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

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In

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Perfluorooctane Sulfonamide in Common Carp (Cyprinus carpio)

3

Meng Chen†, Liwen Qiang†, Xiaoyu Pan‡, Shuhong Fang†, Yuwei Han†, Lingyan

4

Zhu†*

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†Key Laboratory of Pollution Processes and Environmental Criteria, Ministry of

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Education, Tianjin Key Laboratory of Environmental Remediation and Pollution

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Control, College of Environmental Science and Engineering, Nankai University,

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Tianjin, P.R. China 300071

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‡ 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

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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

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this study, isomeric degradation of PFOSA was concomitantly investigated by in vivo

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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-)

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isomers, leading to enrichment of n-PFOSA in the fish. In contrast, Br-PFOS was

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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

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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.

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Keyword: PFOSA, PFOS, in vivo, in vitro, isomers, fish

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 INTRODUCTION

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Perfluorooctane sulfonate (PFOS; C8F17SO3-) and PFOS-precursors (PreFOS,

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which can degrade to PFOS) have been widely used in a variety of commercial and

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household products, due to their special physicochemical properties. Many

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toxicological studies on animals documented that PFOS and PreFOS could display

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various adverse effects to animals.1-5 Due to their global occurrence,6-9 environmental

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persistence, bioaccumulation potential,10-12 and adverse effects to biota and humans,

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PFOS and perfluorooctanesulfonyl fluoride (PFOSF; C8F17SO2F) were added in the

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list of Annex B of the Stockholm Convention on Persistent Organic Pollutants in

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2009.13

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Despite that PFOS and PFOSF were voluntarily phased out by 3M in 2000,

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PreFOS and PFOS are still being produced in China.14 It was reported that the

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production of PFOS was 200-250 t/year during 2008-2011 and that of PFOSF was up

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to 200 t/year by 2006 in China.14, 15 Perfluorooctane sulfonate (PFOS) is still one of

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the major perfluoroalkyl substances (PFASs) which are widely present in the

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environment.16-18 There are two major manufacturing methods, electrochemical

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fluorination (ECF) and telomerization, used to produce PFASs and their precursors.

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Electrochemical fluorination (ECF) had been used to synthesize PFOS and PreFOS,

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producing a mixture of around 30% of branched and 70% linear isomers in the final

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commercial products.19, 20 Perfluorooctane sulfonate (PFOS) in the environment could

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originate from direct emission or from degradation of PreFOS. Paul et al.20 estimated

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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

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degradation of PreFOS to PFOS could make a great contribution to the environmental

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burden of PFOS. Perfluorooctane sulfonamide (PFOSA; C8F17SO2NH2), which is a

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form of PreFOS,14 was more frequently detected in environmental matrices, wildlife

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and human than other PreFOS.7, 21, 22 Perfluorooctane sulfonamide (PFOSA) is usually

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the major metabolite of higher molecular weight PreFOS, such as N-EtFOSA, and it

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was then finally metabolized to PFOS in rat and rainbow trout.23, 24 It was believed

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that the degradation of PFOSA to PFOS was the rate-limiting step of the metabolism

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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

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eliminated preferentially in rats and fish,25,

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bioaccumulated.27 However, field studies on wildlife and humans always found that

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the percentage of Br-PFOS isomers (%Br-PFOS) was much higher (30-52%) than that

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in the commercial ECF-PFOS, in which the %Br-PFOS was consistently close to

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30%.22,

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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

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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

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(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

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mechanisms involved in the isomeric biotransformation of PFOSA in aquatic

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organisms.

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This study aimed to investigate the isomer specific biotransformation of PFOSA

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using common carp as a test animal. In vivo studies were conducted by exposing the

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carp to water spiked with PFOSA. The uptake, elimination, and transformation of

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PFOSA isomers in the carp tissues were extensively investigated to understand the

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isomeric accumulation and metabolism of PFOSA in the carp. To further understand

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the organs in the carp where the metabolism took place and the underlying

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mechanisms involved in the metabolism, in vitro studies were also performed by

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incubating PFOSA with the S9 fractions extracted from the carp liver and kidney.

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 MATERIALS AND METHODS

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Chemicals and Reagents

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Electrochemical fluorination (ECF) PFOS (i.e. 70% linear and 30% branched,

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by 19F NMR) was obtained from the 3M Co (St. Paul, MN, USA). All other native and

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mass labeled PFASs standards, including Br-PFOSK, PFAC–MXB, MPFAC–MXA,

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PFOSA and M8PFOSA-M were purchased from Wellington Laboratories (Guelph,

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ON, Canada). The relative percentage of linear and branched components in

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Br-PFOSK (78.8% linear, 10% iso-PFOS, 1.2% 1m-PFOS, 1.9% 3m-PFOS, 2.2%

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4m-PFOS, 4.5% 5m-PFOS, and 0.71% m2-PFOS) was provided by the Wellington

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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),

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perfluorooctanoate (PFOA), perfluorononanoate (PFNA), perfluorodecanoate (PFDA),

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perfluoroundecanoate

(PFUdA),

perfluorododecanoate

(PFDoA),

99

perfluorotetridecanoate

(PFTrDA),

perflurotetradecanoate

(PFTeDA),

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perfluorohexanesulfonate (PFHxS), PFOS and perfluorodecanesulfonate (PFDS).

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MPFAC–MXA is a mixture of mass labeled internal standards of linear PFHxA,

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PFOA, PFNA, PFDA, PFUdA, PFDoA, PFHxS and PFOS. M8PFOSA-M is a mass

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labeled internal standard for linear PFOSA. The technical product of PFOSA (~ 90%

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purity) was purchased from J & K Co. (Beijin, China).

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The bovine serum albumin (BSA), β-Nicotinamide adenine dinucleotide (NADP+),

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phosphate

107

dehydrogenase (G6PDH), magnesium chloride, phosphate buffers were purchased

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from Sigma Chemical Co. (Tianjin, China). Methanol and formic acid were of high

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performance liquid chromatography (HPLC) grade and obtained from Dikma

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Technology Inc. (Beijing, China). Sodium hydroxide (NaOH, 96.0%) and ammonium

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hydroxide solution (NH4OH, 25%) were purchased from Guangfu Fine Chemical

112

Research Institute (Tianjin, China). Methyl tert-butyl ether (MTBE) and tetrabutyl

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ammonium hydrogen sulfate (TBAH) were purchased from Concord Science and

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Technology (Tianjin, China). Other chemicals were bought from Weida Chemical

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Commercial Ltd. (Tianjin, China). Milli-Q water was used throughout the study.

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Isomer nomenclature

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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

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perfluoromethyl branch, and the number preceding m- represents the carbon number

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on which the perfluoromethyl branch resides. For example, 1m-PFOS refers to

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1-perfluoromethyl-PFOS. The sum of all diperfluoromethyl isomers, which could not

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be easily distinguished, was abbreviated as m2-PFOS. When referring to the total sum

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of all branched and linear isomers, the term PFOS was used. Due to the lack of

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authentic branched standards of PFOSA, the percentages of the isomers of PFOSA

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were estimated based on their chromatographic peak areas relative to the total peak

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areas detected with m/z 499 to 78 transitions, as reported by Asher et al.35 It was

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determined that the technical PFOSA used in the present study consisted of 78%

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n-PFOSA and 22% Br-PFOSA.

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Fish exposure tests

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Common carp, about 8 cm in length and 5-6 g in weight, were purchased from a

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local market and acclimatized in the laboratory for two weeks prior to the exposure

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tests. Filtered dechlorinated water with a hardness of 91.0±2.0 mg/L CaCO3, pH of

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7.6±0.5, dissolved oxygen of 7.0±0.4 mg/L, was maintained at 20±1oC and slightly

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aerated. Four 80 L aquariums with a flow-through system (0.01 L/min) were used for

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the tests: two for control and the other two for exposure tests. Forty fish were added in

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each aquarium and a 12 h light/12 h dark photoperiod was applied. Fish were fed

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daily at a rate of 1.0% body weight. Stock solution of technical PFOSA at 200 mg/L

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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

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technical PFOSA was set at 20 µg/L and the exposure lasted for 10 days. Two fish

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were sampled from each aquarium on days 0, 2, 4, 6, 8, and 10. At the end of

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exposure, all the remaining fish were taken out and transferred to individual tanks

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with clean filtered dechlorinated tap water for depuration, which lasted for another 10

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days. Two fish were sampled from each aquarium on days 12, 14, 16, 18, and 20.

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Upon sampling, the fish were anesthetized with tricaine methane sulfonate (MS-222).

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Blood samples were immediately taken from the fish. All the fish were subsequently

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dissected for liver, kidney and muscle. Other parts such as bones, intestines and skins

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were discarded. At each sampling time, 300 mL of water was sampled. The fish

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samples were stored at -20°C, and the water samples were stored at 4°C until

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extraction.

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In vitro incubation

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Hepatic and renal cytosol fractions, which were denoted as S9, were prepared

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from the same common carp which were acclimatized in the laboratory but were not

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exposed to PFOSA. The preparation method for liver and kidney S9 was adopted

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from Butt et al.36 and the detailed information is provided in SI. Catalase (CAT)

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activity was used as an indicator of S9 enzymatic capacity.

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The in vitro incubations were conducted in a series of polypropylene (PP) tubes,

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in which 10 µL of PFOSA solution in methanol (2 ng/µL), 790 µL of 0.05 M

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phosphate buffer (pH 7.4), 100 µL of premixed NADPH (nicotinamide adenine

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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

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at 20 °C in a water bath with shaking. Three types of experimental controls were

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applied accompanying with the tests. The first control contained active S9 and all

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reaction components except PFOSA to correct background contamination (Control I).

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In the second control, the incubation was the same as the test group except that S9

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was deactivated by heating it at 100 °C for 5 min in a water bath, which was designed

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to correct for abiotic and microbial transformation of PFOSA (Control II). The other

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blank contained all the reaction components and was incubated in the dark to exclude

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the possibility of photo-transformation (Control III).

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At each sampling time, one PP tube was sacrificed and the reaction was

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terminated by adding 0.5 ml of methanol in the reaction solution. The solution was

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vortexed for 1 min, and was immediately stored at -20 °C until extraction and analysis

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for target analytes. All the experiments were repeated in duplicate and the results were

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reported as the means of the two replicates.

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Extraction and instrumental analysis

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The analysis of PFASs in the fish was performed following the procedure

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described by Hansen et al.37 and water samples were extracted using the method

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provided by Fang et al.21 Further details about the extraction are supplied in SI.

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Analyses of the individual PFASs and isomers of PFOSA and PFOS were

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performed on a Waters HPLC system coupled with a Waters Xevo TQ_S tandem mass

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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

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onto a FluoroSep RP Octyl HPLC column (ES Industries, West Berlin, NJ) at 38 °C.

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The flow rate was 150 µL/min, and the program started from 60% A (water adjusted

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to pH 4.0 with ammonium formate) and 40% B (methanol). The initial condition was

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held for 0.3 min and then ramped to 64% B by 1.9 min; increased to 66% B by 5.9

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min, 70% B by 7. 9 min, 74% B by 26 min, and finally to 100% B by 30 min, held

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until 37 min; returned to initial conditions by 38 min, and the column equilibrated for

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another 19 min. Chromatograms were recorded by multiple reaction monitoring

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(MRM) with 1 to 9 transitions per analyte (Table S1).

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Quality assurance and quality control

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For the chemical analyses, a method blank (HPLC grade water) was extracted

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with each batch of 12 samples to check background contamination, and one solvent

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blank (HPLC grade methanol) was injected after 10 samples to monitor any

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instrument carryover. Two quality control standard solutions (2 ng/mL MXB, 5 ng/mL

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Br-PFOS) were run to monitor sensitivity drift along with 8-10 real samples. The

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method detection limits (MDLs) were defined as the concentration with a

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signal-to-noise ratio of 3 if the specific PFASs were not detected in the blanks. For the

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analytes detected in the blanks, MDLs were defined as the mean blank concentration

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plus three times the standard deviation of the blank (Table S2). Recoveries were

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calculated relative to the internal standards in both the samples and standards after

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subtracting the response of the unspiked samples. The matrix spiked recoveries of

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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).

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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

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a first-order decay model39 using a nonlinear regression technique provided by Origin

217

V 8.5 (Origin Lab, USA):

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C e = C t = 0 e − ke t

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Where Ce and Ct=0 are the concentrations of PFASs in the fish (µg/kg ww) at time

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(2)

t and the beginning of the depuration, ke is the elimination rate constant (1/d).

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The uptake rate constant (ku) was estimated by fitting the uptake data to a

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first-order bioaccumulation model.39 Using an interactive nonlinear regression

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technique provided by Origin V 8.5 (Origin Lab, USA).

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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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Depuration half-life (t1/2) was calculated using the following Eqn:

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t1/2 =

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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

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Paired Student’s t-test was conducted to assess the difference in the growth rates,

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HSI (hepatosomatic index) factor and the concentrations of PFOS and PFOSA

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between the control and exposed fish samples. One way analysis of variance

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(ANOVA) was used to assess the difference in the concentrations of PFOS and

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PFOSA among different tissues. All statistical analyses were performed with IBM©

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SPSS Statistics version 20 (Chicago, IL), and significance was set as p< 0.05.

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 RESULTS AND DISCUSSION

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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

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that the spiked PFOSA did not show obvious toxicity to the fish during the exposure

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period.

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Uptake and elimination of PFOSA

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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

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technical PFOSA product. No other PreFOSs were detected neither in the technical

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mixture or the exposure solution. PFOS was detected at 0.01 ug/L in the technical

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mixture and its concentration was three orders of magnitude lower than the PFOSA

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concentration.

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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

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carp. Its uptake did not reach steady state in the 10 d exposure. It is interesting that

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PFOS also displayed a similar increase in fish whole body concentration as PFOSA

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did during the uptake phase. The concentrations of PFOS and PFOSA in the fish of

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the control groups were always less than 1% of those in the exposed fish. The increase

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of PFOS concentration in the fish indicates that common carp has the capability to

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metabolize PFOSA, and PFOS in the exposed fish was due to the degradation of

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PFOSA in fish body. Previous studies reported that PFOS was the end product in

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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

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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

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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

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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

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negligible, which agreed with the results of the in vivo experiments.

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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

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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

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

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rainbow

trout

to

8:2

and

10:2

fluorotelomer

alcohols

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

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

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

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

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

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

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

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

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

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

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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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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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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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(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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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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609 610 611

Figure 1

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612 613 614 615 616

Figure 2

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Figure 3

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622 623 624 625

Figure 4

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Figure 5

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Figure 6

635

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TOC/Abstract art

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