Marine ecosystems, particularly lagoons, are of great ecological and economic importance becausethey support vital habitats for numerous marine organisms. However, they sustain several anthropogenic pressures (Barhoumi et al. 2014). In fact, increasing industrialization has led to a massive release of pollutants into these enclosed ecosystems (Di Salvatore et al. 2013). During the last few decades, the aquatic environment in Tunisia, especially the north coast, has been contaminated by trace element (TE) pollutants as a result of rapid industrialization and urbanization (Mzoughi and Chouba 2012, Daldoul et al. 2015). In recent years, the discharge of industrial wastewater containing a high level of TEs has increased (Ghribi et al. 2020). The TE content in sediments from the Tunisian gulfhas been observed to reach a considerable level that could be released into the water by flow changes or benthic agitation, causinga sustained impact on aquatic organisms and eventually on human beings through the food chain (Ennouri et al. 2016).

Among these contaminants, metals and metalloids are a particular concern because of their toxic effects and their persistence through bioaccumulation and biomagnification along the food chain (Leung et al. 2016, Faggio et al. 2018). From an ecotoxicological point of view, it has been demonstrated that TEs can adversely affect aquatic organisms mainly through an excessive generation of reactive oxygen species (ROS) (Telahigue et al. 2018, Yang et al. 2018). Indeed, ROS are proven to cause metabolic and oxidative homeostasis imbalances because of their high reactivity towards cellular components such as lipids, proteins and nucleic acids (Bejaoui et al. 2020). To protect cells against ROS, some specific antioxidants such as superoxide dismutase, catalase (CAT), glutathione peroxidase (GPx), thiols as glutathione (GSH) and ascorbic acid (Vit C) are also elevated in the detoxification of free radicals (Barhoumi et al. 2014). Oxidative stress can also be reduced through the complication of free TEs by metallothionein (MT). The latter is an oxyradicalscavenger playing an important role inthe homeostasis of essential TEs and in the detoxification of non-essential ones (Gagné et al. 2008). Histopathology is also a powerful tool for monitoring anthropogenic contamination (El-Shenawy et al. 2009). According to Bejaoui et al (2020), the histological study could provide information on the adaptive response to environmental pollution and cell damage. In marine ecosystems, histopathological alterations have been associated either with deterioration of environmental conditions or with pollution (Fanta et al. 2003, Marchand et al. 2009).

During the last few decades, marine invertebrates, especially bivalve molluscs, have been widely used as sentinel species for aquatic pollutants associated with ROS generation (Tsangaris et al. 2016, Uluturhan et al. 2019, Bejaoui et al. 2020). These sessile filter-feeder organisms are commonly used to assess the biological effects of contaminated ecosystems (Cravo et al. 2012). The clam Ruditapes decussatus is among the most common marine molluscs in Tunisia and around the world. It is an important worldwide economic resource and has been widely used as a bioindicator for monitoring water quality in numerous environmental investigations (Campillo et al. 2013, Mansour et al. 2020, Bejaoui et al. 2020).

There are numerous studies focusing on TEs contamination in sediments and marine organisms from the northern coast of Tunisia (including the Tunisian Gulf and the Bizerte lagoon) (Chalghmi et al. 2016, Ghannem et al. 2016). However, investigation of the effect of TE contamination in bivalves from Ghar el Melh and the Northern and Southern Lagoons of Tunisia has so far been insufficient. Previous studies have integrated the toxicity responses provided by the clam R. decussatus and contamination to assess the quality of some Tunisian lagoons (Chalghmi et al. 2016). The current study is aimed at comparing the environmental quality of three lagoons with different contamination levels, a less contaminated area (LCA), a moderately contaminated area (MCA) and a highly contaminated area (HCA) on the northern Tunisian coastline by integrating water parameters, TE accumulation, biological responses, macromolecules and tissue injuries using the digestive gland of R. decussatus.

Owing to their sedentary nature and filter-feeding behaviour, bivalves are particularly exposed to anthropogenic contaminants that enter coastal environments. These organisms have a great ability to accumulate TEs in their tissues and some species may even thrive in highly polluted environments (Cantillo 1998). In the current study, the recorded levels of TEs (Zn, Cu, Pb, Fe and Cd) in R. decussatus digestive glands collected from three sites impacted by different human activities provide evidence for the ability/capacity of this organ to accumulate TEs. The inter-relationship for essential (Fe and Zn) and non-essential (Pb, Cd, and Cu) TEs accumulated in R. decussatus digestive gland strongly indicates a similar pathway of TE uptake. The results obtained showed that TE concentrations in R. decussatus were significantly higher in clams collected from the MCA and the HCA. Bivalves can absorb TEs directly from the surrounding environment, both by absorption and/or adsorption from interstitial water and by direct ingestion from sediment, and these TEs may influence their metabolism and other species via the food chain (Chen et al. 2007).

Several investigations have demonstrated that sediments from the HCA are highly impacted by TEs. Their accumulation rate in R. decussatus digestive gland must depend on their filtration, ingestion rates, gut fluid quality and detoxification strategies (Chouba et al. 2010, Bejaoui et al. 2020). Our data match well with previous studies that reported a major accumulation of TEs, especially Pb, in the sediments of the central area of the Gulf of Tunis (Ennouri et al. 2010, Chalghmi et al. 2016) and in others organisms such as green macroalgae (Chouba et al. 2010) and mollusks (Brahim et al. 2015, Lahbib et al. 2016, Bejaoui et al. 2017) following the discharge of TEs from several anthropogenic activities such as harbours, industrial and electrical discharges (Bejaoui et al. 2020).Similar results have demonstrated that TE accumulations in R. decussatus is associated with the anthropogenic pollution of the Tunisian coasts (Chalghmi et al. 2016, Bejaoui et al. 2020) and other Mediterranean areas (Figueira and Freitas 2013, Gabr et al. 2020).

The current study has also revealed that TE concentrations in the digestive glands of R. decussatus from the MCA and the HCA vary considerably depending on seasons, showing higher trends of Cd, Pb, Cu, Zn and Fe in summer and winter. Such findings could be explained by the fact that in summer the low hydrodynamics of these lagoons and the high evaporation rate might contribute to the concentration of TEs in the water column and their deposition on the surface sediments and consequently on the bivalve tissues (Ghribi et al. 2020). However, in winter the current and wave activity agitates the sediments and transports the TEs in the aqueous phase, leading to their bioavailability and their uptake by the bivalves. This increase in winter could be due to the possible discharge of TE concentrations into the waters of the MCA and the HCA or to runoff from adjacent farmland, because these sampling areas are located near the conurbation of Tunis city (Chouba et al. 2010, Chalghmi et al. 2016). Our results were confirmed by the characteristics of the lagoon, which is a laminar medium with an average bathymetry not exceeding one metre and prevailing drastic conditions, such as abrupt and very great spatiotemporal fluctuations of abiotic parameters. In addition, exchanges with the sea are weak and the hydrodynamics are highly attenuated, especially in the coves, where the waters are almost stagnant (Ben Mosbah et al. 2010). Furthermore, recent anthropogenic activities such as industrial wastewater discharges (e.g. CHOTRANA) and sediment excavation can also alter the hydraulic loading and TE source at these two sites (Ben Mosbah et al. 2010).

In addition, the environmental conditions such as temperature, salinity, pH and chlorophyll a are considered the main factors influencing TE concentrations in bivalves, affecting their speciation, solubility and reaction rates in the water column (Frías-Espericueta et al. 1999, Ghribi et al. 2020). As shown in our present work, TE showed positive correlations with T°C, salinity and suspended particulate matter (p<0.01; r≤0.451) (see Supplementary Material Table S4). Similar works have reported on Mytilus galloprovinciallis from Algerian coasts (Rouane Hacene et al. 2015) and Bizerte lagoon (Kefi et al. 2015) and on Arca noae from Bizerte lagoon (Ghribi et al. 2020). The seasonal variations in TE concentrations could also be related to the physiological status of bivalves and their reproductive cycle (Bordin et al. 1992, Regoli and Orlando 1994). According to several studies, bivalves accumulate high TE contents during maturation and less during the spawning period. The reproductive cycle of R. Decussatus is characterized by a maturation phase followed by an extended spawning phase during summer (Fradi 2012). A new ripening phase takes place in winter, especially for the clams from the HCA, followed by a short massive spawning in late November and early December (Hmida 2004). These dynamisms of clam maturation may explain the higher accumulation of TEs during the summer and winter, especially for these collected from the HCA.

The bioaccumulation of TEs in bivalve species depends not only on direct exposure to the contaminant and its environment, but also on different physiological and biochemical activities through which a specific organism treats micronutrients (Nicholson and Lam 2005). Likewise, bivalves have seasonal changes in their biometric parameters, especially during the warm season. Previous findings on bivalves demonstrate that the decline of biometric indices, such as the CI, is a consequence of anthropogenic activities in the ecosystem (Nicholson and Lam 2005, Bejaoui et al. 2017). It appears that TE uptake could be one of several factors influencing the CI and GI of clams sampled from the MCA and the LCA. Our results agree with several studies carried out on TE accumulation in bivalve tissues (Sabatini et al. 2011, Marques et al. 2016).

The application of the TESVI and THQ could possibly demonstrate suitable tools for the monitoring programmes, in order to define the anthropogenic pollution sources of the study sites. In fact, the TESVI has been determined in R. decussatus for the first time, providing an operational system that highlights the most environmentally challenging elements and allows comparisons of TEs based on the overall spatial variability of their environmental levels throughout the study areas (Richir and Gobert 2014). Our results indicate that the high TESVI values recorded for Pb correlate with our previous data, showing that it has a larger spatial variation than the other TEs. Regarding the THQ, our data indicate that all R. decussatus investigated were safe for human consumption regarding the levels of Zn, Cu, Fe, Cd and Pb (which were below 1), as already stated by Vieira et al. (2011).

It has been widely reported that TEs in bivalves are capable of stimulating ROS production and inducing MT production (Cipro et al. 2017). MTs are low molecular weight peptides rich in cysteine, which work against the toxicity caused by TEs and are involved in detoxification processes (Viarengo et al. 1985). These metallo-proteins are known for their protective free radical scavenging activity, playing an active role in the capture of harmful oxidant radical species (Kumari et al. 1998, Telahigue et al. 2018). The observed induction of MTs in R. decussatus when exposed to environmental pollution probably shows an adaptive response of the digestive gland to the toxicological manifestation induced by TEs, especially Cd and Pb. Our results are in agreement with previous studies carried out by Rabei et al. (2018) on Donax trunculus collected seasonally from polluted sites in the gulf of Annaba and Bejaoui et al. (2020) on R. decussatus collected from Tunisian lagoons.

Stimulation of MTs is related to the protection against oxidative stress and macromolecule damage (Baltaci et al. 2017). TE accumulation generates an intracellular imbalance and stimulates ROS, leading to the appearance of lipids, proteins and DNA damage through the generation of hydroxyl and free radicals from H₂O₂ via the Fenton reaction (Thomas et al. 2013). As a result of massive free radical generation, MDA performs as second toxic messengers and can form during lipid peroxidation (Cravo et al. 2012, Telahigue et al. 2018). Our results showed an increase in the MDA level in the digestive gland of R. decussatus collected from the HCA and the MCA, particularly during the warm season. This increase appears to be due to TE uptake, which accelerates the ability to scavenge ROS through the extreme production of lipid peroxides in the digestive gland. In earlier studies, similar increases in MDA levels have been reported in Fulvia fragilis from the Bizerte lagoon (Mahmoud et al. 2010), R. decussatus from the Ria Formosa lagoon (Cravo et al. 2012) and Aulacomya atra from the South Atlantic Patagonian coast (Di Salvatore et al. 2013). Furthermore, many studies have established a clear link between lipid peroxidation and genotoxic diseases (Marnett 2002, Javed et al. 2016, Bejaoui et al. 2020). In fact, MDA appears to be an important contributor to DNA damage and mutations, as reported by Ayala et al. (2014). In our study, the increase in MDA levels may be considered a significant endogenous source of DNA damage. This finding was confirmed by a significant degradation of the DNA contents in the R. decussatus digestive glands collected from the MCA and the HCA. The alteration of the DNA in clams’ digestive glands from the HCA indicates how bivalves are experiencing pollution stress, in view of the fact that this area has long been receiving untreated waters and harbour discharges (Ennouri et al. 2016). Similarly, high DNA alteration rates in aquatic organisms collected from different contaminated sites have been observed (Bejaoui et al. 2018).

The potential of TE accumulation causing adverse effects in the environment and its constituents has been well reported for the tissues of organisms (Mahmoud et al. 2010, Bejaoui et al. 2018). Proteins are one of the main targets of the effects of TEs (Bejaoui et al. 2020). Oxidation is the major consequence of protein damage both externally and within cells. The occurrence of protein oxidation in the digestive gland of R. decussatus was also confirmed by the level of AOPP, which reflects an excess of free radical generation (Prevodnik et al. 2007). The comparison between sites showed higher AOPP levels in the digestive gland of clams from the MCA and the HCA than in those from the LCA, suggesting the important role of TEs in the exertion of protein oxidation (Rabei et al. 2018).

Protein oxidation can lead to amino acid changes leading to the formation of carbonyl and other oxidized moieties, as well as to the failure of sulfhydryl groups (Bainy et al. 1996). These groups, including thiols, can react with free radicals and with products of lipid peroxidation to protect cells against the development of oxidative stress (Tandon et al. 2002, Carmeli et al. 2008). Among them, the glutathione status is one of the first lines of defence including GSH and NPSH. These antioxidants are the most abundant thiols in the cell and are considered the main cellular redox barrier. In our study, specimens from the MCA and the HCA exhibited higher GSH and NPSH levels over the year than those from the LCA. This increase can be explained by the new synthesis of GSH and NPSH as an adaptive response in addition to ROS generation. Our results are in accordance with those of previous studies carried out on bivalves and show the increase of the GSH and NPSH levels in relation to seasonal change and TE exposure (Mahmoud et al. 2010, Augustyniak et al. 2009).

Vitamin C is a powerful reducing agent and an important hydrophilic vitamin for animals and humans. It plays an important protective role in the damage induced by free radicals scavenging hydroxyls, radicals and singlet oxygen (Tandon et al. 2002). The increase in Vit C levels in the digestive gland of R. decussatus from the MCA and the HCA could be due to the crucial role of this antioxidant in protecting the physical status of organisms exposed to TE pollution (Chetoui et al. 2019). In line with our data, Bejaoui et al (2020) showed a similar variation in Vit C levels in R. decussatus sampled from the HCA. Conversely, Jena et al. (2009) showed a decrease in the Vit C level in P. viridis digestive gland collected from a polluted site. In fact, these authors explained the decrease in Vit C by the availability of glutathione to reduce the dehydroascorbate into ascorbate as a result of the high ROS generation.

To mitigate damage to the lipid membrane, bivalves are capable of enhancing their antioxidant defence systems, such as CAT and GPx enzymes (Regoli and Giuliani 2014). Several studies have demonstrated that antioxidant enzymes could protect bivalves from the toxicity of environmental pollutants. In the present work, clams collected from the MCA and the HCA exhibited increases in GPx and CAT activities. These findings highlight the ability of R. decussatus to quench ROS overproduction and to alleviate cellular injuries. Our results are in total agreement with the increase in CAT and GPx activities in Tapes philippinarum from the Adriatic Sea (Bocchetti et al. 2008), in Aulacomya atra from the Nuevo Gulf in Northern Patagonia (Giarratano et al. 2014) and in Fulvia fragilis in the Bizerte lagoon (Mahmoud et al. 2010) in relation to high TE concentrations and chemical pollutants.

The effect of environmental pollution on digestive gland mechanisms was also assessed through the AChE activity. In fact, AChE is present in the neuromuscular junctions and cholinergic synapses of the central nervous system and terminates the signal transmission by hydrolysing acetylcholine (Ach), a neurotransmitter that conducts nerve impulses across neuromuscular junctions (Lodish et al. 2000). TEs show a noticeable affinity with the sulphur donor, which binds to the thiol residues of proteins (Viarengo 1985). Our results confirm that the metallic pollution found in the MCA and the HCA affected the cholinergic system, as demonstrated by a significant inhibition of AChE in the digestive gland of R. decussatus. Tankoua et al (2012) suggested that the decrease in AChE activities could be induced by the accumulation of TEs (such as Cd, Cu and Zn) in Scrobicularia plana on the French coast.

Oxidative stress responses in R. decussatus digestive glands resulting from the combination of TE accumulation and abiotic factors may cause injuries in tissues. The study of histological injuries induced by prolonged exposure to environmental conditions is considered a successful approach for the assessment of the environmental quality of an area (Cappello et al. 2013, Vajargah et al. 2018). It is well established that the enhancement of antioxidant responses in the digestive gland may trigger a cascade of events, eventually leading to cell death. Clams collected from each of the three sampling areas exhibited pathological changes. The occurrence and severity of abnormalities showed fluctuations throughout the sampling seasons. The investigation of the interaction between the location and pollution type showed that the rate of histopathological lesions in the digestive glands was highest in clams from the HCA moderate in clams from the MCA and lowest in clams from the LCA.

The two affected clam populations were characterized by the presence of haemocyte infiltration and epithelial cell degradation during winter and autumn. This phenomenon has been well described as one of the responses to diverse environmental stressors (Pirrone et al. 2018). It has previously been established that haemocyte infiltration is the cause of the inflammatory process due to a rapid release of pro-inflammatory and vasoactive mediators (Bouallegui et al. 2018). Furthermore, atrophic tubules were observed in the same seasons and were characterized by a decrease in the density of the epithelia followed by the extension of the digestive lumen. Also, necrosis and fibrosis were observed, confirming the cell membrane rupture and the inadequate DNA degradation in the digestive gland of clams from the HCA. These abnormalities are known to be induced by exposure to a variety of pollutants (such as polycyclic aromatic hydrocarbons and TEs) and untreated municipal wastewater (Schlacher et al. 2007, Sonawane 2015, Osman et al. 2017). These abnormalities could be correlated to TE uptake but also to seasonal variability of water parameters (Kolyuchkina et al. 2017). Our results were confirmed by the existence of several probable sources of contaminants within the HCA, including the watershed, a wastewater treatment plant, harbour activities and industrial and domestic waste (Ben Ayed et al. 2012, Ennouri et al. 2016). In addition, our results were similar to those of previous reports showing that the histological abnormalities can be harmful when the non-essential TE levels exceed those required for the normal metabolic function (Yee-Duarte et al. 2018). Similar studies reported changes in the histological structure of some bivalves in relation to TE pollution and seasonal variation (Costa et al. 2013, Bejaoui et al. 2020).

The general analyses of data by PCA indicate a significant separation among sampling periods and lagoons. This study established a clear seasonality of the abiotic factors, TEs and biological parameters tested as biomarkers in clams from different lagoons. Indeed, clams’ digestive glands from the HCA were characterized by elevated concentrations of TE, confirming their metabolic responses in relation to TE accumulation. Homogeneity in response to TE may also be due to a combination of geomorphologic and hydrologic factors characterizing the HCA. Indeed, this lagoon is a semi-enclosed basin with a high input of anthropic discharges, backwater and relatively high water mixing between sites (Chouari 2015, Ennouri et al. 2016). Previous reports have described that the circulation of water in the HCA area is strongly limited by the shallow depth and by the very irregular shape of the banks (Chouari 2015). Furthermore, these important oxidative responses to TEs in the clam digestive glands from the HCA could be associated with the filtration rate of the sediment particles, which have a high level of TEs, brought from the Tunis Gulf by currents (Ennouri et al. 2010, Zaaboub et al. 2014). In addition, a marked effect of seasonality was detected for clams from the HCA, especially during the summer season. These results indicate that R. decussatus was more influenced by the seasonal variations in environmental parameters such as temperature, salinity and food availability (suspended matter, etc.). Indeed, the HCA was characterized by a black sticky mud with a strong smell of hydrogen sulphide and rich seaweeds with some fragments of shells and water stagnation (Kochlef 2003).

Based on the results of the investigated biochemical biomarkers (MDA, MTs, AOPP, GSH, NPSH and Vit C), we conclude that the oxidative stress generated in the digestive gland of R. decussatus is probably related to the seasonal variation in TEs and environmental parameters. However, in the future, similar studies should be conducted for other lagoons along the Tunisian coasts using R. decussatus as a sentinel species, reflecting the impact of organic and inorganic pollutants according to seasonal variation. Further investigations on the organic and inorganic pollutants in the sediments are still required to provide a global view of the health status of these coastal environments. These data could also provide us with a time-integrated picture concerning the toxicity of TEs in R. decussatus and the capacity of this bivalve to regulate or accumulate TEs.