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In vivo and in vitro isomer-specific biotransformation of perfluorooctane sulfonamide in common carp (Cyprinus carpio) Meng Chen, Liwen Qiang, Xiaoyu Pan, Shuhong Fang, Yuwei Han, and Ling-Yan Zhu Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b00488 • Publication Date (Web): 08 Jun 2015 Downloaded from http://pubs.acs.org on July 1, 2015
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Environmental Science & Technology
1
In
2
Perfluorooctane Sulfonamide in Common Carp (Cyprinus carpio)
3
Meng Chen†, Liwen Qiang†, Xiaoyu Pan‡, Shuhong Fang†, Yuwei Han†, Lingyan
4
Zhu†*
5
†Key Laboratory of Pollution Processes and Environmental Criteria, Ministry of
6
Education, Tianjin Key Laboratory of Environmental Remediation and Pollution
7
Control, College of Environmental Science and Engineering, Nankai University,
8
Tianjin, P.R. China 300071
9
‡ College of Marine Science of Engineering, Tianjin University of Science and
10
Vivo
and
In
Vitro
Isomer-Specific
Biotransformation
of
Technology, Tianjin, P.R. China 300457
∗
To whom correspondence should be addressed. E-mail: [email protected]. Phone: +86-22-23500791. Fax: +86-22-23503722. 1
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ABSTRACT
12
Biotransformation of PFOS-precursors (PreFOS) may contribute significantly to
13
the level of perfluorooctanesulfonate(PFOS) in the environment. Perfluorooctane
14
sulfonamide (PFOSA) is one of the major intermediates of higher molecular weight
15
PreFOS. Its further degradation to PFOS could be isomer specific and thereby explain
16
unexpected high percentages of branched (Br-) PFOS isomers observed in wildlife. In
17
this study, isomeric degradation of PFOSA was concomitantly investigated by in vivo
18
and in vitro tests using common carp as an animal model. In the in vivo tests branched
19
isomers of PFOSA and PFOS were eliminated faster than the corresponding linear (n-)
20
isomers, leading to enrichment of n-PFOSA in the fish. In contrast, Br-PFOS was
21
enriched in the fish, suggesting that Br-PFOSA isomers were preferentially
22
metabolized to Br-PFOS than n-PFOSA. This was confirmed by the in vitro test. The
23
exception was 1m-PFOSA, which could be the most difficult to be metabolized due to
24
its α-branched structure, resulting in the deficiency of 1m-PFOS in the fish. The in
25
vitro tests indicated that the metabolism mainly took place in the fish liver instead of
26
its kidney, and it was mainly a Phase I reaction. The results may help to explain the
27
special PFOS isomer profile observed in wildlife.
28
Keyword: PFOSA, PFOS, in vivo, in vitro, isomers, fish
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INTRODUCTION
31
Perfluorooctane sulfonate (PFOS; C8F17SO3-) and PFOS-precursors (PreFOS,
32
which can degrade to PFOS) have been widely used in a variety of commercial and
33
household products, due to their special physicochemical properties. Many
34
toxicological studies on animals documented that PFOS and PreFOS could display
35
various adverse effects to animals.1-5 Due to their global occurrence,6-9 environmental
36
persistence, bioaccumulation potential,10-12 and adverse effects to biota and humans,
37
PFOS and perfluorooctanesulfonyl fluoride (PFOSF; C8F17SO2F) were added in the
38
list of Annex B of the Stockholm Convention on Persistent Organic Pollutants in
39
2009.13
40
Despite that PFOS and PFOSF were voluntarily phased out by 3M in 2000,
41
PreFOS and PFOS are still being produced in China.14 It was reported that the
42
production of PFOS was 200-250 t/year during 2008-2011 and that of PFOSF was up
43
to 200 t/year by 2006 in China.14, 15 Perfluorooctane sulfonate (PFOS) is still one of
44
the major perfluoroalkyl substances (PFASs) which are widely present in the
45
environment.16-18 There are two major manufacturing methods, electrochemical
46
fluorination (ECF) and telomerization, used to produce PFASs and their precursors.
47
Electrochemical fluorination (ECF) had been used to synthesize PFOS and PreFOS,
48
producing a mixture of around 30% of branched and 70% linear isomers in the final
49
commercial products.19, 20 Perfluorooctane sulfonate (PFOS) in the environment could
50
originate from direct emission or from degradation of PreFOS. Paul et al.20 estimated
51
that the maximum direct historical emission of PFOS in the environment was 3
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450-2700 t, while the emissions of PreFOS were 6800-45250 t.14 Thus, the
53
degradation of PreFOS to PFOS could make a great contribution to the environmental
54
burden of PFOS. Perfluorooctane sulfonamide (PFOSA; C8F17SO2NH2), which is a
55
form of PreFOS,14 was more frequently detected in environmental matrices, wildlife
56
and human than other PreFOS.7, 21, 22 Perfluorooctane sulfonamide (PFOSA) is usually
57
the major metabolite of higher molecular weight PreFOS, such as N-EtFOSA, and it
58
was then finally metabolized to PFOS in rat and rainbow trout.23, 24 It was believed
59
that the degradation of PFOSA to PFOS was the rate-limiting step of the metabolism
60
of high molecular PreFOS, and it was assumed that the toxicokinetics of PFOSA
61
metabolism would affect the isomer profiles of PFOS in the environment.24
62
Some studies have reported that the toxicity and toxicokinetics of PFASs was
63
isomer-specific. Laboratory studies demonstrated that most Br-PFOS isomers were
64
eliminated preferentially in rats and fish,25,
65
bioaccumulated.27 However, field studies on wildlife and humans always found that
66
the percentage of Br-PFOS isomers (%Br-PFOS) was much higher (30-52%) than that
67
in the commercial ECF-PFOS, in which the %Br-PFOS was consistently close to
68
30%.22,
69
enriched in wildlife.32 One plausible explanation was that the metabolism of PreFOS
70
was isomer-specific.33 Martin et al.24 observed the biotransformation of PFOSA in rats
71
was also isomer-specific, leading to enrichment of Br-PFOS isomers in the rats. In
72
vitro study, it was found that the N-deethylation of branched isomers of N-EtFOSA
73
(Br-N-EtFOSA) was faster than the linear N-EtFOSA.34 Since PFOSA plays an
28-31
26
and n-PFOS was preferentially
Thus, it is very difficult to explain why the Br-PFOS isomers were
4
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important role in the degradation of higher molecular PreFOS and its further
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degradation could be isomeric specific, it is very important to uncover the underlying
76
mechanisms involved in the isomeric biotransformation of PFOSA in aquatic
77
organisms.
78
This study aimed to investigate the isomer specific biotransformation of PFOSA
79
using common carp as a test animal. In vivo studies were conducted by exposing the
80
carp to water spiked with PFOSA. The uptake, elimination, and transformation of
81
PFOSA isomers in the carp tissues were extensively investigated to understand the
82
isomeric accumulation and metabolism of PFOSA in the carp. To further understand
83
the organs in the carp where the metabolism took place and the underlying
84
mechanisms involved in the metabolism, in vitro studies were also performed by
85
incubating PFOSA with the S9 fractions extracted from the carp liver and kidney.
86
MATERIALS AND METHODS
87
Chemicals and Reagents
88
Electrochemical fluorination (ECF) PFOS (i.e. 70% linear and 30% branched,
89
by 19F NMR) was obtained from the 3M Co (St. Paul, MN, USA). All other native and
90
mass labeled PFASs standards, including Br-PFOSK, PFAC–MXB, MPFAC–MXA,
91
PFOSA and M8PFOSA-M were purchased from Wellington Laboratories (Guelph,
92
ON, Canada). The relative percentage of linear and branched components in
93
Br-PFOSK (78.8% linear, 10% iso-PFOS, 1.2% 1m-PFOS, 1.9% 3m-PFOS, 2.2%
94
4m-PFOS, 4.5% 5m-PFOS, and 0.71% m2-PFOS) was provided by the Wellington
95
Laboratories based on
19
F NMR analysis. PFAC–MXB is a mixture of linear 5
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of
perfluorohexanoate
(PFHxA),
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standards
perfluoroheptanoate
(PFHpA),
97
perfluorooctanoate (PFOA), perfluorononanoate (PFNA), perfluorodecanoate (PFDA),
98
perfluoroundecanoate
(PFUdA),
perfluorododecanoate
(PFDoA),
99
perfluorotetridecanoate
(PFTrDA),
perflurotetradecanoate
(PFTeDA),
100
perfluorohexanesulfonate (PFHxS), PFOS and perfluorodecanesulfonate (PFDS).
101
MPFAC–MXA is a mixture of mass labeled internal standards of linear PFHxA,
102
PFOA, PFNA, PFDA, PFUdA, PFDoA, PFHxS and PFOS. M8PFOSA-M is a mass
103
labeled internal standard for linear PFOSA. The technical product of PFOSA (~ 90%
104
purity) was purchased from J & K Co. (Beijin, China).
105
The bovine serum albumin (BSA), β-Nicotinamide adenine dinucleotide (NADP+),
106
phosphate
107
dehydrogenase (G6PDH), magnesium chloride, phosphate buffers were purchased
108
from Sigma Chemical Co. (Tianjin, China). Methanol and formic acid were of high
109
performance liquid chromatography (HPLC) grade and obtained from Dikma
110
Technology Inc. (Beijing, China). Sodium hydroxide (NaOH, 96.0%) and ammonium
111
hydroxide solution (NH4OH, 25%) were purchased from Guangfu Fine Chemical
112
Research Institute (Tianjin, China). Methyl tert-butyl ether (MTBE) and tetrabutyl
113
ammonium hydrogen sulfate (TBAH) were purchased from Concord Science and
114
Technology (Tianjin, China). Other chemicals were bought from Weida Chemical
115
Commercial Ltd. (Tianjin, China). Milli-Q water was used throughout the study.
116
Isomer nomenclature
117
glucose-6-phosphate
(Glc-6-PO4),
glucose-6-phosphate
The nomenclature for specific PFOS isomers (Table S1) was adopted from 6
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Benskin et al.29 Briefly, the linear and isopropyl isomers were abbreviated as n- and
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iso-PFOS, respectively. For the other monomethyl branched isomers, m- refers to a
120
perfluoromethyl branch, and the number preceding m- represents the carbon number
121
on which the perfluoromethyl branch resides. For example, 1m-PFOS refers to
122
1-perfluoromethyl-PFOS. The sum of all diperfluoromethyl isomers, which could not
123
be easily distinguished, was abbreviated as m2-PFOS. When referring to the total sum
124
of all branched and linear isomers, the term PFOS was used. Due to the lack of
125
authentic branched standards of PFOSA, the percentages of the isomers of PFOSA
126
were estimated based on their chromatographic peak areas relative to the total peak
127
areas detected with m/z 499 to 78 transitions, as reported by Asher et al.35 It was
128
determined that the technical PFOSA used in the present study consisted of 78%
129
n-PFOSA and 22% Br-PFOSA.
130
Fish exposure tests
131
Common carp, about 8 cm in length and 5-6 g in weight, were purchased from a
132
local market and acclimatized in the laboratory for two weeks prior to the exposure
133
tests. Filtered dechlorinated water with a hardness of 91.0±2.0 mg/L CaCO3, pH of
134
7.6±0.5, dissolved oxygen of 7.0±0.4 mg/L, was maintained at 20±1oC and slightly
135
aerated. Four 80 L aquariums with a flow-through system (0.01 L/min) were used for
136
the tests: two for control and the other two for exposure tests. Forty fish were added in
137
each aquarium and a 12 h light/12 h dark photoperiod was applied. Fish were fed
138
daily at a rate of 1.0% body weight. Stock solution of technical PFOSA at 200 mg/L
139
was prepared in methanol, which was diluted with water to the desired concentration 7
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with methanol less than 0.01% (v/v). In the exposure tests, the concentration of the
141
technical PFOSA was set at 20 µg/L and the exposure lasted for 10 days. Two fish
142
were sampled from each aquarium on days 0, 2, 4, 6, 8, and 10. At the end of
143
exposure, all the remaining fish were taken out and transferred to individual tanks
144
with clean filtered dechlorinated tap water for depuration, which lasted for another 10
145
days. Two fish were sampled from each aquarium on days 12, 14, 16, 18, and 20.
146
Upon sampling, the fish were anesthetized with tricaine methane sulfonate (MS-222).
147
Blood samples were immediately taken from the fish. All the fish were subsequently
148
dissected for liver, kidney and muscle. Other parts such as bones, intestines and skins
149
were discarded. At each sampling time, 300 mL of water was sampled. The fish
150
samples were stored at -20°C, and the water samples were stored at 4°C until
151
extraction.
152
In vitro incubation
153
Hepatic and renal cytosol fractions, which were denoted as S9, were prepared
154
from the same common carp which were acclimatized in the laboratory but were not
155
exposed to PFOSA. The preparation method for liver and kidney S9 was adopted
156
from Butt et al.36 and the detailed information is provided in SI. Catalase (CAT)
157
activity was used as an indicator of S9 enzymatic capacity.
158
The in vitro incubations were conducted in a series of polypropylene (PP) tubes,
159
in which 10 µL of PFOSA solution in methanol (2 ng/µL), 790 µL of 0.05 M
160
phosphate buffer (pH 7.4), 100 µL of premixed NADPH (nicotinamide adenine
161
dinucleotide phosphate) regenerating solution (containing 1.6 mM NADP+, 3.3 mM 8
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Glc-6-PO4, 0.4 U/mL G6PDH, and 3.3 mM magnesium chloride), and 100 µL of S9
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fraction (total protein was 0.5 ± 0.01 mg) were added. All incubations were conducted
164
at 20 °C in a water bath with shaking. Three types of experimental controls were
165
applied accompanying with the tests. The first control contained active S9 and all
166
reaction components except PFOSA to correct background contamination (Control I).
167
In the second control, the incubation was the same as the test group except that S9
168
was deactivated by heating it at 100 °C for 5 min in a water bath, which was designed
169
to correct for abiotic and microbial transformation of PFOSA (Control II). The other
170
blank contained all the reaction components and was incubated in the dark to exclude
171
the possibility of photo-transformation (Control III).
172
At each sampling time, one PP tube was sacrificed and the reaction was
173
terminated by adding 0.5 ml of methanol in the reaction solution. The solution was
174
vortexed for 1 min, and was immediately stored at -20 °C until extraction and analysis
175
for target analytes. All the experiments were repeated in duplicate and the results were
176
reported as the means of the two replicates.
177
Extraction and instrumental analysis
178
The analysis of PFASs in the fish was performed following the procedure
179
described by Hansen et al.37 and water samples were extracted using the method
180
provided by Fang et al.21 Further details about the extraction are supplied in SI.
181
Analyses of the individual PFASs and isomers of PFOSA and PFOS were
182
performed on a Waters HPLC system coupled with a Waters Xevo TQ_S tandem mass
183
spectrometry (MS/MS) operated in negative electrospray ionization (ESI) mode using 9
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the method developed by Benskin et al.29 Briefly, 10 µL of the extract was injected
185
onto a FluoroSep RP Octyl HPLC column (ES Industries, West Berlin, NJ) at 38 °C.
186
The flow rate was 150 µL/min, and the program started from 60% A (water adjusted
187
to pH 4.0 with ammonium formate) and 40% B (methanol). The initial condition was
188
held for 0.3 min and then ramped to 64% B by 1.9 min; increased to 66% B by 5.9
189
min, 70% B by 7. 9 min, 74% B by 26 min, and finally to 100% B by 30 min, held
190
until 37 min; returned to initial conditions by 38 min, and the column equilibrated for
191
another 19 min. Chromatograms were recorded by multiple reaction monitoring
192
(MRM) with 1 to 9 transitions per analyte (Table S1).
193
Quality assurance and quality control
194
For the chemical analyses, a method blank (HPLC grade water) was extracted
195
with each batch of 12 samples to check background contamination, and one solvent
196
blank (HPLC grade methanol) was injected after 10 samples to monitor any
197
instrument carryover. Two quality control standard solutions (2 ng/mL MXB, 5 ng/mL
198
Br-PFOS) were run to monitor sensitivity drift along with 8-10 real samples. The
199
method detection limits (MDLs) were defined as the concentration with a
200
signal-to-noise ratio of 3 if the specific PFASs were not detected in the blanks. For the
201
analytes detected in the blanks, MDLs were defined as the mean blank concentration
202
plus three times the standard deviation of the blank (Table S2). Recoveries were
203
calculated relative to the internal standards in both the samples and standards after
204
subtracting the response of the unspiked samples. The matrix spiked recoveries of
205
water (10 ng/L) and fish whole body homogenate (5 ng/g, ww) ranged in 98-109% 10
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and 74-93%, respectively (Table S2).
207
Data analysis
208 209 210 211 212 213 214
The PFOSA branched isomers could not be baseline separated and thus were reported as the sum of branched isomers (Br-PFOSA). The growth rate was calculated using an exponential model over the exposure time: Wt = ae bt
(1)
Where Wt is the fish weight (g) at time t (d), a is the initial fish weight (g), and b is the growth rate.
215
The elimination rate constant (ke) was calculated by fitting the depuration data to
216
a first-order decay model39 using a nonlinear regression technique provided by Origin
217
V 8.5 (Origin Lab, USA):
218
C e = C t = 0 e − ke t
219
Where Ce and Ct=0 are the concentrations of PFASs in the fish (µg/kg ww) at time
220
(2)
t and the beginning of the depuration, ke is the elimination rate constant (1/d).
221
The uptake rate constant (ku) was estimated by fitting the uptake data to a
222
first-order bioaccumulation model.39 Using an interactive nonlinear regression
223
technique provided by Origin V 8.5 (Origin Lab, USA).
224
225 226
Ce =
ku C s (1 − e − ket ) ke
(3)
Where Ce is the concentration of PFASs in the fish at time t (µg/kg ww), Cs is PFASs concentration in water (µg/L), ku is the uptake rate coefficient (kg/L×d). 11
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227
Depuration half-life (t1/2) was calculated using the following Eqn:
228
t1/2 =
229
The kinetic bioaccumulation concentration factor (BCF) was estimated as the
230
231
232
ln 2 ke
(4)
quotient of uptake (ku) and elimination rate (ke) constants.40 BCF =
ku ke
(5)
Statistical analysis
233
Paired Student’s t-test was conducted to assess the difference in the growth rates,
234
HSI (hepatosomatic index) factor and the concentrations of PFOS and PFOSA
235
between the control and exposed fish samples. One way analysis of variance
236
(ANOVA) was used to assess the difference in the concentrations of PFOS and
237
PFOSA among different tissues. All statistical analyses were performed with IBM©
238
SPSS Statistics version 20 (Chicago, IL), and significance was set as p< 0.05.
239
RESULTS AND DISCUSSION
240
Fish mortality, growth rate, and HSI
241
No mortality of fish occurred in the control and exposure tests throughout the
242
experiments. At each sampling time, the fish mass and HSI were measured, and the
243
data are listed in Table S3. No significant differences in fish mass and HSI were
244
observed between the control and exposure tests (p> 0.05). The HSIs in both the
245
control and exposure tests were constant, suggesting that the fish liver functioned
246
normally during the exposure. In all the tests, the fish growth followed an exponential 12
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kinetic (Eqn 1) with a very low growth rate of 2.1~2.8×10-3g/d. The results indicate
248
that the spiked PFOSA did not show obvious toxicity to the fish during the exposure
249
period.
250
Uptake and elimination of PFOSA
251
During the uptake period, the concentration of PFOSA in the exposure solution
252
was relatively stable at 15.5±0.18 µg/L, and the percentage of Br-PFOSA in total
253
PFOSA (%Br-PFOSA) was also very stable at 22±1%, which was consistent with the
254
technical PFOSA product. No other PreFOSs were detected neither in the technical
255
mixture or the exposure solution. PFOS was detected at 0.01 ug/L in the technical
256
mixture and its concentration was three orders of magnitude lower than the PFOSA
257
concentration.
258
As shown in Figure 1, the fish whole body concentration of PFOSA increased
259
rapidly during the uptake phase, suggesting that PFOSA could be well accumulated in
260
carp. Its uptake did not reach steady state in the 10 d exposure. It is interesting that
261
PFOS also displayed a similar increase in fish whole body concentration as PFOSA
262
did during the uptake phase. The concentrations of PFOS and PFOSA in the fish of
263
the control groups were always less than 1% of those in the exposed fish. The increase
264
of PFOS concentration in the fish indicates that common carp has the capability to
265
metabolize PFOSA, and PFOS in the exposed fish was due to the degradation of
266
PFOSA in fish body. Previous studies reported that PFOS was the end product in
267
rainbow trout and rat following dietary exposure to PFOSA.23, 24
13
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In the current study, some PFASs, such as PFPeA, PFHxA, PFHpA, PFOA and
269
PFHxS (liver>muscle. However, in the present study, the PFOS concentration was
368
higher in the carp liver than kidney, though the total protein concentration in the carp
369
kidney (43.0 mg/g) was slightly higher than in the liver (37.7 mg/g). In the current
370
study, PFOS was originated from the degradation of PFOSA in the carp. The
371
difference in the tissue distribution (liver and kidney) of PFOSA and PFOS suggests
372
that PFOS was mainly formed in the fish liver, resulting in higher concentration in the
373
liver than in the kidney. To ascertain this assumption, in vitro tests were performed by
374
incubation of PFOSA with the carp liver and kidney S9 fractions individually, which
375
will be discussed later.
of
PFOS
in
rainbow
trout
decreased
in
the
order
of
376
In agreement with the whole body burden, all tissues contained a
377
lower %Br-PFOSA than in water (Figure 3B). The mean %Br-PFOSA in the fish 18
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tissues ranged from 6.61±2.05% in the kidney to 11.6±1.45% in liver, but there was
379
no statistically significant differences among the tissues (p>0.05). On the contrary, all
380
tissues contained a higher %Br-PFOS than the water, mostly probably due to the
381
preferential
382
The %Br-PFOS was the highest in the carp liver and generally followed the order of
383
liver>blood~kidney>muscle (Figure 3B). This further supports that the transformation
384
of PFOSA mainly occurred in the carp liver. Similar to the PFOS isomer profile in the
385
fish whole body, all branched isomers were enriched in the carp tissues compared to
386
the ECF PFOS, with the exception of 1m-PFOS, which was depleted (Figure 4).
387
Relative deficiency of 1m-PFOS in rat which was exposed to PFOSA was also
388
observed in a previous study.24 These were contradicting to the fact that 1m-PFOS
389
was the most slowly eliminated among the PFOS isomers in the animals which were
390
exposed to PFOS directly.25, 26 The most possible reason for this discrepancy is the
391
absorption or metabolism of the α-branched PFOSA isomer was much lower than
392
other branched isomers in the carp.
393
In vitro experiments
biotransformation
of
Br-PFOSA
than
n-PFOSA
(Figure
3B).
394
For Control I, the concentrations of PFOSA and PFOS in the incubation solution
395
were two order of magnitude lower than those in the test groups. The PFOSA and
396
PFOS concentrations in the test groups were corrected by subtracting the background
397
levels. PFPeA, PFHxA, PFHpA, PFOA and PFHxS (0.48×10-3~2.11×1-3 pmol)
398
were also detected both in the control and test groups, but there was no significant
399
difference between the control and test groups (p>0.05). In addition, their levels were 19
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one or several magnitudes lower than PFOS and PFOSA in the exposed fish, and did
401
not show any increasing trend. These suggest that the presence of these compounds
402
mainly from background contamination rather than degradation of PFOSA. In the
403
Control II, the PFOSA concentration was constant during the course of incubation. No
404
significant difference in the concentrations of PFOSA and PFOS was observed
405
between the Control III and the test group (p>0.05). These suggest that there was no
406
phototransformation of PFOSA during the incubation. A mass balance (Table S4) was
407
calculated, and the molar mass of PFOSA at the beginning and the total molar masses
408
of PFOSA and PFOS at the end of the experiment was consistent. This strongly
409
suggests that no other metabolic products were produced or their production was
410
negligible, which agreed with the results of the in vivo experiments.
411
Figure 5A shows the variation of the concentrations of PFOSA and PFOS during
412
the incubation. A rapid decrease in PFOSA concentration and a significant increase in
413
PFOS were observed during the 64 h incubation. The results were similar with a
414
previous study which used rainbow trout microsomes to investigate the transformation
415
of N-EtPFOSA and both PFOSA and PFOS were observed.28 In previous in vitro
416
study with rat liver microsomes, cytosol or S9 fractions, no formation of PFOS from
417
PFOSA was observed, although this was observed in the rat liver slices but with very
418
low biotransformation rate.34, 44 However, PFOSA could undergo N-glucuronidation
419
when it was incubated with rat or monkey liver microsomes in the presence of
420
UDP-glucuronic acid (UDPGA).44,
421
PFOSA in rat and monkey was mediated by Phase II metabolism instead of Phase I.44
45
It was speculated that the transformation of
20
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422
The significant transformation of PFOSA to PFOS in our in vitro incubation which
423
was mediated by liver S9 without UDPGA suggests that it was a Phase I reaction,
424
although Phase II mediated transformation could not be ruled out. The results suggest
425
that fish have different mechanisms to metabolize PFOSA compared with rat and
426
monkeys, and further studies are warranted to shed light on the difference.
427
In the test group with kidney S9, the concentration of PFOSA remained constant
428
during the entire course of incubation (Figure S1). This indicates that the
429
biotransformation of PFOSA did occur in the carp liver instead of in its kidney, which
430
supports the results obtained in the in vivo tests.
431
Figure 5B illustrates the change of the branched isomers of PFOS and PFOSA
432
during the incubation period. The %Br-PFOSA declined from 21.8±0.12% gradually
433
to 8.24±2.59% at the end of incubation, while the %Br-PFOS increased slightly
434
during the course of incubation. The biotransformation of PFOSA might be described
435
by a first-order kinetics with R2 of 0.80 and 0.84 for Br-PFOSA and n-PFOSA
436
respectively (Figure 6). The reaction rate of Br-PFOSA was significantly higher than
437
n-PFOSA, indicating that the branched PFOSA isomers were preferentially
438
transformed than n-PFOSA in the fish liver, which was consist with the in vivo tests.
439
ASSOCIATED CONTENTS
440
Supporting Information
441
Description of the S9 fraction preparation, sample extraction and tables giving
442
the MRM transition of PFASs and PFOSA, recoveries and MDLs of PFASs and
21
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443
PFOSA in fish samples, fish physical parameters, and mass balance in vitro
444
experiments, figures illustrating the variation of PFOSA and PFOS in the kidney S9
445
experiment. This material is available free of charge via the internet at
446
http://pubs.acs.org.
447
ACKNOWLEDGMENTS
448
We acknowledge financial support from the Natural Science Foundation of
449
China (NSFC 21325730, 21277077), Ministry of Education (20130031130005),
450
Ministry of Environmental Protection (201009026) and the Ministry of Education
451
innovation team (IRT 13024).
452
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diester in Japanese medaka (Oryzias latipes). Environ. Sci. Technol. 2014, 48 (2), 1058-1066.
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sulfonate (PFOS)-precursor by Cytochrome P450 isozymes and human liver microsomes.
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contribution of PFOS-precursors to the Lake Ontario foodweb. Environ. Sci. Technol. 2012, 46
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Chem. 2010, 29 (12), 2736-41.
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Hamers, T. Competitive binding of poly- and perfluorinated compounds to the thyroid hormone
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transport protein transthyretin. Toxicol. Sci. 2009, 109 (2), 206-216.
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(39) O'brien, J. M.; Kennedy, S. W.; Chu, S. G.; Letcher, R. J. Isomer-specific accumulation of
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perfluorooctane sulfonate in the liver of chicken embryos exposed in ovo to a technical mixture.
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Environ. Toxicol. Chem. 2011, 30 (1), 226-231.
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(40) Zhang, Y.; Beesoon, S.; Zhu, L.; Martin, J. W. Biomonitoring of perfluoroalkyl acids in human urine and estimates of biological half-life. Environ. Sci. Technol. 2013, 47 (18), 10619-27.
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(41) Martin, J. W.; Mabury, S. A.; Solomon, K. R.; Muir, D. C. G. Bioconcentration and tissue
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distribution of perfluorinated acids in rainbow trout (Oncorhynchus mykiss). Environ. Toxicol. 25
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Chem. 2003, 22 (1), 196-204. (42) Jones, P. D.; Hu, W.; De Coen, W.; Newsted, J. L.; Giesy, J. P. Binding of perfluorinated fatty acids to serum proteins. Environ. Toxicol. Chem. 2003, 22 (11), 2639-49.
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Identification of perfluorooctane sulfonate binding protein in the plasma of tiger pufferfish
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Takifugu rubripes. Ecotoxicol. Environ. Saf. 2014, 104, 409-13.
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(44) Xu, L.; Krenitsky, D. M.; Seacat, A. M.; Butenhoff, J. L.; Anders, M. W. Biotransformation of
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N-ethyl-N-(2-hydroxyethyl) perfluorooetanesulfonamide by rat liver microsomes, cytosol, and
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slices and by expressed rat and human cytochromes P450. Chem. Res. Toxicol. 2004, 17 (6),
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767-775.
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(45) Xu, L.; Krenitsky, D. M.; Seacat, A. M.; Butenhoff, J. L.; Tephly, T. R.; Anders, M. W.
580
N-glucuronidation of perfluorooctanesulfonamide by human, rat, dog, and monkey liver
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microsomes and by expressed rat and human UDP-glucuronosyltransferases. Drug. Metab.
582
Dispos. 2006, 34 (8), 1406-10.
583
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584
Table 1. The uptake rate (ku), elimination rate (ke), half-life (t1/2) and dynamic
585
bioconcentration factors of PFOSA and PFOS isomers in the carp whole body ke(1/d)b
Compounds
R2
Half-life(d)b
ku(L/Kg/d)b
R2
BCF(L/Kg)b
PFOSA n-PFOSA
0.10±0.01
0.92
6.93±0.70
18.5±0.96
0.92
185±8.98
Br-PFOSA
0.19±0.05
0.82
3.64±1.03
8.57±1.39
0.70
45.1±12.8
∑PFOSA
0.10±0.01
0.91
6.93±0.70
25.12±1.77
0.89
251±7.50
PFOSA (not considering the biotransformation of PFOSA) n-PFOSAa
0.10±0.01
0.92
6.93±0.70
13.4±0.65
0.88
134±7.01
Br-PFOSAa
0.19±0.05
0.82
3.64±1.03
2.04±0.19
0.70
10.7±1.96
∑PFOSAa
0.10±0.01
0.91
6.93±0.70
15.0±0.82
0.83
150±6.87
PFOS n-PFOS
0.06±0.01
0.86
11.4±1.98
1m-PFOS
0.16±0.02
0.89
4.23±0.55
3+5m-PFOS 0.14±0.02
0.70
4.78±0.72
4m-PFOS
0.15±0.02
0.73
4.68±0.63
iso-PFOS
0.10±0.02
0.88
7.29±1.44
m2-PFOS
0.09±0.02
0.95
7.96±1.80
∑PFOS
0.09±0.01
0.84
8.02±0.87
586
a
587
b
the parameters calculated without including PFOS. Mean value ± standard deviation
27
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588
Figure Captions:
589
Figure 1. The whole body concentrations of PFOSA and PFOS in the common carp
590
during the uptake and elimination phases. Each point represents the mean
591
±1 standard error. Vertical dashed line delineates the end of the uptake
592
phase.
593
Figure 2. The percentage of total branched isomers of PFOS and PFOSA in the carp
594
whole body during the uptake and elimination phases. Each point represents
595
the mean ±1 standard error. Vertical dashed line delineates the end of the
596
uptake phase.
597
Figure 3. A, The concentrations of PFOSA and PFOS in the carp tissues and blood
598
after 10 days exposure to the ECF-PFOSA. B, The percentages of branched
599
isomers of PFOSA in the carp tissues and blood after 10 days exposure to
600
the ECF-PFOSA. Each point represents the mean ±1 standard error.
601
Figure 4. Percentages of individual PFOS isomers in the tissues and whole body of
602
the common carp and in a 3M manufactured ECF-PFOS product.
603
Figure 5. Variation of the concentrations of PFOSA and PFOS (A) and percentage of
604
the %Br-PFOS and %Br-PFOSA (B) over time in the treatments with carp
605
liver S9. Each point represents the mean ±1 standard error.
606 607
Figure 6. The reaction kinetics for the biotransformation of n-PFOSA and Br-PFOSA in the treatments with carp liver S9.
28
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Figure 1
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Figure 2
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Article
In vivo and in vitro isomer-specific biotransformation of perfluorooctane sulfonamide in common carp (Cyprinus carpio) Meng Chen, Liwen Qiang, Xiaoyu Pan, Shuhong Fang, Yuwei Han, and Ling-Yan Zhu Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b00488 • Publication Date (Web): 08 Jun 2015 Downloaded from http://pubs.acs.org on July 1, 2015
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
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Page 1 of 35
Environmental Science & Technology
1
In
2
Perfluorooctane Sulfonamide in Common Carp (Cyprinus carpio)
3
Meng Chen†, Liwen Qiang†, Xiaoyu Pan‡, Shuhong Fang†, Yuwei Han†, Lingyan
4
Zhu†*
5
†Key Laboratory of Pollution Processes and Environmental Criteria, Ministry of
6
Education, Tianjin Key Laboratory of Environmental Remediation and Pollution
7
Control, College of Environmental Science and Engineering, Nankai University,
8
Tianjin, P.R. China 300071
9
‡ College of Marine Science of Engineering, Tianjin University of Science and
10
Vivo
and
In
Vitro
Isomer-Specific
Biotransformation
of
Technology, Tianjin, P.R. China 300457
∗
To whom correspondence should be addressed. E-mail: [email protected]. Phone: +86-22-23500791. Fax: +86-22-23503722. 1
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11
ABSTRACT
12
Biotransformation of PFOS-precursors (PreFOS) may contribute significantly to
13
the level of perfluorooctanesulfonate(PFOS) in the environment. Perfluorooctane
14
sulfonamide (PFOSA) is one of the major intermediates of higher molecular weight
15
PreFOS. Its further degradation to PFOS could be isomer specific and thereby explain
16
unexpected high percentages of branched (Br-) PFOS isomers observed in wildlife. In
17
this study, isomeric degradation of PFOSA was concomitantly investigated by in vivo
18
and in vitro tests using common carp as an animal model. In the in vivo tests branched
19
isomers of PFOSA and PFOS were eliminated faster than the corresponding linear (n-)
20
isomers, leading to enrichment of n-PFOSA in the fish. In contrast, Br-PFOS was
21
enriched in the fish, suggesting that Br-PFOSA isomers were preferentially
22
metabolized to Br-PFOS than n-PFOSA. This was confirmed by the in vitro test. The
23
exception was 1m-PFOSA, which could be the most difficult to be metabolized due to
24
its α-branched structure, resulting in the deficiency of 1m-PFOS in the fish. The in
25
vitro tests indicated that the metabolism mainly took place in the fish liver instead of
26
its kidney, and it was mainly a Phase I reaction. The results may help to explain the
27
special PFOS isomer profile observed in wildlife.
28
Keyword: PFOSA, PFOS, in vivo, in vitro, isomers, fish
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INTRODUCTION
31
Perfluorooctane sulfonate (PFOS; C8F17SO3-) and PFOS-precursors (PreFOS,
32
which can degrade to PFOS) have been widely used in a variety of commercial and
33
household products, due to their special physicochemical properties. Many
34
toxicological studies on animals documented that PFOS and PreFOS could display
35
various adverse effects to animals.1-5 Due to their global occurrence,6-9 environmental
36
persistence, bioaccumulation potential,10-12 and adverse effects to biota and humans,
37
PFOS and perfluorooctanesulfonyl fluoride (PFOSF; C8F17SO2F) were added in the
38
list of Annex B of the Stockholm Convention on Persistent Organic Pollutants in
39
2009.13
40
Despite that PFOS and PFOSF were voluntarily phased out by 3M in 2000,
41
PreFOS and PFOS are still being produced in China.14 It was reported that the
42
production of PFOS was 200-250 t/year during 2008-2011 and that of PFOSF was up
43
to 200 t/year by 2006 in China.14, 15 Perfluorooctane sulfonate (PFOS) is still one of
44
the major perfluoroalkyl substances (PFASs) which are widely present in the
45
environment.16-18 There are two major manufacturing methods, electrochemical
46
fluorination (ECF) and telomerization, used to produce PFASs and their precursors.
47
Electrochemical fluorination (ECF) had been used to synthesize PFOS and PreFOS,
48
producing a mixture of around 30% of branched and 70% linear isomers in the final
49
commercial products.19, 20 Perfluorooctane sulfonate (PFOS) in the environment could
50
originate from direct emission or from degradation of PreFOS. Paul et al.20 estimated
51
that the maximum direct historical emission of PFOS in the environment was 3
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450-2700 t, while the emissions of PreFOS were 6800-45250 t.14 Thus, the
53
degradation of PreFOS to PFOS could make a great contribution to the environmental
54
burden of PFOS. Perfluorooctane sulfonamide (PFOSA; C8F17SO2NH2), which is a
55
form of PreFOS,14 was more frequently detected in environmental matrices, wildlife
56
and human than other PreFOS.7, 21, 22 Perfluorooctane sulfonamide (PFOSA) is usually
57
the major metabolite of higher molecular weight PreFOS, such as N-EtFOSA, and it
58
was then finally metabolized to PFOS in rat and rainbow trout.23, 24 It was believed
59
that the degradation of PFOSA to PFOS was the rate-limiting step of the metabolism
60
of high molecular PreFOS, and it was assumed that the toxicokinetics of PFOSA
61
metabolism would affect the isomer profiles of PFOS in the environment.24
62
Some studies have reported that the toxicity and toxicokinetics of PFASs was
63
isomer-specific. Laboratory studies demonstrated that most Br-PFOS isomers were
64
eliminated preferentially in rats and fish,25,
65
bioaccumulated.27 However, field studies on wildlife and humans always found that
66
the percentage of Br-PFOS isomers (%Br-PFOS) was much higher (30-52%) than that
67
in the commercial ECF-PFOS, in which the %Br-PFOS was consistently close to
68
30%.22,
69
enriched in wildlife.32 One plausible explanation was that the metabolism of PreFOS
70
was isomer-specific.33 Martin et al.24 observed the biotransformation of PFOSA in rats
71
was also isomer-specific, leading to enrichment of Br-PFOS isomers in the rats. In
72
vitro study, it was found that the N-deethylation of branched isomers of N-EtFOSA
73
(Br-N-EtFOSA) was faster than the linear N-EtFOSA.34 Since PFOSA plays an
28-31
26
and n-PFOS was preferentially
Thus, it is very difficult to explain why the Br-PFOS isomers were
4
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important role in the degradation of higher molecular PreFOS and its further
75
degradation could be isomeric specific, it is very important to uncover the underlying
76
mechanisms involved in the isomeric biotransformation of PFOSA in aquatic
77
organisms.
78
This study aimed to investigate the isomer specific biotransformation of PFOSA
79
using common carp as a test animal. In vivo studies were conducted by exposing the
80
carp to water spiked with PFOSA. The uptake, elimination, and transformation of
81
PFOSA isomers in the carp tissues were extensively investigated to understand the
82
isomeric accumulation and metabolism of PFOSA in the carp. To further understand
83
the organs in the carp where the metabolism took place and the underlying
84
mechanisms involved in the metabolism, in vitro studies were also performed by
85
incubating PFOSA with the S9 fractions extracted from the carp liver and kidney.
86
MATERIALS AND METHODS
87
Chemicals and Reagents
88
Electrochemical fluorination (ECF) PFOS (i.e. 70% linear and 30% branched,
89
by 19F NMR) was obtained from the 3M Co (St. Paul, MN, USA). All other native and
90
mass labeled PFASs standards, including Br-PFOSK, PFAC–MXB, MPFAC–MXA,
91
PFOSA and M8PFOSA-M were purchased from Wellington Laboratories (Guelph,
92
ON, Canada). The relative percentage of linear and branched components in
93
Br-PFOSK (78.8% linear, 10% iso-PFOS, 1.2% 1m-PFOS, 1.9% 3m-PFOS, 2.2%
94
4m-PFOS, 4.5% 5m-PFOS, and 0.71% m2-PFOS) was provided by the Wellington
95
Laboratories based on
19
F NMR analysis. PFAC–MXB is a mixture of linear 5
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perfluorohexanoate
(PFHxA),
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96
standards
perfluoroheptanoate
(PFHpA),
97
perfluorooctanoate (PFOA), perfluorononanoate (PFNA), perfluorodecanoate (PFDA),
98
perfluoroundecanoate
(PFUdA),
perfluorododecanoate
(PFDoA),
99
perfluorotetridecanoate
(PFTrDA),
perflurotetradecanoate
(PFTeDA),
100
perfluorohexanesulfonate (PFHxS), PFOS and perfluorodecanesulfonate (PFDS).
101
MPFAC–MXA is a mixture of mass labeled internal standards of linear PFHxA,
102
PFOA, PFNA, PFDA, PFUdA, PFDoA, PFHxS and PFOS. M8PFOSA-M is a mass
103
labeled internal standard for linear PFOSA. The technical product of PFOSA (~ 90%
104
purity) was purchased from J & K Co. (Beijin, China).
105
The bovine serum albumin (BSA), β-Nicotinamide adenine dinucleotide (NADP+),
106
phosphate
107
dehydrogenase (G6PDH), magnesium chloride, phosphate buffers were purchased
108
from Sigma Chemical Co. (Tianjin, China). Methanol and formic acid were of high
109
performance liquid chromatography (HPLC) grade and obtained from Dikma
110
Technology Inc. (Beijing, China). Sodium hydroxide (NaOH, 96.0%) and ammonium
111
hydroxide solution (NH4OH, 25%) were purchased from Guangfu Fine Chemical
112
Research Institute (Tianjin, China). Methyl tert-butyl ether (MTBE) and tetrabutyl
113
ammonium hydrogen sulfate (TBAH) were purchased from Concord Science and
114
Technology (Tianjin, China). Other chemicals were bought from Weida Chemical
115
Commercial Ltd. (Tianjin, China). Milli-Q water was used throughout the study.
116
Isomer nomenclature
117
glucose-6-phosphate
(Glc-6-PO4),
glucose-6-phosphate
The nomenclature for specific PFOS isomers (Table S1) was adopted from 6
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Benskin et al.29 Briefly, the linear and isopropyl isomers were abbreviated as n- and
119
iso-PFOS, respectively. For the other monomethyl branched isomers, m- refers to a
120
perfluoromethyl branch, and the number preceding m- represents the carbon number
121
on which the perfluoromethyl branch resides. For example, 1m-PFOS refers to
122
1-perfluoromethyl-PFOS. The sum of all diperfluoromethyl isomers, which could not
123
be easily distinguished, was abbreviated as m2-PFOS. When referring to the total sum
124
of all branched and linear isomers, the term PFOS was used. Due to the lack of
125
authentic branched standards of PFOSA, the percentages of the isomers of PFOSA
126
were estimated based on their chromatographic peak areas relative to the total peak
127
areas detected with m/z 499 to 78 transitions, as reported by Asher et al.35 It was
128
determined that the technical PFOSA used in the present study consisted of 78%
129
n-PFOSA and 22% Br-PFOSA.
130
Fish exposure tests
131
Common carp, about 8 cm in length and 5-6 g in weight, were purchased from a
132
local market and acclimatized in the laboratory for two weeks prior to the exposure
133
tests. Filtered dechlorinated water with a hardness of 91.0±2.0 mg/L CaCO3, pH of
134
7.6±0.5, dissolved oxygen of 7.0±0.4 mg/L, was maintained at 20±1oC and slightly
135
aerated. Four 80 L aquariums with a flow-through system (0.01 L/min) were used for
136
the tests: two for control and the other two for exposure tests. Forty fish were added in
137
each aquarium and a 12 h light/12 h dark photoperiod was applied. Fish were fed
138
daily at a rate of 1.0% body weight. Stock solution of technical PFOSA at 200 mg/L
139
was prepared in methanol, which was diluted with water to the desired concentration 7
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with methanol less than 0.01% (v/v). In the exposure tests, the concentration of the
141
technical PFOSA was set at 20 µg/L and the exposure lasted for 10 days. Two fish
142
were sampled from each aquarium on days 0, 2, 4, 6, 8, and 10. At the end of
143
exposure, all the remaining fish were taken out and transferred to individual tanks
144
with clean filtered dechlorinated tap water for depuration, which lasted for another 10
145
days. Two fish were sampled from each aquarium on days 12, 14, 16, 18, and 20.
146
Upon sampling, the fish were anesthetized with tricaine methane sulfonate (MS-222).
147
Blood samples were immediately taken from the fish. All the fish were subsequently
148
dissected for liver, kidney and muscle. Other parts such as bones, intestines and skins
149
were discarded. At each sampling time, 300 mL of water was sampled. The fish
150
samples were stored at -20°C, and the water samples were stored at 4°C until
151
extraction.
152
In vitro incubation
153
Hepatic and renal cytosol fractions, which were denoted as S9, were prepared
154
from the same common carp which were acclimatized in the laboratory but were not
155
exposed to PFOSA. The preparation method for liver and kidney S9 was adopted
156
from Butt et al.36 and the detailed information is provided in SI. Catalase (CAT)
157
activity was used as an indicator of S9 enzymatic capacity.
158
The in vitro incubations were conducted in a series of polypropylene (PP) tubes,
159
in which 10 µL of PFOSA solution in methanol (2 ng/µL), 790 µL of 0.05 M
160
phosphate buffer (pH 7.4), 100 µL of premixed NADPH (nicotinamide adenine
161
dinucleotide phosphate) regenerating solution (containing 1.6 mM NADP+, 3.3 mM 8
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Glc-6-PO4, 0.4 U/mL G6PDH, and 3.3 mM magnesium chloride), and 100 µL of S9
163
fraction (total protein was 0.5 ± 0.01 mg) were added. All incubations were conducted
164
at 20 °C in a water bath with shaking. Three types of experimental controls were
165
applied accompanying with the tests. The first control contained active S9 and all
166
reaction components except PFOSA to correct background contamination (Control I).
167
In the second control, the incubation was the same as the test group except that S9
168
was deactivated by heating it at 100 °C for 5 min in a water bath, which was designed
169
to correct for abiotic and microbial transformation of PFOSA (Control II). The other
170
blank contained all the reaction components and was incubated in the dark to exclude
171
the possibility of photo-transformation (Control III).
172
At each sampling time, one PP tube was sacrificed and the reaction was
173
terminated by adding 0.5 ml of methanol in the reaction solution. The solution was
174
vortexed for 1 min, and was immediately stored at -20 °C until extraction and analysis
175
for target analytes. All the experiments were repeated in duplicate and the results were
176
reported as the means of the two replicates.
177
Extraction and instrumental analysis
178
The analysis of PFASs in the fish was performed following the procedure
179
described by Hansen et al.37 and water samples were extracted using the method
180
provided by Fang et al.21 Further details about the extraction are supplied in SI.
181
Analyses of the individual PFASs and isomers of PFOSA and PFOS were
182
performed on a Waters HPLC system coupled with a Waters Xevo TQ_S tandem mass
183
spectrometry (MS/MS) operated in negative electrospray ionization (ESI) mode using 9
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the method developed by Benskin et al.29 Briefly, 10 µL of the extract was injected
185
onto a FluoroSep RP Octyl HPLC column (ES Industries, West Berlin, NJ) at 38 °C.
186
The flow rate was 150 µL/min, and the program started from 60% A (water adjusted
187
to pH 4.0 with ammonium formate) and 40% B (methanol). The initial condition was
188
held for 0.3 min and then ramped to 64% B by 1.9 min; increased to 66% B by 5.9
189
min, 70% B by 7. 9 min, 74% B by 26 min, and finally to 100% B by 30 min, held
190
until 37 min; returned to initial conditions by 38 min, and the column equilibrated for
191
another 19 min. Chromatograms were recorded by multiple reaction monitoring
192
(MRM) with 1 to 9 transitions per analyte (Table S1).
193
Quality assurance and quality control
194
For the chemical analyses, a method blank (HPLC grade water) was extracted
195
with each batch of 12 samples to check background contamination, and one solvent
196
blank (HPLC grade methanol) was injected after 10 samples to monitor any
197
instrument carryover. Two quality control standard solutions (2 ng/mL MXB, 5 ng/mL
198
Br-PFOS) were run to monitor sensitivity drift along with 8-10 real samples. The
199
method detection limits (MDLs) were defined as the concentration with a
200
signal-to-noise ratio of 3 if the specific PFASs were not detected in the blanks. For the
201
analytes detected in the blanks, MDLs were defined as the mean blank concentration
202
plus three times the standard deviation of the blank (Table S2). Recoveries were
203
calculated relative to the internal standards in both the samples and standards after
204
subtracting the response of the unspiked samples. The matrix spiked recoveries of
205
water (10 ng/L) and fish whole body homogenate (5 ng/g, ww) ranged in 98-109% 10
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and 74-93%, respectively (Table S2).
207
Data analysis
208 209 210 211 212 213 214
The PFOSA branched isomers could not be baseline separated and thus were reported as the sum of branched isomers (Br-PFOSA). The growth rate was calculated using an exponential model over the exposure time: Wt = ae bt
(1)
Where Wt is the fish weight (g) at time t (d), a is the initial fish weight (g), and b is the growth rate.
215
The elimination rate constant (ke) was calculated by fitting the depuration data to
216
a first-order decay model39 using a nonlinear regression technique provided by Origin
217
V 8.5 (Origin Lab, USA):
218
C e = C t = 0 e − ke t
219
Where Ce and Ct=0 are the concentrations of PFASs in the fish (µg/kg ww) at time
220
(2)
t and the beginning of the depuration, ke is the elimination rate constant (1/d).
221
The uptake rate constant (ku) was estimated by fitting the uptake data to a
222
first-order bioaccumulation model.39 Using an interactive nonlinear regression
223
technique provided by Origin V 8.5 (Origin Lab, USA).
224
225 226
Ce =
ku C s (1 − e − ket ) ke
(3)
Where Ce is the concentration of PFASs in the fish at time t (µg/kg ww), Cs is PFASs concentration in water (µg/L), ku is the uptake rate coefficient (kg/L×d). 11
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227
Depuration half-life (t1/2) was calculated using the following Eqn:
228
t1/2 =
229
The kinetic bioaccumulation concentration factor (BCF) was estimated as the
230
231
232
ln 2 ke
(4)
quotient of uptake (ku) and elimination rate (ke) constants.40 BCF =
ku ke
(5)
Statistical analysis
233
Paired Student’s t-test was conducted to assess the difference in the growth rates,
234
HSI (hepatosomatic index) factor and the concentrations of PFOS and PFOSA
235
between the control and exposed fish samples. One way analysis of variance
236
(ANOVA) was used to assess the difference in the concentrations of PFOS and
237
PFOSA among different tissues. All statistical analyses were performed with IBM©
238
SPSS Statistics version 20 (Chicago, IL), and significance was set as p< 0.05.
239
RESULTS AND DISCUSSION
240
Fish mortality, growth rate, and HSI
241
No mortality of fish occurred in the control and exposure tests throughout the
242
experiments. At each sampling time, the fish mass and HSI were measured, and the
243
data are listed in Table S3. No significant differences in fish mass and HSI were
244
observed between the control and exposure tests (p> 0.05). The HSIs in both the
245
control and exposure tests were constant, suggesting that the fish liver functioned
246
normally during the exposure. In all the tests, the fish growth followed an exponential 12
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247
kinetic (Eqn 1) with a very low growth rate of 2.1~2.8×10-3g/d. The results indicate
248
that the spiked PFOSA did not show obvious toxicity to the fish during the exposure
249
period.
250
Uptake and elimination of PFOSA
251
During the uptake period, the concentration of PFOSA in the exposure solution
252
was relatively stable at 15.5±0.18 µg/L, and the percentage of Br-PFOSA in total
253
PFOSA (%Br-PFOSA) was also very stable at 22±1%, which was consistent with the
254
technical PFOSA product. No other PreFOSs were detected neither in the technical
255
mixture or the exposure solution. PFOS was detected at 0.01 ug/L in the technical
256
mixture and its concentration was three orders of magnitude lower than the PFOSA
257
concentration.
258
As shown in Figure 1, the fish whole body concentration of PFOSA increased
259
rapidly during the uptake phase, suggesting that PFOSA could be well accumulated in
260
carp. Its uptake did not reach steady state in the 10 d exposure. It is interesting that
261
PFOS also displayed a similar increase in fish whole body concentration as PFOSA
262
did during the uptake phase. The concentrations of PFOS and PFOSA in the fish of
263
the control groups were always less than 1% of those in the exposed fish. The increase
264
of PFOS concentration in the fish indicates that common carp has the capability to
265
metabolize PFOSA, and PFOS in the exposed fish was due to the degradation of
266
PFOSA in fish body. Previous studies reported that PFOS was the end product in
267
rainbow trout and rat following dietary exposure to PFOSA.23, 24
13
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In the current study, some PFASs, such as PFPeA, PFHxA, PFHpA, PFOA and
269
PFHxS (liver>muscle. However, in the present study, the PFOS concentration was
368
higher in the carp liver than kidney, though the total protein concentration in the carp
369
kidney (43.0 mg/g) was slightly higher than in the liver (37.7 mg/g). In the current
370
study, PFOS was originated from the degradation of PFOSA in the carp. The
371
difference in the tissue distribution (liver and kidney) of PFOSA and PFOS suggests
372
that PFOS was mainly formed in the fish liver, resulting in higher concentration in the
373
liver than in the kidney. To ascertain this assumption, in vitro tests were performed by
374
incubation of PFOSA with the carp liver and kidney S9 fractions individually, which
375
will be discussed later.
of
PFOS
in
rainbow
trout
decreased
in
the
order
of
376
In agreement with the whole body burden, all tissues contained a
377
lower %Br-PFOSA than in water (Figure 3B). The mean %Br-PFOSA in the fish 18
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tissues ranged from 6.61±2.05% in the kidney to 11.6±1.45% in liver, but there was
379
no statistically significant differences among the tissues (p>0.05). On the contrary, all
380
tissues contained a higher %Br-PFOS than the water, mostly probably due to the
381
preferential
382
The %Br-PFOS was the highest in the carp liver and generally followed the order of
383
liver>blood~kidney>muscle (Figure 3B). This further supports that the transformation
384
of PFOSA mainly occurred in the carp liver. Similar to the PFOS isomer profile in the
385
fish whole body, all branched isomers were enriched in the carp tissues compared to
386
the ECF PFOS, with the exception of 1m-PFOS, which was depleted (Figure 4).
387
Relative deficiency of 1m-PFOS in rat which was exposed to PFOSA was also
388
observed in a previous study.24 These were contradicting to the fact that 1m-PFOS
389
was the most slowly eliminated among the PFOS isomers in the animals which were
390
exposed to PFOS directly.25, 26 The most possible reason for this discrepancy is the
391
absorption or metabolism of the α-branched PFOSA isomer was much lower than
392
other branched isomers in the carp.
393
In vitro experiments
biotransformation
of
Br-PFOSA
than
n-PFOSA
(Figure
3B).
394
For Control I, the concentrations of PFOSA and PFOS in the incubation solution
395
were two order of magnitude lower than those in the test groups. The PFOSA and
396
PFOS concentrations in the test groups were corrected by subtracting the background
397
levels. PFPeA, PFHxA, PFHpA, PFOA and PFHxS (0.48×10-3~2.11×1-3 pmol)
398
were also detected both in the control and test groups, but there was no significant
399
difference between the control and test groups (p>0.05). In addition, their levels were 19
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one or several magnitudes lower than PFOS and PFOSA in the exposed fish, and did
401
not show any increasing trend. These suggest that the presence of these compounds
402
mainly from background contamination rather than degradation of PFOSA. In the
403
Control II, the PFOSA concentration was constant during the course of incubation. No
404
significant difference in the concentrations of PFOSA and PFOS was observed
405
between the Control III and the test group (p>0.05). These suggest that there was no
406
phototransformation of PFOSA during the incubation. A mass balance (Table S4) was
407
calculated, and the molar mass of PFOSA at the beginning and the total molar masses
408
of PFOSA and PFOS at the end of the experiment was consistent. This strongly
409
suggests that no other metabolic products were produced or their production was
410
negligible, which agreed with the results of the in vivo experiments.
411
Figure 5A shows the variation of the concentrations of PFOSA and PFOS during
412
the incubation. A rapid decrease in PFOSA concentration and a significant increase in
413
PFOS were observed during the 64 h incubation. The results were similar with a
414
previous study which used rainbow trout microsomes to investigate the transformation
415
of N-EtPFOSA and both PFOSA and PFOS were observed.28 In previous in vitro
416
study with rat liver microsomes, cytosol or S9 fractions, no formation of PFOS from
417
PFOSA was observed, although this was observed in the rat liver slices but with very
418
low biotransformation rate.34, 44 However, PFOSA could undergo N-glucuronidation
419
when it was incubated with rat or monkey liver microsomes in the presence of
420
UDP-glucuronic acid (UDPGA).44,
421
PFOSA in rat and monkey was mediated by Phase II metabolism instead of Phase I.44
45
It was speculated that the transformation of
20
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422
The significant transformation of PFOSA to PFOS in our in vitro incubation which
423
was mediated by liver S9 without UDPGA suggests that it was a Phase I reaction,
424
although Phase II mediated transformation could not be ruled out. The results suggest
425
that fish have different mechanisms to metabolize PFOSA compared with rat and
426
monkeys, and further studies are warranted to shed light on the difference.
427
In the test group with kidney S9, the concentration of PFOSA remained constant
428
during the entire course of incubation (Figure S1). This indicates that the
429
biotransformation of PFOSA did occur in the carp liver instead of in its kidney, which
430
supports the results obtained in the in vivo tests.
431
Figure 5B illustrates the change of the branched isomers of PFOS and PFOSA
432
during the incubation period. The %Br-PFOSA declined from 21.8±0.12% gradually
433
to 8.24±2.59% at the end of incubation, while the %Br-PFOS increased slightly
434
during the course of incubation. The biotransformation of PFOSA might be described
435
by a first-order kinetics with R2 of 0.80 and 0.84 for Br-PFOSA and n-PFOSA
436
respectively (Figure 6). The reaction rate of Br-PFOSA was significantly higher than
437
n-PFOSA, indicating that the branched PFOSA isomers were preferentially
438
transformed than n-PFOSA in the fish liver, which was consist with the in vivo tests.
439
ASSOCIATED CONTENTS
440
Supporting Information
441
Description of the S9 fraction preparation, sample extraction and tables giving
442
the MRM transition of PFASs and PFOSA, recoveries and MDLs of PFASs and
21
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PFOSA in fish samples, fish physical parameters, and mass balance in vitro
444
experiments, figures illustrating the variation of PFOSA and PFOS in the kidney S9
445
experiment. This material is available free of charge via the internet at
446
http://pubs.acs.org.
447
ACKNOWLEDGMENTS
448
We acknowledge financial support from the Natural Science Foundation of
449
China (NSFC 21325730, 21277077), Ministry of Education (20130031130005),
450
Ministry of Environmental Protection (201009026) and the Ministry of Education
451
innovation team (IRT 13024).
452
22
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454
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Muir, D. C. G. Biomagnification of perfluoroalkyl compounds in the bottlenose dolphin (Tursiops
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of
rainbow
trout
to
8:2
and
10:2
fluorotelomer
alcohols
and
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Single dose. Environ. Toxicol. Chem. 2009, 28 (3), 542-554.
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G. G. Perfluorooctane sulfonate toxicity, isomer-specific accumulation, and maternal transfer in
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Friesen, K. Biotransformation of N-ethyl perfluorooctanesulfonamide by rainbow trout
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Chem. 2003, 22 (1), 196-204. (42) Jones, P. D.; Hu, W.; De Coen, W.; Newsted, J. L.; Giesy, J. P. Binding of perfluorinated fatty acids to serum proteins. Environ. Toxicol. Chem. 2003, 22 (11), 2639-49.
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Table 1. The uptake rate (ku), elimination rate (ke), half-life (t1/2) and dynamic
585
bioconcentration factors of PFOSA and PFOS isomers in the carp whole body ke(1/d)b
Compounds
R2
Half-life(d)b
ku(L/Kg/d)b
R2
BCF(L/Kg)b
PFOSA n-PFOSA
0.10±0.01
0.92
6.93±0.70
18.5±0.96
0.92
185±8.98
Br-PFOSA
0.19±0.05
0.82
3.64±1.03
8.57±1.39
0.70
45.1±12.8
∑PFOSA
0.10±0.01
0.91
6.93±0.70
25.12±1.77
0.89
251±7.50
PFOSA (not considering the biotransformation of PFOSA) n-PFOSAa
0.10±0.01
0.92
6.93±0.70
13.4±0.65
0.88
134±7.01
Br-PFOSAa
0.19±0.05
0.82
3.64±1.03
2.04±0.19
0.70
10.7±1.96
∑PFOSAa
0.10±0.01
0.91
6.93±0.70
15.0±0.82
0.83
150±6.87
PFOS n-PFOS
0.06±0.01
0.86
11.4±1.98
1m-PFOS
0.16±0.02
0.89
4.23±0.55
3+5m-PFOS 0.14±0.02
0.70
4.78±0.72
4m-PFOS
0.15±0.02
0.73
4.68±0.63
iso-PFOS
0.10±0.02
0.88
7.29±1.44
m2-PFOS
0.09±0.02
0.95
7.96±1.80
∑PFOS
0.09±0.01
0.84
8.02±0.87
586
a
587
b
the parameters calculated without including PFOS. Mean value ± standard deviation
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Figure Captions:
589
Figure 1. The whole body concentrations of PFOSA and PFOS in the common carp
590
during the uptake and elimination phases. Each point represents the mean
591
±1 standard error. Vertical dashed line delineates the end of the uptake
592
phase.
593
Figure 2. The percentage of total branched isomers of PFOS and PFOSA in the carp
594
whole body during the uptake and elimination phases. Each point represents
595
the mean ±1 standard error. Vertical dashed line delineates the end of the
596
uptake phase.
597
Figure 3. A, The concentrations of PFOSA and PFOS in the carp tissues and blood
598
after 10 days exposure to the ECF-PFOSA. B, The percentages of branched
599
isomers of PFOSA in the carp tissues and blood after 10 days exposure to
600
the ECF-PFOSA. Each point represents the mean ±1 standard error.
601
Figure 4. Percentages of individual PFOS isomers in the tissues and whole body of
602
the common carp and in a 3M manufactured ECF-PFOS product.
603
Figure 5. Variation of the concentrations of PFOSA and PFOS (A) and percentage of
604
the %Br-PFOS and %Br-PFOSA (B) over time in the treatments with carp
605
liver S9. Each point represents the mean ±1 standard error.
606 607
Figure 6. The reaction kinetics for the biotransformation of n-PFOSA and Br-PFOSA in the treatments with carp liver S9.
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Figure 1
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Figure 4
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Figure 5
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Figure 6
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