Abstract
Participants in the coastal socio-economy of the Mediterranean Sea, such as industries, aquaculture, urban populations, conglomerates, and tourists, create intense anthropogenic pressures on marine ecosystems (such as the release of trace metals). This raises concerns about their impact on the surrounding environment and on marine organisms, including those collected for human consumption. This study introduces the possibility of using Patella caerulea (Linnaeus 1758), indigenous to the Mediterranean Sea, as a biosentinel of marine pollution. This study proposes coupling environmental (bioaccumulation) and toxicological (redox homeostasis) measures of bioavailability with genetic variability (COI mtDNA) assessments. Concentrations of six trace metals (cadmium, copper, iron, lead, nickel, and zinc) were measured in surface seawater and in P. caerulea individuals collected from four coastal stations on the Tunisian coast where different levels of metal contamination have occurred. The quantified biomarkers involved the determination of antioxidant defense enzymes, catalase (CAT), glutathione peroxidase (GPX), superoxide dismutase (SOD), and the measurement of lipid peroxidation indicated by malondialdehyde (MDA) levels. Our study identified critical levels of metal contamination among locations in the Gulf of Gabes. Concomitantly, the induction of antioxidant biomarkers (especially SOD and GPX) was observed, highlighting the potential of P. caerulea to acclimate to stressful pollution conditions. Molecular analysis of COI (mtDNA) revealed low discrimination between the four P. caerulea populations, highlighting the role of marine currents in the Mediterranean Sea in the dispersal and passive transportation of limpet larvae, allowing an exchange of individuals among physically separated, P. caerulea populations.
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Introduction
Most coastal developments in the Mediterranean do not address their long-term impact (WWF 2020). More than 50% of the human population of Mediterranean countries is concentrated on the coast. The region attracts more than 30% of world tourism and the trend is increasing (WWF 2020). Moreover, the Mediterranean Sea has (MS) a semienclosed configuration, inducing circular marine currents (counterclockwise from the Strait of Gibraltar) (El-Geziry and Bryden 2010). Therefore, the MS is considered one of the first seas suffering harm from anthropogenic activities, such as high metal emissions (Milano et al. 2012; Guittonny-Philippe et al. 2014). In Tunisia, coastal areas host 64% of the total human population and concentrate most of the country’s economic activities (WWF 2020). Several towns (Sousse, Monastir, Sfax, Gabes) are located along the coast, inducing intense anthropogenic pollution (urbanization, industrialization, and agricultural activities), often including metals (Ladhar-Chaabouni et al. 2012; Ghannem et al. 2014; Harrabi et al. 2018).
During recent decades, anthropogenic activities (such as industry, landfills, wastewater, and mineral extraction) have increased environmental metal emissions (Nriagu 1996), promoting their accumulation within the MS hydrological system (Debnath et al. 2021). Many studies have reported high metal contamination levels in rivers, oceans, and seas (Wang et al. 2013; Kaste et al. 2018; La Colla et al. 2021).
Metal pollution in marine ecosystems is currently among the chief environmental concerns. Metals affect the biotic community either through genetic alteration of the population (including genetic bottlenecks and species extinctions) or via physiological disruptions (e.g., growth and reproduction) (Belfiore and Anderson 2001; Ross et al. 2002; Kim et al. 2003; Matson et al. 2006; Ensibi et al. 2015; Rumisha et al. 2017). This is particularly true for species having a benthic period in their life cycle, including fish and many invertebrates that are in contact with bioavailable metals in sediments and surface waters (Matson et al. 2006). High Cd, Cu, Fe, Pb, and Zn concentrations are commonly reported in sediment, water, and biotic ecosystem components (Ansari et al. 2004; Mezghani-Chaari et al. 2015; Ayari et al. 2016). However, metals, such as Cd, Cr, Hg, Ni, and Pb, can be toxic to marine organisms even at very low concentrations (Pavalaki et al. 2016). Conversely, other metals, such as Fe, Cu, Mn, Se, and Zn, not only are essential for hormesis but also can be ecotoxic at excessive bioavailable concentrations (Calabrese and Baldwin 2003).
Trace elements persisting in ecological niches (e.g., sediments, water, or marshes) accumulate in organisms (bioaccumulation) and are passed upward through trophic networks to top predators (biomagnification) (Readman et al. 1993; Ettajani et al. 2001). Hence, these environmental contaminants are considered a threat not only to marine biodiversity but also to humans through the ingestion of contaminated marine food products (Amiard et al. 2008; Rabaoui et al. 2013; Bashir et al. 2020).
In addition to chemical measurements, biological approaches using recognized biosentinels can assist in assessing environmental risks to various species caused by pollution (Reguera et al. 2018; Louzon et al. 2020, 2021).
The most widely used species for assessing marine pollution are bivalves, such as mussels and oysters (Mytillus sp. and Crassostrea sp.), because they are easy-to-obtain filter feeders that bioaccumulate pollutants and are abundant on most coasts worldwide (Beliaeff et al. 1998; Cantillo 1998; Solé et al. 2000; Oros et al. 2005; Viñas et al. 2018). However, bivalves can be rare in some areas, underscoring the need for alternative biosentinel species especially those with different sensitivities to pollutants, belonging to different trophic levels, and exhibiting different paths of exposure (Reguera et al. 2018). In recent years, studies on marine gastropods have contributed to a better knowledge on metal accumulation in these species and simultaneously have allowed the evaluation of human health risks resulting from their consumption (Ahn et al. 2002; Bergasa et al. 2007; Yüzereroğlu et al. 2010; Shefer et al. 2015; Conti et al. 2017; Barchiesi et al. 2020).
Among intertidal grazers inhabiting rocky shores, limpets (Patella sp.) present increased opportunities for assessing environmental and toxicological bioavailability and impact (De Pirro et al. 2001; Reguera et al. 2018). Interest in the use of these organisms in biomonitoring programs is based on their wide distribution in the Northeast Atlantic and the Mediterranean (Poppe and Goto 1991; Jenkins et al. 2001), well-documented biology (Fernández et al. 2016; Reguera et al. 2018), abundance, and sedentary lifestyle (Fretter and Graham 1972). In addition, these grazer gastropods play a key role in coastal ecosystems by regulating the degree of algal coverage and even succession processes in rocky intertidal communities (Coleman et al. 2006; Reguera et al. 2018; Palladino et al. 2021). Lozano-Bilbao et al. (2021) studied the ability of P. aspera and P. candei crenata to accumulate trace metals, and these two limpet species were found to be useful as bioindicators of marine pollution based on their propensity for bioaccumulating metals, especially cadmium (Cd) and copper (Cu). Patella caerulea is a common wild species abundant on exposed shores of all Mediterranean coasts (Della Santina and Chelazzi 1991; Palladino et al. 2021). Consequently, the study of pollutant accumulation in this species can be relevant for predicting the potential transfer of contaminants through food webs by predation on contaminated limpets (Viñas et al. 2018).
Advances in cell biology support possibilities for assessing the biological and ecological significance of contaminants based on biological responses, or biomarkers, induced by exposure to xenobiotics (Douhri and Sayah 2009; Al Kaddissi 2012). Consequently, biomarkers are early warning tools that allow earlier detection of pollutants and their biological effects before perturbation at the level of entire marine communities (Moreira and Guilhermino 2005; Douhri and Sayah 2009). Biomarkers are measured in species capable of accumulating contaminants (e.g., biosentinels) (Al Kaddissi 2012). To date, only biomarkers of exposure have been studied in P. caerulea (cytochrome P450 enzymes (Yawetz et al. 1992; Bonacci et al. 2007) and ethoxyresorufin-O-deethylase (EROD) (Bresler et al. 2003)). Studies of pollution effects at the organismic level have been rare, although interest has been shown in integrated biomarker response (IBR) approaches for various biosentinel organisms used in ecotoxicology (Beliaeff and Burgeot 2002). In addition, these IBRs present a growing potential (Mleiki et al. 2020) for improving ecotoxicological risk assessment methodologies with pertinent biosentinels, such as P. caerulea.
Bioavailable metals can induce the production of reactive oxygen species (ROS) (O2−, OH−, NO−, H2O2, HOCl, O2, ONOO−) in living cells (Vlahogianni et al. 2007). H2O2 is considered a primary cellular precursor of the hydroxyl radical (HO.) and consists of a highly reactive and toxic form of ROS. Its removal is considered an important defense strategy against oxidative stress in marine organisms (Regoli et al. 2002a; Vlahogianni et al. 2007). To counteract the negative effects of ROS (lipid peroxidation, protein and DNA damage, etc.), marine organisms exhibit multiple enzymatic antioxidant defense systems (Legeay et al. 2005). The antioxidant activity of catalase (CAT) has great significance, as it prevents the conversion of Η2Ο2 to harmful OH- (Cutler 1984). CAT breaks hydrogen peroxide down into water and oxygen. This reaction is an important strategy of marine organisms against oxidative stress (Regoli et al. 2002a, 2002b). Superoxide dismutase (SOD) breaks down hydrogen peroxide and hydroperoxide radicals to harmless molecules (H2O2/alcohol and O2) (Silvestre 2005). Glutathione peroxidase (GPx), which is a selenium-containing antioxidant enzyme, effectively reduces H2O2 and lipid peroxides to water and lipid alcohols, respectively, and oxidizes glutathione to glutathione disulfide (Touyz 2004). Oxidative stress can also result in lipid peroxidation, which can be used as a biomarker (Mylonas and Kouretas 1999). Malondialdehyde (MDA) is the main active aldehyde resulting from the peroxidation of polyunsaturated fatty acids from membranes and, therefore, is used to predict cellular damage caused by ROS (Vlahogianni et al. 2007). Although we know the mechanisms of homeostasis perturbation in marine mollusks, understanding the relationship between their induction and the environmental bioavailability of metals in P. caerulea is needed.
To provide this knowledge, the first aim of this study is to look for relationships between known environmental bioavailability of metals (i.e., measured bioaccumulation) and the induction of toxicological effects using a multi-marker approach based on measures of Redox homeostasis (CAT, SOD, GPX, and MDA). The goal was to evaluate the usefulness of this species in environmental risk assessment on Mediterranean coasts. The first step toward achieving these objectives was to determine, by investigating mitochondrial COI variability, that all limpets used in the study belong to the species P. caerulea, thereby ensuring the relevance of conclusions regarding this species, and to evaluate whether distinct genetic variability patterns may exist between limpets at the different stations according to local metal pollution.
Materials and methods
Description of the stations
Among the 10,000 industrial companies officially registered in Tunisia that are categorized as highly polluting, 12% of these are located in Tunis, Bizerte, Sousse, Mahdia, Sfax, Gabes, and Gafsa (INS 2013) (Fig. 1). Among the polluting industries, large-scale phosphate production plants based in Gabes (and previously in Sfax), discharging approximately 12,000 tons of phosphogypsum per day, are a significant threat to the equilibrium of the marine ecosystem in Gabes city. And, beyond this, since 1970, all of the bays surrounding the Gulf of Gabes have been increasingly impacted by expanding industrialization (Bejaoui et al. 2004; Ghannem et al. 2010; Drira 2018).
Stations for this study were selected after ensuring that the selected species was present and sufficiently abundant to sample (Palladino et al. 2021). In addition, the sampling locations represent sites of ecological and economic importance with varying degrees of exposure to contaminants from discharges from neighboring urban, industrial, and agricultural regions (Amari 1984; Ben charrada 1997; INS 2013). Hence, the strategy in choosing the sampling sites assumed a gradient of pollution proceeding from north to south (Amari 1984). Four stations were selected along the Tunisian coast to include different levels of water contamination (Table 1). Station locations, from north to south, included Cap Bon (CB), belonging to the gulf of Tunis, and three distant stations belonging to the gulf of Gabes: Chebba (C), Kerkennah Islands (K), and Gabes (G) (Fig. 1). The GPS coordinates of the sampling stations are provided in the supplementary information (Table S1).
The physico-chemical properties of the water at each station were measured using a CTD multiparameter probe, YSI Pro30 CTD handmeter, as suggested by the UNESCO protocols (UNESCO 1994; UNEP/MAP/MED 2020) and also as described in ISO 10523 (2008) (Table S2). Triplicate sampling of surface seawater was performed for each station in March 2018 for the analysis of trace metals. Samples were filtered using a thin membrane (0.45 μm), acidified and stored at 4 °C for metal analyses using absorption flame emission spectroscopy (AAS) (Kremling 1976). The limits of detection (LODs) and limits of quantification (LOQs) for each metal are mentioned (Table 1). The levels of metals detected at each station (Table 1) were indicative of anthropogenic activities. Moreover, the precision and accuracy of the analytical method were validated by including certified standard reference material TORT-1 Lobster Hepatopancreas (National Research Council of Canada) (Table S3).
Sample handling
Marine gastropods accumulate pollutants, including heavy metals, from the water column and via the ingestion of contaminants accumulated in phytoplankton or adsorbed on detritus and on sediment particles. Because they occupy a benthic niche, they present a relevant model to reflect local pollution to which other taxa may be exposed including, for example, crustaceans, arthropods, corals, and free-swimming finfish (Wang et al. 1996; Griscom et al. 2000).
At each sampling station, individuals of the marine gastropod P. caerulea were collected by hand at low tide and transported in cool boxes (4 °C) to the laboratory. At least 30 individuals were sampled in each station.
Surface seawater was sampled likewise at the four stations. Seawater samples were collected in triplicate in sampling bottles at each station at a 1-m depth according to HELCOM (2019) guidelines for sampling requirements. Once in the laboratory, limpet samples were rinsed in previously filtered seawater to remove the residues of sediment and particles present in the mantle and on the visceral masses. Samples were stored at − 20 °C until measurements for bioaccumulation (“Trace metals assessment” section) and enzymatic activity (“Enzymatic biomarkers” section) were conducted in the laboratory of Biodiversity and Aquatic Ecosystems (BEA) in the Faculty of Science at Sfax University in Tunisia. Individuals used for molecular analysis (“Molecular identification of Patella caerulea” section) were stored in 70% ethanol (Pereira et al. 2011) until being transported to France for further analysis in Mer Molecules Santé laboratory (MMS) at Le Mans University in France.
Laboratory analyses
Once the samples were defrosted, they were dissected on ice plates (4 °C). Calcareous shells were gently removed using a grooved probe. Before being homogenized, samples of soft tissues were weighed (Average wet weight = 2.3 ± 0.3 g). Six samples were divided into two equal aliquots of 1.0 g per sample, one for the determination of trace metals and the other for further oxidative stress biomarker analysis. As the visceral masses were too small to allow measurements in different parts of the body, all the soft parts of P. caerulea were considered when analyzing trace metals and enzymatic biomarkers (Cravo et al. 2004; Rabaoui et al. 2013).
Molecular identification of Patella caerulea
Molecular identification of samples was conducted in the MMS laboratory at Le Mans University in France. Total genomic DNA of ten selected individuals per station was extracted from the soft bodies of samples preserved and transported to France in 70% ethanol. For DNA analyses on the individual specimens, the shell was gently removed with a sterile grooved probe, and individuals were dissected using a sterile scalpel blade. DNA extraction was performed according to the hexadecyltrimethylammonium bromide (CTAB) method adapted from Doyle and Doyle (1987). The DNA was then visualized in a 1% agarose gel, and the DNA concentration was evaluated using a NanoDrop spectrophotometer, Thermo scientific (2000). DNA identification was conducted on each individual specimen used in this study based on the mitochondrial COI sequence. The COI amplifications (481 bp) respected the following conditions: 150 ng DNA added to GoTaq®DNA polymerase kit (M3001, Promega), for PCR conducted in 25 µL total volume and including 35 cycles (1 min, 94 °C; 1 min, 49.5 °C; 1 min, 72 °C) and final elongation (10 min, 72 °C). The primers used, COIpatellaF (5′-GAATATGAGCAGGTTTAG-3′, Tm: 49.1 °C, 38.9% GC) and COIpatellaR (5′-CRGCTAAAACAGGTAAWG-3′, Tm: 50.3 °C, 41.7% GC), were designed from a multiple alignment of COI sequences of several Patella species (P. aspera: KU566778, P. caerulea: EU073896, P. depressa: KU566787, P. ferruginea: GQ469876, P. lugubris: EU073889, P. pellucida: KU566788, P. rustica: GQ469882, P. ulyssiponensis: GQ469889; P. vulgata: KU566795). The sequences were aligned using Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/). The phylogenetic tree was built using MEGA X.10.1.8 software (Kumar et al. 2018). Genetic variability was estimated by the nucleotide diversity (π) and the number of variable sites (S) using DNASP 5.0. (http://www.ub.edu/dnasp/). Tajima’s D test was also performed to evaluate the hypothesis that all mutations were selectively neutral.
Trace metals assessment
Measurements of trace metals were conducted in the laboratory of Chemistry in the Faculty of Science at Sfax University in Tunisia. Six samples per location were analyzed. For each limpet sample, an amount of 1.0 g was dissected using a grooved probe and a scalpel blade, and the samples were oven dried overnight at 60 °C in porcelain crucibles to remove moisture and thereafter were weighed (average weight 0.23 ± 0.01 g) (Cravo and Bebianno 2005). The percentage moisture was calculated as the loss of weight after drying. The remaining 1.0 g portions were labeled and stored at − 20 °C until enzymatic analyses were conducted. The dried samples were placed in a muffle furnace at 500 °C for 12 h to induce calcination (Fonollosa et al. 2017; Primost et al. 2017). The ashes obtained were filtered using a solution of nitric acid HNO3 (1%) adjusted to 30 mL with deionized water. Metal concentrations of these samples were determined using atomic absorption flame emission spectroscopy (AAS) Perkin Elmer, AAnalyst 200 (SAAF) using the (UNEP/IAE/FAO 1984) method. The targeted trace metals are cadmium (Cd), copper (Cu), iron (Fe), lead (Pb), nickel (Ni), and zinc (Zn). The accuracy and precision of the analytical methods were assessed by the analysis of one aliquot of reference material, as well as one laboratory reagent blank. All samples were analyzed in triplicate, and the average metal concentration was calculated. The blank did not contain any detectable concentration of the studied metals. Recovery rates for metals were determined with certified reference materials (TORT-1, lobster hepatopancreas) between 90 and 100%, indicating the absence of analytical bias (Table S3).
Enzymatic biomarkers
Measurements of enzymatic biomarkers were conducted in the laboratory of Biodiversity and Aquatic Systems in the Faculty of Science at Sfax University in Tunisia. The labeled 1.0 g portions left from each sample used for metal assessment (six analyzed samples per station) were thawed on ice and then ground (special electric homogenizer ULTRA TURRAX) in 2 ml of tris-buffered saline (TBS) (pH = 7.4). The homogenate was centrifuged twice at 10,000 × g at 4 °C for 20 min, and the collected supernatants were kept at − 20 °C for further short-term measurements of enzymatic activities. Proteins were quantified as mg/ml according to the Lowry method (Lowry et al. 1951) using a bovine serum albumin (BSA) calibration curve (R2 = 1). Absorbance was measured at 490 nm. SOD activity was measured indirectly by the colorimetric method developed by Beauchamp and Fridovich (1971) using the riboflavin/methionine complex producing superoxide anions and nitro blue tetrazolium (NBT). NBT oxidation by the superoxide O2− anion was measured at 560 nm, and the results are expressed as units (U) of SOD activity.mg−1 protein. CAT activity was measured following the decrease in absorbance at 240 nm due to H2O2 consumption (Aebi 1984). The reaction took place in phosphate buffer (100 mM) at pH 7.0 at 25 °C with H2O2 (500 mM) and total protein (1–1.5 mg protein/ml). The CAT activity is the difference in the absorbance at that wavelength per unit of time (µmoles/min/mg protein) according to the protocol proposed by Aebi (1984). Total GPx activity was measured at 412 nm according to the protocol described by Flohé and Günzler (1984). The supernatant (v) was mixed with 1 mM GSH (2v). H2O2 (1.3 mM) was added to initiate the reaction. The reaction was stopped after 10 min by the addition of 1% trichloroacetic acid (TCA). After centrifugation, Na2HPO4 (320 mM) and Ellman’s reagent (5,5′-dithiobis-(2-nitrobenzoic acid)) (DNTB) (1 mM) were added to the supernatant. GPx activity was expressed in μmoles of oxidized GSH/min/mg protein.
Lipid peroxidation
The malondialdehyde (MDA) level in limpets was measured using the protocol defined by Niehaus and Samuelsson (1968). Briefly, 0.5 mL of tissue supernatant from a previous experiment (“Enzymatic biomarkers” section) was mixed with TBS and TCA (20%)-BHT (0.01%) and incubated at 100 °C for 30 min. Samples were then vortexed, cooled, and finally centrifuged at 10,000 × g for 10 min. Then, 1 mL of thiobarbituric acid (TBA) solution (0.67%), 40 µl of HCl (0.6 M) and 0.5 mL of supernatant were incubated for 15 min at 90 °C. Finally, the absorbance of the TBA–MDA complex was measured at 532 nm. The MDA concentration was calculated using its molar extinction coefficient (ε = 155 mM/cm). MDA was expressed in nmoles/mg protein.
Data analysis
Stations are presented in maps drawn with QGIS software version 3.0.2. Data analysis was performed with R studio (version 4.0.3). Data are presented as the mean ± standard deviation. Normality and homogeneity of the variance were checked with the Shapiro–Wilk test and Bartlett test, respectively. Comparison of contamination degrees of the studied stations and metal concentrations was conducted using one-way ANOVA followed by a post hoc test (Tukey HSD). The differences were considered significant when the p value was less than 0.05.
Results and discussion
Molecular identification and genetic diversity of Patella caerulea
The evolutionary history is inferred by using the maximum likelihood method and the Hasegawa-Kishino-Yano model (Hasegawa et al. 1985). The tree with the highest log likelihood (− 1709.28) is presented in Fig. 2. The percentage of trees in which the associated taxa clustered together is shown next to their respective branches. Initial tree(s) for the heuristic search were obtained automatically by applying neighbor-joining and BioNJ algorithms to a matrix of pairwise distances estimated using the maximum composite likelihood (MCL) approach and then selecting the topology with superior log likelihood value. A discrete gamma distribution was used to model evolutionary rate differences among sites (5 categories (+ G, parameter = 1.5614)). The rate variation model allowed some sites to be invariable evolutionarily ([+ I], 54.13% sites). Codon positions included were 1st + 2nd + 3rd + Noncoding. There was a total of 353 positions in the final dataset. The phylogenetic tree (Fig. 2) showed that all individuals used in this study were included in the P. caerulea group (robust monophyletic group), confirming the species identity across all sample stations. The nucleotide diversity (π: 0.00381), the number of polymorphic sites (S: 19 on 481 sites) and Tajima ‘D statistic (D: − 1.73907) indicated a low intraspecific polymorphism. The haplotype network of COI (481 bp) sequences of P. caerulea inhabiting the Mediterranean Sea (Fig. 3) highlights the presence of shared haplotypes (haplogroups) between individuals from at least four different countries. Tunisian individuals are found in shared haplotypes (for example, with individuals from Morocco, Greece, and Spain) but also in separate haplotypes. This seems to show a great mixing of populations within the semienclosed environment of the Mediterranean. This low P. caerulea variability could be explained by marine currents in the Mediterranean Sea, which rotate counterclockwise and allow larval limpet dispersion along the Tunisian coasts (El-Geziry and Bryden 2010), transporting them far from their place of origin and thus creating genetic mixing between Mediterranean P. caerulea populations. This result of shared haplotypes between individuals from different Mediterranean locations is in agreement with the findings of Wesselmann et al. (2018) on Pinna nobilis, who highlighted the importance of ocean currents and pelagic larval transport time in shaping the population connectivity of the species in the Mediterranean basin. This genetic connectivity has also been described for several Mediterranean fish species (Galarza et al. 2009).
Metal concentrations in limpets
The average concentrations and the bioaccumulation factors (BAFs) (calculated as the ratio between concentrations in limpets and concentrations in water samples for the same metal) of each metal assessed in the soft tissues of limpet P. caerulea from different stations are summarized in Table 2.
Concerning the variation between stations, most of the highest concentrations were found in limpets from Gabes city, for Cu (0.06 ± 0.07 µg/g DW), Cd (0.23 ± 0.21 µg/g DW), Ni (0.60 ± 0.25 µg/g DW), Pb (1.29 ± 0.36 µg/g DW), and Zn (8.21 ± 7.48 µg/g DW) (Table 2), but not for iron, which was slightly higher in limpets belonging to Cap Bon station (9.10 ± 4.72 µg/g DW) than in those coming from Gabes (8.03 ± 5.71 µg/g DW), although there was no significant difference (p value = 0.2 > 0.05). This is because Gabes city is under strong anthropogenic aggression from the phosphoric acid industry (Bejaoui et al. 2004; Ghannem et al. 2010; Drira 2018).
On the other hand, the lowest mean values of the studied metals were scattered among the different stations. Fe, Pb, and Zn (0.73 ± 0.31, 0.21 ± 0.11, and 1.54 ± 1.24 µg/g DW, respectively) were found at Chebba station, and Cu and Ni (0.014 ± 0.01 µg/g DW for Cu and under the limit of detection (0.07) for Ni, respectively) were found at Cap Bon station (Table 2).
Cd, which is considered one of the most toxic heavy metals (Burger 2008; Rabaoui et al. 2013), was detected only in limpets from Gabes station (0.23 ± 0.21 µg/g DW) with a BAF = 1.
The highest concentrations of iron (9.10 ± 4.72 µg/g DW) and second highest concentration of lead (1.08 ± 0.37 µg/g DW) were recorded in limpets collected at Cap Bon station but this station was presumed to be the control site (Belkhodja et al. 2012).
Significant interstation differences were noted for each trace metal assessed in P. caerulea, except for copper, which did not present any difference between stations and which was represented by the lowest concentrations found in limpet tissues from each location, with 0.014 ± 0.01 µg/g DW at Cap Bon station, 0.03 ± 0.04 µg/g DW at Chebba station, 0.04 ± 0.03 µg/g DW at Kerkennah Islands station, and 0.06 ± 0.07 µg/g DW at Gabes station. Considering the values of BAFs, copper also presented the lowest BAFs at all the studied stations (0.004 at both the Chebba and Cap Bon stations, 0.023 at the Gabes station, and 0.007 at the Kerkennah station). However, Zn and Fe appear to have the highest BAFs among all the studied metals per studied station except for Gabes station, where the highest BAFs were represented by Zn and Cd.
Comparing our results (Table 3) with levels reported for other geographical areas for P. caerulea and other Patella species (Table 3), cadmium concentrations recorded within the present study are the lowest compared to the results of previous studies in the Gulf of Gabes (0.605 ± 0.0252–1.065 ± 0.0858 µg/g DW) by Rabaoui et al. (2013), in the north coast of Tunisia (0.78 ± 0.24–1.63 ± 0.27 µg/g DW) by Belkhodja and Romdhane (2013), in Iskenderun Gulf in Turkey (0.24 ± 0.03–0.68 ± 0.03 µg/g DW) by Yüzereroğlu et al. (2010), and in Italian islands (3.30 ± 0.37–6.30 ± 2.08, 1.7–11.8 and 0.104 ± 0.03–2.318 ± 2.99 µg/g DW) by Campanella et al. (2001), Cubadda et al. (2001) and Conti et al. (2017), respectively. However, our Cd levels were higher than those determined in the Ionian Sea in Italy (0.15 ± 0.06–0.16 ± 0.05 µg/g DW) by Storelli and Marcotrigiano (2005). Studies on other limpet species showed lower Cd concentrations for Patella piperata in the Canary Islands (0.039 ± 0.011 µg/g DW) by Bergasa et al. (2007) and for Patella candei and Patella aspera (0.041 ± 0.013–0.102 ± 0.011 µg/g DW, respectively) by Lozano-Bilbao et al. (2021). Based on the above-mentioned results, Cubadda et al. (2001) and Yüzereroğlu et al. (2010) suggested that limpets of the genus Patella may be suitable species for biomonitoring Cd availability. However, the data from the study of Espinosa and Rivera-Ingraham (2016) on Patella ferruginea in Ceuta, Spain, do not fit with previous reports for Cd, suggesting that P. ferruginea was an exception since the Cd concentrations detected in soft tissues were independent of the existing pollution gradient.
Belkhodja et al. (2012) considered Cap Bon station as a control station in their study on limpets in 2012. The present study was conducted 6 years later and showed that waters around the Cap station are becoming more polluted with metals. This could be explained by the fact that this station receives domestic wastewater and effluent discharges from the three treatment plants in the surrounding areas through Wadi Soltane, Wadi Meliane, and Wadi el Bey (Ennouri et al. 2010; Belkhodja et al. 2012). For example, Wadi el Bey drains 60% of the pollutants discharged from several urban centers in northeastern Tunisia. The main sources of pollution in Wadi El Bey are tannery, stationery, brewery, tomato processing, and slaughterhouse discharges (COMETE engineering-Bceom-IHE 2008). Pollution impacts linked to increasing urbanization and tourism are significant along this part of the coast (WWF 2020).
Our range levels for lead (0.21 ± 0.11–1.29 ± 0.36 µg/g DW) were clearly lower than those reported earlier on the north coast of Tunisia (3.51 ± 0.67–3.61 ± 0.8 µg/g DW) (Belkhodja and Romdhane 2013) and on Pontine Islands in Italy (3.58 ± 0.94–0.479 ± 0.14 µg/g DW) (Conti et al. 2017), although our results for Pb were comparable with Pb ranges reported for Patella specimens in other studies (Campanella et al. 2001; Cubadda et al. 2001; Storelli and Marcotrigiano 2005; Bergasa et al. 2007; Yüzereroğlu et al. 2010; Rabaoui et al. 2013; Lozano-Bilbao et al. 2021) (Table 3).
Our range levels (Table 2) for Ni (0.11 ± 0.04–0.60 ± 0.25 µg/g DW) were clearly lower than those reported earlier in P. caerulea in contaminated areas from the north coast of Tunisia (Belkhodja et al. 2012) and on Pontine Islands in Italy (Conti et al. 2017). However, our range levels for Ni were generally comparable to those reported for P. candei and P. aspera in the Canary Islands (Lozano-Bilbao et al. 2021) and for P. caerulea in the Iskenderum Gulf in Turkey (Yüzereroğlu et al. 2010) (Table 3).
Our range levels for Cu are clearly lower than those reported in Patella specimens from other geographic areas (i.e., those previously reported in P. caerulea in the Italian Islands by Campanella et al. (2001) and Cubadda et al. (2001) and in P. candei and P. aspera in the Canary Islands by Lozano-Bilbao et al. (2021)) (Table 3). The lower-than-expected differences between our copper levels assessed in tissues from areas potentially expected to have varying degrees of pollution can be explained, as suggested by Cubadda et al. 2001, by the strong capacity of the species P. caerulea to regulate their body levels of copper. This regulatory mechanism in marine invertebrates might involve enhanced metal elimination, resistance to metal uptake, or some combination of the two, or storing excess metals in a nontoxic form until further use (Depledge and Rainbow 1990). However, laboratory experiments of copper exposure of P. aspera, P. caerulea, and P. rustica did not mention any copper regulation ability in the tested individuals, even though they showed interspecific differences in short-term copper intake and excretion dynamics (De Pirro et al. 2001; Reguera et al. 2018). This may also be linked to the stressful effect generated by cadmium (BAF = 1) as a replacement for copper in various proteins, such as ferritin, by promoting its release from biological membranes, thus contributing to an increase in its intracellular concentrations (Casalino et al. 1997; Dorta et al. 2003; Watjen and Beyersmann 2004; Al Kaddissi 2012).
Our results for Zn and Fe concentration ranges were generally comparable to those reported for Patella specimens in other localities (Table 3), while for Fe, concentration ranges were lower than those recently reported in P. candei and P. aspera on Canary Islands (Lozano-Bilbao et al. 2021), and for Zn, concentration ranges were lower than those reported in P. caerulea in Pontine Islands, Italy, (Conti et al. 2017) and along the Iranian coasts (Bordbar et al., 2015). High bioaccumulation levels of Zn (Table 2), especially at the Gabes station (BAF = 1.546), can be explained by the presence of large quantities of Zn in the Mediterranean environment and in sediments (Drira 2018). However, Devez (2004) showed that zinc accumulation is not necessarily correlated with environmental contamination. Another possibility is that this metal is a biologically essential element in sea organisms (Trevizani et al. 2016).
Studies on limpets indicate that, regardless of environmental quality, these animals tend to preferentially accumulate Fe and Zn (Cravo and Bebianno 2005; Belkhodja et al. 2012; Espinosa and Rivera-Ingraham 2016), and, as seen in the present study, P. caerulea is not an exception (highest concentrations in limpets and highest BAFs are recorded with these two metals (Table 2)). Apparently, the high concentration of Fe in marine invertebrate soft tissues is not surprising (Espinosa and Rivera-Ingraham 2016), and for the specific case of limpets, their teeth (radula) are 1.46% formed by an iron-based mineral material called goethite (Davies and Cliffe 2000). Zn, on the other hand, easily accumulates in soft tissues through passive uptake and is a constituent of metabolically significant enzymes and pigments, along with Fe (Rivera Ingraham et al. 2013).
Limpets represent the most important herbivorous grazers of temperate rocky shores (Palladino et al. 2021). They accumulate pollutants, including heavy metals, from the water column and via the ingestion of contaminants accumulated in phytoplankton or adsorbed on detritus and on sediment particles (Wang et al. 1996; Griscom et al. 2000). Therefore, it would be important to consider food composition and contaminant bioavailability in resources when studying bioavailability for limpet species, as recommended by Espinosa and Rivera-Ingraham (2016). Ozaki (2019) demonstrated that diet richness and/or composition can be affected by pollution-induced changes in trophic resources and that the type of food items consumed by mice affected their trophic exposure to trace metals. In the same context of factors determining the environmental bioavailability of contaminants, Espinosa and Rivera-Ingraham (2016) showed that females of P. ferruginea in Spain presented the highest values for trace metals, and this result was due only to sex and not to the size differences between males and females. Lozano-Bilbao et al. (2021) showed that environmental bioavailability can also be species-dependent when they compared the ability of accumulating metals between two limpet species and revealed that P. aspera showed a higher concentration of elements than P. candei crenata. From this perspective, in future studies considering the influence of various ecophysiological factors on metal bioaccumulation, it would be relevant to normalize the sampling protocol and suggest threshold guide values (TGVs) in assessing what may be considered abnormal internal concentrations in Patella, as well as the environmental bioavailability of metals, such as those existing for Gastropoda subadults in terrestrial ecosystems (ISO 24032:2021; Louzon et al. 2021).
Measurement of antioxidant responses and lipid peroxidation levels in P. caerulea
Protein quantification
The statistical analysis of total levels of protein in P. caerulea did not reveal any significant difference between stations, except for Gabes station (p > 0.05), where the highest protein concentration (8.33 ± 4.48 mg/ml) was reported, which seems similar to the cadmium contamination level at the same station. This agrees with the findings of Khebbeb et al. (2010), who noted a positive correlation between total levels of protein and cadmium concentration in the clam Ruditapes decussatus contaminated for a period of 21 days. However, Douhri and Sayah (2009) observed a protein concentration decrease in Patella vulgata collected from impacted sites in the Bay of Tangier in Morocco, resulting from their degradation or a reduction in their biosynthesis by xenobiotics (Douhri and Sayah 2009). Several studies related the measurement of the total level of protein not as a biomarker, but for the expression of enzymatic activities such as CAT, SOD, and GPx (de Almeida et al. 2004; Attig et al. 2010; Buffet et al. 2011; Soltani et al. 2012). For our study, the concentrations of proteins SOD, CAT, GPx, and MDA are represented in Fig. 4.
SOD activity
The findings, represented in Fig. 4, showed that the SOD activities (U/mg protein) measured in P. caerulea collected at the Kerkennah and Gabes stations (6 ± 1.9 U/mg protein and 5.25 ± 0.7 U/mg protein, respectively) were significantly higher (p = 0.01) than the SOD activities in limpets from Cap Bon and Chebba stations (2.9 ± 0.4 U/mg protein and 3.9 ± 0.7 U/mg protein, respectively). Zelko et al. (2002) suggested that active sites of SOD isoenzymes require the presence of catalytic metals (Cu, Fe, Mn, Ni, Zn) for their activation. This confirms our results because the highest BAFs for Cu, Fe, Ni, and Zn were recorded in limpets from Gabes and Kerkennah stations (except for BAF (Fe), which was the highest in Gabes but not in Kerkennah station (Table 2). Our results are in agreement with those of Espinosa and Rivera-Ingraham (2016) on P. ferruginea in Spain, who found significant SOD activity in the area with high levels of trace metals. Vlahogianni et al. (2007) showed the same result of increased SOD activity in mussels (M. galloprovincialis) challenged by high levels of Cu, Ni, and Zn. Several authors have shown that the increase in SOD activity in marine organisms is linked to the presence of heavy metals (Farombi et al. 2007; Fernández et al. 2010; Lushchak 2011; Batista et al. 2014). Farombi et al. (2007) explained that the accumulation of trace metals causes oxidative stress, which leads to the production of superoxide anions that induce SOD to catalytically scavenge superoxide radicals to H2O2, providing a defense against this type of oxygen toxicity.
CAT activity
No significant difference was shown for CAT activity measurements in limpets among the sites. Even though the Gabes station presented the highest CAT activity (16.32 ± 9.95 μmol/min/mg protein), it did not present any significant difference, as seen in Fig. 4.
In the same context, it was reported previously that CAT activity was lower in individuals of P. ferruginea inhabiting polluted sites (Espinosa and Rivera-Ingraham 2016). Lionetto et al. (2003) showed a similar result of no significance in the variation of CAT activity in mussels from sites exposed to the highest anthropogenic activities on Italian coasts. Similarly, da Silva et al. (2005) reported a lack of CAT activity change in the oyster Crassostrea rhizophorae exposed to diesel fuel. In contrast, individuals of P. vulgata inhabiting marine polluted areas of Moroccan coasts showed a significant increase in CAT activity (Douhri and Sayah 2009). Vlahogianni et al. (2007) and Jebali et al. (2007) also observed an increase in CAT activity in mussels (Mytilus galloprovincialis) from polluted areas of the Saronikos Gulf of Greece and in clams (Ruditapes decussatus) from Tunisian polluted marine ecosystems. In the present study, the lack of marked differences in this enzymatic activity between limpets from the different stations seems to confirm the suggestion of previous field (Regoli 1998) and laboratory (Cajaraville et al. 1997; Lionetto et al. 2003) studies that reported the possibility of compensatory mechanisms occurring in the CAT response of mussels (M. galloprovincialis) from chronically polluted populations that may not show expected biological responses. This could be the case for the present study and could explain the lack of significant differences in CAT activities among the studied stations. Another explanation by Regoli et al. (2002b) suggested that complex interactions between prooxidants and their cellular targets may confound the assessment of oxidative stress conditions based solely on antioxidant levels. Thus, it is not uncommon to see how, in the face of a certain stressor, some antioxidants increase while others remain unchanged or even decrease (Regoli et al. 2002b). Therefore, chemically induced perturbation of the cellular redox status was suggested to demonstrate cell damage as an impact of degraded environmental quality (Espinosa and Rivera-Ingraham 2016).
Other studies found that CAT activity and Fe concentration are interdependent because iron is an essential element of the active center of some proteins (Jurczuk et al. 2004; Al Kaddissi 2012).
GPx activity
Similar to SOD, the results (Fig. 4) showed that GPx activities (nmoles GSH/min/mg of proteins) measured at Gabes (9.77 ± 1.9 nmoles GSH/min/mg of proteins) and Kerkennah (10.85 ± 2.72 nmoles GSH/min/mg of proteins) stations were significantly higher than the other stations. GPx activity recorded in limpets from Cap Bon station (2.9 ± 0.8 nmoles GSH/min/mg of proteins) was significantly lower than that of all the other stations. Thus, the pollution trend from north to south is supported by the increasing GPx activity in P. caerulea from Cap Bon toward the southern stations.
Espinosa and Rivera-Ingraham (2016) reported comparable results while assessing heavy metals in the endangered limpet species P. ferruginea from the strait of Gibraltar, where individuals sampled from the most polluted areas presented the highest levels of GPx activity. Elevated GPx activity was also reported in mussels exposed to high metal pollution on the coasts of Ria de Vigo (Vidal-Liñán et al. 2014) and the North Tyrrhenian Sea (Regoli and Principato 1995).
Lipid peroxidation
Induced MDA concentrations (nmols/mg protein) were noted in all studied Tunisian stations (Fig. 4), indicating cellular stress resulting from changes in the environment, most likely through chemical contamination (Vlahogianni et al. 2007). However, no significant differences were recorded between the different stations. Our results are in agreement with those of Viarengo et al. (1991) and Pampanin et al. (2005) in Italy and Vlahogianni et al. (2007) in the Saronikos Gulf in Athens, who studied the toxic effects of heavy metals on lipid peroxidation in Mytilus galloprovincialis. They demonstrated an increase in MDA levels after metal exposure. Moreover, the absence of a significant difference between our four sampling stations is in agreement with the explanation supplied by Viarengo et al. (1991), who measured MDA in the digestive gland of mussels from Italy and showed that MDA levels fluctuate significantly according to time of year and that no significant difference in MDA activity is observed during spring (and particularly in March). This is in agreement with our findings, especially because our sampling was conducted during March. This finding is presumably linked to the seasonal metabolic status of the animals, which depends on gametogenesis (Gabbott 1975; Lowe et al. 1982) and food availability (Hawkins and Bayne 1984).
Conclusions
The Tunisian coast has increasing levels of pollution from the north (Cap Bon) to the south (the city of Gabes), with Gabes being the only station where we detected Cd in both water and animal tissue. Moreover, levels of the most toxic metals (e.g., Cd and Pb) have reached critical limits at the four investigated stations. Our results confirmed that stations belonging to the Gulf of Gabes (Chebba, Kerkennah, and Gabes) were the most polluted (the highest BAFs and highest total metal concentrations). These results may be explained either by the specific hydrodynamic features of the Gulf of Gabes, which are responsible for transporting pollutants between the different localities of the Gulf, and/or by the specific characteristics of domestic and industrial wastes. This region is industrially exploited and is the location of the largest chemical industries in Tunisia, including the phosphoric acid industry. The area is also frequented by tourists, which means that all domestic waste of urban areas, including tourist resorts, is channeled directly into the sea. Domestic waste was reported to be a source of heavy metal pollution (Wei and Yang 2010).
The present study confirms that limpets can be used as biosentinel organisms for identifying early warning responses to long-term ecological damage. It is known that risk assessment of environmental pollution cannot be based solely on chemical analysis, which does not provide a clear indication of toxic effects of xenobiotics on aquatic biota (Livingstone 2001). The interpretation of risk linked to pollution was supported by our study of biomarkers. Based on the results obtained in this study, there is a correlation between the level of pollution in the investigated sites and the response of animal-related biochemical parameters (CAT, GPx, SOD, MDA). It would be useful to measure trace metal content in sediments to identify contaminant increases in animal tissues (e.g., gills, muscle, digestive glands), especially considering ecophysiological factors (e.g., seasons, sexual maturity), in determining whether there are species-specific mechanisms involved in the bioaccumulation of trace metals. However, in the present study, the lack of marked differences in the enzymatic activity of CAT between limpets from the different stations appears to confirm the possibility of compensatory or adaptive mechanisms operating in chronically polluted marine animal populations.
Our molecular COI investigation on intraspecific variability allowed us to suggest that P. caerulea presents a genetic continuum along the Tunisian coasts, which can be explained by the Mediterranean Sea’s counterclockwise currents, resulting in larval limpet dispersion along the Tunisian coasts (El-Geziry and Bryden 2010). This transports larvae far from their place of origin, thus creating genetic mixing between Mediterranean P. caerulea populations. The same approach is being developed on genetically close but geographically distinct species to determine whether genetic variability exists between different populations exposed to the same pollution. Nevertheless, we suggest that additional experimental research should be conducted in considering factors of sex, size, and age of the individuals tested, carrying out exposure experiments in conditions of mono- or multi-metallic contamination to estimate with more precise approaches (such as qPCR and transcriptomic pattern) for understanding the genetic expression rates of stress biomarkers (CAT, HSP70, GPx, SOD, metallothioneins, etc.), and establishing lethal dose standards for various metals and species. This research is a work in progress. Limpets, being grazer gastropods, play a key role in coastal ecosystems by regulating the degree of algal coverage. Trophic resources are also susceptible to pollution, and we suggest that trophic transfers of trace metals should be considered in further works on environmental contamination and marine animals.
Overall, in the context of developing evidence, methodologies (combining chemical and biological approaches) can assist decision-makers in better managing coastal pollution. This work demonstrates that P. caerulea can be used as a good bioindicator in Mediterranean ecosystems and offers promising perspectives for the development of integrated biomarker indices and threshold guide values (TGVs) for evaluating environmental bioavailability (Beliaeff and Burgeot 2002; Ciliberti et al.2017; Louzon et al. 2021).
Data availability
All data generated and analyzed during this study are available from the corresponding author on reasonable request.
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Acknowledgements
The authors are grateful to Pr. Ayadi for allowing us to do the measurements of trace metals in his laboratory at the University of Sfax in Tunisia. We kindly thank Christophe Buyse, English teacher at Le Mans University, for the time devoted to proofreading our work and Dr. Louzon for his valuable contribution in the revision of the manuscript.
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Part of the PhD is financed by the Eiffel Scholarship Program of Excellence to insure MZ’s (PhD student) stay in Le Mans University, France. Otherwise, no funding was received to assist with the preparation of this manuscript.
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MZ: Conceptualization, Formal Analysis, Investigation, Methodology, Visualization, Validation, Writing – Original draft, Writing – Review and Editing; KA: Methodology; IM: Review; HA: Resources, Supervision; VL: Conceptualization, Methodology, Project Administration, Supervision, Writing—Original Draft, Writing – Review and Editing.
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Zaidi, M., Athmouni, K., Metais, I. et al. The Mediterranean limpet Patella caerulea (Gastropoda, Mollusca) to assess marine ecotoxicological risk: a case study of Tunisian coasts contaminated by metals. Environ Sci Pollut Res 29, 28339–28358 (2022). https://doi.org/10.1007/s11356-021-18490-3
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DOI: https://doi.org/10.1007/s11356-021-18490-3