- Email: [email protected]
Evaluation of single and joint toxicity of perfluorooctane sulfonate, perfluorooctanoic acid, and copper to Carassius auratus using oxidative stress biomarkers
Accepted Manuscript Title: Evaluation of single and joint toxicity of perfluorooctane sulfonate, perfluorooctanoic acid and copper to Carassius auratus using oxidative stress biomarkers Author: Mingbao Feng Qun He Lingjun Meng Xiaoling Zhang Zunyao Wang PII: DOI: Reference:
S0166-445X(15)00033-8 http://dx.doi.org/doi:10.1016/j.aquatox.2015.01.025 AQTOX 4041
To appear in:
Aquatic Toxicology
Received date: Revised date: Accepted date:
8-11-2014 22-1-2015 28-1-2015
Please cite this article as: Feng, Mingbao, He, Qun, Meng, Lingjun, Zhang, Xiaoling, Wang, Zunyao, Evaluation of single and joint toxicity of perfluorooctane sulfonate, perfluorooctanoic acid and copper to Carassius auratus using oxidative stress biomarkers.Aquatic Toxicology http://dx.doi.org/10.1016/j.aquatox.2015.01.025 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Evaluation of single and joint toxicity of perfluorooctane sulfonate, perfluorooctanoic acid and copper to Carassius auratus using oxidative stress biomarkers Mingbao Feng, Qun He, Lingjun Meng, Xiaoling Zhang, Zunyao Wang* State Key Laboratory of Pollution Control and Resources Reuse, School of the Environment, Nanjing University, Jiangsu Nanjing 210023, P.R. China
Highlights
► The toxicity of PFOS, PFOA, Cu and their mixtures in fish was evaluated. ►
Oxidative stress biomarkers and trace element homeostasis were determined. ► Concentration distributions in fish tissues were measured. ► Toxicity order was proposed via the IBR index.
ABSTRACT
Perfluorooctane sulfonate, perfluorooctanoic acid and copper have been recently regarded as ubiquitous environmental contaminants in aquatic ecosystems worldwide. *
Corresponding author: Zunyao Wang. Address: State Key Laboratory of Pollution Control and Resources Reuse, School of the Environment, Xianlin Campus, Nanjing University, Jiangsu Nanjing 210023, P.R. China. Tel: +86-25-89680358; Fax: +86-25-89680358. E-mail address: [email protected].
However, data on their possible combined toxic effects on aquatic organisms are still lacking. In this study, a systematic experimental approach was used to assess the impacts of these chemicals and their mixtures on hepatic antioxidant status of Carassius auratus after 4 days. Oxidative stress was apparently observed for joint exposure by determining biochemical parameters (superoxide dismutase, catalase, glutathione peroxidase, reduced glutathione and malondialdehyde). The integrated biomarker response index was calculated to rank the toxicity order, from which the synergistic effect was tentatively proposed for joint-toxicity action. In addition, these treatments significantly altered trace element homeostasis in different fish tissues, and the concentration distribution of these test chemicals was also measured. Taken together, these results provided some valuable toxicological data on the joint effects of perfluorinated compounds and heavy metals on aquatic species, which can facilitate further understanding on the potential risks of other coexisting pollutants in the natural aquatic environment. Keywords: Perfluorinated compounds; copper; joint toxicity; hepatic oxidative stress; trace element homeostasis.
1. Introduction
Perfluorinated compounds (PFCs) are a class of persistent pollutants that have been extensively used as surfactants, lubricants, polymers, paints and fire-fighting foams in various industry and consumer products for over 60 years (Giesy and Kannan, 2001; Lindstrom et al., 2011). The widespread use and unique properties of PFCs have contributed to their ubiquitous occurrence in the environment, wildlife and humans worldwide (Giesy and Kannan, 2001; Dai et al., 2006; Houde et al., 2006; Lau et al., 2007). Recently, they have been regarded as a group of emerging persistent organic pollutants (Lindstrom et al., 2011; Corsini et al., 2014). Among PFCs, perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) have been routinely detected as the predominant pollutants (Giesy and Kannan, 2001; Dai et al., 2006; Lindstrom et al., 2011), and increasing concern has arisen regarding their environmental behavior and fate (Lindstrom et al., 2011). Although PFOS and PFOA are generally found in surface waters at low levels (0.1-100 ng/L), these compounds could bioaccumulate and biomagnify through the food chains (Taniyasu et al., 2003; Kannan et al., 2005). Consequently, higher concentrations of PFOS and PFOA have been detected in fish, such as plaice (Pleuronectes platessa) (7760 ng PFOS/g wet weight) in Belgium (Hoff et al., 2003) and tilapia (Oreochromis niloticus) (1100 ng PFOS and PFOA/g dry weight) in Taiwan (Tseng et al., 2006). In light of the widespread distribution and persistence of PFOS and PFOA in the aquatic environment, their potential adverse impacts on fish species have been widely investigated. Previous studies have revealed that exposure to PFOS and PFOA could induce hepatotoxicity and disturbances of DNA metabolism homeostasis (Hoff et al.,
2003; Liu et al., 2009; Wei et al., 2009), developmental and reproductive toxicity (Ankley et al., 2005; Oakes et al., 2005; Du et al., 2009). Oakes et al. (2005) reported that short-term exposure of fathead minnow (Pimephales promelas) and rainbow trout (Oncorhynchus mykiss) to PFOS could increase the hepatic fatty acyl-CoA oxidase activity and oxidative damage as well as alter hormone levels. Especially, oxidative stress, caused by the imbalance of cellular redox homeostasis, has been commonly indicated in fish exposed to PFOS and PFOA (Liu et al., 2007; Kim et al., 2010; Shi and Zhou, 2010; Arukwe and Mortensen, 2011), and has been suspected to be one of the main causes for their hepatotoxicity and developmental toxicity (Liu et al., 2007; Shi and Zhou, 2010). However, previous toxicological researches on PFOS and PFOA mainly focused on their single toxic effects, whereas the possible joint-toxicity action with other contaminants such as heavy metals has been seldom investigated. Contamination with heavy metals in freshwater ecosystem has always been of great concern, since they are bioaccumulative, nonbiodegradable and toxic to aquatic biota. Copper (Cu), released from a variety of anthropologic activities such as mining, waste emission and application of fertilizers and pesticides, has been extensively detected in aquatic environment, with concentrations up to 152 µg/L (Roy, 1997; Mansour and Sidky, 2002). Functioning as a cofactor for many enzymes involved in some vital biological processes, Cu is known as an essential micronutrient in living organisms (Jiang et al., 2014). However, it may become inhibitory and even toxic at high levels (Sanchez et al., 2005; Eyckmans et al., 2011), and Cu-induced oxidative damage to fish species has been recorded in these studies. Under natural conditions, fish can be
simultaneously exposed to multiple anthropogenic stressors such as organic pollutants and heavy metals. Recent researches have increasingly focused on the possible joint effects of different toxicants on aquatic species, such as petroleum hydrocarbons and Cu on polychaete (Perinereis aibuhitensis) (Sun et al., 2009), polycyclic musks and cadmium (Cd) on Carassius auratus (Chen et al., 2012), as well as methyl parathion and Cd on zebrafish (Danio rerio) (Ling et al., 2012). Unfortunately, little information is currently available regarding the joint toxic effects of PFCs and Cu on fish, since they have been ubiquitously detected in natural water worldwide. Generally, xenobiotics entering into aquatic species can be metabolized by different detoxifying enzymes, converting them into more water-soluble substances and typically facilitating their elimination (Kumar and Surapaneni, 2001). Traditionally, the biotransformation processes are broadly divided into phase I and phase II metabolism (Kumar and Surapaneni, 2001). The most important enzymes involved in phase I metabolism are cytochrome P450 monooxygenases, whereas some reactive oxygen species (ROS) can be generated during intracellular metabolism (Hayes and McLellan, 1999; Kumar and Surapaneni, 2001). Also some xenobiotics can release ROS in the cell. Normally, these ROS are continually detoxified by antioxidant defense system, comprising of antioxidant enzymes such as superoxide dismutase (SOD),
catalase
(CAT)
and
glutathione
peroxidase
(GPx),
as
well
as
low-molecular-weight scavengers such as reduced glutathione (GSH) (Li et al., 2011). However, oxidative stress occurs in organisms when steady-state ROS generation is transiently or chronically enhanced during xenobiotic metabolism, causing increased
damage to cellular constituents such as membrane lipids, termed as lipid peroxidation and measured by malondialdehyde (MDA) (Lushchak et al., 2011; Feng et al., 2013). In the present study, a systematic experimental approach was used to evaluate the single and joint toxicity of PFOS, PFOA and Cu on antioxidant status in fish liver. A panel of oxidative stress biomarkers (SOD, CAT, GPx, GSH and MDA) was selected for biochemical measurements, and the integrated biomarker response index was calculated to improve their discriminatory power. The impacts of these test chemicals on trace element homeostasis, such as iron (Fe), zinc (Zn) and selenium (Se), in liver, gill and muscle were assessed. Furthermore, the distribution of PFOS, PFOA and Cu in these three fish tissues was also determined. Therefore, the goals of this study were to: (1) determine the tissue distribution of PFOS, PFOA and Cu in fish; (2) assess and compare their single and joint toxicity on aquatic species. 2. Materials and methods 2.1. Chemicals PFOS (> 98%) and PFOA (> 98%) were obtained from Energy-Chemical Company (Shanghai, China) and Tokyo Chemical Industry Co. Ltd. (Tokyo, Japan), respectively. Some important physico-chemical properties of these PFCs were listed in Table S1. Analytical grade copper(II) sulfate pentahydrate (CuSO4•5H2O, ≥ 99%) was acquired from Guangdong Guanghua Chemical Factory Co. Ltd. (Guangzhou, China). For the toxicity evaluations, PFOS and PFOA were dissolved in dimethylsulfoxide (DMSO) to prepare the stock solutions (100 mmol/L for PFOS and 120.77 mmol/L for PFOA). CuSO4 stock solution (15.74 mmol/L) was prepared with ultrapure water (> 18.2 MΩ
cm), obtained from a Milli-Q Plus system (Millipore, Bedford, MA, USA). All these stock solutions were further diluted with carbon-filtered and dechlorinated tap water to give their nominal concentrations. Kits for oxidative stress biomarker assays were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). 2.2. Fish Juvenile Carassius auratus weighting 27.85 ± 3.25 g (mean ± SD) were purchased from a local aquatic breeding base. 100 fish were initially acclimatized in aquaria containing 150 L dechlorinated and aerated freshwater at least for 10 days, with the total mortality near zero. During acclimatization, fish were fed twice a day with fish pellets and food residues were removed. The fish were starved for 24 h prior to the experiment and dissection to avoid prandial effects. Throughout the exposure period, water conditions were measured as: temperature, 23 ± 2 ℃; pH, 7.25 ± 0.25 units; dissolved oxygen, 6.5 ± 0.5 mg/L; conductivity, 352.4 ± 12.5 µS/cm; total hardness, 174.3 ± 10.2 mg CaCO3/L. Ion levels were determined as: Na+, 12.1 ± 0.4 mg/L; K+, 2.40 ± 0.05 mg/L; Ca2+, 42.12 ± 0.92 mg/L; Mg2+, 7.84 ± 0.04 mg/L; and Cl-, 27.9 ± 1.3 mg/L. In addition, the test metals in water were assayed as: Cu, 1.42 ± 0.17 µg/L; Fe, 10.27 ± 2.37 µg/L; Zn, 5.88 ± 0.72 µg/L; and Se, 0.78 ± 0.08 µg/L. The tests were conducted in accordance with national and institutional guidelines for animal welfare. 2.3. Experimental design Eleven glass tanks were used to conduct the experiment. After acclimatization, 5 fish per group were randomly selected and placed in each tank containing 20 L of test solutions or 20 L of dechlorinated tap water. Regarding experimental treatments, fish
were individually exposed to three waterborne pollutants with two different doses or their mixtures, i.e. PFOS (1 and 10 µmol/L), PFOA (1.21 and 12.10 µmol/L), Cu (0.79 and 3.15 µmol/L), PFOS + Cu (1 + 0.79 µmol/L and 10 + 3.15 µmol/L), PFOA + Cu (1.21 + 0.79 µmol/L and 12.10 + 3.15 µmol/L). The exposure concentrations of PFOS and PFOA were chosen according to their recent toxicological researches on different fish species (Liu et al., 2007; Shi et al., 2008; Kim et al., 2010; Shi and Zhou, 2010). Their features of high bioaccumulation and negligible elimination also supported the high doses in exploring toxicity mechanisms. The concentrations of Cu were chosen to be representative of environmental levels in polluted freshwaters and similar to previous studies (Sanchez et al., 2005; Eyckmans et al., 2011). The aqueous concentrations of these chemicals were monitored during the exposure period. Besides, Cu speciation prediction was performed by Visual MINTEQ software (ver. 3.0, Stockholm, Sweden), and Cu was mainly present as Cu3(OH)42+ under the presented exposure conditions. Another group was chosen as the control, with exposure to dechlorinated tap water. Fish were exposed to these conditions for 4 days, an exposure time point which was also studied in previous toxicological evaluations of PFOS and PFOA in fish (Kim et al., 2010; Shi and Zhou, 2010). The tanks were continuously aerated and water were refreshed every 24 h to minimize the contamination from metabolic wastes. No fish mortality occurred during toxicity tests. 2.4. Sampling procedures On completion of the exposure, 5 fish in each treatment were killed by sharp blow on the head, and their tissues (liver, gill and muscle) were quickly dissected out on an
ice-cold plate, cleaned of extraneous tissues in physiological saline solution (0.9% NaCl), and stored at -80 ℃ until assays. A part of liver tissues (0.2 g) were homogenized (1:10, w/v) using an Ultra Turrax homogenizer (Ika, Germany) in cold physiological saline solution. The homogenates were centrifuged (Eppendorf, Germany) at 4000 g for 15 min at 4 ℃, and the supernatants were collected for biochemical determination. 2.5. Biochemical analysis The supernatants were assayed for oxidative stress biomarkers (SOD, CAT, GPx, GSH and MDA) using the Diagnostic Reagent Kits according to the manufacturer’s instructions. SOD (EC 1.15.1.1) activity was determined by the inhibition of cytochrome c reduction in the presence of hypoxanthine/xanthine oxidase O2-• generator system (McCord and Fridovich, 1969). CAT (EC 1.11.1.6) activity, measured by H2O2 breakdown, was estimated following the method of Claiborne (1985). GPx (EC 1.11.1.9) activity was evaluated based on the rate of NADPH oxidation by the coupled reaction with glutathione reductase (Lawrence and Burk, 1976). GSH level was assayed according to the method of Jollow et al. (1974). MDA content was measured using the method described by Jain et al. (1989), which was conducted based on 2-thiobarbituric acid (2,6-dihydroxypyrimidine-2-thiol; TBA) reactivity. Specific activity of enzymes was defined as units of activity per mg of protein, while GSH level and MDA content were expressed as µmol/g protein and nmol/mg protein, respectively. Protein content was examined by the method of Bradford using bovine serum albumin as a standard (Bradford, 1976).
2.6. Integrated biomarker response (IBR) A method for combining all measured biomarker responses into one general “stress index” termed “Integrated Biomarker Response” (IBR) (Beliaeff and Burgeot, 2002) was used to compare the potential toxic effects of these treatments on fish liver. The detailed calculations are shown in the Supporting Information. 2.7. Quantification of PFOS and PFOA in exposure solutions and fish tissues The concentrations of PFOS and PFOA in exposure solutions and fish tissues were measured by Agilent 1260 infinity high performance liquid chromatography (HPLC) coupled with API 4000 triple quadrupole mass spectrometer (AB Sciex, Concord, ON, Canada). The specific analysis procedures are provided in the Supporting Information. 2.8. Quantification of Cu and trace elements (Fe, Zn and Se) in exposure solutions and fish tissues After above determinations, some remaining tissues (liver, gill and muscle) were adequately digested with nitric acid and sulfuric acid (4:1, v/v) at 120 ℃ for at least 2 h. The digestates were cooled to room temperature, and then diluted to 10 mL with 1% HNO3 solution. Together with the exposure water samples, tissue samples were measured for concentrations of Cu, Fe, Zn and Se using an inductive coupled plasma mass spectrometer (NexION 300X ICP-MS Spectrometers, PerkinElmer, Shelton, CT, USA). Digestion blanks indicated negligible contamination. All measurements were performed in duplicated and metal levels were expressed as mg/kg dry weight (d.w.). 2.9. Statistical analysis All data were expressed as means ± SD (standard deviation) and analyzed using
SPSS statistical package (ver. 16.0, SPSS Company, Chicago, USA). Experimental values were checked for normality by Shapiro-Wilk one-sample test and homogeneity of variance by Levene’s test. One-way ANOVA followed by Duncan’s test and Post Hoc LSD multiple comparison test was performed to signal the significant intergroup differences, and their respective significance limits were set at p < 0.05 and p < 0.01. 3. Results 3.1. Quantification of PFOS, PFOA and Cu in exposure solutions and fish tissues Together with the nominal doses, the measured concentrations of PFOS, PFOA and Cu in exposure solutions were listed in Table 1. It can be seen that the latter values were largely similar to the former (within ± 20%). Hence, the nominal concentrations were used in the following descriptions.
S0166-445X(15)00033-8 http://dx.doi.org/doi:10.1016/j.aquatox.2015.01.025 AQTOX 4041
To appear in:
Aquatic Toxicology
Received date: Revised date: Accepted date:
8-11-2014 22-1-2015 28-1-2015
Please cite this article as: Feng, Mingbao, He, Qun, Meng, Lingjun, Zhang, Xiaoling, Wang, Zunyao, Evaluation of single and joint toxicity of perfluorooctane sulfonate, perfluorooctanoic acid and copper to Carassius auratus using oxidative stress biomarkers.Aquatic Toxicology http://dx.doi.org/10.1016/j.aquatox.2015.01.025 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Evaluation of single and joint toxicity of perfluorooctane sulfonate, perfluorooctanoic acid and copper to Carassius auratus using oxidative stress biomarkers Mingbao Feng, Qun He, Lingjun Meng, Xiaoling Zhang, Zunyao Wang* State Key Laboratory of Pollution Control and Resources Reuse, School of the Environment, Nanjing University, Jiangsu Nanjing 210023, P.R. China
Highlights
► The toxicity of PFOS, PFOA, Cu and their mixtures in fish was evaluated. ►
Oxidative stress biomarkers and trace element homeostasis were determined. ► Concentration distributions in fish tissues were measured. ► Toxicity order was proposed via the IBR index.
ABSTRACT
Perfluorooctane sulfonate, perfluorooctanoic acid and copper have been recently regarded as ubiquitous environmental contaminants in aquatic ecosystems worldwide. *
Corresponding author: Zunyao Wang. Address: State Key Laboratory of Pollution Control and Resources Reuse, School of the Environment, Xianlin Campus, Nanjing University, Jiangsu Nanjing 210023, P.R. China. Tel: +86-25-89680358; Fax: +86-25-89680358. E-mail address: [email protected].
However, data on their possible combined toxic effects on aquatic organisms are still lacking. In this study, a systematic experimental approach was used to assess the impacts of these chemicals and their mixtures on hepatic antioxidant status of Carassius auratus after 4 days. Oxidative stress was apparently observed for joint exposure by determining biochemical parameters (superoxide dismutase, catalase, glutathione peroxidase, reduced glutathione and malondialdehyde). The integrated biomarker response index was calculated to rank the toxicity order, from which the synergistic effect was tentatively proposed for joint-toxicity action. In addition, these treatments significantly altered trace element homeostasis in different fish tissues, and the concentration distribution of these test chemicals was also measured. Taken together, these results provided some valuable toxicological data on the joint effects of perfluorinated compounds and heavy metals on aquatic species, which can facilitate further understanding on the potential risks of other coexisting pollutants in the natural aquatic environment. Keywords: Perfluorinated compounds; copper; joint toxicity; hepatic oxidative stress; trace element homeostasis.
1. Introduction
Perfluorinated compounds (PFCs) are a class of persistent pollutants that have been extensively used as surfactants, lubricants, polymers, paints and fire-fighting foams in various industry and consumer products for over 60 years (Giesy and Kannan, 2001; Lindstrom et al., 2011). The widespread use and unique properties of PFCs have contributed to their ubiquitous occurrence in the environment, wildlife and humans worldwide (Giesy and Kannan, 2001; Dai et al., 2006; Houde et al., 2006; Lau et al., 2007). Recently, they have been regarded as a group of emerging persistent organic pollutants (Lindstrom et al., 2011; Corsini et al., 2014). Among PFCs, perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) have been routinely detected as the predominant pollutants (Giesy and Kannan, 2001; Dai et al., 2006; Lindstrom et al., 2011), and increasing concern has arisen regarding their environmental behavior and fate (Lindstrom et al., 2011). Although PFOS and PFOA are generally found in surface waters at low levels (0.1-100 ng/L), these compounds could bioaccumulate and biomagnify through the food chains (Taniyasu et al., 2003; Kannan et al., 2005). Consequently, higher concentrations of PFOS and PFOA have been detected in fish, such as plaice (Pleuronectes platessa) (7760 ng PFOS/g wet weight) in Belgium (Hoff et al., 2003) and tilapia (Oreochromis niloticus) (1100 ng PFOS and PFOA/g dry weight) in Taiwan (Tseng et al., 2006). In light of the widespread distribution and persistence of PFOS and PFOA in the aquatic environment, their potential adverse impacts on fish species have been widely investigated. Previous studies have revealed that exposure to PFOS and PFOA could induce hepatotoxicity and disturbances of DNA metabolism homeostasis (Hoff et al.,
2003; Liu et al., 2009; Wei et al., 2009), developmental and reproductive toxicity (Ankley et al., 2005; Oakes et al., 2005; Du et al., 2009). Oakes et al. (2005) reported that short-term exposure of fathead minnow (Pimephales promelas) and rainbow trout (Oncorhynchus mykiss) to PFOS could increase the hepatic fatty acyl-CoA oxidase activity and oxidative damage as well as alter hormone levels. Especially, oxidative stress, caused by the imbalance of cellular redox homeostasis, has been commonly indicated in fish exposed to PFOS and PFOA (Liu et al., 2007; Kim et al., 2010; Shi and Zhou, 2010; Arukwe and Mortensen, 2011), and has been suspected to be one of the main causes for their hepatotoxicity and developmental toxicity (Liu et al., 2007; Shi and Zhou, 2010). However, previous toxicological researches on PFOS and PFOA mainly focused on their single toxic effects, whereas the possible joint-toxicity action with other contaminants such as heavy metals has been seldom investigated. Contamination with heavy metals in freshwater ecosystem has always been of great concern, since they are bioaccumulative, nonbiodegradable and toxic to aquatic biota. Copper (Cu), released from a variety of anthropologic activities such as mining, waste emission and application of fertilizers and pesticides, has been extensively detected in aquatic environment, with concentrations up to 152 µg/L (Roy, 1997; Mansour and Sidky, 2002). Functioning as a cofactor for many enzymes involved in some vital biological processes, Cu is known as an essential micronutrient in living organisms (Jiang et al., 2014). However, it may become inhibitory and even toxic at high levels (Sanchez et al., 2005; Eyckmans et al., 2011), and Cu-induced oxidative damage to fish species has been recorded in these studies. Under natural conditions, fish can be
simultaneously exposed to multiple anthropogenic stressors such as organic pollutants and heavy metals. Recent researches have increasingly focused on the possible joint effects of different toxicants on aquatic species, such as petroleum hydrocarbons and Cu on polychaete (Perinereis aibuhitensis) (Sun et al., 2009), polycyclic musks and cadmium (Cd) on Carassius auratus (Chen et al., 2012), as well as methyl parathion and Cd on zebrafish (Danio rerio) (Ling et al., 2012). Unfortunately, little information is currently available regarding the joint toxic effects of PFCs and Cu on fish, since they have been ubiquitously detected in natural water worldwide. Generally, xenobiotics entering into aquatic species can be metabolized by different detoxifying enzymes, converting them into more water-soluble substances and typically facilitating their elimination (Kumar and Surapaneni, 2001). Traditionally, the biotransformation processes are broadly divided into phase I and phase II metabolism (Kumar and Surapaneni, 2001). The most important enzymes involved in phase I metabolism are cytochrome P450 monooxygenases, whereas some reactive oxygen species (ROS) can be generated during intracellular metabolism (Hayes and McLellan, 1999; Kumar and Surapaneni, 2001). Also some xenobiotics can release ROS in the cell. Normally, these ROS are continually detoxified by antioxidant defense system, comprising of antioxidant enzymes such as superoxide dismutase (SOD),
catalase
(CAT)
and
glutathione
peroxidase
(GPx),
as
well
as
low-molecular-weight scavengers such as reduced glutathione (GSH) (Li et al., 2011). However, oxidative stress occurs in organisms when steady-state ROS generation is transiently or chronically enhanced during xenobiotic metabolism, causing increased
damage to cellular constituents such as membrane lipids, termed as lipid peroxidation and measured by malondialdehyde (MDA) (Lushchak et al., 2011; Feng et al., 2013). In the present study, a systematic experimental approach was used to evaluate the single and joint toxicity of PFOS, PFOA and Cu on antioxidant status in fish liver. A panel of oxidative stress biomarkers (SOD, CAT, GPx, GSH and MDA) was selected for biochemical measurements, and the integrated biomarker response index was calculated to improve their discriminatory power. The impacts of these test chemicals on trace element homeostasis, such as iron (Fe), zinc (Zn) and selenium (Se), in liver, gill and muscle were assessed. Furthermore, the distribution of PFOS, PFOA and Cu in these three fish tissues was also determined. Therefore, the goals of this study were to: (1) determine the tissue distribution of PFOS, PFOA and Cu in fish; (2) assess and compare their single and joint toxicity on aquatic species. 2. Materials and methods 2.1. Chemicals PFOS (> 98%) and PFOA (> 98%) were obtained from Energy-Chemical Company (Shanghai, China) and Tokyo Chemical Industry Co. Ltd. (Tokyo, Japan), respectively. Some important physico-chemical properties of these PFCs were listed in Table S1. Analytical grade copper(II) sulfate pentahydrate (CuSO4•5H2O, ≥ 99%) was acquired from Guangdong Guanghua Chemical Factory Co. Ltd. (Guangzhou, China). For the toxicity evaluations, PFOS and PFOA were dissolved in dimethylsulfoxide (DMSO) to prepare the stock solutions (100 mmol/L for PFOS and 120.77 mmol/L for PFOA). CuSO4 stock solution (15.74 mmol/L) was prepared with ultrapure water (> 18.2 MΩ
cm), obtained from a Milli-Q Plus system (Millipore, Bedford, MA, USA). All these stock solutions were further diluted with carbon-filtered and dechlorinated tap water to give their nominal concentrations. Kits for oxidative stress biomarker assays were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). 2.2. Fish Juvenile Carassius auratus weighting 27.85 ± 3.25 g (mean ± SD) were purchased from a local aquatic breeding base. 100 fish were initially acclimatized in aquaria containing 150 L dechlorinated and aerated freshwater at least for 10 days, with the total mortality near zero. During acclimatization, fish were fed twice a day with fish pellets and food residues were removed. The fish were starved for 24 h prior to the experiment and dissection to avoid prandial effects. Throughout the exposure period, water conditions were measured as: temperature, 23 ± 2 ℃; pH, 7.25 ± 0.25 units; dissolved oxygen, 6.5 ± 0.5 mg/L; conductivity, 352.4 ± 12.5 µS/cm; total hardness, 174.3 ± 10.2 mg CaCO3/L. Ion levels were determined as: Na+, 12.1 ± 0.4 mg/L; K+, 2.40 ± 0.05 mg/L; Ca2+, 42.12 ± 0.92 mg/L; Mg2+, 7.84 ± 0.04 mg/L; and Cl-, 27.9 ± 1.3 mg/L. In addition, the test metals in water were assayed as: Cu, 1.42 ± 0.17 µg/L; Fe, 10.27 ± 2.37 µg/L; Zn, 5.88 ± 0.72 µg/L; and Se, 0.78 ± 0.08 µg/L. The tests were conducted in accordance with national and institutional guidelines for animal welfare. 2.3. Experimental design Eleven glass tanks were used to conduct the experiment. After acclimatization, 5 fish per group were randomly selected and placed in each tank containing 20 L of test solutions or 20 L of dechlorinated tap water. Regarding experimental treatments, fish
were individually exposed to three waterborne pollutants with two different doses or their mixtures, i.e. PFOS (1 and 10 µmol/L), PFOA (1.21 and 12.10 µmol/L), Cu (0.79 and 3.15 µmol/L), PFOS + Cu (1 + 0.79 µmol/L and 10 + 3.15 µmol/L), PFOA + Cu (1.21 + 0.79 µmol/L and 12.10 + 3.15 µmol/L). The exposure concentrations of PFOS and PFOA were chosen according to their recent toxicological researches on different fish species (Liu et al., 2007; Shi et al., 2008; Kim et al., 2010; Shi and Zhou, 2010). Their features of high bioaccumulation and negligible elimination also supported the high doses in exploring toxicity mechanisms. The concentrations of Cu were chosen to be representative of environmental levels in polluted freshwaters and similar to previous studies (Sanchez et al., 2005; Eyckmans et al., 2011). The aqueous concentrations of these chemicals were monitored during the exposure period. Besides, Cu speciation prediction was performed by Visual MINTEQ software (ver. 3.0, Stockholm, Sweden), and Cu was mainly present as Cu3(OH)42+ under the presented exposure conditions. Another group was chosen as the control, with exposure to dechlorinated tap water. Fish were exposed to these conditions for 4 days, an exposure time point which was also studied in previous toxicological evaluations of PFOS and PFOA in fish (Kim et al., 2010; Shi and Zhou, 2010). The tanks were continuously aerated and water were refreshed every 24 h to minimize the contamination from metabolic wastes. No fish mortality occurred during toxicity tests. 2.4. Sampling procedures On completion of the exposure, 5 fish in each treatment were killed by sharp blow on the head, and their tissues (liver, gill and muscle) were quickly dissected out on an
ice-cold plate, cleaned of extraneous tissues in physiological saline solution (0.9% NaCl), and stored at -80 ℃ until assays. A part of liver tissues (0.2 g) were homogenized (1:10, w/v) using an Ultra Turrax homogenizer (Ika, Germany) in cold physiological saline solution. The homogenates were centrifuged (Eppendorf, Germany) at 4000 g for 15 min at 4 ℃, and the supernatants were collected for biochemical determination. 2.5. Biochemical analysis The supernatants were assayed for oxidative stress biomarkers (SOD, CAT, GPx, GSH and MDA) using the Diagnostic Reagent Kits according to the manufacturer’s instructions. SOD (EC 1.15.1.1) activity was determined by the inhibition of cytochrome c reduction in the presence of hypoxanthine/xanthine oxidase O2-• generator system (McCord and Fridovich, 1969). CAT (EC 1.11.1.6) activity, measured by H2O2 breakdown, was estimated following the method of Claiborne (1985). GPx (EC 1.11.1.9) activity was evaluated based on the rate of NADPH oxidation by the coupled reaction with glutathione reductase (Lawrence and Burk, 1976). GSH level was assayed according to the method of Jollow et al. (1974). MDA content was measured using the method described by Jain et al. (1989), which was conducted based on 2-thiobarbituric acid (2,6-dihydroxypyrimidine-2-thiol; TBA) reactivity. Specific activity of enzymes was defined as units of activity per mg of protein, while GSH level and MDA content were expressed as µmol/g protein and nmol/mg protein, respectively. Protein content was examined by the method of Bradford using bovine serum albumin as a standard (Bradford, 1976).
2.6. Integrated biomarker response (IBR) A method for combining all measured biomarker responses into one general “stress index” termed “Integrated Biomarker Response” (IBR) (Beliaeff and Burgeot, 2002) was used to compare the potential toxic effects of these treatments on fish liver. The detailed calculations are shown in the Supporting Information. 2.7. Quantification of PFOS and PFOA in exposure solutions and fish tissues The concentrations of PFOS and PFOA in exposure solutions and fish tissues were measured by Agilent 1260 infinity high performance liquid chromatography (HPLC) coupled with API 4000 triple quadrupole mass spectrometer (AB Sciex, Concord, ON, Canada). The specific analysis procedures are provided in the Supporting Information. 2.8. Quantification of Cu and trace elements (Fe, Zn and Se) in exposure solutions and fish tissues After above determinations, some remaining tissues (liver, gill and muscle) were adequately digested with nitric acid and sulfuric acid (4:1, v/v) at 120 ℃ for at least 2 h. The digestates were cooled to room temperature, and then diluted to 10 mL with 1% HNO3 solution. Together with the exposure water samples, tissue samples were measured for concentrations of Cu, Fe, Zn and Se using an inductive coupled plasma mass spectrometer (NexION 300X ICP-MS Spectrometers, PerkinElmer, Shelton, CT, USA). Digestion blanks indicated negligible contamination. All measurements were performed in duplicated and metal levels were expressed as mg/kg dry weight (d.w.). 2.9. Statistical analysis All data were expressed as means ± SD (standard deviation) and analyzed using
SPSS statistical package (ver. 16.0, SPSS Company, Chicago, USA). Experimental values were checked for normality by Shapiro-Wilk one-sample test and homogeneity of variance by Levene’s test. One-way ANOVA followed by Duncan’s test and Post Hoc LSD multiple comparison test was performed to signal the significant intergroup differences, and their respective significance limits were set at p < 0.05 and p < 0.01. 3. Results 3.1. Quantification of PFOS, PFOA and Cu in exposure solutions and fish tissues Together with the nominal doses, the measured concentrations of PFOS, PFOA and Cu in exposure solutions were listed in Table 1. It can be seen that the latter values were largely similar to the former (within ± 20%). Hence, the nominal concentrations were used in the following descriptions.