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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
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Environmental Science & Technology is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
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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
4 5
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
11
increase with their chain length. Therefore, we hypothesized that long-chain PFAAs will affect the
12
bioconcentration of short-chain PFAAs in organisms. To testify this hypothesis, the bioconcentration
13
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
15
concentrations of the short-chain PFAAs in zebrafish tissues increased with exposure time till steady
16
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
18
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.
21
These results suggest that the effect of long-chain PFAAs on the bioconcentration of short-chain
22
PFAAs should be taken into account in assessing the ecological and environmental effects of short-
23
chain PFAAs.
24 25
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
Article
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
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Environmental Science & Technology is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
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Environmental Science & Technology
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Long-chain perfluoroalkyl acids (PFAAs) affect the bioconcentration and
2
tissue distribution of short-chain PFAAs in zebrafish (Danio rerio)
3
Wu Wen, Xinghui Xia*, Diexuan Hu, Dong Zhou, Haotian Wang, Yawei Zhai, Hui Lin
4 5
School of Environment, Beijing Normal University, State Key Laboratory of Water Environment
6
Simulation, Beijing 100875, China
7
*
Corresponding author. Tel./fax: +86 10 58805314.
E-mail address: [email protected] (X. Xia) 1 / 34
ACS Paragon Plus Environment
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Page 2 of 34
8
Abstract
9
Short- and long-chain perfluoroalkyl acids (PFAAs), ubiquitously co-existing in the environment,
10
can be accumulated in organisms by binding with proteins and their binding affinities generally
11
increase with their chain length. Therefore, we hypothesized that long-chain PFAAs will affect the
12
bioconcentration of short-chain PFAAs in organisms. To testify this hypothesis, the bioconcentration
13
and tissue distribution of five short-chain PFAAs (linear C-F=3-6) were investigated in zebrafish in
14
the absence and presence of six long-chain PFAAs (linear C-F=7-11). The results showed that the
15
concentrations of the short-chain PFAAs in zebrafish tissues increased with exposure time till steady
16
states reached in the absence of long-chain PFAAs. However, in the presence of long-chain PFAAs,
17
these short-chain PFAAs in tissues increased till peak values reached and then decreased till steady
18
states, and the uptake and elimination rate constants of short-chain PFAAs declined in all tissues and
19
their BCFss decreased by 24-89%. The inhibitive effect of long-chain PFAAs may be attributed to
20
their competition for transporters and binding sites of proteins in zebrafish with short-chain PFAAs.
21
These results suggest that the effect of long-chain PFAAs on the bioconcentration of short-chain
22
PFAAs should be taken into account in assessing the ecological and environmental effects of short-
23
chain PFAAs.
24 25
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.
34
Because of their special physical and chemical properties (thermal and chemical stability,
35
hydrophobicity and lipophobicity), the long-chain PFAAs (no less than seven fluorinated carbons)
36
have been manufactured and used in numerous industrial and commercial applications for over half
37
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
41
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
44
than seven fluorinated carbons),9 which have the similar physiochemical properties to long-chain
45
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
65
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
140
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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of methanol and filtered for injection. 8 / 34
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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
218
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
