Uptake, depuration and bioconcentration of bisphenol AF (BPAF) in whole-body and tissues of zebrafish (Danio rerio)

Ecotoxicology and Environmental Safety 132 (2016) 339–344

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Uptake, depuration and bioconcentration of bisphenol AF (BPAF) in whole-body and tissues of zebrafish (Danio rerio) Jiachen Shi, Yunjia Yang, Jing Zhang, Yixing Feng, Bing Shao n Beijing Key Laboratory of Diagnostic and Traceability Technologies for Food Poisoning, Beijing Center for Disease Control and Prevention, Beijing 100013, China

art ic l e i nf o

a b s t r a c t

Article history: Received 17 March 2016 Received in revised form 25 May 2016 Accepted 26 May 2016 Available online 27 June 2016

Bisphenol AF (BPAF) is an analog of Bisphenol A (BPA) and is widely used as a raw material in the plastics industry. However, an understanding of the potential risks posed by BPAF in the aquatic environment is lacking. The bioconcentration factor (BCF) is a measure used to assess the secondary poisoning potential as well as risks to human health. In this work we measured the accumulation and elimination of BPAF in the whole-body and in liver, muscle and gonad tissues of zebrafish. BPAF uptake was relatively rapid with equilibrium concentrations reached after 24–72 h of exposure. We observed gender differences both in whole-body and in tissue accumulation. Muscle was the primary BPAF storage tissue during the uptake phase in this study. In the elimination phase, BPAF concentrations declined rapidly during depuration, especially during the initial 2 h, and the rate of elimination in males was faster than females from the whole-body and from tissues. The appearance of BPAF glucuronide (BPAF-G) at the start of the uptake phase indicated the rapid biotransformation of BPAF to BPAF-G in vivo. The high lipid content of female gonad could act to delay the diffusion of the xenobiotic within the body in a contaminated environment, but it also acts to delay xenobiotic elimination from the body. & 2016 Elsevier Inc. All rights reserved.

Keywords: Bisphenol AF Bioconcentration Uptake Depuration Zebrafish

1. Introduction Bisphenol AF (BPAF) is a fluorinated derivative of bisphenol A (BPA) and is used widely as both a crosslinking agent and as a monomer in the plastics industry (Feng et al., 2012). It is used in the manufacture of fluoroelastomers such as gaskets and hoses in food processing equipment and with polycarbonate copolymers used in high temperature composites, electronic materials and other plastics in contact with the environment. With the widely used of BPAF in industrial, the occurrences of BPAF in the environmental matrices have raised the concerns of researchers. About 46% of sewage sludge samples from United States were detected with BPAF in the survey of U.S. Environmental Protection Agency (EPA) and the sediments from industrialized areas in Korea were also detected with BPAF (Liao et al., 2012; Yu et al., 2015). In China, the concentrations of BPAF in sediments and soils near ecosystems were also up to several micrograms per kilogram and in river water in the micrograms per liter range (Song et al., 2012; Yang et al., 2014b). Hydrophobicity at Abbreviations: BPAF, Bisphenol AF; BPA, Bisphenol A; BCF, Bioconcentration factor; BPAF-G, BPAF glucuronide; OECD, Organization for Economic Cooperation and Development; PFOA, Perfluorooctanoic Acid n Corresponding author. E-mail address: [email protected] (B. Shao). http://dx.doi.org/10.1016/j.ecoenv.2016.05.025 0147-6513/& 2016 Elsevier Inc. All rights reserved.

the methylene bridge of BPA derivatives is an important factor for their estrogenic activity. Hydrophobic substituents in place of the 1-methyl group of the propane moiety, as seen in BPAF, increase its hormonal activity (Kitamura et al., 2005). The binding affinity of BPAF was approximately 20 times stronger and 48 times stronger than that of BPA as a ligand for ERα and ERβ, respectively (Matsushima et al., 2010). In addition to the in vitro toxicity of BPAF, there are numerous reports of its potential ecotoxicity. In studies with zebrafish, embryo-larvae treated with 2.0 mg/L of BPAF showed 100% mortality after 120 h post-fertilization (Song et al., 2014). A 28-day exposure to 2-month-old zebrafish with 1 mg/L BPAF caused liver damage and disrupted sex hormone levels as well as vitellogenin expression in males (Yang et al., 2016). We recently reported that BPAF exposure to zebrafish decreased their fertilization success and the survival rates of offspring (Shi et al., 2015). However, an understanding of the potential risks posed by BPAF in the aquatic environment is lacking. The bioconcentration factor (BCF) is the ratio of the concentration of a substance in an organism to the concentration of the substance in the surrounding water. BCF can be a measure for considering secondary poisoning potential and assessing risks to human health (Regoli et al., 2012). The BCF of BPA was reported to be as high as 144 in the freshwater clam Pisidium amnicum and even higher levels (2800
13,000) have been found in algae

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(Heinonen et al., 2002; Yang et al., 2014a). Lee et al. (2015) detected low amounts of BPA from wild freshwater fish with concentrations varying from non-detectable to 25.2 μg/kg and BCF values ranging from 1 to 274. However, to our far knowledge, information concerning the bioconcentration of BPA and its analogs are limited and there is a complete lack of information on the bioconcentration of BPAF in fish. Zebrafish (Danio rerio) is a commonly used model for the analysis of sublethal effects of toxicants in vertebrates (Segner, 2009; Tokarz et al., 2013). In this study we measured BPAF bioconcentration, tissue uptake, distribution and elimination in zebrafish exposed to different concentrations of the chemical. Since our previous research indicated that BPAF glucuronide (BPAF-G) was a major metabolite, we also measured BPAF-G in this study (Li et al., 2013).

2. Materials and methods 2.1. Chemicals BPAF (CAS No. 1478-61-1, 99% purity) was purchased from Sigma-Aldrich (St. Louis, MO, USA) and dissolved in ethanol (Sigma-Aldrich, St. Louis, MO, USA, Z99.8% purity) to obtain a concentration of 10 mg/mL. Deuterated BPAF (98 atom D) was purchased from CDN Isotopes Inc. Quebec, Canada. BPAF-G was isolated and purified from the urine of Sprague-Dawley rats by semipreparative HPLC as described in a previous report (Li et al., 2013). 2.2. Instrumentation and analytical procedures Analyte identification and quantitation were performed using an Acquity ultra performance liquid chromatography system (UPLC) coupled to a Xevo TQ-S triple quadrupole mass spectrometer (Waters, Milford, MA, USA). UPLC separation was conducted on an Acquity BEH C18 column (2.1 mm  100 mm; 1.7 mm; Waters). The mobile phases were LC-MS grade methanol and water. The flow rate was set at 0.4 mL/min, and the injection volume was 5 μL. The initial gradient conditions were 40% methanol for 1 min, followed by a linear increase to 80% methanol over 5 min. Methanol was increased to 100% at 6.1 min, held for 2.0 min, and finally returned to the initial state to equilibrate for 2 min before the next injection. MS/MS acquisition was operated in negative-ion mode with multiple reaction monitoring (MRM). The capillary voltage was 2.9 kV. The source temperature and desolvation temperatures were 150 °C and 400 °C, respectively. Nitrogen gas (purity 99%) was used as the cone and desolvation gas at flow rates of 150 L/h and 1000 L/h, respectively. For each analyte, two transitions were selected for identification and the corresponding cone voltage and collision energy were optimized for maximum intensity. This data is shown in Table S1 (Supplementary Information). To ensure that the samples could be accurately analyzed, an isotopic internal standard curve using deuterated BPAF at 0.05, 0.1, 0.2, 0.5, 1.0, 2.0 and 5.0 ng/mL were prepared for the determination of BPAF. The standard addition method was applied for the analyzing of BPAF-G because an isotopic version was not commercially available. 2.3. Zebrafish maintenance Zebrafish (Danio rerio, AB strain) were obtained from the Department of Biological Sciences and Biotechnology, Tsinghua University (Beijing, China). The fish were cultured in a flow-through system (Esen, Beijing, China) with conditioned water at 27 71 °C with a photoperiod of 14:10 h light/dark cycles. To generate conditioned water, 75 g NaHCO3, 18 g sea salt and 8.4 g CaSO4 were

added to 1000 L reverse osmosis-generated water. 10–20% of the total fish water volume was changed with conditioned water on a daily basis. All the fish were fed with freshly hatched Artemia nauplii (Fengnian Aquaculture Co., Ltd. Tianjin, China) twice and flake food (Tetra, Germany) once daily. In order to ensure high water quality, food remains and debris were removed daily. 2.4. Experimental overview Zebrafish were grown until 6 months old and divided into groups based on sex and kept in separate tanks for use in two different formal experimental studies. The experiments were totally conducted under the guidance of the Organization for Economic Cooperation and Development (OECD) test number 305 (http://www.oecd-ilibrary.org/environment/test-no-305-bioaccu mulation-in-fish-aqueous-and-dietary-exposure_9789264185296en). The procedures were performed in compliance with the guidelines of Institutional Animal and Care and Use Committees (IACUC) of Beijing Center for Disease Control and Prevention, Beijing of China. In the first experiment, male and female zebrafish were exposed separately to five different exposure concentrations of BPAF for studies of bioconcentration and the metabolite BPAF-G levels in whole-body fish. In the second experiment, males and females were exposed to a single concentration of BPAF in aquariums to study uptake, distribution and elimination. However, before the formal experiments, preliminary experiments were conducted to investigate the water exchange rate and the duration of the uptake phase. To determine the optimal water exchange rate, approximately 30 fish were distributed into one tank containing 10 L exposure solution with 20 mg/L BPAF. According to the guidance of OECD 305 (Paragraph 51), the 20 μg/L was selected as the highest concentration in this study based on the 144 h half-lethal-concentration (LC50) of BPAF for zebrafish larvae for long-term exposure (Song et al., 2014). The water sample of each tank was collected and measured every 12 h for 48 h without any renewal. To investigate of the duration of the uptake phase, 16 male and 16 female fish were distributed into one tank containing 10 L exposure solution with 20 mg/L BPAF. Every seven days, 4 male and 4 female fish were sampled from the tank and plunged into icewater for euthanasia and then slightly dried on absorbent paper and weighed (wet weight). The whole-body fish were suspended in 1 mL acetonitrile and homogenized at a vertical velocity of 6 m/ s for 40 s or longer using a FastPrep-24 Tissue and Cell Homogenizer (MP Biomedicals, Santa Ana, California, USA) (Bellamy et al., 2014). This procedure resulted in complete homogenization of the whole-body fish. After sonication for 10 min and centrifugation at 12,000  g for 10 min, the supernatants were collected and the analytes measured. The exposure experiment was run 28 days and the fish were sampled four times. In the first formal experiment, male and female zebrafish were exposed separately to different BPAF concentrations for 168 h in 10 L aquariums. Control fish were kept in clean water. The aquariums used were one piece glass tanks with a capacity of 15 L (18 cm  30 cm  30 cm). Whole-body BPAF concentrations were then determined from six males and six females from each exposure group. BPAF concentrations of 1, 2, 5, 10 and 20 mg/L used in this study were based on the guidance of OECD 305 (Paragraph 51). The 1 μg/L as the lowest concentration in this study was based on the determined concentrations in aquatic systems (ranged from ND (not detected) to 15 μg/L with a median value of 3 μg/L) (Song et al., 2012). The water was renewed every 12 h and the exposure time was prolonged to 168 h based on our preliminary studies. In the second formal experiment, males and females were exposed to a single concentration of 20 μg/L of BPAF in aquariums to

J. Shi et al. / Ecotoxicology and Environmental Safety 132 (2016) 339–344

2.5. Data analysis Statistical analyses were performed using the measured concentrations of BPAF in the fish (whole-body or tissues) and water (as dissolved BPAF) at each sampling date. According to OECD method number 305, the BCF was calculated under steady-state conditions. BCF ¼CB/CW where CB and CW are the BPAF concentrations in the fish and the tank water at steady state conditions, respectively (Ulhaq et al., 2015). The residual ratio of BPAF in zebrafish during the depuration phase was calculated as the residual concentration of BPAF in zebrafish at sample time divided by the concentration of BPAF in zebrafish at the beginning of the depuration phase. All data were analyzed using SPSS for Windows 13.0 Software and presented as mean 7 standard deviation (SD). One-way analysis of variance (ANOVA) was applied to calculate statistical significance between each male and female group. A P o0.05 was considered statistically significant.

3. Results 3.1. Results of the preliminary experiment In the preliminary experiment, the concentrations of BPAF in the chambers with fish declined about 10% after 12 h and declined about 20% after 24 h. If the exposure time was prolonged to 48 h, the concentrations declined about 40% and the water changed to turbid and was not suitable for the fish (data not shown). Therefore, during the formal experiments, the water was renewed every 12 h and throughout the exposure period so BPAF concentrations could be maintained while conforming to the requirements of OECD 305. There were no obvious differences between the BPAF concentrations in whole-body fish during the four sample times in the preliminary experiment (data not shown). The BPAF concentrations in the fish were constant after seven days (168 h). We therefore set the duration of the formal experiments at 168 h in this study. BPAF was not detected in control aquariums in either experiment and no mortalities were observed in either experiment.

250

Whole-body concentration of BPAF (μg/kg)

200 y = 8.7363x + 1.8372 R² = 0.9983 150 male female 100

50 y = 5.2219x - 0.3341 R² = 0.9996 0 0

4

8

12

16

20

24

Exposure concentration (μg/L)

1800

Whole-body concentration of BPAF-G (μg/kg)

study uptake, distribution and elimination. Approximately 30 fish of each gender were randomly distributed into one tank containing 10 L exposure solution and this was replicated four times in total. All the fish were exposed for 168 h followed by a washout period of 24 h. During the test, fish were fed daily except for the 24 h prior to sacrifice. Tank water was totally renewed every 12 h with water containing the original BPAF concentration. In order to assess trends in BPAF bioconcentration, fish were sampled at 0, 3, 6, 12, 24, 72, 120 and 168 h after exposure. After an uptake phase, the exposure media was replaced by a clean, non-contaminated exposure media and the remaining fish were sampled at 0, 2, 4, 6 and 24 h after depuration. Each time, 2 fish were sampled from each tank (8 fish at each time for each gender). Four fish from each gender were used for the whole-body concentration determination and another four were used for the tissue determinations. Livers, gonads and muscles were surgically dissected and weighed. The organs were then transferred to Lysing Matrix-D Tubes (MP Biomedicals, Solon, Ohio, USA) for tissue processing. The wholebody fish and tissue samples were homogenized using the FastPrep-24 Tissue and Cell Homogenizer as described in the preliminary experiments above. The water samples were obtained during each fish collection period.

341

1500

1200 y = 59.854x + 3.3002 R² = 0.9973 900 male 600

female

300 y = 26.949x - 14.74 R² = 0.9973 0 0

4

8

12

16

20

24

Exposure concentration (μg/L)

Fig. 1. Concentration of BPAF and BPAF-G in whole-body fish after exposed with different BPAF concentrations for 168 h (A) Concentration of BPAF (B) Concentration of BPAF-G. The results are shown as mean 7 standard deviation (SD) (n ¼6).

3.2. Results of the first experiment In the first experiment, there was a linear relationship between exposure concentration and the BPAF levels in whole-body tissue homogenates. The BPAF concentrations measured in whole-body samples of fish exposed to 1–20 μg/L BPAF ranged between 8.5 70.5 to 1747 6 μg/kg in males and 5.0 7 1.1 to 1057 12 μg/kg in females after 168 h of exposure. The mean BCF for males was 9.0 70.4 and for females was 5.2 70.2 (Fig. 1A). The major metabolite that we observed in our previous study, BPAF-G, was also the primary metabolite we detected in the zebrafish in this study. BPAF-G levels in whole-body samples of fish exposed to 1–20 μg/L BPAF ranged between 23.8 715.0 to 5337208 μg/kg in males and 54.3 715.8 to 11807 398 μg/kg in females after 168 h exposure (Fig. 1B). The linearity in Fig. 1 indicated the BCF of BPAF in zebrafish was constant and the metabolic activity of zebrafish to BPAF was stable. With the increase of the exposure concentration, the concentration levels of whole-body fish presented a heteroscedacity in standard deviation. This phenomenon is frequently encountered in studies of xenobiotic bioconcentration in zebrafish and is due to individual fish differences. In a study of PFOA uptake in zebrafish, the concentration levels of whole-body fish also presented a heteroscedacity in standard deviation (Ulhaq et al., 2015). On the other hand, the relative standard deviation (RSD) of the BPAF levels in whole-body fish with different exposure concentrations were similar in the different concentrations. This data is presented in Supporting Information (Table S2, Table S3). 3.3. Results of the second experiment In the second experiment, both male and female zebrafish

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14

16

12

14 12

10 BCF of muscle

BCF of whole-body fish

342

8 6 4

10 8

male

6

female

4

2

2

0

0 0

30

60

90

120

150

180

0

30

60

90

120

150

180

Exposure time (h)

Exposure time (h)

45

60

40 50 35 30 BCF of gonad

BCF of liver

40

30

25

male

20

female

15

20

10 10 5 0

0 0

30

60

90

120

150

180

0

30

Exposure time (h)

60

90

120

150

180

Exposure time (h)

Fig. 2. Bioconcentration factor (BCF) of whole-body fish and tissues exposed to 20 μg/L BPAF (A) whole-body fish (B) muscle (C) liver (D) gonad. The results are shown as mean 7 standard deviation (SD) (n ¼4).

absorbed BPAF relatively rapidly from the water. The equilibrium concentrations in the whole fish body occurred between 24 and 72 h of exposure. After 72 h of exposure, the mean BCFs were 9.8 71.0 in males and 5.3 7 0.8 in females (Fig. 2A). The equilibrium concentrations of the muscle, liver and gonad occurred between 24 and 72 h of exposure. After equilibrium, the mean BCFs of muscle were 9.4 71.0 in males and 4.6 71.2 in females (Fig. 2B). The mean BCFs of livers were 36.9 78.5 in males and 10.3 73.0 in females after 72 h exposure (Fig. 2C). The mean BCFs of gonads were 27.7711.6 in males and 10.2 7 1.4 in females after 72 h exposure (Fig. 2D). A slight rise of the BCF content in livers and gonads occurred in males during this exposure period. In the accumulation phase, BPAF could be detected in the gonads at all sampling times. The equilibrium concentrations in the female gonads occurred after 6 h exposure which was more rapid than with the other tissues we tested. BPAF-G was detected in all whole-body fish and muscle samples at the first 3-hour sample time (Table 1). The levels in females were significantly higher than those in males. However, BPAF-G was detected in only a few liver and gonad samples and the concentrations were much lower than those in muscle (data not shown).

Table 1 Concentrations of BPAF-G in whole-body fish and muscle of zebrafish during the second experiment. Concentrations of BPAF-G (mg/Kg) Sample time

Whole-body fish

muscle

hours

male

female

male

female

3 6 12 24 72 120 168

301 7 38 383 7 99 402 7 96 4407 50 5137 164 595 7 114 5337 108

503 7 42* 5377 124* 5737 138* 749 7 206* 999 7 226* 1090 7 290* 1080 7 298*

677 25 1057 36 120 746 1277 55 677 35 1817 70 1367 42

1177 39* 607 20* 91 724* 597 19* 757 29* 697 37* 587 21*

The results are shown as mean 7 standard deviation (SD) (n ¼ 4). Asterisk indicates a significant difference between male and female at the same sample time (P o0.05).

24 h of wash-out, almost 80% of the fish-associated BPAF was cleared. On the other hand, residual BPAF levels in both the wholebody and tissues of females were higher than in males at comparable sample times. The levels in female gonads decreased more slowly than from other organs in female fish (Table 2).

3.4. Results of the depuration phase 4. Discussion The residual BPAF ratios during the depuration phase of wholebody fish and tissues are shown in Table 2. BPAF was rapidly eliminated in the initial 2 h during the depuration phase. After

The present study utilized a two-step experimental design for assessing the uptake and depuration of BPAF in zebrafish. BPAF

J. Shi et al. / Ecotoxicology and Environmental Safety 132 (2016) 339–344

343

Table 2 The residual ratio of BPAF during the depuration phase. Residual ratio of BPAF (%) Sample Time (h)

0

2

4

6

24

Whole-body

Male Female

100.0 100.0

22.9 712.2 38.0 721.5*

13.2 7 7.8 30.2 717.6*

10.4 7 5.6 26.17 10.5*

5.2 7 2.5 14.2 7 29.5*

muscle

Male Female

100.0 100.0

25.5 716.1 46.6 714.1*

16.0 7 7.8 33.3 7 16.2*

12.6 7 6.9 28.7 714.1*

7.9 73.9 21.3 7 10.5*

liver

Male Female

100.0 100.0

56.3 721.2 80.0 718.5*

33.7 7 9.8 43.6 717.7*

25.7 74.1 36.17 10.8*

11.9 78.9 19.3 7 4.8*

gonad

Male Female

100.0 100.0

ND 51.7 7 23.6

ND 34.0 79.1

ND 30.2 76.1

ND 22.4 7 7.3

The results are shown as mean 7 standard deviation (SD) (n ¼4). Asterisk indicates a significant difference between male and female at the same sample time (Po 0.05). ND: not detected

uptake was relatively rapid with equilibrium concentrations reached after 24–72 h of exposure. Gender differences between levels in both whole-body fish and tissues were observed. In general, the whole-body concentrations of BPAF in male fish was consistently higher than those in females in all exposure groups. This gender bias in whole-body accumulation is frequently observed in studies of the accumulation of xenobiotic pollutants in aquatic ecosystems. In a study of perfluorooctanoic acid (PFOA) accumulation, there were clear differences in PFOA levels between male and female zebrafish using a short exposure period. The gender differences were attributed to differences in hormone levels between males and females and the altered expression of solute carriers (Hagenaars et al., 2013). However, the gender bias was minimal when the experiment was extended from 4 to 28 days. The authors considered this result might be due to disturbances in sex steroid metabolism which subsequently altered renal excretion (Hagenaars et al., 2013). Such phenomena were also observed in the studies of PFOA with fathead minnows and rats (Vanden Heuvel et al., 1992; Oakes et al., 2004). Differences in hormone levels between males and females with or without exposure were also observed in our previous study (Shi et al., 2015). As the gender difference of BCF was observed during the uptake phase, and the BPAF concentrations in zebrafish was stable in the 28-day preliminary experiments, differences in hormone levels could be a factor in the gender bias seen in the present study. BPAF was detected in all tissues selected in this study and the BCFs of different tissues differed between sexes. The similarity of BCF in muscle and whole-body fish in males and females indicated that the muscle contained the highest BPAF levels during the uptake phase. Liver is an essential organ that plays a pivotal role in metabolism, digestion and nutrient storage (Cox and Goessling, 2015). The high concentration of BPAF in the liver is probably due to the enterohepatic circulation of this chemical (Ulhaq et al., 2015). In addition, female gonads are larger than those of males and have a higher lipid content. This may increase the accumulation potential of the female gonad (Chang et al., 2013; Orias et al., 2015). Previous studies have shown that if the level of xenobiotic pollutants exceeds the capacity of the liver to metabolize them, these compounds could potentially diffuse into other organs (Orias et al., 2015). Female gonads had a higher BCF and a shorter equilibrium time than whole-body fish in this study. The average BPAF concentrations in the liver and gonad in the males were similar after equilibrium during the accumulation phase. Although the BCF of the male liver was highest in this study, considering the smaller masses of liver and gonads in the male, it may be that the male fish lacked an important storage organ compared with

female. The female gonad could act to delay the diffusion of the xenobiotic within the body in a contaminated environment. On the other hand, the high levels of BPAF in female gonads indicated that BPAF binds to the ovary for uptake into the oocytes. This maternal transfer pathway indicates a potential for adverse effects on early embryonic developmental and offspring health (Yu et al., 2011). In fact, in long-term studies with zebrafish exposed to BPAF, adverse effects on reproduction, embryonic growth and offspring development have been reported from our laboratory (Shi et al., 2015). The elimination phase in the present study demonstrated that BPAF levels declined rapidly at the depuration phase and especially during the initial 2 h. However, the elimination rate from males was faster than females from whole-body and from tissues. There are other instances of gender differences in xenobiotic pollutant elimination. For example, the PFOA half-life is genderrelated and female rats excrete PFOA almost 70 times faster than males (Kudo et al., 2002). In fish, the female fathead minnows and the Nile tilapia have shorter PFOA half-lives compared to males. Elimination rate is influenced by body size but other anatomical and physiological factors can be of importance (Ulhaq et al., 2015). In our study, the elimination rate of female gonads was slower than in muscle and whole-body in males and females. Considering the higher lipid content of female gonad and the high lipophilicity of BPAF (Log Kow: 2.82 70.77), the elimination rate of BPAF in female gonads was slower than the rates in other tissues. This delayed BPAF elimination in females. During the uptake phase, BPAF-G was the major BPAF metabolite and could be detected from the beginning of the accumulation phase. The appearance of BPAF-G at the start of the uptake phase indicated a rapid biotransformation of BPAF to BPAF-G in vivo. This was also observed during BPAF exposure in mice (Li et al., 2013). Gender-specific bioconcentration of different types of toxicants is common and can be attributed to differences in uptake, metabolism, distribution and elimination (Wang et al., 2015). In our study, BPAF-G levels in female zebrafish were higher than males during the uptake phase indicating a more rapid metabolic activity in females. On the other hand, only a small number of liver and gonad samples had detectable levels of BPAF-G. These concentrations were also much lower than muscle levels. Since the hydrophilicity of BPAF-G is higher than BPAF, BPAF-G would tend to accumulate in fish body water. Accordingly, females will have a higher water content resulting in greater BPAF-G levels due to their greater body size than males of the same age. However, this trend did not follow for muscle tissue. On the other hand, BPAF elimination in females was slower than males and could reflect a larger specific surface area for males to excrete BPAF. This

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suggests that the physical excretion of BPAF from the body is the primary elimination mode in fish. Future research is needed to determine the reasons for these differences and whether they can be reflected in differences in gene expression between the sexes.

5. Conclusions In this work, the accumulation and elimination of BPAF in the whole-body and tissues (liver, muscle and gonad) of zebrafish with different exposure concentrations were measured for the first time. A linear relationship was observed between exposure concentrations and BPAF levels in whole-body fish homogenates in both male and female fish. There was an obvious gender difference in the BCF between males and females and muscle was the primary storage tissue. The differences in hormone levels could be a factor in the gender bias seen in the present study. The elimination study showed that BPAF levels declined rapidly at the depuration phase. This was especially apparent during the initial 2 h and was more rapid in males. The high lipid content of female gonad could act to delay the diffusion of the xenobiotic within the body in a contaminated environment, but it also delayed the elimination of the xenobiotic from the body. The appearance of BPAF-G at the start of the uptake phase indicated the rapid biotransformation of BPAF to BPAF-G in vivo. However, even though BPAF-G levels in female zebrafish were higher than males during the uptake phase, the elimination rates in females was slower than males. Male body size and weight of male was usually smaller than females of the same age, which resulted in greater specific surface area for BPAF excretion. Physical excretion of BPAF from body is the main mode of elimination in fish. The high concentration of BPAF in female gonads indicated a potential for maternal transfer as we previously predicted.

Acknowledgments This work was financially supported by the National Natural Science Foundation of China (41273132).

Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.ecoenv.2016.05. 025.

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