First Report on the Occurrence and Bioaccumulation of

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First Report on the Occurrence and Bioaccumulation of Hexafluoropropylene Oxide Trimer Acid (HFPO-TA): An Emerging Concern Yitao Pan, Hongxia Zhang, Qianqian Cui, Nan Sheng, Leo Wai-Yin Yeung, Yong Guo, Yan Sun, and Jiayin Dai Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b02259 • Publication Date (Web): 07 Aug 2017 Downloaded from http://pubs.acs.org on August 9, 2017

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Environmental Science & Technology

First Report on the Occurrence and Bioaccumulation of Hexafluoropropylene Oxide Trimer Acid (HFPO-TA): An Emerging Concern

Yitao Pan,1,2 Hongxia Zhang,1 Qianqian Cui,1 Nan Sheng,1 Leo W.Y. Yeung,3 Yong Guo,4 Yan Sun,4 and Jiayin Dai1,*

1

Key Laboratory of Animal Ecology and Conservation Biology, Institute of Zoology, Chinese

Academy of Sciences, Beijing 100101, P. R. China; 2University of Chinese Academy of Sciences, Beijing 100049, China; 3Man-Technology-Environment Research Centre (MTM), School of Science and Technology, Örebro University, SE-70182, Örebro, Sweden; 4Key Laboratory of Organofluorine Chemistry Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai, P. R. China

*Correspondence author: Jiayin Dai, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, P. R. China. Telephone: +86-10-64807185. E-mail: [email protected]

Competing financial interests: The authors declare no conflicts of interest.

1

ACS Paragon Plus Environment


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ABSTRACT

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Here we report on the occurrence of a novel perfluoroalkyl ether carboxylic acid (PFECA),

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ammonium perfluoro-2-[(propoxy)propoxy]-1-propanoate (HFPO-TA), in surface water and

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common carps (Cyprinus carpio) collected from Xiaoqing River and in residents residing

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near a fluoropolymer production plant in Huantai County, China. Compared with the levels in

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the upstream of Xiaoqing River, HFPO-TA concentrations (5200–68500 ng/L) were

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approximately 120–1600 times higher at the downstream, after receiving fluoropolymer plant

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effluent from a tributary. The riverine discharge of HFPO-TA was estimated to be 4.6 t/yr,

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accounting for 22% of total PFAS discharge. In the wild common carp collected downstream

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from the point source, HFPO-TA was detected in the blood (median: 1510 ng/mL), liver (587

11

ng/g ww), and muscle (118 ng/g ww). The log BCFblood of HFPO-TA (2.18) was significantly

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higher than that of PFOA (1.93). Detectable levels of HFPO-TA were also found in the sera

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of residents (median: 2.93 ng/mL). This is the first report on the environmental occurrence

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and bioaccumulation of this novel chemical. Our results indicate an emerging usage of

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HFPO-TA in the fluoropolymer manufacturing industry and raise concerns about the toxicity

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and potential health risks of HFPO-TA to aquatic organisms and humans.

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INTRODUCTION

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Per- and polyfluoroalkyl substances (PFASs) are synthetic fluorinated chemicals that

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have been used since the 1950s.1 The unique amphiphilic properties of PFASs have made

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them useful in a wide variety of industrial applications, such as the production of

21

fluoropolymers, surface repellent coatings, metal plating, and fire-fighting foam.2 Legacy

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PFASs, typically long-chain (seven perfluorinated carbons or longer) perfluoroalkyl

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carboxylic acids (PFCAs) and perfluoroalkane sulfonic acids (PFSAs),1 are of great concern

24

due to their environmental persistence, bioaccumulation potential, and possible toxicity.3,4 As

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a result, global regulations have been issued to reduce the production and use of these

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compounds.3 In 2006, eight major fluorochemical companies participated in the 2010/2015

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Perfluorooctanoate (PFOA) Stewardship Program proposed by the US Environmental

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Protection Agency, which aimed to eliminate the production and emission of PFOA by 2015.5

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In 2009, the Stockholm Convention on Persistent Organic Pollutants initiated regulation of

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the use of perfluorooctanesulfonate (PFOS), its salts, and related substances.6 In 2015, the

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Risk Assessment Committee from the European Union adopted the German and Norwegian

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proposal to restrict the manufacture, use, and marketing of PFOA, its salts, and related

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substances.7

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Since these restrictions, manufacturers have started to produce shorter-chain

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perfluorinated8 and other fluorinated compounds9 as alternatives, which include

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functionalized perfluoropolyethers (PFPEs) such as perfluoroether carboxylic and sulfonic

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acids (PFECAs and PFESAs).4 By inserting one or more ether oxygens into the

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perfluorinated carbon backbone, PFECAs and PFESAs are hoped to be more degradable10 3



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and have replaced PFCAs and PFSAs in many applications.11-13 In chrome plating,

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chlorinated polyfluorinated ether sulfonic acids (6:2 and 8:2 Cl-PFESAs) have been used as

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mist suppressants to replace PFOS in China,11 and have since been widely detected in abiotic

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and biotic environments.11,14-18 In fluoropolymer manufacture, certain PFECAs, such as

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perfluoro-2-propoxypropanoic acid (HFPO-DA), have been used as an alternative to PFOA.

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Since 2010, the ammonium salt of HFPO-DA (GenX™ produced by DuPont)12 has been

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produced at 10-100 tons per year in Europe,13 and has subsequently been observed in river

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waters downstream of fluorochemical industrial parks in Germany (107.6 ng/L),19 China

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(3825 ng/L)19, and the US (631 ng/L).20 In addition, several other structurally similar

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chemicals have also been identified in the US, suggesting varied and widespread usage of

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PFECA homologues.10,20

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Hexafluoropropylene oxide (HFPO) is a well-known key compound in organofluorine

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chemistry.21 Including HFPO-DA, which is the dimer acid of HFPO (structure shown in

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Figure S1), oligomeric HFPO can be applied as a monomer or intermediate in the synthesis of

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fluorinated chemicals.21 The trimer acid of HFPO, HFPO-TA (Figure S1), is used as a

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processing aid in the manufacture of fluorinated polymers, such as polytetrafluoroethylene

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and polyvinylidene fluoride,22 and is an important building block in the synthesis of other

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fluorinated products, including surface active agents,23 oil repellent agents,24 ionic liquids,25

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and industrial additives.26-28 Available information on the physical and chemical properties of

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HFPO-TA are shown in Table S1. However, information is scarce in regards to its annual

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production, environmental occurrence, wildlife or human exposure, bioaccumulation

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potential, and toxic effects. 4


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In the present investigation, water and fish samples were collected from various sites in

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Xiaoqing River, China. Elevated concentrations of PFCAs have been reported previously in

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water19,29 and sediment samples of Xiaoqing River,30 which is likely due to discharge from

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one of the largest fluoropolymer production facilities in Asia,29 which has a reported annual

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production of approximately 37000 t of polytetrafluoroethylene (PTFE), 500 t of

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perfluorinated ethylene-propylene co-polymers, 300 t of polyvinylidenefluoride (PVDF), and

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40 t of ammonium perfluorooctanoate.31 Human blood samples from local residents in

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Huantai County, where the fluoropolymer production facility is located, were also collected.

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The objectives of the present investigation were (1) to investigate whether novel alternative

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HFPO-TA was present in freshwater and wild freshwater fish of Xiaoqing River; (2) if so, to

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determine the tissue distribution and bioaccumulation potential in wild fish; and (3) to

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evaluate human exposure to HFPO-TA, as well as other legacy PFASs, in local residents.

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MATERIALS AND METHODS

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Sample collection. Xiaoqing River is located in Shandong Province, China, with a

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length of approximately 233 km and a catchment area of 13000 km2. Parallel to the Yellow

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River, Xiaoqing River flows through four industrialized cities (Jinan, Binzhou, Zibo, and

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Dongying) before finally entering Laizhou Bay of the Bohai Sea. From November 29 to

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December 1 2015, a total of 18 water samples were collected upstream (S1–S6), from the

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tributary receiving fluoropolymer plant effluent (S7–S10), and downstream (S11–18) of

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Xiaoqing River (Figure 1 and Table S2). Approximately 1 L of water from a depth of 1 m was

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collected in methanol rinsed polypropylene bottles and stored at −20 °C until analysis.

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Common carp (Cyprinus carpio) were captured in the area between S12–S13 on 5



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December 1 2015 (n = 15). Information on gender, body weight, and length can be found in

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Table S3. Approximately 2–4 mL of whole blood was collected immediately in EDTA-coated

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vacutainer tubes (BD Biosciences, USA). Liver and muscle samples were carefully dissected

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from the fish, wrapped with aluminum foil, and maintained at −20 °C.

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Human subjects (22 male and 26 female) were recruited at Huantai County Hospital,

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located 8 km from the fluoropolymer plant. Participants were residents recruited at their first

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presentation to the hospital in January 2016. All subjects had lived in Huantai for at least two

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years, and had never worked in the fluoropolymer plant. Blood samples were centrifuged

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immediately after collection, with sera transferred and stored at −80 °C until analysis. The

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research protocol was approved by the Ethics Committee of the Institute of Zoology, Chinese

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Academy of Sciences, and the study hospital.

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Sample extraction. The water and biota samples were extracted based on previously

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published methods.32,33 Details of the extraction method on different matrices are provided in

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the Supporting Information (SI). In brief, water samples were extracted using a solid phase

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extraction (SPE) cartridge (Phenomenex strata X-AW, 200 mg/6 mL);32 whereas fish blood,

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fish liver and human serum were extracted using an ion-pair extraction method.33 An alkaline

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digestion method was used for fish muscle samples.32 Additional cleanup using the SPE

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method was applied to fish liver and muscle samples.

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Instrument analysis. Target PFASs (structures shown in Figure S1), including PFCAs

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(C4-C14), PFSAs (C4, C6, C8), and Cl-PFESAs (4:2. 6:2, 8:2), were quantified using an

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Acquity UPLC coupled to a Xevo TQ-S triple quadrupole mass spectrometer (Waters,

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Milford, MA, USA). Due to the poor sensitivity (limit of quantification, LOQ: 5–20 ng/mL) 6


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of HFPO-TA and HFPO-DA with the Xevo TQ-S, they were quantified using an API 5500

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triple-quadrupole mass spectrometer (AB SCIEX, Framingham, MA, USA), which showed

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much better quantification limits (0.05–0.1 ng/mL). Multiple reaction-monitoring (MRM) in

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ESI- mode was used in both mass spectrometers. Chromatographic separation was

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accomplished using an Acquity BEH C18 column (100 mm × 2.1 mm, 1.7 µm, Waters, MA,

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USA) with mobile phases of 2 mM ammonium acetate in water (A) and methanol (B) at a

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flow rate of 0.3 mL/min.

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Quality assurance and quality control. Extraction blanks, method detection limits

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(MDL), quality control samples, and matrix recovery tests were conducted to ensure accurate

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quantification of PFASs. All labware, solvents, and sampling equipment were prescreened to

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reduce possible contamination. In daily operation, two extraction blanks were included in

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every batch. No detectable contamination was found for most PFASs, except for consistent

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low levels of PFBA and HFPO-DA (from the SPE cartridge). Therefore, the levels for these

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compounds were reported on a blank corrected basis, and the MDLs were defined as the

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average plus three times the standard deviation of extraction blanks (shown in Table S5). Two

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QC samples (SRM1957, non-fortified human blood serum, National Institute of Standards

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and Technology, USA) were used in every ten human serum samples, and the measured mean

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levels of PFHpA (0.270 ± 0.024), PFOA (4.963 ± 0.369), PFNA (0.843 ± 0.040) and PFHxS

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(3.854 ± 0.279) were within the reported range (Table S6). Matrix recoveries (n = 4) were

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validated by spiking 2 ng of standard into a blank matrix and subjected to the extraction

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method discussed above, with values within 93%–109% in water, 77%–109% in serum,

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72%–124% in liver, and 80%–125% in muscle (Table S7). The 1/x weighted calibration curve 7



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was verified daily and exhibited excellent linearity (R2 > 0.99).

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The confirmation of the occurrence of HFPO-TA in the samples (i.e., some of the water

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and biota samples) were conducted using a X500R Q-TOF System (AB SCIEX, Framingham,

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MA, USA) in ESI- mode. The instrument was operated in full scan MS (100−1000 m/z) and

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MS/MS mode (50−1000 m/z) simultaneously through information dependent acquisition

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(IDA). The detailed parameters are provided in the SI. The molecular ion and fragment ion in

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water (m/z = 495.9507, ∆m = −2.627 ppm, and m/z = 184.9824, ∆m = −4.325 ppm) and fish

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blood (m/z = 494.9509, ∆m = −2.222 ppm, and m/z = 184.9827, ∆m = −2.703 ppm) suggested

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the presence of HFPO-TA in corresponding matrices. These observations were further

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confirmed by the identical retention time with that in HFPO-TA standard (Figure 2).

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Data analysis. Descriptive statistics are provided for PFAS concentrations in water and

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biota samples. When the concentrations of the PFASs were below the MDL, a value of

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MDL/2 was employed. Riverine mass discharge (t/yr) of PFASs from Xiaoqing River was

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calculated by multiplying the measured concentration (ng/L) with the annual water flux

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(m3/yr), and multiplying by 10–12 to harmonize with the units. Measured PFAS concentration

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was derived from the average levels in water samples close to the river mouth (S15–S18),

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whereas annual water flux was acquired from the hydrological station adjacent to site S16,

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with a value of 6.5 × 108 m3/yr.34 The bioconcentration factor (BCF) was calculated as the

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measured PFAS concentrations in fish blood and tissue (on a wet weight basis) divided by

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those in corresponding water samples (mean levels of S12 and S13). Tissue/blood ratios were

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calculated to describe the distribution pattern of HFPO-TA in common carp. One-way

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analysis of variance (ANOVA) followed by Duncan’s multiple range tests were used to test 8


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for differences in the BCF of PFASs. All statistical analyses were performed using IBM

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PASW statistics 18.0 (SPSS Inc., USA) with a statistical significance threshold of p < 0.05.

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RESULTS AND DISCUSSION

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Concentrations in Xiaoqing River. The concentrations and spatial distributions of

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PFASs in Xiaoqing River are presented in Figure 3 and Table S8. Alternatives of PFASs,

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including HFPO-TA, HFPO-DA, and 6:2 Cl-PFESA, and 12 legacy PFASs were all detected

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in the water samples. Results showed that PFOA was the predominant compound, accounting

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for 60 ± 18% of all PFASs, followed by HFPO-TA (24 ± 12%); PFBA, PFHxA, PFPeA,

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PFHpA, and HFPO-DA (1.3 ± 1.0%). Along the main stream of Xiaoqing River, the ΣPFAS

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concentration increased by three orders of magnitude, from 48.4 ng/L (S2) to 81900 ng/L

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(S11), and then decreased 2.5-fold to 32800 ng/L (S18) before entering Laizhou Bay. The

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sharp increases in PFAS levels were attributed to the Dongzhulong tributary, where a peak

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level of 282000 ng/L was observed at sampling site S8, approximately 800-fold higher than

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that upstream (355 ng/L, S7). This contamination was likely caused by the fluoropolymer

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production plant located between sites S7 and S8, which has also been identified as a point

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source of PFASs in previous studies;19,29,30 PFOA was found to be the major compound in

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previous and current investigations. The peak level of PFOA in the present investigation

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(197000 ng/L) were lower than the values collected in the same location (e.g., 396000 ng/L30

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and 724000 ng/L19 in April 2014), possibly due to the fluctuations in emissions and

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hydrological conditions over time. Our results also showed that novel alternative HFPO-TA

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ranked second highest after PFOA, with a maximum level of 68500 ng/L at site S8, whereas

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HFPO-DA was observed with a peak level of 2100 ng/L, comparable to that reported by 9



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Heydebreck et al. at the same location (3800 ng/L).19 The spatial distributions of HFPO-TA

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and HFPO-DA were highly associated with the industrial point source (e.g., fluoropolymer

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manufacturer). In contrast, no observable spatial trends for PFSAs, PFESAs, or C9-C14

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PFCAs were observed.

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Our results revealed that the studied fluoropolymer plant impacted the Dongzhulong

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tributary, and consequently the majority of the Xiaoqing Basin. Other fluoropolymer facilities

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with smaller production capacities might also exacerbate the PFAS pollution.29,30 For

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example, the occurrence of HFPO-TA and HFPO-DA at sampling sites S1–S6 implied other

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point sources upstream. The levels of ΣPFASs downstream of Xiaoqing River remained

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relatively stable at 31600–35200 ng/L, which was possibly attributed by other tributaries.

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Based on an annual river water flux of 6.5 × 108 m3/yr,34 the riverine discharge of ΣPFASs

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was estimated to be 21.3 t/yr (15.5 t/yr of PFOA, 4.6 t/yr of HFPO-TA; Table S9). Although

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instantaneous concentrations might result in a biased estimate, they can provide an

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approximation of HFPO-TA mass flux in Xiaoqing River. Sea waters were not collected in

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the current study; however, it is plausible that HFPO-TA could be detected in Laizhou Bay.

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Based on earlier studies,19,30 PFAS concentrations in Laizhou Bay were 3–10 times more

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diluted than that in the river mouth. If that is the case, the level of HFPO-TA could be

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approximately 3000–10000 ng/L, still one to two orders of magnitude higher than that before

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the point source input. Such high levels might be harmful to aquatic life in Laizhou Bay;

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however, there are no aquatic toxicity data available on this novel HFPO-TA compound.

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Tissue distribution. The levels of total and individual PFASs in fish tissue are shown in

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Figure 4A and Table S10. All PFASs were detected in most blood and liver samples (> 94%), 10


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but lower detection rates (0−47%) were found for C4–C6 PFCAs, PFBS, PFHxS, and 4:2

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Cl-PFESA in muscle. The blood samples contained the greatest concentration of ΣPFASs

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(mean value 4350 ng/mL), followed by the liver samples (1200 ng/g ww), and then the

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muscle samples (225 ng/g ww). The composition profiles of PFASs in different tissues are

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shown in Figure 4B. Similar to water samples, the concentrations of PFOA and HFPO-TA

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were at least one to two orders of magnitude higher than that of other PFASs. PFOA was the

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predominant component in blood (median: 2190 ng/mL, accounting for 56 ± 15% of

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ΣPFASs), whereas HFPO-TA was dominant in the liver (587 ng/mL, 47 ± 17%) and muscle

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samples (118 ng/mL, 51 ± 16%). The ratios of HFPO-TA between tissue and blood were

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calculated to further clarify its distribution, and were then compared with other PFCAs with

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similar molecular chain lengths (e.g., PFOA and PFNA; Figure 4C). Tissue/blood ratios of

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HFPO-TA in the liver samples (45 ± 31%) were 5-fold greater than that in the muscle

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samples (9 ± 7%). Compared with PFOA and PFNA, HFPO-TA had higher tissue/blood ratios,

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but only reached statistical significance in the muscles.

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As expected, the levels of PFASs in common carp captured downstream of Xiaoqing

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River near the emission source were tens to hundreds of times higher than those of other fish

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species from different regions.35,36 However, the PFAS distribution and tissue/blood ratios

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were generally consistent with other studies.35-38 The observed concentrations in tissues in

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descending order (blood > liver > muscle) were in good agreement with previous

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researches,15,35,37,38 suggesting that all PFASs, including HFPO-TA, share similar mechanisms

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of distribution. However, HFPO-TA tended to be more accumulative in liver and muscle

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compared with that of PFOA and PFNA (Figure 4B and 4C). This discrepancy might be due 11



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to the differences in protein binding affinity and/or hydrophobic properties. Since liver and

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muscle are rich in proteins and phospholipids, greater binding affinity or hydrophobicity may

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lead to additional sorption,39,40 consequently leading to a higher distribution in liver and

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muscle. This hypothesis was supported by our recent findings that HFPO-TA was more

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strongly bound than PFOA to human liver fatty acid binding protein (hL-FABP), one of the

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most abundant proteins in the liver.41 The dissociation constant of HFPO-TA (Kd = 4.36 ±

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1.17) was found to be much lower than that of PFOA (Kd = 8.03 ± 2.10), indicating a much

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stronger binding affinity of HFPO-TA to hL-FABP than PFOA.41 Additionally, although the

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lone pair electrons of the O atom at the insertion of ester bonds in HFPO-TA might have

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decreased the hydrophobicity, the larger molecular size consequently increased its

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hydrophobicity,42 compared with similar molecular structures of PFOA and PFNA (Table S11,

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EPI Suite V4.11).

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Bioaccumulation. The tissue-specific bioconcentration factors (BCFs) for common carp

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are listed in Table S12. The log BCFs for all PFASs ranged from 0.49 to 5.93 in the fish blood

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samples, approximately 0.4 and 1.0 log units higher than those in the liver and muscle

230

samples, respectively. Since all target PFASs were frequently detected in blood, but not in

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liver and muscle, log BCFblood was used to better reflect the differences in bioaccumulation

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potential among PFASs (Figure 5). Log BCF increased significantly with increasing

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molecular chain length in each category of PFAS, which was in good agreement with

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previous studies focusing on PFCAs and PFSAs.37,38,43 For the first time, increasing trends

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were also observed in PFECAs and PFESAs. The log BCFs for PFESAs were higher than

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those for PFSAs with the same number of carbons in the backbones (i.e., 4:2 Cl-PFESA > 12


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PFHxS; 6:2 Cl-PFESA > PFOS), suggesting that the inserted ester oxygen and/or the chlorine

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atom increased the bioaccumulation potential of PFASs. No clear pattern was found between

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PFECAs and PFCAs having the same number of carbons; HFPO-DA (C6) had higher BCF

240

than PFHxA (C6), whereas HFPO-TA (C9) had a lower BCF than that of PFNA (C9). The

241

reason might be the branched carbons in HFPO-TA (Figure S1) that lead to less

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hydrophobicity and complicate the comparison. The log BCFs for HFPO-DA, PFBA, PFPeA,

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and PFHxA were all relatively low (< 1), suggesting lower bioaccumulation potential for

244

these compounds. In general, log Kow and log BCF are used to predict bioaccumulation

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potential.37 The higher estimated log Kow (5.555) of HFPO-TA by EPI Suite V4.11 suggested

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it was more bioaccumulative than PFOA (log Kow = 4.814) and PFNA (log Kow = 5.483,

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Table S11). However, the log BCFblood value for HFPO-TA (2.18 ± 0.44) fell between that for

248

PFOA (1.93 ± 0.34) and PFNA (3.01 ± 0.37), suggesting that HFPO-TA was more

249

bioaccumulative than PFOA but less bioaccumulative than PFNA. This deviation might be

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due to the high concentrations of HFPO-TA in Xiaoqing River, since the absorption from

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water into biota might be partly saturated at this high concentration.44-46

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Human exposure. Human exposure to PFASs was evaluated in 48 Huantai residents,

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with an average age of 53 years (Table 1). Detectable levels of HFPO-TA, C7-C13 PFCAs,

254

PFHxS, PFOS, and 4:2, 6:2, and 8:2 Cl-PFESAs were measured in most serum samples (>

255

97.9%). PFBA and PFTeDA were detected in 87.5% and 62.5% of serum samples, whereas

256

the detection rates for HFPO-DA, PFPeA, PFHxA, and PFBS ranged between 16.7–39.6%.

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Results also showed that PFOA was dominant, and accounted for 86 ± 9% of total PFASs. We

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previously reported a median PFOA level of 284.34 ng/mL in residents from Changshu, 13



259

another important fluorochemical industrial zone in China.47 The median level of PFOA (126

260

ng/mL) here was approximately 50% lower than that detected in our previous study,47 but

261

was 5 times higher than the reported levels by C8 Health Project (median 24 ng/mL), which

262

focused on the residents living near DuPont Washington Works plant in West Virginia,

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US.48,49 Additionally, level in this study was still 40–100 times higher than that recorded in

264

other populations from China (1.39 ng/mL),50 Canada (2.17 ng/mL)51 and the US (3.07

265

ng/mL).52 Such elevated PFOA levels suggest strong PFAS exposure from the nearby

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fluoropolymer industrial plant.

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Results also showed that HFPO-TA was the fourth highest in median level (2.93 ng/mL),

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next to PFOA, PFOS, and 8:2 Cl-PFESA (Table 1). The skewness of HFPO-TA distribution

269

was the greatest among all PFASs; 80% of subjects had HFPO-TA levels between

270

non-detectable and 9.23 (mean 2.63 ng/mL), whereas 20% ranged within 12.0–55.0 (mean

271

36.8 ng/mL). The high variability in the HFPO-TA levels suggests that certain factors are

272

affecting the extent of exposure in the study subjects. No subject reported an employment

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history related to fluoropolymer production, and no age or gender differences in residents

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were observed in HFPO-TA levels (data not shown). Fish consumption frequency might be an

275

important predictor, since relatively high levels of HFPO-TA (median 118 ng/g ww, Table

276

S10) were observed in the muscle of common carp, and more frequent consumption of

277

contaminated fish from Xiaoqing River might result in higher HFPO-TA exposure. According

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to Shandong Statistic Year Book, the average fish consumption in the studied area was 17.0

279

g/day.53 Applying an average body weight of 60 kg for adults, the daily intake of HFPO-TA

280

was estimated to be 33.4 ng/kg/day based on following equation: daily intake (ng/kg/day) = 14


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281

HFPO-TA level in muscle (ng/g) × fish consumption (g/day) / body weight (kg). Another

282

important factor might be residence distance from the fluoropolymer facility. A previous

283

study has shown that PFASs generated from a point source can lead to PFAS exposure in

284

humans via dust ingestion and dermal absorption, with the estimated daily intake for residents

285

inversely associated with distance.54 Another evidence is that the levels of PFCAs in home

286

produced chicken eggs declined with increasing distance from the same fluoropolymer plant

287

in this study.55 Location information for the residents were not acquired here, which hampers

288

further exploration on the relationship between serum PFAS levels and residence distance.

289

Future study is needed to evaluate this relationship.

290

Environmental implications. Earlier research has shown that large proportions of

291

extractable organic fluorine in biota and humans cannot be explained by known PFASs.56,57

292

Thus, the identification of unknown fractions is of great importance, and will improve our

293

understanding of the current situation regarding the manufacture, usage, and release of PFASs.

294

In the current study, relatively high levels of HFPO-TA were measured in the surface water

295

and fish samples downstream from a fluoropolymer production plant, accounting for 24–51%

296

of total PFASs. The estimated annual riverine discharge of HFPO-TA (4.6 t/yr) was

297

approximately 30% of that for PFOA, indicated an emerging, significant amount of

298

HFPO-TA being used in fluoropolymer manufacture. With rapidly increasing demands in

299

China and more stringent regulations for PFOA use, it is reasonable to believe that the

300

production and usage of HFPO-TA as an alternative will continue to increase. We evaluated

301

the bioaccumulation potential for HFPO-TA in common carp. Although HFPO-TA (BCFblood

302

= 204 L/kg) was not bioaccumulative according to the range of promulgated bioaccumulation 15



303

“B” 1000–5000 L/kg,43 it could be regarded as having a “tendency to accumulate in

304

organisms” based on the regulatory criteria of 1–1000 L/kg.58 In addition, the BCF of

305

HFPO-TA was significantly higher than that of legacy PFOA, suggesting greater

306

bioaccumulation potential in aquatic organisms. Thus, more attention should be paid to its

307

aquatic toxicity and ecological risk, especially in regions suspected of being polluted, such as

308

Laizhou Bay. The presence of HFPO-TA in the sera of local residents also raises concern

309

about the potential health risks related to exposure. The replacement of PFOA with HFPO-TA

310

or other polyfluorinated chemicals need to be treated cautiously until further investigations

311

regarding its metabolism, toxicity, and health risk are fully explored.

312

ACKNOWLEDGMENTS

313

This work was supported by the National Natural Science Foundation of China

314

(31320103915 and 21377128) and the Strategic Priority Research Program of the Chinese

315

Academy of Sciences (XDB14040202).

316

Supporting Information

317

Additional information included standards and reagents, synthesis of HFPO-TA, PFASs

318

analysis, qualitative analysis of HFPO-TA, and other materials in Tables S1−S12 and Figures

319

S1−S3.

16


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REFERENCES (1) Buck, R. C.; Franklin, J.; Berger, U.; Conder, J. M.; Cousins, I. T.; de Voogt, P.; Jensen, A. A.; Kannan, K.; Mabury, S. A.; van Leeuwen, S. P. Perfluoroalkyl and polyfluoroalkyl substances in the environment: Terminology, classification, and origins. Integr. Environ. Assess. Manage. 2011, 7 (4), 513-41. (2) Kissa, E. Fluorinated surfactants and repellents. 2nd ed.; Marcel Dekker Inc.: New York, 2001. (3) Lindstrom, A. B.; Strynar, M. J.; Libelo, E. L. Polyfluorinated compounds: Past, present, and future. Environ. Sci. Technol. 2011, 45 (19), 7954-7961. (4) Wang, Z. Y.; Cousins, I. T.; Scheringer, M.; Hungerbuehler, K. Hazard assessment of fluorinated alternatives to long-chain perfluoroalkyl acids (PFAAs) and their precursors: Status quo, ongoing challenges and possible solutions. Environ. Int. 2015, 75, 172-179. (5) U.S. Environmental Protection Agency. 2010/2015 PFOA stewardship program; 2006. (6) Stockholm Convention. Governments unite to step-up reduction on global DDT reliance and add nine new chemicals under international treaty; 2009. (7) Committee for Risk Assessment. Background document to the opinion on the Annex XV dossier proposing restrictions on perfluorooctanoic acid (PFOA), PFOA salts and PFOA-related substances; 2015. (8) Ritter, S. K. Fluorochemicals go short. Chem. Eng. News. 2010, 88 (5), 12-17. (9) United Nations Environment Programme. Technical paper on the identification and assessment of alternatives to the use of perfluorooctane sulfonic acid in open applications; 2012. 17



(10) Strynar, M.; Dagnino, S.; McMahen, R.; Liang, S.; Lindstrom, A.; Andersen, E.; McMillan, L.; Thurman, M.; Ferrer, I.; Ball, C. Identification of novel perfluoroalkyl ether carboxylic acids (PFECAS) and sulfonic acids (PFESAs) in natural waters using accurate mass time-of-flight mass spectrometry (TOFMS). Environ. Sci. Technol. 2015, 49 (19), 11622-11630. (11) Wang, S. W.; Huang, J.; Yang, Y.; Hui, Y. M.; Ge, Y. X.; Larssen, T.; Yu, G.; Deng, S. B.; Wang, B.; Harman, C. First report of a chinese PFOS alternative overlooked for 30 years: Its toxicity, persistence, and presence in the environment. Environ. Sci. Technol. 2013, 47 (18), 10163-10170. (12) Dupont. DupontTM GenX processing aid for making fluoropolymer resins; 2010. (13) European Chemicals Agency. Registered substances; 2013. (14) Ruan, T.; Lin, Y. F.; Wang, T.; Liu, R. Z.; Jiang, G. B. Identification of novel polyfluorinated ether sulfonates as PFOS alternatives in municipal sewage sludge in China. Environ. Sci. Technol. 2015, 49 (11), 6519-6527. (15) Shi, Y. L.; Vestergren, R.; Zhou, Z.; Song, X. W.; Xu, L.; Liang, Y.; Cai, Y. Q. Tissue distribution and whole body burden of the chlorinated polyfluoroalkyl ether sulfonic acid F-53B in crucian carp (Carassius carassius): Evidence for a highly bioaccumulative contaminant of emerging concern. Environ. Sci. Technol. 2015, 49 (24), 14156-14165. (16) Shi, Y. L.; Vestergren, R.; Xu, L.; Zhou, Z.; Li, C. X.; Liang, Y.; Cai, Y. Q. Human exposure and elimination kinetics of chlorinated polyfluoroalkyl ether sulfonic acids (Cl-PFESAs). Environ. Sci. Technol. 2016, 50 (5), 2396-2404. 18


Page 18 of 32

Page 19 of 32


(17) Pan, Y. T.; Zhu, Y. S.; Zheng, T. Z.; Cui, Q. Q.; Buka, S. L.; Bin, Z.; Guo, Y.; Xia, W.; Yeung, L. W. Y.; Li, Y. R.; Zhou, A. F.; Qiu, L.; Liu, H. X.; Jiang, M. M.; Wu, C. S.; Xu, S. Q.; Dai, J. Y. Novel chlorinated polyfluorinated ether sulfonates and legacy per-/polyfluoroalkyl substances: Placental transfer and relationship with serum albumin and glomerular filtration rate. Environ. Sci. Technol. 2017, 51 (1), 634-644. (18) Liu, Y. W.; Ruan, T.; Lin, Y. F.; Liu, A. F.; Yu, M.; Liu, R. Z.; Meng, M.; Wang, Y. W.; Liu, J. Y.; Jiang, G. B. Chlorinated polyfluoroalkyl ether sulfonic acids in marine organisms from Bohai Sea, China: Occurrence, temporal variations, and trophic transfer behavior. Environ. Sci. Technol. 2017, 51 (8), 4407-4414. (19) Heydebreck, F.; Tang, J. H.; Xie, Z. Y.; Ebinghaus, R. Alternative and legacy perfluoroalkyl substances: Differences between European and Chinese river/estuary systems. Environ. Sci. Technol. 2015, 49 (14), 8386-8395. (20) Sun, M.; Arevalo, E.; Strynar, M.; Lindstrom, A.; Richardson, M.; Kearns, B.; Pickett, A.; Smith, C.; Knappe, D. R. U. Legacy and emerging perfluoroalkyl substances are important drinking water contaminants in the Cape Fear River watershed of North Carolina. Environ. Sci. Tech. Let. 2016, 3 (12), 415-419. (21) Millauer, H.; Schwertfeger, W.; Siegemund, G. Hexafluoropropene oxide - a key compound in organofluorine chemistry. Angew. Chem. Int. Edit. 1985, 24 (3), 161-179. (22) Han, G. Synthesis and application of hexafluoropropene oxide polymer. Zhejiang Chemical Industry (in Chinese) 2010, 41 (11), 1-4. (23) Zhang, W.; Zhang, Y. M. Synthesis and surfactivity of non-ionic fluorocarbon surfactant. Modern Chemical Industry (in Chinese) 2011, 31 (4), 49-52. 19



Page 20 of 32

(24) Zhou, X. Synthesis and application of the HFPO trimer-based polymer oil repellent. Chemical Production & Technology (in Chinese) 2007, 14 (4), 5-8. (25) Kysilka, O.; Rybackova, M.; Skalicky, M.; Kvicalova, M.; Cvacka, J.; Kvicala, J. Fluorous imidazolium room-temperature ionic liquids based on HFPO trimer. J. Fluorine Chem. 2009, 130 (7), 629-639. (26) Kysilka, O.; Rybackova, M.; Skalicky, M.; Kvicalova, M.; Cvacka, J.; Kvicala, J. HFPO trimer-based alkyl triflate, a novel building block for fluorous chemistry. Preparation, reactions and F-19 gcosy analysis. Collect. Czech. Chem. C. 2008, 73 (12), 1799-1813. (27) Meissner, E.; Wróblewska, A. Oligomerization of hexafluoropropylene oxide in the presence of alkali metal halides. Polish J. Chem. Technol. 2007, 9 (3), 95-97. (28) Bazhin, D. N.; Gorbunova, T. I.; Zapevalov, A. Y.; Beketov, I. V.; Saloutin, V. I. A study of the physico-chemical features of the [(perfluoroalkyl)methyl]oxirane amino derivatives based on the hexafluoropropylene oxide trimer. Russ. J. Gen. Chem. 2011, 81 (9), 1829-1833. (29) Wang, P.; Lu, Y. L.; Wang, T. Y.; Fu, Y. N.; Zhu, Z. Y.; Liu, S. J.; Xie, S. W.; Xiao, Y.; Giesy, J. P. Occurrence and transport of 17 perfluoroalkyl acids in 12 coastal rivers in south Bohai coastal region of China with concentrated fluoropolymer facilities. Environ. Pollut. 2014, 190, 115-122. (30) Shi, Y. L.; Vestergren, R.; Xu, L.; Song, X. W.; Niu, X. M.; Zhang, C. H.; Cai, Y. Q. Characterizing

direct

emissions

of

perfluoroalkyl

substances

from

ongoing

fluoropolymer production sources: A spatial trend study of Xiaoqing river, China. Environ. Pollut. 2015, 206, 104-112. 20


Page 21 of 32


(31) Dongyue Group Limited. 2012 annual report; 2012. (32) Taniyasu, S.; Kannan, K.; So, M. K.; Gulkowska, A.; Sinclair, E.; Okazawa, T.; Yamashita, N. Analysis of fluorotelomer alcohols, fluorotelorner acids, and short- and long-chain perfluorinated acids in water and biota. J. Chromatogr. A 2005, 1093 (1-2), 89-97. (33) Hansen, K. J.; Clemen, L. A.; Ellefson, M. E.; Johnson, H. O. Compound-specific, quantitative characterization of organic: Fluorochemicals in biological matrices. Environ. Sci. Technol. 2001, 35 (4), 766-770. (34) Ministry of Water Resources of the People's Republic of China. Hydrological data of Huaihe River Basin, annual hydrological report; 2014. China Water & Power Press. (35) Murakami, M.; Adachi, N.; Saha, M.; Morita, C.; Takada, H. Levels, temporal trends, and tissue distribution of perfluorinated surfactants in freshwater fish from Asian countries. Arch. Environ. Con. Tox. 2011, 61 (4), 631-641. (36) Lam, N. H.; Cho, C. R.; Kannan, K.; Cho, H. S. A nationwide survey of perfluorinated alkyl substances in waters, sediment and biota collected from aquatic environment in Vietnam: Distributions and bioconcentration profiles. J. Hazard. Mater. 2017, 323, 116-127. (37) Martin, J. W.; Mabury, S. A.; Solomon, K. R.; Muir, D. C. G. Bioconcentration and tissue distribution of perfluorinated acids in rainbow trout (oncorhynchus mykiss). Environ. Toxicol. Chem. 2003, 22 (1), 196-204. (38) Labadie, P.; Chevreuil, M. Partitioning behaviour of perfluorinated alkyl contaminants between water, sediment and fish in the Urge River (nearby Paris, France). Environ. 21



Pollut. 2011, 159 (2), 391-397. (39) Zhang, L. Y.; Ren, X. M.; Guo, L. H. Structure-based investigation on the interaction of perfluorinated compounds with human liver fatty acid binding protein. Environ. Sci. Technol. 2013, 47 (19), 11293-11301. (40) Xie, W.; Ludewig, G.; Wang, K.; Lehmler, H. J. Model and cell membrane partitioning of perfluorooctanesulfonate is independent of the lipid chain length. Colloid. Surface B 2010, 76 (1), 128-136. (41) Sheng, N.; Cui, R. N.; Wang, J. H.; Guo, Y.; Wang, J. S.; Dai, J. Y. Cytotoxicity of novel fluorinated alternatives to long-chain perfluoroalkyl substances to human liver cell line and their binding capacity to human liver fatty acid binding protein. Arch. Toxicol. 2017 (under minor revision). (42) Gomis, M. I.; Wang, Z. Y.; Scheringer, M.; Cousins, I. T. A modeling assessment of the physicochemical properties and environmental fate of emerging and novel per- and polyfluoroalkyl substances. Sci. Total Environ. 2015, 505, 981-991. (43) Conder, J. M.; Hoke, R. A.; De Wolf, W.; Russell, M. H.; Buck, R. C. Are PFCAs bioaccumulative? A critical review and comparison with regulatory lipophilic compounds. Environ. Sci. Technol. 2008, 42 (4), 995-1003. (44) Kudo, N.; Katakura, M.; Sato, Y.; Kawashima, Y. Sex hormone-regulated renal transport of perfluorooctanoic acid. Chem-Biol. Interact. 2002, 139 (3), 301-316. (45) Kudo, N.; Sakai, A.; Mitsumoto, A.; Hibino, Y.; Tsuda, T.; Kawashima, Y. Tissue distribution and hepatic subcellular distribution of perfluorooctanoic acid at low dose are different from those at high dose in rats. Biol. Pharm. Bull. 2007, 30 (8), 22


Page 22 of 32

Page 23 of 32


1535-1540. (46) Liu, C. H.; Gin, K. Y. H.; Chang, V. W. C.; Goh, B. P. L.; Reinhard, M. Novel perspectives on the bioaccumulation of PFCs - the concentration dependency. Environ. Sci. Technol. 2011, 45 (22), 9758-9764. (47) Wang, J. S.; Zhang, Y. T.; Zhang, W.; Jin, Y. H.; Dai, J. Y. Association of perfluorooctanoic acid with hdl cholesterol and circulating miR-26b and miR-199-3p in workers of a fluorochemical plant and nearby residents. Environ. Sci. Technol. 2012, 46 (17), 9274-9281. (48) Barry, V.; Winquist, A.; Steenland, K. Perfluorooctanoic acid (PFOA) exposures and incident cancers among adults living near a chemical plant. Environ. Health Perspect. 2013, 121 (11-12), 1313-1318. (49) Steenland, K.; Zhao, L. P.; Winquist, A.; Parks, C. Ulcerative colitis and perfluorooctanoic acid (PFOA) in a highly exposed population of community residents and workers in the Mid-Ohio Valley. Environ. Health Perspect. 2013, 121 (8), 900-905. (50) Pan, Y. Y.; Shi, Y. L.; Wang, J. M.; Cai, Y. Q.; Wu, Y. N. Concentrations of perfluorinated compounds in human blood from twelve cities in China. Environ. Toxicol. Chem. 2010, 29 (12), 2695-2701. (51) Haines, D. A.; Murray, J. Human biomonitoring of environmental chemicals-early results of the 2007-2009 Canadian Health Measures Survey for males and females. Int. J. Hyg. Envir. Heal. 2012, 215 (2), 133-137. (52) Centers for Disease Control and Prevention. Fourth national report on human exposure to environmental chemicals, February 2015. 23



(53) Shandong Statistical Bureau. Shandong statistical yearbook; 2015. (54) Su, H. Q.; Lu, Y. L.; Wang, P.; Shi, Y. J.; Li, Q. F.; Zhou, Y. Q.; Johnson, A. C. Perfluoroalkyl acids (PFAAs) in indoor and outdoor dusts around a mega fluorochemical industrial park in China: Implications for human exposure. Environ. Int. 2016, 94, 667-673. (55) Su, H. Q.; Shi, Y. J.; Lu, Y. L.; Wang, P.; Zhang, M.; Sweetman, A.; Jones, K.; Johnson, A. Home produced eggs: An important pathway of human exposure to perfluorobutanoic acid (PFBA) and perfluorooctanoic acid (PFOA) around a fluorochemical industrial park in China. Environ. Int. 2017, 101, 1-6. (56) Yeung, L. W. Y.; Miyake, Y.; Taniyasu, S.; Wang, Y.; Yu, H. X.; So, M. K.; Jiang, G. B.; Wu, Y. N.; Li, J. G.; Giesy, J. P.; Yamashita, N.; Lam, P. K. S. Perfluorinated compounds and total and extractable organic fluorine in human blood samples from China. Environ. Sci. Technol. 2008, 42 (21), 8140-8145. (57) Yeung, L. W. Y.; Miyake, Y.; Wang, Y.; Taniyasu, S.; Yamashita, N.; Lam, P. K. S. Total fluorine, extractable organic fluorine, perfluorooctane sulfonate and other related fluorochemicals in liver of Indo-Pacific humpback dolphins (Sousa chinensis) and finless porpoises (Neophocaena phocaenoides) from South China. Environ. Pollut. 2009, 157 (1), 17-23. (58) Gao, Y. P.; Ji, Y. M.; Li, G. Y.; Mai, B. X.; An, T. C. Bioaccumulation and ecotoxicity increase during indirect photochemical transformation of polycyclic musk tonalide: A modeling study. Water Res. 2016, 105, 47-55.

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Table 1 Serum PFAS levels in local residents from Huantai (n = 48)

HFPO-DA HFPO-TA PFBA PFPeA PFHxA PFHpA PFOA PFNA PFDA PFUnDA PFDoDA PFTriDA PFTeDA PFBS PFHxS PFOS 4:2 Cl-PFESA 6:2 Cl-PFESA 8:2 Cl-PFESA ΣPFASs

Detection rate (%) 37.5 97.9 87.5 16.7 16.7 100 100 100 100 100 100 100 62.5 39.6 100 100 97.9 100 100

Geometric mean 0.13 2.41 0.29 0.03 0.03 0.25 134 1.24 0.96 0.53 0.06 0.07 0.01 0.01 0.46 5.79 0.04 4.04 0.06 158

Median n.d. 2.93 0.35 n.d. n.d. 0.25 126 1.31 1.01 0.60 0.06 0.08 0.01 0.01 0.51 6.54 0.04 4.19 0.06 147

n.d., not detected

25


Percentile 5th n.d. 0.18 n.d. n.d. n.d. 0.05 22.0 0.48 0.26 0.19 0.02 0.03 n.d. n.d. 0.10 1.95 0.01 1.49 0.02 29.3

Percentile 95th 1.72 53.4 3.05 0.16 0.17 1.75 638 3.46 3.87 1.30 0.20 0.17 0.03 0.04 1.29 13.7 0.10 9.86 0.19 725


Figure legends Figure 1. Sampling sites in Xiaoqing River

Figure 2. Accurate mass measurement, LC retention time, and MS2 fragmentation patterns of HFPO-TA in standard (A), water (B), and fish blood samples (C).

Figure 3. PFAS concentrations (ng/L) in water samples along Xiaoqing River.

Figure 4. (A) Concentrations of ΣPFASs, (B) composition profiles, and (C) tissue:blood ratios in common carp. Variables with different letters indicate statistically significant differences by Duncan’s multiple range test at p < 0.05.

Figure 5. Log BCFblood of PFASs with increasing molecular chain length. Different letters indicate statistically significant differences in BCFs by Duncan’s multiple range test at p < 0.05.

26


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Figure 1. Sampling sites in Xiaoqing River 65x53mm (600 x 600 DPI)



Figure 2. Accurate mass measurement, LC retention time, and MS2 fragmentation patterns of HFPO-TA in standard (A), water (B), and fish blood samples (C). 165x119mm (300 x 300 DPI)


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Figure 3. PFAS concentrations (ng/L) in water samples along Xiaoqing River. 177x88mm (300 x 300 DPI)



Figure 4. (A) Concentrations of ΣPFASs, (B) composition profiles, and (C) tissue:blood ratios in common carp. Variables with different letters indicate statistically significant differences by Duncan’s multiple range test at p < 0.05. 190x178mm (300 x 300 DPI)


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Figure 5. Log BCFblood of PFASs with increasing molecular chain length. Different letters indicate statistically significant differences in BCFs by Duncan’s multiple range test at p < 0.05. 127x63mm (300 x 300 DPI)



TOC 47x26mm (300 x 300 DPI)


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First Report on the Occurrence and Bioaccumulation of

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