The daily discharge of pollutants from human activities such as industry, agriculture, shipping, ports, domestic waste and anthropogenic pressure is a serious problem that has harmful effects on aquatic organisms (Paraskevopoulou et al. 2014). Many animals and plants living in polluted environments are often exposed to complex mixtures of chemical contaminants (Sureda et al. 2011), and these species have been proposed as a biomonitor of pollutants in coastal areas (Bodin et al. 2011). Polychaetes (Annelida) have been used as sentinels in coastal studies for monitoring purposes, especially in soft-bottom habitats (Li et al. 2012). These worms are one of the most important groups in terms of diversity and abundance in macro-benthic communities in coastal and marine environments, (Chouikh et al. 2019). They play a vital role in ecosystem functioning, not only because of their dominance, but also because of their different feeding modes and reproductive strategies (Han et al. 2016).

Scientists and environmental managers are currently working for a more specific concept of biomonitoring of the marine environment based on the study of the biological response by analysing bioaccumulation of metals, which are of particular concern due to their toxic effect, their persistence and their tendency to bioaccumulate and spread throughout food chains (Yi et al. 2008). Due to the complex stress responses influenced by metals, metalloids and their mixtures, it is important to create a battery of biomarkers that represent different roles and biological levels, such as the variation of oxidative stress (Di Salvatore et al. 2013, Bejaoui et al. 2017) and trace element concentration (Di Salvatore et al. 2013, Sureda et al. 2011), which can be linked to whole-organism responses and ecological responses to indicate the presence of harmful substances in the marine environment (Cravo et al. 2012). The toxicity of metals has been attributed mainly to their capacity to increase the generation of reactive oxygen species (ROS), which are thought to be the main cause of lipid and protein peroxidation (Chetoui et al. 2019). To neutralize free radicals and counteract the deleterious effects of ROS, cells possess an antioxidant defence system (Halliwell 2007).

In this study, the marine polychaete Perinereis cultrifera (Grube, 1840) was used as a biological model. It is present on the northwestern coasts of Europe, the Mediterranean Sea, the Atlantic, and the Indian and Pacific Oceans (Durchon 1957, Wu et al. 1985). In Tunisia, it is widespread but poorly documented (Zghal and Ben Amor 1989). According to the literature, P. cultrifera is an intertidal species, inhabiting rocky coasts under stones and boulders, usually where there is an accumulation of sediment (Scaps et al. 1992). Other studies have shown that this species can live in muddy sediments (Ghirardini et al. 1999). The mode of reproduction of P. cultrifera differs widely according to the geographical situation of the populations. It has a lifespan of three years and reproduces by epitokous or atokous (Scaps et al. 1992, Rouabah and Scaps 2003). In Tunisia, no studies on the reproduction of P. cultrifera have been carried out.

Several studies have reported that P. cultrifera is a suitable bioindicator species of marine pollution (Guemouda et al. 2014, Snani et al. 2015, Meghlaoui et al. 2015). However, no study has been carried out on P. cultrifera responses to anthropogenic pollution on the Tunisian coast. The present study is the first to evaluate the effect of environmental trace element bioaccumulation on the redox status system (lipid peroxidation, neurotoxicity and biomarker responses) and the biochemical reserves in whole tissues of P. cultrifera from two sites in relation to seasonal variation.

Over the past decades, aquatic ecosystems have suffered from an increase in anthropogenic pollution due to the presence of toxic contaminants (hydrocarbons, pesticides and heavy metals) and other environmental factors (hypoxia, changes in temperature and salinity) (Campillo et al. 2013). Several studies have revealed the effectiveness of marine invertebrates such as P. cultrifera as a bioindicator and sentinel organisms for toxicological studies in coastal waters (Guemouda et al. 2014, Snani et al. 2015, Meghlaoui et al. 2015). They are known to accumulate metals in their tissues and survive in contaminated environments, and they are herbivores, have sensitivity and resistance to contaminants and are relatively easy to collect, handle, culture and transport (King et al. 2004).

Spatial and temporal variations in metal concentrations were observed between the two sampling sites of the port of Rades (S1) and the Punic port of Carthage (S2), most likely resulting from the high temperature and evaporation, because in summer metal accumulation shows a significant positive correlation with water parameters such as temperature (p<0.05). The difference in accumulation of metals in the two populations probably indicated the anthropogenic discharges of industrial activities and maritime traffic in the port of Rades. In agreement with this hypothesis, several studies have shown a high accumulation of metals in polychaetes during the warm season. Rouhi et al. (2007) demonstrated that in spring and summer metals were accumulated mostly in the whole soft tissues of two polychaete species (Sabellaria alveolata and Arenicola grubii) collected from the coastline of Jorf Lasfar, Morocco. These findings are probably related to environmental factors and the physiological activity of the animals. Furthermore, Bat et al. (2019) showed a significant difference of metal accumulation between seasons in polychaetes on the shores of Sinop in the Black Sea.

As a metal binding protein, metallothionein (MT), a non-enzymatic antioxidant rich in cysteine and with a low molecular weight, plays a key role in cell defence in addition to ROS generation (Viarengo et al. 1999). Our data showed a higher MT level in S1 than in S2, probably reflecting an adaptative response of the sampled species to the toxic effects induced by trace metal accumulation. This result was confirmed by a positive correlation between MT level and the tested metals in P. cultrifera tissue (Cd: 0.510; Zn: 0.418). Our results were correlated with those of Suriya et al. (2012) and Won et al. (2008), who demonstrated that trace metal accumulation can generate ROS in Capitella capitela and Perinereis nunta, which in turn contributes to an enhancement of MT level.

Environmental agents such as metals generate intracellular imbalance and stimulate ROS, causing oxidative stress. The biological targets for these highly reactive oxygen species are lipids and proteins, which cause oxidations through the generation of hydroxyl and free radicals from H₂O₂ via the Fenton reaction, and can adversely affect cells by producing lipid peroxidation of intracellular membranes (Bejaoui et al. 2019). There is considerable evidence that lipid peroxidation, as measured by TBARS levels, could be proposed as a biomarker of membrane lipid damage because it is considered a by-product of lipid oxidative damage (Freitas et al. 2012). Our data show lower lipid content in S1 than in S2, which correlates with the increase in the TBARS level mainly in summer. This increase in the TBARS level and decreases in lipid content appears to be due to metal uptake, which accelerates the ability to scavenge ROS by extreme production of lipid peroxides resulting from alteration of membrane fluidity and integrity (Chetoui et al. 2019). Previous studies conducted on polychaetes such as Hediste diversicolor from contaminated sites on the southwestern Iberian coast (Gomes et al. 2013) and the Oued Souss estuary in the Bay of Agadir, Morocco (Ait Alla et al. 2006) revealed similar increases in TBARS levels. This may be linked to several factors, including metal concentration (Cd, Pb, etc.) and environmental changes (temperature, salinity, etc.), as described by Sun and Zhou (2008) and Freitas et al. (2015) on Hediste diversicolor and Diopatra neapolitana, respectively. Also, Zhao et al. (2013) found that a high salinity level may increase the potential ecological risk of metal accumulation by generating chemical mobility, thus crossing the biological barriers and interacting with the components of the cellular membranes, causing alterations in their permeability and lipid peroxidation. Our results also showed a significant positive correlation between TBARS levels and Cd (0.735), salinity (0.420) and temperature (0.641) for polychaetes collected from S2.

Several studies have demonstrated that trace metal accumulation in marine organisms alters their biochemical composition, including proteins and glycogen (Bejaoui et al. 2019). Among these macromolecules, proteins are abundant in biological tissues, having high rate constants for reaction with many species, which is why these proteins are the main target for biological oxidants (Wang et al. 2019). Therefore, oxidant formation is the main consequence of protein damage both externally and within cells. The occurrence of protein reduction in the P. cultrifera tissues from S1 was probably due to excess free radical generation under metal accumulation. Both high lipid peroxidation and protein oxidation may be due to metal contamination, as reported in Mytillus galloprovincialis by Bouki et al. (2013), who suggested that metal contamination affects the amount of protein and increases TBARS levels. Our data show a similar glycogen content between the two sampled harbours in summer and winter, whereas in spring and autumn content was higher in S2. Our results were in accordance with those of previous studies carried out on aquatic organisms, which showed that proximate composition decreased when species were exposed to anthropogenic pollution (Douhri and Sayah 2009, Bejaoui et al. 2019).

The effect of environmental metal pollution on P. cultrifera tissue mechanism was also assessed through AChE activity. 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). Its inhibition can be seen as an early warning biomarker of contaminant risks (Cravo et al. 2012). Our results showed inhibition of AChE activity in specimens taken from the port of Rades. Snani et al. (2015) suggest that the decrease in AChE release could be induced by the accumulation of trace metals in Perinereis cultrifera from the eastern coast of Algeria. Similar observations have been reported in Hediste diversicolor and Patella vulgate collected from polluted sites of Bay of Tangier in Morocco (Douhri and Sayah 2009). In addition, our results showed increases in AChE activity in winter and decreases in summer. Moulton et al. (1996) showed that high environmental temperature caused the inhibition of AChE activity in Elliptio complenata mussels. Additionally, Scaps and Borot (2000) demonstrated that salinity and temperature can also affect acetylcholinesterase activity in Hediste diversicolor.

At the cellular level, the functional changes in redox statistics induced by ROS production leads to alterations in cellular metabolism. This damaging action greatly increases the responses of enzymes antioxidants such as SOD, GST CAT and GSH levels (Cravo et al. 2012). SOD, CAT and GST are three major antioxidant enzymes in the cells. #SOD is the primary defence line against oxygen‐derived free radicals and catalyses the dismutation of the superoxide anion (O₂ ^−·) into hydrogen peroxide (H₂O₂), which changes into H₂O and O₂ through CAT# (Ma et al. 2017). CAT is one of the enzymes involved in reduction of H₂O₂ via the Fenton reaction (Chance et al. 1979). GST protects cells against trace element-induced toxicity by deactivating the cytotoxic and genotoxic compounds, catalysing the S-conjugation and reducing GSH (Salinas and Wong 1999).

We found higher SOD and CAT activity in S1 than in S2, most probably reflecting an adaptive response of those species to remove the harmful effects of ROS generation against environmental stressors, as reported by Carregosa et al. (2014). In addition, increased activities have been reported in several fish and invertebrate species collected from polluted sites (Jebali et al. 2007, Bejaoui et al. 2020). As shown in the study of Ait Alla et al. (2006), these enzymatic activities increased in Hediste diversicolor collected from the Oued Souss (Bay of Agadir, Morocco) before implantation of wastewater treatment. Douhri and Sayah (2009) also reported an increase in CAT activity in two marine invertebrates, Hediste diversicolor and Patella vulgate, taken from polluted sites of the Bay of Tangier in Morocco. Our findings are also similar to those obtained for P. cultrifera collected from three sites impacted with different levels of anthropogenic pollution on the eastern coasts of Algeria (Guemouda et al. 2014). The increase in enzymatic activities could also be explained by the higher concentration of bio-accumulated metals, which causes oxidative stress and an antioxidant response (Gaete et al. 2017). However, a significant correlation was observed between Cd, Zn and SOD activity as well as Zn and GST activity (Table 4), suggesting that these metals are bioavailable and responsible for the variation in enhancement of the activities of these antioxidants.

The results of seasonal variations in GST activity at each site showed that it was highest in summer and higher in the port of Rades (S1) than the Punic port of Carthage (S2) throughout the study period (p<0.05). In addition, this enzyme plays a role in cellular protection against oxidative stress, which can be triggered by pollutants such as metals, PCBs and PAHs (Michel et al. 1998). These findings are similar to those obtained for the worms Hediste diversicolor collected from the polluted estuary of the Seine (Durouet al. 2007). To substantiate the mechanisms underlying trace metal accumulation, GSH was also examined. This non-enzymatic antioxidant is the most important cytosolic sulfhydryl compound that acts as a reducing and protective agent against intensive toxic substances, including trace metals through their -SH groups (Das et al. 2017). Our results showed a higher GSH level in S1 than in S2, which could be a result of increased synthesis and neutralization of ROS via the thiol group. Dissimilar studies on polychaetes have shown a decrease in GSH level in relation to trace metal accumulation (Lv et al. 2016). According to the above results, the significant relationship between antioxidant biomarkers (mainly GSH, SOD and GST) and the concentrations of metals (mainly Cd and Zn) in P. cultrifera tissue suggest the activation of the mechanisms of detoxification due to the generation of ROS. These findings are similar to those obtained by Cataldo et al. (2011) in earthworms at sites contaminated by metals and by Gaete et al. (2017) in Perinereis gualpensis from estuaries of central Chile.

In conclusion, in different seasons and geographical locations P. cultrifera shows different physiological responses. In this study, we have assessed the effects of anthropogenic pollution in terms of metal accumulation, antioxidant response and alteration in proximate composition in relation to seasonal change in P. cultrifera tissues. The results clearly show that S1 is more affected by anthropic pollution than S2. Our study has identified some insights into anthropogenic pollution at the harbour sites, but further studies on metal accumulation in sediments and exposure of P. cultrifera to metals are still required to provide general information about the health status of these coastal harbours.