The use of Fulvia fragilis (Mollusca: Cardiidae) in the biomonitoring of Bizerta lagoon: A mutimarkers approach

Ecological Indicators 10 (2010) 696–702

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The use of Fulvia fragilis (Mollusca: Cardiidae) in the biomonitoring of Bizerta lagoon: A mutimarkers approach Naima Mahmoud a, Mohamed Dellali a,*, Monia El Bour b, Patricia Aissa a, Ezzeddine Mahmoudi a a b

Laboratory of Environment Biomonitoring, Coastal Ecology and Ecotoxicology Unit, Faculty of Sciences of Bizerta, 7021, Zarzouna, Tunisia Laboratory of Bacteriology and Pathology, National Institute of Marine Sciences and Technologies of Salammboˆ, 2025, Salammbo, Tunisia

A R T I C L E I N F O

A B S T R A C T

Article history: Received 3 August 2009 Received in revised form 15 November 2009 Accepted 23 November 2009

Five biomarkers, catalase (CAT) activity, glutathione-S-transferase (GST) activity, the neural transmitter enzyme acetylcholinesterase (AChE), reduced glutathione (GSH) and malondialdehyde (MDA) levels were measured in specimens of Fulvia fragilis collected from Bizerta lagoon (Tunisia). Results demonstrated that F. fragilis showed differential biomarker response according to the importance of the anthropogenic pressure and the nature of pollutants that affect the lagoon. A clear organotropism was also observed with a higher biomarker response in digestive gland than in gills of this bivalve. These results indicate that F. fragilis constitutes a useful tool as sentinel organism for biomonitoring of aquatic pollution. ß 2009 Elsevier Ltd. All rights reserved.

Keywords: Fulvia fragilis Bizerta lagoon Biomarkers GSH CAT GST MDA AChE

1. Introduction The presence of toxic agents in the ecosystems has increased in recent decades, especially in aquatic environments. Among Mediterranean coastal ecosystems, many lagoons are subject to human-induced pressures resulting from urbanization, industrialization and intensive agriculture. Pollutants endanger the health of organisms including humans (e.g. Ternes et al., 2007), but their toxicity is difficult to measure. Thus, it is necessary to develop strategies to assess if a given ecosystem is under stress or not. The effect of xenobiotic substances on aquatic organisms is currently taken into account when carrying out quality assessments of the aquatic environment. For the monitoring of aquatic environment, it is becoming increasingly necessary to assess the potential impact of substances entering estuarine and marine waters. Marine pollution has been traditionally documented in terms of chemical concentrations of contaminants; however, these measurements did not provide estimations of the deleterious effects on living organisms and are now complemented with biological criteria, especially with the measurement of biomarkers. By the mid-1980s, a wide range of biomarkers, constituting an early warning system,

* Corresponding author. E-mail address: [email protected] (M. Dellali). 1470-160X/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.ecolind.2009.11.010

have been developed and suggested for use in monitoring programs (Lam and Gray, 2003). Different categories of biomarkers have been identified, e.g. biomarkers of exposure and response, or general and specific biomarkers (De Lafontaine et al., 2000). Antioxidant enzymes such as catalase (CAT), reduced glutathione (GSH) and glutathione-S-transferase (GST) could be useful as biomarkers reflecting not only exposure to contaminants, but also their toxicity (Mosleh et al., 2007) especially when the body is exposed to pollutants that generate oxidative stress. The AChE activity has been used as a biomarker of exposure to several chemicals such as organophosphate and carbamate insecticides in aquatic environments (Oliveira et al., 2007). Elevated concentrations of MDA are an expression of lipid peroxidation (Sunderman, 1987) that indicates cell damage. Biomarkers, validated in bivalves for pollution biomonitoring (Ringwood et al., 1999), have led to new approaches in fundamental and applied research. Among marine invertebrates, bivalves are well suited to assess contaminant impacts. These organisms are able to accumulate several classes of pollutants because they are sessile, filter feeding, widely distributed and abundant in coastal and estuarine areas (Bocchetti and Regoli, 2006). In Tunisia, the Bizerta lagoon, located near some industrial units and agricultural area, exploited in conchyliculture since 1964 (Beji, 2000), represented a receptor of several industrial wastes,

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pesticides and chemical fertilizers through soil erosion and runoff leading to a decrease in bivalves and fish production (ANPE, 1990). In this aquatic area, many studies have investigated the application of biochemical tools, but these biomarker approaches have been restricted to certain species such as Mediterranean clam Ruditapes decussatus (Dellali et al., 2001, 2004), the mussel Mytilus galloprovincialis (Dellali et al., 2001; Khessiba et al., 2001, 2005) and Hexaplex trunculus (Rome´o et al., 2006). However, no studies have assessed the use of Fulvia fragilis, an abundant Cardiidae in the Bizeta lagoon, as a sentinel species and tool for biomonitoring this coastal ecosystem. In this paper, we present the results of the responses of five biomarkers [reduced glutathione (GSH), catalase (CAT, EC 1.11.1.6), glutathione-S-transferase (GST), malondialdehyde (MDA) and acetylcholinesterase (AChE, EC 3.1.1.7)] in Fulvia fragilis in a Mediterranean coastal lagoon (Bizerta lagoon, Tunisia). 2. Materials and methods 2.1. Investigated area and field sampling Specimens of Fulvia fagilis (Forsska˚l in Niebuhr, 1775) of shell length 50  3 mm were collected from two sites (Menzel Abderrahmen and Jwawda) in Bizerta lagoon (Northern Tunisia) in June and December 2006 (Fig. 1). These two sampling sites are differently impacted by pollution. In fact, Menzel Abderrahmen (N378130 27800 E98510 15600 ) (S1) is continuously effected by urban effluent but Jwawda (N378110 91700 E98550 65900 ) (S2), a site far from any known source of pollution and located in the southeast of the Bizerta lagoon (Fig. 1). The collected specimens of F. fragilis were dissected upon return to the laboratory. Digestive gland and gills were dissected and stored at 20 8C until analyses. 2.2. Hydrological parameters The physico-chemical quality of the Bizerta lagoon waters was monitored in situ over 2 months (June and December). Salinity (PSU), temperature (8C), dissolved oxygen (mg/l), and pH were

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measured at the two sampling sites with a WTW-197i multimeter. Samples for nutrient analysis (1000 ml) were filtered through Whatman GF/F filters. The filtrates were collected in acid-washed vials and kept frozen (20 8C) until analysis (within 2 weeks). Nutrient concentrations were determined by spectrophotometric methods (NO3 NO2: Wood et al., 1967; PO43: Murphy and Riley, 1962; NH4+: Aminot and Chaussepied, 1983). 2.3. Biomarker measurements For biochemical measurements, F. fragilis were dissected and digestive gland and gills were homogenized with an ultra-turrax in a TRIS buffer (50 mM Tris, 150 mM NaCl, pH 7.4). The homogenates were centrifuged at 9000  g for 30 min. All procedures were carried at 4 8C. The resulting supernatants were frozen at 30 8C until biochemical analysis. The variation in optical density was quantified using a Beckman DU500 spectrophotometer. Total proteins were determined according to Bradford (1976). AChE activity was determined using the method of Ellman et al. (1961) adapted to a microplate reader by Galgani and Bocquene´ (1991). GST activity was measured according to the method of Habig et al. (1974), using 1-chloro-2,4-dinitrobenzene (CDNB) and GSH as substrates (Habig et al., 1974). Absorbance was measured at 340 nm, and activities were expressed as nanomoles of conjugated product formed per minute and per milligram of protein. Catalase activities were assayed as described by Claiborne (1985). The variations of absorbance at 240 nm, caused by the dismutation of hydrogen peroxide, were measured as a function of time (e = 40 M). Lipid peroxidation was estimated by the formation of TBARS. TBARS, considered as ‘‘malonedialdehyde (MDA)-like peroxide products’’, were quantified by reference to MDA absorbance (e = 156  103 M1 cm1). Reduced glutathione (GSH) level was determined according to the method described by Moron et al. (1979). Homogenates were immediately precipitated with 0.1 ml of 25% TCA (trichloroacetic acid) and the precipitate was removed after centrifugation. FreeSH groups were assayed in a total 3 ml volume by the addition of

Fig. 1. Bizerta lagoon (Northern Tunisia): (S1) Menzel Abderrahmen and (S2) Jwawda.

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Table 1 Environmental parameters measured at investigated stations of Bizerta lagoon (June–December 2006). Station

Parameters

June

December

S1

Salinity (PSU) Temp. (8C) pH O2 (mg/l) NO3 (mg/l) NO2 (mg/l) NH44+ (mg/l) PO43 (mg/l) Ntot./Ptot. NO3/PO43 Chl. a (mg/m3)

34.40 27.60 8.25 6.92 159.27 11.32 15.21 43.32 5.36 3.67 4.56

31.30 15.10 7.65 7.40 148.67 22.03 106.61 22.42 4.30 6.63 1.23

S2

Salinity (PSU) Temp. (8C) pH O2 (mg/l) NO3 (mg/l) NO2 (mg/l) NH44+ (mg/l) PO43 (mg/l) Ntot./Ptot. NO3/PO43 Chl. a (mg/m3)

34.30 27.60 8.31 7.06 77.87 5.95 42.40 23.27 4.53 3.34 2.35

31.20 15.00 7.76 7.60 77.87 11.76 54.10 22.70 4.51 3.42 2.85

2 ml of 0.6 mM DTNB (Acide 5,50 -DiThio-bis2-NitroBenzoı¨que) and 0.9 ml of 0.2 mM sodium phosphate buffer (pH 8.0) to 0.1 ml of the supernatant and the absorbance was measured at 412 nm using a UV–vis Systronics spectrophotometer. Glutathione was used as a standard to calculate nmol GSH/mg prot. 2.4. Statistical analysis The one-way ANOVA was used to test differences between the measured biomarker activities in each station and the Tukey HSD test was used in pairwise comparisons between sites. A significant difference was assumed when p < 0.05. The Bravais– Pearson correlation coefficient was calculated to determine correlations between environmental parameters and biomarkers responses. 3. Results 3.1. Physico-chemical parameters Salinity varied from 31.20 to 34.40 PSU (Table 1) with minimum values measured in December and maximum values recorded in June. Water temperature at the two sampled stations (Table 1) exhibited comparable temporal fluctuations from 15 to 27.60 8C. The monthly pH values remained relatively constant throughout the sampling period and ranged from 7.65 to 8.31, with minimum values during December. Monthly dissolved oxygen levels (Table 1), all stations considered, varied between a minimal value of 6.92 mg/l in June (station S1) and a maximum of 7.60 mg/l in December at station S2. In S1, nitrate values ranged from 148.67 mg/l in December to 159.27 mg/l in June. Lowest nitrate values (<78 mg/l) were recorded at S2. Nitrite concentrations were relatively low at the two sites. The highest value of nitrite (5.95 mg/ l) was observed at station S2 in June. Ammonia concentrations were relatively higher at S1 (a maximum of 106.61 mg/l was recorded in December) compared to the levels measured in S2 (Table 1). Phosphate concentrations were higher at S1 in summer with a maximum of 43.32 mg/l recorded in June. Chlorophyll a concentrations were low (<5 mg/m3) at the both stations.

3.2. Biochemical responses In S1, the summer level of GSH registered in digestive gland was approximately two times higher than observed in the gill, whereas in winter, the activity in digestive gland decreased significantly (Fig. 2A). In December as in June, the values of GSH are the lowest in station S2 (Jwawda); this result was obtained in gills as in digestive gland. This suggests the consumption of the GSH in animals from station S2 or an induction of synthesis of this biomarker in animals collected from the station S1. The CAT activity was significantly higher in the digestive gland compared to that of gills in the both sites (p < 0.05) (Fig. 2B). The CAT activity was higher in the digestive gland and gills of specimens from Menzel Abderrahmen (S1) compared to those of Jwawda (p < 0.05) (Fig. 2B). The maximum of this enzymatic activity (29.20  0.65 mmol/min/mg prot.) was noted in bivalve’s digestive gland collected from S1. A minimum of 2.84  0.73 mmol/ min/mg prot. was measured in gills of the specimens collected in the station S2. In December, the induction of catalase activity was observed in bivalves of S1 with a maximum of 10.32  0.70 mmol/ min/mg prot. recorded in the digestive gland. A significant positive correlation was recorded between the CAT activity in gills, and the pH (r = 0.993, p < 0.05) and a significant negative correlation with dissolved oxygen (r = 0.951, p < 0.05). In June as in December, significantly higher GST activity values were found in digestive gland and gills of F. fragilis collected in S2 compared to the specimens from S1. The values of GST activity recorded in digestive glands of F. fragilis collected from Jwawda (S2) were significantly higher than those recorded in F. fragilis from Menzel Abderrahmen (S1) sampling site (9.18  1.37 mmol/min/ mg prot.). Compared to 4.54  0.29 mmol/min/mg prot., measured in June, specimens from S2 exhibited significantly higher activity levels than those from S1. In gills, significantly higher levels of GST activity were found in specimens collected from S2 compared to those from station S1 (5.60  0.84 mmol/min/mg prot. vs. 2.88  0.20 mmol/ min/mg prot. noted in June) (Fig. 2C). In the digestive gland, significantly higher values of GST activity were only observed in F. fragilis from Jwawda in June as in December (Fig. 2C). The GST activity in digestive gland was correlated negatively with dissolved oxygen (r = 0.997, p < 0.05). The level of MDA was higher in digestive gland than in gills of F. fragilis collected from the both stations S1 and S2. In gills, no significant differences were found in MDA levels among samples of June (Fig. 2D). In December, significantly higher MDA levels were found in the digestive glands of F. fragilis collected from S2. Our results confirm that the rate of lipid peroxidation was higher in the digestive gland than in gills. Bivalves from S2 presented significantly higher MDA levels relatively to S1. Negative correlation was recorded between the MDA in gills and some abiotic factors (temperature, r = 0.998, p < 0.01; salinity, r = 0.999, p < 0.01 and pH, r = 0.999, p < 0.01). In June as in December, compared to the specimens from station S2, F. fragilis sampled from S1 exhibited lower AChE activity (p < 0.001) with minimum values (1.61  0.43 nmol/min/mg prot. in June and 6.29  0.42 nmol/min/mg protein in December) recorded in digestive gland (Fig. 2E). 4. Discussion In the Bizerta lagoon, salinity varied both spatially and temporally from 31.20 to 34.40 PSU with a minimum in December and a maximum in June. Low salinity values measured in December can be explained by the large freshwater discharge (20 Mm3 year1) from Ichkeul Lake through the Tinja River (Harzallah, 2002). Water temperature at both stations exhibited comparable seasonal fluctuations from 15 to 29.6 8C. In S1,

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Fig. 2. (A) Reduced glutathione (GSH) (expressed as nmol/mg prot.), (B) catalase activity (expressed as mmol/min/mg prot.), (C) glutathione-S-transferase (GST) (expressed as mmol/min/mg prot.), (D) malonedialdehyde (as nmol de MDA/mg prot.) and (E) acetylcholinesterase (AChE) (expressed as nmol/min/mg prot.) in digestive gland and gills of F. fragilis collected from the Bizerta lagoon in June and December 2006 (mean  SD, n = 10). Significant differences were determined by one-way ANOVA followed by Tukey HSD test; differences between sites attributed with the same letters were not significant and sites with different letters were significantly different at p < 0.05. S1: Menzel Abderrahmen, S2: Jwawda.

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temperature was always higher than at the S2 station. Conversely, monthly dissolved oxygen levels at the two stations considered, varied between a maximum measured in December (S2) and a minimum recorded in S1 in June. The increase in temperature and salinity associated with the relative decrease in dissolved oxygen that we have observed in summer is due to the eutrophication of the Bizerta lagoon (Khessiba et al., 2005). The monthly pH values remained relatively constant throughout the sampling period and ranged from 7.65 to 8.41, with minimum values in December. Oxygen availability is one of the most limiting growth factors and chronic hypoxia may be an important environmental stressor influencing the growth of mollusc (Harris et al., 1999; Willson and Burnett, 2000; Baker and Mann, 1992). Recent progress in invertebrate immunology has established that application of anoxic and hypoxic stress can alter immune responses in mollusk, and may cause increased susceptibility to disease (Boyd and Burnett, 1999; Malham et al., 2002; Pampanin et al., 2002; Cheng et al., 2004; Matozzo et al., 2005; Monari et al., 2005). The hypoxia conditions can also cause depression of immune activities in bivalve haemocytes (Chen et al., 2007). The lowest nitrate values were recorded in S2. Low concentrations of NO3, proved its significant utilization by the plankton community and post-bloom conditions (Verity et al., 2002). Nitrite concentrations were relatively low in the two sites. The highest value of nitrites (22.03 mg/l) was observed in station S1 in December. Ammonia concentrations were higher in S1 with a maximum of 106.61 mg/l in December. Phosphates concentrations were also higher in S1 with a maximum of 23.27 mg/l recorded in summer. The highest value of chlorophyll a concentrations (5.30 mg/m3) was recorded in S1 in June. In summer, the increase in water temperature could stimulate bacterial activity and benthic metabolism, which in turn enhanced the mineralization. Nutrient concentrations are frequently used as water quality indicators because they represent the most influenced chemical factors by human activities (Stumm and Baccini, 1983). Mediterranean coastal lagoons are generally characterized by weak discharge of sea water and can be subject to recurrent eutrophication, caused by increased nutrient loading from expanding anthropogenic activities (De Casabianca et al., 1997; Solidoro et al., 2005). In these eutrophic systems, diatom blooms may occur mainly in spring and summer (Gilabert, 2001; Nuccio et al., 2003). Considering its nutrients and chlorophyll a (Chl. a) status, the Bizerte lagoon was classified in the lower range of eutrophicated systems (Sakka Hlaili et al., 2008). However, the enclosed nature of the lagoon and the long renewal time of water (several months, Harzallah, 2003) could accentuate its eutrophication state in the future (Sakka Hlaili et al., 2008). The Bizerta lagoon is subjected to many anthropogenic pressures including urbanisation and industrial activities. The direct and indirect discharges of urban and industrial wastes and runoff lead to the chemical contamination of the lagoon by various toxic compounds such as heavy metals (Zn, Cd, Ni, Pb and Cu) (Yoshida et al., 2004) organo-chlorinated pesticides (Cheikh et al., 2002), halogenated aromatics compounds like polychlorobiphenyls (PCBs) (Derouiche et al., 2004), organotins (Mzoughi et al., 2005) and polycyclic aromatic hydrocarbons (PAHs) (Trabelsi and Driss, 2005; Ben Said et al., 2008). Biomarkers are biological parameters, known to vary in response to environmental pollutants, and participate in the normal metabolism of organisms. The contamination of an environment provoke the generation of the reactive oxygen species (ROS) (Lima et al., 2007) that induce or inhibit the activity of some biological composites called biomarkers (e.g. GSH, CAT). In this paper, the lowest GSH activity was measured in digestive gland of animals collected from S1 in December indicating an

increase of reactive oxygen species in this organ. However, in S2, the values of GSH showed a decrease in June. In June as in December, the values of GSH are highest in station S1. The increase of GSH detected at the two sites cannot be explained by the increased regeneration of oxidized glutathione carried out by this enzyme, but represents a GSH new synthesis as an adaptive response towards its increased use. In addition, changes in membrane permeability induced by MDA can decrease GSH cellular levels by allowing faster ROS intake and GSH loss (Ault and Lawrence, 2003). Our results revealed that the CAT activity was higher in the digestive gland and gills of the specimens from Menzel Abderrahmen (S1) compared to those from Jwawda (p < 0.05). Wastewaters from many sewers are discharged directly into the eastern sector of the lagoon; the city of Menzel Abderrahmen may also be responsible for the enrichment of the S1 station in various pollutants. In the two sampled stations, the values of CAT showed temporal fluctuations. These biological responses can be modulated also by seasonal changes of both environmental and biological factors, potentially influencing responsiveness and sensitivity to pollutants (Pellerin-Massicotte, 1994; Dellali et al., 2001). Catalase activity that we have recorded in F. fragilis at the two sampled stations increased during June. Our study confirmed previous findings that catalase activity is influenced by temperature (Khessiba et al., 2005; Dellali et al., 2001). In summer, bivalves collected from the two stations (S1 and S2) showed an increased catalase activity. Our results are in total agreement with the experimental variations of CAT activity with temperature in Mytilus galloprovincialis (Khessiba et al., 2005) and the fluctuations found in situ in the Bizerta lagoon for mussels M. galloprovincialis and clams Ruditapes decussatus (Dellali et al., 2001, 2004). The GST activity, as a biomarker of defense, participates also in anti-oxidative defenses (Michel et al., 1998; Hajime et al., 2005) and can be triggered by some pollutants (Goldberg and Bertine, 2000; Pennec and Pennec, 2003; Moreiro and Guilhmino, 2005). In the two sampled stations, GST activity measured in digestive gland as in gills was generally low in December and high in June. The increase in GST activity observed in summer suggests an activation of digestive gland detoxification processes probably due to the contamination of the lagoon by PAHs (Ben Said et al., 2008). The values of GST activity recorded in digestive glands of F. fragilis collected from Jwawda were significantly higher than those measured in animals from Menzel Abderrahmen. This induction of the GST despite weak value of the CAT with animals of Menzel Abderrahmen station can be related to specific pollutants that affect the lagoon. When the defense by the enzymes of the phase I of cell metabolism is insufficient, toxic effects appear and the protection at this stage is assured by the GST activity grouping together many isoenzymes of conjugation of the phase II. The MDA level is proportional to the extent of lipid peroxidation (Aust, 1985) and serves as a marker for oxidation of membrane lipids. Many studies have reported that the MDA concentration is an important parameter in evaluating the level of oxidative stress in the organisms (Thomas and Wofford, 1993; Rome´o and GnassiaBarelli, 1997). In general, organisms with lowered antioxidant status could be more susceptible to lipid peroxidation, and therefore presenting higher levels of MDA (Doyotte et al., 1997; Cossu et al., 2000). In our study, the level of MDA of F. fragilis collected from S2 was high in December. This increase of MDA level seems to be caused by the increase of provision in rainwaters washing out the agricultural bordering lands and enriching this sector on various pollutants (pesticides and heavy metals) inducing the lipid peroxidation. Our results are comparable to those of Pellerin-Massicotte (1997) that revealed an in situ increase

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of lipid peroxidation in mussel (Mytilus edulis) collected, in winter, from the Canadian coast. Temporal evolution of acetylcholinesterase showed a low activity in June. This suggests that potential pollutants like pesticides may be highly used in summer in domestic use. In fact, AChE activity is commonly used to diagnose pesticide exposure in environmental monitoring studies (Mora et al., 1999; Davies et al., 2001). The lowest acetylcholinesterase activity observed in this paper with animals collected in the station of Menzel Abderrahmen is partly explained by the higher levels in various pesticides [hexachlorobenzene (HCB (0.12 ng/g dry weight), heptachlore (0.06 ng/g dry weight), polychlorobiphenyl (PCB) congeners, p,p0 DDE (0.13 ng/g dry weight); o,p0 -DDD (0.35 ng/g dry weight), and dichlorodiphe´nyltrichloroe´thane (DDT) (0.48 ng/g dry weight)] recorded in sediment of Menzel Abderrahmen station (Cheikh et al., 2004). In addition, Yoshida et al. (2004) had also reported higher values of Zn (35.20–189.20 ppm), Cd (0.07–0.63 ppm), Ni (7.80–36.30 ppm), Pb (2.35–73.8 ppm) and Cu (7.40–36.21 ppm) in the sediments of this station. Among the two sampled stations, S1 appeared to be the most anthropogenically affected. In F. fragilis, the biochemical responses were generally higher in digestive gland, the major organ of metal accumulation in bivalves (Pipe et al., 1999), than in gills, the main interface between the organism and its environment (Rajalakshmi and Mohandas, 2005). Our results suggest that digestive gland is advantageous than gills for biomarkers’ response in this bivalve. 5. Conclusion The battery of parameters applied in the present work including F. fragilis’ oxidative stress, measured in two distinct tissues, provided a discrimination of sites with different levels of contamination after redundancy analysis. A global analysis of F. fragilis responses indicates that the station S1 (Menzel Abderrahmen) is more polluted with ‘‘cocktail’’ of pollutants than the station S2 (Jwawda). The oxidative stress was confirmed by the elevation of GSH, CAT, GST activities, MDA level and inhibition of AChE activity. These results demonstrate that F. fragilis constitutes a useful tool in biomonitoring of aquatic pollution and can be employed as a sentinel species in Bizerta lagoon. This work represents the first stage of a monitoring program that is being developed in wild populations of F. fragilis in the Bizerta lagoon. In future surveys, we will separate effects due to chemical contamination from those due to natural fluctuations of both abiotic parameters and F. fragilis’ annual physiological cycle. References Aminot, A., Chaussepied, M., 1983. Manuel des analyses chimiques en milieu marin. Centre National pour l’Exploitation des Oce´ans, 395 p. ANPE, 1990. Diagnostic pre´liminaire pour l’Etude de l’Equilibre Ecologique du lac de Bizerte. GIC-NNEA-TECI, ANPE, Tunisie. Ault, J.G., Lawrence, D.A., 2003. Glutathione distribution in normal and oxidatively stressed cells. Exp. Cell. Res. 285, 9–14. Aust, S.D., 1985. Lipid oxidation. In: Greenwald, R.A. (Ed.), CRC Handbook of Methods for Oxygen Radical Research. CRC Press, Boca Raton, FL, pp. 203–207. Baker, S.M., Mann, R., 1992. Effects of hypoxia and anoxia on larval settlement, juvenile growth, and juvenile survival of the oysters Crassostrea virginica. Biol. Bull. 9, 182–265. Beji, O., 2000. Les ressources vivantes exportables du lac de Bizerte: Etat actuel et potentialite´s (premie`re partie). Bull. Inst. Nat. Sci. Tech. Mer Salammboˆ 27, 45– 60. ˜ i-Urriza, M.S., El Bour, M., Dellali, M., Aissa, P., Duran, R., 2008. Ben Said, O., Gon Characterization of aerobic polycyclic aromatic hydrocarbondegrading bacteria from Bizerte lagoon sediments, Tunisia. J. Appl. Microbiol. 104, 987–997. Bocchetti, R., Regoli, F., 2006. Seasonal variability of oxidative biomarkers, lysosomal parameters, metallothioneins and peroxisomal enzymes in the Mediterranean mussel Mytilus galloprovincialis from Adriatic Sea. Chemosphere 65, 913–921.

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