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6:2 Fluorotelomer alcohol aerobic biotransformation in activated sludge from two domestic wastewater treatment plants
Chemosphere 92 (2013) 464–470
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6:2 Fluorotelomer alcohol aerobic biotransformation in activated sludge from two domestic wastewater treatment plants Lijie Zhao a, Patricia K. McCausland b, Patrick W. Folsom b, Barry W. Wolstenholme b, Hongwen Sun a,⇑, Ning Wang b,⇑, Robert C. Buck b a b
MOE Key Laboratory of Pollution Processes and Environmental Criteria, Nankai University, Tianjin, China E.I. du Pont de Nemours & Company, Inc., Newark, DE, USA
h i g h l i g h t s " 5:2 sFTOH is the dominant volatile product of 6:2 FTOH aerobic biotransformation. " 5:3 Acid and PFHxA are the major non-volatile transformation products. " 6:2 FTOH aerobic biotransformation is not a source of PFBA and PFHpA.
a r t i c l e
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Article history: Received 23 September 2012 Received in revised form 13 February 2013 Accepted 16 February 2013 Available online 26 March 2013 Keywords: 6:2 Fluorotelomer alcohol (6:2 FTOH) 5:3 Polyfluorinated acid (5:3 acid) Perfluorohexanoic acid (PFHxA) 5:2 Secondary polyfluorinated alcohol (5:2 sFTOH) Activated sludge Aerobic biotransformation
a b s t r a c t 6:2 Fluorotelomer alcohol [6:2 FTOH, F(CF2)6CH2CH2OH] is a major basic chemical being used to manufacture FTOH-based products. After the end of use, 6:2 FTOH-based products may be released to domestic wastewater treatment plants (WWTPs) as a first major environmental entry point. Activated sludge collected from two WWTPs was dosed with 6:2 FTOH to investigate its biotransformation rate and to identify major transformation products. The volatile 5:2 sFTOH [F(CF2)5CH(OH)CH3] is the most abundant transformation product and accounted for an average of 40 mol% of initially dosed 6:2 FTOH after two months of incubation with activated sludge, with 30 mol% detected in the headspace. PFPeA [F(CF2)4 COOH] averaged 4.4 mol% after two months, 2.4–7 times lower than that in sediment and soils. The much lower level of PFPeA formed in activated sludge compared with soil indicates that microbial populations in activated sludge may lack enzymes or suitable environment conditions to promote rapid 5:2 sFTOH decarboxylation to form PFPeA, resulting in more 5:2 sFTOH partitioned to the headspace. PFHxA [F(CF2)5COOH] and 5:3 [F(CF2)5CH2CH2COOH] acid are major non-volatile transformation products in activated sludge. For example, PFHxA averaged 11 mol% after two months, which is about 30% higher compared with sediment and soils, suggesting that microbes in WWTPs may utilize similar pathways as that in sediment and soils to convert 5:2 sFTOH to PFHxA. 5:3 Acid averaged 14 mol% after two months, comparable to that in soils and slightly lower than in sediment, further confirming that 5:3 acid is a unique product of 6:2 FTOH biotransformation in the environment. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Perfluoroalkyl acids (PFAAs) such as perfluoroalkane sulfonates (PFSAs) and perfluoroalkyl carboxylates (PFCAs) are widely detected in the environment and biota (Buck et al., 2011; Houde et al., 2011; Benskin et al., 2011) because of their wide use and ⇑ Corresponding authors. Addresses: College of Environmental Science and Engineering, Nankai University, 94 Weijin Street, Tianjin 300071, China. Tel.: +86 22 23509241 (H. Sun), DuPont Haskell Global Centers for Health and Environmental Sciences, Glasgow 300, PO Box 6300, Newark, DE 19714-6300, USA. Tel.: +1 302 366 6665 (N. Wang). E-mail addresses: [email protected] (H. Sun), ning.wang@usa. dupont.com (N. Wang). 0045-6535/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.chemosphere.2013.02.032
historical applications in industrial and consumer products (Prevedouros et al., 2006). For example, PFSAs and PFCAs are detected in soil (Naile et al., 2010), sediment (Li et al., 2011; Benskin et al., 2011), activated sludge from WWTPs (Schultz et al., 2006; Ahrens et al., 2011; Kunacheva et al., 2011; Sun et al., 2011), and in biota (Naile et al., 2010; Houde et al., 2011). The sources of PFSAs and PFCAs are from their historical direct environment emissions and to a less extent from precursor degradation (Ellis et al., 2004; Prevedouros et al., 2006; Martin et al., 2006; Cousins et al., 2011). The long-chain PFSAs (P6 carbon chain length) and longchain PFCAs (P8 carbon chain length) have much longer elimination half-life in exposed animals and humans compared with their short-chain homologs (Lau, 2012). For example, perfluorohexane
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sulfonate (PFHxS) and perfluorooctanoic acid (PFOA) have a serum/ plasma elimination half-life of 8.5 and 3 years, respectively, in human, compared with 30 and 3 d, respectively, for perfluorobutane sulfonate (PFBS) and perfluorobutanoic acid (PFBA) (Lau, 2012). Because of the potential long elimination time and potential toxicity associated with long-chain PFSAs and PFCAs, major manufacturers in the developed world are in the process of replacing the longchain chemistry with short-chain based chemistry (US EPA, 2006). 6:2 FTOH is a major raw material being used to replace 8:2 FTOH, a potential PFOA precursor when subjected to microbial biotransformation in activated sludge (Wang et al., 2005a,b) and soils (Liu et al., 2007; Wang et al., 2009). 6:2 FTOH biodegradation in soil (Liu et al., 2010a,b) and sediment (Zhao et al., 2012) leads to PFPeA, PFHxA, PFBA [F(CF2)3COOH], 5:3 acid, and 4:3 acid [F(CF2)4 CH2CH2COOH] as major non-volatile transformation products. Intermediate products such as 6:2 FTCA [F(CF2)6CH2COOH] and 6:2 FTUCA [F(CF2)5CF = CHCOOH] were also observed in sediment but not in soils dosed with 6:2 FTOH. The volatile intermediate products 5:2 ketone [F(CF2)5C(O)CH3] and 5:2 sFTOH were observed in both sediment and soil (Liu et al., 2010a; Zhao et al., 2012). Currently, little information is available on 6:2 FTOH biotransformation in activated sludge from WWTPs. WWTPs are important sites for anthropogenic chemicals to be transformed or degraded by microbes before the effluent and sludge solids are being discharged to the environment along with un-reacted precursors, intermediates, and stable transformation products. The typical sludge retention time in WWTPs in the US states of Delaware (DE) and Pennsylvania (PA) is less than 14 d and ranges from 2 to 60 d (Bitton, 2011) in US. 6:2 FTOH-based monomeric products or impurities may be degraded by microbes in activated sludge. 6:2 FTOH primary biotransformation to intermediates and eventually to major stable products are relatively fast (within days) as shown in an earlier study in bacterial culture (Liu et al., 2010a). It is important to identify 6:2 FTOH transformation products and understand its transformation rates. Such practical knowledge can help environmental modeling and monitoring, and predict environmental concentrations of 6:2 FTOH stable transformation products for risk assessment to human and environmental health. 6:2 FTOH conversion rates to other major products may be different in activated sludge from those observed in aerobic soils and sediment because of differences in microbial populations and environmental conditions. For example, WWTPs contain mainly bacteria associated with digested plant and animal wastes (Bitton, 2011) whereas soils contain both bacteria and fungi (Tate III, 2000) and sediment contains mainly bacteria associated with animal materials (Nealson, 1997). These differences may affect 6:2 FTOH partitioning behavior, bioavailability, and ultimately microbial ability to transform or degrade 6:2 FTOH in different environmental matrices as discussed above. When [1,2-14C] 6:2 FTOH was applied to Sassafras soil, about 25% of initially applied 14C was conjugated with soil organic materials catalyzed by microbial enzymes to form soil-bound residues that may not be available for further biodegradation (Liu et al., 2010b). The solid content in activated sludge is relatively low (1– 3 dry wt.% in WWTPs from PA, DE, and MD states) due to the presence of the predominant aqueous phase compared with soil. It is not known to what extent 6:2 FTOH can be bound to sludge organic materials and whether such bound residues can affect 6:2 FTOH biotransformation. Diluted activated sludge from WWTPs was successfully used to study 6:2 fluorotelomer sulfonate [6:2 FTSA, F(CF2)6CH2CH2SO3K+, Wang et al., 2011] and 5:3 acid (Wang et al., 2012) biotransformation. The use of diluted activated sludge decreased potential matrix effects of the sludge organic materials on LC/MS/MS quantitative analysis of 6:2 FTOH transformation products and also ensured aeration of activated sludge media dosed with 6:2 FTSA and 5:3
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acid in glass serum bottles as mentioned above. Also, our preliminary work with both diluted and non-diluted sludge dosed with 6:2 FTOH did not show significant differences in 6:2 FTOH primary biotransformation and molar yields of major transformation products. Overall, the diluted sludge system helped achieve good mass balance (i.e., good recovery of starting materials and transformation products (Wang et al., 2011, 2012), thus assuring study integrity. The aerated bottles with both diluted and native sludge in this study were used to measure 6:2 FTOH relative transformation ratios (molar yields) to different transformation products. Such information is necessary for further studying in situ 6:2 FTOH biotransformation rates in WWTPs using on-site simulation test. The objectives of this study were to determine 6:2 FTOH biotransformation potential from different WWTPs, to identify major transformation products, and to compare 6:2 FTOH biotransformation outcomes with those reported in soils and sediment. The information sought in this study can help understand the sources and potential FTOH precursor contribution to PFCAs detected in WWTPs and in other environmental compartments such as soils and sediment. 2. Materials and methods 2.1. Chemicals 6:2 FTOH purity was 99% from Sigma–Aldrich (St. Louis, MO). The custom-synthesized [1,2-14C] 6:2 FTOH [F(CF2)614CH214CH2OH] had a radiochemical purity of +99%. The purities of 4:3 acid and a newly synthesized 5:3 Uacid [F(CF2)5CH = CHCOOH] standards were 96% and 95%, respectively. The other fluorinated chemical standards were all above 97% pure. The details of these chemical standards including acronyms, structure, CAS numbers and sources were listed in an early study (Liu et al., 2010a). Stable isotope quantification internal standards used in LC/MS/MS analysis were [1,1,2,2-D; 3-13C] 6:2 FTOH [F(CF2)513CF2CD2CD2OH] (DuPont, Wilmington, DE) and [1,2-13C] PFHxA [F(CF2)413CF213 COOH] (Wellington Laboratories, Ontario, Canada). All solvents were HPLC grade or higher and all other chemicals referenced were at least reagent grade. De-ionized water (18 MX cm) was from a Barnstead E-Pure system used throughout all the experiment. Omsolv water (EMD Chemicals, Gibbstown, NJ) was used for LC/MS/ MS analysis. 2.2. Sludge collection and experiment set up The activated sludge was collected from the aeration basins immediately after the primary clarifiers from two domestic WWTPs in Delaware and Pennsylvania. The sludge was brought directly to the laboratory, was aerated and stirred in a magnetic plate at room temperature to suspend the microorganisms to be used as the inoculums or autoclaved as the sterile controls. The activated sludge was dosed with 6:2 FTOH or [1,2-14C] 6:2 FTOH to initiate the experiments in the same day when the sludge was collected. Glass serum bottles (119-mL volume) were used as experimental vessels. Before dosing with 6:2 FTOH, the live or sterile sludge was mixed with mineral media in two dilution ratios (v/v), one part of sludge to 9 parts of mineral media (referred as diluted sludge) and 9 parts of sludge to one part of mineral media (referred as native sludge). Each glass serum sample bottle contains 30 mL of the sludge and mineral media mixture with 85 mg L 1 of KH2PO4, 218 mg L 1 of K2HPO4, 334 mg L 1 of Na2HPO42H2O, 5 mg L 1 of NH4Cl, 36.4 mg L 1 of CaCl22H2O, 22.5 mg L 1 of MgSO47H2O, and 0.25 mg L 1 of FeCl36H2O with a pH of 7.0 (OECD, 1992). Antibiotics (kanamycin, chloramphenicol and cycloheximide) were applied to the sterile sludge at a final concentration of 200 mg L 1 to retard potential microbial growth.
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Each bottle with the 30-mL sludge/mineral media mixture was crimp-sealed with a butyl rubber stopper and aluminum cap. Ten microliter of 6:2 FTOH or [1,2-14C] 6:2 FTOH stock solution made in 50% ethanol (pure ethanol: water = 1:1) was injected into each of the live or sterile sludge bottles through a glass microsyringe after each bottle was inverted. Some of the sample bottles containing live sludge/mineral media mixture were only dosed with 10 lL of 50% ethanol for each bottle to serve as live matrix control for monitoring headspace oxygen content to approximate the aeration condition in the live sludge sample bottles dosed with 6:2 FTOH. One C18 SPE cartridge (0.6 g sorbent, Alltech, Deerfield, IL) pre-activated with acetonitrile was connected with a 18-gauge needle, which was inserted into the headspace of the sample bottles to ensure aeration and to capture volatile transformation products during 6:2 FTOH biotransformation (Zhao et al., 2012). All the testing vessels were incubated for up to 60 d inside an orbital shaker in upright position with 150 rpm shaking at room temperature (20 °C). It is noted that the room temperature at which this study was conducted is higher than temperature at which activated sludge resides in WWTP in winter but is lower than the typical temperature of activated sludge in a WWTP in summer. 2.3. Sample processing At each sampling time point up to 60 d, the oxygen gas content in the headspace of live matrix control bottles (n = 2) was measured with a headspace oxygen analyzer model 905 (Quantek Instruments, Grafton, MA). Prior to opening the sample bottles for adding solvent to extract 6:2 FTOH and transformation products, the headspace of each sample bottle of live sludge (n = 2–3), sterile sludge (n = 2–3), and live matrix control (n = 2) was purged through the C18 cartridge for 1–2 min with an air pump at 1– 2 L min 1. Each of the C18 cartridges was eluted with 5 mL of acetonitrile to recover volatile 6:2 FTOH and transformation products such as 5:2 ketone and 5:2 sFTOH. After the headspace air purging as described above, the septum from each sample bottle was transferred into a 20-mL glass vial and 5 mL acetonitrile was immediately added to the vial to extract 6:2 FTOH and transformation products. Thirty milliliter of acetonitrile was added to the remaining 30-mL sample mixture of each bottle, which was crimp-sealed with a fresh butyl rubber stopper and aluminum cap to extract 6:2 FTOH and transformation products from the sludge. The sample extraction was carried out with 200 rpm shaking at 50 °C for 1–4 d inside an orbital shaker with longer extraction time (e.g., 3–4 d) to accommodate working schedule. Previous work showed that 6:2 FTOH and transformation products are stable for at least 7 d at 50 °C and extraction efficiency did not differ much from 1 d to 4 d incubation at 50 °C (Liu et al., 2010a). The sludge extracts were centrifuged at 2000 rpm (950g force) for 20 min to collect the supernatant (1st extract). The extracts/supernatant from C18 cartridges, septa, and sludge samples were filtered through 0.45 lm-pore nylon filters before LC/MS/MS analysis. To determine the formation of non-extractable or sludge-bound 14 C residues after dosing with [1,2-14C] 6:2 FTOH, the sludge pellets after centrifugation were collected after decanting the supernatant (1st extract). The sludge pellets were then washed 1–2 times by repeatedly rinsing with 20 mL acetonitrile, centrifugation, and decanting to remove the remaining free 14C in the aqueous phase. The washed pellet in each sample bottle was mixed with 10 mL of concentrated HCl plus acetonitrile (concentrated HCl: acetonitrile = 1:4) and incubated at 50 °C overnight with 200 rpm shaking. The pellet extract (0.1 mL) from each sample bottle was mixed with 5 mL liquid scintillation cocktail for counting 14 C activities with a LS 5000TD liquid scintillation counter (Beckman, Fullerton, CA). Based on the 14C counts in the sludge pellet
and total 14C applied at day 0, the percent of sludge-bound 14C residues per sample can then be calculated. Previous work showed that 5:3 acid in the 1st acetonitrile extract of soil and sediment (Liu et al., 2010b; Zhao et al., 2012) can only be quantitatively analyzed after post treatment of the 1st extract with NaOH plus EnviCarb graphitized carbon. Similar clean-up procedures were also used to process the 1st extract from the sludge samples. In a 2-mL polypropylene tube, 1.2 mL acetonitrile, 30 mg Envi-Carb, and 45 lL of 1 M NaOH were added to 0.4 mL of the 1st extract. The mixture was incubated at 50 °C for 3–4 h with 200 rpm shaking. After centrifugation at 12,000 rpm (9000g force) for 10–20 min, the supernatant from each sample was filtered through a 0.45 um-pore nylon filter before LC/MS/ MS analysis. All the processed samples were stored at 10 to 20 °C if they were not analyzed immediately. 2.4. LC/MS/MS quantitative analysis The C18 cartridge eluent from the headspace, septum extracts, 1st sludge extracts, and the NaOH-Envicarb treated 1st extracts were all analyzed by LC/MS/MS to estimate the molar yields of transformation products over a period of up to 60 d. For each sample, 950 lL eluent or extract was spiked with 50 lL inter-standards containing 200 lg L 1 of [1,2-13C] PFHxA and 5000 lg L 1 of [1,1,2,2-D; 3-13C] 6–2 FTOH. The [1,2-13C] PFHxA was used to correct potential matrix effects on PFCAs and polyfluorinated acids, and [1,1,2,2-D; 3-13C] 6–2 FTOH was used to correct potential matrix effects on 6:2 FTOH, 5:2 ketone, and 5:2 sFTOH. The LC/MS/MS quantitative analysis was done by a Waters 2795 HPLC–Micromass Quattro Micro tandem mass spectrometry system (Milford, MA) performed at negative electro spray ionization mode with multiple reaction monitoring. Detailed instrumental methods and experimental conditions were included in Table S1. The mobile phase consisted of two parts: A, 0.15% acetic acid in Omnisov water; B, 0.15% acetic acid in acetonitrile. The separation was accomplished by an Agilent Zorbax RX-C8 column (150 mm 2.1 mm, 5 lm particle size, pore size = 80 Å, not end-capped, and carbon loading = 5.5%) under a gradient mobile phase. The detection limits for 6:2 FTOH and transformation products were listed in Table S1 of Supplementary data. 3. Results and discussion 3.1. Experiment system integrity and mass balance The oxygen content in the headspace of live matrix control bottles was always above 14% in the activated sludge from two WWTPs, indicating an aerobic environment in live sludge dosed with 6:2 FTOH. C18 cartridge use allowed air exchange between headspace and ambient air to ensure an aerobic environment in live activated sludge just as occurred in a river sediment system with similar aeration method (Zhao et al., 2012). The total recovery of 6:2 FTOH plus transformation products in live Pennsylvania sludge samples (including sludge extracts, septum extracts, and C18 cartridge eluents) ranged from 82 to 122 mol% of initially applied 6:2 FTOH from day 2 to day 56 (Fig. 1). The total recovery of 6:2 FTOH plus transformation products in Delaware sludge samples in an initial study ranged from 47 to 81 mol% for live sludge and 58% to 91 mol% for sterile sludge from day 1 to day 28 (Fig. 2, Delaware I experiment). The lower recovery may be due to the potential loss of volatile 6:2 FTOH, 5:2 ketone, and 5:2 sFTOH because of possible incomplete trapping by the C18 cartridges used in this experiment. To improve the recovery, a new experiment with [1,2-14C] 6:2 FTOH as a test substance was conducted by using a new batch of C18 cartridges to
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6:2 FTOH
6:2 FTCA
6:2 FTUCA
6:2 FTOH
6:2 FTCA
6:2 FTUCA
5:3 acid
4:3 acid
5:2 ketone
5:3 acid
4:3 acid
5:2 ketone
5:2 sFTOH PFHxA
PFBA Sum
PFPeA
5:2 sFTOH PFHxA
PFBA Sum
PFPeA Sterile control
120
mol% of 6:2 FTOH applied at day 0
mol% of 6:2 FTOH applied at day 0
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A Pennsylvania Delaware I
100 80 60 40 20
100
0 0
10
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A Delaware DelawareII
80
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40
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0 0
60
5
10
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5:2 sFTOH in activated sludge
5:2 sFTOH in the headspace
25
mol% of 6:2 FTOH applied at day 0
mol% of 6:2 FTOH applied at day 0
5:2 sFTOH in activated sludge
B Pennsylvania 20
15
10
5
0
2
7
14
15
20
25
30
Time (d)
28
56
capture the aforementioned volatile compounds in the headspace of the sample bottles. The total recovery of 6:2 FTOH plus transformation products in Delaware sludge samples (Delaware II experiment) ranged from 79 to 115 mol% for live sludge and from 81 to 93 mol% for sterile sludge from day 3 to day 60 (Fig. 3). Lower levels of non-extractable fraction or sludge-bound 14C residues were formed during [1,2-14C] 6:2 FTOH biotransformation in activated sludge compared with soil dosed with [1,2-14C] 6:2 FTOH, in which soil-bound 14C residues accounted for 25 mol% of initially applied [1,2-14C] 6:2 FTOH to live soil. The sludge-bound 14 C residues were less than 1.6 mol% of initially applied [1,2-14C] 6:2 FTOH at day 5 in both diluted and native activated sludge (Fig. 4). The sludge-bound 14C residues ranged from 8 to 9.6 mol% of initially applied [1,2-14C] 6:2 FTOH from day 10 to day 28 in diluted activated sludge and ranged from 9.9 to 11.3 mol% from the same period in native sludge (Fig. 4). These results demonstrated that no discernible differences existed between diluted and native sludge regarding the extent of sludge-bound 14 C residue formation. No 14C-bound-residues was observed in sterile sludge dosed with [1,2-14C] 6:2 FTOH, as the formation of
B Delaware I 10 8 6 4 2 0
1
2
7
14
28
Time (d)
Time (d) Fig. 1. The molar yields of 6:2 FTOH transformation products in activated sludge collected from a WWTP in Pennsylvania. The sludge was diluted 10 times with mineral media. The 6:2 FTOH starting concentration was 1.57 mg L 1 for the live sludge (n = 2). Graph A shows the molar yields of all individual transformation products recovered from the aqueous phase and headspace in comparison to moles of 6:2 FTOH applied at day 0. Graph B shows the molar yield of volatile intermediate product, 5:2 sFTOH, the major precursor to PFCAs.
5:2 sFTOH in the headspace
12
Fig. 2. The molar yields of 6:2 FTOH transformation products in activated sludge collected from a WWTP in Delaware (Delaware I experiment). The sludge was diluted 10 times with mineral media (one part sludge was mixed with 9 parts of mineral media). The 6:2 FTOH starting concentration was 2.85 mg L 1 for live sludge (n = 3) and 3.13 mg L 1 for sterile control (n = 3). Graph A shows the molar yields of all individual transformation products recovered from the aqueous phase and headspace in comparison to moles of 6:2 FTOH applied at day 0. Graph B shows the molar yield of volatile intermediate product, 5:2 sFTOH, the major precursor to PFCAs.
14
C-bound residues between 14C-labeled fluorinated compounds and organic matters of environmental matrices is an enzymecatalyzed process (Liu et al., 2010b). The above observations of aerobic environment and quantitative recovery of 6:2 FTOH and potential transformation products as well as sludge-bound residues in diluted sludge demonstrated the integrity of the experimental systems and analytical methods used in this study. On the other hand, the oxygen content in the headspace of native sludge from a WWTP in Delaware dropped to below 2% within 1 d and repeatedly headspace-purging with fresh air to replenish oxygen resulted in poor recovery of 6:2 FTOH and transformation products (total recovery = 49 mol% at day 28 versus 78 mol% for diluted sludge, Table S3). After giving a correction factor of 1.6 (78 mol% divided by 49 mol%) for native sludge, the molar yields of major stable and transient intermediates are generally similar except PFPeA, which is much higher in native sludge (Table S3). Because of the poor mass balance, no further detailed discussion will be presented for the native sludge.
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6:2 FTOH
6:2 FTCA
6:2 FTUCA
5:3 acid
4:3 acid
5:2 ketone
5:2 sFTOH PFHxA
5:3 Uacid Sum
PFPeA Sterile control
mol% of [1,2-14C] 6:2 FTOH applied at day 0
A A Delaware DelawareIII 100 80 60 40
10-fold diluted sludge
10 8 6
Non-extractable 14C fractions (Sludge-bound 14C residues)
4 2 0 0
20
0
10
20
30
40
50
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Time (d) 5:2 sFTOH in activated sludge
5:2 sFTOH in the headspace
40
B Delaware II 30
20
10
0
5
10
15
20
25
30
Time (d)
0
mol% of [1,2-14C] 6:2 FTOH applied at day 0
Original activated sludge
12
% of 14C counts applied at day 0
468
3
7
14
28
60
Time (d) Fig. 3. The molar yields of [1,2-14C] 6:2 FTOH transformation products in activated sludge collected from a WWTP in Delaware (Delaware II experiment). The sludge was diluted 10 times with mineral media. The [1,2-14C] 6:2 FTOH starting concentration was 2.90 mg L 1 for both live sludge (n = 2) and sterile controls (n = 2). Graph A shows the molar yields of all individual transformation products recovered from the aqueous phase and headspace in comparison to moles of 6:2 FTOH applied at day 0. Graph B shows the molar yield of volatile intermediate product, 5:2 sFTOH, the major precursor to PFCAs.
3.2. 5:2 sFTOH is the most abundant intermediate product formed during 6:2 FTOH biotransformation Primary 6:2 FTOH biotransformation in activated sludge was rapid, with more than 97 mol% converted within 3 d to at least nine transformation products (Figs. 1 and 3). 6:2 FTCA was observed in the early stage of 6:2 FTOH biotransformation via two enzymatic oxidation steps (Liu et al., 2010a), peaked at 6–24 mol% on days 2–3 in activated sludge from the two WWTPs and became nondetectable on day 7 due to its rapid transformation (Figs. 1 and 3). This result is similar to what was observed in sediment (Zhao et al., 2012) and in contrast to soil, where no 6:2 FTCA was observed over 90-d 6:2 FTOH biotransformation (Liu et al., 2010a). 6:2 FTUCA, formed via a facile one-step enzymatic HF elimination of 6:2 FTCA, peaked on days 2–3 at 17–27 mol% with only a low level detected on days 28–60 (Figs. 1 and 3). Thus only low quantities of 6:2 FTUCA may be potentially transported to other environment matrices via sludge amendment to surface soil (Sepulvado et al., 2011) or via direct WWTP effluent discharge to surface waters. 6:2 FTUCA was only briefly detected in sediment
Fig. 4. Recovery of the non-extractable (or sludge-bound) 14C fractions from the sludge solids after dosing with [1,2-14C] 6:2 FTOH. The activated sludge (n = 3) was collected from a Delaware WWTP. The [1,2-14C] 6:2 FTOH starting concentrations was 1.17 mg L 1 for the 10-fold diluted live sludge (n = 3) and 1.33 mg L 1 for the native sludge (n = 3). The non-extractable 14C fraction was defined as the 14C recovered from the sludge solids after first acetonitrile extraction and additional acetonitrile wash. The open circles represent the% recovery of non-extractable 14C fractions from the 10-fold diluted sludge and the solid circles represent the % recovery of non-extractable 14C fractions from the native sludge. No non-extractable 14C was detected in the diluted sterile sludge solids (n = 3) and native sterile sludge solids (n = 3).
on day 1 (Zhao et al., 2012) and not detected at any time over 90 d in soil due to its rapid biotransformation to downstream products (Liu et al., 2010a). 5:3 Uacid, formed via one-step enzymatic defluorination of 6:2 FTUCA, for the first time was quantified in activated sludge using a newly synthesized standard. The 5:3 Uacid level peaked at 20 mol% on day 3 and declined to 0.9 mol% on day 60 in activated sludge from Delaware (Fig. 3) with a LC/MS/MS detection limit of 3 lg L 1 using an ion transition of 339 > 295 (new method, Table S1). For Pennsylvania activated sludge (Fig. 1), no 5:3 Uacid was detected over 56 d with a LC/MS/MS detection limit of 40 lg L 1 (or 5 mol% of initially applied 6:2 FTOH) using an ion transition of 339 > 255 (old method, Table S1), suggesting that the 5:3 Uacid level is lower than 5 mol% if it is ever formed. This major difference in 5:3 Uacid molar yields indicates that microorganisms in WWTP from Pennsylvania may have higher catalytic capacity than that from Delaware to rapidly convert 5:3 Uacid to downstream transformation products. The 5:3 Uacid was not quantitatively analyzed for soil and sediment samples dosed with 6:2 FTOH due to lack of analytical standard at the time (Liu et al., 2010a; Zhao et al., 2012). The levels and time trends of intermediates such as 6:2 FTCA, 6:2 FTUCA, and 5:3 Uacid described above exhibited considerable variations between activated sludge from different WWTPs (this study), sediment (Zhao et al., 2012), and soil (Liu et al., 2010a). Such variations are expected. The 6:2 FTOH biotransformation intermediates are bioavailable and readily converted to corresponding stable transformation products in the environment. The volatile intermediate 5:2 sFTOH is the most abundant transformation product observed in this study and averaged 40 mol% on days 56–60 of initially applied 6:2 FTOH in activated sludge from the two WWTPs (Figs. 1 and 3). About 30 mol% 5:2 sFTOH was observed in the headspace of the sample bottles on days 56–60, suggesting that the majority was not available for further biotransformation to PFPeA and PFHxA by microbes in activated sludge. Rather, a substantial portion of this volatile intermediate would emit to ambient air and be subject to further abiotic atmospheric degradation. This is in sharp contrast to 6:2 FTOH biotransformation in soil, where less than 16 mol% 5:2
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Table 1 Comparison of the major stable transformation products and the ratioa of PFPeA plus PFHxA versus 5:3 acid in different environmental matrices during 6:2 FTOH biotransformation. Environ. matrix
PFBA (%)
PFPeA (%)
PFHxA (%)
5–3 Acid (%)
R(PFPeA + PFHxA)a/5–3
Soilb Bacterial cultureb Sedimentc Activated Sludged
1.8 <0.5 1.5 <0.5
30 <0.5 10 4.4
8.1 5.1 8.4 11
15 5.5 22 14
2.5 0.9 0.8 1.1
Acid
a b c d
Calculated by using the molar yield of PFPeA plus PFHxA to divide the molar yield of 5:3 acid. Liu et al. (2010a). Zhao et al. (2012). Average of activated sludge samples from two WWTPs in Delaware and Pennsylvania of this study.
sFTOH was observed on day 60, most of which was detected in the soil phase and available for biotransformation to PFPeA and PFHxA (Liu et al., 2010a). Thus, 5:2 sFTOH bioavailability and the ability of microbial enzymes to decarboxylate it may be the rate-limiting factors in determining PFCA molar yields in activated sludge containing 6:2 FTOH. 5:2 Ketone averaged 9.4 mol% on days 56–60 (Figs. 1 and 3) of the initially applied 6:2 FTOH in activated sludge from the two WWTPs. 5:2 ketone is the direct 5:2 sFTOH precursor and can be rapidly converted to 5:2 sFTOH in soil via one enzymatic reduction step (Liu et al., 2010a). The much lower level of 5:2 ketone on days 56–60 compared with that of 5:2 sFTOH implies that 5:2 ketone was also rapidly converted to 5:2 sFTOH as occurred in soil. 5:2 Ketone was formed by decarboxylation of 6:2 FTUCA via multiple enzymatic steps yet to be elucidated (Liu et al., 2010a). 3.3. 5:3 Acid and PFHxA are the major nonvolatile transformation products during 6:2 FTOH biotransformation 5:3 Acid is the most abundant nonvolatile biotransformation product and averaged 14.1 mol% in activated sludge from the two WWTPs on days 56–60 (Figs. 1 and 3). 5:3 Acid was formed by reducing 5:3 Uacid via one enzymatic step (Liu et al., 2010a). The 5:3 acid molar yield in activated sludge was very similar for the two WWTPs from Delaware and Pennsylvania, accounting for 13 mol% and 15 mol% of initially applied 6:2 FTOH on days 56– 60, respectively (Figs. 1 and 3). Moreover, 5:3 acid molar yield in activated sludge was similar to that reported in soil (Liu et al., 2010a) and slightly lower than that in sediment (Zhao et al., 2012), reinforcing the notion that 5:3 acid is a unique 6:2 FTOH biotransformation product. 5:3 Acid is relatively stable compared with other intermediates and it can be slowly degraded to 4:3 acid in activated sludge via one carbon removal pathways (Wang et al., 2012). 4:3 acid accounted for 1.3 mol% on days 56–60 of initially applied 6:2 FTOH (Figs. 1 and 3). This represents a 9.2% conversion (1.3 mol%/14.1 mol%) from 5:3 acid to 4:3 acid in activated sludge. The PFPeA molar yield averaged 4.4 mol% in the activated sludge studied (Figs. 1 and 3), which is 2.4–7 time lower than that reported in sediment and soil (Liu et al., 2010a; Zhao et al., 2012). The lower conversion of 6:2 FTOH to PFPeA in activated sludge may be partly due to the low bioavailability of its direct precursor, 5:2 sFTOH, most of which partitioned to the headspace. Bacterial populations are dominant in WWTP activated sludge and are mostly responsible for degradation of environment pollutants (Bitton, 2011). However, a pure bacterial strain had very limited ability to degrade 5:2 sFTOH to PFPeA (Kim et al., 2012). On the other hand, surface soils contain active fungal populations (Tate III, 2000) and can rapidly degrade 5:2 sFTOH to PFPeA with a 30% molar yield (Liu et al., 2010a). Taken together, these experiments suggest that fungal enzymes are more efficient at decarboxylating 5:2 sFTOH to PFPeA. No PFBA or PFHpA was observed in this study. These results suggest that biotransformation of 6:2 FTOH-based
products in activated sludge of WWTPs is not expected to be a major source of PFBA, PFPeA, and PFHpA. PFHxA is the major PFCA formed during 6:2 FTOH biotransformation in activated sludge and averaged 11 mol% of initially applied 6:2 FTOH, about 30% higher than that reported in soil (Liu et al., 2010a) and sediment (Zhao et al., 2012). The PFHxA molar yield also varied little in activated sludge of different WWTPs from Delaware and Pennsylvania, averaged 11 mol% of initially applied 6:2 FTOH on days 56–60 in both sites. The relative consistent molar yields of 5:3 acid and PFHxA in activated sludge from different WWTPs may help predict mass flow of these two important transformation products from WWTPs to other environments if 6:2 FTOH-based products were discharged to WWTPs. This consistency also suggests that microbes in activated sludge from different WWTPs deployed very similar enzymatic pathways to convert 6:2 FTOH to 5:3 acid and to PFHxA, respectively. 3.4. Using the R(PFPeA+PFHxA)/5:3 acid ratio to discern the potential origin of PFPeA and PFHxA 5:3 Acid is a unique 6:2 FTOH biotransformation product and was formed along with shorter-chain PFCAs such as PFPeA and PFHxA in soil (Liu et al., 2010a), sediment (Zhao et al., 2012), and activated sludge of this study. 5:3 Acid can only be formed via 6:2 FTOH biotransformation by microbes in the environment. No 5:3 acid was detected as an impurity in other polyfluorinated chemicals and no abiotic pathways are known to convert FTOHs or other poly-fluorinated chemicals to 5:3 acid. The level of 5:3 acid is in the same order of magnitude as that of the sum of PFPeA plus PFHxA in laboratory studies conducted in various environmental matrices (Table 1). As Table 1 illustrates, the ratio of the molar yield of PFPeA plus PFHxA divided by the molar yield of 5:3 acid is close to 1.0 in bacterial culture, sediment, and activated sludge, whereas the ratio is 2.5 in soil. Thus 5:3 acid may serve as an ‘‘internal standard’’ to estimate the relative contribution of 6:2 FTOH versus direct PFCA emissions to PFPeA and PFHxA present in an environmental matrix. For example, if the sum of PFPeA plus PFHxA were found to be an order of magnitude higher than that of 5:3 acid, then direct PFCA emissions would be suggested as the primary source for detected PFPeA and PFHxA. On the other hand, if the sum of PFPeA plus PFHxA is similar to that of 5:3 acid, then 6:2 FTOH microbial biotransformation would be suggested as the primary source of detected PFPeA and PFHxA. 4. Conclusions 6:2 FTOH biotransformation in activated sludge from two domestic WWTPs led to 5:2 sFTOH as the most abundant biotransformation product on days 56–60, predominantly in the sample bottle headspace and not available for further biodegradation. 6:2 FTOH biotransformation in activated sludge also generated 5:3 acid and PFHxA as the major biotransformation products on
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days 56–60. PFBA and PFHpA were not observed and PFPeA was formed in small quantities. These results imply that WWTPs could be potential sources of 5:2 sFTOH to the ambient air and of 5:3 acid and PFHxA to sludge effluent and sludge solids, but not major sources of PFBA, PFPeA, and PFHpA if 6:2 FTOH-based products were discharged to WWTPs. The markedly lower PFPeA yield compared with that in soil during 6:2 FTOH biotransformation in activated sludge may be due to the limited bioavailability of 5:2 sFTOH in the aqueous phase to be further degraded and lower efficiency of bacterial enzymes in activated sludge to decarboxylate 5:2 sFTOH to PFPeA compared with that of soil fungal enzymes. Together with the outcomes that 6:2 FTOH biotransformation produced PFPeA as the dominant product in soil and 5:3 acid as the major product in sediment, our work infer that 6:2 FTOH biotransformation rates and product profiles are different in major environmental matrices studied so far. Such variations between the activated sludge, soil, and sediment may be attributed to differences in microbial populations, bioavailability, and enzymatic catalytic capacities towards the major intermediates for further conversion to major stable products such as PFCAs and 5:3 acid. These differences may provide opportunities for optimizing microbial growth conditions, isolating microbial populations, and identifying microbial enzymes with enhanced biodegradability toward polyfluorinated chemicals. Acknowledgements We would like to thank Dr. Alexander Shtarov for synthesis of 5:3 Uacid and other intermediates and analytical standards used in this research. We thank the Nature Science Foundation (No. 41225014) and Ministry of Science and Technology (No. 2009DFA92390) of China to provide funding to support Lijie Zhao. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.chemosphere. 2013.02.032. References Ahrens, L., Shoeib, M., Harner, T., Lee, S.C., Guo, R., Reiner, E.J., 2011. Wastewater treatment plant and landfills as sources of polyfluoroalkyl compounds to the atmosphere. Environ. Sci. Technol. 45, 8098–8105. Benskin, J.P., Phillips, V., St. Louis, V.L., Martin, J.W., 2011. Source elucidation of perfluorinated carboxylic acids in remote alpine lake sediment cores. Environ. Sci. Technol. 45, 7188–7194. Bitton, 2011. Wastewater Microbiology, fourth ed. Wiley-Blackwell, Hoboken, NJ. Buck, R.C., Franklin, J., Berger, U., Conder, J.M., Cousins, I.T., Voogt, P., Jensen, A.A., Kannan, K., Mabury, S.A., Leeuwen, S.P.J., 2011. Perfluoroalkyl and polyfluoroalkyl substances in the environment: terminology, classification, and origins. Integr. Environ. Assess. Manage. 7, 513–541. Cousins, I.T., Kong, D., Vestergren, R., 2011. Reconciling measurement and modelling studies of the sources and fate of perfluorinated carboxylates. Environ. Chem. 8, 339–354. Ellis, D.A., Martin, J.W., De Silva, A.O., Mabury, S.A., Hurley, M.D., Sulbaek Andersen, M.P., Wallington, T.J., 2004. Degradation of fluorotelomer alcohols: a likely atmospheric source of perfluorinated carboxylic acids. Environ. Sci. Technol. 38, 3316–3321.
Houde, M., De Silva, A.O., Muir, D.C.G., Letcher, R.J., 2011. Monitoring of perfluorinated compounds in aquatic biota: an updated review on PFCs in aquatic biota. Environ. Sci. Technol. 45, 7962–7973. Kim, M.H., Wang, N., McDonald, T., Chu, K.H., 2012. Biodefluorination and biotransformation of fluorotelomer alcohols by two alkane degrading Pseudomonas strains. Biotechnol. Bioeng. 109, 3041–3048. Kunacheva, C., Tanaka, S., Fujii, S., Boontanon, S.K., Musirat, C., Wongwattana, T., Shivakoti, B.R., 2011. Mass flows of perfluorinated compounds (PFCs) in central wastewater treatment plants of industrial zones in Thailand. Chemosphere 83, 737–744. Lau, C., 2012. Perfluoroalkyl acids: recent research highlights. Reprod. Toxicol. 33, 405–409. Li, F., Sun, H., Hao, Z., He, N., Zhao, L., Zhang, T., Sun, T., 2011. Perfluorinated compounds in Haihe River and Dagu drainage canal in Tianjin, China. Chemosphere 84, 265–271. Liu, J., Lee, L.S., Nies, L.F., Nakatsu, C.H., Turco, R.F., 2007. Biotransformation of 8:2 fluorotelomer alcohol in soil and by soil bacterial isolates. Environ. Sci. Technol. 41, 8024–8030. Liu, J., Wang, N., Szostek, B., Buck, R.C., Panciroli, P.K., Folsom, P.W., Sulecki, L.M., Bellin, C.A., 2010a. 6–2 Fluorotelomer alcohol aerobic biodegradation in soil and mixed bacterial culture. Chemosphere 78, 437–444. Liu, J., Wang, N., Buck, R.C., Wolstenholme, B.W., Folsom, P.W., Sulecki, L.M., Bellin, C.A., 2010b. Aerobic biodegradation of [14C] 6:2 fluorotelomer alcohol in a flowthrough soil incubation system. Chemosphere 80, 716–723. Martin, J.W., Ellis, D.A., Mabury, S.A., Hurley, M.D., Wallington, T.J., 2006. Atmospheric chemistry of perfluoroalkanesulfonamides: kinetic and product studies of the OH radical and Cl atom initiated oxidation of N-Ethyl perfluorobutanesulfonamide. Environ. Sci. Technol. 40, 864–872. Naile, J.E., Khim, J.S., Wang, T., Chen, C., Luo, W., Kwond, B.O., Park, J., Koh, C.H., Jones, P.D., Lu, Y., Giesy, J.P., 2010. Perfluorinated compounds in water, sediment, soil and biota from estuarine and coastal areas of Korea. Environ. Pollut. 158, 1237–1244. Nealson, K.H., 1997. Sediment bacteria: who’s there, what are they doing, and what is new. Annu. Rev. Earth Planet. Sci. 25, 403–434. OECD, 1992. Organization for Economic Cooperation and Development, Guideline for the Testing of Chemicals 301: Ready Biodegradability, July 17. Prevedouros, K., Cousins, I.T., Buck, R.C., Korzeniowski, S.H., 2006. Sources, fate and transport of perfluorocarboxylates. Environ. Sci. Technol. 40, 32–44. Schultz, M.M., Higgins, C.P., Huset, C.A., Luthy, R.G., Barofsky, D.F., Field, J.A., 2006. Fluorochemical mass flows in a municipal wastewater treatment facility. Environ. Sci. Technol. 40, 7350–7357. Sepulvado, J.G., Blaine, A.C., Hundal, L.S., Higgins, C.P., 2011. Occurrence and fate of perfluorochemicals in soil following the land application of municipal biosolids. Environ. Sci. Technol. 45, 8106–8112. Sun, H., Li, F., Zhang, T., Zhang, X., He, N., Song, Q., Zhao, L., Sun, L., Sun, T., 2011. Perfluorinated compounds in surface waters and WWTPs in Shenyang, China: mass flows and source analysis. Water Res. 45, 4483–4490. Tate III, 2000. Soil Microbiology, second ed. John Wiley & Sons, Inc., New York, NY. U.S. Environmental Protection Agency (EPA), 2006. 2010/2015 PFOA Stewardship Program. EPA-HQ-2003-0012-1071.. Wang, N., Szostek, B., Folsom, P.W., Sulecki, L.M., Capka, V., Buck, R.C., Berti, W.R., Gannon, J.T., 2005a. Aerobic biotransformation of 14C-labeled 8–2 Telomer B Alcohol by activated sludge from a domestic sewage treatment plant. Environ. Sci. Technol. 39, 531–538. Wang, N., Szostek, B., Buck, R.C., Folsom, P.W., Sulecki, L.M., Capka, V., Berti, W.R., Gannon, J.T., 2005b. Fluorotelomer alcohol biodegradation – direct evidence that perfluorinated carbon chains breakdown. Environ. Sci. Technol. 39, 7516– 7528. Wang, N., Szostek, B., Buck, R.C., Folsom, P.W., Sulecki, L.M., Gannon, J.T., 2009. 8–2 Fluorotelomer alcohol aerobic soil biodegradation: Pathways, metabolites, and metabolite yields. Chemosphere 75, 1089–1096. Wang, N., Liu, J., Buck, R.C., Korzeniowski, S.H., Wolstenholme, B.W., Folsom, P.W., Sulecki, L.M., 2011. 6:2 Fluorotelomer sulfonate aerobic biotransformation in activated sludge of waste water treatment plants. Chemosphere 82, 853–858. Wang, N., Buck, R.C., Szostek, B., Sulecki, L.M., Wolstenholme, B.W., 2012. 5:3 Polyfluorinated acid aerobic biotransformation in activated sludge via novel one-carbon removal pathways. Chemosphere 87, 527–534. Zhao, L., Folsom, P.W., Wolstenholme, B.W., Sun, H., Wang, N., Buck, R.C., 2012. 6:2 Fluorotelomer alcohol biotransformation in an aerobic river sediment system. Chemosphere 90, 203–209.
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6:2 Fluorotelomer alcohol aerobic biotransformation in activated sludge from two domestic wastewater treatment plants Lijie Zhao a, Patricia K. McCausland b, Patrick W. Folsom b, Barry W. Wolstenholme b, Hongwen Sun a,⇑, Ning Wang b,⇑, Robert C. Buck b a b
MOE Key Laboratory of Pollution Processes and Environmental Criteria, Nankai University, Tianjin, China E.I. du Pont de Nemours & Company, Inc., Newark, DE, USA
h i g h l i g h t s " 5:2 sFTOH is the dominant volatile product of 6:2 FTOH aerobic biotransformation. " 5:3 Acid and PFHxA are the major non-volatile transformation products. " 6:2 FTOH aerobic biotransformation is not a source of PFBA and PFHpA.
a r t i c l e
i n f o
Article history: Received 23 September 2012 Received in revised form 13 February 2013 Accepted 16 February 2013 Available online 26 March 2013 Keywords: 6:2 Fluorotelomer alcohol (6:2 FTOH) 5:3 Polyfluorinated acid (5:3 acid) Perfluorohexanoic acid (PFHxA) 5:2 Secondary polyfluorinated alcohol (5:2 sFTOH) Activated sludge Aerobic biotransformation
a b s t r a c t 6:2 Fluorotelomer alcohol [6:2 FTOH, F(CF2)6CH2CH2OH] is a major basic chemical being used to manufacture FTOH-based products. After the end of use, 6:2 FTOH-based products may be released to domestic wastewater treatment plants (WWTPs) as a first major environmental entry point. Activated sludge collected from two WWTPs was dosed with 6:2 FTOH to investigate its biotransformation rate and to identify major transformation products. The volatile 5:2 sFTOH [F(CF2)5CH(OH)CH3] is the most abundant transformation product and accounted for an average of 40 mol% of initially dosed 6:2 FTOH after two months of incubation with activated sludge, with 30 mol% detected in the headspace. PFPeA [F(CF2)4 COOH] averaged 4.4 mol% after two months, 2.4–7 times lower than that in sediment and soils. The much lower level of PFPeA formed in activated sludge compared with soil indicates that microbial populations in activated sludge may lack enzymes or suitable environment conditions to promote rapid 5:2 sFTOH decarboxylation to form PFPeA, resulting in more 5:2 sFTOH partitioned to the headspace. PFHxA [F(CF2)5COOH] and 5:3 [F(CF2)5CH2CH2COOH] acid are major non-volatile transformation products in activated sludge. For example, PFHxA averaged 11 mol% after two months, which is about 30% higher compared with sediment and soils, suggesting that microbes in WWTPs may utilize similar pathways as that in sediment and soils to convert 5:2 sFTOH to PFHxA. 5:3 Acid averaged 14 mol% after two months, comparable to that in soils and slightly lower than in sediment, further confirming that 5:3 acid is a unique product of 6:2 FTOH biotransformation in the environment. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Perfluoroalkyl acids (PFAAs) such as perfluoroalkane sulfonates (PFSAs) and perfluoroalkyl carboxylates (PFCAs) are widely detected in the environment and biota (Buck et al., 2011; Houde et al., 2011; Benskin et al., 2011) because of their wide use and ⇑ Corresponding authors. Addresses: College of Environmental Science and Engineering, Nankai University, 94 Weijin Street, Tianjin 300071, China. Tel.: +86 22 23509241 (H. Sun), DuPont Haskell Global Centers for Health and Environmental Sciences, Glasgow 300, PO Box 6300, Newark, DE 19714-6300, USA. Tel.: +1 302 366 6665 (N. Wang). E-mail addresses: [email protected] (H. Sun), ning.wang@usa. dupont.com (N. Wang). 0045-6535/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.chemosphere.2013.02.032
historical applications in industrial and consumer products (Prevedouros et al., 2006). For example, PFSAs and PFCAs are detected in soil (Naile et al., 2010), sediment (Li et al., 2011; Benskin et al., 2011), activated sludge from WWTPs (Schultz et al., 2006; Ahrens et al., 2011; Kunacheva et al., 2011; Sun et al., 2011), and in biota (Naile et al., 2010; Houde et al., 2011). The sources of PFSAs and PFCAs are from their historical direct environment emissions and to a less extent from precursor degradation (Ellis et al., 2004; Prevedouros et al., 2006; Martin et al., 2006; Cousins et al., 2011). The long-chain PFSAs (P6 carbon chain length) and longchain PFCAs (P8 carbon chain length) have much longer elimination half-life in exposed animals and humans compared with their short-chain homologs (Lau, 2012). For example, perfluorohexane
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sulfonate (PFHxS) and perfluorooctanoic acid (PFOA) have a serum/ plasma elimination half-life of 8.5 and 3 years, respectively, in human, compared with 30 and 3 d, respectively, for perfluorobutane sulfonate (PFBS) and perfluorobutanoic acid (PFBA) (Lau, 2012). Because of the potential long elimination time and potential toxicity associated with long-chain PFSAs and PFCAs, major manufacturers in the developed world are in the process of replacing the longchain chemistry with short-chain based chemistry (US EPA, 2006). 6:2 FTOH is a major raw material being used to replace 8:2 FTOH, a potential PFOA precursor when subjected to microbial biotransformation in activated sludge (Wang et al., 2005a,b) and soils (Liu et al., 2007; Wang et al., 2009). 6:2 FTOH biodegradation in soil (Liu et al., 2010a,b) and sediment (Zhao et al., 2012) leads to PFPeA, PFHxA, PFBA [F(CF2)3COOH], 5:3 acid, and 4:3 acid [F(CF2)4 CH2CH2COOH] as major non-volatile transformation products. Intermediate products such as 6:2 FTCA [F(CF2)6CH2COOH] and 6:2 FTUCA [F(CF2)5CF = CHCOOH] were also observed in sediment but not in soils dosed with 6:2 FTOH. The volatile intermediate products 5:2 ketone [F(CF2)5C(O)CH3] and 5:2 sFTOH were observed in both sediment and soil (Liu et al., 2010a; Zhao et al., 2012). Currently, little information is available on 6:2 FTOH biotransformation in activated sludge from WWTPs. WWTPs are important sites for anthropogenic chemicals to be transformed or degraded by microbes before the effluent and sludge solids are being discharged to the environment along with un-reacted precursors, intermediates, and stable transformation products. The typical sludge retention time in WWTPs in the US states of Delaware (DE) and Pennsylvania (PA) is less than 14 d and ranges from 2 to 60 d (Bitton, 2011) in US. 6:2 FTOH-based monomeric products or impurities may be degraded by microbes in activated sludge. 6:2 FTOH primary biotransformation to intermediates and eventually to major stable products are relatively fast (within days) as shown in an earlier study in bacterial culture (Liu et al., 2010a). It is important to identify 6:2 FTOH transformation products and understand its transformation rates. Such practical knowledge can help environmental modeling and monitoring, and predict environmental concentrations of 6:2 FTOH stable transformation products for risk assessment to human and environmental health. 6:2 FTOH conversion rates to other major products may be different in activated sludge from those observed in aerobic soils and sediment because of differences in microbial populations and environmental conditions. For example, WWTPs contain mainly bacteria associated with digested plant and animal wastes (Bitton, 2011) whereas soils contain both bacteria and fungi (Tate III, 2000) and sediment contains mainly bacteria associated with animal materials (Nealson, 1997). These differences may affect 6:2 FTOH partitioning behavior, bioavailability, and ultimately microbial ability to transform or degrade 6:2 FTOH in different environmental matrices as discussed above. When [1,2-14C] 6:2 FTOH was applied to Sassafras soil, about 25% of initially applied 14C was conjugated with soil organic materials catalyzed by microbial enzymes to form soil-bound residues that may not be available for further biodegradation (Liu et al., 2010b). The solid content in activated sludge is relatively low (1– 3 dry wt.% in WWTPs from PA, DE, and MD states) due to the presence of the predominant aqueous phase compared with soil. It is not known to what extent 6:2 FTOH can be bound to sludge organic materials and whether such bound residues can affect 6:2 FTOH biotransformation. Diluted activated sludge from WWTPs was successfully used to study 6:2 fluorotelomer sulfonate [6:2 FTSA, F(CF2)6CH2CH2SO3K+, Wang et al., 2011] and 5:3 acid (Wang et al., 2012) biotransformation. The use of diluted activated sludge decreased potential matrix effects of the sludge organic materials on LC/MS/MS quantitative analysis of 6:2 FTOH transformation products and also ensured aeration of activated sludge media dosed with 6:2 FTSA and 5:3
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acid in glass serum bottles as mentioned above. Also, our preliminary work with both diluted and non-diluted sludge dosed with 6:2 FTOH did not show significant differences in 6:2 FTOH primary biotransformation and molar yields of major transformation products. Overall, the diluted sludge system helped achieve good mass balance (i.e., good recovery of starting materials and transformation products (Wang et al., 2011, 2012), thus assuring study integrity. The aerated bottles with both diluted and native sludge in this study were used to measure 6:2 FTOH relative transformation ratios (molar yields) to different transformation products. Such information is necessary for further studying in situ 6:2 FTOH biotransformation rates in WWTPs using on-site simulation test. The objectives of this study were to determine 6:2 FTOH biotransformation potential from different WWTPs, to identify major transformation products, and to compare 6:2 FTOH biotransformation outcomes with those reported in soils and sediment. The information sought in this study can help understand the sources and potential FTOH precursor contribution to PFCAs detected in WWTPs and in other environmental compartments such as soils and sediment. 2. Materials and methods 2.1. Chemicals 6:2 FTOH purity was 99% from Sigma–Aldrich (St. Louis, MO). The custom-synthesized [1,2-14C] 6:2 FTOH [F(CF2)614CH214CH2OH] had a radiochemical purity of +99%. The purities of 4:3 acid and a newly synthesized 5:3 Uacid [F(CF2)5CH = CHCOOH] standards were 96% and 95%, respectively. The other fluorinated chemical standards were all above 97% pure. The details of these chemical standards including acronyms, structure, CAS numbers and sources were listed in an early study (Liu et al., 2010a). Stable isotope quantification internal standards used in LC/MS/MS analysis were [1,1,2,2-D; 3-13C] 6:2 FTOH [F(CF2)513CF2CD2CD2OH] (DuPont, Wilmington, DE) and [1,2-13C] PFHxA [F(CF2)413CF213 COOH] (Wellington Laboratories, Ontario, Canada). All solvents were HPLC grade or higher and all other chemicals referenced were at least reagent grade. De-ionized water (18 MX cm) was from a Barnstead E-Pure system used throughout all the experiment. Omsolv water (EMD Chemicals, Gibbstown, NJ) was used for LC/MS/ MS analysis. 2.2. Sludge collection and experiment set up The activated sludge was collected from the aeration basins immediately after the primary clarifiers from two domestic WWTPs in Delaware and Pennsylvania. The sludge was brought directly to the laboratory, was aerated and stirred in a magnetic plate at room temperature to suspend the microorganisms to be used as the inoculums or autoclaved as the sterile controls. The activated sludge was dosed with 6:2 FTOH or [1,2-14C] 6:2 FTOH to initiate the experiments in the same day when the sludge was collected. Glass serum bottles (119-mL volume) were used as experimental vessels. Before dosing with 6:2 FTOH, the live or sterile sludge was mixed with mineral media in two dilution ratios (v/v), one part of sludge to 9 parts of mineral media (referred as diluted sludge) and 9 parts of sludge to one part of mineral media (referred as native sludge). Each glass serum sample bottle contains 30 mL of the sludge and mineral media mixture with 85 mg L 1 of KH2PO4, 218 mg L 1 of K2HPO4, 334 mg L 1 of Na2HPO42H2O, 5 mg L 1 of NH4Cl, 36.4 mg L 1 of CaCl22H2O, 22.5 mg L 1 of MgSO47H2O, and 0.25 mg L 1 of FeCl36H2O with a pH of 7.0 (OECD, 1992). Antibiotics (kanamycin, chloramphenicol and cycloheximide) were applied to the sterile sludge at a final concentration of 200 mg L 1 to retard potential microbial growth.
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Each bottle with the 30-mL sludge/mineral media mixture was crimp-sealed with a butyl rubber stopper and aluminum cap. Ten microliter of 6:2 FTOH or [1,2-14C] 6:2 FTOH stock solution made in 50% ethanol (pure ethanol: water = 1:1) was injected into each of the live or sterile sludge bottles through a glass microsyringe after each bottle was inverted. Some of the sample bottles containing live sludge/mineral media mixture were only dosed with 10 lL of 50% ethanol for each bottle to serve as live matrix control for monitoring headspace oxygen content to approximate the aeration condition in the live sludge sample bottles dosed with 6:2 FTOH. One C18 SPE cartridge (0.6 g sorbent, Alltech, Deerfield, IL) pre-activated with acetonitrile was connected with a 18-gauge needle, which was inserted into the headspace of the sample bottles to ensure aeration and to capture volatile transformation products during 6:2 FTOH biotransformation (Zhao et al., 2012). All the testing vessels were incubated for up to 60 d inside an orbital shaker in upright position with 150 rpm shaking at room temperature (20 °C). It is noted that the room temperature at which this study was conducted is higher than temperature at which activated sludge resides in WWTP in winter but is lower than the typical temperature of activated sludge in a WWTP in summer. 2.3. Sample processing At each sampling time point up to 60 d, the oxygen gas content in the headspace of live matrix control bottles (n = 2) was measured with a headspace oxygen analyzer model 905 (Quantek Instruments, Grafton, MA). Prior to opening the sample bottles for adding solvent to extract 6:2 FTOH and transformation products, the headspace of each sample bottle of live sludge (n = 2–3), sterile sludge (n = 2–3), and live matrix control (n = 2) was purged through the C18 cartridge for 1–2 min with an air pump at 1– 2 L min 1. Each of the C18 cartridges was eluted with 5 mL of acetonitrile to recover volatile 6:2 FTOH and transformation products such as 5:2 ketone and 5:2 sFTOH. After the headspace air purging as described above, the septum from each sample bottle was transferred into a 20-mL glass vial and 5 mL acetonitrile was immediately added to the vial to extract 6:2 FTOH and transformation products. Thirty milliliter of acetonitrile was added to the remaining 30-mL sample mixture of each bottle, which was crimp-sealed with a fresh butyl rubber stopper and aluminum cap to extract 6:2 FTOH and transformation products from the sludge. The sample extraction was carried out with 200 rpm shaking at 50 °C for 1–4 d inside an orbital shaker with longer extraction time (e.g., 3–4 d) to accommodate working schedule. Previous work showed that 6:2 FTOH and transformation products are stable for at least 7 d at 50 °C and extraction efficiency did not differ much from 1 d to 4 d incubation at 50 °C (Liu et al., 2010a). The sludge extracts were centrifuged at 2000 rpm (950g force) for 20 min to collect the supernatant (1st extract). The extracts/supernatant from C18 cartridges, septa, and sludge samples were filtered through 0.45 lm-pore nylon filters before LC/MS/MS analysis. To determine the formation of non-extractable or sludge-bound 14 C residues after dosing with [1,2-14C] 6:2 FTOH, the sludge pellets after centrifugation were collected after decanting the supernatant (1st extract). The sludge pellets were then washed 1–2 times by repeatedly rinsing with 20 mL acetonitrile, centrifugation, and decanting to remove the remaining free 14C in the aqueous phase. The washed pellet in each sample bottle was mixed with 10 mL of concentrated HCl plus acetonitrile (concentrated HCl: acetonitrile = 1:4) and incubated at 50 °C overnight with 200 rpm shaking. The pellet extract (0.1 mL) from each sample bottle was mixed with 5 mL liquid scintillation cocktail for counting 14 C activities with a LS 5000TD liquid scintillation counter (Beckman, Fullerton, CA). Based on the 14C counts in the sludge pellet
and total 14C applied at day 0, the percent of sludge-bound 14C residues per sample can then be calculated. Previous work showed that 5:3 acid in the 1st acetonitrile extract of soil and sediment (Liu et al., 2010b; Zhao et al., 2012) can only be quantitatively analyzed after post treatment of the 1st extract with NaOH plus EnviCarb graphitized carbon. Similar clean-up procedures were also used to process the 1st extract from the sludge samples. In a 2-mL polypropylene tube, 1.2 mL acetonitrile, 30 mg Envi-Carb, and 45 lL of 1 M NaOH were added to 0.4 mL of the 1st extract. The mixture was incubated at 50 °C for 3–4 h with 200 rpm shaking. After centrifugation at 12,000 rpm (9000g force) for 10–20 min, the supernatant from each sample was filtered through a 0.45 um-pore nylon filter before LC/MS/ MS analysis. All the processed samples were stored at 10 to 20 °C if they were not analyzed immediately. 2.4. LC/MS/MS quantitative analysis The C18 cartridge eluent from the headspace, septum extracts, 1st sludge extracts, and the NaOH-Envicarb treated 1st extracts were all analyzed by LC/MS/MS to estimate the molar yields of transformation products over a period of up to 60 d. For each sample, 950 lL eluent or extract was spiked with 50 lL inter-standards containing 200 lg L 1 of [1,2-13C] PFHxA and 5000 lg L 1 of [1,1,2,2-D; 3-13C] 6–2 FTOH. The [1,2-13C] PFHxA was used to correct potential matrix effects on PFCAs and polyfluorinated acids, and [1,1,2,2-D; 3-13C] 6–2 FTOH was used to correct potential matrix effects on 6:2 FTOH, 5:2 ketone, and 5:2 sFTOH. The LC/MS/MS quantitative analysis was done by a Waters 2795 HPLC–Micromass Quattro Micro tandem mass spectrometry system (Milford, MA) performed at negative electro spray ionization mode with multiple reaction monitoring. Detailed instrumental methods and experimental conditions were included in Table S1. The mobile phase consisted of two parts: A, 0.15% acetic acid in Omnisov water; B, 0.15% acetic acid in acetonitrile. The separation was accomplished by an Agilent Zorbax RX-C8 column (150 mm 2.1 mm, 5 lm particle size, pore size = 80 Å, not end-capped, and carbon loading = 5.5%) under a gradient mobile phase. The detection limits for 6:2 FTOH and transformation products were listed in Table S1 of Supplementary data. 3. Results and discussion 3.1. Experiment system integrity and mass balance The oxygen content in the headspace of live matrix control bottles was always above 14% in the activated sludge from two WWTPs, indicating an aerobic environment in live sludge dosed with 6:2 FTOH. C18 cartridge use allowed air exchange between headspace and ambient air to ensure an aerobic environment in live activated sludge just as occurred in a river sediment system with similar aeration method (Zhao et al., 2012). The total recovery of 6:2 FTOH plus transformation products in live Pennsylvania sludge samples (including sludge extracts, septum extracts, and C18 cartridge eluents) ranged from 82 to 122 mol% of initially applied 6:2 FTOH from day 2 to day 56 (Fig. 1). The total recovery of 6:2 FTOH plus transformation products in Delaware sludge samples in an initial study ranged from 47 to 81 mol% for live sludge and 58% to 91 mol% for sterile sludge from day 1 to day 28 (Fig. 2, Delaware I experiment). The lower recovery may be due to the potential loss of volatile 6:2 FTOH, 5:2 ketone, and 5:2 sFTOH because of possible incomplete trapping by the C18 cartridges used in this experiment. To improve the recovery, a new experiment with [1,2-14C] 6:2 FTOH as a test substance was conducted by using a new batch of C18 cartridges to
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6:2 FTCA
6:2 FTUCA
5:3 acid
4:3 acid
5:2 ketone
5:3 acid
4:3 acid
5:2 ketone
5:2 sFTOH PFHxA
PFBA Sum
PFPeA
5:2 sFTOH PFHxA
PFBA Sum
PFPeA Sterile control
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capture the aforementioned volatile compounds in the headspace of the sample bottles. The total recovery of 6:2 FTOH plus transformation products in Delaware sludge samples (Delaware II experiment) ranged from 79 to 115 mol% for live sludge and from 81 to 93 mol% for sterile sludge from day 3 to day 60 (Fig. 3). Lower levels of non-extractable fraction or sludge-bound 14C residues were formed during [1,2-14C] 6:2 FTOH biotransformation in activated sludge compared with soil dosed with [1,2-14C] 6:2 FTOH, in which soil-bound 14C residues accounted for 25 mol% of initially applied [1,2-14C] 6:2 FTOH to live soil. The sludge-bound 14 C residues were less than 1.6 mol% of initially applied [1,2-14C] 6:2 FTOH at day 5 in both diluted and native activated sludge (Fig. 4). The sludge-bound 14C residues ranged from 8 to 9.6 mol% of initially applied [1,2-14C] 6:2 FTOH from day 10 to day 28 in diluted activated sludge and ranged from 9.9 to 11.3 mol% from the same period in native sludge (Fig. 4). These results demonstrated that no discernible differences existed between diluted and native sludge regarding the extent of sludge-bound 14 C residue formation. No 14C-bound-residues was observed in sterile sludge dosed with [1,2-14C] 6:2 FTOH, as the formation of
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Time (d) Fig. 1. The molar yields of 6:2 FTOH transformation products in activated sludge collected from a WWTP in Pennsylvania. The sludge was diluted 10 times with mineral media. The 6:2 FTOH starting concentration was 1.57 mg L 1 for the live sludge (n = 2). Graph A shows the molar yields of all individual transformation products recovered from the aqueous phase and headspace in comparison to moles of 6:2 FTOH applied at day 0. Graph B shows the molar yield of volatile intermediate product, 5:2 sFTOH, the major precursor to PFCAs.
5:2 sFTOH in the headspace
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Fig. 2. The molar yields of 6:2 FTOH transformation products in activated sludge collected from a WWTP in Delaware (Delaware I experiment). The sludge was diluted 10 times with mineral media (one part sludge was mixed with 9 parts of mineral media). The 6:2 FTOH starting concentration was 2.85 mg L 1 for live sludge (n = 3) and 3.13 mg L 1 for sterile control (n = 3). Graph A shows the molar yields of all individual transformation products recovered from the aqueous phase and headspace in comparison to moles of 6:2 FTOH applied at day 0. Graph B shows the molar yield of volatile intermediate product, 5:2 sFTOH, the major precursor to PFCAs.
14
C-bound residues between 14C-labeled fluorinated compounds and organic matters of environmental matrices is an enzymecatalyzed process (Liu et al., 2010b). The above observations of aerobic environment and quantitative recovery of 6:2 FTOH and potential transformation products as well as sludge-bound residues in diluted sludge demonstrated the integrity of the experimental systems and analytical methods used in this study. On the other hand, the oxygen content in the headspace of native sludge from a WWTP in Delaware dropped to below 2% within 1 d and repeatedly headspace-purging with fresh air to replenish oxygen resulted in poor recovery of 6:2 FTOH and transformation products (total recovery = 49 mol% at day 28 versus 78 mol% for diluted sludge, Table S3). After giving a correction factor of 1.6 (78 mol% divided by 49 mol%) for native sludge, the molar yields of major stable and transient intermediates are generally similar except PFPeA, which is much higher in native sludge (Table S3). Because of the poor mass balance, no further detailed discussion will be presented for the native sludge.
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3.2. 5:2 sFTOH is the most abundant intermediate product formed during 6:2 FTOH biotransformation Primary 6:2 FTOH biotransformation in activated sludge was rapid, with more than 97 mol% converted within 3 d to at least nine transformation products (Figs. 1 and 3). 6:2 FTCA was observed in the early stage of 6:2 FTOH biotransformation via two enzymatic oxidation steps (Liu et al., 2010a), peaked at 6–24 mol% on days 2–3 in activated sludge from the two WWTPs and became nondetectable on day 7 due to its rapid transformation (Figs. 1 and 3). This result is similar to what was observed in sediment (Zhao et al., 2012) and in contrast to soil, where no 6:2 FTCA was observed over 90-d 6:2 FTOH biotransformation (Liu et al., 2010a). 6:2 FTUCA, formed via a facile one-step enzymatic HF elimination of 6:2 FTCA, peaked on days 2–3 at 17–27 mol% with only a low level detected on days 28–60 (Figs. 1 and 3). Thus only low quantities of 6:2 FTUCA may be potentially transported to other environment matrices via sludge amendment to surface soil (Sepulvado et al., 2011) or via direct WWTP effluent discharge to surface waters. 6:2 FTUCA was only briefly detected in sediment
Fig. 4. Recovery of the non-extractable (or sludge-bound) 14C fractions from the sludge solids after dosing with [1,2-14C] 6:2 FTOH. The activated sludge (n = 3) was collected from a Delaware WWTP. The [1,2-14C] 6:2 FTOH starting concentrations was 1.17 mg L 1 for the 10-fold diluted live sludge (n = 3) and 1.33 mg L 1 for the native sludge (n = 3). The non-extractable 14C fraction was defined as the 14C recovered from the sludge solids after first acetonitrile extraction and additional acetonitrile wash. The open circles represent the% recovery of non-extractable 14C fractions from the 10-fold diluted sludge and the solid circles represent the % recovery of non-extractable 14C fractions from the native sludge. No non-extractable 14C was detected in the diluted sterile sludge solids (n = 3) and native sterile sludge solids (n = 3).
on day 1 (Zhao et al., 2012) and not detected at any time over 90 d in soil due to its rapid biotransformation to downstream products (Liu et al., 2010a). 5:3 Uacid, formed via one-step enzymatic defluorination of 6:2 FTUCA, for the first time was quantified in activated sludge using a newly synthesized standard. The 5:3 Uacid level peaked at 20 mol% on day 3 and declined to 0.9 mol% on day 60 in activated sludge from Delaware (Fig. 3) with a LC/MS/MS detection limit of 3 lg L 1 using an ion transition of 339 > 295 (new method, Table S1). For Pennsylvania activated sludge (Fig. 1), no 5:3 Uacid was detected over 56 d with a LC/MS/MS detection limit of 40 lg L 1 (or 5 mol% of initially applied 6:2 FTOH) using an ion transition of 339 > 255 (old method, Table S1), suggesting that the 5:3 Uacid level is lower than 5 mol% if it is ever formed. This major difference in 5:3 Uacid molar yields indicates that microorganisms in WWTP from Pennsylvania may have higher catalytic capacity than that from Delaware to rapidly convert 5:3 Uacid to downstream transformation products. The 5:3 Uacid was not quantitatively analyzed for soil and sediment samples dosed with 6:2 FTOH due to lack of analytical standard at the time (Liu et al., 2010a; Zhao et al., 2012). The levels and time trends of intermediates such as 6:2 FTCA, 6:2 FTUCA, and 5:3 Uacid described above exhibited considerable variations between activated sludge from different WWTPs (this study), sediment (Zhao et al., 2012), and soil (Liu et al., 2010a). Such variations are expected. The 6:2 FTOH biotransformation intermediates are bioavailable and readily converted to corresponding stable transformation products in the environment. The volatile intermediate 5:2 sFTOH is the most abundant transformation product observed in this study and averaged 40 mol% on days 56–60 of initially applied 6:2 FTOH in activated sludge from the two WWTPs (Figs. 1 and 3). About 30 mol% 5:2 sFTOH was observed in the headspace of the sample bottles on days 56–60, suggesting that the majority was not available for further biotransformation to PFPeA and PFHxA by microbes in activated sludge. Rather, a substantial portion of this volatile intermediate would emit to ambient air and be subject to further abiotic atmospheric degradation. This is in sharp contrast to 6:2 FTOH biotransformation in soil, where less than 16 mol% 5:2
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Table 1 Comparison of the major stable transformation products and the ratioa of PFPeA plus PFHxA versus 5:3 acid in different environmental matrices during 6:2 FTOH biotransformation. Environ. matrix
PFBA (%)
PFPeA (%)
PFHxA (%)
5–3 Acid (%)
R(PFPeA + PFHxA)a/5–3
Soilb Bacterial cultureb Sedimentc Activated Sludged
1.8 <0.5 1.5 <0.5
30 <0.5 10 4.4
8.1 5.1 8.4 11
15 5.5 22 14
2.5 0.9 0.8 1.1
Acid
a b c d
Calculated by using the molar yield of PFPeA plus PFHxA to divide the molar yield of 5:3 acid. Liu et al. (2010a). Zhao et al. (2012). Average of activated sludge samples from two WWTPs in Delaware and Pennsylvania of this study.
sFTOH was observed on day 60, most of which was detected in the soil phase and available for biotransformation to PFPeA and PFHxA (Liu et al., 2010a). Thus, 5:2 sFTOH bioavailability and the ability of microbial enzymes to decarboxylate it may be the rate-limiting factors in determining PFCA molar yields in activated sludge containing 6:2 FTOH. 5:2 Ketone averaged 9.4 mol% on days 56–60 (Figs. 1 and 3) of the initially applied 6:2 FTOH in activated sludge from the two WWTPs. 5:2 ketone is the direct 5:2 sFTOH precursor and can be rapidly converted to 5:2 sFTOH in soil via one enzymatic reduction step (Liu et al., 2010a). The much lower level of 5:2 ketone on days 56–60 compared with that of 5:2 sFTOH implies that 5:2 ketone was also rapidly converted to 5:2 sFTOH as occurred in soil. 5:2 Ketone was formed by decarboxylation of 6:2 FTUCA via multiple enzymatic steps yet to be elucidated (Liu et al., 2010a). 3.3. 5:3 Acid and PFHxA are the major nonvolatile transformation products during 6:2 FTOH biotransformation 5:3 Acid is the most abundant nonvolatile biotransformation product and averaged 14.1 mol% in activated sludge from the two WWTPs on days 56–60 (Figs. 1 and 3). 5:3 Acid was formed by reducing 5:3 Uacid via one enzymatic step (Liu et al., 2010a). The 5:3 acid molar yield in activated sludge was very similar for the two WWTPs from Delaware and Pennsylvania, accounting for 13 mol% and 15 mol% of initially applied 6:2 FTOH on days 56– 60, respectively (Figs. 1 and 3). Moreover, 5:3 acid molar yield in activated sludge was similar to that reported in soil (Liu et al., 2010a) and slightly lower than that in sediment (Zhao et al., 2012), reinforcing the notion that 5:3 acid is a unique 6:2 FTOH biotransformation product. 5:3 Acid is relatively stable compared with other intermediates and it can be slowly degraded to 4:3 acid in activated sludge via one carbon removal pathways (Wang et al., 2012). 4:3 acid accounted for 1.3 mol% on days 56–60 of initially applied 6:2 FTOH (Figs. 1 and 3). This represents a 9.2% conversion (1.3 mol%/14.1 mol%) from 5:3 acid to 4:3 acid in activated sludge. The PFPeA molar yield averaged 4.4 mol% in the activated sludge studied (Figs. 1 and 3), which is 2.4–7 time lower than that reported in sediment and soil (Liu et al., 2010a; Zhao et al., 2012). The lower conversion of 6:2 FTOH to PFPeA in activated sludge may be partly due to the low bioavailability of its direct precursor, 5:2 sFTOH, most of which partitioned to the headspace. Bacterial populations are dominant in WWTP activated sludge and are mostly responsible for degradation of environment pollutants (Bitton, 2011). However, a pure bacterial strain had very limited ability to degrade 5:2 sFTOH to PFPeA (Kim et al., 2012). On the other hand, surface soils contain active fungal populations (Tate III, 2000) and can rapidly degrade 5:2 sFTOH to PFPeA with a 30% molar yield (Liu et al., 2010a). Taken together, these experiments suggest that fungal enzymes are more efficient at decarboxylating 5:2 sFTOH to PFPeA. No PFBA or PFHpA was observed in this study. These results suggest that biotransformation of 6:2 FTOH-based
products in activated sludge of WWTPs is not expected to be a major source of PFBA, PFPeA, and PFHpA. PFHxA is the major PFCA formed during 6:2 FTOH biotransformation in activated sludge and averaged 11 mol% of initially applied 6:2 FTOH, about 30% higher than that reported in soil (Liu et al., 2010a) and sediment (Zhao et al., 2012). The PFHxA molar yield also varied little in activated sludge of different WWTPs from Delaware and Pennsylvania, averaged 11 mol% of initially applied 6:2 FTOH on days 56–60 in both sites. The relative consistent molar yields of 5:3 acid and PFHxA in activated sludge from different WWTPs may help predict mass flow of these two important transformation products from WWTPs to other environments if 6:2 FTOH-based products were discharged to WWTPs. This consistency also suggests that microbes in activated sludge from different WWTPs deployed very similar enzymatic pathways to convert 6:2 FTOH to 5:3 acid and to PFHxA, respectively. 3.4. Using the R(PFPeA+PFHxA)/5:3 acid ratio to discern the potential origin of PFPeA and PFHxA 5:3 Acid is a unique 6:2 FTOH biotransformation product and was formed along with shorter-chain PFCAs such as PFPeA and PFHxA in soil (Liu et al., 2010a), sediment (Zhao et al., 2012), and activated sludge of this study. 5:3 Acid can only be formed via 6:2 FTOH biotransformation by microbes in the environment. No 5:3 acid was detected as an impurity in other polyfluorinated chemicals and no abiotic pathways are known to convert FTOHs or other poly-fluorinated chemicals to 5:3 acid. The level of 5:3 acid is in the same order of magnitude as that of the sum of PFPeA plus PFHxA in laboratory studies conducted in various environmental matrices (Table 1). As Table 1 illustrates, the ratio of the molar yield of PFPeA plus PFHxA divided by the molar yield of 5:3 acid is close to 1.0 in bacterial culture, sediment, and activated sludge, whereas the ratio is 2.5 in soil. Thus 5:3 acid may serve as an ‘‘internal standard’’ to estimate the relative contribution of 6:2 FTOH versus direct PFCA emissions to PFPeA and PFHxA present in an environmental matrix. For example, if the sum of PFPeA plus PFHxA were found to be an order of magnitude higher than that of 5:3 acid, then direct PFCA emissions would be suggested as the primary source for detected PFPeA and PFHxA. On the other hand, if the sum of PFPeA plus PFHxA is similar to that of 5:3 acid, then 6:2 FTOH microbial biotransformation would be suggested as the primary source of detected PFPeA and PFHxA. 4. Conclusions 6:2 FTOH biotransformation in activated sludge from two domestic WWTPs led to 5:2 sFTOH as the most abundant biotransformation product on days 56–60, predominantly in the sample bottle headspace and not available for further biodegradation. 6:2 FTOH biotransformation in activated sludge also generated 5:3 acid and PFHxA as the major biotransformation products on
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days 56–60. PFBA and PFHpA were not observed and PFPeA was formed in small quantities. These results imply that WWTPs could be potential sources of 5:2 sFTOH to the ambient air and of 5:3 acid and PFHxA to sludge effluent and sludge solids, but not major sources of PFBA, PFPeA, and PFHpA if 6:2 FTOH-based products were discharged to WWTPs. The markedly lower PFPeA yield compared with that in soil during 6:2 FTOH biotransformation in activated sludge may be due to the limited bioavailability of 5:2 sFTOH in the aqueous phase to be further degraded and lower efficiency of bacterial enzymes in activated sludge to decarboxylate 5:2 sFTOH to PFPeA compared with that of soil fungal enzymes. Together with the outcomes that 6:2 FTOH biotransformation produced PFPeA as the dominant product in soil and 5:3 acid as the major product in sediment, our work infer that 6:2 FTOH biotransformation rates and product profiles are different in major environmental matrices studied so far. Such variations between the activated sludge, soil, and sediment may be attributed to differences in microbial populations, bioavailability, and enzymatic catalytic capacities towards the major intermediates for further conversion to major stable products such as PFCAs and 5:3 acid. These differences may provide opportunities for optimizing microbial growth conditions, isolating microbial populations, and identifying microbial enzymes with enhanced biodegradability toward polyfluorinated chemicals. Acknowledgements We would like to thank Dr. Alexander Shtarov for synthesis of 5:3 Uacid and other intermediates and analytical standards used in this research. We thank the Nature Science Foundation (No. 41225014) and Ministry of Science and Technology (No. 2009DFA92390) of China to provide funding to support Lijie Zhao. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.chemosphere. 2013.02.032. References Ahrens, L., Shoeib, M., Harner, T., Lee, S.C., Guo, R., Reiner, E.J., 2011. Wastewater treatment plant and landfills as sources of polyfluoroalkyl compounds to the atmosphere. Environ. Sci. Technol. 45, 8098–8105. Benskin, J.P., Phillips, V., St. Louis, V.L., Martin, J.W., 2011. Source elucidation of perfluorinated carboxylic acids in remote alpine lake sediment cores. Environ. Sci. Technol. 45, 7188–7194. Bitton, 2011. Wastewater Microbiology, fourth ed. Wiley-Blackwell, Hoboken, NJ. Buck, R.C., Franklin, J., Berger, U., Conder, J.M., Cousins, I.T., Voogt, P., Jensen, A.A., Kannan, K., Mabury, S.A., Leeuwen, S.P.J., 2011. Perfluoroalkyl and polyfluoroalkyl substances in the environment: terminology, classification, and origins. Integr. Environ. Assess. Manage. 7, 513–541. Cousins, I.T., Kong, D., Vestergren, R., 2011. Reconciling measurement and modelling studies of the sources and fate of perfluorinated carboxylates. Environ. Chem. 8, 339–354. Ellis, D.A., Martin, J.W., De Silva, A.O., Mabury, S.A., Hurley, M.D., Sulbaek Andersen, M.P., Wallington, T.J., 2004. Degradation of fluorotelomer alcohols: a likely atmospheric source of perfluorinated carboxylic acids. Environ. Sci. Technol. 38, 3316–3321.
Houde, M., De Silva, A.O., Muir, D.C.G., Letcher, R.J., 2011. Monitoring of perfluorinated compounds in aquatic biota: an updated review on PFCs in aquatic biota. Environ. Sci. Technol. 45, 7962–7973. Kim, M.H., Wang, N., McDonald, T., Chu, K.H., 2012. Biodefluorination and biotransformation of fluorotelomer alcohols by two alkane degrading Pseudomonas strains. Biotechnol. Bioeng. 109, 3041–3048. Kunacheva, C., Tanaka, S., Fujii, S., Boontanon, S.K., Musirat, C., Wongwattana, T., Shivakoti, B.R., 2011. Mass flows of perfluorinated compounds (PFCs) in central wastewater treatment plants of industrial zones in Thailand. Chemosphere 83, 737–744. Lau, C., 2012. Perfluoroalkyl acids: recent research highlights. Reprod. Toxicol. 33, 405–409. Li, F., Sun, H., Hao, Z., He, N., Zhao, L., Zhang, T., Sun, T., 2011. Perfluorinated compounds in Haihe River and Dagu drainage canal in Tianjin, China. Chemosphere 84, 265–271. Liu, J., Lee, L.S., Nies, L.F., Nakatsu, C.H., Turco, R.F., 2007. Biotransformation of 8:2 fluorotelomer alcohol in soil and by soil bacterial isolates. Environ. Sci. Technol. 41, 8024–8030. Liu, J., Wang, N., Szostek, B., Buck, R.C., Panciroli, P.K., Folsom, P.W., Sulecki, L.M., Bellin, C.A., 2010a. 6–2 Fluorotelomer alcohol aerobic biodegradation in soil and mixed bacterial culture. Chemosphere 78, 437–444. Liu, J., Wang, N., Buck, R.C., Wolstenholme, B.W., Folsom, P.W., Sulecki, L.M., Bellin, C.A., 2010b. Aerobic biodegradation of [14C] 6:2 fluorotelomer alcohol in a flowthrough soil incubation system. Chemosphere 80, 716–723. Martin, J.W., Ellis, D.A., Mabury, S.A., Hurley, M.D., Wallington, T.J., 2006. Atmospheric chemistry of perfluoroalkanesulfonamides: kinetic and product studies of the OH radical and Cl atom initiated oxidation of N-Ethyl perfluorobutanesulfonamide. Environ. Sci. Technol. 40, 864–872. Naile, J.E., Khim, J.S., Wang, T., Chen, C., Luo, W., Kwond, B.O., Park, J., Koh, C.H., Jones, P.D., Lu, Y., Giesy, J.P., 2010. Perfluorinated compounds in water, sediment, soil and biota from estuarine and coastal areas of Korea. Environ. Pollut. 158, 1237–1244. Nealson, K.H., 1997. Sediment bacteria: who’s there, what are they doing, and what is new. Annu. Rev. Earth Planet. Sci. 25, 403–434. OECD, 1992. Organization for Economic Cooperation and Development, Guideline for the Testing of Chemicals 301: Ready Biodegradability, July 17. Prevedouros, K., Cousins, I.T., Buck, R.C., Korzeniowski, S.H., 2006. Sources, fate and transport of perfluorocarboxylates. Environ. Sci. Technol. 40, 32–44. Schultz, M.M., Higgins, C.P., Huset, C.A., Luthy, R.G., Barofsky, D.F., Field, J.A., 2006. Fluorochemical mass flows in a municipal wastewater treatment facility. Environ. Sci. Technol. 40, 7350–7357. Sepulvado, J.G., Blaine, A.C., Hundal, L.S., Higgins, C.P., 2011. Occurrence and fate of perfluorochemicals in soil following the land application of municipal biosolids. Environ. Sci. Technol. 45, 8106–8112. Sun, H., Li, F., Zhang, T., Zhang, X., He, N., Song, Q., Zhao, L., Sun, L., Sun, T., 2011. Perfluorinated compounds in surface waters and WWTPs in Shenyang, China: mass flows and source analysis. Water Res. 45, 4483–4490. Tate III, 2000. Soil Microbiology, second ed. John Wiley & Sons, Inc., New York, NY. U.S. Environmental Protection Agency (EPA), 2006. 2010/2015 PFOA Stewardship Program. EPA-HQ-2003-0012-1071.