Oxidative stress responses of the mussel Mytilus galloprovincialis exposed to emissary's pollution in coastal areas of Casablanca 

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 Lout fi ^a , Mostafa Kabine ^a

a

Department of Biology, Laboratory of Biochemistry and Molecular Biology, Faculty of Science Ain Chock, University Hassan II Casablanca, Morocco

b

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

Article history:

Received 13 May 2016 Received in revised form 11 October 2016

Accepted 9 November 2016

Keywords:

Biomarkers Oxidative stress

Mussels Mytilus galloprovincialis Antioxidant defence enzymes Lipid peroxidation

Heavy metals

a b s t r a c t

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 (tem- perature, 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.

Contents

1. Introduction . . . 96

2. 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

3. 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

4. Discussion . . . 101

* Corresponding author.

E-mail address: z_mejdoub@hotmail.com (Z. Mejdoub).

Contents lists available at ScienceDirect

Ocean & Coastal Management

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / o ce c o a m a n

http://dx.doi.org/10.1016/j.ocecoaman.2016.11.018

0964-5691/© 2016 Elsevier Ltd. All rights reserved.

5. 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 in fl uences that can be exerted on the environ- ment 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 insuf fi cient 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 envi- ronmental 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 com- plementarities between the chemical and biochemical analysis, al- lows 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 fi sh 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 in- terest 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 pol- lutants 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 expo- sure causes their depletion, leading to disruption of core metabolic and regulatory process, enhancing oxidative damage to bio- molecules, such as lipid peroxidation, protein and DNA damage (Bebianno et al., 2005; Lushchak, 2011). Currently, this multi- biomarker approach in invertebrates has been supported by inter- national 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 (Sol e 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 in fl uenced 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 ef fl uents 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 met- allurgy, and fi nally 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 fi rst study of systematic biomonitoring, using indigenous mussels M. galloprovincialis as bioindicators and the seasonal var- iations of GST and CAT activities and LP concentrations as bio- markers 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 environ- ment 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 (Fig. 1): S1 (Grande Zen^ ata beach, Casablanca's upstream part, with a geographical position of NR 33

39

06.7 ” /W 007

28

48.3

), S2 (Saada beach, 3 km south of the fi rst station, to a geographical position N 33

37

23,4 " /W 007

31

'53,2

), S3 (ONE seaside, 8 km south of the second station with a geographical position N 33

36

27,9 " /W 007

34

28,3

), S4 (Sidi Abderrahmane beach, 17 km

south of the third site, with a geographic position of N 33

34

52,1

" /W 007

42

41,4

). The site S1 is from a beach with strong hy-

drodynamics extending on a rocky reef. It is located next to the discharge point of a petroleum re fi nery, 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 ef fl uent 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 Z. Mejdoub et al. / Ocean & Coastal Management 136 (2017) 95e103

96

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

51

36

et W 7

03

00

).

2.2. Sampling and sample preservation

All samples were collected at low tide in one day during the year 2011 e 2012. 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 (April e May), summer (July e September), autumn (October e November) and winter (December e January). 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 fl ow area of the sewer, where the water movement is most active, and a few centimetres below the surface. Environ- mental parameters including water temperature, conductivity, and pH were measured in situ at all locations for sea water and

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

Metals concentrations were measured simultaneously in seawater and wastewater. Samples volume was preserved by the addition of 2 ml concentrated H

SO

, in order to avoid precipita- tion. 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 scienti fi c research (UATRS) of the National Center of Scienti fi c Researches and Techniques (CNRST) in Rabat, Morocco.

2.4. Enzymatic biomarkers and total protein quanti fi cation

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,

Fig. 1. Study area and sampling stations in Casablanca coast (Morocco).

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 al- bumin (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 H

O

(0.036% w/w, immediately prepared before used). CAT activity was evaluated by kinetic measurement at 25

C and results was expressed as m ^moles 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 ac- tivity expressed as nmol CDNB conjugate formed per minute per milligram of proteins, using a molar extinction coef fi cient of 9.6 mM

cm

. All chemicals were products of Sigma.

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 milli- gram of proteins.

2.6. Statistical analysis

Data are expressed as mean ± standard deviation (SD) of fi ve 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 fi gures, all experimental groups were compared to the control. Statistical comparisons were made assuming homogeneity of variance. Values of *p < 0.05 were considered signi fi cant. All analyses were performed by Excel soft- ware 2013 for Windows.

3. Results

Generally, the sea water analysis reveal some stressful envi- ronmental 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 signi fi cant. 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 ef fl uents discharge standards.

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 Physico-chimical parameters

Temperature (

C) pH Conductivity (ms/cm) Seawater S

17.5 (13.1e25) 8,2 (7.9e8,9) 54.6 (54.8e55.2)

S

18.1 (15.3e25) 8.1 (8.01e8.3) 53.1 (52.2e53.4) S

19.02 (15.8e25) 7.7 (7.3e8.4) 53.4 (52.7e55) S

20.6 (16.3e26.8) 8.4 (8.3e8.6) 53.2 (53.1e53.3) C 17 (15.7e20) 8 (7.9e8.1) 54.2 (54e54.5) Wastewater S

19.5 (17.4e25) 7.6 (7.6e7.7) 21.5

(16.7e35.2)

S

23

(21.5e25) 5.8

(5.6e6.3) 5 (3.7e7.7)

a

Parameters exceeding the limit values of effluent's standard norms.

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.

Z. Mejdoub et al. / Ocean & Coastal Management 136 (2017) 95e103

98

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 Casa- blanca 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

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

sites. Mussel's hepatopancreas expressed a signi fi cant CAT activity relative to the control mussels, in all sampling sites. The maximum value (289 m mol/min/mg of proteins) reached in mussels from the Fig. 3. Concentrations of heavy metals (Hg, Pb and Cu) in seawater (S1, S2, S3, S4 and

C) and wastewater samples (S2

and S3

).

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 Pb Cu

Seawater S

0.6

(0.04e1.02) 0.2 (0.03e0.24) 0.03 (0.02e0.04) S

0.5

(0.3e0.67) 0.19 (0.07e0.23) 0.02 (0.01e0.03) S

1.5

(0.85e2.2) 0.22 (0.15e0.3) 0.02 (0.015e0.025) S

0.3

(0.03e0.7) 0.22 (0.15e0.3) 0.035 (0.03e0.04)

C 0.001 0.03 0.007

Wastewater S

0.55

(0.4e0.7) 0.2 (0.16e0.24) 0.025 (0.02e0.03) S

1.04

(0.5e1.6) 0.3 (0.3e0.4) 0.03 (0.03e0.04)

a

Concentrations exceeding the limit values by standards norms in seawater and effluents.

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).

most polluted site S3 at spring. Whereas, mussel's gills from polluted sites (S2 and S3) showed a two-fold decrease of CAT ac- tivity, 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 signi fi cant level of CAT activities in the three organs (223 m mol/min/mg of protein in gills, 164 m mol/min/mg of protein in hepatopancreas, 75 m mol/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 signi fi cantly the highest GST levels in all organs tested, when compared to reference site: S3 > S2 > S1 > S4 > C in all seasons.

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 fi sh and in in- vertebrates (Fitzpatrick et al., 1997; Cheung et al., 2001; Gowland et al., 2002). Our fi ndings 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 re fl ect the state of per- oxidation of cell membranes and the impairment of antioxidant

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).

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).

Z. Mejdoub et al. / Ocean & Coastal Management 136 (2017) 95e103

100

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 hepatopan- creas 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 0,35 e 2,4 nmol g

1 w. w.t (Muscles), compared to 0,15 e 0,55 (Gills and hepato- pancreas) and 0,15 e 0,4 (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 in fl uence of environmental abiotic factors on various bio- logical 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 toxi- cology. 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 signi fi cant mechanism of toxicity to aquatic organisms sub- jected to metallic pollution (Regoli and Principato, 1995;

Livingstone, 2003; Lushchak, 2011). Accordingly, the high concen- trations 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 concen- tration of the country. Moreover, the S1 station recorded the highest values of Hg. This is presumably due to the direct dis- charges to the sea from a re fi ning 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 contami- nation 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 gallo- provincialis to acute emissaries pollution is con fi rmed from the measurements of CAT and GST activities, even if the cause-effect relationship between the oxy-radical metabolism and cellular damage is dif fi cult 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 detoxi fi cation of H

O

, 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

that gold fi sh C. auratus expressed a reduced catalase activity after 40 day exposure to 0.005 e 0.025 mg/l copper (Liu et al., 2006).

Recently, publication on the mercury effects on free radical pro- cesses found that mercury exposure causes depletion of antioxi- dant in fi sh Liza aurata. The activities of catalase, glutathione peroxidase, and glutathione reductase signi fi cantly 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 hepa- topancreas expressed the highest GST activity that is rather related to detoxi fi cation, especially in mussels from S2 and S3. Actually, the glutathione S-transferase family (GST) belongs to the Phase II detoxi fi cation enzymes, and plays an important role in the com- bination of a wide variety of substances with glutathione to facili- tate 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 fi eld conditions (Durou et al., 2007) and laboratory exposure (Livingstone, 1991). Moreover, the induction of GST can be regarded as an adaptive response to an altered environment (Vidal-Li~ n an et al., 2010a). Our results for GST activity presented the most uni- form pattern. The signi fi cant levels of GST activities in mussels could be a consequences of the environmental contamination by heavy metals detected in the seawater. Similar results were pre- sented 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 labo- ratory conditions or in situ (Narbonne et al., 2005). Also, a signi fi - cant 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, “ S2

and “ S3

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

detoxi fi cation 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 antioxi-

dant enzymes such as CAT, will increase the content of superoxide

radical and H

O

. They will reacting together to produce hydroxyl

radical that can attacks all biomolecules and disturbs cell meta-

bolism (Esfandiari et al., 2007; Shao et al., 2005). The antioxidant

defense enzyme induced were not enough to reduce lipid peroxi-

dation levels in mussels from those polluted sites, whereas

inhibition of CAT has been suggested as a transitory response to acute pollution (Regoli and Principato, 1995; Pampanin et al., 2005).

Many studies con fi rmed this negative relationship between CAT activity and MDA. Similar results were presented by Seonock et al.

(2013) in mussels Mytilus galloprovincialis. They revealed signi fi - cant 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 suf fi cient 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 dif- fi culties related to the integration of ecotoxicological approach with in situ biomonitoring studies. Signi fi cant 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 pollu- tion biomonitoring (Manduzio et al., 2004; Lima et al., 2007).

However, biomarkers showed signi fi cant 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.

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