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Facilitated bioaccumulation of perfluorooctane sulfonate in common carp (Cyprinus carpio) by graphene oxide and remission mechanism of fulvic acid Liwen Qiang, Meng Chen, Ling-Yan Zhu, Wei Wu, and Qiang Wang Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b02100 • Publication Date (Web): 10 Oct 2016 Downloaded from http://pubs.acs.org on October 10, 2016
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Facilitated bioaccumulation of perfluorooctane sulfonate in common carp (Cyprinus
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carpio) by graphene oxide and remission mechanism of fulvic acid
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Liwen Qiang†, Meng Chen†, Lingyan Zhu ,†,‡ , Wei Wu†, Qiang Wang†
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†College of Environmental Science and Engineering, Tianjin Key Laboratory of Environmental
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Remediation and Pollution Control, Key Laboratory of Pollution Processes and Environmental
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Criteria, Ministry of Education, Nankai University, Tianjin, P.R. China 300071
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‡College of natural resources and environment, Northwest A&F University, Yangling, Shaanxi, P.R.
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China 712100
*
9 10
∗
To whom correspondence should be addressed. E-mail:
[email protected]. Phone: +86-22-23500791. Fax: +86-22-23503722. 1
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ABSTRACT
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As one of the most popular carbon-based nano-materials, graphene oxide (GO) has the potential
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to be released in aquatic environment and interact with some coexistent organic pollutants, such as
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perfluorooctane sulfonate (PFOS), which is an emerging persistent organic pollutant. In this study,
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the adsorption of PFOS on GO in presence of fulvic acid (FA), the impacts of GO and FA on PFOS
16
toxicokinetics in carp (Cyprinus carpio), and in vitro digestion behaviors were examined. The results
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indicated that PFOS could be strongly adsorbed on GO with a Freundlich affinity coefficient KF of
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580 ± 205 (mg/g)/(mg/L)n, while the adsorption was suppressed by FA due to competitive adsorption.
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GO significantly enhanced the bioaccumulation of PFOS in blood, kidney, liver, gill, intestine and
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muscle of carp, and the corresponding BAF was in the range of 2 026 ~ 53 513 L/kg. The
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enhancement was greatest for liver and intestine, which was 10.3 and 9.33 times of that without GO,
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respectively. In vivo toxicokinetic and in vitro digestion-absorption experiments indicated that GO
23
could carry PFOS to penetrate the intestine cells. There herein, PFOS absorption, especially via
24
intestine, and the uptake rate coefficient (ku) were greatly enhanced, leading to distinctly promoted
25
bioaccumulation of PFOS in fish. However, FA could facilitate the flocculation of GO in the intestine,
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and also accelerate excretion of GO-PFOS complex. Thus, in the presence of FA, PFOS absorption
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was reduced and the promotion effect of GO on PFOS accumulation was remitted.
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Keywords: graphene oxide, PFOS, fulvic acid, bioaccumulation, fish
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INTRODUCTION
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Graphene oxide (GO) is a graphene sheet with carboxylic groups at its edges and phenol
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hydroxyl and epoxide groups on its basal plane.1 Due to its excellent water dispersion and 2
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amphiphilic characteristics,2 it has found wide applications in energy storage, catalysis, cell imaging,
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biochemical sensing, drug delivery and pollutant remediation.3 Although the production volume of
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GO is less than some metallic nanomaterials, such as nano-TiO2,4 nano-Ag,5 it is now considered to
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be a potential emerging environmental pollutant, and its adverse effects on aquatic organisms have
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received increasing attentions.6 Many studies documented that GO could interact with cells and
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bacteria via specific interactions, and produced some toxicological effects (including genotoxicity
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and cytotoxicity).7-10 Studies on biodistribution of GO in mice/rat demonstrated that GO was prone
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to accumulate in liver, lungs and spleen through intravenous injection.11-14 Giving that GO has
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abundant π-π electrons and surface carboxylic, phenol hydroxylic groups, it could adsorb a variety of
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pollutants, such as organic molecules (phenanthrene, tetracycline),15, 16 inorganic metal ions (Pb2+,
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Cd2+),17, 18 macromolecules (DNA, humic acid)19, 20 and particles (single-walled carbon nanotubes),21
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forming GO-associated complexes in aquatic environment.
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As an emerging persistent organic pollutant (POP), perfluorooctane sulfonate (PFOS) has been
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recognized as a global contaminant and widely present in humans, wildlives and waters all over the
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world.22 It is extremely persistent in the environment and shows toxicities on mammals and fish.23, 24
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Owing to the hydrophobic carbon chain and hydrophilic sulfonate functional group,25 PFOS could be
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adsorbed by a variety of adsorbents,25 such as multiwalled carbon nanotubes26 and carbon nitride.27 It
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is assumed that PFOS may also be adsorbed on GO to form GO-PFOS complex given that GO has
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abundant surface groups. In our previous study, it was found that nano-TiO2 could promote PFOS
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accumulation in fish by carrying PFOS into intestine and promote PFOS absorption. But TiO2 could
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not be accumulated in fish tissues since the aggregated nano-TiO2 particles were too large to
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penetrate the cell membranes.28 Considering that GO was distinctly different from nano-TiO2 in 3
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chemico-physical properties, it is hypothesized that GO may affect PFOS bioavailability and
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bioaccumulation via special mechanisms.
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Natural organic matter (NOM) is widely present in aquatic environment
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and it could affect 29-32
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the fate and transport as well as bioavailabilities and toxicities of co-exist contaminants.
For
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example, NOM might reduce the toxicities of co-exist AgNPs in Shewanella oneidensis MR-1 33 and
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Daphnia,32 GO in zebrafish embryogenesis,34 but enhance the toxicity of TiO2 in developing
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zebrafish.35 Humic substances are complex organic molecules that represent the largest constituent of
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NOM, while fulvic acid (FA) is an important constituent of humic substances, which is soluble in
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water under all pH conditions.36 It has many O-containing functional groups such as hydroxyl,
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carboxyl and phenolic groups,37 and is able to interact with a variety of organic and inorganic
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compounds.
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The objectives of the present study were to investigate: 1) adsorption and desorption of PFOS
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on GO and the influence of FA on these processes; 2) impacts of GO on uptake and elimination of
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PFOS in fish tissues with/without FA; 3) absorption behaviors of PFOS and GO using an in vitro
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digestion and absorption model. The results are beneficial to assess the impacts of engineered GO on
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the environmental behaviors of organic pollutants in natural aquatic environment.
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MATERIALS AND METHODS
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Chemicals and Reagents
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Potassium perfluorooctane sulfonate (PFOS, 98%), and sodium perfluoro-1-[1, 2, 3,
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4-13C4]octanesulfonate (MPFOS, 99%) were purchased from Wellington Laboratories (Guelph, ON,
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Canada). The stock solution of PFOS was prepared at 500 mg/L in Milli-Q water, and stored at 4 ℃.
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Graphene oxide nanoplates (GO) (> 99%) was purchased from Nano Materials Tech Co. (Tianjin, 4
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China). Fulvic acid (FA) (> 90%) was purchased from Aladdin Chemistry Co. Ltd. (Shanghai, China).
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Pepsin (from porcine gastric mucosa lyophilized powder, 3 200 ~ 4 500 U/mg protein), pancreatin
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(from porcine pancreas > 7 500 U/mg), sodium taurocholate and sodium glycodeoxycholate were
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purchased from Sigma Chemical Co. (Sigma-Aldrich China Ltd., Shanghai, China). All of the
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solvents used for chromatography were of high-performance liquid chromatography (HPLC) grade.
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Milli-Q water was used for the sample pretreatment.
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Preparation and Characterization of GO and FA
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Based on the information provided by the supplier, GO was synthesized using a modified
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Hummers method.38 A stock suspension of GO (300 mg/L) was prepared by sonicating 0.3 g of GO
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powder in 1 000 mL Milli-Q water for 4 h. The obtained stock suspension was stored in dark at 4 ℃.
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The hydrodynamic size of GO nanoplates was measured by dynamic light scattering (Zetasizer
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nano-ZS90, Malvern Instruments, United Kingdom) and was in the range of 246 ~ 257 nm. The
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representative transmission electron microscope (TEM) images (JEM-2 100, JEOL, Tokyo, Japan)
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and field emission scanning electron microscopy images (FE-SEM, LEO, 1530 vp, Germany) of GO
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are shown in Figure 1 a, b, c. The thickness of the GO material, which was 0.7 ~ 1.8 nm, was
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determined by atomic force microscopy imaging (AFM, Santa Barbara, CA) (Figure 1 d). The
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surface C/O atomic ratio was determined as 2.14 with an X-ray photoelectron spectroscopy (PHI 5
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400 ESCA System). The Brunauer-Emmer-Teller (BET, Micromertics ASAP 2010 Accelerated
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Surface Area and Poresimetry System, Micromeritics Instrument Corporation) surface area was
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measured as 208.6 m2/g. The Raman spectra (Renishew in Via Raman spectrometer, RM2 000, UK)
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is shown in Figure 1 e (the 2 D/G intensity ratio further indicated that the product consisted of more
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than one layer of nanosheets);39 and the Fourier transform infrared (FTIR) transmission spectrum 5
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(110 Bruker TENSOR 27 apparatus, Bruker Optics Inc., Germany) is shown in Figure 1 f. The FA
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stock solution was prepared by dissolving 200 mg of FA in 1 L of Milli-Q water, which was stirred
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for 6 h, and centrifuged at 2 739 g for 1 h. The supernatant solution was filtered through a 0.45 µm
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membrane to remove particulates. The obtained FA stock solution was measured with a total organic
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carbon analyzer (TOC-VCSN, Shimadzu, Japan) and it contained 80.3 ± 5.5 mg of C/L (n=3). The
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FTIR spectrum of obtained FA is shown in Figure S1 a, and was characterized by X-ray
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photoelectron spectroscopy to contain 77.56% C 1s, 19.53% O 1s, and 2.91% Na 1s. The result is
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shown in Figure S1 b.
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Experimental Design
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Exposure tests were conducted in a series of 12 L of glass aquariums (the diameter was 20 cm,
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height was 40 cm, water depth was 30 cm). To ensure accurate analysis of PFOS in different tissues,
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three-month old juvenile common carp (Cyprinus carpio) were chosen for the experiment. They
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were about 10 cm in length and 8.68 ~ 11.5 g in weight, and obtained from a local fish market. The
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fish were acclimated in aerated and dechlorinated tap water (pH 7.1 ~ 7.5) at 25 ± 1℃ (14:10 h
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light/dark photoperiod) for at least one month and were fed with fish feed twice a day.
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A certain amount of PFOS, GO or FA stock solutions were added in the aquariums with 9 L
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water and dispersed with ultrasonication and mechanical stirring for 30 min. The aquariums were
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lightly aerated (Oxygen aeration pump ACO-002, 35 W, 40 L/min) throughout the experiment.
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Filtered dechlorinated water with a hardness of 98.0 ± 2.6 mg/L CaCO3, pH of 7.1 ± 0.3, dissolved
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oxygen of 7.9 ± 0.2 mg/L, was maintained at 25 ± 1 °C. The nominal PFOS concentration in all test
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groups was 500 ng/L, except for the control group in which PFOS, GO and FA were not added. The
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concentrations of PFOS and FA were set to be as close as possible to potential environmental levels 6
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near source area.
For GO, its concentration was set to be as lower as possible, meanwhile
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ensuring operability and accuracy in experimental tests. Five exposure groups were designed: (1)
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Blank group (no PFOS, GO, or FA); (2) PF group (PFOS and 2 mg C/L FA); (3) P group (PFOS only,
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without GO or FA); (4) PG group (PFOS and 1 mg/L GO); (5) PFG group (PFOS, 2 mg C/L FA and
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1 mg/L GO). The exposure lasted for 28 days. One aquarium in each group was sacrificed and four
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fish were sampled on the day of 0, 1, 2, 6, 10, 16, 22, and 28 (Figure S2). At the end of exposure, all
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the remaining fish were taken out and transferred to clean aquariums with dechlorinated tap water for
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depuration, which lasted for another 54 days. Four fish were sampled on the day of 29, 30, 34, 38, 44,
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50, 56, 62, and 82 for each group. The exposure solution or clean water was completely renewed
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every two days. To minimize the influence of fish feed, fishes were fed in clean water when the
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solution was renewed. Upon sampling, the fish were anesthetized with tricaine methanesulfonate
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(MS-222). It was reported that PFOS was prone to accumulated in blood, liver, kidney and gill,22
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while intestine is one of the major uptake routes for pollutants. Thus blood was immediately taken
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from the fish. Subsequently the fish were dissected for liver, kidney, intestine, gill and muscle, which
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were homogenized separately. At each sampling time, 200 mL of water was sampled from the middle
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of the aquariums. The fish samples were stored at −54 °C, and the water samples were stored at 4 °C
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until pretreatment.
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Simulated Gastric and Intestinal Digestion Model
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Preparation of digestive fluids. The simulated digestive fluids were prepared on a daily basis as
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described in the United States Pharmacopeia with minor modifications.42 The simulated gastric
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control fluid (SGFc) contained 2 mg/mL NaCl in Milli-Q water (pH 2.0). The simulated gastric fluid
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(SGF) was prepared by mixing pepsin with the SGFc (stored at 4 ℃) to achieve a concentration of 4 7
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mg/mL. The simulated intestinal control solution (SIFc) (pH 6.8) was composed of 6.8 mg/mL of
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KH2PO4 and 0.616 mg/ mL of NaOH in Milli-Q water. The simulated intestinal fluid (SIF) consisted
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of 10 mg/mL pancreatin in SIFc while the simulated bile solution (SBS) consisted of 4 mM sodium
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taurocholate and 4 mM sodium glycodeoxycholate in SIFc.
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Simulated Gastric Digestion. Two mL of solution of the P, PF, PG, or PFG group (the mixture of
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PG or PFG group was allowed to reach sorption equilibrium before the in vitro experiment) was
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incubated with SGFc (25 mL) in a 50 mL polypropylene (PP) centrifuge tube (50 mL, CNW, China)
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for 10 min at 37℃ in a shaking water bath (Crystal Technology & Industries, Inc. USA). The
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solution was brought to pH 2.0 ± 0.1 with 1 M HCl, and topped up to 29 mL with SGFc. Freshly
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prepared 5 mL of SGF was added, and the mixture was subsequently incubated in a shaking water
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bath (167 rpm) at 37℃ for 2 h. The gastric digestion was terminated by adding 1 M NaOH solution
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to adjust the solution pH to 6.8, at which the pepsin was inactivated.
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Simulated Intestinal Digestion. The dynamic model described by Marambe et al. was used in
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this study.43 In this model, a dialysis bag made of Spectra/Por dialysis membrane (flat width, 45 mm;
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diameter, 29 mm) was used to simulate an intestinal compartment, based on the gastric
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small-intestinal model (TIM-1).
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membrane was 5 KDa. The volume of the gastric digest (pH 6.8) was adjusted to 35 mL using SIFc
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and transferred to the dialysis bag. One mL of bile solution and 4 mL of SIF containing pancreatin,
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previously maintained at 37 ℃, were added, and digestion continued for 0, 0.5, 1, 2, 4 and 6 h with
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continuous stirring, respectively. In this setup, the dialysis bag was immersed in a buffer (similar in
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composition to SIFc pH 6.8, 1 000 mL) which was maintained at 37℃. The buffer was replenished at
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a delivering rate of 1.6 mL/min using a peristaltic pump (Baoding Longer Precision Pump Co., Ltd.
44-46
The molecular weight cutoff (MWCO) of the dialysis
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China), and it carried the dialysed products to a receiving flask. At the end of digestion, the buffer in
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the receiving flask and surrounding the dialysis bag was combined and labeled as dialysate. The
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mixture remaining in the dialysis bag was collected in a 50 mL PP centrifuge tube and centrifuged at
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10 956 g for 10 min at 4 °C. The supernatant was labeled as retentate A, while the precipitate was
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labeled as retentate B.
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Analysis of PFOS and GO
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All the samples were pretreated for PFOS and GO analysis. The information about sample
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pretreatment and analyses of PFOS and GO in the samples are provided in Supporting Information
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(SI).
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Batch Adsorption-Desorption Experiments
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Sorption and desorption of PFOS on GO with/without FA were conducted. The details about the
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sorption and desorption experiments are provided in SI.
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Data Analysis
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In the sorption experiments, the mass of PFOS adsorbed on GO could be calculated using the following equation: =
0 − ×
(1)
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where, qe is the mass of PFOS adsorbed on GO (mg/g), C0 and Ce are PFOS concentrations in the
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aqueous phase at the beginning and end of adsorption experiment (mg/L), V is the volume of the
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solution (40 ml), and m is the mass of GO particles (g).
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For desorption experiment, the mass of desorbed PFOS can be calculated as follows: ∆ =
×
(2)
where, ∆ is the mass of PFOS desorbed from GO (mg/g), C1 is PFOS concentration in the 9
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aqueous phase at the end of the desorption step (mg/L), and r is the fraction of supernatant replaced
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at desorption, which was 0.75 in this study. Freundlich isotherm model is commonly used and usually applied to describe PFOS
189 190
adsorption.26
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=
192
Where, KF [(mg/g) / (mg/L) n] is the Freundlich affinity coefficient, which denotes the adsorption
193
capacity, and n (unitless) is the Freundlich linearity index.47, 48 The bioaccessibility of PFOS (%PFOS) is defined as the fraction of PFOS present in the
194 195
(3)
dialysate in the in vitro model. It was calculated using the following equation.49, 50 %PFOS =
196
!"#$%"&'()*+ &,&"#'()*+
×100
(4)
The elimination rate constant (ke) of PFOS in fish was calculated by fitting the depuration data
197 198
to a first-order decay model using a nonlinear regression technique provided by Origin V 8.5 (Origin
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Lab, USA), = .=0 −/.
200
(5)
201
Where Ce and Ct=0 are the concentrations of PFOS in the fish (ng/g ww) at time t and the beginning
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of depuration, and ke is the elimination rate constant (1/d).51
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The uptake rate constant (ku) was estimated by fitting the uptake data to a first-order
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bioaccumulation model using an interactive nonlinear regression technique provided by Origin V
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8.5.
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=
/0 1 1 − −/ . /
207
Where Ce is the concentration of PFOS in the fish at time t (ng/g ww), Cs is the free PFOS
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concentration in water (ng/L), and ku is the uptake rate coefficient (L/Kg/d). 10
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Depuration half-life (t1/2) was calculated using the following equation,
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.1/2 =
210
(7)
After ku and ke were obtained, kinetic bioaccumulation factor (BAF, L/kg) was estimated as
211 212
52 /
ku/ke.22
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For PFOS concentrations in fish, they were expressed as the mean values of four replicates and
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standard deviation. One-way analysis of variance (ANOVA) was used to compare the differences
215
between groups, and the differences were considered statistically significant when p was 0.05) in fish body weight was observed during the experimental period. In the
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P group, the uptake and depuration kinetics of PFOS in the fish tissues are shown in Figure 2. PFOS
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concentrations in various tissues increased with exposure time, up to maximum on day 28. The
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PFOS concentrations in the fish tissues followed the order of: blood > kidney > liver > gill >
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intestine > muscle, which was in agreement with previous studies.22 The PFOS concentrations
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decreased exponentially in the depuration process. The bioaccumulation parameters were calculated
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and are shown in Table 1. The calculated BAFs of PFOS in the fish tissues were in the range of 900 ~
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16 623 L/kg. The large BAFs in blood, kidney and liver were related to the high uptake rates in these
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tissues. The half-life was in the range of 19.2 ~ 34.9 d, and the half-life in the liver and blood was
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longer than that reported in rainbow trout,22 which could be due to different fish species.
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In the presence of GO, the measured total PFOS concentration in water was similar to the P
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group (Figure S5 a), while the dissolved PFOS concentration in PG group was much lower than that
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of P group (p liver > kidney >
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intestine > gill > muscle, which was different from that in P group. Gill absorption is an important
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uptake route for dissolved toxicants to enter blood by passive diffusion through the lamellar
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blood-water interface of gill. 22, 58, 59 As shown in Table 1, the uptake rate constant (ku) of PFOS in
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gill in PG group was 2.88 times of that in P group. This was in agreement with the results studied by 13
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Zielińska et al, who reported that the rate of the solid/water partition equilibration of diclofenac was
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enhanced in the presence of sorbing nanoparticles.60 However, the kus of PFOS in liver, blood and
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intestine in the PG group were more promoted, which were 10.3, 3.64 and 9.33 times of those in P
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group. This suggested that apart from absorption via gills, PFOS was also greatly taken-up by
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intestine in the form of GO-PFOS complexes. This hypothesis was testified by the large amount of
280
black GO residue in intestine, which will be discussed later. As a result, PFOS entered enterohepatic
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recirculation in fish, whereby it was continuously recycled between blood, liver, and intestine.22 It is
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worth noting that the greatest facilitation of ku mainly happened in the liver and intestine. Previous
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studies using mice as model animal via tail vein injection demonstrated most GO mainly located in
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liver and other reticuloendothelial system.11, 12, 14 The results suggested that GO might carry PFOS
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and circulate from intestine to liver and blood, leading to promoted accumulation of PFOS in these
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tissues.
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In the PF group, the dissolved PFOS level was similar to that of P group, suggesting that there
288
was no significant interaction between them. As a consequence, the BAFs of PFOS in fish tissues
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(except in kidney) were similar to the P group, implying that FA alone did not distinctly affect PFOS
290
accumulation. In the PFG group, the dissolved PFOS in water column was slightly lower than P
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group, but higher than PG group (Figure S5). This was explained by the alleviated adsorption of
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PFOS on GO in the presence of FA. The PFOS concentration in the fish tissues in the PFG group
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was lower than the PG group during the uptake period, and in some tissues, such as in blood,
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intestine, and muscle, was even lower than the P group (Figure 2). This suggested that FA inhibited
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PFOS accumulation in fish in the presence of GO. Similar phenomena was reported in previous
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studies, which demonstrated that HA could reduce the toxicities of co-exist pollutants.33, 34 As shown 14
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in Table 1, the ku of PFOS in the PFG group was significantly lower than that in PG group, but
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similar to that in P group. Similarly, the BAFs in the PFG group were also lower than those in PG but
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similar to P group. These results suggested that PFOS accumulation in fish was highly related to the
300
uptake routes of PFOS.
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In Vitro Simulated Digestion and Absorption Behaviors
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As reported in our previous study,28 nano-TiO2 promoted accumulation of PFOS in fish by
303
acting as a vehicle of PFOS. However, nano-TiO2 is prone to aggregate and precipitate in water, and
304
it could not be absorbed in fish but depurated from fish directly. The greater promotion effect of GO
305
on PFOS accumulation could be related to its better absorption and stronger binding ability to PFOS
306
than nano-TiO2. An in vitro digestion test was conducted to understand the absorption of PFOS in the
307
presence of GO and FA, and the PFOS profiles in the different groups are shown in Figure 3. The
308
PFOS profile in the dialysate increased gradually, while decreased in the retentates over time in all
309
groups. The %PFOS was 69.7, 70.3%, 84.4, and 54.4% in P, PF, PG, and PFG group after 6 h
310
digestion, respectively. In the PF group, FA itself did not affect PFOS bioaccessibility in digestive
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system (Figure S7), which well explained the negligible effect of single FA on PFOS
312
bioaccumulation in fish. In the presence of GO, %PFOS was facilitated to a greater extent compared
313
with P group. It was reported that the monolayer or a few-layer GO sheets were able to cut and
314
penetrate the membranes by the sharp edges.6, 34 The sharp zigzag edges of GO are visible in Figure
315
1b. Thus, GO acted vehicle to carry more PFOS into dialysate giving that GO could adsorb PFOS
316
strongly. In order to further illustrate the effect of GO in digestion process, absorptivity of GO in
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different groups was also assessed. As shown in Figure 4, the absorptivity of GO increased with
318
digestion time, which could reach 45.8 ± 1.8 % in 6 h. These results indicated that GO might 15
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penetrate cell membranes and circulate in body fluid. As a consequence, PFOS accumulation in fish
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tissues was promoted.
321
In the presence of FA, %PFOS was lower than the P and PG groups. As shown in Figure S4, FA
322
modified the surface of GO and made the edges of GO sheets become much blunter and thicker,
323
agreeing with the hydrodynamic size, which was 253 and 386 nm in PG and PFG group, respectively.
324
In addition, serious aggregation of GO was observed in PFG group in the digestion process (Figure
325
S8). Most GO flocculated in the presence of FA in digestive system, which could happen in fish
326
intestine. As a consequence, it became hard for GO to penetrate the membranes, leading to reduced
327
bioaccessibility of PFOS in intestine. Hence, the %PFOS in PFG was lower than that in P and PG
328
groups.
329
Based on the in vitro experiment, the great facilitation effect of GO on PFOS accumulation in
330
fish was correlated to the great uptake rate and facilitated absorption of PFOS by GO in intestine. On
331
the other hand, FA suppressed the bioavailability of GO and PFOS, leading to reduced accumulation
332
of PFOS in fish in the presence of FA.
333
Uptake Mechanism of PFOS in Fish
334
To further explore the contribution of uptake routes, including gill absorption and ingestion of
335
GO, to PFOS accumulation, concentration ratios of PFOS in gill and blood (G/B ratio), and intestine
336
and blood (I/B ratio) during the uptake period were calculated and the results are presented in Figure
337
5. In P group, it was obvious that G/B and I/B ratios increased with exposure time and achieved the
338
maximum on day 28. G/B was always higher than I/B ratio during the uptake period, indicating that
339
gill absorption made greater contribution to the total uptake of PFOS by fish. In the presence of GO,
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both I/B and G/B were higher than the P group, suggesting that uptakes by gill and intestine were all 16
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promoted. Fish takes up free PFOS via gill absorption. In addition, GO binding with PFOS might be
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absorbed via gill and promoted PFOS absorption. However, the GO sheets were very flexible and
343
may flat out on the gill surface, thus occupying absorption sites
344
exposure time went on. Thus, after 16 d, the G/B ratio decreased. During the whole exposure period,
345
I/B was close or higher than G/B and increased all the time. This suggested that ingestion of GO and
346
the following absorption via intestine made more contribution to blood accumulation of PFOS than
347
gill.
30
and blocking passages in gill as
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Some black residues adhering to the intestine wall were observed even after purging for one
349
week in PG group, suggesting that GO could interact with the cellular membranes intensely. Since
350
GO is amphiphilic, it could easily pass through phospholipid bilayer.61 Therefore, it is potential for
351
GO which carried PFOS to adsorb on the intestine wall for a longer time and even pass through cell
352
membranes, leading to enhanced assimilation of PFOS by intestine wall.
353
In occurrence of FA, G/B and I/B were lower than those in PG group, especially for I/B, even
354
lower than P group. This implied that the remission effect of FA was mainly due to the suppression of
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PFOS absorption via intestine. It was reported that NOM could facilitate graphene elimination in
356
Daphnia magna.62 Unlike P group, the SEMs of feces in PG and PFG groups showed apparent sheet
357
and corrugation structure, indicating large amount of GO was depurated in the feces (Figure S9).
358
Figure 6 illustrates the color of the fish feces in PG and PFG groups during the depuration period. It
359
is clear that GO was depurated from fish much faster in the presence of FA. Thus, the residence time
360
of GO-PFOS in intestine was much shorter, leading to less absorption of PFOS in the presence of FA.
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As shown in Figure S9, PFOS level in feces was the highest in PFG group, suggesting that FA acted
362
as a lubricant and reduced adherence of GO on intestine wall, then facilitated the excretion of PFOS. 17
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ASSOCIATED CONTENTS
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Supporting Information
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Details of sample pretreatment, analysis of PFOS and GO, batch adsorption-desorption
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experiments, experiment design, characterization of FA, PFOS concentration in water column, BAFs
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of PFOS in fish tissues, PFOS profile and experimental phenomena in vitro digestion model, SEM
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images and PFOS concentration in feces. This material can be found in the online version.
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ACKNOWLEDGMENTS
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We acknowledge financial support from Ministry of Science and Technology (2014CB932001),
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the Natural Science Foundation of China (NSFC 21325730, 21577067, 21277077), Ministry of
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Education (20130031130005), the Ministry of Education innovation team (IRT 13024) and Yangtze
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River scholar program.
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375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393
(1) Liu, S. B.; Hu, M.; Zeng, T. H.; Wu, R.; Jiang, R. R.; Wei, J.; Wang, L.; Kong, J.; Chen, Y. Lateral
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Table 1 Uptake rate (ku), elimination rate (ke), half-life (t1/2) and dynamic bioaccumulation factors of PFOS in fish tissues in different groups Tissues
ke (1/d)
Rke2
BAF (L/kg)
ku (L/Kg/d)
Rku2
Half-life (d)
492±34 168±31 191±27 228±84 106±16 19.8±1.7
0.976 0.940 0.934 0.893 0.901 0.964
23.4±2.2 19.2±2.8 31.6±3.9 34.3±3.4 34.9±5.9 31.4±4.6
1793±270 1568±307 1961±388 613±125 305±23 36.1±1.8
0.904 0.918 0.909 0.945 0.919 0.907
20.7±2.5 11.4±0.5 18.4±2.1 34.7±4.7 43.8±4.0 38.9±4.4
260±137 250±60 322±87 243±32 87.6±24.7 23.3±6.1
0.816 0.869 0.932 0.995 0.962 0.886
50.9±4.1 10.7±1.8 25.0±1.8 31.7±3.3 76.4±4.2 28.3±2.4
P group
Blood Intestine Liver Kidney Gill Muscle
0.0296±0.0028 0.0360±0.0037 0.0219±0.0033 0.0202±0.0034 0.0199±0.0038 0.0220±0.0022
0.635 0.921 0.824 0.786 0.761 0.906
16623±1168 4671±867 8715±1246 11292±4147 5349±794 900±76 PG group
Blood Intestine Liver Kidney Gill Muscle
0.0335±0.0029 0.0608±0.0030 0.0377±0.0030 0.0200±0.0004 0.0158±0.0022 0.0178±0.0014
0.936 0.979 0.964 0.692 0.747 0.943
53513±8072 26082±5049 51983±10284 30712±6268 19308±1434 2026±102 PFG group
Blood Intestine Liver Kidney Gill Muscle
0.0136±0.0026 0.0650±0.0032 0.0277±0.0014 0.0219±0.003 0.00907±0.00324 0.0245±0.0027
0.766 0.901 0.980 0.814 0.410 0.856
19137±10063 3846±932 11650±3134 11134±1486 9666±2720 953±251
525 526
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Figure Captions:
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Figure 1 Characterization of GO. a) TEM image of GO in lower magnification, b) TEM image of GO
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in higher magnification, c) SEM image of GO, d) AFM image of GO, e) Raman spectra of
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GO, f) FTIR spectra of GO
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Figure 2 PFOS concentration in fish tissues during uptake and depuration phases. a) blood, b) liver, c)
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kidney, d) muscle, e) gill, f) intestine. Each data point represents the mean concentration of
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the quadruplicate (n=4). The error bar represents standard deviation for four replicates (n=4)
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Figure 3 PFOS profile of dialysate, retentate A and retentate B in different groups. a) PFOS profile in
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P group, b) PFOS profile in PG group, c) PFOS profile in PFG group
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Figure 4 Absorptivity rate of GO in the digestion model of different groups
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Figure 5 The concentration ratios of PFOS in gill or intestine and blood (G/B or I/B) during the
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uptake period in different groups. The error bar represents the standard deviation for four
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replicates (n=4)
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Figure 6 Pictures of fish feces in PG and PFG groups in depuration period
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