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Environmental Measurements Methods
Non-Target Mass Spectrometry Reveals New Perfluoroalkyl Substances in Fish from the Yangtze River and Tangxun Lake, China Yanna Liu, Manli Qian, Xinxin Ma, Lingyan Zhu, and Jonathan W. Martin Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b00779 • Publication Date (Web): 16 Apr 2018 Downloaded from http://pubs.acs.org on April 16, 2018
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Environmental Science & Technology
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Non-Target Mass Spectrometry Reveals New Perfluoroalkyl Substances
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in Fish from the Yangtze River and Tangxun Lake, China
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Yanna Liu,1 Manli Qian,2 Xinxin Ma,3 Lingyan Zhu,3 Jonathan W. Martin1,4*
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1
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Pathology, University of Alberta, Edmonton, AB, Canada, T6G 2G3
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2
Analytical Chemistry Laboratory, Wuxi AppTec, Suzhou, China, 215104
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3
Key Laboratory of Pollution Processes and Environmental Criteria, Ministry of Education,
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College of Environmental Science and Engineering, Nankai University, Tianjin, China, 300071
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4
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Division of Analytical and Environmental Toxicity, Department of Laboratory Medicine and
Science for Life Laboratory, Department of Environmental Science and Analytical Chemistry,
Stockholm University, Stockholm, Sweden, 10691
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ABSTRACT
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Non-target high resolution mass spectrometry (Nt-HRMS) has proven useful for
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identification of unknown poly- and perfluoroalkyl substances (PFASs) in commercial products
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and water, but applications to biological samples are limited. China is the major PFAS
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manufacturing nation, thus here we adapted our Nt-HRMS methods to fish collected from
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Yangtze River and Tangxun Lake to discover potentially bioaccumulative PFASs in aquatic
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organisms destined for human consumption. In addition to traditional PFASs, over 330 other
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fluorinated analytes belonging to 10 classes of PFASs were detected among the pooled fish
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livers, including 6 sulfonate classes, 2 amine classes, 1 carboxylate class, and 1 N-heterocycle
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class. One class was detected in samples from both locations, 8 classes were detected exclusively
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in Tangxun Lake fish, and 1 class was detected exclusively in Yangtze River fish – 10 km
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downstream of a fluorochemical manufacturing site where we first reported these substances in
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wastewater three years ago. Four of the PFAS classes (>165 analytes) are reported for the first
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time here. Wider monitoring and toxicological testing should be a priority for understanding the
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health
risks
posed
to
people
and
wildlife
exposed
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these
substances.
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Environmental Science & Technology
INTRODUCTION
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Many long chain poly- and perfluoroalkyl substances (PFASs) are now globally
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distributed as environmental contaminants.1, 2 Since their first discovery in the environment 18
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years ago,1 voluntary industrial phase-outs3-5 and domestic or international restrictions and
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regulations4-11 have been introduced to limit future emissions. However, there has been a global
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geographic shift of PFAS manufacturing to countries with fewer restrictions, and a simultaneous
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shift towards alternative PFASs12-14 with uncertain toxicity and environmental fate. Owing to the
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environmental persistence of many perfluorinated compounds, there is little evidence that
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environmental concentrations of legacy PFASs are yet declining,15 thus it is important to identify
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and prevent new alternative PFASs from spreading globally.
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After the 3M Company phased out its C8 production in North America and Europe
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between 2000 and 2002, developing countries (e.g. China) increased their PFAS production,16
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and contamination of local environments17,
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between a contaminated environment and local people is well described for fisheries around
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Tangxun Lake in China.18 Here, wastewater from an industrial region is known to have
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contaminated the lake water, such that it now contains very high concentrations of long chain
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PFASs, including perfluorooctanesulfonate (PFOS, 73-1700 ng/L) and perfluorooctanoate
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(PFOA, 71-1400 ng/L) as well as known alternative-PFASs such as perfluorobutanesulfonate
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(PFBS, 2200-4500 ng/L) and perfluorobutanoate (PFBA, 1800-6300 ng/L). In blood of fishery
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employees who live around this lake, and who also likely consume the fish, serum PFOS
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concentrations were extremely high, up to 31,000 ng/mL.20 Moreover, use of PFASs in China
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may have global consequences, as shown for F-53B (ClC6F12OC2F4SO3K, CAS No. 73606-19-6),
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a metal plating mist suppressant chemical used only in China since the 1970s.21-23 F-53B is
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and of human serum19 has followed. The link
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detectable in Chinese environmental22-24 and biological samples,24-27 but was also recently
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detected in Greenland ringed seals and polar bears.28
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Shorter-chain PFASs and structurally modified long-chain perfluorinated acids, such as
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those with ether bonds or non-fluorinated carbons, are two broad categories of known
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alternative-PFASs.13 Manufacturers claim that such alternatives are less bioaccumulative and
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“safe for their intended use”,29,
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environmental fate data for these. In many cases the alternative-PFASs, or their predicted
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breakdown products, do not avoid the problem of environmental persistence. For example,
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trifluoroacetic acid and PFBS were similarly resistant to microbial degradation as PFOS,31, 32 and
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perfluoroether chains were as resistant as perfluoroalkyl chains to abiotic and biotic
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degradation.33 Moreover, shorter-chain alternatives do not necessarily avoid unwanted
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bioaccumulative properties. Perfluorobutanesulfonamide (a PFBS precursor) was recently
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detected at 80 ng/g in fish in the Netherlands,34 suggestive of its bioaccumulation potential.
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but there are few publicly available toxicity studies nor
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Another concern is that unknown PFASs are now being intentionally manufactured as
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alternatives or unintentionally produced as byproducts, and that these may already be adding to
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environmental contamination. At a fluorochemical manufacturing site on the Yangtze River in
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China, our previous work by non-target high-resolution mass spectrometry (Nt-HRMS) revealed
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36 new PFASs in wastewater,35 including three PFAS classes that had not previously been
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reported. Moreover, it is generally reported that >90% of the total mass balance of organic
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fluorine in environmental36, 37 and biological samples27, 38, 39 is from unknown compounds. These
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unknowns may be attributable to unidentified legacy PFASs, or to contemporary alternative-
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PFASs, and it is important to identify these unknowns to evaluate or mitigate risks of PFAS
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exposure.
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Environmental Science & Technology
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Nt-HRMS techniques are valuable tools for chemical discovery owing to their high mass
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spectral resolving power, which reduce interferences, and their high mass accuracy which
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enables accurate empirical formula prediction. Their successful application to PFAS discovery in
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water
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HRMS method for PFAS discovery in water35 to the discovery of potentially bioaccumulative
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PFASs in fish tissue collected at two commercial fishery locations in China. Details of each
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chemical discovery and of related environmental and toxicological significance are discussed.
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or in firefighters’ serum43 are already demonstrated. Here we adapted our Nt-
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MATERIALS AND METHODS
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Samples Collection
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In October 2015, a commercial fisherman was recruited at each location (Figure S1) to
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collect fish on their regular routes. One location was on the Yangtze River, in a region
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approximately 10 km downstream of a wastewater treatment plant (WWTP) that services a major
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fluorochemical manufacturing park in Changshu, Jiangsu.35 We previously identified new PFASs
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in influent to this WWTP, and the effluent is known to be released to the Yangtze River.44 The
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other location was in Tangxun Lake in Wuhan, Hubei. This lake receives discharge from a
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WWTP that processes both domestic and industrial wastewater and has been identified as a
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contaminated water body.18
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Three fish species were obtained at the Yangtze River site, including common carp
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(Cyprinus carpio), silver carp (Hypophthaimichthys molitrix) and bighead carp (Aristichthys
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nobilis). The same three species were obtained at Tangxun Lake, as well as a fourth species,
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white Amur bream (Parabramis pekinensis). Between 3 and 7 individual whole fish were
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obtained and weighed (whole-fish wet weight) for each species at each location. Whole liver was
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collected from each individual fish, weighed (wet liver weight), and placed into a clean
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polyethylene bag with changing of gloves between fish to avoid cross-contamination. Livers
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were shipped overnight in an ice box to Nankai University (Tianjin, China) where all liver
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samples, and 10 g potassium chloride granules, as quality control for possible laboratory
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contamination, were freeze dried, weighed (dry liver weight, Table S1) and shipped to the
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University of Alberta for analysis.
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Sample Extraction and Nt-HRMS Analysis
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The same number of freeze-dried individual livers from the same species at each location
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were pooled, resulting in 7 fish samples (3 for the Yangtze River and 4 for Tangxun Lake, Table
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S1). All fish samples (one pooled sample per species at each site) with the procedural blank salts
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were extracted with acidified acetonitrile and purified with weak anion exchange cartridges
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(detailed in SI). After successively washing with 2% formic acid, water and methanol, the
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cartridges were eluted with 1% NH4OH in methanol, and the eluents were subjected to a
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previously developed non-target HPLC-Orbitrap Nt-HRMS method for PFAS discovery.35
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Briefly, a combination of in-source fragmentation flagging scan, full-scan and MSn experiments
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were used to detect and characterize unknown PFASs. Fluorinated fragment ions (e.g. [C2F5]-)
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detected by in-source fragmentation flagging were used to flag retention times of PFAS
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molecular ions in full-scan mode. Exact masses (±5 ppm) of the molecular ions, isotopic patterns
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and in-source fragments were used to assign empirical formulae.
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For each non-target class of PFAS homologues discovered, plausible structures were
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proposed based on MSn analyses, and confidence levels (CL=1-4) were assigned according to
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established criteria.45 Briefly, CL=1 represents confirmed structures by match to reference
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standards, CL= 2 represents probable structures by comparing to library spectra or by diagnostic
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Environmental Science & Technology
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evidence, CL=3 represents tentative candidates whose possible structure can be proposed but
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lack sufficient information to assign an exact structure (e.g. position of a chlorine atom on an
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aromatic ring), and CL=4 is used when the unknown analyte ion can be assigned an
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unambiguous formula, but no structural information is available.45
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Quality Control and Quantification
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Procedural blanks were injected between samples to check for background responses.
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Instrumental quantification limits (IQLs), method quantification limits (MQLs), absolute
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recoveries, absolute matrix effects and absolute total extraction efficiencies were evaluated with
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a mixed fish liver matrix containing equal amounts of the 7 pooled fish liver samples (detailed in
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SI). Legacy PFASs were quantified using authentic standards, and non-target PFASs were semi-
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quantified against structurally similar authentic standards as described in SI and in Table S3.
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RESULTS AND DISCUSSION
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Method Performance
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Absolute recoveries of the solid phase extraction method for model mass-labeled PFASs
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were
