Chemosphere xxx (2010) xxx–xxx
Contents lists available at ScienceDirect
Chemosphere
journal homepage: www.elsevier.com/locate/chemosphere
6:2 Fluorotelomer sulfonate aerobic biotransformation in activated sludge
of waste water treatment plants
Ning Wang ⇑, Jinxia Liu 1, Robert C. Buck, Stephen H Korzeniowski, Barry W. Wolstenholme,
Patrick W. Folsom, Lisa M. Sulecki
E.I. du Pont De Nemours, Co., Inc., Wilmington, DE, USA
a r t i c l e
i n f o
Article history:
Received 19 July 2010
Received in revised form 29 October 2010
Accepted 1 November 2010
Available online xxxx
Keywords:
6:2 Fluorotelomer sulfonate (6:2 FTS)
Aerobic biotransformation
Perfluorocarboxylic acids
Perfluoropentanoic acid (PFPeA)
Perfluorohexanoic acid (PFHxA)
Activated sludge
a b s t r a c t
+
The aerobic biotransformation of 6:2 FTS salt [F(CF2)6CH2CH2SO
3 K ] was determined in closed bottles for
90 d in diluted activated sludge from three waste water treatment plants (WWTPs) to compare its biotransformation potential with that of 6:2 FTOH [F(CF2)6CH2CH2OH]. The 6:2 FTS biotransformation was
relatively slow, with 63.7% remaining at day 90 and all observed transformation products together
accounting for 6.3% of the initial 6:2 FTS applied. The overall mass balance (6:2 FTS plus observed transformation products) at day 90 in live and sterile treatments averaged 70% and 94%, respectively. At day
90, the stable transformation products observed were 5:3 acid [F(CF2)5CH2CH2COOH, 0.12%], PFBA
[F(CF2)3COOH, 0.14%], PFPeA [F(CF2)4COOH, 1.5%], and PFHxA [F(CF2)5COOH 1.1%]. In addition, 5:2 ketone
[F(CF2)5C(O)CH3] and 5:2 sFTOH [F(CF2)5CH(OH)CH3] together accounted for 3.4% at day 90. The yield of
all the stable transformation products noted above (2.9%) was 19 times lower than that of 6:2 FTOH in
aerobic soil. Thus 6:2 FTS is not likely to be a major source of PFCAs and polyfluorinated acids in WWTPs.
6:2 FTOH, 6:2 FTA [F(CF2)6CH2COOH], and PFHpA [F(CF2)6COOH] were not observed during the 90-d incubation. 6:2 FTS primary biotransformation bypassed 6:2 FTOH to form 6:2 FTUA [F(CF2)5CF = CHCOOH],
which was subsequently degraded via pathways similar to 6:2 FTOH biotransformation. A substantial
fraction of initially dosed 6:2 FTS (24%) may be irreversibly bound to diluted activated sludge catalyzed
by microbial enzymes. The relatively slow 6:2 FTS degradation in activated sludge may be due to microbial aerobic de-sulfonation of 6:2 FTS, required for 6:2 FTS further biotransformation, being a rate-limiting step in microorganisms of activated sludge in WWTPs.
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
Perfluoroalkyl-carboxylic (PFCA) and sulfonic (PFSA) acids such
as perfluorooctanoic acid (PFOA), perfluorohexane sulfonate
(PFHxS) and perfluorooctane sulfonate (PFOS) have been widely
detected in the environment and biota (Dreyer and Ebinghaus,
2009; Jin et al., 2009; Loos et al., 2009; Quinete et al., 2009; Ahrens
et al., 2010; Schuetze et al., 2010). PFOA, PFHxS, PFOS and their
longer carbon chain length homologues are persistent in the
environment and in biota. The major historic global manufacturer
of perfluoroalkyl sulfonyl chemistry terminated manufacture of
PFHxS and higher homologues in 2002 (3M Company, 2000) and
a group of companies have recently committed to work to
⇑ Corresponding author. Address: DuPont Haskell Global Centers for Health and
Environmental Sciences, Glasgow 300, P.O. Box 6300, Newark, DE 19714-6300, USA.
Tel.: +1 302 366 6665; fax: +1 302 366 6602.
E-mail address: [email protected] (N. Wang).
1
Present address: Chesapeake Biological Laboratory, University of Maryland,
Center for Environmental Science, Solomons, MD 20688, USA.
essentially eliminate the manufacture and use of perfluorooctanoic
acid (PFOA), higher homologues and their potential precursors by
2015 (US Environmental Protection Agency (EPA), 2006). Alternative short chain products based on perfluorobutane sulfonyl (PFBS)
and six-carbon fluorotelomer raw materials are commercially
available (Ritter, 2010).
Fluorinated organics have many unique and useful properties
and have been broadly used (Kissa, 2001). In addition to PFCAs
and PFSAs, researchers have identified other fluorinated substances present in the environment. For example, fluorinated
surfactants have been used for decades as critical ingredients in
fire-fighting foam (aqueous film-forming foam, AFFF) products because of their unparalleled surface tension lowering, wetting and
spreading properties (Kissa, 2001; Schultz et al., 2003). Historically,
perfluoroalkyl sulfonates (PFSAs) such as PFOS and PFSA-based
surfactant derivatives [e.g., F(CF2)nSO2N(R)R0 where R = H, CH3,
C2H5, R0 = additional functional group] were the most widely used
surfactants in AFFF (Cortina and Korzeniowski, 2008a). Alternatively, fluorinated surfactants based on fluorotelomer thiol [(Falk,
1984), e.g., F(CF2)nCH2CH2SCH2CH(OH)CH2N+H(CH3)CH2CO
2 ] and
0045-6535/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.chemosphere.2010.11.003
Please cite this article in press as: Wang, N., et al. 6:2 Fluorotelomer sulfonate aerobic biotransformation in activated sludge of waste water treatment
plants. Chemosphere (2010), doi:10.1016/j.chemosphere.2010.11.003
2
N. Wang et al. / Chemosphere xxx (2010) xxx–xxx
sulfonyl [e.g., F(CF2)nCH2CH2SO2NHCH2CH2N+(CH3)2CH2CH2CO
2]
chemistry have also been used in AFFF. The perfluoroalkyl sulfonyl
surfactants degrade to PFOS and PFHxS (Moody et al., 2003; Rhoads
et al., 2008). The fluorotelomer thiol and sulfonyl surfactants degrade to fluorotelomer sulfonates [e.g., F(CF2)nCH2CH2SO
3 , n = 4,
6, 8] (Schultz et al., 2004). As a result of these fluorinated surfactants being used in AFFF at fire training facilities and to put out major fires, their degradation products have been found in ground
water, soil and biota (Moody et al., 2003; Schultz et al., 2004;
Oakes et al., 2010).
Fluorinated surfactants based on six-carbon fluorotelomer raw
materials have largely replaced PFOS and PFSA-based surfactants
in AFFF products (Cortina and Korzeniowski, 2008a). There are significant physical, chemical and biological differences between the
degradation products PFOS, PFHxS and 6:2 fluorotelomer sulfonate
[6:2 FTS, F(CF2)6CH2CH2SO
3 ] (Cortina and Korzeniowski, 2008b).
From an environmental fate perspective, PFOS and PFHxS are
chemically and biologically inert. However, 6:2 FTS is analogous
to fluorotelomer alcohol, F(CF2)nCH2CH2OH. 8:2 FTOH [F(CF2)8CH2
CH2OH] degrades under aerobic conditions in numerous environmental matrices with multiple –CF2– groups being removed (Wang
et al., 2005a,b; Liu et al., 2007; Wang et al., 2009). Likewise, 6:2
FTOH degrades under aerobic conditions in soil and bacterial
culture to form even higher yields of transformation products
including perfluorobutanoic acid with more –CF2– groups being
removed (Liu et al., 2010a,b).
The biodegradability of fluorinated compounds has recently
been reviewed (Parsons et al., 2008). Of keen interest is the desire
to identify microbes that will degrade fluorinated materials (Iwai
et al., 2009; Murphy, 2010). Current literature indicates that microbial biotransformation of fluorinated chemicals varied among
different species and populations. For example, microbial biotransformation of fluorotelomer-based ethoxylate (Frömel and Knepper,
2010) and phosphate surfactants (Lee et al., 2010) has been reported. In a study where a mixture of fluorinated substances was
incubated in municipal sewage sludge, no biotransformation was
observed (Saez et al., 2008). Currently, little published information
is available regarding 6:2 FTS biotransformation potential and its
environmental behavior. Based on 6:2 FTS molecular structure
and previous literature regarding biotransformation of non-fluorinated straight-chain alkyl sulfonates (Swisher, 1987), 6:2 FTS must
be de-sulfonated first for further biotransformation to occur. The
de-sulfonation and de-fluorination of 6:2 FTS were observed when
it was incubated in laboratory bacterial isolate, Pseudomonas sp.
Strain 2 (Key et al., 1998). Six volatile fluorinated products were
detected by GC/AED. While none contained sulfur, they were not
structurally identified or quantified.
The objective of this study was to determine 6:2 FTS aerobic
biotransformation rate, identify major transformation products,
and establish 6:2 FTS biotransformation pathways. 6:2 FTS is structurally similar to 6:2 FTOH. This study was designed to evaluate
whether 6:2 FTS would biodegrade in the environment at similar
rates and with similar biotransformation pathways as those of
6:2 FTOH and to determine the extent to which 6:2 FTS degradation may lead to PFCAs. Moreover, the study sought to assess
whether 6:2 FTS would be strongly absorbed to environmental
matrices and thereby reduce its potential mobility and bioavailability for further biotransformation as has been observed for 8:2
FTOH and 6:2 FTOH (Wang et al., 2009; Liu et al., 2010a). 6:2 FTOH
is strongly absorbed to soil (Liu et al., 2007) and 23% of initially applied 14C-labeled 6:2 FTOH in live soil can be irreversibly bound to
soil (Liu et al., 2010a). 6:2 FTS, due to the sulfonate group, may
have an increased absorption tendency (Higgins and Luthy, 2006)
and be even more sorptive than 6:2 FTOH in soil. Fluorinated
chemicals can be strongly absorbed to soil organic matters such
as humic acid constituents (Longstaffe et al., 2010). Such potential
strong absorption can hinder transformation product quantification and identification due to reduced recovery of precursor and
potential transformation products to achieve good mass balance
in a biotransformation study, unless a radioisotope labeled test
chemical is available. To alleviate this potential problem, we used
diluted activated sludge to investigate 6:2 FTS biotransformation.
The diluted sludge reduced organic matters that can potentially
bind to 6:2 FTS and potential transformation products. Furthermore, WWTPs are potential sites for fluorinated chemicals and
their transformation products to enter into the environment. Diluted activated sludge from WWTPs was used to study biotransformation of 8:2 FTOH (Wang et al., 2005b), N-ethyl perfluorooctane
sulfonamidoethanol (Rhoads et al., 2008), and polyfluorinated
phosphates (Lee et al., 2010). Thus, 6:2 FTS biotransformation potential in activated sludge can be compared with other fluorinated
chemicals to gain perspective on its potential contribution to
poly- and per-fluorinated chemicals that may be detected in the
environment.
2. Materials and methods
2.1. Chemicals
6:2 Fluorotelomer sulfonate, potassium salt, 6:2 FTS,
+
F(CF2)6CH2CH2SO
3 K , was synthesized by DuPont with +99% purity by NMR analysis. No potential transformation products were
observed for the 6:2 FTS testing material. Additional fluorinated
standards utilized for quantitative analysis were the same as described previously (Liu et al., 2010b). Stable isotope quantitation
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)413CF213COOH] (Wellington Laboratories, Ontario, Canada). All solvents were HPLC grade or higher
and all other chemicals were reagent grade or higher. De-ionized
water (18 MX cm) was from a Barnstead E-Pure system.
2.2. Activated sludge biotransformation
Activated sludge (2 L in a 4-L container) was collected the same
day the experiment was initiated from waste water treatment
plants (WWTPs) in the states of Pennsylvania, Maryland, and Delaware. The sludge was aerated at room temperature to suspend the
microorganisms and used as the inoculums or was autoclaved as
sterile controls. 129-mL glass serum sample bottles were used as
test vessels. In each test vessel, 3 mL of activated sludge suspension was mixed with 27 mL of mineral media. The 10-fold dilution
of the activated sludge was aimed to minimize potential absorption of 6:2 FTS and potential transformation products by activated
sludge. The mineral media consisted of 85 mg L1 of KH2PO4,
218 mg L1 of K2HPO4, 334 mg L1 of Na2HPO42H2O, 5 mg L1 of
NH4Cl, 36.4 mg L1 of CaCl22H2O, 22.5 mg L1 of MgSO47H2O,
and 0.25 mg L1 of FeCl36H2O with a pH of 7.0. The 6:2 FTS starting concentrations in live and sterile control ranged from 1.8 to
2.6 mg L1 sludge/mineral medium mixture dosed as 0.01 mL ethanol/H2O (50:50, v/v) stock solution. The sterile control was also
supplemented with a triple antibiotics solution to further control
microbial activity as described previously (Liu et al., 2010b). After
each test vessel was filled with appropriate test media, the bottle
was crimp-sealed with an butyl rubber septum/aluminum cap.
All the glass serum bottles were shaken horizontally at 250 rpm
in an environmental incubator in the dark at room temperature
prior to being sacrificed for sample processing and analysis. Each
sampling time (days 0, 7, 14, 28, 56, and 90) included three replicates of live sludge, three replicates of sterile sludge and two replicates of live sludge matrix (only 0.01 mL of ethanol/H2O (50:50,
Please cite this article in press as: Wang, N., et al. 6:2 Fluorotelomer sulfonate aerobic biotransformation in activated sludge of waste water treatment
plants. Chemosphere (2010), doi:10.1016/j.chemosphere.2010.11.003
3
N. Wang et al. / Chemosphere xxx (2010) xxx–xxx
v/v) was added to the sludge/mineral medium mixture). The day 0
samples were immediately extracted with a solvent as soon as all
the live and sterile sample bottles were dosed with 6:2 FTS, typically within 10 min. To assess the activated sludge viability,
approximately 2.0 mg 6:2 or 8:2 FTOH L1 sludge/mineral solution
were added to separate sample bottles to determine the de-fluorination potential, an indicator of sludge ability to degrade fluorotelomer-based chemicals (Wang et al., 2005a; Liu et al., 2007).
leased comparable to previous studies (Wang et al., 2005a; Liu et
al., 2007), indicating that the microorganisms in the sludge were
able to degrade 8:2 FTOH or 6:2 FTOH. The oxygen content in
the live bottle headspace was approximately 18% at day one, and
12% thereafter from days 7 to 90, higher than that in the bacterial
culture systems that are able to degrade 8:2 and 6:2 FTOH (Wang
et al., 2005a; Liu et al., 2010b).
3.2. Observed transformation products and mass balance
2.3. Sampling and sample preparation procedures
The sludge extracts and the C18 cartridge eluents were combined and analyzed by LC/MS/MS with methods as previously described (Liu et al., 2010b). The LC/MS/MS system, consisted of a
Micromass Quattro Micro and either an Agilent 1100 or a Waters
2795 HPLC, was operated in negative electrospray ionization mode
with multiple reaction monitoring (see Table S1 in Supplementary
material for ion transitions monitored). The column for HPLC separation was an Agilent Zorbax RX-C8 (150 mm 2.1 mm, 5 lm
particle size, pore size 80 Å, not end-capped, and with 5.5% carbon
loading). The mobile phases consisted of 0.15% acetic acid in nanopure water and 0.15% acetic acid in acetonitrile in a gradient manner with a flow rate of 0.4 mL/min. The sample injection volume
was 10–20 lL. Before LC/MS/MS analysis, all samples were acidified with concentrated HCl to a final concentration of 0.6% HCl
(w/v%) and then spiked at 50 lL mL1 sample of an internal standard solution containing 100 or 200 ng mL1 of [1,2-13C] PFHxA
and 5000 ng mL1 of [1,1,2,2-D; 3-13C] 6:2 FTOH. When an analyte
concentration exceeded the upper limit of calibration range, dilutions were made such that the final solution composition was as
close to 1:1 of acetonitrile:water as possible. Detailed information
on the instrumental methods and detection limits is provided in
the Supplementary material.
3. Results and discussion
3.1. Viability of the activated sludge
The activated sludge collected from three sites de-fluorinated
8:2 or 6:2 FTOH (Fig. S1, Supplementary material) with fluoride re-
120
6:2 FTS - Live
5:2 ketone
PFHxA
Sum - Live
5:3 acid
PFPeA
6:2 FTS - Sterile
6:2 FTUA
5:2 sFTOH
% of 6:2 FTS applied at day 0
2.4. LC/MS/MS analysis
The average total molar yield (6.3%) of all observed transformation products at day 90 from 6:2 FTS biotransformation (Figs. 1–3)
from the three WWTPs is much lower than that from 6:2 FTOH biotransformation in bacterial culture and soil (Liu et al., 2010b),
where more than 55% of 6:2 FTOH was converted to other transformation products. No transformation products were observed in
activated sludge collected from Delaware (Fig. 3), even though
the sludge was able to de-fluorinate 8:2 FTOH (Fig. S1, Supplementary material). This reflects variations in microbial populations
from the WWTPs studied and their ability to degrade 6:2 FTS. At
day 90, 5:2 sFTOH and 5:2 ketone together averaged 3.4% molar
yields. Stable transformation products PFPeA and PFHxA accounted
for 1.5% and 1.1%, respectively, and PFBA and 5:3 acid each
A
Pennsylvania
100
80
60
40
20
0
0
% of 6:2 FTS applied at day 0
At sampling time, the headspace of the live, sterile, and matrix
bottles was purged with approximately 1.5 L of air through a C18
cartridge (0.6 g sorbent, Alltech, Deerfield, IL) to capture potential
volatile transformation products. Each C18 cartridge was eluted
with 5 mL acetonitrile (CH3CN) and the eluant stored at below
10 °C before LC/MS/MS analysis. Oxygen content in the headspace of matrix bottles was measured with an oxygen meter before
the purging. After the headspace purging, the septum was pushed
into the bottle and 30 mL of CH3CN was added. The bottle was then
sealed with a fresh septum to extract the remaining 6:2 FTS and
transformation products in the sludge for 2–7 d at 50 °C. The
sludge extracts then were centrifuged at approximately 9000 g
for 20 min to collect the supernatant, which was filtered through
0.45 lm-pore nylon filters and stored below 10 °C before LC/
MS/MS analysis.
To measure fluoride in the sample bottles dosed with 6:2 or 8:2
FTOH, 3 ml of the activated sludge test solution was withdrawn
from each of the sample bottles at each time point and was mixed
with 3 mL of TISAB II solution (VWR, West Chester, PA). The mixture was centrifuged and the supernatant was collected for fluoride
analysis with a fluoride-selective electrode as described previously
(Wang et al., 2005b).
4
20
40
60
80
Pennsylvania
B
3
2
1
0
0
20
40
60
80
Time (d)
Fig. 1. Individual study transformation products versus time in activated sludge
(n = 3) collected from Pennsylvania (A). Some error bars are not visible if they are
smaller than the symbol height. Transformation products were only observed in
live sludge. The average initial 6:2 FTS concentration in activated sludge was
1.8 mg L1. Graph (B) is a zoom view of (A) to show the trend of individual
transformation products over time.
Please cite this article in press as: Wang, N., et al. 6:2 Fluorotelomer sulfonate aerobic biotransformation in activated sludge of waste water treatment
plants. Chemosphere (2010), doi:10.1016/j.chemosphere.2010.11.003
4
N. Wang et al. / Chemosphere xxx (2010) xxx–xxx
A 140
6:2 FTS - Live
5:2 ketone
PFHxA
Sum - Live
5:3 acid
PFPeA
6:2 FTS - Sterile
6:2 FTUA
5:2 sFTOH
% of 6:2 FTS applied at day 0
Maryland
120
100
80
60
40
20
0
0
20
40
60
B
80
% of 6:2 FTS applied at day 0
Maryland
8
6
4
2
accounted for 0.1% of the initial 6:2 FTS applied at day 0 (Table 1).
The 5:3 acid levels peaked between day 14 and day 28 with an
average yield of about 0.44% and decreased thereafter (Figs. 1–3).
The decrease may be due to strong absorption of 5:3 acid to the
sludge as was observed in soil. A small fraction (<15%) of 5:3 acid
can be further transformed to 4:3 acid and other transformation
products (Liu et al., 2010b and unpublished results). PFPeA and
PFHxA concentrations increased steadily over the 90-d incubation,
corresponding to the presence of their direct precursor, 5:2 sFTOH,
at day 90. Table 1 shows that the total stable transformation product yields for 6:2 FTS are 19 times lower than for 6:2 FTOH biotransformation in aerobic soil (Liu et al., 2010b) within the same
time frame. In other words, PFCAs, particularly PFPeA and PFHxA,
generated from potential 6:2 FTS biotransformation in WWTPs
are much lower compared with that from 6:2 FTOH. No PFHpA
[F(CF2)6COOH] was detected in the activated sludge from the
three sampling sites after 90 d. Other potential transient transformation products such as 6:2 FTOH and 6:2 fluorotelomer acid
[F(CF2)6CH2COOH, 6:2 FTA] were not detected.
The overall mass balance (6:2 FTS plus observed transformation
products) at day 90 in live and sterile treatments averaged 70% and
94%, respectively (Figs. 1–3). This indicates that the extraction
method used was sufficient to recover 6:2 FTS and transformation
products from diluted activated sludge. The higher recovery of 6:2
FTS from sterile sludge compared with live sludge was mainly due
to the lack of microbial enzymatic activity that may catalyze the
formation of non-extractable complex between 6:2 FTS and sludge
components. The autoclave sterilization procedure did not reduce
the absorption capability of the sterile sludge, which was able to
strongly absorb 8:2 FTOH (Wang et al., 2005a).
3.3. Microbe-catalyzed covalent binding of 6:2 FTS to activated sludge
0
0
20
40
60
80
Time (d)
Fig. 2. Individual study transformation products versus time in activated sludge
(n = 3) collected from Maryland (A). Some error bars are not visible if they are
smaller than the symbol height. Transformation products were only observed in
live sludge. The average initial 6:2 FTS concentration in activated sludge was
2.2 mg L1. Graph (B) is a zoom view of (A) to show the trend of individual
transformation products over time.
6:2 FTS - Live
% of 6:2 FTS applied at day 0
6:2 FTS - Sterile
Delaware
100
80
The 24% (94% 70%) unaccounted mass in live treatment in
comparison to the sterile controls may be due to complex formation between 6:2 FTS and organic components of activated sludge
catalyzed by enzymes, as occurred between 5:3 acid and soil components (Liu et al., 2010a). The exact enzymatic mechanisms are
not understood. Base (e.g., NaOH) treatment plus Envicarb™ activated carbon clean-up at high temperature (50 °C) with soil can
help only partially recover the soil-bound 14C when dosed with
14
C-labeled 6:2 FTOH (Liu et al. 2010a). This suggests relatively
strong covalent bond(s) between the fluorinated chemicals and organic components. Otherwise, a weak hydrogen or ester bond can
be easily broken-up by NaOH treatment, resulting in comparable
recovery of the fluorinated chemicals from live soil as with sterile
samples. This complex via covalent binding was not extractable by
organic solvent such as acetonitrile at elevated temperature (50 °C)
and is likely to have limited availability for further biotransformation in the environment. In comparison with 6:2 FTOH, the sulfonate group (SO
3 ) may increase absorption potential (Higgins and
Luthy, 2006) of 6:2 FTS to organic matters of environmental matrices in their native status (e.g., undiluted sludge).
3.4. The rate-limiting step in 6:2 FTS biotransformation
0
0
20
40
60
80
Time (d)
Fig. 3. Individual study transformation products versus time in activated sludge
(n = 2) collected from Delaware. No transformation products were observed in live
sludge over 90-d incubation. The average initial 6:2 FTS concentration in activated
sludge was 2.6 mg L1.
Microbial aerobic de-sulfonation is most likely the rate-limiting
step in 6:2 FTS biotransformation. For biotransformation to occur,
the sulfonate group needs to be removed from 6:2 FTS prior to
further degradation to form other transformation products. For
non-fluorinated alkane sulfonates with carbon chain length ranged
8–12, the de-sulfonation reaction is facile, catalyzed by alkane
sulfonate-a-hydroxylase, leading to their eventual mineralization
to CO2 (Swisher, 1987). It is not known why 6:2 FTS is relatively
resistant to microbial de-sulfonation. 6:2 FTS is larger and more rigid
(due to the polyfluorinated carbon chain) than its non-fluorinated
Please cite this article in press as: Wang, N., et al. 6:2 Fluorotelomer sulfonate aerobic biotransformation in activated sludge of waste water treatment
plants. Chemosphere (2010), doi:10.1016/j.chemosphere.2010.11.003
N. Wang et al. / Chemosphere xxx (2010) xxx–xxx
Table 1
Comparison of 6:2 FTS stable transformation product yields in activated sludge and of
6:2 FTOH in Sassafras soil (Liu et al., 2010b) at day 90.
6:2 FTOH in soil
Loss of 2C (two –CH2– groups)
Loss of 3C (one –CF2– group, two
–CH2– groups)
Loss of 4C (two –CF2– groups,
two –CH2– groups)
5:3 acid
Sum of the stable transformation
products
(Liu et al., 2010b)
Transformation
product yield
6:2 FTS in activated
sludge
(This study)
Transformation
product yield
8.1% (PFHxA)
30% (PFPeA)
1.1% (PFHxA)
1.5% (PFPeA)
1.8% (PFBA)
0.14% (PFBA)
15%
55%
0.12%
2.9%
alkane sulfonate counterparts. Such possible steric hindrance may
make it more difficult for 6:2 FTS to reach the active site of an alkane sulfonate-a-hydroxylase and result in the observed low 6:2
FTS primary transformation rate. By comparison, potential aerobic
microbial de-sulfonation of perfluorinated alkane sulfonyl substances [e.g., F(CF2)nSO2N(R)R0 where R = H, CH3, C2H5, R0 = additional functional group] is even more difficult. No PFOA was
detected from aerobic biotransformation of N-ethyl perfluorooctane sulfonamidoethanol [C8F17SO2N(Et)CH2CH2OH] and PFOS
was the major stable transformation product observed (Rhoads
et al., 2008).
3.5. 6:2 FTS biotransformation pathways
Fig. 4 presents the proposed 6:2 FTS biotransformation pathways based on observed transformation products in this study
and prior knowledge of 6:2 FTOH biotransformation pathways
(Liu et al., 2010b). 6:2 FTS biotransformation generally follows
some of the major pathways for 6:2 FTOH with several distinctions.
As described earlier, the initial 6:2 FTS de-sulfonation may be catalyzed by alkane sulfonate-a-hydroxylase to form an unstable
intermediate, 1-hydroxy 6:2 FTS [F(CF2)6CH2CH(OH)SO
3 ], which
is then rapidly converted to 6:2 fluorotelomer aldehyde
[F(CF2)6CH2CHO, 6:2 FTAL] while releasing sulfonic acid (HSO
3 ).
This indicates that primary 6:2 FTS biotransformation bypasses
6:2 FTOH, consistent with the result that no 6:2 FTOH was observed in activated sludge dosed with 6:2 FTS. 6:2 FTAL is a tran-
Non-extractable
bound residue
(6:2 FTS)
F(CF2)6CH2CH2SO3H
Desulfonation
Oxidation reactions
Minor pathways
F(CF2)5CF=CHCO2H
(6:2 FTUA)
F(CF2)5C(O)CH3
(5:2 ketone)
F(CF2)5CH(OH)CH3
(5:2 sFTOH)
F(CF2)5CH2CH2CO2H
(5:3 acid)
F(CF2)4CO2H
(PFPeA)
F(CF2)5CO2H
(PFHxA)
Fig. 4. Proposed 6:2 FTS aerobic biotransformation pathways. The double arrows
indicate multiple transformation steps. The solid arrows indicate proposed transformation steps based on observed transformation products in this study.
5
sient transformation product that undergoes rapid oxidation to
6:2 FTA [F(CF2)6CH2CO2H]. 6:2 FTAL was not observed in soil and
bacterial culture dosed with 6:2 FTOH (Liu et al., 2010b). Similarly,
6:2 FTA was not observed in this study as well as in an earlier study
(Liu et al., 2010b) due to its rapid microbial conversion to 6:2 FTUA
[F(CF2)5CF@CHCO2H].
6:2 FTUA is the immediate measurable transformation product
from 6:2 FTS de-sulfonation and oxidation. Its level peaked between 7 and 28 d and decreased thereafter with coincident increasing levels of 5:2 ketone, 5:2 sFTOH, PFPHxA, and PFPeA. These four
transformation products were also observed in soil and bacterial
culture dosed with 6:2 FTOH (Liu et al., 2010b). This suggests that
6:2 FTUA is further metabolized following similar biotransformation pathways as previously discussed for 6:2 FTOH (Liu et al.,
2010b). For example, 6:2 FTUA can be metabolized to 5:2 ketone
via decarboxylation and other reactions. The 5:2 ketone is then converted to 5:2 sFTOH catalyzed by a dehydrogenase. 5:2 sFTOH is the
direct precursor to PFHxA and PFPeA (Liu et al., 2010a,b). For 5:2
sFTOH to be converted to PFHxA and PFPeA, multiple enzymatic
reactions are involved in removing fluorine and carbon atoms from
the 5:2 sFTOH molecules. Such molecular shortening mechanisms
are currently not well understood. We speculate that various enzymes (e.g., dehydrogenase, hydratase, monooxygenase, and decarboxylase) were involved in converting 5:2 sFTOH to PFHxA and
PFPeA. The 5:2 sFTOH and 5:2 ketone are likely two of the volatile
transformation products reported but not identified in an earlier
study of 6:2 FTS biotransformation (Key et al., 1998).
The pathways leading to 5:3 acid from 6:2 FTUA seemed to be
operational at an earlier stage of 6:2 FTS metabolism, since the
5:3 acid level diminished at day 90 after peaking between days
14 and 28. Perhaps this is due to 5:3 acid absorption/complexation
to activated sludge. This is in sharp contrast to 6:2 FTOH biotransformation in bacterial culture and soil (Liu et al., 2010a,b), where
the 5:3 acid level remained constant after day 14. Nonetheless,
6:2 FTUA can be de-fluorinated to a transient transformation product, 5:3 unsaturated acid [F(CF2)5CH@CHCOOH], which was rapidly
converted to 5:3 acid catalyzed by a dehydrogenase.
Microbial a-oxidation of 6:2 FTS to PFHpA was not operational
in activated sludge since no PFHpA was ever detected over 90-d
incubation. PFHpA was also not detected in bacterial culture and
soil dosed with 6:2 FTOH (Liu et al., 2010a,b). Similarly, PFNA
[F(CF2)8COOH] was not detected in 8:2 FTOH-dosed bacterial culture, activated sludge, and soil (Dinglasan et al., 2004; Wang
et al., 2005a,b; Liu et al., 2007; Wang et al., 2009). These results
demonstrate that microbial a-oxidation of 6:2 FTS, 6:2 FTOH, and
8:2 FTOH to PFHpA and PFNA, respectively, may not be operational
in the environment, although such oxidation reactions occur at low
yields in mammalian systems (Kudo et al., 2005; Martin et al.,
2005; Fasano et al., 2006, 2009). PFHpA was observed as a major
transformation product in phosphate-depleted activated sludge
mixture dosed with 6:2 monoPAP and 6:2 diPAP (Lee et al.,
2010), presumably via a-oxidation of the major intermediate
transformation product, 6:2 FTOH. Activated sludge in WWTPs in
general is rich in phosphate. Likewise, phosphate is also abundant
in most soil and sediment. It is difficult to find an environmental
compartment with depleted phosphate, except maybe in pure
snow or rainfall with limited microbial populations. The observed
microbial a-oxidation by Lee et al. under specific laboratory conditions with depleted phosphate may not be representative what
would occur in the environment, at least not in the WWTPs.
4. Conclusions
6:2 FTS aerobic biotransformation in activated sludge from
WWTPs is relatively slow with less than 7% of initially dosed 6:2
Please cite this article in press as: Wang, N., et al. 6:2 Fluorotelomer sulfonate aerobic biotransformation in activated sludge of waste water treatment
plants. Chemosphere (2010), doi:10.1016/j.chemosphere.2010.11.003
6
N. Wang et al. / Chemosphere xxx (2010) xxx–xxx
FTS converted to various transformation products after 90 d. The
initial microbial aerobic de-sulfonation of 6:2 FTS may be the
rate-limiting step in determining 6:2 FTS biotransformation potential. The major stable transformation products PFPeA and PFHxA
together accounted for 2.6% of initially dosed 6:2 FTS after 90 d.
Substantial amounts of 6:2 FTS (24%) may be irreversibly bound
to organic components of the activated sludge and became less
available for further biotransformation. Primary biotransformation
of 6:2 FTS in activated sludge bypassed 6:2 FTOH to form 6:2 FTUA
directly, which was then degraded following the pathways similar
to that of 6:2 FTOH in bacterial culture and soil to form PFPeA and
PFHxA eventually. No microbial a-oxidation of 6:2 FTS to PFHpA
was observed in activated sludge. To better understand the environmental fate of poly- and per-fluorinated sulfonates, future studies may be needed to determine 6:2 FTS biotransformation rates
and degradation pathways in other environmental compartments
such as soil and sediment and under anaerobic conditions. The potential microbial de-sulfonation of perfluorinated sulfonamido
alcohols to form PFCAs in different environmental compartments
is also yet to be determined.
Acknowledgement
The authors wish to thank Dr. Mark Russell for helpful comments on this paper and Dr. Alexander Shtarov for synthesis of
the 13C-labeled internal standard used in this study.
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.chemosphere.2010.11.003.
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