90
13
0
100
17
0
100
20
60
40
20 min
10 min
10 VL
LC/MS parameters
Experiment information:
Scan type:
Q I , selected ion monitoring
Scan time:
2.01 sec
Peak Hopping:
Disabled
Mass defect:
0 mmu/l00 amu
Pause time:
2 msec
Dwell time:
400 msec
Masses
scanned (am@*: 413 (PFOA), 427 (THPFOS), 499 (PFOS), 616, 630 (N-EtFOSE-
alcohol)
* 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 OC -20 volts air (high purity) nitrogen
4.0 TEST PROCEDURES, RESULTS, AND DISCUS-ION 4.1 Toxicity Test 4.1 .I 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
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(Table I ) , 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]sodiumbenzoate 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-WLsample 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 14C0, 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 ''C0, from the sodium benzoate control flask was not substantially different in the presence of PFOS based on comparison of the rate of 14C02 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 would not be an issue since all measured concentrations still in the medium (see Table 4)
were below 100% of the 20.8 mg/L nominal concentration.
4.2 Activated SludgelSediment Biodegradation Study - Acclimation Phase
4.2.1 Test Procedures. The acclimation phase of the biodegradation study in activated sludgekediment was conducted in 300-mL baffled Erlenmeyerflasks. Inoculumwas collected from several sites: the aeration basin from the Wareham, Massachusetts activated sludge plant, the secondary return activated sludge linefrom the New Bedford,Massachusettswastewater 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
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collected by personnel at American Agricultural Services, Inc., Lucama, North Carolina and was added to provide a surface (protection) for microbialgrowth and soil microorganisms. The soil was characterized byAgvise 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/lOO g. Aerobic sludge from each wastewater treatment plant was added to the acclimation flasks at a concentration of 1.OO mg/mL (dry weight basis). Sandy soil was added at 10.0 mg/mL medium, and sediment at 1.OO 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 concentrationspecified in the OECD ready biodegradation test guidelines, Table I ) , trace minerals (Trace Minerals Corp., 0.020 mL/100 mL), yeast extract (Difco, 5.00 mg/l00 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 ( l - 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 incubatedat 22 f 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,
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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 RBCwastewater 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.OL 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.O-pmGelman filter by injection of 1.O-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 muflask) 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 phasewere saved in 1OO-mL amber Wheaton bottles with Teflon@-linedrubber septa under refrigeration priorto analysis and completion of the PFOS method validation. At week 10 andl2, approximately 2% 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 Resultsand 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
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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 assumedthat 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 progressedwas 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 (CO,) could be evolved from PFOS,
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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-mi 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 I O 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 I O , 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 biodegradationvials 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 mL of the 1mg/mL
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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 CO, production 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 CO, was performed on duplicate samples using a ThermoGlass 12OOTM 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.
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PFOS - Activated SludgelSediment
Page 20
Table 7 presents data for CO, analyses of the headspace gas from these systems. Very little difference in headspace CO, production was noted between the blank and PFOS containing test vessels. This is also indicative of PFOS recalcitrance. On day 63, CO, in 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 CO, 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 sludgelsediment 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.
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PFOS - Activated Sludae/Sediment REFERENCES
Paae 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.
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PFOS - Activated Sludge/Sediment
Table 1.
Composition of OECD inorganic salts medium.a
Page 22
Solution A"
B
C D
Reagentsb KH,PO, K,HPO,
Na,HPO, .2H,O NH,CI CaCI,
MgSO,. 7H,O FeCI, .6H,O
HCI
Concentration, glL 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 8,C, and D, and then bringing to a volume of 1 L with water. Proportionate amounts of salts with different waters of hydration may be substituted. The pH of this solution should be 7.4.
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PFOS - Activated Sludge/Sediment
Table 2.
Toxicity assay of PFOS in activated sludge.
Page 23
Test Vessel
Blank Controlb
Time (hours) 6
24 48
[14C]Radioactivity in CO, Trap
DPM"
Percent Applied Dose
<LOGIC
0
341 00
31 .O
46700
42.4
PFOSd
6
<LOQ
0
24
33400
30.4
48
47000
42.7
a DPM = dissociations per minute Blank Control vessels were comprised of 100 mL of mineral salts medium which contained 5 mg/L ['4C]sodium benzoate <LOQ = less than the Limit of Quantitation (60 dpm) PFOSvessels were comprised of 100 mLof mineralsalts mediumwhich contained 20 mg/L of PFOS and 5 mg/L [`4C]sodiumbenzoate
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PFOS - Activated Sludge/Sediment
Table 3.
Total organic carbon (TOC) analyses during the pre- and post dosing phases of the activated sludgelsediment acclimation study."
Page 24
Test Day
Pre Dose -27 -2 0 -1 6 -1 3 -8 -1
Post Dose 0 7 14 21 28 35 42 49 56 63 70
~~~
~
Blank Control
NAb 42.35 35.67 24.45 21.67 11.98
NA 10.07 14.93 11.17 15.54 21.22 26.04 16.62 16.31 14.10 12.06
TOC (rnglL) PFOS
~
Analytical Control
NA 45.75 38.16 24.76 17.48 11.39
---C
10.23 33.85 17.44 14.86 14.94d 15.78 16.91 18.56 19.61 18.25
NA NA NA NA NA NA
NA 1.699 1.467 1.604 1.293 0.999 0.876 0.703 1.297 1.224 0.899
a Samples were filtered through a 1.O-pm Gelman filter and duplicate 1.O mL samples were analyzed on the
Dohrman DC-80 carbon analyzer. Not analyzed. Initial TOC measurement at the start of the post-dose phase (To). Remainder of analyses indicated after 7 days of incubation prior to feeding. One sample was an outlier and that data point was not used.
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PFOS - Activated Sludge/Sediment
Table 4.
PFOS concentration measurements from the activated sludge/sediment acclimation flasks.
Page 25
Sample No./Ty pea
Nominal (mg'L)
PFOS Concentration
Measured (mglL)
Measured (%)
Biomass Medium Total
Biomass Medium
Total (Mass BaIance)
Test Samples
AS399-01-1lDay 7
23.5
10.0
16.5
26.5
42.6
70.0
113
AS399-01-2IDay 14
24.4
11.5
14.3
25.8
47.1
58.6
106
AS399-01-3IDay 21
24.8
19.6
9.43
29.0
78.9
38.0
117
AS399-01-4IDay 28
25.0
33.3
11.6
44.8
133
46.2
179
AS399-01-5IDay 35
25.0
21.2
5.47
26.7
84.8
21.9
107
AS399-01IDay 112
22.0
24.7
3.64
28.4
112
16.6
129
Blank Samples
AS399-06-011Day 7
0.00
< 0.01OOb < 0.0100 < 0.0100
NA"
NA
NA
AS399-06-02lDay 14
0.00
< 0.0100 < 0.0100 < 0.0100
NA
NA
NA
AS399-06-03IDay 21
0.00
< 0.0100 < 0.0100 < 0.0100
NA
NA
NA
AS399-06-04IDay 28
0.00
< 0.0100 < 0.0100 < 0.0100
NA
NA
NA
AS399-06-05IDay 35
0.00
< 0.0100 < 0.0100 < 0.0100
NA
NA
NA
QC Medium Samplesd
C599-19lDays 7-28"
10.0
NA
11.3
NA
NA
113
NA
C599-20lDays 7-28% 20.0
NA
22.6
NA
NA
113
NA
C599-21/Days 7-28"
20.0
NA
22.1
NA
NA
110
NA
C599-59lDay 35"
10.0
NA
10.5
NA
NA
105
NA
C599-60lDay 35'
20.0
NA
22.7
NA
NA
114
NA
C599-61IDay 35"
20.0
NA
20.9
NA
NA
105
NA
C899-14IDay 112"
5.00
NA
5.02
NA
NA
100
NA
C899-15IDay 112"
10.0
NA
10.3
NA
NA
103
NA
C899-16lDay 112e
20.0
NA
22.5
NA
NA
112
NA
QC Biomass Samples
C599-95IDays 7-35'
2.50
2.56
NA
NA
102
NA
NA
C599-961Days 7-35'
2.50
2.58
NA
NA
103
NA
NA
C599-97IDays 7-35'
5.00
5.33
NA
NA
107
NA
NA
C899-111Day 112'
2.50
2.61
NA
NA
104
NA
NA
C899-12IDav 112'
2.50
2.60
NA
NA
104
NA
NA
C899-13lDa; 112'
5.00
5.76
NA
NA
115
NA
NA
a Day 0 samples were not taken.
Values expressed as < 0,0100 were below the limit of quantitation
NA = not applicable
QC = quality control sample.
" QC sample fortified in control medium.
QC sample fortified in blank biomass.
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PFOS - Activated Sludge/Sediment
Table 5.
PFOS concentration measurements from the analytical control flasks.
Page 26
Sample No./Type*
Nominal (mg'L)
PFOS Concentration
Measured (mglL)
Measured (%)
Biomass Medium Total
Biomass Medium
Total (Mass Balance)
Test Samples
AS399-07-011Day 7
21.2
NAb
21 .I
21.1
NA
99.6
100
AS399-07-021Day 14
21.2
NA
19.8
19.8
NA
93.2
93.2
AS399-07-031Day 21
21.2
NA
11.1
11.1
NA
52.4
52.4
AS399-07-041Day 28
21.2
NA
17.8
17.8
NA
83.9
83.9
AS399-07-051Day 35
21.2
NA
10.2
10.2
NA
48.0
48.0
AS399-071Day 112
21.2
NA
5.74
5.74
NA
27.1
27.1
Blank Samples
AS399-09-011Day 7
0.00
0.0100
0.0100 0.0100
NA
NA
NA
AS399-09-021Day 14
0.00
0.0100
0.0100 0.0100
NA
NA
NA
AS399-09-031Day 21
0.00
0.0100
0.0100 0.0100
NA
NA
NA
AS399-09-041Day 28
0.00
0.0100
0.0100 0.0100
NA
NA
NA
AS399-09-05lDay 35
0.00
< 0.0100
0.0100 < 0.0100
NA
NA
NA
QC Samplesd
(2599-191Days 7-28
10.0
NA
11.3
NA
NA
113
NA
C599-201Days 7-28
20.0
NA
22.6
NA
NA
113
NA
C599-211Days 7-28
20.0
NA
22.1
NA
NA
110
NA
C599-59lDay 35
10.0
NA
10.5
NA
NA
105
NA
C599-601Day 35
20.0
NA
22.7
NA
NA
114
NA
C599-611Day 35
20.0
NA
20.9
NA
NA
105
NA
C899-14lDay 112
5.00
NA
5.02
NA
NA
100
NA
C899-15IDay 112
10.0
NA
10.3
NA
NA
103
NA
C899-16lDav 112
20.0
NA
22.5
NA
NA
112
NA
a Day 0 samples were not taken.
Values expressed as < 0.0100 were below the limit of quantitation
NA = not applicable QC = quality control sample.
e QC sample fortified in control medium.
QC sample fortified in blank biomass.
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PFOS - Activated Sludge/Sediment
Table 6.
Selection of test conditions for the sealed PFOS CO, evolution study.a
Page 27
Solids Concentration, mgIL (dry weight basis)b
Full Strength Medium
'I,, Strength Medium
Day
10
30
0 +++
4
+++
5 +++
6 +/-+I-+
7 -+/-+I-
9
--+/-
10
--+/-
12 ---
14
---
16
---
17
-
21
---
100 666 2000 6666
+++ +++ +++ +++
+/-+/-+/--+/---
-------
---
---
10 30 100
666
2000 6666
+++ +++ +++ +++
+++ +++ +++ +++
+++ +++ +++ +I-+/-+/-
+++ +++ +++ +/-+/-+/-
+++ +++ +++ +/-+/-+I-
+++ +++ +++ +/-+/-+/-
+++ +++ +++ +/-+/-+/-
+++ +++ +++ +/-+/-+/-
+++ +++ +++ +I-+/-+/-
+++ +++ +++ --+/-
+i+ +++ +++ --+/-
+++ +++ +++ ---
a Test system = 2-mL serum vials, 15 mL medium consisting of OECD salts, Wareham raw sewage, 100 mg CIL sludge extract, resazurin and activated sludge. For 'Irostrength medium, raw sewage and sludge extract were diluted 1 to 10 in the test system.
Aerobicity was judged by observing medium color; + = pink color and presence of 0,; - = colorless and no 0,; +/- =
faint pink color.
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PFOS - Activated Sludge/Sediment
Table 7.
CO, analysis of the headspace in sample vials during the closed vial (headspace) aerobic biodegradation test with PFOS."
Page 28
Test Vessel
Replicate
Headspace C, mglL
Day
0
15 30
63
% Theoretical
Day
0
15
30
63
Inoculum Controlb
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 ControlC
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 Fortifiedd
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. Contains "acclimated'activatedsludge in mineralsalts medium. Contains 79.3 mg/L (20 mg C/L) PFOS in mineral salts medium. Contains "acclimated' activated sludge in mineralsalts mediumand 79.3 mg/L PFOS.
e NA = not applicable
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PFOS - Activated Sludge/Sediment
Page 29
Table 8.
PFOS concentration measurements for the 0.200 mglL test samples from the sealed vessels (headspace).
Sample No./ Sample Day
Nominal (mglL)
Inoculum Control HS0699-1010 HS799-03/16 HS799-04 I16 HS799-121130 HS799-122130 HS899-69/61 HS899-7016 1
Avg .
PFOS + Inoculum HS0699-1510 HS799-08/16 HS799-09/16 HS799-10116 HS799-126130 HS799-127130 HS799-128130 H 5899-7416 1 HS899-75161 HS899-76/61
Avg
Blank Analytical Control HS0699-08 HS799-01/16 HS799-02/16
HS799-119130 HS799-120130 HS899-67161
HS899-68/61
0.000 0.000 0.000 0.000 0.000 0.000 0.000
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 (mglL)
Measured (%)
Total
Biomass Medium Total
Biomass Medium
(Mass
BaI ance)
0.0400 0.300 0.340
NA"
NA
NA
0.023 0.365 0.388
NA
NA
NA
0.020 0.373 0.393
NA
NA
NA
0.005 0.341 0.346
NA
NA
NA
0.019 0.316 0.335
NA
NA
NA
0.0309 0.352 0.395
NA
NA
NA
0.0222 0.310 0.340
NA
NA
NA
0.0229 0.337 0.362
NA
NA
NA
0.0841 0.249 0.333
39.7
117
157
0.0321 0.175 0.207
15.1
82.5
97.7
0.0131 0.207 0.220
6.18
97.6
104
0.0351 0.271 0.306
16.6
128
144
0.0521 0.188 0.240
24.6
88.7
113
0.0251 0.108 0.133
11.8
50.9
62.8
0.0471 0.202 0.249
22.2
95.3
118
0.0066 0.101 0.108
3.11
47.6
50.8
0.0748 0.220 0.295
35.3
104
139
0.0273 0.100 0.127
12.9
47.2
60.0
0.0397 0.180 0.219
79.9
85.9
105
NA <0.0100d NA
NA
<0.0100
NA
NA <0.0100 NA
NA
<0.0100
NA
NA ~0.0100 NA
NA
<0.0100
NA
NA <0.0100 NA
NA
<0.0100
NA
NA <0.0100 NA
NA
~0.0100
NA
0.0100 0.0100 0.0100
NA
NA
NA
< 0.0100 -= 0.0100 < 0.0100
NA
NA
NA
Analytical Control
HS0699-1210
0.212
NA
0.232 0.232
NA
109
109
HS799-05/16
0.212
NA
0.197 0.197
NA
92.9
92.9
HS799-06/16
0.21 2
NA
0.198 0.198
NA
93.4
93.4
HS799-07/16
0.212
NA
0.196 0.196
NA
92.5
92.5
HS799-123130
0.212
NA
0.179 0.179
NA
84.4
84.4
HS799-124130
0.212
NA
0.187 0.187
NA
88.2
88.2
HS799-125130
0.212
NA
0.188 0.188
NA
88.7
88.7
HS899-71I6 1
0.212
NA
0.159 0.163
NA
75.0
75.0
H S899-7216 1
0.212
NA
0.142 0.144
NA
67.0
67.0
HS899-73/61
0.212
NA
0.152 0.154
NA
71.7
71.7
a NA = not applicable Net values after subtraction of inoculum control average values.
Ratio equivalent to 18% PFOS on solids and 82% in medium.
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
QC" Medium
C699-26610 C699-26710 C799-40116 C799-41/16 C799-42/16 C799-43/16 c799-145130 C799-146130 C799-I47130 C799-I48130 C899-94/61 C899-95/61 C899-96/61 C899-97/61
Nominal (mglL)
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
0.195
NA
NA
97.5
NA
NA
0.186
NA
NA
93.0
NA
NAb
0.195
NA
NA
106
NA
NA
0.186
NA
NA
111
NA
NA
0.195
NA
NA
105
NA
NA
0.186
NA
NA
98.5
NA
NA
0.162
NA
NA
81 .O
NA
NA
0.176
NA
NA
88.0
NA
NA
0.170
NA
NA
85.0
NA
NA
0.163
NA
NA
81.5
NA
NA
0.221
NA
NA
111
NA
NA
0.207
NA
NA
104
NA
NA
0.229
NA
NA
114
NA
NA
0.218
NA
NA
109
NA
QC solids
C699-26210
0.200
0.219
NA
NA
110
NA
NA
C699-26310
0.200
0.213
NA
NA
107
NA
NA
C799-38/16
0.200
0.219
NA
NA
115
NA
NA
C799-39/16
0.200
0.213
NA
NA
110
NA
NA
C799-I49130
0.200
0.21 1
NA
NA
106
NA
NA
C799-I50130
0.200
0.21 0
NA
NA
105
NA
NA
C899-98/61
0.200
0.21 1
NA
NA
106
NA
NA
C899-99/61
0.200
0.205
NA
NA
103
NA
NA
a QC = quality control sample NA = not applicable
BACK TO MAIN
PFOS - Activated Sludge/Sediment
Page 31
Table 9.
PFOS concentration measurements for the 105 mglL test samples from the sealed vessels (headspace).
Sample No./ Sample Day
Blank Inoculum
HS799-41IO
HS799-4210 HS799-135/15 HS799-136/15 HS899-25/30 HS899-26/30 HS999-25/63 HS999-26/63
PFOS + Inoculum HS799-4510 HS799-4610
HS799-139/15 HS799-140/15 HS899-29/30 HS899-30130 HS999-29/63 HS999-30163
Analytical Control HS799-4310 HS99-4410
HS799-I37115 HS799-138115 HS899-27/30 HS899-28/30 HS999-27/63 HS999-28/63
Nominal (mglL)
PFOS Concentration
Measured (mg/L)
Measured (%)
Biomass Medium Total
Biomass Medium
Total (Mass
0.000
0.0100" 0.0100 0.0100
NAb
NA
NA
0.000
0.0100 0.0100 0.0100
NA
NA
NA
0.000
0.0100 0.0100 0.0100
NA
NA
NA
0.000
0.0100 0.0100 0.0100
NA
NA
NA
0.000
0.0100 0.0100 0.0100
NA
NA
NA
0.000
0.0100 0.0100 0.0100
NA
NA
NA
0.000
0.0100 0.0100 0.0100
NA
NA
NA
0.000
0.0100 0.0100 0.0100
NA
NA
NA
105
1.31
151
152
1.25
144
145
105
2.86
134
136
2.72
127
130
105
0.912
138
139
0.869
131
132
105
1.10
145
146
1.05
138
139
105
1.25
142
143
1.19
135
136
105
2.50
136
139
2.34
130
132
105
1.42
125
127
1.35
119
121
105
2.27
130
132
2.17
124
126
105
NA
120
120
NAa
114
114
105
NA
115
115
NA
109
109
105
NA
129
129
NA
123
123
105
NA
109
109
NA
103
103
105
NA
123
123
NA
117
117
105
NA
124
124
NA
118
118
105
NA
108
108
NA
103
103
105
NA
99.5
99.5
NA
94.7
94.7
QC" Medium
C799-13110
25.0
NA
25.3
NA
NA
101
NA
C799-13210
50.0
NA
53.7
NA
NA
107
NA
C799-13310
100
NA
104
NA
NA
104
NA
C799-165115
25.0
NA
29.5
NA
NA
118
NA
C799-166/15
50.0
NA
59.1
NA
NA
118
NA
C799-167115
100
NA
115
NA
NA
116
NA
C899-02/30
25.0
NA
27.5
NA
NA
110
NA
C899-03/30
50.0
NA
52.6
NA
NA
105
NA
C899-04/30
100
NA
106
NA
NA
106
NA
C999-103163
25.0
NA
26.8
NA
NA
107
NA
C999-104163
50.0
NA
53.6
NA
NA
107
NA
C999-105/63
100
NA
110
NA
NA
110
NA
" Values presented as less than were below the limit of quantitation.
NA = not applicable
QC = quality control sample
BACK TO MAIN
PFOS - Activated Sludae/Sediment
Paae 32
Table 9.
Continued. PFOS concentration measurements for the 105 mglL test samples from the sealed vessels (headspace).
Sample No./ Sample Day
Nominal (mglL)
PFOS Concentration
Measured (mg/L)
Measured (X)
Total
Biomass Medium Total
Biomass Medium
(Mass
QC" Solids
C799-13410
33.3
35.7
NAb
NA
107
NA
NA
C799-13510
66.7
69.9
NA
NA
105
NA
NA
C799-13610
100
99.0
NA
NA
99.0
NA
NA
C799-I68115
33.3
37.1
NA
NA
111
NA
NA
C799-169115
66.7
68.9
NA
NA
103
NA
NA
C799-170/15
100
107
NA
NA
107
NA
NA
ca99-05/30
33.3
37.7
NA
NA
113
NA
NA
C899-06/30
66.7
75.4
NA
NA
113
NA
NA
C899-07/30
100
102
NA
NA
102
NA
NA
C999-106163
66.7
68.1
NA
NA
102
NA
NA
C999-107163
66.7
72.3
NA
NA
108
NA
NA
C999-108163
100
103
NA
NA
103
NA
NA
a QC = quality control sample NA = not applicable
PFOS - Activated Sludge/Sediment
Figure 1. Flow chart of extraction procedures.
BACK TO MAIN Page 33
SLUDGEISEDIMENT SAMPLES AND ANALYTICAL CONTROL
All samples that contain solids:
Centrifuge @ 1200 x g for 30 min
filter
0.200 mg/l
Addn 1 volume 0.5M TBA-HS04
Extract w/MeOH
Extract 2x300ml EtOAcIhexane
3.
3.
3.
(discard)
LOW VOLUME
-l LC-MS ANALYSIS
RECONSTITUTE IN MEOH
filter
ANALYSIS
BACK TO MAIN
PFOS - Activated Sludge/Sediment
Figure 2.
Total measured PFOS concentration during acclimation in
activated sludgelsediment.'
Page 34
160 140
i cn 120
E
100 80 60 40 20 0
0
T
1 1 +Measured
-m- Nominal
i
I
I
3
6
9
12
15
Week Number
a 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.
PFOS - Activated SludgeKediment
Figure 3. PFOS concentration in the analytical control flask.
BACK TO MAIN Page 35
80
70
T
60
+Measured
-.- Nominal
50
40
30
20
10
0
0
3
6
9
12
15
Week Number
BACK TO MAIN
PFOS - Activated Sludge/Sediment
Figure 4.
Concentration of PFOS versus time during the closed vial (headspace) aerobic biodegradation test.
Page 36
100 90 80 70 60 50 40 30 20 10 0
0
1 ~ +Nominal
~ +Inoculated +Analytical Control
L
10
20
30
40
50
60
Day Number