Occurrence and Tissue Distribution of Novel Perfluoroether Carboxylic

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Occurrence and Tissue Distribution of Novel Perfluoroether Carboxylic and Sulfonic Acids and Legacy Per/Polyfluoroalkyl Substances in Black-Spotted Frog (Pelophylax nigromaculatus) Qianqian Cui, Yitao Pan, Hongxia Zhang, Nan Sheng, Jianshe Wang, Yong Guo, and Jiayin Dai Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b03662 • Publication Date (Web): 09 Jan 2018 Downloaded from http://pubs.acs.org on January 10, 2018

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Environmental Science & Technology

Occurrence and Tissue Distribution of Novel Perfluoroether Carboxylic and Sulfonic Acids and Legacy Per/Polyfluoroalkyl Substances in Black-Spotted Frog (Pelophylax nigromaculatus)

Qianqian Cui,1,2,# Yitao Pan,1,# Hongxia Zhang,1 Nan Sheng,1 Jianshe Wang,1 Yong Guo,3 and Jiayin Dai1,*

1

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

Academy of Sciences, Beijing 100101, China; 2University of Chinese Academy of Sciences, Beijing 100049, P. R. China; 3Key Laboratory of Organofluorine Chemistry Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai, P. R. China

# These authors contributed to this work equally.

*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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Research on perfluoroalkyl substances (PFASs) continues to grow. However, very little is

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known about these substances in amphibians. Here we report for the first time on the

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occurrence, tissue distribution, and bioaccumulation of two novel PFASs, chlorinated

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polyfluorinated ether sulfonic acid (6:2 Cl-PFESA) and hexafluoropropylene oxide trimer

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acid (HFPO-TA), in the black-spotted frog (Pelophylax nigromaculatus) from China. Frogs

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from cities with large-scale fluorochemical industries had significantly greater liver ΣPFAS

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levels (mean 54.28 ng/g in Changshu; 31.22 ng/g in Huantai) than those from cities without

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similar industry (9.91 ng/g in Zhoushan; 7.68 ng/g in Quzhou). Females had significantly

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lower liver PFAS levels than males, and older frogs tended to have lower PFAS levels than

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younger frogs. Skin, liver, and muscle contributed nearly 80% to the whole body burden of

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6:2 Cl-PFESA in males, whereas the female ovary alone accounted for 58.4%. These results

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suggest substantial maternal transfer of 6:2 Cl-PFESA to eggs, raising concern regarding its

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developmental toxicity on frogs and other species. The bioaccumulation factor results (6:2

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Cl-PFESA ˃ PFOS; HFPO-TA ˃ PFOA) suggest a stronger accumulative potential in the

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black-spotted frog for these alternative substances compared to their predecessors. Future

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studies on their toxicity and ecology risk are warranted.

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INTRODUCTION

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Per- and polyfluoroalkyl substances (PFASs) are a diverse group of chemicals used in

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various industrial and specialized consumer products, such as the production of

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fluoropolymers, fire-retarding foams, hard metal plating, pesticides, and surface repellent

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coatings of textiles and paper.1 Currently, there are at least 3000 PFASs on the global

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market.2 Among them, long-chain PFASs, including perfluoroalkyl carboxylic acids (PFCAs)

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with seven or more perfluorinated carbons and perfluoroalkane sulfonic acids (PFSAs) with

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six or more perfluorinated carbons, have received worldwide concern due to their propensity

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for bioaccumulation, ubiquitous distribution in the environment, biopersistence, and possible

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toxicity for wildlife and humans.3,4 As a result, numerous actions to reduce the release of

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long-chain PFASs and their precursors have been carried out since 2000.5-11 After years of

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effort by global regulators, governments, and industry, perfluorooctanesulfonate (PFOS) and

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related substances have been listed as Persistent Organic Pollutants under the Stockholm

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Convention,11 with perfluorooctanoate (PFOA) and related compounds being evaluated for

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listing.12

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During the phase out of long-chain PFASs, some novel fluorinated compounds have

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emerged as replacements. These alternatives include shorter-chain homologues of long-chain

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PFASs,13 and functionalized perfluoropolyethers such as perfluorinated perfluoroether

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carboxylic (PFECAs) and sulfonic acids (PFESAs).4 By insertion of oxygen atoms into

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perfluoroalkyl chains, the backbones of PFECAs and PFESAs can be divided into two or

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more shorter perfluorinated chains, which are hoped to be more degradable.14 Among the

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PFESAs, chlorinated polyfluorinated ether sulfonic acids (6:2 and 8:2 Cl-PFESAs, i.e. 3



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Cl(CF2)xO(CF2)2SO3-, where x = 6 and 8, respectively, as shown in Supporting Information

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(SI) Figure S1), have been used to replace PFOS as mist suppressants (commercial name

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F-53B) in the chromium plating industry.15 The annual production of F-53B is estimated to be

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20–30 t/year in China.15 For PFECAs, hexafluoropropylene oxide dimer acid (HFPO-DA)

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has replaced PFOA as a processing aid in the manufacture of fluoropolymers.16 Recently,

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hexafluoropropylene oxide trimer acid (HFPO-TA) has also been used in China.17 The

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production volume of HFPO-DA is currently 10–100 t/year in Europe,7 though the volume

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for HFPO-TA remains unclear.

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The occurrences of these alternatives in abiotic and biotic environments have been

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examined, with 6:2 and 8:2 Cl-PFESAs found to be ubiquitous in Chinese surface water,18

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municipal sewage sludge,19 wildlife,20,21 and humans.22,23 Recent studies have shown that 6:2

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Cl-PFESA can be biomagnified along the food chain in marine organisms,20 and its

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bioaccumulative potential may be even higher than that of PFOS in certain freshwater species

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such as crucian carp.21 The detection of 6:2 Cl-PFESA in remote Arctic wildlife24 (e.g., polar

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bears and killer whales) further implies the long-range transport of this chemical, which is of

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global concern. For PFECAs, HFPO-DA and HFPO-TA have been detected in rivers close to

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fluorochemical plants in the US,25 Germany,26 and China.17,26 Our recent study also reported

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on the presence of HFPO-TA in wild common carp, and found HFPO-TA to be potentially

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more accumulative than PFOA.17 Biomonitoring research on these alternative substances, as

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well as legacy PFASs, continues to grow, but information gaps remain. Studies on the

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occurrence and bioaccumulation of PFASs have predominately focused on fish, aquatic birds,

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and mammals,27-30 but rarely on amphibians.31 Although limited research has reported on the 4


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presence of PFASs in amphibians, such studies only included amphibians as part of a larger

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set of animal classes to explore the distribution of PFASs along the food chain.28,32 Study

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solely on amphibians, especially on the tissue distribution and whole body burden of PFASs,

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is lacking. Cutaneous respiration is a supplementary respiratory mode for many amphibians,

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though also a primary mode in some species. The skin of most frogs is highly vascular and

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permeable to allow the exchange of gas and small molecules.33 This may influence the tissue

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distribution and bioaccumulation of PFASs compared with that in aquatic fish and terrestrial

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animals.

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Amphibians are currently among the world’s most endangered animals.34 With their

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permeable skins, low mobility, and reliance on accessible water bodies, amphibians are more

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vulnerable to threats such as habitat destruction, chemical contaminants, introduced species,

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and pathogenic diseases.35 It is reported that 42% of amphibian species are declining in

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population, with environmental pollution the second most common threat, affecting 19% of

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all amphibian species.34 The black-spotted frog (Pelophylax nigromaculatus, formerly Rana

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nigromaculata) is a common and widespread species in Eastern Asia, with a maximum

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longevity of 13 years. It is listed in the IUCN Red List as Near Threatened (NT) due to its

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rapid population decline in China.34 Growing industrial activities in China have caused

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increasing pollution, which is likely an important threat to the black-spotted frog, as it is to

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other amphibian species.34 Therefore, it is necessary to investigate the exposure levels of

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chemical contaminants, such as PFASs, and explore their distribution profiles in the

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black-spotted frog.

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In the present study, wild black-spotted frogs were collected in four cities of China with 5



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or without fluorochemical plants nearby. The main objectives were (1) to investigate whether

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novel chemical alternatives Cl-PFESAs and HFPO-TA are present in frogs; (2) to explore the

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relationships of PFAS exposure with fluorochemical industrial activities, as well as sex- and

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age-related differences among frogs; and (3) to generate currently missing data for the tissue

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distribution and whole body burden of legacy and alternative PFASs in frogs.

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MATERIALS AND METHODS

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Locations and Sample Collection. From May to September 2016, a total of 56

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black-spotted frogs (P. nigromaculatus) were captured alive in rice paddy fields in Changshu

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(n = 29), Quzhou (n = 12), and Zhoushan (n = 11), and in corn fields in Huantai (n = 4) from

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China (SI Table S1 and Figure S2). Large-scale fluorochemical industries are found in

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Changshu and Huantai, but not in Quzhou and Zhoushan. In Changshu, a fluorine chemical

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industrial zone with over 27 fluorochemical plants is situated in a suburban area of the city.36

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One of the largest polytetrafluoroethylene (PTFE) production plants in Asia is located in

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Huantai, with a reported production volume of 37000 t/year.37 The sampling sites in

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Changshu and Huantai were approximately 10–20 km from the fluorochemical plants. In

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each sampling site, five water samples (each approximately 200 mL) were collected in the

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rice paddy fields or corn field ditches in parallel with frog collection, and were pooled into a

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1 L methanol-rinsed polypropylene bottle. Sex, snout-vent length, and whole body weight of

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the frogs were recorded before sacrifice (SI Table S1). Age was assessed by

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skeletochronology (details in SI). Tissue samples, including the liver, kidney, heart, lung,

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stomach, intestine, gonad, skin, and muscle, were carefully dissected from the frogs,

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subsequently weighed (SI Table S2), cleaned with Milli-Q water, homogenized, and 6


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maintained at −20°C. All tissues were extracted separately without being pooled. Large egg

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masses were observed in all ovaries from Changshu (captured in June) and Quzhou frogs

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(May), but not in any ovaries from the Zhoushan frogs (August), as the reproductive period

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for black-spotted frog ranges from April to July. The entire ovary was homogenized, and the

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unlaid eggs were not separated. It was not possible to obtain a sufficient volume of blood for

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PFAS analysis; therefore, blood samples were not collected in this study. All research

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protocols were approved by the Ethics Committee of the Institute of Zoology, Chinese

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Academy of Sciences.

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Standards. A total of 17 target PFASs were included in this study (see the SI for full

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names). Native and mass-labeled standards for PFCAs (C4–C14) and PFSAs (C4, C6, and C8)

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were purchased from Wellington Laboratories Inc., (Ontario, Canada). Native standards of

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HFPO-TA and 6:2 and 8:2 Cl-PFESAs were synthesized based on our earlier published

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methods, with all purities above 98%.17

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Sample Extraction. The samples were extracted based on previously published

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methods.38,39 Details of the extraction methods for different matrices are provided in the SI.

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Briefly, water samples were extracted by a weak anion exchange solid phase extraction (SPE)

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cartridge (strata X-AW, 200 mg/6 mL, Phenomenex, CA, USA). Frog tissue samples, except

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for muscle and carcass, were extracted using ion-pair extraction. Alkaline digestion was used

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for muscle and carcass samples to achieve a more effective digestion. Additional cleanup

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using an Oasis weak anion exchange (WAX) cartridge (200 mg/6 mL Waters, MA, USA)

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was applied to all frog samples.

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Instrument Analysis. An Acquity UPLC coupled to a Xevo TQ-S triple quadrupole 7



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mass spectrometer (Waters, Milford, MA, USA) was used to quantify target PFASs, except

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for HFPO-DA and HFPO-TA. The quantification of these two PFECAs was conducted using

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an API 5500 triple-quadrupole mass spectrometer (AB SCIEX, Framingham, MA, USA) due

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to its much higher sensitivity than Xevo TQ-S. Details of chromatographic column and

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instrument parameters are presented in the SI (Table S3).

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Quality Assurance and Quality Control. To reduce possible contamination, all labware

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and dissection tools were prescreened and rinsed with methanol before use. Two extraction

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blanks were included in every 18 samples to monitor background contamination. The

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matrix-specific limits of quantification (LOQs) and spike-recoveries are provided in SI Table

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S4 and S5, respectively. The LOQs were evaluated based on three criteria: (1) concentration

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resulting in a signal-to-noise ratio of 10 in different matrices; (2) lowest concentration of

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standard in the calibration curve with measured concentrations within ±20% of its theoretical

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value; and (3) concentration or dilution factor. Spike recoveries (n = 3) were validated by

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spiking 2 ng of standard into a blank matrix (laboratory-bred black-spotted frogs) and

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subjected to the extraction method mentioned above. An eleven-point calibration curve was

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verified daily and exhibited excellent linearity in the range of 0.01−50 ng/mL (R2 > 0.99).

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Instrumental drift was checked by injecting a 0.1 ng/mL standard in every 10 samples. A new

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calibration curve was reconstructed if a deviation of greater than ±20% from its theoretical

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concentration was observed. However, no instrumental drift was observed during any batch.

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Data Analysis. When the concentrations of the PFASs were below the LOQs, a value of

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LOQ/√2 was employed for the descriptive statistics and comparisons. Analytes with detection

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rates of less than 50% were excluded from the statistics. As the livers of fish, frogs, birds, and 8


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mammals typically accumulate high levels of PFASs and are commonly used as the

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representative tissue in many earlier studies,28,40-42 we used the concentration of PFASs in the

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liver to compare the geographical as well as age- and sex-related differences in frogs. The

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non-parametric Mann-Whitney U test was used to test for differences within groups. The

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relationship between PFAS liver concentration and frog age was examined by linear

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regression. Fourteen frogs (ten from Changshu and four from Huantai) were selected to

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explore the tissue distribution and whole body burden of PFASs. The whole body burden (ng)

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was calculated by the following equation:

ℎ = × = "#$

,"

× %

,"

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Where mfrog (g) is the total body weight, Cwhole body (ng/g) is the estimated PFAS concentration

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for the whole body, Ctissue (ng/g) is the concentration of PFAS in certain tissue, and ftissue is

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the mass fraction of individual tissues. The contribution of each target tissue was

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subsequently calculated from the whole body burden of PFASs. The bioaccumulation factor

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(BAF) was calculated as the concentration of PFASs in the whole frog body over the

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concentration in the corresponding water sample. &A( =

∑"#$ ," × % = ) )

,"

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All statistical analyses were performed using IBM PASW statistics 18.0 (SPSS Inc., USA)

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with a statistical significance threshold of p < 0.05.

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RESULTS AND DISCUSSION

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Geographical Differences in Liver PFAS Concentrations. The concentrations of

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legacy and alternative PFASs in frog liver samples are presented in Figure 1 and SI Table S6. 9



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Of the 17 PFASs, including 11 PFCAs, three PFSAs, two PFESAs, and one PFECA, all were

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detected, except for PFBA, PFPeA, PFHpA, and PFBS. The detection frequencies for PFOA,

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PFDA, PFUnDA, PFDoDA, PFTriDA, and 6:2 Cl-PFESA were 100%, and ranged from 7.1%

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to 98% for other substances. Geographical differences in PFAS levels were observed (Figure

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1). For legacy PFASs (sum of all detected PFCAs and PFSAs), the liver concentrations from

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Changshu (33.6 ± 24.4 ng/g ww) and Huantai (13.2 ± 6.6 ng/g ww) were significantly higher

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than those from Zhoushan (6.7 ± 2.8 ng/g ww) and Quzhou (6.9 ± 4.5 ng/g ww). The highest

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mean ΣPFESA concentration (sum of 6:2 and 8:2 Cl-PFESAs) was also observed in

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Changshu (20.7 ± 25.9 ng/g ww), which was 5−26 times greater than that from the other

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cities. HFPO-TA was only detected in samples from Huantai, with a mean value of 13.4 ± 9.4

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ng/g ww.

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The PFAS composition patterns in the liver also varied among different sampling sites

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(SI Figure S3). No PFAS was consistently predominant in the samples from the four cities. In

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Changshu and Zhoushan, 6:2 Cl-PFESA was the predominant compound, accounting for 29.7%

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and 30.6% of total PFASs, respectively, and its proportions (9.2−30.6%) were all

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significantly greater than those of PFOS (7.6−18.8%) in corresponding locations. HFPO-TA

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was the most abundant in samples from Huantai, accounting for 40.9% of total PFASs,

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whereas PFTriDA was predominant in Quzhou, accounting for 26.2% of total PFASs.

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The discrepancies between the level and composition of PFASs in the frog liver were

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probably due to the level of PFAS exposure in the local environments. In Changshu and

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Huantai, although the farmland water samples were collected at least 10 km from the

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fluoropolymer plants, the concentrations of ΣPFAS (1.35 µg/L in Changshu; 54.7 µg/L in 10


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Huantai) were one to three orders of magnitude higher than those in Quzhou (21.3 ng/L) and

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Zhoushan (26.1 ng/L, SI Table S7). We did not observe direct wastewater discharge in the

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rice paddy fields of Changshu, but the high levels of PFASs in the corn field ditches in

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Huantai probably came from the strongly interconnected waterway network of the Xiaoqing

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River basin. Large-scale manufacture of PTFE and other fluoropolymers is reported to be the

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main source of pollution in Xiaoqing River.37 Furthermore, it is likely that PFOA and novel

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replacement HFPO-TA, which are used as processing aids, are not fully consumed in the

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fluoropolymer manufacture process, with inefficient treatment resulting in final release into

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the river basin.17 It is worth noting that PFOA and PFHxA accounted for a large proportion of

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ΣPFAS in waters from Changshu and Huantai, but not in the liver of frogs (SI Figure S3). On

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the contrary, the proportions of novel Cl-PFESAs and HFPO-TA were relatively small in the

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water, but were significantly enhanced in the frog livers. These results may represent different

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accumulative potentials for these chemicals, as discussed below. Taken together the results

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from the four locations indicated a tendency for frogs living closer to fluorochemical

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industries to have higher and more novel PFAS exposure compared with those living in

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locations without fluorochemical industries. It should be noted that water samples from each

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location were collected only once, and all water samples have only one time point. Analysis

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of a water sample can be used to evaluate broad differences in PFAS levels spatially, however

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a larger sample size would be necessary to confirm these trends. As the black-spotted frog is

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a near threatened species in China, whether PFASs cause adverse effects on frogs at current

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concentrations should be of concern. We did not observe any significant associations between

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PFAS levels and liver somatic indices (data not shown). Further information regarding the 11



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toxicity of PFASs on frogs, especially the novel alternatives, is urgently needed.

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Sex- and Age-Related Differences in PFAS Levels. Significant sex-related differences

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in liver ΣPFAS levels were observed in frogs from Changshu, Zhoushan, and Quzhou (Figure

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2). The mean ΣPFAS concentrations in males were 85.2 ng/g ww, 12.0 ng/g ww, 10.9 ng/g

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ww in Changshu, Zhoushan, and Quzhou, respectively, which were approximately 1.6−2.3

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times higher than levels in the corresponding females. No female was captured in Huantai,

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which hampered our exploration of sex-specific accumulation of HFPO-TA. Sex-related

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differences were also explored in other tissues in frogs from Changshu. Most tissues showed

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higher PFAS levels in males than females, although the opposite pattern was observed in the

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gonad and kidney (SI Table S8). Mean ΣPFAS concentration in the female ovary (95.4 ng/g

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ww) was three times higher than that in the male testis (30.9 ng/g ww), whereas the mean

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ΣPFAS level in the kidney was 132.5 ng/g ww in females versus 91.7 ng/g ww in males.

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Sex-related differences have also been observed in turtles,28 fish,40 birds,41 and

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mammals.42 In most studies, males have shown higher PFAS concentrations in liver samples

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compared to those in females.40-42 These differences could be attributed to factors such as

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maternal transfer through eggs,28 cord blood,22 and breast-feeding,43 and the sex-related

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expression of organic anion transporter proteins, resulting in faster PFAS elimination rates in

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female organisms.44,45 For black-spotted frogs, maternal transfer through eggs may be the

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most important contributor to sex-related differences. As PFASs preferentially accumulate in

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protein rich matrices, like the egg,46 the conception of large numbers of eggs during

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reproductive cycles could accumulate a considerable proportion of the PFAS burden in

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female frogs, consequently leading to the observed differences in tissue PFAS levels 12


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compared to males. Here, necropsy revealed the presence of large masses of eggs in the

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ovaries of female frogs in the reproductive season. It is reasonable to believe that a large

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proportion of PFASs would be released along with eggs before the end of the reproductive

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period. The higher PFAS concentrations in female kidneys may indicate another contributor

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to the sex-related differences, with female frogs possibly having a higher renal elimination

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rate, and thus accelerated clearance of PFASs and decreased body burden. This hypothesis

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could be supported by earlier literatures, that some female marine mammals (e.g., harbor

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porpoises47 and harbor seals48) had higher kidney PFAS concentrations than males. Faster

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renal elimination rate of PFASs was also observed in females than males in rats,49 fish,50

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monkeys51 and humans.52

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In the linear regression model, a significantly negative association was found between

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liver ΣPFAS concentration and frog age (p = 0.028, SI Table S9). When sampling sites and

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sex were included in the model as covariates, the association between ΣPFAS level and age

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remained statistically significant (p = 0.047, SI Table S9). Age-related differences in the

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ΣPFAS levels among cities are shown in Figure 3. In Changshu and Quzhou, the ΣPFAS

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concentrations in 1-year-old frogs were significantly higher than those in 3-year-old frogs.

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These results demonstrate possible age-related differences in PFAS accumulation for

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black-spotted frogs, in accordance with our previous study on the Chinese alligator, in which

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serum levels of long-chain PFCAs were lower in adults than in juveniles.53 Age-related

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similarities have also been observed in a variety of mammals, including harbor porpoises,47

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beluga whales,54 bottlenose dolphins,55 and Baikal seals.56 It is not yet clear why age shows

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an influence on the bioaccumulation of PFASs in these wildlife. A possible factor may be 13



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somatic growth dilution, which has occurred in methyl-mercury studies in aquatic

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organisms.57 However, conflicting results have also been obtained from Chinese sturgeon46

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and Ridley turtles,58 which present positive relationships between age and PFAS

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accumulation. These inconsistent results indicate that the process of PFAS bioaccumulation

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in wildlife is complicated, and demonstrate the importance of studying species-specific PFAS

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accumulation.

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Tissue Distribution and Whole Body Burden. The tissue distribution patterns of

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PFASs in the black-spotted frogs are shown in Figure 4. The detection rates of target PFASs

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in different frog tissues are shown in SI Table S10. The accumulation of PFASs varied among

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different tissues. The highest mean ΣPFAS concentrations were observed in the kidney (112.1

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ng/g ww in Changshu, 140.0 ng/g ww in Huantai), followed by, in descending order, the

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ovary > liver > testis ≈ skin ≈ lung ≈ heart ≈ intestine ≈ stomach ≈ muscle ≈ carcass. The

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distributions of ΣPFCAs and ΣPFSAs among tissues were identical with that of ΣPFASs (SI

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Table S11 and S12), though the mean concentration of ΣPFESAs in the ovary was even

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higher than that in the kidney of frogs from Changshu (SI Table S11). HFPO-TA was

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uniquely detected in Huantai, with the highest level observed in the kidney (59.3 ng/g ww)

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followed by the liver (13.4 ng/g ww, SI Table S12). Regrettably, the level in the ovary was

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unknown as no female was collected in Huantai. Whole body burdens of 6:2 Cl-PFESA and

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HFPO-TA were explored and compared to their corresponding predecessors PFOS and PFOA,

277

respectively (Figure 5). Although the concentrations of PFOS and 6:2 Cl-PFESA were high in

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the kidney, the contribution to the whole body burden was fairly low (1.6−4.0%) when

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considering the body fraction of tissues. Over 70% of the whole body burden of PFOS and 14


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6:2 Cl-PFESA was from the skin, liver, and muscle for males, whereas the female ovary

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accounted for 70.3% and 58.4% of the whole body burden for PFOS and 6:2 Cl-PFESA,

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respectively (Figure 5). Muscle was the primary body compartment for HFPO-TA (32.4% of

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the total body burden), followed by the liver (24.0%) and skin (19.6%) in male frogs from

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Huantai (Figure 5). Furthermore, the whole body ΣPFAS level was lower in males from

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Huantai (mean 7.14 ng/g ww) than males from Changshu (9.50 ng/g ww), although the

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discrepancy did not reach statistical significance (Figure 6). The body burden of ΣPFAS was

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significantly higher in females from Changshu (16.82 ng/g ww) than in males, although in

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most tissues males tended to have higher ΣPFAS levels (SI Table S8).

289

Previous studies on the tissue distribution of PFASs in other species have shown that the

290

kidney generally contains the highest levels of PFASs in fish,46,59 whereas the liver usually

291

shows the highest PFAS concentration in birds and marine mammals.60-62 Frogs are perched

292

midway on the evolutionary tree between fish, and birds and mammals. The highest PFAS

293

levels observed in the frog kidney (Figure 4) indicate that the PFAS tissue distribution pattern

294

in the frog is, to some extent, much closer to that in fish. The ovary had the second highest

295

PFAS levels, and highest whole body burden proportion. As frogs in Changshu were

296

collected during the mating period, all ovary samples were obviously enlarged (SI Table S2)

297

and full of unlaid eggs. It is reasonable to consider that the abundance of PFASs in the ovary

298

was mainly attributable to PFASs in the eggs, which helps explain the sex-related differences

299

observed in tissue PFAS concentrations discussed above. A predominant proportion of PFASs

300

was accumulated in the ovary, which decreased the burden in other tissues. Furthermore, the

301

inconsistent results between tissue and whole body ΣPFAS levels in females versus males 15



302

indicate that the level of ΣPFAS in a single tissue (e.g., liver) does not well-represent the

303

whole body burden for gravid females. The pattern in the whole body might accord with that

304

in the liver after the reproductive period, as 68.5% of the ΣPFAS burden was released with

305

the eggs (Figure 6).

306

The relatively high fraction of PFASs, especially PFOS and 6:2 Cl PFESA, stored in the

307

skin (about one third of total burden in males) is an interesting finding. To the best of our

308

knowledge, no study has previously investigated the body burden of PFASs in skin. Therefore,

309

it is difficult to judge whether similar patterns also occur in other species, or if it is a special

310

feature of the black-spotted frog and other amphibians. The cutaneous respiration of frogs

311

allows the exchange of gas and small molecules through their permeable skins,33 thus

312

indicating a possibly significant exposure route for contaminants like PFASs.

313

Overall, a similar distribution pattern was observed for PFOS and 6:2 Cl-PFESA, as well

314

as for PFOA and HFPO-TA, suggesting that these compounds share similar mechanisms of

315

distribution. However, some obscure but interesting findings are noteworthy. The liver

316

accounted for a greater proportion of the whole body burden for 6:2 Cl-PFESA and

317

HFPO-TA compared to their corresponding predecessors PFOS and PFOA (Figure 5). A

318

recent investigation on the binding affinity to hepatic fatty acid binding protein (h-FABP, one

319

of the most abundant proteins in the liver) may help explain this phenomenon. Both 6:2

320

Cl-PFESA and HFPO-TA are more strongly bound to h-FABP than PFOS and PFOA,63

321

probably leading to additional sorption and higher distribution in the liver. Contrarily, the

322

kidney showed a lower burden of the PFAS alternatives than their predecessors (6:2

323

Cl-PFESA ˂ PFOS; HFPO-TA ˂ PFOA, Figure 5). Thus, we speculated that 6:2 Cl-PFESA 16


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Page 17 of 38


324

and HFPO-TA might exhibit lower excretion by renal clearance. Earlier literature on PFASs

325

has shown that higher hydrophobicity generally leads to lower renal elimination.23,64,65

326

Additionally, the insertion of ester bonds or chlorine atoms might enlarge the molecular size,

327

and hence increase the hydrophobic properties of 6:2 Cl-PFESA (log Kow = 5.24, estimated

328

by EPI Suite 4.11) and HFPO-TA (log Kow = 5.56) compared with those of PFOS (log Kow

329

= 4.49) and PFOA (log Kow = 4.81).15,17 The ester bonds may also lead to structural

330

distortion on the molecular backbone, which could affect the polarity and hydrophobicity of

331

the acidic functional group. This hypothesis is in accordance with an earlier human study,

332

suggesting slower renal clearance for Cl-PFESAs than PFOS.23 However, additional

333

empirical evidence is still needed.

334

Bioaccumulation Factors. The bioaccumulation factor (BAF) for the black-spotted frog

335

was estimated based on PFAS concentration in the whole body compared with that in

336

corresponding water sample. Although adult frogs do not inhabit water exclusively, which

337

might result in bias, the calculation of BAF based on water concentration still provides a

338

useful estimate on the bioaccumulative potential of contaminants, and has been widely used

339

in other research on frogs.66-68 The mean log BAFwhole body for PFASs ranged from −0.68 to

340

4.57 in frogs from Changshu (SI Figure S4), approximately 0.5−1.0 log units higher than

341

those from Huantai (SI Figure S5). In both locations, the BAFwhole body of PFASs increased

342

significantly with increasing carbon chain length, which is in good agreement with previous

343

studies.59,69,70 The mean BAFwhole body for 6:2 Cl-PFESA (1304 L/kg) was higher than that for

344

PFOS (1050 L/kg, SI Figure S4), whereas the value for HFPO-TA (0.76 L/kg) was higher

345

than that for PFOA (0.37 L/kg, SI Figure S5). The BAF rankings for the two alternatives 17



346

versus their predecessors (6:2 Cl-PFESA ˃ PFOS; HFPO-TA ˃ PFOA) were the same as our

347

earlier investigation on common carp,17 although the values were one to two orders of

348

magnitude lower than before. This discrepancy might be species related, or possibly due to

349

different subjects being used to estimate BAF. We used BAFblood in common carp in our

350

previous study,17 which might overestimate the bioaccumulation of PFASs in the whole body.

351

In the present study, Cl-PFESAs showed a higher bioaccumulation than PFOS. The median

352

BAFwhole

353

bioaccumulative in frogs. However, this finding should not be discounted. PFOA does not

354

bioaccumulate in aquatic animals,70 but does in air-breathing mammals.71 Similar (or more

355

adverse) patterns might also apply to HFPO-TA, considering its higher bioaccumulative

356

potential in common carp17 and black-spotted frog than that of PFOA.

body

for HFPO-TA was relatively low (< 1), suggesting it may not be

357

In conclusion, the occurrence of novel PFAS alternatives Cl-PFESAs and HFPO-TA,

358

along with legacy PFASs, were detected in black-spotted frogs from China. HFPO-TA was

359

distinctively found in frogs from fluoropolymer industrial regions, whereas the presence of

360

Cl-PFESAs appeared ubiquitous. Frogs living closer to fluorochemical industries tended to

361

have higher PFAS exposure compared to those living in fluorochemical industry-free regions,

362

indicating a potential threat to the black-spotted frog. Our study provides useful information

363

on the tissue distribution and total body burden of PFASs in frogs. The relatively large

364

proportion of PFAS burden in the skin might be distinctive in amphibians, and the greatest

365

abundance in the ovary could explain the sex-related differences observed in PFAS

366

concentrations. The findings from the ovary further imply that high levels of PFASs can be

367

transferred from mother to eggs, and increase concern about the developmental toxicity of 18


Page 18 of 38

Page 19 of 38


368

novel PFAS alternatives on frogs and other species. As these alternatives are alike in

369

molecular structure with their predecessors, similar toxicity should not be surprising. A recent

370

study showed that acute exposure to 6:2 Cl-PFESA led to multiple dysplasia in embryo

371

zebrafish,72 while the toxicity of HFPO-TA remains unknown. Further study is necessary to

372

understand the species-specific accumulation, toxicity, and ecological risk of Cl-PFESAs,

373

HFPO-TA, and other fluorinated alternatives.

374

ACKNOWLEDGMENTS

375

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

376

31320103915 and 21737004) and the Strategic Priority Research Program of the Chinese

377

Academy of Sciences (XDB14040202) for Dr. Dai; and the National Natural Science

378

Foundation of China (grant 21421002) for Dr. Guo.

379

Supporting Information

380

Additional information includes standards and reagents, sample extraction, QA/QCs,

381

skeletochronological age determination, and other materials in Figures S1−S5 and Tables

382

S1−S12.

19



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29



Figure legends

Figure 1. Concentrations of PFASs in frog livers from Changshu (CS, n = 29), Huantai (HT, n = 4), Zhoushan (ZS, n = 11), and Quzhou (QZ, n = 12). Different letters indicate statistically significant differences among locations. Bars and whiskers indicate means ± standard deviation.

Figure 2. Sex-related ΣPFAS concentrations in frog livers from Changshu (CS, n = 29), Zhoushan (ZS, n = 11), and Quzhou (QZ, n = 12). Data are expressed as means ± SD. * p < 0.05; ** p < 0.01.

Figure 3. Age-related ΣPFAS concentrations in frog livers from Changshu (CS, n = 29), Huantai (HT, n = 4), Zhoushan (ZS, n = 11), and Quzhou (QZ, n = 12). Bars and whiskers indicate means ± standard deviation. Only one frog in Changshu was 4-years-old, hence it was excluded from the comparison among groups. * p < 0.05; ** p < 0.01.

Figure 4. Tissue distribution of ΣPFAS concentration (mean ± standard deviation) in black-spotted frog from Changshu (n = 10) and Huantai (n = 4).

Figure 5. Whole body burden of 6:2 Cl-PFESA (up) and HFPO-TA (down) compared to legacy PFOS and PFOA in black-spotted frogs from Changshu (n = 10) and Huantai (n = 4).

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Figure 6. ΣPFAS body burden levels in frogs from Huantai (n = 4) and Changshu (n = 10). Bars and whiskers indicate means ± standard deviation. Different letters indicate statistically significant differences among locations. The dashed line bar represents an estimated body burden of ΣPFAS for female frogs in Changshu, with all eggs released after the reproductive period.

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Figure 1. Concentrations of PFASs in frog livers from Changshu (CS, n = 29), Huantai (HT, n = 4), Zhoushan (ZS, n = 11), and Quzhou (QZ, n = 12). Different letters indicate statistically significant differences among locations. Bars and whiskers indicate means ± standard deviation. 381x201mm (300 x 300 DPI)


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Figure 2. Sex-related ΣPFAS concentrations in frog livers from Changshu (CS, n = 29), Zhoushan (ZS, n = 11), and Quzhou (QZ, n = 12). Data are expressed as means ± SD. * p < 0.05; ** p < 0.01. 152x127mm (300 x 300 DPI)



Figure 3. Age-related ΣPFAS concentrations in frog livers from Changshu (CS, n = 29), Huantai (HT, n = 4), Zhoushan (ZS, n = 11), and Quzhou (QZ, n = 12). Bars and whiskers indicate means ± standard deviation. Only one frog in Changshu was 4-years-old, hence it was excluded from the comparison among groups. * p < 0.05; ** p < 0.01. 131x72mm (220 x 220 DPI)


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Figure 4. Tissue distribution of ΣPFAS concentration (mean ± standard deviation) in black-spotted frog from Changshu (n = 10) and Huantai (n = 4). 317x139mm (300 x 300 DPI)



Figure 5. Whole body burden of 6:2 Cl-PFESA (up) and HFPO-TA (down) compared to legacy PFOS and PFOA in black-spotted frogs from Changshu (n = 10) and Huantai (n = 4). 82x119mm (220 x 220 DPI)


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Figure 6. ΣPFAS body burden levels in frogs from Huantai (n = 4) and Changshu (n = 10). Bars and whiskers indicate means ± standard deviation. Different letters indicate statistically significant differences among locations. The dashed line bar represents an estimated body burden of ΣPFAS for female frogs in Changshu, with all eggs released after the reproductive period. 215x127mm (300 x 300 DPI)



TOC 84x47mm (300 x 300 DPI)


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Occurrence and Tissue Distribution of Novel Perfluoroether Carboxylic

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