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Oxidative stress responses of the mussel Mytilus galloprovincialis exposed to emissary's pollution in coastal areas of Casablanca
Ocean & Coastal Management 136 (2017) 95e103
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Ocean & Coastal Management journal homepage: www.elsevier.com/locate/ocecoaman
Review
Oxidative stress responses of the mussel Mytilus galloprovincialis exposed to emissary's pollution in coastal areas of Casablanca Zineb Mejdoub a, *, Abdelilah Fahde b, Mohammed Loutfi a, Mostafa Kabine a a b
Department of Biology, Laboratory of Biochemistry and Molecular Biology, Faculty of Science Ain Chock, University Hassan II Casablanca, Morocco Department of Biology, Laboratory of Environment and Aquatic Ecology, Faculty of Science Ain Chock, University Hassan II Casablanca, Morocco
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 May 2016 Received in revised form 11 October 2016 Accepted 9 November 2016
The bivalve mollusks are among the aquatic bioindicators that are commonly used in monitoring water pollution studies, thanks to their behavior and metabolism. They are directly affected by the level of pollution in a given site. During this research, the study of the biological response in gills, hepatopancreas and muscles of indigenous mussels Mytilus galloprovincialis were used for monitoring emissary's pollution in four polluted sites in the coastal environment of Casablanca. Seasonal variations of the activity of antioxidant defence enzymes, catalase (CAT), glutathione S-transferase (GST), as well as lipid peroxidation (LP) were measured as biomarkers within a one year period and compared to mussels from an unpolluted sampling site. This study was completed by analysing a series of abiotic factors (temperature, pH and conductivity) and chemicals (heavy metals; Hg, Pb, Cu) into seawater. Our result showed that the availability of metallic contamination and the environmental stress conditions causes relatively an oxidative stress in this species at each station studied. While the pollution's level clearly varies according to the sampling campaign. Furthermore, they revealed a significant increase in GST activities and LP concentrations and significant decrease in CAT activities in mussels collected in sites with industrial contamination. This negative correlation suggested that the organisms at this location are exposed to a relatively higher level of oxidative stress. This first study in the area confirm that variations of antioxidant defence enzymes activities and LP concentrations in mussels could be used as prospective biomarkers of toxicity in environmental monitoring programs. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Biomarkers Oxidative stress Mussels Mytilus galloprovincialis Antioxidant defence enzymes Lipid peroxidation Heavy metals
Contents 1. 2.
3.
4.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96 Materials & methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96 2.1. Site description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96 2.2. Sampling and sample preservation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97 2.3. Trace metal concentrations in surface seawater and wastewater . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97 2.4. Enzymatic biomarkers and total protein quantification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97 2.5. Lipid peroxidation concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98 2.6. Statistical analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98 3.1. Trace metal concentrations in surface seawater (Casablanca coast) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99 3.2. Seasonal measurements of CAT activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99 3.3. Seasonal measurements of GST activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100 3.4. LP concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
* Corresponding author. E-mail address: [email protected] (Z. Mejdoub). http://dx.doi.org/10.1016/j.ocecoaman.2016.11.018 0964-5691/© 2016 Elsevier Ltd. All rights reserved.
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Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102 Competing interests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
1. Introduction The intensive anthropogenic activities causes the accumulation of different xenobiotics in the aquatic environment. Nowadays, the major concerns of scientists and environmental managers are the evaluation of their influences that can be exerted on the environment and its resources. Admittedly, chemical analyses of the various environment compartments (water, soil, sediment) inform about the presence of a contaminant and about its biogeochemical cycle. Nevertheless, this information is insufficient to know the real impact of toxic substances on organisms (Michel, 1993; Livingstone, 2001). One-time measures have little meaning because of the temporal variability of the medium. Many studies have been developed for the use of biochemical, physiological and histological changes to assess the organism's exposure to contaminants. They proposed to highlight the concept of biomarkers as a good environmental monitoring tool (Stegeman et al., 1992; Calow, 1993; Valavanidis et al., 2006). Actually, biochemical indicators could be the early warning systems for contamination, which effects are still reversible. However, they cannot be used as the only approach to detect or predict a risk of contamination. Therefore, numerous additional markers are needed to assess the biological impact of contaminants. (Lafaurie et al., 1992; Lagadic et al., 1997). The complementarities between the chemical and biochemical analysis, allows a full diagnosis of the source and the impact of contamination on an ecosystem during a lapse of time (Michel, 1993). These biomonitoring techniques employ the ability of some species to concentrate a large range of chemical contaminants in their tissues (Phillips, 1976; Parant, 1998). Marine organisms, such as fish and invertebrate, have been successfully used as sentinel organisms and sensitive bioindicators for pollutants (Viarengo, 1989; Sheehan and Power, 1999; Corsi et al., 2003; Frenzilli et al., 2004). Although, many pollutants are known to enhance the reactive oxygen species (ROS) production, disturbing organism's redox status (Gomez-Mendikute and Cajaraville, 2003; Livingstone, 2001; Lushchak, 2011). Recently, there has been an increasing interest in studies of oxidative toxicity in aquatic organisms by different pollutants (Livingstone, 1998; Orbea et al., 2002; Lushchak, 2011). Especially in bivalves because of their range of changes in enzymatic antioxidant defences after exposure to pollutants with oxidative potential (Regoli et al., 2002). The cellular antioxidant system is an important element for free radical process monitoring (Santovito et al., 2005). They play a crucial role to maintain the cellular balance between prooxidant challenge and antioxydant defences, providing a cellular homeostasis (Winston, and Di Giulio, 1991; Livingstone, 2001; Valavanidis et al., 2006). Their inductions counteract oxidative stress, but prolonged exposure causes their depletion, leading to disruption of core metabolic and regulatory process, enhancing oxidative damage to biomolecules, such as lipid peroxidation, protein and DNA damage (Bebianno et al., 2005; Lushchak, 2011). Currently, this multibiomarker approach in invertebrates has been supported by international conventions like ICES, OSPAR. Also governmental institutions such as French Research Institute for Exploitation of the Sea (IFREMER) and United Nations Environment Programme (UNEP) recommend this tool for pollution monitoring studies (Sole et al., 2009). Therefore, the present study use essentially the mussel
M. galloprovincialis from Casablanca coast as a bioindicators of emissaries contamination in various coastal ecosystems which are influenced by anthropogenic activities. Casablanca coast is threatened by strong demographic and economic pressures that are increasingly growing that impacts coastal environments. The population estimated at more than 5 million habitants which represent a demographic concentration of more than 21.65% of the Moroccan population (Sbai, 2001). Therefore, anthropogenic pollution has led to an adverse effect on coastal environments and as a result in human health. In fact, one of the major problems is the direct discharge of wastewater into marine environment, without any treatment in most cases. In fact, 80% of the Grand Casablanca industry discharge their effluents directly into the sea via 7 collectors (Menioui, 2007). There are all kinds of industries with high polluting potential such as chemistry and chemicals, food processing, mechanical engineering and metallurgy, and finally the textile and leather. Many published works reported that Casablanca collector's are also responsible for an intense polluting load, especially an organic matter, suspended material, nutrients, and toxic metals (Bouthir et al., 2004; Benbrahim et al., 2006). Which raised the concern about the impact of these discharges on the coastal ecosystems. This first study of systematic biomonitoring, using indigenous mussels M. galloprovincialis as bioindicators and the seasonal variations of GST and CAT activities and LP concentrations as biomarkers in four polluted sites of Casablanca coast, was performed in order to assess emissary's pollution effects on marine organisms. Such a monitoring approach is able to highlight the relationship between the presence of chemical contaminants in the environment and biological responses. Alternatively, these stress indicators sensitive to pollutants in benthic populations would be able to provide additional information about ecosystem state. 2. Materials & methods 2.1. Site description At the beginning of the study, we investigated various locations in Casablanca coast, aiming for indigenous mussels and seasonal availability for their collection. Mussels from wild populations were collected at four following localities within the Casablanca coast ^ta beach, Casablanca's upstream part, with (Fig. 1): S1 (Grande Zena a geographical position of NR 33 390 06.7”/W 007 280 48.300 ), S2 (Saada beach, 3 km south of the first station, to a geographical position N 33 370 23,4 "/W 007 310 '53,200 ), S3 (ONE seaside, 8 km south of the second station with a geographical position N 33 360 27,9 "/W 007 340 28,300 ), S4 (Sidi Abderrahmane beach, 17 km south of the third site, with a geographic position of N 33 340 52,1 "/W 007 420 41,400 ). The site S1 is from a beach with strong hydrodynamics extending on a rocky reef. It is located next to the discharge point of a petroleum refinery, and considered an area with moderate pollution. While, S2 and S3 sites are from an urban area, showing strong industrial activity. They were especially threatened by the presence of an effluent collectors opening directly on the sea, and carrying multicolour wastewater. Many of these hazardous substances are scarcely degradable. Hydrobionts develop a number of physiological mechanisms for their adaptation
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Fig. 1. Study area and sampling stations in Casablanca coast (Morocco).
to these surroundings. The site S4 was located within an urban area, presenting domestic pollution both in the water and in sediment. The control site was located in a small touristic city far-off any source of pollution (beach located in the area of Skhirat city, in the Moroccan Atlantic coast, with a geographic position of N 33 510 3600 et W 7 030 0000 ).
wastewater, with a WTW (Wissenschaft lich Technische Werkst€ atten, Weilheim, Germany) multilab system. Immediately, the different samples collected were transported in insulated coolers to the laboratory. 2.3. Trace metal concentrations in surface seawater and wastewater
2.2. Sampling and sample preservation All samples were collected at low tide in one day during the year 2011e2012. From the intertidal zone, about 150 indigenous mussels M. galloprovincialis clinging to rocks were taken from each of the studied sites, and placed in sterile plastic bags. They were collected in the spring (AprileMay), summer (JulyeSeptember), autumn (OctobereNovember) and winter (DecembereJanuary). They were characterized by similar maximum length: 55 ± 10 mm shell length. Simultaneously, seawater and wastewater samples was collected using polythene bottles (1 L). The were sampled directly in the coast while wastewater samples are taken upstream of the collector in the flow area of the sewer, where the water movement is most active, and a few centimetres below the surface. Environmental parameters including water temperature, conductivity, and pH were measured in situ at all locations for sea water and
Metals concentrations were measured simultaneously in seawater and wastewater. Samples volume was preserved by the addition of 2 ml concentrated H2SO4, in order to avoid precipitation. Then, samples were stored in an ice cooler, and transported to the laboratory. Analysis of trace metals was performed by ICP-AES (Inductively coupled plasma atomic emission spectrometry) at the technical unit support to scientific research (UATRS) of the National Center of Scientific Researches and Techniques (CNRST) in Rabat, Morocco. 2.4. Enzymatic biomarkers and total protein quantification Every season of the study, the sampled mussels were collected at a rate of thirty animals per station, 15 for the determination of protein concentration and 15 for biomarkers analysis. Immediately,
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gills, hepatopancreas and muscles were dissected on ice. The soft various tissues were weighed and then homogenized (using a potter homogeniser) in 1:5 w/v homogenization buffer (0,05 phosphate buffer, pH 7.4). The homogenate was centrifuged twice at 10,000 g at 4 C for 20 min. The supernatants were diluted 1:5 v/ v with phosphate buffer, saved in aliquots and kept at 20 C until enzymatic analysis. Before each enzyme assay, all prepared samples were assayed in amount of protein according to the Bradford (1976) method, using the Coomassie blue reagent and bovine serum albumin (BSA Sigma) as a standard to normalize all biochemical results. Catalase activity (CAT, EC 1.11.1.6) was assayed following the method of hydrogen peroxide consumption at 240 nm (Aebi, 1984; Greenwald, 1985). The reaction mixture consisted of 0.05 M phosphate buffer (pH 7.0), and H2O2 (0.036% w/w, immediately prepared before used). CAT activity was evaluated by kinetic measurement at 25 C and results was expressed as mmoles hydrogen peroxide transformed per minute and per milligram of proteins. Glutathione S transferase activity (GST, EC 2.5.1.18) was measured toward the Habig method, using glutathione (GSH) and 1-chloro-2,4-dinitrobenzene (CDNB) as substrate (Habig et al., 1974). The reaction rate was recorded at 340 nm, and enzyme activity expressed as nmol CDNB conjugate formed per minute per milligram of proteins, using a molar extinction coefficient of 9.6 mM-1 cm-1. All chemicals were products of Sigma.
Table 1 Seawater and wastewater physico-chimical parameter's means in the studied sites (S1, S2, S3, S4) of the Casablanca coast and control (C). Stations
Seawater
Wastewater a
Physico-chimical parameters
S1 S2 S3 S4 C S2 S3
Temperature ( C)
pH
Conductivity (ms/cm)
17.5 (13.1e25) 18.1 (15.3e25) 19.02 (15.8e25) 20.6 (16.3e26.8) 17 (15.7e20) 19.5 (17.4e25) 23a (21.5e25)
8,2 (7.9e8,9) 8.1 (8.01e8.3) 7.7 (7.3e8.4) 8.4 (8.3e8.6) 8 (7.9e8.1) 7.6 (7.6e7.7) 5.8a (5.6e6.3)
54.6 (54.8e55.2) 53.1 (52.2e53.4) 53.4 (52.7e55) 53.2 (53.1e53.3) 54.2 (54e54.5) 21.5a (16.7e35.2) 5 (3.7e7.7)
Parameters exceeding the limit values of effluent's standard norms.
2.5. Lipid peroxidation concentration Enzymatic activities and MDA were determined with a BioMate 3 UV spectrophotometer at 37 C. MDA concentration and all enzyme activities were measured in triplicate for each sample. For the determination of lipid peroxidation (LP), we measured Malondialdehyde (MDA) by the TBARs method (Samokyszyn and Marnett, 1990; Buege and Aust, 1978). This method is based on the property of certain compounds, in particular MDA, to react with thiobarbituric acid and regenerate a pink chromophore absorbing at 535 nm. Thus, 1 ml of the heat denatured supernatant is added to 1 ml of the reaction mixture in 1:1:1 [thiobarbituric acid (0.375%) and trichloroacetic acid (15%) in hydrochloric acid (0.25 N)]. The LP concentrations unit were expressed as nmoles of MDA per milligram of proteins. 2.6. Statistical analysis Data are expressed as mean ± standard deviation (SD) of five independent experiments, each performed in triplicate. One-way analysis of variance ANOVA and Tukey Kramer's test were used for testing differences among sites. In the figures, all experimental groups were compared to the control. Statistical comparisons were made assuming homogeneity of variance. Values of *p < 0.05 were considered significant. All analyses were performed by Excel software 2013 for Windows. 3. Results
Fig. 2. Seasonal variations of (a) temperature ( C), (b) pH and (c) conductivity (ms/cm) of seawater and wastewater along the study area in the Casablanca coast.
Generally, the sea water analysis reveal some stressful environmental conditions for aquatic organisms at the four sampling sites of Casablanca coast. The physico-chimical parameter's values vary with sensible range between stations of sampling. Even if, the S3 wastewaters expressed an acidic pH value, and showed a strong mineralization in discharges. The pH and conductivity of seawater remained characteristic of the marine environment at all locations (Table 1). However, seasonal variations of seawater temperature were significant. The highest mean value (26.8 C) was recorded in
summer period, whereas the lowest value (13.1 C) was recorded in autumn (Fig. 2). The wastewater collected at the two stations S2 and S3 record the highest temperature values, with a maximum value of about 23.3 C at the station S3 (Table 1). Still, these values remain below 30 C and complying with the effluents discharge standards.
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3.1. Trace metal concentrations in surface seawater (Casablanca coast) Heavy metals analysis showed that lead, mercury and copper were at high concentrations at the 4 selected locations of Casablanca Coast during all sampling campaigns (Fig. 3). Regarding the sites variation, the highest levels for all metals were recorded at S3 and S2 in the center parts of the Casablanca Coast (Table 2). Those high amounts are mainly related to emissaries that discharge sewages from the industrial zone. While, S4 and S1 were placed in slight industrial activity, those stations showed considerable levels of heavy metals. 3.2. Seasonal measurements of CAT activity
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Table 2 Indicative mean values of the traces metal's concentrations (mg/l) in seawater and wastewater of four area (S1, S2, S3, S4) of the Casablanca coast and control (C). Stations
Heavy metal concentrations (mg/l) Hg
Seawater
Wastewater
S1 S2 S3 S4 C S2 S3
a
0.6 (0.04e1.02) 0.5a (0.3e0.67) 1.5a (0.85e2.2) 0.3a (0.03e0.7) 0.001 0.55a (0.4e0.7) 1.04a (0.5e1.6)
Pb
Cu
0.2 (0.03e0.24) 0.19 (0.07e0.23) 0.22 (0.15e0.3) 0.22 (0.15e0.3) 0.03 0.2 (0.16e0.24) 0.3 (0.3e0.4)
0.03 (0.02e0.04) 0.02 (0.01e0.03) 0.02 (0.015e0.025) 0.035 (0.03e0.04) 0.007 0.025 (0.02e0.03) 0.03 (0.03e0.04)
a Concentrations exceeding the limit values by standards norms in seawater and effluents.
The summary of results for seasonal CAT activity in mussels from the different sites was presented in Fig. 4. The levels of CAT activity in the three organs showed considerable differences among
Fig. 4. Seasonal variations of catalase (CAT) activity in gills (A), Hepatopancreas (B), and muscles (C) of mussels Mytilus galloprovincialis from the sampling sites S1, S2, S3, S4 and Control. Mean values ± SD. (ANOVA, Tukey HSD, *p < 0.05).
Fig. 3. Concentrations of heavy metals (Hg, Pb and Cu) in seawater (S1, S2, S3, S4 and C) and wastewater samples (S20 and S30 ).
sites. Mussel's hepatopancreas expressed a significant CAT activity relative to the control mussels, in all sampling sites. The maximum value (289 mmol/min/mg of proteins) reached in mussels from the
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most polluted site S3 at spring. Whereas, mussel's gills from polluted sites (S2 and S3) showed a two-fold decrease of CAT activity, compared to other sites: S1 > S4 > C > S2 > S3 in all the seasons (p < 0.05, Fig. 4a). In addition, only mussel's muscles from S4 showed similar CAT activity to control. When, mussels from S1 recorded significant level of CAT activities in the three organs (223 mmol/min/mg of protein in gills, 164 mmol/min/mg of protein in hepatopancreas, 75 mmol/min/mg of protein in muscles). 3.3. Seasonal measurements of GST activity Measurements of seasonal GST activity in mussels in the different sites was presented in Fig. 5. GST did not show seasonal variations and presented the highest values in mussels from the most polluted areas. Mussels from contaminated sites (S2 and S3) has significantly the highest GST levels in all organs tested, when compared to reference site: S3 > S2 > S1 > S4 > C in all seasons.
Fig. 5. Seasonal variations of glutathione S-transferase (GST) activity in gills (A), Hepatopancreas (B), and muscles (C) of mussels Mytilus galloprovincialis from the sampling sites S1, S2, S3, S4 and Control. Mean values ± SD. (ANOVA, Tukey HSD,*p < 0.05).
Thus, the several-fold higher GST activity in S3 mussel's could show that these animals are under higher pollution stress conditions (Fig. 3). The assessment of GST activity after exposure to certain pollutants has already been demonstrated both in fish and in invertebrates (Fitzpatrick et al., 1997; Cheung et al., 2001; Gowland et al., 2002). Our findings were consistent with other studies, which also showed higher GST activity in organisms from polluted sites when compared to those from reference sites (Bainy et al., 2000; Lau and Wong, 2003; Manduzio et al., 2004; Saenz et al., 2010). 3.4. LP concentration Seasonal measurements of LP concentration in the three organs in mussels are presented in Fig. 6. LP represent the most type of cell damage widely studied. The levels of MDA reflect the state of peroxidation of cell membranes and the impairment of antioxidant
Fig. 6. Seasonal variations of catalase (MDA) activity in gills (A), Hepatopancreas (B), and muscles (C) of mussels Mytilus galloprovincialis from the sampling sites S1, S2, S3, S4 and Control. Mean values ± SD. (ANOVA, Tukey HSD, *p < 0.05).
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defense systems to remove excess reactive oxygen species (Nzengue, 2008). LP concentration showed a consistent trend for upper levels in the polluted sites S2 and S3, where smaller increases were detected in the S1 site. By comparison, the control mussels had much lower levels of MDA, as expected. Gills and hepatopancreas showed a 2 fold increase in concentration of LP compared to mussels' muscles. The LP concentrations varied from 0,45 to 3,7 nmol g-1 w. w.t (Gills and hepatopancreas) and 0,35e2,4 nmol g-1 w. w.t (Muscles), compared to 0,15e0,55 (Gills and hepatopancreas) and 0,15e0,4 (Muscles) from the control. Our result were similar with other studies, where mussels exposed to polluted areas express an increased concentration of LP, comparing to less polluted sites (Lau and Wong, 2003; Pampanin et al., 2005). The higher MDA accumulation (3,8 nmol/mg protein) was obtained in gill's mussels from S2 during spring period. 4. Discussion The influence of environmental abiotic factors on various biological activities has been reported in many published works. Particularly, the clout on oxidative processes in aquatic ecosystems has been shown (Parihar et al., 1997; Robillard et al., 2003; Borkovic et al., 2005). Many studies have shown that increasing temperature also increases the basal cell metabolism inducing oxidative stress in the species studied (Bocchetti et al., 2008; Verlecar et al., 2007). Thus, the high increases of seawater temperature could be a stressful conditions for mussels from industrial sites (S2 and S3)in our study. In addition, the contamination of the coastal ecosystem by heavy metals represent the major crisis in environmental toxicology. Especially, some traces metals is not biodegradable and they can be accumulated by various aquatic organisms with toxic levels. They are well known to induce oxidative stress by their stimulation of ROS generation. Several published works reported that the stimulus of the ROS production and oxidative damage resulting, can be a significant mechanism of toxicity to aquatic organisms subjected to metallic pollution (Regoli and Principato, 1995; Livingstone, 2003; Lushchak, 2011). Accordingly, the high concentrations of non-essential metals (as Hg and Pb) detected in seawater samples above natural charges, could be consequential to oxidative stress in mussels of this aquatic ecosystems. Contamination by these elements is attributed mainly to industrial liquid waste, denoting an important input in the receiving environment. Similar results were obtained by Bouthir et al. (2004) in the two sites (S2 and S3), which recorded the highest urban and industrial concentration of the country. Moreover, the S1 station recorded the highest values of Hg. This is presumably due to the direct discharges to the sea from a refining industry located upstream of this station. Especially, as coastal ocean currents have N-S orientation. Also, the “S4” station showed considerable levels of these heavy metals while is placed in slight industrial activity. This contamination is most probably due to liquid discharges from a company located upstream of this region, that uses mercury cathodes for the production of chlorine and sodium hydroxide. The biological response of indigenous mussels Mytilus galloprovincialis to acute emissaries pollution is confirmed from the measurements of CAT and GST activities, even if the cause-effect relationship between the oxy-radical metabolism and cellular damage is difficult to highlight. Many scientist consider CAT as a biomarker need to be monitored in case of oxidative contaminants. CAT is the primary antioxidant defense involved in detoxification of H2O2, which is the major cellular precursor of the toxic hydroxyl radical (HO-). Particularly, numerous study suggested that CAT is sensitive to some oxidative stress-inducing contaminants in cell membranes, as metals (Labrot et al., 1996). Recent study revealed
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that goldfish C. auratus expressed a reduced catalase activity after 40 day exposure to 0.005e0.025 mg/l copper (Liu et al., 2006). Recently, publication on the mercury effects on free radical processes found that mercury exposure causes depletion of antioxidant in fish Liza aurata. The activities of catalase, glutathione peroxidase, and glutathione reductase significantly decreased (Mieiro et al., 2010). Several studies consider the inhibition of catalase as a temporary response to severe pollution (Regoli and Principato, 1995). Our data for reduced CAT activity in gills of M. galloprovincialis compared to control values, is presumably due to increased levels of pollution, indicating the critical pollution level in two sites (S2 and S3). While, the increased values of CAT activity in mussels from S1 and S4, indicating that pollution levels are relative moderate. This organisms are facing an oxidative stress, possibly associated with the presence of heavy metals (Hg, Pb and Cu) in the environment. Furthermore, the major tissue of xenobiotics uptake as hepatopancreas expressed the highest GST activity that is rather related to detoxification, especially in mussels from S2 and S3. Actually, the glutathione S-transferase family (GST) belongs to the Phase II detoxification enzymes, and plays an important role in the combination of a wide variety of substances with glutathione to facilitate their solubility and excretion (Livingstone, 1998). This result explain the low CAT activities obtained in mussel's gills from those sites. GST proved more useful for pollution monitoring compared to alternative biomarkers, and several authors have proposed this enzyme as biomarkers of exposure to pollution. GST activities were found to be modulated by metals or organic contaminants under both field conditions (Durou et al., 2007) and laboratory exposure (Livingstone, 1991). Moreover, the induction of GST can be regarded ~a n as an adaptive response to an altered environment (Vidal-Lin et al., 2010a). Our results for GST activity presented the most uniform pattern. The significant levels of GST activities in mussels could be a consequences of the environmental contamination by heavy metals detected in the seawater. Similar results were presented in a study of petrochemical contamination in M. galloprovincialis. While, high concentrations of coastal waters with heavy metals can cause poisoning, leading to oxidative stress conditions found in individuals of these aquatic ecosystems (Lima et al., 2007). Many authors note that lipid peroxidation appears to be modulated by chemical and organic contaminants tested in laboratory conditions or in situ (Narbonne et al., 2005). Also, a significant induction of MDA in different clam tissues exposed to cadmium solutions has been reported by Khebbeb et al. (2010). Also, the induction of MDA has been noted in clams Donax trunculus taken from a polluted site in Gulf of Annaba in eastern Algeria (Soltani et al., 2012). Actually, the corresponding results of high LP concentrations explain the low rate of CAT activities recorded in mussels from the both releases stations, “S200 and “S300 in which all stress conditions are met. We suggest that this tissues shows a strong alteration of antioxidant defense system, marked by high production of reactive oxygen species. Some studies reported that biomarker generates a primary response to a limited adaptation, after an exposure to a pollutant. However, an alteration of the detoxification system will be observed in case of stress persists, leading to uncompensated damage (Livingstone, 2003; Vasseur and Leguille, 2004). In fact, the cooperation from antioxidant enzymes is essential for the scavenging of ROS. The low activity of antioxidant enzymes such as CAT, will increase the content of superoxide radical and H2O2. They will reacting together to produce hydroxyl radical that can attacks all biomolecules and disturbs cell metabolism (Esfandiari et al., 2007; Shao et al., 2005). The antioxidant defense enzyme induced were not enough to reduce lipid peroxidation levels in mussels from those polluted sites, whereas
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inhibition of CAT has been suggested as a transitory response to acute pollution (Regoli and Principato, 1995; Pampanin et al., 2005). Many studies confirmed this negative relationship between CAT activity and MDA. Similar results were presented by Seonock et al. (2013) in mussels Mytilus galloprovincialis. They revealed significant differences in CAT, GST and LP activities. However, only CAT activity negatively correlated with relevant stress. They suggested that reduction in CAT activity caused redox imbalance, which was sufficient to induce further oxidative stress that led to increased activities of GST and LP in mussels. Indeed, emissaries are responsible for the contaminations of aquatic environment, by many chemicals that have been shown to be pro-oxidants in aquatic organisms such as metals (Phillips, 1976; Livingstone, 2003; Nzengue, 2008). And, they are easily taken up from the sediment, water-column and food into the tissues of resident organisms. The raw sewerage implantations causes metabolic disturbances occurring into indigenes mussels Mytilus galloprovincialis from the coastal environment of Casablanca. This agents responsible for oxidative stress in marine organisms, caused changes that can range from the molecular level (metabolism) to the whole organism scale (population dynamics). An effective management system need to be establish ensuring the protection of this aquatic environment. 5. Conclusions Our research, regardless of its restrictions, provides an example of antioxidant multibiomarkers potentiality, emphasizing the difficulties related to the integration of ecotoxicological approach with in situ biomonitoring studies. Significant biochemical changes occurred in gills, hepatopancreas and muscles tissues. Those biomarker responses could be allocated to differences in both pollution levels and seasonal variability. The evaluation of oxidative stress in Mytilus galloprovincialis species revealed promising results in accordance with the availability of contaminations (heavy metals) and environmental stress conditions at each station. From a methodological standpoint, this work affords another effectiveness evidence of the use of multibiomarker approach in mussels for biomonitoring study. The use of mussel gills and hepatopancreas as bioindicators tissues has been well-developed revealing site-effects. Our results were in similar trend with other published work that revealing them as organs of choice for pollution biomonitoring (Manduzio et al., 2004; Lima et al., 2007). However, biomarkers showed significant levels in mussel muscles from the most polluted areas (S2 and S3), whereas trivial levels in the less polluted site (S4), when compared to control. This fact suggest that muscles seems to be a responsive organ in case of acute pollution. Competing interests The authors declare that they have no competing interests. References Aebi, H., 1984. Catalase in vivo. Methods Enzymol. 105, 121e126. Bainy, A.C.D., Almeida, E.A., Muller, I.C., et al., 2000. Biochemical responses in farmed mussel Perna perna transplanted to contaminated sites on Santa Catarina Island, SC. Braz. Mar. Environ. Res. 50, 411e416. Bebianno, M.J., Company, R., Serafim, A., Cosson, R.P., Fiala-Medoni, A., 2005. Antioxidant systems and lipid peroxidation in Bathymodiolusazoricus from midAtlantic ridge hydrothermal vent fields. Aquat. Toxicol. 75, 354e373. Benbrahim, S., Chafik, A., Chfiri, R., Bouthir, F.Z., Siefeddine, M., Makaoui, A., 2006. Survey of the carriers influencing the geographical and temporal distribution of contamination by heavy metals along the Atlantic Moroccan coasts: the case of mercury, lead and cadmium. Mar. Life 16 (1e2), 37e47. Bocchetti, R., Lamberti, C.V., Pisanelli, B., Razzetti, E.M., Maggi, C., Catalano, B., Sesta, G., Martuccio, G., Gabellini, M., Regoli, F., 2008. Seasonal variations of
exposure biomarkers, oxidative stress responses and cell damage in the clams. Tapes philippinarum, and mussels, Mytilus galloprovincialis, from Adriatic sea. Mar. Environ. Res. 66, 24e26. Borkovic, S.S.S., aponjic, J.S., Pavlovic, S.Z., Blagojevic, D.P., Milosevic, S.M., Kovacevic, T.B., Radojicic, R.M., Spasic, M.B., Zikic, R.V., Saicic, Z.S., 2005. The activity of antioxidant defence enzymes in the mussel Mytilus galloprovincialis from the Adriatic Sea. Comp. Biochem. Physiol. C Toxicol. Pharmacol. 141, 366e374. Bouthir, F.Z., Chafik, A., Benbrahim, S., Souabi, S., El Merdhy, H., Messoudi, A., physico-chimique des eaux co ^ tie res du littoral de la Sifeddine, M., 2004. Qualite an Atlantique marocain) utilisant la moule Wilaya du grand Casablanca (oce tallique, Mytilus galloprovincialis comme indicateur de la contamination me 2004 Mar. Life 14, 1e2, 59-70. Bradford, M.M., 1976. A rapid and sensitive method for the quantification of microgram quantited of protein utilizing the principe of protein-dye binding. Anal. Biochem. 72, 248e254. Buege, J.A., Aust, S.D., 1978. Microsomal lipid peroxidation. Methods Enzymol. 52, 302e310. Calow, P., 1993. General principles and overview. In: Calow, P. (Ed.), Handbook of Ecotoxicology, 1, pp. 1e5. Cheung, C.C.C., Zheng, G.J., Li, A.M.Y., et al., 2001. Relationships between tissue concentrations of polycyclic aromatic hydrocarbons and oxidative responses of marine mussels, perna viridis. Aquat. Toxicol. 52, 189e203. Corsi, I., Mariottini, M., Sensini, C., Lancini, L., Focardi, S., 2003. Fish as bioindicators of brackish ecosystem health: integrating biomarker responses and target pollutant concentrations. Oceanol. Acta 26, 129e138. Durou, C., Poirier, L., Amiard, J.C., Budzinski, H., Gnassia-Barelli, M., Lemenach, K., Peluhet, L., Mouneyrac, C., Romeo, M., Amiard-Triquet, C., 2007. Environ. Pollut. 148, 445e458. Esfandiari, E., Shakiba, M.R., Mahboob, S., Alyari, H., Toorchi, M., 2007. Water stress, antioxidant enzyme activity and lipid peroxidation in wheat seedling. J. Food, Agric. Environ. 5, 149e153. €rlin, L., Bolognesi, C., Sturve, J., Frenzilli, G., Scarcelli, V., Del Barga, I., Nigro, M., Fo € teborg harbour. 2004. DNA damage in eelpout (Zoarces viviparus) from Go Mutat. Res. 552, 187e195. Fitzpatrick, P.J., O'Hallaran, J., Sheehan, D., Walsh, A.R., 1997. Biomarkers 2, 51e56. Gomez-Mendikute, A., Cajaraville, M.P., 2003. Comparative effects of cadmium, copper, paraquat and benzo[a]pyrene on the actin cytoskeleton and production of reactive oxygen species (ROS) in mussel haemocytes. Toxicol. Vitro 17, 539e546. Gowland, B.T., McIntosh, A.D., Davies, I.M., Moffat, C.F., Webster, L., 2002. Implications from a field study regarding the relationship between polycyclic aromatic hydrocarbons and glutathione S-transferase activity in mussels. Mar. Environ. Res. 54, 231e235. Greenwald, R.A., 1985. Handbook of Methods for Oxygen Radicals in Biology and Medicine. Oxford University Press, New York. Habig, W.H., Pabst, M.J., Jakoby, W.B., 1974. Glutathione S-transferase. J. Biol. Chem. 249, 7130e7139. Khebbeb, M.E.H., Nadji, S., Amrani, A., 2010. The effect of cadmium exposure on malonedialdehyde and reduced glutathione concentrations in several tissues of a bivalve mollusc (Ruditapes decussatus) fished from Mellah lagoon (North East of Algeria). Ann. Biol. Res. 1, 166e173. Labrot, F., Ribera, D., Saint-Denis, M., Narbonne, J.F., 1996. In vitro and in vivo studies of potential biomarkers of lead and uranium contamination: lipid peroxidation, acetylcolinesterase, catalase and glutathione peroxidase activities in three nonmammalian species. Biomarkers 1, 21e28. Lafaurie, M., Narbonne, J.F., Galgani, F., 1992. Indicateurs biochimiques de contamination de l'environnement marin. Anal. Mag. 20 (6), 27e33. cotoxicologie : principes Lagadic, L., Caquet, T., Amiard, J.C., 1997. Biomarqueurs en e finitions. In: Lagadic, L., Caquet, T., Amiard, J.C., Masson, Ramade F. (Eds.), et de cotoxicologie, aspects fondamentaux, pp. 1e9. in Biomarqueurs en e Lau, P.S., Wong, H.L., 2003. Effect of size, tissue parts, and location on six biochemical markers in the green-lipped mussel, perna viridis. Mar. Poll. Bull. 46, 1563e1572. Lima, I., Moreira, S.M., Rendon-Von Osten, J., et al., 2007. Biochemical responses of the marine mussel Mytilus galloprovincialis to petrochemical environmental contamination along the northwestern coast of Portugal. Chemosphere 66, 1230e1242. Liu, H., Wang, W., Zhang, J.F., Wang, X.R., 2006. Effects of copper and its ethylenediaminetetraacetate complex on the antioxidant defenses of the goldfish, Carassius auratus. Ecotoxicol. Environ. Saf. 65, 350e354. Livingstone, D.R., 1991. Adv. Comp. Environ. Physiology 7, 145e187. Livingstone, D.R., 1998. Organic xenobiotics in aquatic ecosystems: quantitative and qualitative differences in biotransformation by invertebrates and fish. Comp. Environ. Physiol. 120A, 43e49. Livingstone, D.R., 2001. Contaminant-stimulated reactive oxygen species production and oxidative damage in aquatic organisms. Mar. Pollut. Bull. 42, 656e666. Livingstone, D.R., 2003. Oxidative stress in aquatic organisms in relation to pollud. Ve t 154 (6), 427e430. tion and aquaculture. Rev. Me Lushchak, V.I., 2011. Environmentally induced oxidative stress in aquatic animals. Aquat. Toxicol. 101, 13e30. Manduzio, H., Monsinjon, T., Galap, C., Leboulenger, F., Rocher, B., 2004. Seasonal variation in antioxydant defences in blue mussels Mytilus edulis collected from a polluted aera: major contributions in gills of an inducible isoform of Cu/Znsuperoxide dismutase and of glutathione-S transferase. Aquat. Toxicol. 70,
Z. Mejdoub et al. / Ocean & Coastal Management 136 (2017) 95e103 83e93. ation d’un cadre politique favorisant Menioui, M., 2007. Sensibilisation et cre gration de l’environnement et du de veloppement avec l’accent sur la l’inte gre e des zones co ^ tie res. Royaume du Maroc. Ministe re de gestion inte nagement du Territoire, de l’Eau et de l’Environnement. Direction de la l’Ame vention des Risques. Surveillance et de la Pre l'e tude des interactions entre les contaminants Michel, X., 1993. Contribution a culaires et applichimiques organiques et les organismes marins : bases mole la biosurveillance de l'environnement co ^ tier. The se. Universite cations a Bordeaux 1. Mieiro, C.L., Ahmad, I., Pereira, M.E., Duarte, A.C., Pacheco, M., 2010. Antioxidant system breakdown in brain of feral golden grey mullet (Liza aurata) as an effect of mercury exposure. Ecotoxicology 19 (6), 1034e1045. Narbonne, J.F., Aarab, N., Clerandeau, C., et al., 2005. Scale of classification based on biochemical markers in mussels: application to pollution monitoring in Mediterranean coasts and temporal trends. Biomarkers 10 (1), 58e71. canismes de toxicite redox du cadmium, du Nzengue, Y., 2008. Comparaison des me cuivre et du zinc : place des metallothioneines et de p53. Life Sciences [q-bio. University Joseph-Fourier -Grenoble I. Orbea, A., Ortiz-Zarragoitia, M., Sole, M., Porte, C., Cajaraville, M.P., 2002. Antioxidant enzymes and peroxisome proliferation in relation to contaminant body burdens of PAHs and PCBs in bivalve molluscs, crabs and fish from the Urdaibai and Plentzia estuaries (Bay of Biscay). Aquat. Toxicol. 58, 75e98. Pampanin, D.M., Camus, L., Gomiero, A., Marangon, I., Volpato, E., Nasci, C., 2005. Susceptibility to oxidative stress of mussels (Mytilus galloprovincialis) in the Venice Lagoon (Italy). Mar. Pollut. Bull. 50, 1548e1557. tabolisme des xe nobiotiques chez les bivalves. C.R. Acade mie Parant, M., 1998. Le me Sci. Paris, Sci. vie 321, 485e494. Parihar, M.S., Javeri, T., Hemnani, T., Dubey, A.K., Prakash, P., 1997. Responses of superoxide dismutase, glutathione peroxidase and reduced glutathione antioxidant defenses in gills of the freshwater catfish (Heteropneustes fossilis) to short-term elevated temperature. J. Therm. Biol. 22 (2), 151e156. Phillips, D.J.H., 1976. The common mussel Mytilus edulis as an indicator of pollution by zinc, cadmium, lead and copper. Effect of environmental variables on uptake of metals. Mar. Biol. 38, 59e69. Regoli, F., Principato, G., 1995. Glutathione, glutathione-dependent and antioxidant enzymes in mussels, Mytilus galloprovincialis, exposed to metal under field and laboratory conditions: implications for the use of biochemical biomarkers. Aquat. Toxicol. 31, 143e164. Regoli, F., Gorbi, S., Frenzilli, G., Nigro, M., et al., 2002. Oxidative stress in ecotoxicology: from the analysis of individual antioxidants to a more integrated approach. Mar. Environ. Res. 54, 419e423. Robillard, S., Beauchamp, G., Laulier, M., 2003. The role of abiotic factors and pesticide levels on enzymatic activity in the freshwater mussel Anodonta cygnea at three different exposure sites. Comp. Biochem. Physiology Part C 135, 49e59. Saenz, L.A., Seibert, E.L., Zanette, J., et al., 2010. Biochemical biomarkers and metals
103
in Perna perna mussels from mariculture zones of Santa Catarina, Brazil. Ecotoxicol. Environ. Saf. 73, 796e804. Samokyszyn, V.M., Marnett, L.J., 1990. Inhibition of liver microsomal lipid peroxidation by 13-cis-retinoic acid. Free RadicBiol Med. 8, 491e496. Santovito, G., Piccinni, E., Cassino, A., Irato, P., Albergoni, V., 2005. Antioxidant responses of the Mediterranean mussel, Mytilus galloprovincialis, to environmental variability of dissolved oxygen. Comp. Biochem. Physiol. 140, 321e329. ^ tier marocain. ISBN 9981Sbai, L., 2001. Le droit de l’environnement marin et co tat du milieu et une le gislation obsole te, Rabat, 889-28-8. Dichotomie entre l’e Maroc, p. 146. Seonock, W., Vianney, D., Hyokyung, W., 2013. Expressions of oxidative stressrelated genes and antioxidant enzyme activities in Mytilus galloprovincialis (Bivalvia, Mollusca) exposed to hypoxia. Zool. Stud. 52, 15. Shao, H.B., Liang, Z.S., Shao, M.A., 2005. Changes of anti-oxidative enzymes and MDA content under soil water deficits among 10 wheat (Triticum aestivum L.) genotypes at maturation stage. Colloids Surfaces B Biointerfaces 45, 7e13. Sheehan, D., Power, A., 1999. Effects of seasonality on xenobiotic and antioxidant defence mechanisms of bivalve molluscs. Comp. Biochem. Physiology Part C 123, 193e199. , M., Kopecka-Pilarczyk, J., Blasco, J., 2009. Pollution biomarkers in two estuaSole rine invertebrates Nereis diversicolor and Scrobicularia plana, from marsh ecosystem in SW Spain. Environ. Int. 35, 523e531. Soltani, N., Amira, A., Sifi, K., Beldi, H., 2012. Environmental monitoring of the Annaba gulf (Algeria): measurement of biomarkers in Donax trunculus and metallic pollution. Bull.Soc. Zool. Fr. 137, 47e56. Stegeman, J.J., Brouwer, M., Di Guilio, T., Forlin, L., Fowler, B.A., Sanders, B.M., Van Veld, P.A., 1992. Molecular responses to environmental contamination: enzyme and protein systems as indicators of chemical exposure and effect. In: Huggett, R.J., Kimerle, R.A., Mehrle, P.M., Bergman, H.L. (Eds.), Biomarkers : Biochemical, Physiological, and Histological Markers of Anthropogenic Stress. Lewis Publishers, Chelsea, Etats-Unis, pp. 235e335. Valavanidis, A., Vlahogianni, T., Dassenakis, M., et al., 2006. Molecular biomarkers of oxidative stress in aquatic organisms in relation to toxic environmental pollutants. Ecotoxicol. Environ. Saf. 64, 178e189. Vasseur, P., Leguille, C., 2004. Defense systems of benthic invertebrates in response to environmental stressors. Environ. Toxicol. 19 (4), 433e436. Verlecar, X.N., Jena, K.B., Chainy, G.B., 2007. Biochemical markers of oxidative stress in Perna viridis exposed to mercury and temperature. Chem. -Biol. Interact. 167, 219e226. Viarengo, A., 1989. Heavy metals in marine invertebrates: mechanisms of regulation and toxicity at the cellular level. CRC Rev. Aquat. Sci. 1, 295e317. ~a n, L., Bellas, J., Campillo, J.A., et al., 2010. Integrated use of antioxidant Vidal-Lin enzymes in mussels, Mytilus galloprovincialis, for monitoring pollution in highly productive coastal areas of Galicia (NW Spain). Chemosphere 78, 265e272. Winston, G.W., Di Giulio, R.T., 1991. Prooxidant and antioxidant mechanisms in aquatic organisms. Aquat. Toxicol. 19, 137e161.
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Review
Oxidative stress responses of the mussel Mytilus galloprovincialis exposed to emissary's pollution in coastal areas of Casablanca Zineb Mejdoub a, *, Abdelilah Fahde b, Mohammed Loutfi a, Mostafa Kabine a a b
Department of Biology, Laboratory of Biochemistry and Molecular Biology, Faculty of Science Ain Chock, University Hassan II Casablanca, Morocco Department of Biology, Laboratory of Environment and Aquatic Ecology, Faculty of Science Ain Chock, University Hassan II Casablanca, Morocco
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 May 2016 Received in revised form 11 October 2016 Accepted 9 November 2016
The bivalve mollusks are among the aquatic bioindicators that are commonly used in monitoring water pollution studies, thanks to their behavior and metabolism. They are directly affected by the level of pollution in a given site. During this research, the study of the biological response in gills, hepatopancreas and muscles of indigenous mussels Mytilus galloprovincialis were used for monitoring emissary's pollution in four polluted sites in the coastal environment of Casablanca. Seasonal variations of the activity of antioxidant defence enzymes, catalase (CAT), glutathione S-transferase (GST), as well as lipid peroxidation (LP) were measured as biomarkers within a one year period and compared to mussels from an unpolluted sampling site. This study was completed by analysing a series of abiotic factors (temperature, pH and conductivity) and chemicals (heavy metals; Hg, Pb, Cu) into seawater. Our result showed that the availability of metallic contamination and the environmental stress conditions causes relatively an oxidative stress in this species at each station studied. While the pollution's level clearly varies according to the sampling campaign. Furthermore, they revealed a significant increase in GST activities and LP concentrations and significant decrease in CAT activities in mussels collected in sites with industrial contamination. This negative correlation suggested that the organisms at this location are exposed to a relatively higher level of oxidative stress. This first study in the area confirm that variations of antioxidant defence enzymes activities and LP concentrations in mussels could be used as prospective biomarkers of toxicity in environmental monitoring programs. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Biomarkers Oxidative stress Mussels Mytilus galloprovincialis Antioxidant defence enzymes Lipid peroxidation Heavy metals
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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96 Materials & methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96 2.1. Site description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96 2.2. Sampling and sample preservation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97 2.3. Trace metal concentrations in surface seawater and wastewater . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97 2.4. Enzymatic biomarkers and total protein quantification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97 2.5. Lipid peroxidation concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98 2.6. Statistical analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98 3.1. Trace metal concentrations in surface seawater (Casablanca coast) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99 3.2. Seasonal measurements of CAT activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99 3.3. Seasonal measurements of GST activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100 3.4. LP concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
* Corresponding author. E-mail address: [email protected] (Z. Mejdoub). http://dx.doi.org/10.1016/j.ocecoaman.2016.11.018 0964-5691/© 2016 Elsevier Ltd. All rights reserved.
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Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102 Competing interests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
1. Introduction The intensive anthropogenic activities causes the accumulation of different xenobiotics in the aquatic environment. Nowadays, the major concerns of scientists and environmental managers are the evaluation of their influences that can be exerted on the environment and its resources. Admittedly, chemical analyses of the various environment compartments (water, soil, sediment) inform about the presence of a contaminant and about its biogeochemical cycle. Nevertheless, this information is insufficient to know the real impact of toxic substances on organisms (Michel, 1993; Livingstone, 2001). One-time measures have little meaning because of the temporal variability of the medium. Many studies have been developed for the use of biochemical, physiological and histological changes to assess the organism's exposure to contaminants. They proposed to highlight the concept of biomarkers as a good environmental monitoring tool (Stegeman et al., 1992; Calow, 1993; Valavanidis et al., 2006). Actually, biochemical indicators could be the early warning systems for contamination, which effects are still reversible. However, they cannot be used as the only approach to detect or predict a risk of contamination. Therefore, numerous additional markers are needed to assess the biological impact of contaminants. (Lafaurie et al., 1992; Lagadic et al., 1997). The complementarities between the chemical and biochemical analysis, allows a full diagnosis of the source and the impact of contamination on an ecosystem during a lapse of time (Michel, 1993). These biomonitoring techniques employ the ability of some species to concentrate a large range of chemical contaminants in their tissues (Phillips, 1976; Parant, 1998). Marine organisms, such as fish and invertebrate, have been successfully used as sentinel organisms and sensitive bioindicators for pollutants (Viarengo, 1989; Sheehan and Power, 1999; Corsi et al., 2003; Frenzilli et al., 2004). Although, many pollutants are known to enhance the reactive oxygen species (ROS) production, disturbing organism's redox status (Gomez-Mendikute and Cajaraville, 2003; Livingstone, 2001; Lushchak, 2011). Recently, there has been an increasing interest in studies of oxidative toxicity in aquatic organisms by different pollutants (Livingstone, 1998; Orbea et al., 2002; Lushchak, 2011). Especially in bivalves because of their range of changes in enzymatic antioxidant defences after exposure to pollutants with oxidative potential (Regoli et al., 2002). The cellular antioxidant system is an important element for free radical process monitoring (Santovito et al., 2005). They play a crucial role to maintain the cellular balance between prooxidant challenge and antioxydant defences, providing a cellular homeostasis (Winston, and Di Giulio, 1991; Livingstone, 2001; Valavanidis et al., 2006). Their inductions counteract oxidative stress, but prolonged exposure causes their depletion, leading to disruption of core metabolic and regulatory process, enhancing oxidative damage to biomolecules, such as lipid peroxidation, protein and DNA damage (Bebianno et al., 2005; Lushchak, 2011). Currently, this multibiomarker approach in invertebrates has been supported by international conventions like ICES, OSPAR. Also governmental institutions such as French Research Institute for Exploitation of the Sea (IFREMER) and United Nations Environment Programme (UNEP) recommend this tool for pollution monitoring studies (Sole et al., 2009). Therefore, the present study use essentially the mussel
M. galloprovincialis from Casablanca coast as a bioindicators of emissaries contamination in various coastal ecosystems which are influenced by anthropogenic activities. Casablanca coast is threatened by strong demographic and economic pressures that are increasingly growing that impacts coastal environments. The population estimated at more than 5 million habitants which represent a demographic concentration of more than 21.65% of the Moroccan population (Sbai, 2001). Therefore, anthropogenic pollution has led to an adverse effect on coastal environments and as a result in human health. In fact, one of the major problems is the direct discharge of wastewater into marine environment, without any treatment in most cases. In fact, 80% of the Grand Casablanca industry discharge their effluents directly into the sea via 7 collectors (Menioui, 2007). There are all kinds of industries with high polluting potential such as chemistry and chemicals, food processing, mechanical engineering and metallurgy, and finally the textile and leather. Many published works reported that Casablanca collector's are also responsible for an intense polluting load, especially an organic matter, suspended material, nutrients, and toxic metals (Bouthir et al., 2004; Benbrahim et al., 2006). Which raised the concern about the impact of these discharges on the coastal ecosystems. This first study of systematic biomonitoring, using indigenous mussels M. galloprovincialis as bioindicators and the seasonal variations of GST and CAT activities and LP concentrations as biomarkers in four polluted sites of Casablanca coast, was performed in order to assess emissary's pollution effects on marine organisms. Such a monitoring approach is able to highlight the relationship between the presence of chemical contaminants in the environment and biological responses. Alternatively, these stress indicators sensitive to pollutants in benthic populations would be able to provide additional information about ecosystem state. 2. Materials & methods 2.1. Site description At the beginning of the study, we investigated various locations in Casablanca coast, aiming for indigenous mussels and seasonal availability for their collection. Mussels from wild populations were collected at four following localities within the Casablanca coast ^ta beach, Casablanca's upstream part, with (Fig. 1): S1 (Grande Zena a geographical position of NR 33 390 06.7”/W 007 280 48.300 ), S2 (Saada beach, 3 km south of the first station, to a geographical position N 33 370 23,4 "/W 007 310 '53,200 ), S3 (ONE seaside, 8 km south of the second station with a geographical position N 33 360 27,9 "/W 007 340 28,300 ), S4 (Sidi Abderrahmane beach, 17 km south of the third site, with a geographic position of N 33 340 52,1 "/W 007 420 41,400 ). The site S1 is from a beach with strong hydrodynamics extending on a rocky reef. It is located next to the discharge point of a petroleum refinery, and considered an area with moderate pollution. While, S2 and S3 sites are from an urban area, showing strong industrial activity. They were especially threatened by the presence of an effluent collectors opening directly on the sea, and carrying multicolour wastewater. Many of these hazardous substances are scarcely degradable. Hydrobionts develop a number of physiological mechanisms for their adaptation
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Fig. 1. Study area and sampling stations in Casablanca coast (Morocco).
to these surroundings. The site S4 was located within an urban area, presenting domestic pollution both in the water and in sediment. The control site was located in a small touristic city far-off any source of pollution (beach located in the area of Skhirat city, in the Moroccan Atlantic coast, with a geographic position of N 33 510 3600 et W 7 030 0000 ).
wastewater, with a WTW (Wissenschaft lich Technische Werkst€ atten, Weilheim, Germany) multilab system. Immediately, the different samples collected were transported in insulated coolers to the laboratory. 2.3. Trace metal concentrations in surface seawater and wastewater
2.2. Sampling and sample preservation All samples were collected at low tide in one day during the year 2011e2012. From the intertidal zone, about 150 indigenous mussels M. galloprovincialis clinging to rocks were taken from each of the studied sites, and placed in sterile plastic bags. They were collected in the spring (AprileMay), summer (JulyeSeptember), autumn (OctobereNovember) and winter (DecembereJanuary). They were characterized by similar maximum length: 55 ± 10 mm shell length. Simultaneously, seawater and wastewater samples was collected using polythene bottles (1 L). The were sampled directly in the coast while wastewater samples are taken upstream of the collector in the flow area of the sewer, where the water movement is most active, and a few centimetres below the surface. Environmental parameters including water temperature, conductivity, and pH were measured in situ at all locations for sea water and
Metals concentrations were measured simultaneously in seawater and wastewater. Samples volume was preserved by the addition of 2 ml concentrated H2SO4, in order to avoid precipitation. Then, samples were stored in an ice cooler, and transported to the laboratory. Analysis of trace metals was performed by ICP-AES (Inductively coupled plasma atomic emission spectrometry) at the technical unit support to scientific research (UATRS) of the National Center of Scientific Researches and Techniques (CNRST) in Rabat, Morocco. 2.4. Enzymatic biomarkers and total protein quantification Every season of the study, the sampled mussels were collected at a rate of thirty animals per station, 15 for the determination of protein concentration and 15 for biomarkers analysis. Immediately,
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gills, hepatopancreas and muscles were dissected on ice. The soft various tissues were weighed and then homogenized (using a potter homogeniser) in 1:5 w/v homogenization buffer (0,05 phosphate buffer, pH 7.4). The homogenate was centrifuged twice at 10,000 g at 4 C for 20 min. The supernatants were diluted 1:5 v/ v with phosphate buffer, saved in aliquots and kept at 20 C until enzymatic analysis. Before each enzyme assay, all prepared samples were assayed in amount of protein according to the Bradford (1976) method, using the Coomassie blue reagent and bovine serum albumin (BSA Sigma) as a standard to normalize all biochemical results. Catalase activity (CAT, EC 1.11.1.6) was assayed following the method of hydrogen peroxide consumption at 240 nm (Aebi, 1984; Greenwald, 1985). The reaction mixture consisted of 0.05 M phosphate buffer (pH 7.0), and H2O2 (0.036% w/w, immediately prepared before used). CAT activity was evaluated by kinetic measurement at 25 C and results was expressed as mmoles hydrogen peroxide transformed per minute and per milligram of proteins. Glutathione S transferase activity (GST, EC 2.5.1.18) was measured toward the Habig method, using glutathione (GSH) and 1-chloro-2,4-dinitrobenzene (CDNB) as substrate (Habig et al., 1974). The reaction rate was recorded at 340 nm, and enzyme activity expressed as nmol CDNB conjugate formed per minute per milligram of proteins, using a molar extinction coefficient of 9.6 mM-1 cm-1. All chemicals were products of Sigma.
Table 1 Seawater and wastewater physico-chimical parameter's means in the studied sites (S1, S2, S3, S4) of the Casablanca coast and control (C). Stations
Seawater
Wastewater a
Physico-chimical parameters
S1 S2 S3 S4 C S2 S3
Temperature ( C)
pH
Conductivity (ms/cm)
17.5 (13.1e25) 18.1 (15.3e25) 19.02 (15.8e25) 20.6 (16.3e26.8) 17 (15.7e20) 19.5 (17.4e25) 23a (21.5e25)
8,2 (7.9e8,9) 8.1 (8.01e8.3) 7.7 (7.3e8.4) 8.4 (8.3e8.6) 8 (7.9e8.1) 7.6 (7.6e7.7) 5.8a (5.6e6.3)
54.6 (54.8e55.2) 53.1 (52.2e53.4) 53.4 (52.7e55) 53.2 (53.1e53.3) 54.2 (54e54.5) 21.5a (16.7e35.2) 5 (3.7e7.7)
Parameters exceeding the limit values of effluent's standard norms.
2.5. Lipid peroxidation concentration Enzymatic activities and MDA were determined with a BioMate 3 UV spectrophotometer at 37 C. MDA concentration and all enzyme activities were measured in triplicate for each sample. For the determination of lipid peroxidation (LP), we measured Malondialdehyde (MDA) by the TBARs method (Samokyszyn and Marnett, 1990; Buege and Aust, 1978). This method is based on the property of certain compounds, in particular MDA, to react with thiobarbituric acid and regenerate a pink chromophore absorbing at 535 nm. Thus, 1 ml of the heat denatured supernatant is added to 1 ml of the reaction mixture in 1:1:1 [thiobarbituric acid (0.375%) and trichloroacetic acid (15%) in hydrochloric acid (0.25 N)]. The LP concentrations unit were expressed as nmoles of MDA per milligram of proteins. 2.6. Statistical analysis Data are expressed as mean ± standard deviation (SD) of five independent experiments, each performed in triplicate. One-way analysis of variance ANOVA and Tukey Kramer's test were used for testing differences among sites. In the figures, all experimental groups were compared to the control. Statistical comparisons were made assuming homogeneity of variance. Values of *p < 0.05 were considered significant. All analyses were performed by Excel software 2013 for Windows. 3. Results
Fig. 2. Seasonal variations of (a) temperature ( C), (b) pH and (c) conductivity (ms/cm) of seawater and wastewater along the study area in the Casablanca coast.
Generally, the sea water analysis reveal some stressful environmental conditions for aquatic organisms at the four sampling sites of Casablanca coast. The physico-chimical parameter's values vary with sensible range between stations of sampling. Even if, the S3 wastewaters expressed an acidic pH value, and showed a strong mineralization in discharges. The pH and conductivity of seawater remained characteristic of the marine environment at all locations (Table 1). However, seasonal variations of seawater temperature were significant. The highest mean value (26.8 C) was recorded in
summer period, whereas the lowest value (13.1 C) was recorded in autumn (Fig. 2). The wastewater collected at the two stations S2 and S3 record the highest temperature values, with a maximum value of about 23.3 C at the station S3 (Table 1). Still, these values remain below 30 C and complying with the effluents discharge standards.
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3.1. Trace metal concentrations in surface seawater (Casablanca coast) Heavy metals analysis showed that lead, mercury and copper were at high concentrations at the 4 selected locations of Casablanca Coast during all sampling campaigns (Fig. 3). Regarding the sites variation, the highest levels for all metals were recorded at S3 and S2 in the center parts of the Casablanca Coast (Table 2). Those high amounts are mainly related to emissaries that discharge sewages from the industrial zone. While, S4 and S1 were placed in slight industrial activity, those stations showed considerable levels of heavy metals. 3.2. Seasonal measurements of CAT activity
99
Table 2 Indicative mean values of the traces metal's concentrations (mg/l) in seawater and wastewater of four area (S1, S2, S3, S4) of the Casablanca coast and control (C). Stations
Heavy metal concentrations (mg/l) Hg
Seawater
Wastewater
S1 S2 S3 S4 C S2 S3
a
0.6 (0.04e1.02) 0.5a (0.3e0.67) 1.5a (0.85e2.2) 0.3a (0.03e0.7) 0.001 0.55a (0.4e0.7) 1.04a (0.5e1.6)
Pb
Cu
0.2 (0.03e0.24) 0.19 (0.07e0.23) 0.22 (0.15e0.3) 0.22 (0.15e0.3) 0.03 0.2 (0.16e0.24) 0.3 (0.3e0.4)
0.03 (0.02e0.04) 0.02 (0.01e0.03) 0.02 (0.015e0.025) 0.035 (0.03e0.04) 0.007 0.025 (0.02e0.03) 0.03 (0.03e0.04)
a Concentrations exceeding the limit values by standards norms in seawater and effluents.
The summary of results for seasonal CAT activity in mussels from the different sites was presented in Fig. 4. The levels of CAT activity in the three organs showed considerable differences among
Fig. 4. Seasonal variations of catalase (CAT) activity in gills (A), Hepatopancreas (B), and muscles (C) of mussels Mytilus galloprovincialis from the sampling sites S1, S2, S3, S4 and Control. Mean values ± SD. (ANOVA, Tukey HSD, *p < 0.05).
Fig. 3. Concentrations of heavy metals (Hg, Pb and Cu) in seawater (S1, S2, S3, S4 and C) and wastewater samples (S20 and S30 ).
sites. Mussel's hepatopancreas expressed a significant CAT activity relative to the control mussels, in all sampling sites. The maximum value (289 mmol/min/mg of proteins) reached in mussels from the
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most polluted site S3 at spring. Whereas, mussel's gills from polluted sites (S2 and S3) showed a two-fold decrease of CAT activity, compared to other sites: S1 > S4 > C > S2 > S3 in all the seasons (p < 0.05, Fig. 4a). In addition, only mussel's muscles from S4 showed similar CAT activity to control. When, mussels from S1 recorded significant level of CAT activities in the three organs (223 mmol/min/mg of protein in gills, 164 mmol/min/mg of protein in hepatopancreas, 75 mmol/min/mg of protein in muscles). 3.3. Seasonal measurements of GST activity Measurements of seasonal GST activity in mussels in the different sites was presented in Fig. 5. GST did not show seasonal variations and presented the highest values in mussels from the most polluted areas. Mussels from contaminated sites (S2 and S3) has significantly the highest GST levels in all organs tested, when compared to reference site: S3 > S2 > S1 > S4 > C in all seasons.
Fig. 5. Seasonal variations of glutathione S-transferase (GST) activity in gills (A), Hepatopancreas (B), and muscles (C) of mussels Mytilus galloprovincialis from the sampling sites S1, S2, S3, S4 and Control. Mean values ± SD. (ANOVA, Tukey HSD,*p < 0.05).
Thus, the several-fold higher GST activity in S3 mussel's could show that these animals are under higher pollution stress conditions (Fig. 3). The assessment of GST activity after exposure to certain pollutants has already been demonstrated both in fish and in invertebrates (Fitzpatrick et al., 1997; Cheung et al., 2001; Gowland et al., 2002). Our findings were consistent with other studies, which also showed higher GST activity in organisms from polluted sites when compared to those from reference sites (Bainy et al., 2000; Lau and Wong, 2003; Manduzio et al., 2004; Saenz et al., 2010). 3.4. LP concentration Seasonal measurements of LP concentration in the three organs in mussels are presented in Fig. 6. LP represent the most type of cell damage widely studied. The levels of MDA reflect the state of peroxidation of cell membranes and the impairment of antioxidant
Fig. 6. Seasonal variations of catalase (MDA) activity in gills (A), Hepatopancreas (B), and muscles (C) of mussels Mytilus galloprovincialis from the sampling sites S1, S2, S3, S4 and Control. Mean values ± SD. (ANOVA, Tukey HSD, *p < 0.05).
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defense systems to remove excess reactive oxygen species (Nzengue, 2008). LP concentration showed a consistent trend for upper levels in the polluted sites S2 and S3, where smaller increases were detected in the S1 site. By comparison, the control mussels had much lower levels of MDA, as expected. Gills and hepatopancreas showed a 2 fold increase in concentration of LP compared to mussels' muscles. The LP concentrations varied from 0,45 to 3,7 nmol g-1 w. w.t (Gills and hepatopancreas) and 0,35e2,4 nmol g-1 w. w.t (Muscles), compared to 0,15e0,55 (Gills and hepatopancreas) and 0,15e0,4 (Muscles) from the control. Our result were similar with other studies, where mussels exposed to polluted areas express an increased concentration of LP, comparing to less polluted sites (Lau and Wong, 2003; Pampanin et al., 2005). The higher MDA accumulation (3,8 nmol/mg protein) was obtained in gill's mussels from S2 during spring period. 4. Discussion The influence of environmental abiotic factors on various biological activities has been reported in many published works. Particularly, the clout on oxidative processes in aquatic ecosystems has been shown (Parihar et al., 1997; Robillard et al., 2003; Borkovic et al., 2005). Many studies have shown that increasing temperature also increases the basal cell metabolism inducing oxidative stress in the species studied (Bocchetti et al., 2008; Verlecar et al., 2007). Thus, the high increases of seawater temperature could be a stressful conditions for mussels from industrial sites (S2 and S3)in our study. In addition, the contamination of the coastal ecosystem by heavy metals represent the major crisis in environmental toxicology. Especially, some traces metals is not biodegradable and they can be accumulated by various aquatic organisms with toxic levels. They are well known to induce oxidative stress by their stimulation of ROS generation. Several published works reported that the stimulus of the ROS production and oxidative damage resulting, can be a significant mechanism of toxicity to aquatic organisms subjected to metallic pollution (Regoli and Principato, 1995; Livingstone, 2003; Lushchak, 2011). Accordingly, the high concentrations of non-essential metals (as Hg and Pb) detected in seawater samples above natural charges, could be consequential to oxidative stress in mussels of this aquatic ecosystems. Contamination by these elements is attributed mainly to industrial liquid waste, denoting an important input in the receiving environment. Similar results were obtained by Bouthir et al. (2004) in the two sites (S2 and S3), which recorded the highest urban and industrial concentration of the country. Moreover, the S1 station recorded the highest values of Hg. This is presumably due to the direct discharges to the sea from a refining industry located upstream of this station. Especially, as coastal ocean currents have N-S orientation. Also, the “S4” station showed considerable levels of these heavy metals while is placed in slight industrial activity. This contamination is most probably due to liquid discharges from a company located upstream of this region, that uses mercury cathodes for the production of chlorine and sodium hydroxide. The biological response of indigenous mussels Mytilus galloprovincialis to acute emissaries pollution is confirmed from the measurements of CAT and GST activities, even if the cause-effect relationship between the oxy-radical metabolism and cellular damage is difficult to highlight. Many scientist consider CAT as a biomarker need to be monitored in case of oxidative contaminants. CAT is the primary antioxidant defense involved in detoxification of H2O2, which is the major cellular precursor of the toxic hydroxyl radical (HO-). Particularly, numerous study suggested that CAT is sensitive to some oxidative stress-inducing contaminants in cell membranes, as metals (Labrot et al., 1996). Recent study revealed
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that goldfish C. auratus expressed a reduced catalase activity after 40 day exposure to 0.005e0.025 mg/l copper (Liu et al., 2006). Recently, publication on the mercury effects on free radical processes found that mercury exposure causes depletion of antioxidant in fish Liza aurata. The activities of catalase, glutathione peroxidase, and glutathione reductase significantly decreased (Mieiro et al., 2010). Several studies consider the inhibition of catalase as a temporary response to severe pollution (Regoli and Principato, 1995). Our data for reduced CAT activity in gills of M. galloprovincialis compared to control values, is presumably due to increased levels of pollution, indicating the critical pollution level in two sites (S2 and S3). While, the increased values of CAT activity in mussels from S1 and S4, indicating that pollution levels are relative moderate. This organisms are facing an oxidative stress, possibly associated with the presence of heavy metals (Hg, Pb and Cu) in the environment. Furthermore, the major tissue of xenobiotics uptake as hepatopancreas expressed the highest GST activity that is rather related to detoxification, especially in mussels from S2 and S3. Actually, the glutathione S-transferase family (GST) belongs to the Phase II detoxification enzymes, and plays an important role in the combination of a wide variety of substances with glutathione to facilitate their solubility and excretion (Livingstone, 1998). This result explain the low CAT activities obtained in mussel's gills from those sites. GST proved more useful for pollution monitoring compared to alternative biomarkers, and several authors have proposed this enzyme as biomarkers of exposure to pollution. GST activities were found to be modulated by metals or organic contaminants under both field conditions (Durou et al., 2007) and laboratory exposure (Livingstone, 1991). Moreover, the induction of GST can be regarded ~a n as an adaptive response to an altered environment (Vidal-Lin et al., 2010a). Our results for GST activity presented the most uniform pattern. The significant levels of GST activities in mussels could be a consequences of the environmental contamination by heavy metals detected in the seawater. Similar results were presented in a study of petrochemical contamination in M. galloprovincialis. While, high concentrations of coastal waters with heavy metals can cause poisoning, leading to oxidative stress conditions found in individuals of these aquatic ecosystems (Lima et al., 2007). Many authors note that lipid peroxidation appears to be modulated by chemical and organic contaminants tested in laboratory conditions or in situ (Narbonne et al., 2005). Also, a significant induction of MDA in different clam tissues exposed to cadmium solutions has been reported by Khebbeb et al. (2010). Also, the induction of MDA has been noted in clams Donax trunculus taken from a polluted site in Gulf of Annaba in eastern Algeria (Soltani et al., 2012). Actually, the corresponding results of high LP concentrations explain the low rate of CAT activities recorded in mussels from the both releases stations, “S200 and “S300 in which all stress conditions are met. We suggest that this tissues shows a strong alteration of antioxidant defense system, marked by high production of reactive oxygen species. Some studies reported that biomarker generates a primary response to a limited adaptation, after an exposure to a pollutant. However, an alteration of the detoxification system will be observed in case of stress persists, leading to uncompensated damage (Livingstone, 2003; Vasseur and Leguille, 2004). In fact, the cooperation from antioxidant enzymes is essential for the scavenging of ROS. The low activity of antioxidant enzymes such as CAT, will increase the content of superoxide radical and H2O2. They will reacting together to produce hydroxyl radical that can attacks all biomolecules and disturbs cell metabolism (Esfandiari et al., 2007; Shao et al., 2005). The antioxidant defense enzyme induced were not enough to reduce lipid peroxidation levels in mussels from those polluted sites, whereas
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inhibition of CAT has been suggested as a transitory response to acute pollution (Regoli and Principato, 1995; Pampanin et al., 2005). Many studies confirmed this negative relationship between CAT activity and MDA. Similar results were presented by Seonock et al. (2013) in mussels Mytilus galloprovincialis. They revealed significant differences in CAT, GST and LP activities. However, only CAT activity negatively correlated with relevant stress. They suggested that reduction in CAT activity caused redox imbalance, which was sufficient to induce further oxidative stress that led to increased activities of GST and LP in mussels. Indeed, emissaries are responsible for the contaminations of aquatic environment, by many chemicals that have been shown to be pro-oxidants in aquatic organisms such as metals (Phillips, 1976; Livingstone, 2003; Nzengue, 2008). And, they are easily taken up from the sediment, water-column and food into the tissues of resident organisms. The raw sewerage implantations causes metabolic disturbances occurring into indigenes mussels Mytilus galloprovincialis from the coastal environment of Casablanca. This agents responsible for oxidative stress in marine organisms, caused changes that can range from the molecular level (metabolism) to the whole organism scale (population dynamics). An effective management system need to be establish ensuring the protection of this aquatic environment. 5. Conclusions Our research, regardless of its restrictions, provides an example of antioxidant multibiomarkers potentiality, emphasizing the difficulties related to the integration of ecotoxicological approach with in situ biomonitoring studies. Significant biochemical changes occurred in gills, hepatopancreas and muscles tissues. Those biomarker responses could be allocated to differences in both pollution levels and seasonal variability. The evaluation of oxidative stress in Mytilus galloprovincialis species revealed promising results in accordance with the availability of contaminations (heavy metals) and environmental stress conditions at each station. From a methodological standpoint, this work affords another effectiveness evidence of the use of multibiomarker approach in mussels for biomonitoring study. The use of mussel gills and hepatopancreas as bioindicators tissues has been well-developed revealing site-effects. Our results were in similar trend with other published work that revealing them as organs of choice for pollution biomonitoring (Manduzio et al., 2004; Lima et al., 2007). However, biomarkers showed significant levels in mussel muscles from the most polluted areas (S2 and S3), whereas trivial levels in the less polluted site (S4), when compared to control. This fact suggest that muscles seems to be a responsive organ in case of acute pollution. Competing interests The authors declare that they have no competing interests. References Aebi, H., 1984. Catalase in vivo. Methods Enzymol. 105, 121e126. Bainy, A.C.D., Almeida, E.A., Muller, I.C., et al., 2000. Biochemical responses in farmed mussel Perna perna transplanted to contaminated sites on Santa Catarina Island, SC. Braz. Mar. Environ. Res. 50, 411e416. Bebianno, M.J., Company, R., Serafim, A., Cosson, R.P., Fiala-Medoni, A., 2005. Antioxidant systems and lipid peroxidation in Bathymodiolusazoricus from midAtlantic ridge hydrothermal vent fields. Aquat. Toxicol. 75, 354e373. Benbrahim, S., Chafik, A., Chfiri, R., Bouthir, F.Z., Siefeddine, M., Makaoui, A., 2006. Survey of the carriers influencing the geographical and temporal distribution of contamination by heavy metals along the Atlantic Moroccan coasts: the case of mercury, lead and cadmium. Mar. Life 16 (1e2), 37e47. Bocchetti, R., Lamberti, C.V., Pisanelli, B., Razzetti, E.M., Maggi, C., Catalano, B., Sesta, G., Martuccio, G., Gabellini, M., Regoli, F., 2008. Seasonal variations of
exposure biomarkers, oxidative stress responses and cell damage in the clams. Tapes philippinarum, and mussels, Mytilus galloprovincialis, from Adriatic sea. Mar. Environ. Res. 66, 24e26. Borkovic, S.S.S., aponjic, J.S., Pavlovic, S.Z., Blagojevic, D.P., Milosevic, S.M., Kovacevic, T.B., Radojicic, R.M., Spasic, M.B., Zikic, R.V., Saicic, Z.S., 2005. The activity of antioxidant defence enzymes in the mussel Mytilus galloprovincialis from the Adriatic Sea. Comp. Biochem. Physiol. C Toxicol. Pharmacol. 141, 366e374. Bouthir, F.Z., Chafik, A., Benbrahim, S., Souabi, S., El Merdhy, H., Messoudi, A., physico-chimique des eaux co ^ tie res du littoral de la Sifeddine, M., 2004. Qualite an Atlantique marocain) utilisant la moule Wilaya du grand Casablanca (oce tallique, Mytilus galloprovincialis comme indicateur de la contamination me 2004 Mar. Life 14, 1e2, 59-70. Bradford, M.M., 1976. A rapid and sensitive method for the quantification of microgram quantited of protein utilizing the principe of protein-dye binding. Anal. Biochem. 72, 248e254. Buege, J.A., Aust, S.D., 1978. Microsomal lipid peroxidation. Methods Enzymol. 52, 302e310. Calow, P., 1993. General principles and overview. In: Calow, P. (Ed.), Handbook of Ecotoxicology, 1, pp. 1e5. Cheung, C.C.C., Zheng, G.J., Li, A.M.Y., et al., 2001. Relationships between tissue concentrations of polycyclic aromatic hydrocarbons and oxidative responses of marine mussels, perna viridis. Aquat. Toxicol. 52, 189e203. Corsi, I., Mariottini, M., Sensini, C., Lancini, L., Focardi, S., 2003. Fish as bioindicators of brackish ecosystem health: integrating biomarker responses and target pollutant concentrations. Oceanol. Acta 26, 129e138. Durou, C., Poirier, L., Amiard, J.C., Budzinski, H., Gnassia-Barelli, M., Lemenach, K., Peluhet, L., Mouneyrac, C., Romeo, M., Amiard-Triquet, C., 2007. Environ. Pollut. 148, 445e458. Esfandiari, E., Shakiba, M.R., Mahboob, S., Alyari, H., Toorchi, M., 2007. Water stress, antioxidant enzyme activity and lipid peroxidation in wheat seedling. J. Food, Agric. Environ. 5, 149e153. €rlin, L., Bolognesi, C., Sturve, J., Frenzilli, G., Scarcelli, V., Del Barga, I., Nigro, M., Fo € teborg harbour. 2004. DNA damage in eelpout (Zoarces viviparus) from Go Mutat. Res. 552, 187e195. Fitzpatrick, P.J., O'Hallaran, J., Sheehan, D., Walsh, A.R., 1997. Biomarkers 2, 51e56. Gomez-Mendikute, A., Cajaraville, M.P., 2003. Comparative effects of cadmium, copper, paraquat and benzo[a]pyrene on the actin cytoskeleton and production of reactive oxygen species (ROS) in mussel haemocytes. Toxicol. Vitro 17, 539e546. Gowland, B.T., McIntosh, A.D., Davies, I.M., Moffat, C.F., Webster, L., 2002. Implications from a field study regarding the relationship between polycyclic aromatic hydrocarbons and glutathione S-transferase activity in mussels. Mar. Environ. Res. 54, 231e235. Greenwald, R.A., 1985. Handbook of Methods for Oxygen Radicals in Biology and Medicine. Oxford University Press, New York. Habig, W.H., Pabst, M.J., Jakoby, W.B., 1974. Glutathione S-transferase. J. Biol. Chem. 249, 7130e7139. Khebbeb, M.E.H., Nadji, S., Amrani, A., 2010. The effect of cadmium exposure on malonedialdehyde and reduced glutathione concentrations in several tissues of a bivalve mollusc (Ruditapes decussatus) fished from Mellah lagoon (North East of Algeria). Ann. Biol. Res. 1, 166e173. Labrot, F., Ribera, D., Saint-Denis, M., Narbonne, J.F., 1996. In vitro and in vivo studies of potential biomarkers of lead and uranium contamination: lipid peroxidation, acetylcolinesterase, catalase and glutathione peroxidase activities in three nonmammalian species. Biomarkers 1, 21e28. Lafaurie, M., Narbonne, J.F., Galgani, F., 1992. Indicateurs biochimiques de contamination de l'environnement marin. Anal. Mag. 20 (6), 27e33. cotoxicologie : principes Lagadic, L., Caquet, T., Amiard, J.C., 1997. Biomarqueurs en e finitions. In: Lagadic, L., Caquet, T., Amiard, J.C., Masson, Ramade F. (Eds.), et de cotoxicologie, aspects fondamentaux, pp. 1e9. in Biomarqueurs en e Lau, P.S., Wong, H.L., 2003. Effect of size, tissue parts, and location on six biochemical markers in the green-lipped mussel, perna viridis. Mar. Poll. Bull. 46, 1563e1572. Lima, I., Moreira, S.M., Rendon-Von Osten, J., et al., 2007. Biochemical responses of the marine mussel Mytilus galloprovincialis to petrochemical environmental contamination along the northwestern coast of Portugal. Chemosphere 66, 1230e1242. Liu, H., Wang, W., Zhang, J.F., Wang, X.R., 2006. Effects of copper and its ethylenediaminetetraacetate complex on the antioxidant defenses of the goldfish, Carassius auratus. Ecotoxicol. Environ. Saf. 65, 350e354. Livingstone, D.R., 1991. Adv. Comp. Environ. Physiology 7, 145e187. Livingstone, D.R., 1998. Organic xenobiotics in aquatic ecosystems: quantitative and qualitative differences in biotransformation by invertebrates and fish. Comp. Environ. Physiol. 120A, 43e49. Livingstone, D.R., 2001. Contaminant-stimulated reactive oxygen species production and oxidative damage in aquatic organisms. Mar. Pollut. Bull. 42, 656e666. Livingstone, D.R., 2003. Oxidative stress in aquatic organisms in relation to pollud. Ve t 154 (6), 427e430. tion and aquaculture. Rev. Me Lushchak, V.I., 2011. Environmentally induced oxidative stress in aquatic animals. Aquat. Toxicol. 101, 13e30. Manduzio, H., Monsinjon, T., Galap, C., Leboulenger, F., Rocher, B., 2004. Seasonal variation in antioxydant defences in blue mussels Mytilus edulis collected from a polluted aera: major contributions in gills of an inducible isoform of Cu/Znsuperoxide dismutase and of glutathione-S transferase. Aquat. Toxicol. 70,
Z. Mejdoub et al. / Ocean & Coastal Management 136 (2017) 95e103 83e93. ation d’un cadre politique favorisant Menioui, M., 2007. Sensibilisation et cre gration de l’environnement et du de veloppement avec l’accent sur la l’inte gre e des zones co ^ tie res. Royaume du Maroc. Ministe re de gestion inte nagement du Territoire, de l’Eau et de l’Environnement. Direction de la l’Ame vention des Risques. Surveillance et de la Pre l'e tude des interactions entre les contaminants Michel, X., 1993. Contribution a culaires et applichimiques organiques et les organismes marins : bases mole la biosurveillance de l'environnement co ^ tier. The se. Universite cations a Bordeaux 1. Mieiro, C.L., Ahmad, I., Pereira, M.E., Duarte, A.C., Pacheco, M., 2010. Antioxidant system breakdown in brain of feral golden grey mullet (Liza aurata) as an effect of mercury exposure. Ecotoxicology 19 (6), 1034e1045. Narbonne, J.F., Aarab, N., Clerandeau, C., et al., 2005. Scale of classification based on biochemical markers in mussels: application to pollution monitoring in Mediterranean coasts and temporal trends. Biomarkers 10 (1), 58e71. canismes de toxicite redox du cadmium, du Nzengue, Y., 2008. Comparaison des me cuivre et du zinc : place des metallothioneines et de p53. Life Sciences [q-bio. University Joseph-Fourier -Grenoble I. Orbea, A., Ortiz-Zarragoitia, M., Sole, M., Porte, C., Cajaraville, M.P., 2002. Antioxidant enzymes and peroxisome proliferation in relation to contaminant body burdens of PAHs and PCBs in bivalve molluscs, crabs and fish from the Urdaibai and Plentzia estuaries (Bay of Biscay). Aquat. Toxicol. 58, 75e98. Pampanin, D.M., Camus, L., Gomiero, A., Marangon, I., Volpato, E., Nasci, C., 2005. Susceptibility to oxidative stress of mussels (Mytilus galloprovincialis) in the Venice Lagoon (Italy). Mar. Pollut. Bull. 50, 1548e1557. tabolisme des xe nobiotiques chez les bivalves. C.R. Acade mie Parant, M., 1998. Le me Sci. Paris, Sci. vie 321, 485e494. Parihar, M.S., Javeri, T., Hemnani, T., Dubey, A.K., Prakash, P., 1997. Responses of superoxide dismutase, glutathione peroxidase and reduced glutathione antioxidant defenses in gills of the freshwater catfish (Heteropneustes fossilis) to short-term elevated temperature. J. Therm. Biol. 22 (2), 151e156. Phillips, D.J.H., 1976. The common mussel Mytilus edulis as an indicator of pollution by zinc, cadmium, lead and copper. Effect of environmental variables on uptake of metals. Mar. Biol. 38, 59e69. Regoli, F., Principato, G., 1995. Glutathione, glutathione-dependent and antioxidant enzymes in mussels, Mytilus galloprovincialis, exposed to metal under field and laboratory conditions: implications for the use of biochemical biomarkers. Aquat. Toxicol. 31, 143e164. Regoli, F., Gorbi, S., Frenzilli, G., Nigro, M., et al., 2002. Oxidative stress in ecotoxicology: from the analysis of individual antioxidants to a more integrated approach. Mar. Environ. Res. 54, 419e423. Robillard, S., Beauchamp, G., Laulier, M., 2003. The role of abiotic factors and pesticide levels on enzymatic activity in the freshwater mussel Anodonta cygnea at three different exposure sites. Comp. Biochem. Physiology Part C 135, 49e59. Saenz, L.A., Seibert, E.L., Zanette, J., et al., 2010. Biochemical biomarkers and metals
103
in Perna perna mussels from mariculture zones of Santa Catarina, Brazil. Ecotoxicol. Environ. Saf. 73, 796e804. Samokyszyn, V.M., Marnett, L.J., 1990. Inhibition of liver microsomal lipid peroxidation by 13-cis-retinoic acid. Free RadicBiol Med. 8, 491e496. Santovito, G., Piccinni, E., Cassino, A., Irato, P., Albergoni, V., 2005. Antioxidant responses of the Mediterranean mussel, Mytilus galloprovincialis, to environmental variability of dissolved oxygen. Comp. Biochem. Physiol. 140, 321e329. ^ tier marocain. ISBN 9981Sbai, L., 2001. Le droit de l’environnement marin et co tat du milieu et une le gislation obsole te, Rabat, 889-28-8. Dichotomie entre l’e Maroc, p. 146. Seonock, W., Vianney, D., Hyokyung, W., 2013. Expressions of oxidative stressrelated genes and antioxidant enzyme activities in Mytilus galloprovincialis (Bivalvia, Mollusca) exposed to hypoxia. Zool. Stud. 52, 15. Shao, H.B., Liang, Z.S., Shao, M.A., 2005. Changes of anti-oxidative enzymes and MDA content under soil water deficits among 10 wheat (Triticum aestivum L.) genotypes at maturation stage. Colloids Surfaces B Biointerfaces 45, 7e13. Sheehan, D., Power, A., 1999. Effects of seasonality on xenobiotic and antioxidant defence mechanisms of bivalve molluscs. Comp. Biochem. Physiology Part C 123, 193e199. , M., Kopecka-Pilarczyk, J., Blasco, J., 2009. Pollution biomarkers in two estuaSole rine invertebrates Nereis diversicolor and Scrobicularia plana, from marsh ecosystem in SW Spain. Environ. Int. 35, 523e531. Soltani, N., Amira, A., Sifi, K., Beldi, H., 2012. Environmental monitoring of the Annaba gulf (Algeria): measurement of biomarkers in Donax trunculus and metallic pollution. Bull.Soc. Zool. Fr. 137, 47e56. Stegeman, J.J., Brouwer, M., Di Guilio, T., Forlin, L., Fowler, B.A., Sanders, B.M., Van Veld, P.A., 1992. Molecular responses to environmental contamination: enzyme and protein systems as indicators of chemical exposure and effect. In: Huggett, R.J., Kimerle, R.A., Mehrle, P.M., Bergman, H.L. (Eds.), Biomarkers : Biochemical, Physiological, and Histological Markers of Anthropogenic Stress. Lewis Publishers, Chelsea, Etats-Unis, pp. 235e335. Valavanidis, A., Vlahogianni, T., Dassenakis, M., et al., 2006. Molecular biomarkers of oxidative stress in aquatic organisms in relation to toxic environmental pollutants. Ecotoxicol. Environ. Saf. 64, 178e189. Vasseur, P., Leguille, C., 2004. Defense systems of benthic invertebrates in response to environmental stressors. Environ. Toxicol. 19 (4), 433e436. Verlecar, X.N., Jena, K.B., Chainy, G.B., 2007. Biochemical markers of oxidative stress in Perna viridis exposed to mercury and temperature. Chem. -Biol. Interact. 167, 219e226. Viarengo, A., 1989. Heavy metals in marine invertebrates: mechanisms of regulation and toxicity at the cellular level. CRC Rev. Aquat. Sci. 1, 295e317. ~a n, L., Bellas, J., Campillo, J.A., et al., 2010. Integrated use of antioxidant Vidal-Lin enzymes in mussels, Mytilus galloprovincialis, for monitoring pollution in highly productive coastal areas of Galicia (NW Spain). Chemosphere 78, 265e272. Winston, G.W., Di Giulio, R.T., 1991. Prooxidant and antioxidant mechanisms in aquatic organisms. Aquat. Toxicol. 19, 137e161.