595 Advances in Environmental Biology, 7(4): 595-604, 2013 ISSN 1995-0756

This is a refereed journal and all articles are professionally screened and reviewed ORIGINAL ARTICLE

Oxidative stress and biochemical composition in trunculus (, ) from the gulf of Annaba (Algeria)

1Karima Sifi, 2Akila Amira and 3Noureddine Soltani

1Laboratory of Applied Biology, Department of Biology, Faculty of Sciences, University Badji-Mokhtar of Annaba 23000-Annaba, Algeria 2Laboratory of Applied Animal Biology Faculty of Sciences, Department of Biology University of Annaba 23000-Annaba, Algeria 3Laboratory of Applied Animal Biology Faculty of Sciences, Department of Biology University of Annaba 23000-Annaba, Algeria

Karima Sifi, Akila Amira and Noureddine Soltani: Oxidative stress and biochemical composition in (Mollusca, Bivalvia) from the gulf of Annaba (Algeria)

ABSTRACT

The gulf of Annaba, the most important touristic and economic coastal zone located in the east of Algeria, is contaminated by several pollutants from urban, agricultural, harbor and industrial activities. The aim of our study consisted to test two biomarkers of oxidative stress, glutathione (GSH) and malondialdehyde (MDA), and to determine the main biochemical components (proteins, lipids, and carbohydrates) in the mantle of an edible species Donax trunculus L. (Mollusca, Bivalvia) associated with environmental pollution. Samples were monthly collected in 2011 at two sites in the Annaba gulf: El Battah, a relatively clean site and Sidi Salem, a polluted site. The results showed a significant decrease in GSH amounts and a significant increase in MDA levels in Sidi Salem compared to El Battah. Furthermore, seasonal evaluation of our biomarkers, indicated that GSH levels are lower and MDA rates are higher in winter, regardless of the sampling site. The biochemical analysis revealed a significant decrease in protein, lipid and carbohydrate amounts in Sidi Salem comparatively with El Battah. In addition, the higher levels in biochemical components are recorded in spring and the lower rates in autumn and winter, this is probably related to respectively the beginning and the end of the reproduction period. Our results suggest an influence of the site quality on the health status of D. trunculus. Thus, the difference noted between the two sites may be due to thier pollution level. A level of pollution in Sidi Salem induced an oxidative stress associated with a mobilization of energetic metabolism for detoxification or for physiological processes such as growth or reproduction. Moreover, D. trunculus appears as a suitable sentinel species for the assessment of the ecotoxicological risk linked to the pollution.

Key words: Donax trunculus, Pollution, Biomonitoring, Oxidative stress, Glutathione, Malondialdehyde, Proteins, Lipids, Carbohydrates.

Introduction endpoint [47, 11]. Donax trunculus L. (Mollusca, Bivalvia) is widely distributed along the Pollution of the coastal ecosystems is a serious Mediterranean coast and frequently used in marine and growing problem. Indeed, different types of pollution studies [8, 46]. In Algeria, this species contamination including industrial, agricultural, exists in higher densities in the sand beaches of the domestic and shipping activities, are discharged into Annaba gulf [50] and is consumed by the local marine ecosystems, and may have a harmful impact population [8]. In addition, this species is used for on aquatic organisms [20, 11]. In Algeria, the gulf of many years ago as a bioindicator species in Annaba is one of the most important touristic and biomonitoring programs of the gulf of Annaba [8, 41, economic coastal areas located in east of Algeria and 5, 25, 42]. various contaminants are detected [39, 1], especially Environment stress caused by pollution may heavy metals [8]. exert toxicity through oxidative stress derived from In this regard, the bivalve mollusks have been increased production of reactive oxygen species widely used as sentinel species for marine health. (ROS). However, when their concentrations exceed They play an important role in the ecosystem the antioxidant capacity of the cell, they could equilibrium and constitute an important economic become harmful, causing serious damage to all bio- Corresponding Author Noureddine Soltani, Laboratory of Applied Animal Biology Faculty of Sciences, Department of Biology University of Annaba 23000-Annaba, Algeria Tel: + 773 38 76 33; E-mail: [email protected] 596 Adv. Environ. Biol., 7(4): 595-604, 2013 molecules of the cell (DNA, proteins, lipids, contents: carbohydrates) and cell death [46,7]. Defense systems that tend to inhibit reactive oxygen species Specimens of D. trunculus of standardized shell formation are include the antioxidant enzymes such size (length 27 ± 1 mm) were collected monthly from as superoxide dismutase (SOD), catalase (CAT), and January to December 2011, from the two selected glutathione S-transferase (GST). Non-enzymatic sites in the Annaba gulf. After collection, D. antioxidants such as numerous low molecular weight trunculus were transported alive to the laboratory, antioxidants, like several vitamins (vitamin B, C, E) and each species was quickly dissected and the and glutathione (GSH) were described [44]. Lipid mantle removed until biochemical analysis. peroxidation is a well-known mechanism of cellular The rate of glutathione was determined injury in vertebrates and invertebrates, and is an according to Weckberker and Cory [54]; whose indicator of an oxidative damage in cells and tissues. principle is based on the colorimetric measurement Therefore, the determination of malondialdehyde of the 2-nitro-5-mercapturic acid, resulting from the (MDA), widely used as biomarker of lipid reduction of 5, 5’-dithio-bis-2-nitrobenzoïc acid peroxidation was made in order to assess adverse (DTNB) by the thiol groups (-SH) of glutathione. effects caused by contaminants [57,53]. Each mantle sample was homogenized individually Changes in biochemical composition may be in 1 ml solution of ethylene diamine tetra-acetic important especially for survival, growth and (EDTA) at 0.02 M. To protect the thiol groups of reproduction. Furthermore, biochemical composition glutathione, the homogenate must undergo a in mollusk’s bivalve has been employed as deproteinization by sulfosalicylic acid (SSA) at 0.25 biomarker in several studies that aimed to evaluate %, where 0.2 ml of SSA was added to 0.8 ml of the impact of anthropogenic activities in the homogenate. After agitation, the mixture was environment [31]. The bivalve's biochemical immersed in an ice bath for 15 min, and then composition used under metabolic reserves forms centrifuged at 1.000 g/min for 5 min. An aliquot (500 begin preferably with the carbohydrates, where the µl) from the supernatant was added to 1 ml of free reducing sugars and polysaccharides have an Tris/EDTA buffer (0.02 M; pH 9.6) and 0.025 ml of energy storage function [56]. The lipids represent an DTNB (0.01 M). The absorbance was read at 412 nm energetic reserve because of their high caloric value wavelength, and the rate of GSH was expressed in and are mainly used in chronic stress conditions. µmol.mg-1 proteins. Phospholipids, also called structural lipids, are Lipid peroxidation was estimated by playing an important role in the cell membrane’s quantification of MDA rates in mantles using the formation [29]. Proteins, the main biochemical method of Draper and Hadley [16]. The principle of component of the organisms may be subject to the method was based on a spectrophotometric reserve or to metabolic transformation [36]. measurement of the color produced during the The present study, aimed to test two biomarkers reaction to thiobarbituric acid (TBA) with MDA. of oxidative stress (GSH, MDA), and to determine Each mantle was homogenized individually in 1 ml the main biochemical components (proteins, lipids of Tris/HCl buffer (50 mM; pH 7.5) and centrifuged and carbohydrates) in the mantle of an edible species during 10 min at 1000 g. For this purpose, 2.5 ml of Donax trunculus L. (Mollusca, Bivalvia) associated trichloroacetic acid (TCA) solution was added to 0.5 with environmental pollution in the gulf of Annaba. ml homogenate and placed in a boiling-water bath The attempt data are useful for the gulf for 15 min. After cooling in tap water, the mixture biomonitoring. was centrifuged at 1000 g for 10 min, and 2 ml of the supernatant was added to 1 ml of TBA solution and Materials And Methods placed in boiling-water bath for 15 min. The solution was then cooled in tap water, and its absorbance was Presentation of sampling sites: measured at 532 nm. The rate of MDA was expressed in µmol.mg-1 proteins. The gulf of Annaba is located in the east of Algeria. It is limited by the Rosa Cap (8° 15' E and Mantle biochemical composition: 36° 38' N) in the East and by the Garde Cap (7° 16' E and 36° 68' N) in the West. El Battah site (36°50' N Proteins, lipids and carbohydrates from mantles and 8° 50' E), is located about 30 km to the East of were extracted from the same sample following the Annaba far away from major human activities, and is procedure of Shibko et al. [40]. In brief, each mantle considered to be a relatively low polluted site. sample (weight: 30 ± 2 mg) was individually Conversely, Sidi Salem site (36° 50' N and 7° 47' E), homogenized in 1 ml of TCA (20%) and then which is located about 1 km to the East of Annaba centrifuged (5.000 g for 10 min). The supernatant city, and collect industrial and domestic wastewater, was used for the carbohydrate’s determination as is considered as a polluted area [1, 2, 8]. described by Duchateau and Florkin [17] using anthrone as reagent and glucose as standard, while Determination of glutathione and malondialdehyde the pellet added with a mixture of ether and

597 Adv. Environ. Biol., 7(4): 595-604, 2013 chloroform (1V/1V) was subjected to a second February, March, April, May, June, July, August, centrifugation (5.000 g for 10 min). The resulted October and November. However, no significant supernatant was used to quantify the lipids based on difference in the level of MDA was detected between the vanillin method of Goldsworthy et al. [22]. the two studied sites in September (p= 0.094) and Finally, a quantification of total proteins was made December (p= 0.431). according to Bradford [10] using Coomassie Bleu The analysis of seasons pooling data of GSH and Brilliant (G250, Merck) as reagent and bovine serum MDA rates by two-way analysis of variance (site, albumin (Sigma) as standard. All biochemical season) shows highly significant (p< 0.001) effects compositions (proteins, lipids, carbohydrates) were of both sites (F1, 112= 26.30; F1, 112 = 13.53) and -1 expressed in µg.mg of wet weight of tissues (w.w). season (F3, 112= 18.03; F3, 112= 11.33) for the two biomarkers GSH and MDA, respectively. Tukey's Data analysis: test made to classify seasons within each site according to their responses of GSH and MDA rates Data were expressed as mean ± standard is summarized in table 3. For GSH rates in El Battah, deviation (SD). Homogeneity of variances was tested two groups are highlighted, the first included spring, (Bartlett and Levene's test), and the normal autumn and winter; and the second included summer. probability test was used (Kolmogorov–Smirnov’s In Sidi Salem tow groups are also shown, the first test). The data that did not correspond to these contains spring, summer, and autumn; and the second features were logarithmically transformed. The contains spring, autumn and winter. The analysis of results of biochemical composition were also MDA rates regrouped into seasons, the Tukey HSD submitted to a simple linear regression to quantify test revealed that the both site’s El Battah and Sidi proteins, lipids and carbohydrates. Comparison of Salem contain similar two groups, one group with mean values was estimated by Student’s t-test. The spring, summer, winter and another group with only effects of both sampling sites (space) and months autumn. (time) were tested by a two-way analysis of variance (ANOVA). After pooling the results by season, the Amounts of proteins, lipids and carbohydrates data were analysis by ANOVA test (site, season), mantle: followed by Tukey's HSD test to classify the seasons according to their responses within each site. All Quantitative evaluation of main biochemical statistical analysis was performed using MINITAB constituents recorded in each mantle of D. trunculus Software (Version 14, Penn State College, PA, collected from two sites (El Battah, Sidi Salem) in USA). The significant level was at p<0.05. the gulf of Annaba are presented in figure 1. Monthly variation of protein amounts showed fluctuations Results: during the different months within the two sites (Fig. 1A). The comparison of protein levels between the Glutathione and malondialdehyde contents: two sites using Student’s t-test revealed a highly significant difference (p< 0.001) with generally The results of monthly variations of GSH lowers quantities of proteins in Sidi Salem amounts in the mantle of D. trunculus are individuals compared to those of El Battah. represented in table 1. The amounts of GSH in El However, the protein amounts are significantly Battah varied from 0.209 ± 0.022 in January to 1.212 higher (p< 0.001) at the Sidi Salem individuals in ± 0.202 µmol.mg-1 proteins in August. In Sidi Salem, May and August. The ANOVA shows significant they range from 0.147 ± 0.017 in June to 0.656 ± effects (p< 0.001) of both site (F1, 96= 759.06) and -1 0.118 µmol.mg proteins in August. A significant month (F11, 96= 947.32). (p<0.05) to highly significant (p< 0.001) lower rate The determination of total lipid amounts showed of GSH has been observed in Sidi Salem as a significant (p< 0.001) difference between the compared with El Battah samples according to samples of the two studied sites. Lower values were different months. Whereas, no significant difference recorded at Sidi Salem samples during the different (p> 0.05) was observed in May (p= 0.258), months except for May and August where the rate at September (p= 0.606) and December (p= 0.301). Sidi Salem became significantly higher (p< 0.001) The rates of MDA (Table 2) recorded in D. than those of El Battah (Fig. 1B). Indeed, the two- trunculus, varied in minimum values from 3.00 ± way ANOVA revealed significant (p< 0.001) effects -1 0.24 µmol.mg proteins in October at El Battah to of both site (F1, 96= 187.41) and month (F11, 96= 5.77 ± 0.61 µmol.mg-1 proteins in September at Sidi 196.70). Salem. A maximum values was observed in March in Data on total carbohydrates quantification both sites El Battah and Sidi Salem with respectively showed a significant (p< 0.001) decrease in Sidi 41.06 ± 2.66 and 47.46 ± 2.41 µmol.mg-1 proteins. A Salem values as compared with those of El Battah comparison of means values showed a highly except for May and August, which inversely the significant (p< 0.05) values in Sidi Salem than in El levels of carbohydrate are significantly higher (p< Battah during the following months: January, 0.001) in Sidi Salem individuals (Fig. 1C). This

598 Adv. Environ. Biol., 7(4): 595-604, 2013 result was confirmed by two-way ANOVA, where autumn. At the site of Sidi Salem, the first significant (p< 0.001) effects of site (F1, 96= 253.83) group contains only spring, the second summer and and month (F11, 96= 214.17) were observed. winter, and finally; the third contains the autumn Total proteins, lipids and carbohydrates (Table 4). The classification of seasons according to regrouped into four seasons (winter, spring, summer, lipid contents showed in El Battah site three groups, autumn) have submitted to variance analysis (site, the first is represented by spring, the second by season) followed by Tukey's test to classify seasons summer, autumn and the third by winter. The site of within each site according to their responses to Sidi Salem showed also three groups; a group biochemical composition of mantles of D. trunculus contains spring and summer; a group contains (Table 4). The results show site and season highly summer and autumn, and finally; a group contains significant effects (p < 0.001) in respectively, winter (Table 4). The carbohydrate contents proteins (F1, 112= 14.04; F3, 112= 32.82), lipids (F1, 112= expressed by seasons and classify by Tukey's test 14.39; F3, 112= 26.85) and carbohydrates (F1, 112= showed tow groups in both sites. In El Battah site, 14.19; F3, 112= 14.26) contents. The ranking of the first group contains spring, and the second the season by the Tukey test according to the rates of contains autumn, summer and winter. In Sidi Salem protein reveals three groups in site, the first group contains spring, autumn and El Battah and Sidi Salem. At the site of El Battah the summer, while the second group contains only winter first group contains spring, the second group summer (Table 4). and winter; the third group contains summer and

Table 1: Monthly variation of GSH (µmol.mg-1 proteins) in D. trunculus mantles collected from El Battah and Sidi Salem sites in the gulf of Annaba during 2011 (mean ± SD; n=5; for each month, values followed by the same letters are not significantly different at p> 0.05). Month El Battah Sidi Salem January 0.209 ± 0.022 a 0.165 ± 0.023 b February. 0.272 ± 0.053 a 0.198 ± 0.027 b March 0.294 ± 0.035 a 0.211 ± 0.011 b April 0.357 ± 0.022 a 0.290 ± 0.006 b May 0.496 ± 0.034 a 0.475 ± 0.021 a June 0.324 ± 0.034 a 0.147 ± 0.017 b July 0.535 ± 0.073 a 0.154 ± 0.025 b August 1.212 ± 0.202 a 0.656 ± 0.118 b September 0.362 ± 0.029 a 0.354 ± 0.008 a October 0.258 ± 0.017 a 0.187 ± 0.007 b November 0.433 ± 0.028 a 0.366 ± 0.027 b December 0.247 ± 0.031 a 0.229 ± 0.016 a

Table 2: Monthly variation of MDA (µmol.mg-1 protein) in D. trunculus mantles collected from El Battah and Sidi Salem sites in the gulf of Annaba during 2011 (mean ± SD; n=5; for each month, values followed by the same letters are not significantly different at p> 0.05). Month El Battah Sidi Salem January 8.90 ± 0.30 a 11.37 ± 1.13 b February 15.68 ± 1.87 a 18.45 ± 1.01 b March 41.06 ± 2.66 a 47.46 ± 2.41 b April 23.13 ± 0.99 a 31.06 ± 0.85 b May 15.22 ± 0.47 a 22.62 ± 0.57 b June 12.81 ± 1.66 a 17.74 ± 2.44 b July 21.77 ± 0.92 a 34.50 ± 3.52 b August 17.13 ± 1.01 a 21.03 ± 1.01 b September 5.11 ± 0.46 a 5.77 ± 0.61 a October 3.00 ± 0.24 a 15.71 ± 1.18 b November 11.58 ± 1.21 a 20.37 ± 2.71 b December 8.16 ± 0.48 a 7.90 ± 0.48 a

Table 3: Seasonal variation of GSH and MDA amounts (µmol.mg-1 protein) in D. trunculus collected from El Battah and Sidi Salem sites in the gulf of Annaba during 2011 (mean ± SD; n=5; for each biomarker and season, values followed by the same letters in minuscule are not significantly different while for each biomarker and site, values followed by the same letters in majuscule are not significantly different at p> 0.05). GSH (µmol.mg-1 protein) MDA (µmol.mg-1 protein) Season El Battah Sidi Salem El Battah Sidi Salem Spring 0.392 ± 0.08 a 0.304 ± 0.14 b 17.05 ± 4.69 a 23.81 ± 5.87 b A AB A A Summer 0.704 ± 0.39 a 0.388 ± 0.12 b 14.67 ± 5.31 a 20.40 ± 12.3 b B A A A Autumn 0.313 ± 0.09 a 0.260 ± 0.08 a 07.58 ± 3.72 a 14.66 ± 5.56 b A AB B B Winter 0.258 ± 0.05 a 0.191 ± 0.02 b 21.90 ± 14.4 a 25.80 ± 16.2 a A B A A

599 Adv. Environ. Biol., 7(4): 595-604, 2013

Table 4: Seasonal variation of main biochemical components (µg.mg-1 w.w) in D. trunculus collected from El Battah and Sidi Salem sites in the gulf of Annaba during 2011 (mean ± SD; n=5; for each biomarker and season, values followed by the same letters in minuscule are not significantly different while for each biomarker and site, values followed by the same letters in majuscule are not significantly different at p> 0.05). Protein (µg.mg-1 w.w) Lipid (µg.mg-1 w.w) Carbohydrate (µg.mg-1 w.w) Season El Battah Sidi Salem El Battah Sidi Salem El Battah Sidi Salem Spring 19.59 ± 3.32 a 17.18 ± 0.55 b 11.82 ± 6.92 a 9.79 ± 4.29 a 16.59 ± 6.21 a 13.87 ± 4.34 a A A A A A A Summer 12.86 ± 5.57 a 11.63 ± 2.78 a 8.30 ± 2.04 a 7.53 ± 1.70 a 11.84 ± 4.90 a 10.00 ± 1.64 a BC B B AB B A Autumn 11.05 ± 2.88 a 8.43 ± 2.45 b 8.65 ± 3.91 a 5.64 ± 2.24 b 13.52 ± 4.78 a 11.32 ± 4.13 a C C B B B A Winter 15.19 ± 1.43 a 11.06 ± 1.96 b 4.98 ± 1.62 a 2.81 ± 0.41 b 10.64 ± 1.63 a 6.36 ± 1.22 b B B C C B B

30 El Battah Sidi Salem 25 A

20 w.w) 1 ‐ 15 proteins

10 (µg.mg Total 5 0

30 25 B 20 w.w) 1 ‐ lipids 15

Total 10 (µg.mg 5 0

30

25 C 20 w.w) 1 ‐ 15 carbohydrates 10 (µg.mg 5 Total 0

Month

Fig. 1: Monthly variations of total proteins (A), lipids (B) and carbohydrates (C) amounts (µg.mg-1 w.w) in D. trunculus collected from El Battah and Sidi Salem sites in the gulf of Annaba during 2011 (mean ± SD; n=5).

Discussion: mixtures of toxic pollutants on marine ecosystem health. Consequently, in recent investigations, the Biological responses in bioindicator species can integration of biochemical data with biomarkers provide more complete and biologically relevant responses was strongly recommended to characterize information on the potential impact of complex the impact of pollution on organisms [19]. The gulf

600 Adv. Environ. Biol., 7(4): 595-604, 2013 of Annaba is a recipient of a large amount of also shown in C. gigas oyster exposed to diesel oil contaminants from urban, agricultural, harbour and [57] and scallop Chlamys farreri under ammonia-N industrial activities [1]. Recently, the concentrations exposition [53]. Several studies have demonstrated of the most important trace metals (Zn, Cu, Pb and that exposure to metal ions can produce lipid Cd) were measured in D. trunculus, sampled at the peroxidation by an increase of MDA rates, as in two same sites as in our present study. The results bivalve, C. farreri exposed to relevant concentrations revealed a significant seasonal effect for all metals. of lead [58]. Higher levels of MDA were also Although, highest levels were detected at Sidi Salem reported in Unio tumidus [13], P. viridis [21] and beach, essentially for Cd [8]. Indeed, Sidi Salem is Bathymodiolus azoricus [12], exposed to pollution. A located near a factory that produces phosphoric, negative correlation similar to that observed in our fertilizers and pesticides, and recent biomonitoring study (GSH vs. MDA concentration) has been studies of Annaba gulf, including several biomarkers reported by authors, such as, Doyotte et al. [15] in have shown an induction of catalase and glutathione bivalve U. tumidus, Torres et al. [48] in the mussel S-transferase, an inhibition of acetylcholinesterase Mytella guyanensis, from polluted sites and activity, and changes in biochemical composition of Richardson et al. [37] in mussel P. viridis exposed to gonads in D. trunculus from Sidi Salem as compared polycyclic aromatic hydrocarbons and to El Battah [5, 25, 41, 42]. organochlorine pesticides. An increase of MDA and Organisms contain a complex network of an depletion of GSH were also detected in oysters C. enzymatic and non-enzymatic antioxidant defense virginica exposed to cadmium [28], and clam S. system that worked together to prevent oxidative solida subjected to anthropogenic pollution [43]. damage to cellular components [44]. Among, non- In the natural environment, the seasonal cycle is enzymatic antioxidant, GSH is a tripeptide thiol, a strong determinate of invertebrate physiology which is found in large concentrations in most cells, (growth, reproduction, immunity). Changes in and plays an important role in protecting these cells environmental factors resulting from seasonal change from oxidative damage. GSH acts as a co-factor in may therefore, powerfully affect the normal phase II biotransformation reactions performed by metabolic activities of mussels [6]. Seasonal GST which cleans cells of free radicals and reactive evaluation of our results indicates that GSH levels oxygen species, and detoxifies hydrogen peroxides are lower and MDA rates higher in winter, regardless and lipid hydroperoxides through reactions of the sampling site. Therefore, it is tempting to catalyzed by glutathione peroxidase [3]. In our study, suppose that an increased of oxygen consumption the rates of GSH have decreased in Sidi Salem rate, as a consequence of energy demands related to samples comparatively to those of El Battah. Our environmental stress, might have caused generation results are agree with those of Srain and Rudolph of ROS and consequently, induction a damages [43] in clam Semele solida from Chile gulf subjected processes in cells [37]. It seems also probable that to anthropogenic pollution. Trevisan et al. [49] the important quantities of Oxygen in winter showed lowering GSH and other antioxidant enzyme increased ROS generation and reducing the cellular activities in gills of Pacific oyster Crassostrea gigas antioxidant capacity for neutralization of free exposed to chlorodinitrobenzene. A decrease of GSH radicals [4]. In most cases, an increase of lipid was also reported in clam Ruditapes philippinarum peroxidation in exposed to air was observed, exposed to combined cadmium and benzo-α-pyrene as reported in the sub-Antartic stone crab Paralomis [52] and in Perna viridis exposed to mercury [51]. granulosa [38], in the Antarctic limpet Nacella Overproduction of ROS in response to cocinna [55], and in the mussel, P. perna [4]. environmental stress can lead to increased lipid Almeida et al. [4] have hypothesized that the peroxidation (LPO) in cell’s membrane. It consists of periodic opening and closure of shells to exposed air, the destruction of the lipid membrane and the allowing to bivalves to maintain of oxygen uptake, production of peroxides and other sub-products such may lead to ROS production and cause oxidative as aldehydes. Among those aldehydes, MDA formed injury. by the rupture of unsaturated fatty acids, is used as an The biochemical composition of organisms can index for lipid peroxidation. Anthropogenic contribute not only to a better understanding of the contaminants such as metals, polycyclic aromatic constraints in the growth and reproduction success, hydrocarbons, polychlorinated biphenyls, and but also for a more effective characterization of the residues from pesticides induce lipid peroxidation organism's condition and quality. Indeed, under [43]. In our study, the rates of MDA have increased stress condition, organisms need more energy to in the tissue of D. trunculus from Sidi Salem site. detoxify the toxicants to minimize the adverse effect Higher levels of MDA in D. trunculus from a of contaminants [56]. The regulation of global polluted site in the gulf of Tunis comparatively with protein synthesis represents an important adaptive a reference site were shown by Tlili et al. [46]. The strategy for a response to cellular stress [27]. same results were reported by Bergayou et al. [9] in Moreover, at the biochemical level, changes in lipids Scrobicularia plana and Cerastoderma edule from may demonstrate the presence of metal inducing the Morocco estuary. An increase of levels of MDA was oxidative stress in organisms [20]. Furthermore,

601 Adv. Environ. Biol., 7(4): 595-604, 2013 carbohydrate metabolisms are the basic metabolic balance of animals such as bivalves as a result of pathways; the intensity and duration of these increased activation of defense and/or repair responses are influenced by the concentration of the mechanisms, which might lead to further reduction in toxicant, the time of exposure and the species studied energy available for reproduction [45]. [56]. The results of our study showed a decrease of Conclusion: proteins, lipids and carbohydrates in Sidi Salem individuals comparatively to El Battah, except in The determination of biomarkers (GSH, MDA) May and August which inversely the levels of these and biochemical composition in D. trunculus from components are higher in Sidi Salem individuals. the gulf of Annaba, show differences between the Indeed, these two months are corresponding to the two sites. Sidi Salem is exposed to anthropogenic beginning and the end of the reproduction period, pollution, while El Battah is a relatively clean site. and the high levels of these components in Sidi These diffrences suggested disturbances in the Salem during these two months may be due to the physiological mechanisms due to an environnmenatl fact that individuals of Sidi Salem which are stress induced by several pollutants. Moreover, D. subjected to pollution, need to mobilize more trunculus is suitable for use of as sentinel species for reserves and energy to assure their reproduction biomonitoring studies. The oxidative stress has been compared to individuals of El Battah. Our results are evaluated by biomarker measures, thus attesting their agree with those of Hamdani and Soltani-Mazouni usefulness as tools for assessment of pollution [25], who showed a decrease of proteins and impacts in situations where complex mixtures of carbohydrates in the gonads of D. trunculus from contaminants are present. Sidi Salem comparatively to El Battah. A decrease in protein contents was reported in several bivalve Acknowledgements species, as Mytilus galloprovincialis, Perna perna and Ruditapes decussatus from polluted sites The authors wish to thank anonymous referees [32,14,23]. Florence et al. [18] showed a lower for useful discussions and critical reading of the protein in Mytilus edulis exposed to several metals. manuscript. This study was supported by the During environmental stress, M. galloprovencialis Algerian Fund for Scientific Research and by the limited their protein synthesis [34]. Agree with our Ministry of Higher Education and Scientific results, the total lipids, were significantly lower in R. Research of Algeria (CNEPRU project F decussatus [45] from the contaminated Portugal gulf. 01120070055 and PNR project 8/u23/1024 to A depletion of lipids was reported in Zebra mussels Professor N. Soltani). Dreissena polymorpha from the polluted site in France [24]. A decrease of carbohydrates was References reported in scallop C. farreri under ammonia-N exposure [53]. Similarly, a decrease of carbohydrate 1. Abdenour, C., B.D. Smith, M.S. Boulakoud, B. metabolism in stressed bivalves has also been Samraoui and P.S. Rainbow, 2000. Trace metals described in previous reports in Anodonta cygnea in shrimps and sediments from Algerian water. [26] and R. philippinarum [33]. J. Catal. Mat. Env. 3: 9-12. The higher levels in biochemical components 2. Abdenour, C., K. Khelili, M.S. Boulakoud and recorded in spring and the lower rates in autumn and P.S. Rainbow, 2004. Trace metals in marine, winter shown in our study is probably in relation to brackish and freshwater prawns (Crustacea, the beginning and the end of the reproduction in D. Decapoda) from North-East Algeria. trunculus [30]. Hamdani and Soltani-Mazouni [25] Hydrobiologia. 432: 217-227. and Tlili et al. [47], have reported the same results in 3. Ahmed, R.G., 2005. Is there a balance between this species during the reproduction period in the gulf oxidative stress and antioxidant defence of Annaba and the gulf of Tunis, respectively. system during development. Medical Journal of Indeed, the biochemical composition data revealed a Islamic World Academy of Sciences, 15: 255- reserve recovery after winter, and a drop after 263. spawning. The reproductive cycle of bivalves is 4. Almeida, E.A., A.C.D. Bainy, A.L. Dafre, O.F regulated by several natural environmental factors Gomes, M.H.G Medeiros and P. Di Mascio, (e.g.; temperature, food availability) but exposure to 2005. Oxidative stress in digestive gland and gill chemical pollutants can also interfere [45]. The of the brown mussel (Perna perna) exposed to energy reserves such as carbohydrates, lipids and air and re-submersed. Journal of Experimental proteins are of considerable importance in Marine Biology and Ecology, 318: 21-30. reproduction and seasonal energy storage and their 5. Amira, A., K. Sifi and N. Soltani, 2011. Measure utilization by a marine bivalve mollusk is closely of environmental stress biomarkers in Donax linked to environmental conditions and the annual trunculus (Mollusca, Bivalvia) from the gulf of gametogenic cycle [47]. Exposures to chemical Annaba (Algeria). European pollutants may also have an impact on energy Journal of Experimental Biology, 1(2): 7-16.

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6. Banni, M., A. Negri, F. Mignone, H. Boussetta, digestive gland of the freshwater bivalve, Unio A. Aldo Viarengo and F. Dondero, 2011. Gene timidus. Aquatic Toxicology, 39: 93-110. expression rhythms in the mussel Mytilus 16. Draper, H.H. and M. Hadley, 1990. galloprovincialis (Lam.) across an annual cycle. Malondialdehyde determination as index of lipid PLoS ONE 6(5): e18904. peroxidation. Methods Enzymology, 186: 421- doi:10.1371/journal.pone.0018904. 431. 7. Béguel, J.P., A. Huvet, V. Quillien, C. Lambert 17. Duchateau, G.H. and M. Florkin, 1959. For and C. Fabioux, 2013. Study of the antioxidant trehalosemie of insects and its signification. capacity in gills of the Pacific oyster Crassostrea Archives of Insect Biochemistry and Physiology, gigas in link with its reproductive investment. 67: 306-314. Comparative Biochemistry and Physiology Part 18. Florence, G., J. Agnés, T. Vincent, J. Maria, P. C, 157: 63-71. Richard and R. Cosson, 2002. Influence of metal 8. Beldi, H., F. Gimbert, S. Maas, R. Scheifler and exposure on metallothionien synthesis and lipid N. Soltani, 2006. Seasonal variations of Cd, Cu, peroxydation in two bivalve molluscs : the Pb and Zn in the edible mollusc Donax trunculus oyster (Crassostrae gigas) and the mussel (Mollusca, Bivalvia) from the gulf of Annaba, (Mytilus edulis). Living resource. 15: 61-66. Algeria. African Journal of 19. Fossi Tankoua, O., P.E. Buffet, J.C. Amiard, B. Agricultural Research, 1(4): 85-90. Berthet, C. Mouneyrac, C. Amiard-Triquet, 9. Bergayou, H., C. Mouneyrac, J. Pellerin and A. 2013. Integrated assessment of estuarine Moukrim, 2009. Oxidative stress responses in sediment quality based on a multi-biomarker bivalves (Scrobicularia plana, Cerastoderma approach in the bivalve Scrobicularia plana. edule) from the Oued Souss estuary (Morocco). Ecotoxicology and Environmental Safety, 88: Ecotoxicology and Environmental Safety, 72: 117-125. 765-769. 20. Freitas, R., E. Costa, C. Velez, J. Santos, A. 10. Bradford, M., 1976. A rapid and sensitive Lima, C. Oliveira, A.M. Rodrigues, V. Quintino method for the quantification of microgram and E. Figueira, 2012. Looking for suitable quantities of protein utilizing the principle of biomarkers in benthic macroinvertebrates protein-dye binding. Analytical Biochemistry, inhabiting coastal areas with low metal 72: 248-254. contamination: Comparison between the bivalve 11. Carreira, S., P.M. Costa, M. Martins, J. Lobo, Cerastoderma edule and the Polychaete M.H. Costa and S. Caeiro, 2013. Diopatra neapolitana. Ecotoxicology and Ecotoxicological heterogeneity in transitional Environmental Safety, 75: 109-118. coastal habitats assessed through the Integration 21. Géret, F., A. Serafim, L. Barreira and M.J. of biomarkers and sediment-contamination Bebianno, 2002. Response of antioxidant profiles: A case study using a commercial clam. systems to copper in the gills of the clam Archives of Ruditapes decussates. Marine Environmental Environmental Contamination and Toxicology, Research, 54: 413-417. 64: 97-109. 22. Goldsworthy, G.J., W. Mordue and J. 12. Company, R., A. Serafim, M.J. Bedianno, R. Guthkelch, 1972. Studies on insect adipokinetic Cosson, B. Shillito and A. Fiala-Medioni, 2004. hormones. General and Effect of cadmium, copper and mercury on Comparative Endocrinology, 18: 545-551. antioxidant enzyme activities and lipid 23. Gorinstein, S., S.T. Jung, S. Moncheva, P. peroxidation in the gills of the hydrothermal Arancibia-Avila, Y.S. Park, S.G. Kang, I. vent mussel Bathymodiolus azoricus. Marine Goshev, S. Trakhtenberg and J. Namiesnik, Environmental Research, 58: 377-381. 2005. Partial characterization of proteins from 13. Cossu, C., A. Doyotte, M. Babut, A. Exinger and mussel Mytilus galloprovincialis as a biomarker P. Vasseur, 2000. Antioxidative biomarkers in of contamination. Archives of freshwater bivalve Unio tumidus in response to Environmental Contamination and Toxicology, different contamination profiles of aquatic 49: 504-510. sediments. Ecotoxicology and 24. Guerlet, E., K. Ledy, A. Meyer and L. Environmental Safety, 45: 106-121. Giamberini, 2007. Towards a validation of a 14. Dellali, M., M. Gnassia-Barelli, M. Roméo and cellular biomarker suite in native and P. Aïssa, 2001. The use of acetylcholinesterase transplanted zebra mussels: A 2-year integrative activity in Ruditapes decussatus and Mytilus field study of seasonal and pollution-induced galloprovincialis in the biomonitoring of Bizerta variations. Aquatic Toxicology, 81: 377-388. lagoon. Comparative Biochemistry and 25. Hamdani, H. and N. Soltani-Mazouni, 2011. Physiology,130 C: 227-235. Changes in biochemical composition of gonads 15. Doyotte, A., C. Cossu, M.C. Jacquin, M. Babut of Donax trunculus L. (Mollusca, Bivalvia) from and P. Vasseur, 1997. Antioxidant enzymes, the gulf of Annaba (Algeria) in relation to glutathione or field exposure in the gills and

603 Adv. Environ. Biol., 7(4): 595-604, 2013

reproductive events and pollution. Jordan. J. Mytilus galleprovindialis to environmental Biol. Sci., 4(3): 149-156. pollution: integrating the responses to oxidative 26. Hemelraad, J., H.J. Herig, E.G. Van Donselaar, stress and other biomarker responses into a D.A. Holwerda and D.I. Zandee, 1990. Effects general stress index. Aquatic Toxicology, 89: of cadmium in freshwater clams. III. Interaction 18-27. with energy metabolism in Anodonta cygnea. 36. Pytharopoulou, S., K. Grintzalis, E. Sazakli, M. Archives of Leotsinidis, C.D. Georgiou and D.L. Kalpaxis, Environmental Contamination and Toxicology, 2011. Translational responses and oxidative 19: 699-703. stress of mussels experimentally exposed to Hg, 27. Ivanina, A.V., C, Taylor and I.M. Sokolova, Cu and Cd: One pattern does not fit at all. 2009. Effects of elevated temperature and Aquatic Toxicology, 105: 157-165. cadmium exposure on stress protein response in 37. Richardson, B., E. Mak, S. de Luca-Abbott, M. eastern oysters Crassostrea virginica (Gmelin). Martin, K. McClellan and P. Lam, 2008. Aquatic Toxicology, 91: 245-254. Antioxidant responses to polycyclic aromatic 28. Lannig, G., J.F. Flores and I.M. Sokolova, 2006. hydrocarbons and organochlorine pesticides in Temperature-dependent stress response in green-lipped mussles (Perna viridis): Do oysters, Crassostrea virginica: Pollution reduces mussels “integrate” biomarker responses?. temperature tolerance in oysters. Aquatic Marine Pollution Bulletin, 57: 503-514. Toxicology, 79: 278-287. 38. Romero, M.C., M. Ansaldo and G.A. Lovrich, 29. Martinez-Pita, I., I. Hachero-Cruzado, C. 2007. Effect of aerial exposure on the Sanchez-Lazo and O. Moreno, 2011. Effect of antioxidant status in the subantarctic stone crab diet on the lipid composition of the commercial Paralomis granulosa (Decapoda: Anomura). clam Donax trunculus (Mollusca: bivalvia): sex- Comparative Biochemistry related differences. Aquatic Research, 1-11. and Physiology Part C: Toxicology and 30. Mouëza, M. and L. Frenkiel-Renault, 1973. Pharmacology, 146: 54-59. Contribution à l'étude de la biologie de Donax 39. Semadi, A. and S. Deruelle, 1993. Lead trunculus L. (Mollusques: Lamellibranches) pollution monitoring by transplanted lichens in dans l'Algérois: la reproduction. Cahiers de Annaba area (Algeria). Rev. Poll. Atmos. (Oct- Biology Marine, 14: 261-283. Dec). 35(140): 86-102. 31. Nahrgang, J., S.J. Brooksb, A. Evenseta, L. 40. Shibko, S., P. Koivistoinen, C. Tratyneck, A. Camusa, M. Jonssona, T.J. Smitha, J. Lukinaa, Newhall and L. Freidman, 1966. A method for M. Frantzena, E. Giarratanoe and P.E. Renaud, the sequential quantitative separation and 2013. Seasonal variation in biomarkers in blue glycogen from a single rat liver homogenate or mussel (Mytilus edulis), Icelandic scallop from a sub cellular fraction. Analytical (Chlamys islandica) and Atlantic cod (Gadus Biochemistry, 19: 415-428. morhua)-Implications for environmental 41. Sifi, K., S. Chouahda and N. Soltani, 2007. monitoring in the Barents Sea. Aquatic Biosurveillance de l’environnement par la Toxicology, 127: 21-35. mesure de biomarqueurs chez Donax trunculus 32. Najimi, S., A. Bouhaimaimi, M. Daubeze, A. dans le golfe d’Annaba (Algérie). Mésogée., 63: Zekhnini, J. Pellerin, J.F. Narbonne and A. 11-18. Moukrim, 1997. Use of acetylcholinesterase in 42. Soltani, N., A. Amira, K. Sifi and H. Beldi, Perna perna and Mytillus galloprovincialis as a 2012. Environmental monitoring of the Annaba biomarker of pollution of Agadir marine bay gulf (Algeria): Measurement of biomarkers in (South of Marocco). Bulletin of Environmental Donax trunculus and metallic pollution. Bulletin Contamination and Toxicology, 58: 901-912. de la Société Zoologique de France, 137(1-4): 33. Plana, S., G. Sinquin, P. Maes, C. Paillard and 47-56. M.L. Pennec, 1996. Variations in biochemical 43. Srain, B. and A. Rudolph, 2010. composition of juvenile Ruditapes Acetylcholinesterase activity, antioxidant philippinarum infected by a Vibrio sp. Diseas. defenses, and lipid peroxidation in the clam Aquatic Organisms, 24: 205-213. Semele solida: Can this species be used as a 34. Pytharopoulou, S., E.C. Kouvela, E. Sazakli, M. bioindicator?. Revista de Biología Marina y Leotsinidis and D.L. Kalpaxis, 2006. Evaluation Oceanografía, 45( Nº2): 227-233. of the global protein synthesis in Mytilus 44. Sussarellu, R., C. Fabioux, M. Camacho galloprovincialis in marine pollution Sanchez, N. Le Goïc, C. Lambert, P. Soudant monitoring: Seasonal variability and correlations and D. Moraga, 2012. Molecular and cellular with other biomarkers. Aquatic Toxicology, 80: response to short-term oxygen variations in the 33-41. Pacific oyster Crassostrea gigas. Journal of 35. Pytharopoulou, S., E. Sazakli, K. Grintzalis, Experimental Marine Biology and Ecology, 412: C.D. Georgiou, M. Leotsinidis and D.L. 87-95. Kalpaxis, 2008. Translational responses of

604 Adv. Environ. Biol., 7(4): 595-604, 2013

45. Teixeira de Sousa, J., D. Matias, S. Joaquim, R. 52. Wang, L., L. Pan, N. Liu, D. Liu, C. Xu and J. Ben-Hamadou and A. Leitão, 2011. Growth Miao, 2011. Biomarkers and bioaccumulation of variation in bivalves: New insights into growth, clam Ruditapes philippinarum in response to physiology and somatic aneuploidy in the carpet combined cadmium and benzo[a]pyrene shell Ruditapes decussatus. Journal of exposure. Food and Chemical Toxicology, 49: Experimental Marine Biology and Ecology, 3407-3417. 406: 46-53. 53. Wang, Z., C. Yan, C.D. Vulpe, Y. Yan and Q. 46. Tlili, S., I. Métais, H. Boussetta and C. Chi, 2012. Incorporation of in situ exposure and Mouneyrac, 2010,. Linking changes at sub- biomarkers response in clams Ruditapes individual and population levels in Donax philippinarum for assessment of metal pollution trunculus: assessment of marine stress. in coastal areas from the Maluan Bay of China. Chemosphere., 81: 692-700. Marine Pollution Bulletin, 64: 90-98. 47. Tlili, S., I. Métais, N. Ayache, Boussetta and C. 54. Weckberker, G. and G. Cory, 1988. Mouneyrac, 2011. Is the reproduction of Donax Ribonucléotide reductase activity and growth of trunculus affected by their sites of origin glutathione depleted mouse leukemial 1210 cells contrasted by their level of contamination?. in vitro. Cacer letters., 40: 257-264. Chemosphere., 84: 1362-1370. 55. Weihe, E., M. Kriews and D. Abele, 2010. 48. Torres, M.A., C.P. Testa, C. Gaspari, M.B. Differences in heavy metal concentrations and in Masutti, C.M.N. Panitz, R. Curi-Pedrosa, E.A. the response of the antioxidant system to Almeida, P. Di Mascio and D.W. Filho, 2002. hypoxia and air exposure in the Antarctic limpet Oxidative stress in the mussel Mytella Nacella concinna. Marine Environmental guyanensis from polluted mangroves on Santa Research, 69: 127-135. Catarina Island, Brazil. 56. Xuan, R., L. Wang, M. Sun, G. Ren and M. Marine Pollution Bulletin, 44: 923-932. Jiang, 2011. Effects of cadmium on 49. Trevisan, R., M. Arl, C.L. Sacchet, C.S. Engel, carbohydrate and protein metabolisms in the N.M. Danielli, D.F. Melloa, C. Brocardob, A.F. freshwater crab Sinopotamon yangtsekiense. Marisc and A.L. Dafrea, 2012. Antioxidant Comparative Biochemistry deficit in gills of Pacific oyster (Crassostrea and Physiology Part C, 154: 268-274. gigas) exposed to chlorodinitrobenzene 57. Zanette, J., E.A. Almeida, A.Z. da Silva, J. increases menadione toxicity. Aquatic Guzenski, J.F. Ferreira, P. Di Mascio, M.R. Toxicology, 108: 85-93. Freire Marques and A.C.D. Bainy, 2011. 50. Vaissiére, R. and G. Fredj, 1963. Contribution à Salinity influences glutathione S-transferase l'étude de la faune benthique du plateau activity and lipid peroxidation responses in the continental de l'Algérie. In: Mouëza M., 1971. Crassostrea gigas oyster exposed to diesel oil. Contribution à l'étude de l'écologie et de la Science of the Total Environment, 409: 1976- biologie de Donax trunculus L. (Mollusque 1983. lamellibranche). Thèse de Doctorat en Sciences 58. Zhang, Y., J. Songa, H. Yuana, Y. Xua, Z. Hec Biologiques. Mention Océanographie., p: 130. and L. Duana, 2010. Biomarker responses in the 51. Verlecar, X., K. Jena and G. Chainy, 2007. bivalve (Chlamys farreri) to exposure of the Biochemical markers of oxidative stress in environmentally relevant concentrations of lead, Perna viridis exposed to mercury and mercury, copper. Environmental temperature. Chemico-Biol Interactions, 167: Toxicology and Pharmacology, 30: 19-25. 219-226.