Environ Sci Pollut Res DOI 10.1007/s11356-013-2485-0
RESEARCH ARTICLE
Effects of chronic exposure to lead, copper, zinc, and cadmium on biomarkers of the European eel, Anguilla anguilla Bruno Nunes & Ricardo Campinho Capela & Tânia Sérgio & Carina Caldeira & Fernando Gonçalves & Alberto Teodorico Correia
Received: 10 July 2013 / Accepted: 19 December 2013 # Springer-Verlag Berlin Heidelberg 2014
Abstract Exposure to specific metallic compounds can cause severe deleterious modifications in organisms. Fishes are particularly prone to toxic effects from exposure to metallic compounds via their environment. Species that inhabit estuaries or freshwater environments can be chronically affected by persistent exposure to a large number of metallic compounds, particularly those released by industrial activities. In this study, we exposed yellow eels (European eel, Anguilla anguilla) for 28 days to environmentally relevant concentrations of four specific metals; lead (300, 600, and 1,200 μg/l), copper (40, 120, and 360 μg/l), zinc (30, 60, and 120 μg/l) and cadmium (50, 150, and 450 μg/l). The selected endpoints to assess the toxicological effects were neurotransmission (cholinesterasic Responsible editor: Markus Hecker B. Nunes (*) : C. Caldeira : F. Gonçalves Departamento de Biologia, Centro de Estudos do Ambiente e do Mar (CESAM), Universidade de Aveiro, Campus Universitário de Santiago, 3810-193 Aveiro, Portugal e-mail: [email protected] C. Caldeira e-mail: [email protected] F. Gonçalves e-mail: [email protected] R. C. Capela : T. Sérgio : A. T. Correia Centro Interdisciplinar de Investigação Marinha e Ambiental (CIIMAR - CIMAR), Rua dos Bragas 289, 450-123 Porto, Portugal R. C. Capela e-mail: [email protected] T. Sérgio e-mail: [email protected] A. T. Correia e-mail: [email protected] A. T. Correia Faculdade de Ciências da Saúde da Universidade Fernando Pessoa (FCS-UFP), Rua Carlos da Maia 296, 4200-150 Porto, Portugal
activity in nervous tissue), antioxidant defense, and phase II metabolism (glutathione-S-transferase [GST] activity, in both gills and liver tissues), and peroxidative damage. The results showed an overall lack of effects on acetylcholinesterase for all tested metals. Lead, copper, and cadmium exposure caused a significant, dose-dependent, increase in GST activity in gill tissue. However, liver GST only significantly increased following zinc exposure. No statistically significant effects were observed for the thiobarbituric acid reactive substances assay, indicating the absence of peroxidative damage. These findings suggest that, despite the occurrence of an oxidative-based response after exposure to lead, copper, and cadmium, this had no consequence in terms of peroxidative membrane damage; furthermore, cholinergic neurotoxicity caused by lead, copper, and cadmium did not occur. The implications of these results are further discussed. Keywords Biomarkers . Oxidative stress . Cholinesterases . Chronic toxicity . Metals . European eel
Introduction Metallic compounds are widespread and their presence in nature has been systematically reported, particularly in the aquatic environment (Förstner and Wittmann 1979). Metals reach the aquatic environment from two major sources: anthropogenic (e.g., mining, domestic and industrial wastes, degradation of metallic plumbing, run-off from urbanized catchments, nuclear plants, smelters, or burning of fossil fuels) and from natural sources (such as volcanoes, erosion, and natural cycling of elements) (Hozhina et al. 2001; Thompson et al. 2005; Rose and Shea 2007; Connan and Tack 2010; Aktar et al. 2011). Metals have a dual importance in nature; they can be both highly toxic, and fundamental for life at the same time (Liu
Environ Sci Pollut Res
and Thiele 1997). Toxic effects documented for metals include neurotoxicity, genotoxicity, carcinogenicity (Florea and Büsselberg 2011), and also enzyme inhibition (El Khalil et al. 2008; Garceau et al. 2010). In fish, metals are known to impair gill function (Playle 1998), thus compromising gas exchange. Metals also elicit oxidative stress, by interfering with oxygen metabolism, disturbing the homeostatic equilibrium between production and inactivation/scavenging of reactive oxygen species (ROS). ROS are short-lived, highly unstable, and chemically reactive intermediates that tend to react with subcellular components, thus disturbing the cell’s functionalities. Production of ROS occurs naturally and/or can be enhanced through several mechanisms, including the interference in electron transport in the mitochondrial membrane (with subsequent accumulation of reactive intermediates), inactivation of antioxidant enzymes, depletion of non-enzymatic antioxidants, and membrane lipid peroxidation (Modesto and Martinez 2010). The reactivity (and consequent deleterious nature) of ROS must be counteracted by antioxidant defense mechanisms, which promptly removes and/or inactivates ROS, to protect organisms from oxidative stress (Li et al. 2010; Modesto and Martinez 2010). Despite the overall efficacy of this defense system, exposure to toxic chemical pollutants (including metals) may cause a permanent imbalance in this state of equilibrium, inducing a decrease in its efficiency. In such cases, oxidative stress can cause irreversible oxidative damage, including oxidation of DNA and other macromolecules, resulting in probable cellular death (Jin et al. 2010; Li et al. 2010; Modesto and Martinez 2010). The biological response of the majority of organisms to metal exposure at toxic levels frequently includes adaptive mechanisms of upregulation and over expression of key enzymes involved in biotransformation/metabolism of a wide range of environmental contaminants (Ognjanovic et al. 2008; Modesto and Martinez 2010) that ultimately favors excretion and consequent reduction in toxicity. Additional responses, when oxidative stress is likely to occur, involve the activation of enzymes of the antioxidant defense system. The isoenzymes glutathione-S-transferase (GSTs) are paradigmatic of the intersection of these two situations, since both are implicated in detoxification and in the antioxidant defense (Jin et al. 2010; Modesto and Martinez 2010; Pereira et al. 2010). Even considering the global efficacy of the antioxidant defense system, peroxidative damage may occur. To quantify the extent of lipoperoxidation, it is possible to measure the amount of malondialdehyde resulting from the oxidative degradation of membrane lipids. Thus, it is possible to assume that measuring LPO is a biomarker of toxic effects (Ognjanovic et al. 2008; Pereira et al. 2010). Inhibition of enzymes by metals is not consensual, as is the case with inhibition of cholinesterasic activity (Nunes 2011). Cholinesterase plays a key role in the termination of chemical
neurotransmission, by cleaving the neurotransmitter acetylcholine after its release at the nervous cleft of cholinergic synapses. Several classes of chemicals (such as organophosphate and carbamate pesticides) act by inhibiting this enzymatic form, causing acute toxicity by overstimulation of postsynaptic membranes, which frequently results in death of the exposed organisms (Nunes et al. 2005). Despite the traditional use of cholinesterasic inhibition to diagnose environmental exposure to the above pesticides, several studies have indicated the potential of using cholinesterasic inhibition to assess the toxic effects of exposure to metallic compounds (Guilhermino et al. 1996; Labrot et al. 1996; Payne et al. 1996). The European eel, Anguilla anguilla, is a facultative catadromous fish species, frequently captured for human consumption, being of considerable economic importance (Daverat et al. 2006). It is a benthic species common in the wild, in temperate areas of the European continent. However, its populations have suffered a considerable decline for the past few years, which can be related to human activities, such as overfishing and habitat loss, in conjunction with natural factors, including parasitism. The combined effects of these factors have been reproductive impairment (Quadroni et al. 2013). However, the contribution of other factors such as pollution (Belpaire and Goemans 2007; Van den Thillart et al. 2009; Geeraerts and Belpaire 2010) appear to have also contributed to the observed population decline. Additionally, eels are capable of inhabiting estuaries in the vicinity of large human population centers, which are typically subjected to high anthropogenic pollution. Moreover, animals captured in heavily polluted areas were significantly more prone to bioaccumulate lipophilic compounds, and bore higher parasite loads (Quadroni et al. 2013). Despite performing an extensive spawning migration, during the feeding stage, yellow eels seem relatively sedentary (Tesch 1977). In fact, because of their high fat content and local benthic feeding behavior, the sub-adult European eel stage is considered extremely prone to the bioaccumulation of pollutants (Linde et al. 1996; Roche et al. 2003). Anguilla anguilla was selected as a test organism for the present study since it presents particular characteristics, such as abundance, easy laboratory rearing, large biological representativity (secondary consumers in freshwater/estuarine areas) and adaptability to varied salinity levels, naturally occurring and thriving in both fresh- and brackish water environments (Costa et al. 2008). Being a benthic species (Prchalová et al. 2013) eels come in contact with metals through distinct routes, including the food chain, water, and porewater. These metals can exhibit different redox potential, leading to the presence of metallic forms of distinct valence (and consequently, distinct toxicity profiles) than those existing in the water column (Marsden and Rainbow 2004). Toxicity that might be expected from interstitial water can thus be different from the toxicity observed for metals dissolved in the water column.
Environ Sci Pollut Res
The objective of the present study was to assess the toxic effects elicited by chronic exposure (28 days) to common anthropogenic metallic contaminants (lead, copper, zinc, and cadmium), in terms of oxidative stress response, phase II metabolism and cholinergic neurotoxicity, in the marine/ brackish fish species Anguilla anguilla (European eel). Cholinergic neurotoxicity was assessed in eye tissue; remaining biomarkers were quantified in liver (target responsible for metabolism) and gills (main target of metals).
percent of the aquaria water was also renewed once every 2 days. Physical-chemical water parameters such as oxygen (9.9±0.2 mg/l), pH (8.5±0.1), and temperature (19.2±0.6 °C) were measured daily during the experimental period using a hand-held multi-probe (YSI, 556 MPS: YSI Inc, Ohio, USA). Ammonium (0.5±0.1 mg/l) and nitrites (0.2±0.0 mg/l) were measured before each medium change using a photometer (YSI, 9300 Photometer: YSI Inc, Ohio, USA) with water test tablets (Palintest, NH3, and NO2: Palintest Ltd, Gateshed, UK). Mortality was less than 10 % for the control group.
Material and methods
Tissue processing
Test organisms
After the 28-day exposure period, animals were euthanized by decapitation on ice-cold phosphate buffer, and eyes, gills, and liver were removed and preserved in liquid nitrogen (−196 °C). Thereafter, eyes, gills, and livers were homogenized in ice-cold phosphate buffer (50 mM, pH 7.0, with 0.1 % Triton X-100) and then the homogenized tissues were centrifuged at 15,000g for 10 min at 4 °C (for GST and thiobarbituric acid reactive substances (TBARS) determinations) and 6,000g for 3 min at 4 °C (for ChE quantification). Supernatants were divided into aliquots and stored at −80 °C.
Juveniles (yellow eel stage) Anguilla anguilla (length, 30– 40 cm) were collected, by professional fishermen in June 2009, from an unpolluted coastal lagoon (Murtosa, North Portugal) (Guilherme et al. 2010). After capture, the eels were transported to laboratory facilities in refrigerated plastic boxes with a 100 % oxygen atmosphere. Upon arrival, the eels were subjected to the following conditions: 12L:12D-controlled photoperiod, water temperature of 18 °C, continuous aeration in a 500-l tank, and artificial seawater at 20 psu. Animals were kept for a period of 2 weeks to acclimatize prior to the onset of exposure experiments. After this period, only healthy animals were selected. Exposure to metals Exposures were chronic, and occurred for periods of 28 days (OECD 2000). Eels were exposed to three distinct sub-lethal concentrations of the metals lead, copper, zinc, and cadmium. Exposure concentrations of metals were based on levels already measured in Portuguese estuaries (Mucha et al. 2003), to simulate a low dose, chronic exposure likely to occur under real environmental scenarios. Four independent experimental assays were performed for different metallic species, namely Pb, Cu, Zn, and Cd. During the experiments, one group (Control Concentration Group) was exposed to uncontaminated water, while the other three groups (Low Concentration Group, Medium Concentration Group, High Concentration Group) were exposed to contaminated water, each with a different concentration of the selected elements. Ten animals were exposed, per concentration, for 28 days (in agreement with the OECD test guideline 215; OECD 2000). Lead concentrations were 300, 600, and 1,200 μg/l; copper concentrations were 40, 120, and 360 μg/l; zinc concentrations were 30, 60, and 120 μg/l; and cadmium concentrations were 50, 150, and 450 μg/l. Exposure was performed in 50-l aquaria. The eels were fed with commercially available mussels (Mytilus galloprovincialis), destined for human consumption, once a day (2 % of fish body weight). Eighty
Biomarker analysis Enzymatic activity was determined for GST in gills and liver tissues, and for ChE in eyes. Lipoperoxidation extent was assessed by the quantification of thiobarbituric acid reactive substances (TBARS; in gills and liver tissue). GST catalyzes the conjugation of the substrate 1-chloro-2, 4dinitrobenzene with glutathione, forming a thioeter that can be followed by the increment of absorbance at 340 nm. GST activity was determined by spectrophotometry according to Habig et al. (1974), and the results were expressed as nanomoles of thioeter produced per minute, per milligram of protein. The activity of ChE was determined by spectrophotometry, according to the protocol of Ellman et al. (1961). In this assay, the ChE activity can be monitored by the formation of the conjugate of thiocholine and DTNB, at a wavelength of 414 nm; thiocholine results from the hydrolytic activity of ChE on the substrate acetylthiocholine. The extent of lipid peroxidation was measured by the quantification of TBARS, according to the protocol described in Buege and Aust (1978). Absorbance readings of each sample were performed in triplicate at 535 nm. This methodology is based on the reaction of compounds such as malondialdehyde (MDA) formed by degradation of initial products of free radical attack, with 2-thiobarbituric acid. TBARS concentrations were expressed as MDA equivalents. Protein quantification of samples was determined according to the Bradford method (Bradford 1976), adapted to flat bottom 96-well microplate.
Environ Sci Pollut Res
Chemicals
Results
The metals (lead, copper, zinc, and cadmium) to which fish were exposed were used in the forms of lead chloride (PbCl2: Sigma-Aldrich, 268690, St Louis, USA), copper sulfate (CuSO4.5H2O: Sigma-Aldrich, 203165, St Louis, USA), zinc sulfate (ZnSO 4.7H 2 O: SigmaAldrich, 204986, St Louis, USA) and cadmium chloride (CdCl2; Acros Organics, Geel, Belgium) in degrees of purity of 98, 99.9, 99, and 99.99 %, respectively. Regular chemical analyses of the food and water during the assays were performed to confirm contaminant levels (OECD, 2010). Water and food analyses were made by Atomic Absorption Spectrophotometry-Flame Atomization (AAS-FA) using a Varian SpectrAA 220FS (Varian Inc, California, USA). Samples were collected from every mussel batch. The collected samples were stored in plastic vials previously decontaminated by washing with 10 % nitric acid (HNO3) and frozen for further analysis. Mussel samples were freeze-dried and pulverized, followed by a HNO3 digestion on a Parr digestion pump (model 4782), then tested for the selected element by AAS-FA. Results were validated according to a certified reference material [NIST SRM 2976: Mussel Tissue (Trace Elements and Methyl mercury) freeze-dried] (National Institute of Standards and Technology, Gaithersburg, USA). LOD=0.34 μg/g dry weight for Pb and Zn; LOD=0.30 μg/g dry weight for Cd and Cu. Concentrations are presented in microgram per gram of dry weight for mussel and microgram per liter for water samples. Measured water metallic contents were within the expected range for control, low, medium, and high concentrations, respectively: Pb (< LOD, 165, 381, and 742 μg/l; LOD=28 μg/l), Cu (< LOD, 55, 107, and 253 μg/l; LOD=5 μg/l), Zn (14, 35, 52, and 101 μg/l; LOD=5 μg/l), and Cd (< LOD, 32, 103, and 328 μg/l; LOD=5 μg/l). For mussels, the measured concentrations were 0.90, 3.30, 161.70, and 0.45 μg/g for Pb, Cu, Zn, and Cd, respectively.
Lead Lead exposure caused a concentration dependent, significant increase in gill GSTs activity (F=6.56; d.f.=3, 32; p0.05; Fig. 1b). Lead did not cause any measurable effect on peroxidative damage in both gills (F=1.21; d.f.=3.34; p>0.05; Fig. 1c) and liver tissues (F=1.55; d.f.=3.26; p>0.05; Fig. 1d). No effect on cholinesterasic inhibition was detected following lead exposure at any of the concentrations (F=1.27; d.f.=3.33; p>0.05; Fig. 1e).
Copper The effects of copper were also concentration dependent, with significant increases in GST activities detected for both gill (F=4.37; d.f.=3.38; p0.05; Fig. 2d), or for cholinesterase inhibition (F=2.10; d.f.= 3.38; p>0.05; Fig. 2e).
Cadmium Cadmium exposure resulted in a significant increase in gill GST activity (F=2.86; d.f.=3.41; p0.05, Fig. 3d, respectively). Cholinesterase activity was also not impaired by cadmium exposure (F=1.53; d.f.=3.41; p>0.05, Fig. 3e).
Statistical analyses Zinc Enzymatic activities were analyzed by comparing results of the metal exposure treatments (i.e., copper, zinc, cadmium, and lead) with the control values. Obtained data was checked for normality and homogeneity of variances prior to statistical analysis. Results were statistically compared with one-way ANOVA, followed by the Dunnet multi-comparison test to determine significant differences between the treatments (metals and exposure levels) and the control. The significance level adopted was p0.05; Fig. 4a); but liver GST activity did significantly increase following zinc exposure (F=12.23; d.f.=3,35; p0.05; Fig. 4c) or liver of Anguilla anguilla (F=1.16; d.f.=3.20; p>0.05; Fig. 4d). Cholinesterase activity was also not affected by zinc exposure at any of the concentrations (F=0.93; d.f=3.32; p>0.05; Fig. 4e).
Environ Sci Pollut Res
Fig. 1 Chronic effects of lead exposure on enzymes of Anguilla anguilla. Values are the mean of ten replicate assays, plus standard error bars; * significant differences, p
RESEARCH ARTICLE
Effects of chronic exposure to lead, copper, zinc, and cadmium on biomarkers of the European eel, Anguilla anguilla Bruno Nunes & Ricardo Campinho Capela & Tânia Sérgio & Carina Caldeira & Fernando Gonçalves & Alberto Teodorico Correia
Received: 10 July 2013 / Accepted: 19 December 2013 # Springer-Verlag Berlin Heidelberg 2014
Abstract Exposure to specific metallic compounds can cause severe deleterious modifications in organisms. Fishes are particularly prone to toxic effects from exposure to metallic compounds via their environment. Species that inhabit estuaries or freshwater environments can be chronically affected by persistent exposure to a large number of metallic compounds, particularly those released by industrial activities. In this study, we exposed yellow eels (European eel, Anguilla anguilla) for 28 days to environmentally relevant concentrations of four specific metals; lead (300, 600, and 1,200 μg/l), copper (40, 120, and 360 μg/l), zinc (30, 60, and 120 μg/l) and cadmium (50, 150, and 450 μg/l). The selected endpoints to assess the toxicological effects were neurotransmission (cholinesterasic Responsible editor: Markus Hecker B. Nunes (*) : C. Caldeira : F. Gonçalves Departamento de Biologia, Centro de Estudos do Ambiente e do Mar (CESAM), Universidade de Aveiro, Campus Universitário de Santiago, 3810-193 Aveiro, Portugal e-mail: [email protected] C. Caldeira e-mail: [email protected] F. Gonçalves e-mail: [email protected] R. C. Capela : T. Sérgio : A. T. Correia Centro Interdisciplinar de Investigação Marinha e Ambiental (CIIMAR - CIMAR), Rua dos Bragas 289, 450-123 Porto, Portugal R. C. Capela e-mail: [email protected] T. Sérgio e-mail: [email protected] A. T. Correia e-mail: [email protected] A. T. Correia Faculdade de Ciências da Saúde da Universidade Fernando Pessoa (FCS-UFP), Rua Carlos da Maia 296, 4200-150 Porto, Portugal
activity in nervous tissue), antioxidant defense, and phase II metabolism (glutathione-S-transferase [GST] activity, in both gills and liver tissues), and peroxidative damage. The results showed an overall lack of effects on acetylcholinesterase for all tested metals. Lead, copper, and cadmium exposure caused a significant, dose-dependent, increase in GST activity in gill tissue. However, liver GST only significantly increased following zinc exposure. No statistically significant effects were observed for the thiobarbituric acid reactive substances assay, indicating the absence of peroxidative damage. These findings suggest that, despite the occurrence of an oxidative-based response after exposure to lead, copper, and cadmium, this had no consequence in terms of peroxidative membrane damage; furthermore, cholinergic neurotoxicity caused by lead, copper, and cadmium did not occur. The implications of these results are further discussed. Keywords Biomarkers . Oxidative stress . Cholinesterases . Chronic toxicity . Metals . European eel
Introduction Metallic compounds are widespread and their presence in nature has been systematically reported, particularly in the aquatic environment (Förstner and Wittmann 1979). Metals reach the aquatic environment from two major sources: anthropogenic (e.g., mining, domestic and industrial wastes, degradation of metallic plumbing, run-off from urbanized catchments, nuclear plants, smelters, or burning of fossil fuels) and from natural sources (such as volcanoes, erosion, and natural cycling of elements) (Hozhina et al. 2001; Thompson et al. 2005; Rose and Shea 2007; Connan and Tack 2010; Aktar et al. 2011). Metals have a dual importance in nature; they can be both highly toxic, and fundamental for life at the same time (Liu
Environ Sci Pollut Res
and Thiele 1997). Toxic effects documented for metals include neurotoxicity, genotoxicity, carcinogenicity (Florea and Büsselberg 2011), and also enzyme inhibition (El Khalil et al. 2008; Garceau et al. 2010). In fish, metals are known to impair gill function (Playle 1998), thus compromising gas exchange. Metals also elicit oxidative stress, by interfering with oxygen metabolism, disturbing the homeostatic equilibrium between production and inactivation/scavenging of reactive oxygen species (ROS). ROS are short-lived, highly unstable, and chemically reactive intermediates that tend to react with subcellular components, thus disturbing the cell’s functionalities. Production of ROS occurs naturally and/or can be enhanced through several mechanisms, including the interference in electron transport in the mitochondrial membrane (with subsequent accumulation of reactive intermediates), inactivation of antioxidant enzymes, depletion of non-enzymatic antioxidants, and membrane lipid peroxidation (Modesto and Martinez 2010). The reactivity (and consequent deleterious nature) of ROS must be counteracted by antioxidant defense mechanisms, which promptly removes and/or inactivates ROS, to protect organisms from oxidative stress (Li et al. 2010; Modesto and Martinez 2010). Despite the overall efficacy of this defense system, exposure to toxic chemical pollutants (including metals) may cause a permanent imbalance in this state of equilibrium, inducing a decrease in its efficiency. In such cases, oxidative stress can cause irreversible oxidative damage, including oxidation of DNA and other macromolecules, resulting in probable cellular death (Jin et al. 2010; Li et al. 2010; Modesto and Martinez 2010). The biological response of the majority of organisms to metal exposure at toxic levels frequently includes adaptive mechanisms of upregulation and over expression of key enzymes involved in biotransformation/metabolism of a wide range of environmental contaminants (Ognjanovic et al. 2008; Modesto and Martinez 2010) that ultimately favors excretion and consequent reduction in toxicity. Additional responses, when oxidative stress is likely to occur, involve the activation of enzymes of the antioxidant defense system. The isoenzymes glutathione-S-transferase (GSTs) are paradigmatic of the intersection of these two situations, since both are implicated in detoxification and in the antioxidant defense (Jin et al. 2010; Modesto and Martinez 2010; Pereira et al. 2010). Even considering the global efficacy of the antioxidant defense system, peroxidative damage may occur. To quantify the extent of lipoperoxidation, it is possible to measure the amount of malondialdehyde resulting from the oxidative degradation of membrane lipids. Thus, it is possible to assume that measuring LPO is a biomarker of toxic effects (Ognjanovic et al. 2008; Pereira et al. 2010). Inhibition of enzymes by metals is not consensual, as is the case with inhibition of cholinesterasic activity (Nunes 2011). Cholinesterase plays a key role in the termination of chemical
neurotransmission, by cleaving the neurotransmitter acetylcholine after its release at the nervous cleft of cholinergic synapses. Several classes of chemicals (such as organophosphate and carbamate pesticides) act by inhibiting this enzymatic form, causing acute toxicity by overstimulation of postsynaptic membranes, which frequently results in death of the exposed organisms (Nunes et al. 2005). Despite the traditional use of cholinesterasic inhibition to diagnose environmental exposure to the above pesticides, several studies have indicated the potential of using cholinesterasic inhibition to assess the toxic effects of exposure to metallic compounds (Guilhermino et al. 1996; Labrot et al. 1996; Payne et al. 1996). The European eel, Anguilla anguilla, is a facultative catadromous fish species, frequently captured for human consumption, being of considerable economic importance (Daverat et al. 2006). It is a benthic species common in the wild, in temperate areas of the European continent. However, its populations have suffered a considerable decline for the past few years, which can be related to human activities, such as overfishing and habitat loss, in conjunction with natural factors, including parasitism. The combined effects of these factors have been reproductive impairment (Quadroni et al. 2013). However, the contribution of other factors such as pollution (Belpaire and Goemans 2007; Van den Thillart et al. 2009; Geeraerts and Belpaire 2010) appear to have also contributed to the observed population decline. Additionally, eels are capable of inhabiting estuaries in the vicinity of large human population centers, which are typically subjected to high anthropogenic pollution. Moreover, animals captured in heavily polluted areas were significantly more prone to bioaccumulate lipophilic compounds, and bore higher parasite loads (Quadroni et al. 2013). Despite performing an extensive spawning migration, during the feeding stage, yellow eels seem relatively sedentary (Tesch 1977). In fact, because of their high fat content and local benthic feeding behavior, the sub-adult European eel stage is considered extremely prone to the bioaccumulation of pollutants (Linde et al. 1996; Roche et al. 2003). Anguilla anguilla was selected as a test organism for the present study since it presents particular characteristics, such as abundance, easy laboratory rearing, large biological representativity (secondary consumers in freshwater/estuarine areas) and adaptability to varied salinity levels, naturally occurring and thriving in both fresh- and brackish water environments (Costa et al. 2008). Being a benthic species (Prchalová et al. 2013) eels come in contact with metals through distinct routes, including the food chain, water, and porewater. These metals can exhibit different redox potential, leading to the presence of metallic forms of distinct valence (and consequently, distinct toxicity profiles) than those existing in the water column (Marsden and Rainbow 2004). Toxicity that might be expected from interstitial water can thus be different from the toxicity observed for metals dissolved in the water column.
Environ Sci Pollut Res
The objective of the present study was to assess the toxic effects elicited by chronic exposure (28 days) to common anthropogenic metallic contaminants (lead, copper, zinc, and cadmium), in terms of oxidative stress response, phase II metabolism and cholinergic neurotoxicity, in the marine/ brackish fish species Anguilla anguilla (European eel). Cholinergic neurotoxicity was assessed in eye tissue; remaining biomarkers were quantified in liver (target responsible for metabolism) and gills (main target of metals).
percent of the aquaria water was also renewed once every 2 days. Physical-chemical water parameters such as oxygen (9.9±0.2 mg/l), pH (8.5±0.1), and temperature (19.2±0.6 °C) were measured daily during the experimental period using a hand-held multi-probe (YSI, 556 MPS: YSI Inc, Ohio, USA). Ammonium (0.5±0.1 mg/l) and nitrites (0.2±0.0 mg/l) were measured before each medium change using a photometer (YSI, 9300 Photometer: YSI Inc, Ohio, USA) with water test tablets (Palintest, NH3, and NO2: Palintest Ltd, Gateshed, UK). Mortality was less than 10 % for the control group.
Material and methods
Tissue processing
Test organisms
After the 28-day exposure period, animals were euthanized by decapitation on ice-cold phosphate buffer, and eyes, gills, and liver were removed and preserved in liquid nitrogen (−196 °C). Thereafter, eyes, gills, and livers were homogenized in ice-cold phosphate buffer (50 mM, pH 7.0, with 0.1 % Triton X-100) and then the homogenized tissues were centrifuged at 15,000g for 10 min at 4 °C (for GST and thiobarbituric acid reactive substances (TBARS) determinations) and 6,000g for 3 min at 4 °C (for ChE quantification). Supernatants were divided into aliquots and stored at −80 °C.
Juveniles (yellow eel stage) Anguilla anguilla (length, 30– 40 cm) were collected, by professional fishermen in June 2009, from an unpolluted coastal lagoon (Murtosa, North Portugal) (Guilherme et al. 2010). After capture, the eels were transported to laboratory facilities in refrigerated plastic boxes with a 100 % oxygen atmosphere. Upon arrival, the eels were subjected to the following conditions: 12L:12D-controlled photoperiod, water temperature of 18 °C, continuous aeration in a 500-l tank, and artificial seawater at 20 psu. Animals were kept for a period of 2 weeks to acclimatize prior to the onset of exposure experiments. After this period, only healthy animals were selected. Exposure to metals Exposures were chronic, and occurred for periods of 28 days (OECD 2000). Eels were exposed to three distinct sub-lethal concentrations of the metals lead, copper, zinc, and cadmium. Exposure concentrations of metals were based on levels already measured in Portuguese estuaries (Mucha et al. 2003), to simulate a low dose, chronic exposure likely to occur under real environmental scenarios. Four independent experimental assays were performed for different metallic species, namely Pb, Cu, Zn, and Cd. During the experiments, one group (Control Concentration Group) was exposed to uncontaminated water, while the other three groups (Low Concentration Group, Medium Concentration Group, High Concentration Group) were exposed to contaminated water, each with a different concentration of the selected elements. Ten animals were exposed, per concentration, for 28 days (in agreement with the OECD test guideline 215; OECD 2000). Lead concentrations were 300, 600, and 1,200 μg/l; copper concentrations were 40, 120, and 360 μg/l; zinc concentrations were 30, 60, and 120 μg/l; and cadmium concentrations were 50, 150, and 450 μg/l. Exposure was performed in 50-l aquaria. The eels were fed with commercially available mussels (Mytilus galloprovincialis), destined for human consumption, once a day (2 % of fish body weight). Eighty
Biomarker analysis Enzymatic activity was determined for GST in gills and liver tissues, and for ChE in eyes. Lipoperoxidation extent was assessed by the quantification of thiobarbituric acid reactive substances (TBARS; in gills and liver tissue). GST catalyzes the conjugation of the substrate 1-chloro-2, 4dinitrobenzene with glutathione, forming a thioeter that can be followed by the increment of absorbance at 340 nm. GST activity was determined by spectrophotometry according to Habig et al. (1974), and the results were expressed as nanomoles of thioeter produced per minute, per milligram of protein. The activity of ChE was determined by spectrophotometry, according to the protocol of Ellman et al. (1961). In this assay, the ChE activity can be monitored by the formation of the conjugate of thiocholine and DTNB, at a wavelength of 414 nm; thiocholine results from the hydrolytic activity of ChE on the substrate acetylthiocholine. The extent of lipid peroxidation was measured by the quantification of TBARS, according to the protocol described in Buege and Aust (1978). Absorbance readings of each sample were performed in triplicate at 535 nm. This methodology is based on the reaction of compounds such as malondialdehyde (MDA) formed by degradation of initial products of free radical attack, with 2-thiobarbituric acid. TBARS concentrations were expressed as MDA equivalents. Protein quantification of samples was determined according to the Bradford method (Bradford 1976), adapted to flat bottom 96-well microplate.
Environ Sci Pollut Res
Chemicals
Results
The metals (lead, copper, zinc, and cadmium) to which fish were exposed were used in the forms of lead chloride (PbCl2: Sigma-Aldrich, 268690, St Louis, USA), copper sulfate (CuSO4.5H2O: Sigma-Aldrich, 203165, St Louis, USA), zinc sulfate (ZnSO 4.7H 2 O: SigmaAldrich, 204986, St Louis, USA) and cadmium chloride (CdCl2; Acros Organics, Geel, Belgium) in degrees of purity of 98, 99.9, 99, and 99.99 %, respectively. Regular chemical analyses of the food and water during the assays were performed to confirm contaminant levels (OECD, 2010). Water and food analyses were made by Atomic Absorption Spectrophotometry-Flame Atomization (AAS-FA) using a Varian SpectrAA 220FS (Varian Inc, California, USA). Samples were collected from every mussel batch. The collected samples were stored in plastic vials previously decontaminated by washing with 10 % nitric acid (HNO3) and frozen for further analysis. Mussel samples were freeze-dried and pulverized, followed by a HNO3 digestion on a Parr digestion pump (model 4782), then tested for the selected element by AAS-FA. Results were validated according to a certified reference material [NIST SRM 2976: Mussel Tissue (Trace Elements and Methyl mercury) freeze-dried] (National Institute of Standards and Technology, Gaithersburg, USA). LOD=0.34 μg/g dry weight for Pb and Zn; LOD=0.30 μg/g dry weight for Cd and Cu. Concentrations are presented in microgram per gram of dry weight for mussel and microgram per liter for water samples. Measured water metallic contents were within the expected range for control, low, medium, and high concentrations, respectively: Pb (< LOD, 165, 381, and 742 μg/l; LOD=28 μg/l), Cu (< LOD, 55, 107, and 253 μg/l; LOD=5 μg/l), Zn (14, 35, 52, and 101 μg/l; LOD=5 μg/l), and Cd (< LOD, 32, 103, and 328 μg/l; LOD=5 μg/l). For mussels, the measured concentrations were 0.90, 3.30, 161.70, and 0.45 μg/g for Pb, Cu, Zn, and Cd, respectively.
Lead Lead exposure caused a concentration dependent, significant increase in gill GSTs activity (F=6.56; d.f.=3, 32; p0.05; Fig. 1b). Lead did not cause any measurable effect on peroxidative damage in both gills (F=1.21; d.f.=3.34; p>0.05; Fig. 1c) and liver tissues (F=1.55; d.f.=3.26; p>0.05; Fig. 1d). No effect on cholinesterasic inhibition was detected following lead exposure at any of the concentrations (F=1.27; d.f.=3.33; p>0.05; Fig. 1e).
Copper The effects of copper were also concentration dependent, with significant increases in GST activities detected for both gill (F=4.37; d.f.=3.38; p0.05; Fig. 2d), or for cholinesterase inhibition (F=2.10; d.f.= 3.38; p>0.05; Fig. 2e).
Cadmium Cadmium exposure resulted in a significant increase in gill GST activity (F=2.86; d.f.=3.41; p0.05, Fig. 3d, respectively). Cholinesterase activity was also not impaired by cadmium exposure (F=1.53; d.f.=3.41; p>0.05, Fig. 3e).
Statistical analyses Zinc Enzymatic activities were analyzed by comparing results of the metal exposure treatments (i.e., copper, zinc, cadmium, and lead) with the control values. Obtained data was checked for normality and homogeneity of variances prior to statistical analysis. Results were statistically compared with one-way ANOVA, followed by the Dunnet multi-comparison test to determine significant differences between the treatments (metals and exposure levels) and the control. The significance level adopted was p0.05; Fig. 4a); but liver GST activity did significantly increase following zinc exposure (F=12.23; d.f.=3,35; p0.05; Fig. 4c) or liver of Anguilla anguilla (F=1.16; d.f.=3.20; p>0.05; Fig. 4d). Cholinesterase activity was also not affected by zinc exposure at any of the concentrations (F=0.93; d.f=3.32; p>0.05; Fig. 4e).
Environ Sci Pollut Res
Fig. 1 Chronic effects of lead exposure on enzymes of Anguilla anguilla. Values are the mean of ten replicate assays, plus standard error bars; * significant differences, p
