In Vivo and in Vitro Isomer-Specific Biotransformation of

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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

ACS Paragon Plus Environment


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

29 2


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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



52

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

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metabolism would affect the isomer profiles of PFOS in the environment.24

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Some studies have reported that the toxicity and toxicokinetics of PFASs was

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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

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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

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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

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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



of

perfluorohexanoate

(PFHxA),

Page 6 of 35

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



140

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



184

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



Page 12 of 35

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



268

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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378

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



400

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



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

22


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453

REFERENCES

454

(1)

Seacat, A. M.; Thomford, P. J.; Hansen, K. J.; Olsen, G. W.; Case, M. T.; Butenhoff, J.

455

L.Subchronic toxicity studies on perfluorooctanesulfonate potassium salt in Cynomolgus

456

monkeys. Toxicol. Sci. 2002, 68 (1), 249-264.

457

(2)

Androl. 2008, 31 (2), 161-169.

458 459

Jensen, A. A.; Leffers, H. Emerging endocrine disrupters: Perfluoroalkylated substances. Int. J.

(3)

Hu, W. Y.; Jones, P. D.; Upham, B. L.; Trosko, J. E.; Lau, C.; Giesy, J. P. Inhibition of gap

460

junctional intercellular communication by perfluorinated compounds in rat liver and dolphin

461

kidney epithelial cell lines in vitro and Sprague-Dawley rats in vivo. Toxicol. Sci. 2002, 68 (2),

462

429-436.

463

(4)

review of monitoring and toxicological findings. Toxicol. Sci. 2007, 99 (2), 366-94.

464 465

(5)

(6)

Giesy, J. P.; Kannan, K. Global distribution of perfluorooctane sulfonate in wildlife. Environ. Sci. Technol. 2001, 35 (7), 1339-42.

468 469

Luebker, D. J.; Hansen, K. J.; Bass, N. M.; Butenhoff, J. L.; Seacat, A. M. Interactions of flurochemicals with rat liver fatty acid-binding protein. Toxicology. 2002, 176 (3), 175-185.

466 467

Lau, C.; Anitole, K.; Hodes, C.; Lai, D.; Pfahles-Hutchens, A.; Seed, J. Perfluoroalkyl acids: A

(7)

Tomy, G. T.; Budakowski, W.; Halldorson, T.; Helm, P. A.; Stern, G. A.; Friesen, K.; Pepper, K.;

470

Tittlemier, S. A.; Fisk, A. T. Fluorinated organic compounds in an eastern Arctic marine food

471

web. Environ. Sci. Technol. 2004, 38 (24), 6475-6481.

472

(8)

Houde, M.; Bujas, T. a. D.; Small, J.; Wells, R. S.; Fair, P. A.; Bossart, G. D.; Solomon, K. R.;

473

Muir, D. C. G. Biomagnification of perfluoroalkyl compounds in the bottlenose dolphin (Tursiops

474

truncatus) food web. Environ. Sci. Technol. 2006, 40 (13), 4138-4144.

475

(9)

Yu, N.; Shi, W.; Zhang, B.; Su, G.; Feng, J.; Zhang, X.; Wei, S.; Yu, H. Occurrence of

476

perfluoroalkyl acids including perfluorooctane sulfonate isomers in Huai River basin and Taihu

477

Lake in Jiangsu province, China. Environ. Sci. Technol. 2013, 47 (2), 710-7.

478 479 480 481

(10) Martin, J. W.; Whittle, D. M.; Muir, D. C. G.; Mabury, S. A. Perfluoroalkyl contaminants in a food web from lake Ontario. Environ. Sci. Technol. 2004, 38 (20), 5379-5385. (11) Houde, M.; Martin, J. W.; Letcher, R. J.; Solomon, K. R.; Muir, D. C. Biological monitoring of polyfluoroalkyl substances: A review. Environ. Sci. Technol. 2006, 40 (11), 3463-73.

482

(12) Tomy, G. T.; Pleskach, K.; Ferguson, S. H.; Hare, J.; Stern, G.; Macinnis, G.; Marvin, C. H.;

483

Loseto, L. Trophodynamics of some PFCs and BFRs in a Western Canadian arctic marine food

484

web. Environ. Sci. Technol. 2009, 43 (11), 4076-4081.

485

(13) Wang, T.; Wang, Y. W.; Liao, C. Y.; Cai, Y. Q.; Jiang, G. B. Perspectives on the inclusion of

486

perfluorooctane sulfonate into the Stockholm Convention on Persistent Organic Pollutants.

487

Environ. Sci. Technol. 2009, 43 (14), 5171-5175.

488

(14) Martin, J. W.; Asher, B. J.; Beesoon, S.; Benskin, J. P.; Ross, M. S. PFOS or PreFOS? Are

489

perfluorooctane sulfonate precursors (PreFOS) important determinants of human and

490

environmental perfluorooctane sulfonate (PFOS) exposure? J. Environ. Monit. 2010, 12 (11), 23



491 492 493

Page 24 of 35

1979-2004. (15) Xie, S.; Wang, T.; Liu, S.; Jones, K. C.; Sweetman, A. J.; Lu, Y. Industrial source identification and emission estimation of perfluorooctane sulfonate in China. Environ. Int. 2013, 52, 1-8.

494

(16) Loewen, M.; Wania, F.; Wang, F. Y.; Tomy, G. Altitudinal transect of atmospheric and aqueous

495

fluorinated organic compounds in Western Canada. Environ. Sci. Technol. 2008, 42 (7),

496

2374-2379.

497 498

(17) Kato, K.; Calafat, A. M.; Needham, L. L. Polyfluoroalkylchemicals in house dust. Environ.Res. 2009, 109, 518-523.

499

(18) Olsen, G. W.; Mair, D. C.; Church, T. R.; Ellefson, M. E.; Reagen, W. K.; Boyd, T. M.; Herron,

500

R. M.; Medhdizadehkashi, Z.; Nobilett, J. B.; Rios, J. A.; Butenhoff, J. L.; Zobel, L. R. Decline in

501

perfluorooctanesulfonate and other polyfluoroalkyl chemicals in American Red Cross adult blood

502

donors, 2000-2006. Environ. Sci. Technol. 2008, 42 (13), 4989-4995.

503 504 505 506

(19) Prevedouros, K.; Cousins, I. T.; Buck, R. C.; Korzeniowski, S. H. Sources, fate and transport of perfluorocarboxylates. Environ. Sci. Technol. 2006, 40 (1), 32-44. (20) Paul, A. G.; Jones, K. C.; Sweetman, A. J. A first global production, emission, and environmental inventory for perfluorooctane sulfonate. Environ. Sci. Technol. 2009, 43 (2), 386-392.

507

(21) Fang, S.; Chen, X.; Zhao, S.; Zhang, Y.; Jiang, W.; Yang, L.; Zhu, L. Trophic magnification and

508

isomer fractionation of perfluoroalkyl substances in the food web of Taihu Lake, China. Environ.

509

Sci. Technol. 2014, 48 (4), 2173-82.

510

(22) Zhang, Y.; Jiang, W.; Fang, S.; Zhu, L.; Deng, J. Perfluoroalkyl acids and the isomers of

511

perfluorooctanesulfonate and perfluorooctanoate in the sera of 50 new couples in Tianjin, China.

512

Environ. Int. 2014, 68, 185-91.

513

(23) Brandsma, S. H.; Smithwick, M.; Solomon, K.; Small, J.; De Boer, J.; Muir, D. C. Dietary

514

exposure

515

perfluorooctanesulfonamide: Uptake, transformation and elimination. Chemosphere. 2011, 82 (2),

516

253-8.

517 518

of

rainbow

trout

to

8:2

and

10:2

fluorotelomer

alcohols

and

(24) Ross, M. S.; Wong, C. S.; Martin, J. W. Isomer-specific biotransformation of perfluorooctane sulfonamide in Sprague-Dawley rats. Environ. Sci. Technol. 2012, 46 (6), 3196-203.

519

(25) Benskin, J. P.; De Silva, A. O.; Martin, L. J.; Arsenault, G.; Mccrindle, R.; Riddell, N.; Mabury, S.

520

A.; Martin, J. W. Disposition of perfluorinated acid isomers in Sprague-Dawley Rats; Part 1:

521

Single dose. Environ. Toxicol. Chem. 2009, 28 (3), 542-554.

522

(26) De Silva, A. O.; Benskin, J. P.; Martin, L. J.; Arsenault, G.; Mccrindle, R.; Riddell, N.; Martin, J.

523

W.; Mabury, S. A. Disposition of perfluorinated acid isomers in Sprague-Dawley rats; Part 2:

524

Subchronic dose. Environ. Toxicol. Chem. 2009, 28 (3), 555-567.

525

(27) Sharpe, R. L.; Benskin, J. P.; Laarman, A. H.; Macleod, S. L.; Martin, J. W.; Wong, C. S.; Goss,

526

G. G. Perfluorooctane sulfonate toxicity, isomer-specific accumulation, and maternal transfer in

527

zebrafish (Danio rerio) and rainbow trout (Oncorhynchus mykiss). Environ. Toxicol. Chem. 2010,

528

29 (9), 1957-66.

529

(28) Tomy, G. T.; Tittlemier, S. A.; Palace, V. P.; Budakowski, W. R.; Braekevelt, E.; Brinkworth, L.; 24


Page 25 of 35


530

Friesen, K. Biotransformation of N-ethyl perfluorooctanesulfonamide by rainbow trout

531

(Onchorhynchus mykiss) liver microsomes. Environ. Sci. Technol. 2004, 38 (3), 758-762.

532

(29) Benskin, J. P.; Bataineh, M.; Martin, J. W. Simultaneous characterization of perfluoroalkyl

533

carboxylate, sulfonate, and sulfonamide isomers by liquid chromatography-tandem mass

534

spectrometry. Anal. Chem. 2007, 79 (17), 6455-6464.

535

(30) Benskin, J. P.; De Silva, A. O.; Martin, J. W. Isomer profiling of perfluorinated substances as a

536

tool for source tracking: A review of early findings and future applications. Rev. Environ.

537

Contam. Toxicol. 2010, 208, 111-60.

538

(31) Haug, L. S.; Thomsen, C.; Bechert, G. Time trends and the influence of age and gender on serum

539

concentrations of perfluorinated compounds in archived human samples. Environ. Sci. Technol.

540

2009, 43 (6), 2131-2136.

541 542

(32) Butt, C. M.; Berger, U.; Bossi, R.; Tomy, G. T. Levels and trends of poly- and perfluorinated compounds in the arctic environment. Sci. Total Environ. 2010, 408 (15), 2936-65.

543

(33) Peng, H.; Zhang, S.; Sun, J.; Zhang, Z.; Giesy, J. P.; Hu, J. Isomer-specific accumulation of

544

perfluorooctanesulfonate from (N-Ethyl perfluorooctanesulfonamido)ethanol-based phosphate

545

diester in Japanese medaka (Oryzias latipes). Environ. Sci. Technol. 2014, 48 (2), 1058-1066.

546

(34) Benskin, J. P.; Holt, A.; Martin, J. W. Isomer-specific biotransformation rates of a perfluorooctane

547

sulfonate (PFOS)-precursor by Cytochrome P450 isozymes and human liver microsomes.

548

Environ. Sci. Technol. 2009, 43 (22), 8566-8572.

549

(35) Asher, B. J.; Wang, Y.; De Silva, A. O.; Backus, S.; Muir, D. C.; Wong, C. S.; Martin, J. W.

550

Enantiospecific perfluorooctane sulfonate (PFOS) analysis reveals evidence for the source

551

contribution of PFOS-precursors to the Lake Ontario foodweb. Environ. Sci. Technol. 2012, 46

552

(14), 7653-60.

553

(36) Butt, C. M.; Muir, D. C.; Mabury, S. A. Biotransformation of the 8:2 fluorotelomer acrylate in

554

rainbow trout. 2. In vitro incubations with liver and stomach S9 fractions. Environ. Toxicol.

555

Chem. 2010, 29 (12), 2736-41.

556

(37) Hansen, K. J.; Clemen, L. A.; Ellefson, M. E.; Johnson, H. O. Compound-specific, quantitative

557

characterization of organic fluorochemicals in biological matrices. Environ. Sci. Technol. 2001,

558

35 (4), 766-770.

559

(38) Weiss, J. M.; Andersson, P. L.; Lamoree, M. H.; Leonards, P. E. G.; Van Leeuwen, S. P. J.;

560

Hamers, T. Competitive binding of poly- and perfluorinated compounds to the thyroid hormone

561

transport protein transthyretin. Toxicol. Sci. 2009, 109 (2), 206-216.

562

(39) O'brien, J. M.; Kennedy, S. W.; Chu, S. G.; Letcher, R. J. Isomer-specific accumulation of

563

perfluorooctane sulfonate in the liver of chicken embryos exposed in ovo to a technical mixture.

564

Environ. Toxicol. Chem. 2011, 30 (1), 226-231.

565 566

(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.

567

(41) Martin, J. W.; Mabury, S. A.; Solomon, K. R.; Muir, D. C. G. Bioconcentration and tissue

568

distribution of perfluorinated acids in rainbow trout (Oncorhynchus mykiss). Environ. Toxicol. 25



569 570 571

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.

572

(43) Honda, M.; Muta, A.; Akasaka, T.; Inoue, Y.; Shimasaki, Y.; Kannan, K.; Okino, N.; Oshima, Y.

573

Identification of perfluorooctane sulfonate binding protein in the plasma of tiger pufferfish

574

Takifugu rubripes. Ecotoxicol. Environ. Saf. 2014, 104, 409-13.

575

(44) Xu, L.; Krenitsky, D. M.; Seacat, A. M.; Butenhoff, J. L.; Anders, M. W. Biotransformation of

576

N-ethyl-N-(2-hydroxyethyl) perfluorooetanesulfonamide by rat liver microsomes, cytosol, and

577

slices and by expressed rat and human cytochromes P450. Chem. Res. Toxicol. 2004, 17 (6),

578

767-775.

579

(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

581

microsomes and by expressed rat and human UDP-glucuronosyltransferases. Drug. Metab.

582

Dispos. 2006, 34 (8), 1406-10.

583

26


Page 26 of 35

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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



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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608

609 610 611

Figure 1

29



612 613 614 615 616

Figure 2

30


Page 30 of 35

Page 31 of 35


617 618 619 620 621

Figure 3

31



622 623 624 625

Figure 4

32


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626 627 628 629 630

Figure 5

33



631 632 633 634

Figure 6

635

34


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636 637 638

639 640

TOC/Abstract art

641 642 643

35


In Vivo and in Vitro Isomer-Specific Biotransformation of

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