Long-Chain Perfluoroalkyl acids (PFAAs) Affect the Bioconcentration

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Long-chain perfluoroalkyl acids (PFAAs) affect the bioconcentration and tissue distribution of short-chain PFAAs in zebrafish (Danio rerio) Wu Wen, Xinghui Xia, Diexuan Hu, Dong Zhou, Haotian Wang, Yawei Zhai, and hui Lin Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b03647 • Publication Date (Web): 09 Oct 2017 Downloaded from http://pubs.acs.org on October 11, 2017

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Long-chain perfluoroalkyl acids (PFAAs) affect the bioconcentration and

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tissue distribution of short-chain PFAAs in zebrafish (Danio rerio)

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Wu Wen, Xinghui Xia*, Diexuan Hu, Dong Zhou, Haotian Wang, Yawei Zhai, Hui Lin

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School of Environment, Beijing Normal University, State Key Laboratory of Water Environment

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Simulation, Beijing 100875, China

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*

Corresponding author. Tel./fax: +86 10 58805314.

E-mail address: [email protected] (X. Xia) 1 / 34

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Abstract

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Short- and long-chain perfluoroalkyl acids (PFAAs), ubiquitously co-existing in the environment,

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can be accumulated in organisms by binding with proteins and their binding affinities generally

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increase with their chain length. Therefore, we hypothesized that long-chain PFAAs will affect the

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bioconcentration of short-chain PFAAs in organisms. To testify this hypothesis, the bioconcentration

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and tissue distribution of five short-chain PFAAs (linear C-F=3-6) were investigated in zebrafish in

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the absence and presence of six long-chain PFAAs (linear C-F=7-11). The results showed that the

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concentrations of the short-chain PFAAs in zebrafish tissues increased with exposure time till steady

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states reached in the absence of long-chain PFAAs. However, in the presence of long-chain PFAAs,

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these short-chain PFAAs in tissues increased till peak values reached and then decreased till steady

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states, and the uptake and elimination rate constants of short-chain PFAAs declined in all tissues and

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their BCFss decreased by 24-89%. The inhibitive effect of long-chain PFAAs may be attributed to

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their competition for transporters and binding sites of proteins in zebrafish with short-chain PFAAs.

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These results suggest that the effect of long-chain PFAAs on the bioconcentration of short-chain

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PFAAs should be taken into account in assessing the ecological and environmental effects of short-

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chain PFAAs.

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Key words: Short-chain perfluoroalkyl acids (PFAAs); Bioconcentration; Tissue distribution;

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Zebrafish (Danio rerio); Perfluorinated substances (PFASs).

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Abstract Art I’m a long-chain PFAA. Protein

Concentration/(ng·g-1, ww)

I’m a short-chain PFAA.

Liver

700 600 500 400 300 200 100 0 0

5

10

15

Time/(d-1)

Protein

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1 Introduction

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Perfluoroalkyl acids (PFAAs) are a class of synthetic organic compounds characterized by a

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hydrophobic perfluoroalkyl chain and a hydrophilic carboxylate or a sulfonate functional group.

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Because of their special physical and chemical properties (thermal and chemical stability,

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hydrophobicity and lipophobicity), the long-chain PFAAs (no less than seven fluorinated carbons)

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have been manufactured and used in numerous industrial and commercial applications for over half

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century,1 which leads to their widespread distribution in the environment.2-4 Additionally, some of

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these pollutants have been testified to be bioaccumulative5, 6 and toxic.7 From 2000, 3M Company

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gradually discontinued the manufacture of some long-chain perfluoroalkyl substances. In 2002, the

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U.S. Environmental Protection Agency published a rule to regulate the use of perfluorooctanoic

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sulfonate(PFOS) and related compounds.8 In 2009, at the 4th Conference of the Parties in the

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Stockholm Convention, PFOS and related compounds were registered under Annex B of the

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Stockholm Convention on Persistent Organic Pollutants. Meanwhile, some short-chain PFAAs (less

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than seven fluorinated carbons),9 which have the similar physiochemical properties to long-chain

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PFAAs, have been synthesized and used as substitutes for long-chain PFAAs to satisfy the

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application demand.9-11

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With their extensive use around the world, the short-chain PFAAs have been detected in water,

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such as rivers and lakes.4, 9, 12, 13 Studies show that perfluorobutyric acid (PFBA) and perfluorobutane

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sulfonate (PFBS) are the most abundant short-chain PFAAs in water,9, 14, 15 and their concentrations

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are sometimes up to μg·L-1.9 In addition, the short-chain PFAAs have also been detected in aquatic

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animals, and even in humans.12, 16-18 Numerous investigations show that some short-chain PFAAs

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have increasing temporal shifting trends in water and aquatic organisms. 19-21 For example, a

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significant increasing trend of PFBS has been found in cetacean and dolphin liver samples from 2002

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to 2014 due to the increasing usage of C4 based alternatives.21 It suggests that some short-chain

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PFAAs show a potential bioconcentration in some kinds of organisms. However, the information

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about the distribution and bioconcentration of short-chain PFAAs in different tissues of aquatic biota,

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such as fish, is limited.5, 22 4 / 34

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Numerous studies suggest that the interaction of PFAAs and proteins plays a key role in the

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PFAA bioaccumulation.23-25 Researchers find that some types of proteins can bind with PFAAs,

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including serum albumin, liver fatty acid-binding protein, some transport proteins, etc.26-30

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According to the result of the molecular docking analysis, hydrophobic fluorinated carbon chain can

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extend to the interior of the binding pocket to make the maximum hydrophobic contact while

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hydrophilic acidic group faces towards the entrance of the pocket.7, 27, 28 Therefore, their binding

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affinities increase with the number of fluorinated carbons.27, 28 For the binding of short-chain PFAAs

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with proteins, the stability may be weaker than long-chain PFAAs. Additionally, the binding sites of

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proteins are reported to be limited for PFAAs.31, 32 Therefore, we hypothesized that the long-chain

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PFAAs will affect the bioconcentration and tissue distribution of short-chain PFAAs in fish.

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To test this hypothesis, the effects of multiple long-chain PFAAs on the bioconcentration and

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tissue distribution of multiple short-chain PFAAs in zebrafish (Danio rerio) were studied in the

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present research, considering that many PFAAs often co-exist in the environment.4, 12, 33 The tested

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short-chain PFAAs included PFBA, PFBS, perfluoropentanoic acid (PFPeA), perfluorohexanoic

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acid (PFHxA), and perfluoroheptanoic acid (PFHpA) which are widely detected in environmental

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media, and the long-chain PFAAs included perfluorooctanoic acid (PFOA), PFOS,

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perfluorononanoic acid (PFNA), perfluorodecanoic acid (PFDA), perfluoroundecanoic acid

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(PFUnA), and perfluorododecanoic acid (PFDoA). The bioconcentration kinetics, tissue distribution

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including blood, brain, gill, intestine, liver, muscle and ovary, and bioconcentration factors (BCFss,

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at steady state) of the short-chain PFAAs were investigated in the absence and presence of the long-

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chain PFAAs. The effect of PFAA properties and tissue types on the bioconcentration was also

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investigated. Furthermore, the effect mechanisms of long-chain PFAAs on the bioconcentration

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kinetics, tissue distribution, and BCFss of short-chain PFAAs were also analyzed.

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2. Materials and Methods

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2.1 Chemicals and reagents

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PFBA (99%) were obtained from Matrix Scientific Trade Co. (Columbia, USA). PFPeA (98%)

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and PFHpA (98%) were supplied by Tokyo Chemical Industries (Tokyo, Japan). PFBS (98%) and

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PFHxA (98%), PFOA (96%), PFOS (98%), PFNA (97%), PFDA (98%), PFUnA (95%), and PFDoA

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(95%) were purchased from Acros Organics (New Jersey, USA). Isotopically labeled standards,

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including 13C4-PFBA (MPFBA, 99%), 13C4-PFOA (MPFOA, 99%), 13C4-PFBA (MPFOS, 99%) and

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13

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Wellington Laboratories (Guelph, Canada). Two stock solutions were prepared for fish exposure.

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The first one contained five short-chain PFAAs at a total concentration of 1000 mg·L-1 in methanol

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(200 mg·L-1 for each PFAA). The second one contained six long-chain PFAAs at a total

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concentration of 1200 mg·L-1 in methanol (200 mg·L-1 for each PFAA). Chromatography grade

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methanol and acetonitrile were supplied by J.T. Baker of Phillipsburg (NJ, USA). Methyl-tert-butyl

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ether (MTBE, 99.5%) was purchased from Acros Organics (New Jersey, USA). Ammonium acetate

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(98%) and tetrabutylammonium hydrogen sulfate (TBA, 99%) were obtained from Amethyst

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Chemicals of J&K Scientific Ltd. (Beijing, China). Any water used in this study was produced by a

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Milli-Q purification system (Millipore, Germany).

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2.2 Zebrafish maintenance and experimental design

C4-PFDoA (MPFDoA, 99%), which were used as recovery indicators, were supplied by

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Given that some PFAAs can be transferred from mother into egg cells and cause toxic effects

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on offspring,34-36 adult female zebrafish were chose as the test fish. They were purchased from a

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local fish farm in Beijing and were allowed to acclimate to laboratory conditions in dechlorinated

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tap water for two weeks prior to PFAA exposure. The acclimation and following exposure

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experiments were conducted with a 14:10 hour light/dark photoperiod at a temperature of 23±2oC

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and the zebrafish were fed with commercial fodder daily.

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All exposure experiments were conducted in polypropylene (PP) tanks. These tanks were

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divided into three groups. The first group was used as the control without any PFAAs. The second

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group was used for the five short-chain PFAA exposure at a total concentration of 50 μg·L-1 (10

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μg·L-1 for each PFAA). The third group was used for the five short-chain and six long-chain PFAA

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exposure at a total concentration of 110 μg·L-1 (10 μg·L-1 for each PFAA). Each tank contained 50 6 / 34

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randomly selected zebrafish. The bioconcentration experiment lasted 28 days. Throughout the

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exposure period, the water in each tank was replaced daily. Uneaten food was siphoned from the

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containers after feeding (30 minutes). The faeces of zebrafish was also siphoned twice a day. The

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control group without any PFAAs was conducted the same as the PFAA-exposed groups. Three

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replicate tanks were available for the control and PFAA-exposed groups.

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To check for the PFAA concentration of the exposure water, fifty-milliliter water from each

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tank was collected at the beginning and end of the experiment, as well as after 24 hours’ exposure.

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Five fish (pooled as a sample) from each tank were sampled at 1, 2, 3, 5, 9, 14, 21, and 28 d for

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PFAA analysis. Sampled fish were anesthetized and rinsed with Milli-Q water, and then dried with

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filter paper. A blood sample was drawn from the caudal vein, and fish were subsequently killed by

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cervical dislocation. Fish tissues (blood, brain, gill, intestine, liver, muscle, and ovary) were collected

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separately and the wet weight (ww) was obtained. All samples were homogenized for the analysis

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of PFAA concentration. All zebrafish were treated humanely with the aim of alleviating any

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suffering according to the OECD Guideline for the Testing of Chemicals.37

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2.3 Sample preparation and PFAA extraction

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For the determination of the short- and long-chain PFAAs in water, all water samples were

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pretreated as previously reported by Taniyasu et al.38 and Zhou et al.9 with some modifications.

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Briefly, the Oasis WAX cartridges (Waters®, 6 cc, 150 mg) were preconditioned with 4 mL of

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methanol containing 0.1% ammonium hydroxide (in methanol), 4 mL of methanol, and 4mL of

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Milli-Q water, sequentially. Then, 4 mL of water sample (without filtering) spiked with 10 ng of

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each recovery indicator was passed through the cartridge at a rate of one drop per second. The

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cartridge was washed with 4 mL of buffer (25 mM acetic acid in Milli-Q water), and then the residual

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water was removed with vacuum pump. The PFAAs were eluted sequentially with 4 mL of methanol

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and 4 mL of methanol with 0.1% ammonium hydroxide. The eluate was evaporated to near dryness

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under a gentle nitrogen stream and reconstituted with 1 mL of methanol and filtered with 0.2-μm

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nylon membranes into PP autosamper vials with PP caps for injection. 7 / 34

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For the determination of the short- and long-chain PFAAs in blood, samples (obtained from 5

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pooled fish) were extracted by the method reported by Yeung et al.39 with some modifications.

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Briefly, the blood sample was spiked with 10 ng of each recovery indicator. A total of 2 mL of

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acetonitrile was added and extracted via sonication for 30 min, followed by centrifugation at 3000

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rpm at 4 oC for 15 min. The supernatant was transferred to another clean tube. The extraction was

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repeated with a further 2 mL of acetonitrile twice. The extracts were combined and evaporated under

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a gentle nitrogen stream to about 0.5 mL and then diluted with 50 mL of Milli-Q water. The diluent

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was then loaded onto a preconditioned Oasis WAX cartridge. The subsequent procedures were the

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same as the water samples. The eluate was reconstituted with 0.4-0.8 mL methanol and filtered for

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injection.

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For the determination of the short- and long-chain PFAAs in muscle, samples (obtained from 5

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pooled fish) were extracted with the method reported by So et al.40 and Taniyasu et al.38 with some

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modifications. Briefly, the homogenized sample spiked with 10 ng of each recovery indicator was

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sonicated in 5 mL of 10 mM KOH (in methanol) at 60 °C for 30 min, and then vortexed, shaken

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(300 rpm, 25 oC, 16 h), and centrifuged (4000 rpm, 15 min). The supernatant was transferred to

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another clean tube. The extraction repeated with a further 5 mL of 10 mM KOH (in methanol) twice.

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The extracts were combined and the subsequent procedures were the same as blood samples. The

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eluate was reconstituted with 0.5-1 mL methanol and filtered for injection.

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For the determination of the short- and long-chain PFAAs in gill, brain, liver, intestine, and

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ovary, samples (obtained from 5 pooled fish) were extracted by iron-pairing method.41 In brief, the

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homogenized tissue sample spiked with 10 ng of each recovery indicator was shaken (250 rpm, 30

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min, 25±0.5 oC) in the iron-pairing solution containing 0.5 mL of 0.5 M TBA (pH=10), 1 mL of 0.25

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M sodium carbonate buffer (pH=10), and 2.5 mL MTBE. The organic layer was separated by

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centrifuging (4000 rmp, 15 min) and transferred to a clean tube. The extraction was repeated with a

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further 2.5 mL of MTBE twice. All extracts were combined in 10-mL PP centrifuge tube. The

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subsequent procedures were the same as blood samples. The eluate was reconstituted with 0.2-1 mL

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2.4 Instrumental analysis of PFAAs

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The short- and long-chain PFAAs were analyzed by liquid chromatography-tandem mass

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spectrometry (LC-MS/MS; Dionex Ultimate 3000 and Applied Biosystems API 3200) in

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electrospray negative ionization mode. Chromatographic separation was in combination with a

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binary pump, an autosampler and a column oven equipped with a Waters Sunfire C18 Column (5

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μm pore size, 4.6×150 mm). The injection volume was 10 μL. Water (5 mM ammonium acetate)

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and methanol were used as mobile phase. At a flow rate of 1 mL·min-1, the mobile phase gradient

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started with a 1 min isocratic step at 70% of methanol, and then ramped to 95% of methanol in 6.5

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min, held at 95% of methanol for 3 min, and then ramped down to 70% of methanol in 0.5 min. For

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quantitative analysis, analyte ions were monitored using multiple reaction monitoring mode. The

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details of instrument parameters are shown in Tables S1 and S2.

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2.5 Data analysis

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All statistical analyses were performed using SPSS 18.0 for windows (SPSS Inc., Chicago

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(IL)., USA) and Microsoft Excel 2016. Bioconcentration data were dynamically fit by Origin 2016

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to obtain the kinetic parameters. Analysis of variance (ANOVA, one factor) was carried out to test

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differences between each two compared groups, and difference was considered significant when

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the significance level was smaller than 0.05.

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2.6 Quality assurance and quality control (QA/QC)

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Throughout the experiments, disposable PP beakers, bottles, tubes, and pipettes were used to

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prevent target compounds adsorption and contamination from glassware and other vessels

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containing fluorochemicals. All vessels were cleaned by methanol three times before use.

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The method detection limits (MDLs) and method quantitation limits(MQLs) for the target

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compounds were defined as tree and ten times of the signal to noise ratio, respectively, and the

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details of the MDLs/MQLs for water and tissues are listed in Table S3. All the target chemicals in

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samples were quantified by external standard calibration. Calibration standards were prepared in 9 / 34

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methanol with the concentrations for each PFAA ranging from 0.2 to 200 μg·L-1. The correlation

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coefficients of the standard curves were higher than 0.99. To ensure the extraction efficiency and

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accuracy of determination, spike recovery experiments for target PFAAs in water and biological

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samples were conducted, and the recoveries ranged from 75.2±3.2% to 105±14.8% and from 73.3

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±1.9% to 104±10.0% (Table S4), respectively. Procedural blanks and calibration verification

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standards were run after every 10 samples to check the background contamination and the validity

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of the calibration. A new calibration curve was run if the quality-control standard was not measured

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within ±20% of its theoretical value. The variations of measured PFAA concentration of the

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exposure solution were lower than 5% (Table S5), which indicated that the aqueous PFAA

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concentration did not change significantly during the exposure experiment. In addition, PFAAs in

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tissues of zebrafish in the control group ranged from no detection to 11.69 ng·gww-1 (PFHpA in

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liver) but they were at least 50 times lower than that in the exposure group. The concentrations of

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target compounds in fish food were tested. Their concentrations were lower than MDLs, except for

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PFOS, which was higher than MDL but lower than MQL.

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3 Results and Discussion

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3.1 Body condition of zebrafish

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Brain-somatic index (BSI), hepatic-somatic index (HSI), and condition factor (K-factor) were

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monitored to assess any toxicological effects on zebrafish.42 As shown in Table S6, no significant

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differences in the length and weight of zebrafish were observed among different groups. No

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significant differences were observed in the BSI, HSI, and K-factor between PFAA-exposed and

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control groups. No mortality was observed in all the control and PFAA-exposed groups. These

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results suggest that the exposure condition did not exert significant toxicological effects on zebrafish.

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3.2 Bioconcentration of the short-chain PFAAs in zebrafish in the absence of the long-

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chain PFAAs

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3.2.1 Bioconcentration kinetic parameters in the absence of the long-chain PFAAs. 10 / 34

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All target compounds were detected in tissues of zebrafish except PFBA in brain and ovary.

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According to the bioconcentration curves (Figure 1), the concentration of the short-chain PFAAs in

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tissues (liver, blood, intestine, gill, ovary, brain, and muscle) increased with exposure time at the

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first few days, and a steady state reached after 5 days’ exposure. Bioconcentration kinetic parameters

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for the short-chain PFAAs in tissues were obtained by fitting the curves with the two-compartment

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kinetic model: 43 k

Cb =Cw ku0 ·(1-e-ke0·t )

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e0

(1)

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Where Cb is the PFAA concentration in a tissue (ng·gww-1) at time t (d); Cw is the PFAA

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concentration in the water (ng·mL-1); ku0 (mL·gww-1·d-1) and ke0 (d-1) are the uptake and elimination

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rate constants of PFAAs in a tissue, respectively; the subscript of “0” refers to the kinetic parameters

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in the absence of the long-chain PFAA exposure. The results are shown in Table 1. The ku0 values

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were influenced by the PFAA properties and the tissue types. Firstly, the ku0 values increased with

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the fluorinated carbons in any tissue, which was consist with the bioconcentration of long-chain

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PFAAs (C-F≥7) in aquatic animals.5, 44 Take the liver for example, the ku0 values for PFBA (C-F=3),

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PFPeA (C-F=4), PFHxA (C-F=5), and PFHpA (C-F=6) were 5.1, 13, 18, and 36 mL·gww-1·d-1

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respectively. Secondly, the acidic headgroup had an influence on ku0 values. Although both PFBS

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and PFPeA had four fluorinated carbons, the ku0 value of PFBS in each tissue was significantly

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higher than that of PFPeA. The ku0 values of PFBS in tissues ranged from0.64 to 16 mL·gww-1·d-1,

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while the ku0 value of PFPeA ranged from 0.23 to 13 mL·gww-1·d-1. Thirdly, the ku0 value of each

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PFAA was different among various tissues, which was highest in liver and blood, followed by

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intestine, gill and ovary, and lowest in brain and muscle. This is similar with the previous research

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that studied the bioconcentration of long-chain PFAAs in rainbow trout (Oncorhynchus mykiss).5 As

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for the ke0, fluorinated carbons or acidic headgroup had no obvious influence on ke0 values of the

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short-chain PFAAs. This is consistent with the study that reported the bioconcentration kinetic

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parameters of three short-chain PFAAs (PFHxA, PFBA, and PFBS) in zebrafish tissues (plasma,

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liver, muscle and Ovary ).22

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3.2.2 Bioconcentration and tissue distribution factors of the short-chain PFAAs at steady state

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in the absence of the long-chain PFAAs.

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The values of BCFss (L·kg-1) for the short-chain PFAAs at steady state (day 28) (Table 1) were

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calculated by the equation: BCFss =Cb/Cw, where Cb is the concentration of PFAA in the tissue of

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zebrafish, ng·gww-1, and Cw is PFAA concentration in water, ng·mL-1. According to the results (Table

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1, Figures 2 and S1), it is similar to the uptake rate constants that the concentrations and BCFss of

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PFAAs in tissues of zebrafish were affected by PFAA properties and tissue types. Initially, the

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concentrations and BCFss of PFAAs increased with fluorinated carbons in each tissue and significant

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positive relationships were observed between logBCFss and fluorinated carbon numbers (P