Antioxidant, genotoxic and lysosomal biomarkers in the freshwater bivalve (Unio pictorum) transplanted in a metal polluted river basin

Aquatic Toxicology 100 (2010) 75–83

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Antioxidant, genotoxic and lysosomal biomarkers in the freshwater bivalve (Unio pictorum) transplanted in a metal polluted river basin Patrizia Guidi a , Giada Frenzilli a , Maura Benedetti b , Margherita Bernardeschi a , Alessandra Falleni a , Daniele Fattorini b , Francesco Regoli b , Vittoria Scarcelli a , Marco Nigro a,∗ a b

Dipartimento di Morfologia Umana e Biologia Applicata, sezione Biologia e Genetica, Università di Pisa, Via A. Volta, 4 56126 Pisa, Italy Dipartimento di Biochimica, Biologia e Genetica, Università Politecnica delle Marche, Via Ranieri, 60121 Ancona, Italy

a r t i c l e

i n f o

Article history: Received 4 May 2010 Received in revised form 23 June 2010 Accepted 6 July 2010 Keywords: Unio pictorum Freshwater bioindicator Bioaccumulation Biomarkers Oxidative stress Genotoxicity Lysosomal alterations

a b s t r a c t The freshwater painter’s mussel (Unio pictorum) was used as sentinel species to assess the chemical disturbance in an Italian river (the river Cecina) characterized by elevated levels of trace metals of both natural and anthropogenic origin. Organisms were transplanted for 4 weeks in different locations of the river basin and the bioaccumulation of metals was integrated with a wide battery of biomarkers consisting of oxidative, genotoxic and lysosomal responses. Such parameters included the levels of individual antioxidants (catalase, glutathione-S-transferases, glutathione reductase, Se-dependent and Se-independent glutathione peroxidases, total glutathione), the total oxyradical scavenging capacity (TOSC), metallothionein-like proteins, the assessment of DNA integrity, chromosomal damages and lysosomal membrane stability. Elevated levels of several metals were measured in sediments, but the relatively low tissue concentrations suggested a moderate bioaccumulation, possibly due to a high excretion efficiency, of U. pictorum and/or to a limited bioavailability of these elements, partly deriving from erosion of bedrocks. Among antioxidant responses, those based on glutathione metabolism and the activity of catalase were mostly affected in bivalves showing a significant accumulation of arsenic, mercury and/or nickel. In these specimens, the content of glutathione and the activities of glutathione reductase and glutathione peroxidases (H2 O2 ) were respectively 9-, 6- and 4-fold lower than in controls, while a 3-fold increase was observed for catalase. Despite some differences in the response of individual antioxidants, a significant reduction of the capability to neutralize peroxyl radicals was observed in bivalves caged in all the impacted sites of the river basin; these organisms also exhibited a significant impairment at the DNA, chromosomal and lysosomal levels. Considering the mild contamination gradient in the investigated area, the overall results suggested that some oxidative biomarkers, as well as those evaluating chromosomal and cell damages, are highly sensitive and could be profitably applied to caged painter’s mussels for environmental quality assessment in freshwater. © 2010 Elsevier B.V. All rights reserved.

1. Introduction Freshwater habitats experience a continuous decline of water quality and biodiversity which parallel the growth of human populations, agricultural exploitation and industrial activities. Notwithstanding, inland waters still represent the main source for drinking water and have a fundamental importance in terms of economic, cultural, aesthetic, scientific and educational values (Dudgeon et al., 2006). In these environments, organisms are exposed to mixtures of pollutants whose interacting effects are hardly interpreted and predicted exclusively from chemical analyses. A more comprehensive approach combining chemistry of

∗ Corresponding author. Tel.: +39 050 2219113; fax: +39 050 2219101. E-mail address: [email protected] (M. Nigro). 0166-445X/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.aquatox.2010.07.009

abiotic matrices with bioaccumulation and biological effects of contaminants is considered as a valuable diagnostic and prognostic tool for environmental quality assessment and management of aquatic ecosystems. Similar monitoring strategies are particularly needed to assess the biological impact of pollutants in river basins (Brack et al., 2007), where the continuous water flow renders analyses of water and sediments poorly effective for describing environmental quality. Among the numerous biological responses proposed in the last decades, those based on variations at the molecular and cellular levels (biomarkers) represent the earliest signals of environmental disturbance and have been increasingly applied in ecotoxicological investigations (Bayne et al., 1985; Depledge, 1994; Regoli et al., 2004). Since single biomarkers do not reflect the impairment of organism health status, the use of a battery of responses is required for a more realistic evaluation of biological impact of chemicals and

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to elucidate the mechanisms underlying the onset of such alterations. Similar to marine bivalves, freshwater species also have characteristics typical of sentinel organisms, like filter feeding and sedentary/sessile habits, and high resistance to environmental disturbance. The freshwater unionids have been profitably used in both laboratory and field investigations (Gaglione and Ravera, 1964; Riccardi and Ravera, 1989; Doyotte et al., 1997; Cossu et al., 2000; Petushok et al., 2002; Lemiere et al., 2005; Labieniec ˇ and Gabryelak, 2007; Stambuk et al., 2008, 2009), but only a few studies reported a multi-biomarker approach in the species Unio tumidus (Doyotte et al., 1997; Cossu et al., 2000). The main limit in field applications with freshwater bivalves is that native populations may be absent or unevenly distributed in the study areas, possibly exhibiting different reproductive status and/or resistance to pollution due to physiological adaptations (Regoli, 1998). In this respect, caged bivalves which permit to overcome most of these problems reducing the influence of genetic and/or physiological variability, are particularly useful for ecotoxicological applications in these environments. In the present study, the freshwater mussels Unio pictorum have been transplanted at different sites of the River Cecina basin (Tuscany, Italy), one of the Italian pilot river basins selected for implementing biomonitoring strategies within the Water Framework Directive (2000/60/EC). This basin has a high naturalistic value, but it is also impacted by human activities including chemical industries, geothermal power plants, agriculture, and urban effluents. Previous surveys by the regional agency for environmental protection (ARPAT) have assessed that mercury, arsenic and boron are the main pollutants deriving from industrial and/or extractive discharges, most of which are mitigated at present. Besides anthropogenic source of metal pollution, the River Cecina basin is also characterized by a high natural background of nickel and chromium, deriving from the erosion of ophiolitic rocks. A previous multi-biomarkers study carried out with the chub (Leuciscus cephalus) detected significant effects of environmental disturbance, including genotoxicity and the expression of vitellogenin gene in male fish, in sites located downstream the main sources of anthropogenic pollution (Frenzilli et al., 2008). Some of these sites were selected in this investigation for translocation experiments with U. pictorum, where bioaccumulation of metals was integrated with the measurement of a wide battery of biomarkers including oxidative stress responses and the assessment of DNA, chromosomal and lysosomal alterations. Changes in the levels and activity of antioxidants have been proposed as biomarkers of contaminantmediated alterations in a variety of aquatic organisms, resulting in useful markers of environmental stress (Regoli and Principato, 1995; Doyotte et al., 1997; Regoli et al., 1998; Cossu et al., 2000). Nevertheless, literature also provides numerous examples of the high variability of antioxidants, with transient or limited changes reported for different species, tissues and pollutants (Livingstone, 2001). Contrasting and not contemporary variations of individual antioxidants are difficult to summarize in an index of varied susceptibility to oxidative stress conditions and have thus been complemented with the measurement of the total oxyradical scavenging capacity (TOSC) which quantifies the overall capacity to neutralize various ROS like hydroxyl and peroxyl radicals (Winston et al., 1998; Regoli and Winston, 1999). TOSC is less sensitive than individual antioxidants but it has a greater biological relevance since a decreased capacity to neutralize specific ROS has been often related with the damage of cellular end points including lysosomal membrane and DNA (Regoli, 2000; Frenzilli et al., 2001; Regoli et al., 2004). These alterations were evaluated in the present work as accumulation of a lipid peroxidation product (malondialdehyde), DNA integrity (comet assay), chromosomal damages (micronucleus frequency) and lysosomal membrane stability (neutral red

retention time). The overall results were expected to validate an ecotoxicological approach and the use of biomarkers in U. pictorum to evaluate the biological disturbance in a river basin, characterized by high natural background of metals combined with an anthropogenic input. 2. Materials and methods 2.1. Mussel sampling, translocation and initial tissue preparation Specimens of the painter’s mussels U. pictorum were handpicked from a relatively unpolluted site of Lake Maggiore (North Italy) (Fig. 1). The day after sampling, bivalves (55–75 mm in shell length) were put in plastic cages (30 cm × 30 cm × 10 cm, 15 specimens per cage) and deployed at 4 sites located in the upper/median course of the River Cecina and its tributary Possera stream (Fig. 1): “Berignone” located upstream, within a natural protected area and relatively unaffected by direct impact of human activities; “Possera 1” and “Possera 2”, along Possera creek a tributary of the River Cecina, downstream Larderello geothermal fields and chemical industry and also potentially impacted by Bulera dumping ground, located upstream Possera 2 site; “Ponteginori” downstream most of the main source of pollution in the median course of the River Cecina, including the industrial settlement at Saline di Volterra (chloro-alkali) which has been associated with Hg enrichment in river sediments and biota during the last decades. Specimens from the Lake Maggiore were used as controls. The translocation experiment was carried out during March 2008. Water temperature was measured three times during caging period and ranged between 6 and 8 ◦ C both along the River Cecina and in the near shore site of Lake Maggiore used as control. Otherwise, the water flow was not monitored but it was actually much lower in the lake than in the river. This difference might have, at least partly, affected biological responses used as biomarkers. After 4 weeks translocation, the haemolymph was withdrawn from the posterior adductor muscle of at least 14 specimens per site and processed for viability assessment, DNA damage, chromosomal alterations and lysosomal membrane destabilization. Whole soft tissues and shell were individually weighed for condition index evaluation (soft part weight × 100/shell weight), the foot was then removed and remaining tissues rapidly frozen in liquid nitrogen and maintained at −80 ◦ C for chemical and biochemical analyses. 2.2. Chemical analysis of sediments and tissues Concentrations of trace metals in sediments and soft tissues of caged bivalves (N. 4 pools of 6–7 specimens each) were assessed by atomic absorption spectrophotometry as previously described (Regoli et al., 2004). Samples were dried at 60 ◦ C until constant weight was reached and digested under pressure with nitric acid and hydrogen peroxide (7:1) in a microwave digestor system (Mars CEM, CEM Corporation, Matthews, NC, USA). Arsenic, cadmium, chromium, copper, iron, manganese, nickel, lead, selenium and zinc were analyzed by flame and flameless atomization (SpectrAA 220FS and SpectrAA-240Z Zeeman, Varian, Mulgrave, VIC, Australia). Mercury content was quantified by cold vapour atomic absorption spectrometry (VGA-76, Vapour Generator Accessory, Varian). Quality assurance and quality control was assessed by processing blank samples and reference standard materials (lyophilized mussel tissue, Standard Reference Material NIST-2977, National Institute of Standards and Technology, Gaithersburg, MD, USA; lyophilized dogfish muscle, Standard Reference Material DORM-2, National Research Council, Ottawa, ON, Canada). Concentrations obtained for standard reference materials were always within the 95% con-

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Fig. 1. Location of the caging sites in the River Cecina basin and reference site (Lake Maggiore). Main pollution sources are also reported.

fidence interval of certified values. All values are expressed as ␮g/g dry weight (d.w.). For analysis of polycyclic aromatic hydrocarbons (PAHs) in caged bivalves, tissues (without foots) were extracted in potassium hydroxide in methanol using a microwave system (55 ◦ C for 15 min) (Mars CEM, CEM Corporation, Matthews, NC) according to Bocchetti et al. (2008). After centrifugation at 1000 × g for 5 min, the methanolic solutions were concentrated and purified with solid phase extraction (Octadecyl C18, 500 mg × 6 mL, Bakerbond; Mallinckrodt Baker, Phillipsburg, NJ, USA). A final volume of 1 mL was recovered with acetonitrile, and HPLC analyses were carried out with water–acetonitrile gradient and fluorimetric detection. PAHs were identified by the retention time of appropriate pure standard solutions (EPA 610 Polynuclear Aromatic Hydrocarbons Mix), and classified as low molecular weight (LMW: naphthalene, acenaphthene, fluorene, phenanthrene, anthracene) and high molecular weight (HMW: fluoranthene, pyrene, benzo[a]anthracene, chrysene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, dibenzo[a,h]anthracene, benzo[g,h,i]perylene). Quality assurance and quality control were tested by processing blank and reference samples (mussel tissues SRM 2977, NIST), and concentrations obtained for the SRM were always within the 95% confidence interval of certified value. The water content in tissues was determined and concentrations of PAHs expressed as ng/g d.w.

2.3. Individual antioxidants, total oxyradical scavenging capacity (TOSC), malondialdehyde and metallothionein-like proteins Biochemical analysis was carried out on 4 pools of mussels per site; each pool was constituted by soft tissues without foots of 6–7 specimens. Antioxidant defenses were measured with specific spectrophotometric assays at a constant temperature of 18 ◦ C, as detailed elsewhere (Regoli et al., 2004). Tissues were homogenized (1:5 w/v ratio) in 100 mM potassium phosphate buffer (pH 7.5), NaCl 1.8%; to which 0.1 mM phenylmethylsulphonyl fluoride (PMSF), 0.1 mg/ml bacitracin, 0.008 TIU/ml aprotinin were added as protease inhibitors. Cytosolic fractions were obtained after centrifugation at 110,000 × g for 1 h at 4 ◦ C.

Catalase (Cat) activity was quantified following the decrease in absorbance at 240 nm (ε = 0.04 mM−1 cm−1 ) due to H2 O2 consumption (12 mM H2 O2 in 100 mM sodium phosphate buffer pH 7.0). Glutathione peroxidase (GPx) activities were measured in a coupled assay where the formed oxidized glutathione (GSSG) is converted to its reduced form (GSH) by glutathione reductase with consumption of NADPH. The resulting decrease of absorbance was monitored at 340 nm (ε = 6.22 mM−1 cm−1 ) in 100 mM potassium phosphate buffer pH 7.5, 1 mM EDTA, 1 mM dithiothreitol (DTT), 1 mM NaN3 (for hydrogen peroxide assay), 2 mM GSH, 1 unit glutathione reductase, 0.24 mM NADPH and 0.5 mM hydrogen peroxide or 0.8 mM cumene hydroperoxide (CHP) as substrates respectively for the selenium-dependent and for the sum of Se-dependent and Seindependent forms. The rate of the blank reaction was subtracted from the total rate. Glutathione-S-transferase (GST) activities were determined at 340 nm using 1-chloro-2,4-dinitrobenzene (CDNB) as substrate in 100 mM sodium phosphate buffer pH 6.5, 1.5 mM CDNB, 1 mM GSH (ε = 9.6 mM−1 cm−1 ). Glutathione reductase activity was quantified at 340 nm by the decrease of absorbance during the oxidation of NADPH used to reduce GSSG to GSH (ε = 6.22 mM−1 cm−1 ). The assay was performed in 100 mM sodium phosphate buffer pH 7.0, 1 mM GSSG and 60 ␮M NADPH. Levels of total glutathione were measured in tissues homogenized (1:5 w/v ratio) in 5% sulphosalicilic acid with 4 mM EDTA and maintained for 45 min on ice before centrifugation at 37,000 × g for 15 min; the resulting supernatants were enzymatically assayed (Regoli et al., 2004). For the measurement of the total oxyradical scavenging capacity (TOSC), samples were homogenized as previously described for other enzymatic analyses except that PMSF and bacitracin were not added in the buffer. The TOSC assay measures the capability of cellular antioxidants to inhibit the oxidation of 0.2 mM ␣-keto-␥-methiolbutyric acid (KMBA) to ethylene gas in the presence of different forms of oxyradicals, artificially generated at constant rate (Regoli, 2000). Peroxyl radicals (ROO·) were generated by the thermal homolysis of 20 mM 2-2 -azobis-(2 methylpropionamidine)-dihydrochloride (ABAP) in 100 mM potassium phosphate buffer, pH 7.4. Hydroxyl radicals were generated from the Fenton reaction of iron–EDTA (1.8 ␮M Fe3+ , 3.6 ␮M EDTA) plus ascorbate (180 ␮M) in 100 mM potassium phosphate

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buffer. Ethylene formation in control and sample reactions was analyzed at 10–12 min time intervals by gas chromatographic analyses according to (Regoli, 2000). from  TOSC  values were quantified   the equation: TOSC = 100 − ( SA/ CA × 100), where SA and CA are the integrated areas calculated under the kinetic curve produced during the reaction course for respective sample (SA) and control (CA) reactions. For all the samples, a specific TOSC (normalized to content of protein) was calculated by dividing the experimental TOSC values by the relative protein concentration contained in the assay. Levels of malondialdehyde (MDA) were spectrophotometrically measured after derivatization in 1-methyl-2-phenylindole (dissolved in acetonitrile/methanol 3:1) with HCl 32%, and calibrated against a malondialdehyde standard curve (Gorbi et al., 2008). Protein concentrations were determined by the Lowry method with bovine serum albumin (BSA) as standard. Metallothionein-like proteins were analyzed in tissues homogenized (1:3, w/v) in 20 mM tris–HCl buffer, pH 8.6, 0.5 M sucrose, 0.006 mM phenylmethylsulphonylfluoride (PMSF) and 0.01% ␤mercaptoethanol. After acidic ethanol/chloroform fractionation of the tissue homogenate, metallothionein-like proteins were spectrophotometrically quantified using GSH as standard and expressed as equivalents of (G)SH (Viarengo et al., 1997).

2.4. Genotoxicity The genotoxic effects were evaluated at molecular level as DNA primary damage by the single cell gel electrophoresis (comet assay) and at chromosomal level through the evaluation of micronucleated cells. The comet assay was performed on haemocytes from 28 specimens per site within 24 h after sampling, according to Singh et al. (1988) with slight modifications. Briefly, cells were embedded in 0.5% low-melting agarose (LMA), spread onto microscope slides pre-coated with 1% normal melting agarose (NMA) and covered with a further layer of LMA. Slides were dipped into a lysing solution (NaCl 2.5 M, Na2 EDTA 100 mM, Trizma Base 10 mM, 10% DMSO, 1% Triton X-100, pH 10) and kept overnight at 4 ◦ C in the dark, in order to solubilise cell membranes and cytoplasm. Successively, slides were treated with alkali (NaOH 300 mM, Na2 EDTA 1 mM, pH > 13) for 10 min and placed in a horizontal electrophoresis apparatus. Electrophoresis was performed for 5 min at 25 V and 300 mA. After run, slides were neutralized with Tris–HCl (0.4 M, pH 7.5), stained with 100 ␮l ethidium bromide and observed under a fluorescence microscope (400×). The amount of DNA fragmentation was quantified as the percentage of DNA migrated into the comet tail (tail DNA), using an image analyzer (Kinetic Imaging, Ltd., Komet, Version 5). At least 50 nuclei per slide and two slides per sample (specimen) were scored and the mean calculated. The micronucleus test (MN) was carried out on haemocytes according to Scarpato et al. (1990). Cells were prefixed for 20 min in a 5% acetic acid, 3% methanol, 92% PBS 20‰ solution, and centrifuged for 5 min at 2000 rpm. The supernatant was removed and 5 ml fixative (from 5:1 to 3:1, depending on air humidity) was added to the suspended pellet; this process was repeated twice. After the last fixation cells were centrifuged, spread onto slides

(two slides per mussel), air dried and stained with 5% Giemsa solution for 10 min. One thousand cells with well preserved cytoplasm per specimen were scored (500 per slide) to determine the frequency of micronuclei according to the following criteria. Micronuclei were defined as round structures, smaller than 1/3 of the main nucleus diameter; micronuclei had to be on the same optical plan and non-refractile as the main nucleus but possess distinguishable boundaries from it (Fenech, 2007). 2.5. Neutral red retention time assay Lysosomal membrane destabilization was assessed by the neutral red retention time (NRRT), according to Lowe et al. (1995), within 24 h after sampling. At least 14 specimens per site were individually analyzed. Haemolymph was incubated on a glass slide with a neutral red (NR) working solution (0.1 mg/ml). Granular haemocytes were microscopically examined at 15 min intervals, for up to 2 h, to evaluate the time at which 50% of cells had leaked to the cytoplasm, the dye previously trapped by lysosomes. 2.6. Statistical analyses Bioaccumulation and biochemical data were compared by analysis of variance, ANOVA (level of significance at p < 0.05); homogeneity of variance was tested by Cochran C, and post hoc comparison (Student–Newman–Keuls) was used to discriminate between means of values. For statistical analysis of genotoxic and lysosomal alteration data, multifactor analysis of variance (MANOVA) or multiple regression analysis (MRA) were performed; multiple range test (MRT) was used to detect differences among sampling sites (level of significance at p < 0.05). 3. Results 3.1. Metals in sediments and bioaccumulation Chemical analyses of sediments showed higher concentrations of all the metals in the sites located along the River Cecina basin compared to those of the reference site, Lake Maggiore (Table 1). Particularly marked differences were observed for iron, manganese and especially chromium and nickel, with levels exceeding by one order of magnitude those of control sediments, and for arsenic, with an hot spot along the two sites of Possera stream (43.06 and 119 ␮g/g dry weight, Table 1). More elevated concentrations of cadmium were measured in sediments of both Possera stations; copper, lead and zinc at Possera 1, mercury at Possera 1 and Ponteginori (Table 1). PAHs were not analyzed in sediments since already available values indicated concentrations ranging from 0.008 to 0.3 ␮g/g. Bioaccumulation of trace metals in transplanted bivalves revealed quite limited variations, with higher concentrations of arsenic, mercury and nickel in molluscs transplanted to Possera and Ponteginori sites (Table 2). Concentrations of PAHs in tissues of bivalves caged along the river basin did not exhibit significant variations compared to those of control specimens, remaining always lower than 0.2 ␮g/g dry weight (Table 2). The condition

Table 1 Metal concentrations (␮g/g dry weight) in sediments collected from the study sites. Sites

As

Cd

Cr

Cu

L. Maggiore Berignone Possera 1 Possera 2 Ponteginori

1.81 8.98 43.06 119 9.53

0.06 0.07 0.15 0.14 0.09

8.98 1427 170.9 790.6 757.2

5.69 13.24 54.8 11.16 16.88

Fe 7740 37,000 26,063 32,921 31,500

Hg

Mn

Ni

Pb

Se

Zn

0.03 0.08 0.11 0.06 0.1

132.9 811.2 716.5 828.6 1576.9

33.73 530.7 163 545.3 240.6

4.38 8.43 42.42 12.44 12.04

0.79 5.56 1.24 2.85 2.13

26.67 48.78 88.6 39.41 52.88

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Table 2 Metals (␮g/g dry weight) and PAHs (ng/g dry weight) bioaccumulation in painter’s mussels after 4 weeks translocation in the study sites. Values are mean ± SD; different letters indicate significant differences among means after post hoc comparison (p < 0.05). L. Maggiore

Berignone

Possera 1

Possera 2

Ponteginori

As Cd Cr Cu Fe Hg Mn Ni Pb Zn

13.9 ± 3.37 a 2.39 ± 0.39 3.48 ± 1.51 17.12 ± 2.75 2588 ± 835 0.04 ± 0.01 a 7535 ± 2266 3.34 ± 0.51 a 3.08 ± 1.22 850 ± 317

11.9 ± 2.31 a 2.11 ± 0.48 3.17 ± 0.41 11.7 ± 2.23 3179 ± 237 0.03 ± 0.002 a 9418 ± 3057 5.25 ± 1.34 ab 3.94 ± 1.80 796 ± 236

12.7 ± 2.01 a 2.40 ± 1.22 1.85 ± 0.53 12.0 ± 1.06 2140 ± 529 0.09 ± 0.01 b 6841 ± 1598 5.38 ± 0.76 ab 2.19 ± 0.30 641 ± 119

18.4 ± 2.98 b 2.01 ± 0.10 2.26 ± 0.51 12.8 ± 1.74 2040 ± 322 0.07 ± 0.01 c 6914 ± 1946 2.86 ± 0.56 a 2.88 ± 0.71 546 ± 163

13.5 ± 1.54 a 2.38 ± 0.69 1.82 ± 0.12 10.9 ± 2.45 2205 ± 194 0.06 ± 0.01 c 7602 ± 1912 6.51 ± 2.54 b 2.75 ± 0.94 563 ± 139

Naphthalene Acenaphthene Fluorene Phenanthrene Anthracene Fluoranthene Pyrene Benzo(a)anthracene Chrysene Benzo(b)fluoranthene Benzo(k)fluoranthene Benzo(a)pyrene Dibenzo(a,h)anthracene Benzo(g,h,i)perylene

55.8 ± 36.3 n.d. 2.23 ± 1.82 18.1 ± 10.2 27.1 ± 12.2 19.3 ± 14.4 16.8 ± 4.44 1.12 ± 1.17 2.29 ± 1.95 0.23 ± 0.13 0.44 ± 0.31 0.63 ± 0.6 n.d. n.d.

38.2 ± 5.73 0.61 ± 0.3 3.11 ± 2.86 16.8 ± 17.9 34.5 ± 11.1 4.65 ± 1.45 8.91 ± 2.58 0.66 ± 0.25 0.53 ± 0.24 0.12 ± 0 0.15 ± 0.1 0.32 ± 0.22 n.d. n.d.

49.8 ± 5.85 0.43 ± 0.3 n.d. 2.66 ± 2.05 33.5 ± 10.5 2.28 ± 1.43 3.05 ± 2.09 0.41 ± 0.16 0.09 ± 0.07 0.05 ± 0.03 0.07 ± 0.02 0.11 ± 0.01 n.d. n.d.

82.5 ± 65.6 0.77 ± 0.52 3.36 ± 2.0 13.6 ± 6.31 56.0 ± 38.4 1.54 ± 0.19 n.d. 0.64 ± 0.45 0.28 ± 0.03 n.d. 0.04 ± 0.01 0.09 ± 0.01 n.d. n.d.

117 ± 60.8 1.21 ± 1.12 5.16 ± 2.05 15.3 ± 6.92 46.7 ± 3.89 4.27 ± 1.93 5.44 ± 1.91 1.03 ± 0.54 0.61 ± 0.47 0.43 ± 0.21 0.22 ± 0.21 0.34 ± 0.28 n.d. 0.2 ± 0.06

LMW PAHs HMW PAHs Total PAHs

103 ± 59.4 40.9 ± 21.7 144 ± 78.9

93.2 ± 11.0 15.4 ± 4.14 108 ± 14.1

86.5 ± 11.3 6.1 ± 3.44 92.6 ± 9.14

156 ± 107 2.61 ± 0.49 159 ± 107

185 ± 69.3 12.6 ± 4.62 198 ± 70.1

index of caged mussels ranged between 76.3 ± 15.8 at Berignone and 83.6 ± 14.7 at Possera 1; however these differences were not statistically significant, suggesting a similar food availability in all the study sites and the absence of interference between biomass variability and bioaccumulation data. 3.2. Biomarkers based on antioxidant system The translocation of U. pictorum in the River Cecina basin did not provoke any mortality but the modulation of numerous biological responses (Fig. 2). Among the biomarkers based on antioxidant responses, those related to glutathione metabolism were mostly affected. The highest depletion of total glutathione was observed in mussels from Possera 2 and, to a lower extent, from the downstream site (Ponteginori), with levels 10-fold and 3-fold lower than in controls, respectively. In organisms caged in Possera 2, the activity of glutathione reductase and Se-dependent glutathione peroxidases were also reduced respectively by 6- and 4-fold compared to control specimens (Fig. 2). Catalase activity was significantly increased in organisms from Possera 1, with mean enzymatic activity approximately 3-fold higher compared to those measured in other investigated sites (Fig. 2). To better evaluate the contribution of individual defenses to the overall antioxidant efficiency, the total oxyradical scavenging capacity (TOSC) was measured toward hydroxyl and peroxyl radicals. The results demonstrated a significant enhancement of the capability to neutralize hydroxyl radicals in specimens from Possera 1 in respect to controls, while the absorbing capacity toward peroxyl radicals was lower in molluscs caged at both Possera sites and Ponteginori than in those from L. Maggiore and Berignone (Fig. 2). Levels of malondialdehyde were higher in bivalves from Possera 2, while values measured in other sites were not significantly different from those of control organisms (Fig. 2). In addition to antioxidant responses, metallothionein-like proteins were also investigated. The highest values were shown by molluscs transplanted in the downstream site Ponteginori. How-

ever, differences with respect to all the other sites were not statistically significant, likely due to the elevated variability (Fig. 2). 3.3. Biomarkers based on cell damage Biomarkers of cell damages exhibited significant differences after 4 weeks transplantation. The lysosomal membrane stability of circulating haemocytes, assessed by the neutral red retention time, was higher in specimens from Lake Maggiore and Berignone and lower in specimens from all the other river sites (Fig. 3). Multiple regression analysis showed that the retention time of the vital dye within lysosomes was largely affected by antioxidant parameters (R-squared 82.8%) (Table 3). The percentage of DNA migrating in the comet assay was higher in specimens deployed in all the river sites, including the relatively uncontaminated site Berignone, depicting a generalized reduction of the DNA integrity in the River Cecina basin compared to Lake Maggiore. In this case the multiple regression analysis indicated that only 30% of DNA migration variance is explained by the fluctuations of antioxidant responses (data not shown). On the other hand, the frequency of micronucleated haemocytes, reflecting chromosomal alterations, was minimum in molluscs caged at Berignone, even lower than in controls, while higher values were observed in specimens caged in other river sites, especially in the downstream area of Ponteginori (Fig. 3). A statistically significant positive correlation was observed between chromosomal damage and mercury bioaccumulation in caged mussels (Fig. 4). 4. Discussion The use of biomarkers has become a major issue in ecotoxicology to evaluate environmental quality through a wide panel of biological responses triggered by contaminants. The use of a multibiomarker approach also allows to cope with the complexity of biochemical and cellular responses to pollution and to explore the mechanisms mediating the appearance of biological alterations. In this study, sediment chemistry was integrated with the measure-

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Fig. 2. Oxidative biomarkers in painter’s mussels from study and reference sites. Values are mean ± SD. Different letters indicate significant differences (p < 0.05).

Table 3 Multiple regression analysis on neutral red retention time values in painter’s mussels after 4 weeks translocation in the study sites. Independent variable

Coefficient

S.E.

t-Value

Significance level

Total glutathione GST Catalase TOSC-ROO· TOSC-HO·

1835.61 −0.849 −4.725 0.448 1.591

245.431 0.125 0.503 0.0678 0.234

7.48 −6.795 −9.393 6.607 6.799

<0.001 <0.001 <0.001 <0.001 <0.001

R-squared = 82.79%; p < 0.001.

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Fig. 3. Biomarkers of cell damage in painter’s mussels from study and reference sites. Values are mean ± SD. Different letters indicate significant differences (p < 0.05).

ment of bioaccumulation and several biomarkers in the painter’s mussels U. pictorum transplanted in various sites along the River Cecina and its tributary Possera stream. Analyses of sediments revealed elevated concentrations of chromium, iron, manganese and nickel in all the river basin, including the “Berignone” site located within a natural protected area, upstream the main sources of anthropogenic impact. These findings reflect a naturally elevated metal background in the entire area associated with a geochemical feature of the basin and abundance of ophiolitic rocks. On the other hand, anthropogenic activities are responsible for some hot spots, like those in the Possera stream, with more elevated sediment concentrations of copper, lead, mercury and especially arsenic, probably deriving from Larderello geothermal power plants, resid-

Fig. 4. Relationships between micronucleus frequency in painter’s mussels haemocytes and Hg tissue levels in caged painter’s mussels. Micronucleus values refer to groups of means while Hg concentrations refer to pooled samples. Correlation coefficient = 0.78, p < 0.01.

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ual industrial depots and the “Bulera” dumping ground. In the past decades, a chloro-alkali industry significantly contributed to Hg contamination in the middle part of Cecina main course, which at present appears mostly mitigated. Although the elevated levels of metals in sediments, bioaccumulation in tissues of transplanted U. pictorum was quite limited, being significantly higher than in controls only for arsenic and mercury in bivalves caged in Possera stream, and for mercury and nickel in specimens deployed at Ponteginori. Previous investigations with U. pictorum already reported only weak relationships between metal levels in tissues of the bivalve and those measured in water and sediments (Ravera et al., 2003a,b). The poor metal bioaccumulation of U. pictorum may reflect a high eliminating efficiency possibly associated with the presence of extra-cellular phosphatic granules in unionids and their role as a major sink for excretion of metal cations (Markich et al., 2001; Bonneris et al., 2005). However, in our study the limited bioaccumulation in transplanted bivalves is likely due to poor bioavailability of those metals deriving from the erosion of bedrocks and which account for the elevated background in the whole Cecina basin. In this respect, arsenic and mercury, which are relevant pollutants of anthropogenic origin in this area, were more readily accumulated by molluscs. The different bioavailability of metals of natural and anthropogenic origin in the river basin is supported by data obtained in the free ranging chubs, L. cephalus (Frenzilli et al., 2008). Mercury levels in fish tissues significantly increased in specimens sampled moving downstream along the River Cecina (from 0.6 up to 2.0 ␮g/g d.w.) while chromium concentrations were always low (0.2–0.4 ␮g/g d.w.) despite the high sediment levels (Frenzilli et al., 2008). Analyses of PAHs in caged bivalves confirmed that metals, both of natural and anthropogenic origin, are the main pollutants of potential environmental concern in the Cecina basin. In this study several biochemical and cellular responses were investigated in U. pictorum caged along the Possera stream and the River Cecina. Levels of metallothionein-like proteins did not exhibit any significant variation in translocated molluscs, in line with the moderate bioaccumulation and the potentially limited importance of these proteins in metal homeostasis of Unionidae compared to phosphatic granules. The impairment of equilibrium between prooxidant forces and antioxidant defenses, is an important and typical pathway mediating toxic effects in aquatic organisms (Winston and Di Giulio, 1991). In this respect, particular attention has been paid to the antioxidant status of caged mussels, by integrating the analysis of individual antioxidant defenses with the measurement of the overall capacity to neutralize specific ROS, as a more comprehensive index of susceptibility to oxidative stress. Accumulation of lipid peroxidation product (MDA), lysosomal membrane destabilization, DNA fragmentation and chromosomal disturbances were assessed being widely used in ecotoxicology as markers of cell toxicity which have been often related with the impairment of oxidative defenses (Winston and Di Giulio, 1991). Consistently with bioaccumulation results, the more evident variations of individual antioxidants were observed in bivalves deployed in the Possera stream and at Ponteginori; however the pattern and/or degree of responses were different in these sites. A clear modulation of glutathione metabolism was measured in molluscs deployed at the Possera 2 site, downstream Larderello geothermal power plants, chemical industry and the Bulera dumping ground. A marked depletion of glutathione was associated with the inhibition of activities of glutathione reductase, Se-dependent glutathione peroxidases and, to a lower extent, of glutathioneS-transferases. Glutathione reductase plays an important role within the cellular antioxidant system, being responsible for the regeneration of reduced glutathione and thus contributing to the maintenance of optimal redox status. The contemporary depletion

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of glutathione levels with the inhibition of glutathione reductase depicts a potential imbalance of the oxidative equilibrium in bivalves from Possera 2, further exacerbated by reduced activities of glutathione peroxidases and glutathione-S-transferases which use glutathione for removing peroxides and for detoxification reactions; the depletion of these enzyme activities render these organisms more susceptible to formed peroxides as also confirmed by the significantly increased content of malondialdehyde. Arsenic is a well known prooxidant metal and exposure to this element has been associated with impairment of antioxidant defenses in aquatic organisms (Schuliga et al., 2002; Kitchin and Ahmad, 2003; Battacharya and Battacharya, 2007; Bagnyukova et al., 2007; Ventura-Lima et al., 2007, 2009). Arsenate impairs the phosphate pentose pathway and decreases NADPH production with adverse implications in the antioxidant responses, since this molecule serves as electron donor for glutathione reductase during the reduction of oxidized glutathione (Morales et al., 2004; Tseng, 2004). Arsenic also forms complexes with GSH which facilitate both the excretion and biotransformation processes through sequential reduction/methylation reactions of the element (Aposhian et al., 2004). The elevated arsenic levels and bioavailability at Possera 2 could thus be responsible for the modulation of antioxidant defenses observed in U. pictorum caged at this site. Different oxidative responses were observed in molluscs deployed at Possera 1, which did not exhibit any modulation of glutathione system, but a significant induction of catalase. The enhancement of this enzymatic activity has been often described in molluscs exposed to chemical stress and interpreted as an adaptive response to counteract the increased oxyradical formation which might have been responsible for the more limited increase of MDA in bivalves caged in Possera 1. Variations of catalase can be modulated also by the intensity and/or duration of chemical disturbance (Regoli, 1998, 2000; Regoli and Principato, 1995; Regoli et al., 1998, 2002, 2004), and the more limited bioaccumulation of arsenic could explain the different pathway of oxidative responses in specimens caged at this site compared to those of the downstream Possera 2 site. The depletion of total glutathione in specimens from Ponteginori confirmed the sensitivity of this oxidative response in revealing a chemical disturbance. This effect did not result in any significant increase of malondialdehyde, suggesting that the normal activity of glutathione peroxidases might have prevented the accumulation of lipid peroxidation products. Similar results on the antioxidant system of Unionidae have been reported for U. tumidus showing a strong decrease of glutathione levels and activity of glutathione-dependent enzymes in response to different contamination profiles both in field and laboratory conditions (Doyotte et al., 1997; Cossu et al., 2000). Although fluctuations of individual antioxidants give useful information on the pathway of oxidative response to chemical impact, the relevance of these results is often difficult to summarize in terms of risk for organisms’ health condition. To partly overcome such uncertainty, in this study the overall capability of caged bivalves to neutralize ROS has been measured against peroxyl and hydroxyl radicals using the TOSC assay. Despite organisms deployed at Possera 1, Possera 2 and Ponteginori exhibited significant but quite different patterns of antioxidant responses, all these organisms revealed a significant decrease in the ability to absorb peroxyl radicals, consistent with the general decrease observed for several antioxidants. On the other hand, the scavenging capacity toward ·OH varied only in U. pictorum deployed at Possera 1, where the significant induction of catalase corroborated the role of this enzyme in protecting against hydroxyl radical toxicity through the removal of hydrogen peroxide, which is one of the main ·OH precursors (Regoli et al., 2003, 2004). The lack of variations in bivalves from other sites, which exhibited depletion of some antioxidants,

would indicate a poor involvement of such defenses in removal of hydroxyl radical. Lysosomal membrane destabilization, DNA fragmentation and chromosomal damages were assessed being widely used in ecotoxicology as marker of cell toxicity which have been often related with the impairment of oxidative defenses (Winston and Di Giulio, 1991). The loss of lysosomal membrane stability clearly discriminated among sites and paralleled the capacity to absorb peroxyl radicals, with higher values detected in both control molluscs and in those deployed at Berignone (within the natural protected area) and shorter in specimens caged at Possera 1, Possera 2 and Ponteginori. Multiple regression analysis showed that more than 80% of NRRT variance is explained by the sum of antioxidant responses confirming the sensitivity of lysosomal membrane to the modulation of cell redox equilibrium and the predictive value of antioxidant responses toward the onset of this kind of cell damage (Regoli, 2000). The comet assay has been largely used for the evaluation of DNA fragmentation in aquatic eco-geno-toxicology (Frenzilli et al., 2009). A decrease of DNA integrity was observed in molluscs caged in all the sites of the Cecina river basin, including the unpolluted Berignone. In line with this finding, the results of the multiple regression analysis showed that only 30% of the DNA migration variance is explained by the modulation of antioxidants. These evidences suggest that the amount of DNA fragmentation detected in painter’s mussel cannot be entirely ascribed to oxidative processes, allowing to hypothesize that a sum of factors, able to impact DNA through different mechanisms, actually co-exist in the study area. The micronucleus test is widely used for the detection of chromosomal fragmentation and/or loss in freshwater and marine sentinel species (Scarpato et al., 1990; Bolognesi et al., 1999; ˇ Stambuk et al., 2009). Micronucleus test in painter’s mussels showed the highest chromosomal integrity in molluscs caged at the unpolluted Berignone site, even lower than in controls, and the lowest in Ponteginori, where DNA and lysosomal membrane integrity also showed the lowest values. In a previous study with U. pictorum ˇ (Stambuk et al., 2009), both the comet assay and the micronucleus test efficiently discriminated between polluted and control sites according to the genotoxic proprieties of contaminants. Also in our investigation the frequency of micronucleated cells was increased in the bivalves deployed at both sites of Possera stream and Ponteginori; however, due to the high inter-individual variability, only in specimens from Ponteginori such an increase was statistically significant. The occurrence of a significant relationship between the frequency of micronucleated cells and Hg bioaccumulation suggests a potential role of this metal in the onset of chromosomal damage. The genotoxic potential of mercury in mussels is well established as inducer of both DNA single-strand breaks and micronuclei frequency (Bolognesi et al., 1999). The relationships between MN frequency and Hg bioaccumulation in painter’s mussel are in accordance with our previous data on free ranging chubs (L. cephalus) sampled from the same environment, which showed a positive correlation between the frequency of micronucleated erythrocytes and total Hg levels in muscle tissue (Frenzilli et al., 2008). In conclusion, the overall results of this study confirm a quite poor metal bioaccumulation capacity in the painter’s mussels U. pictorum, suggesting a certain caution when adopting this species for evaluating bioavailability of these elements in freshwater ecosystems. On the other hand, the multi-biomarker approach confirmed an elevated sensitivity and the possibility to evaluate different forms of biological disturbance among sites with different levels and/or nature of chemical impact. The painter’s mussel U. pictorum appeared as a proper species for caging experiments, thus resulting a potentially very useful sentinel organism for biomonitoring freshwater ecosystems devoid of naturally occurring bivalve populations. However, further studies are needed to explore the

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