Hydrocarbon levels and biochemical biomarkers in the clam Ruditapes decussatus

Abstract

Biomarkers are used as tools to measure physiological, biochemical, and/or molecular changes, reflecting the level of disturbances and alterations caused by the exposure of organisms to chemical pollutants present in In the present study, a multi-biomarker approach was used to assess the effects of hydrocarbon contamination in the southern lagoon of Tunis (Tunisia), .Sediments and clams were collected from four locations: three of them were located within the polluted lagoon (S1, S2 and S3) and another ted in a clean site on the Mediterranean coast (SR).Total hydrocarbons were extracted from clam On the other hand, biomarkers of lipid peroxidation were Hydrocarbon analysis revealed a high pollution agoon of Tunis with a spatial results showed activation of antioxidant enzymes in clams sampled from the polluted area as well as neurotoxicity.These results reveal the alteration of the health status in these organisms generated by the anthropogenic pollution in this area.Overall, the present work clearly showed that affected biomarkers could be useful tools for biomonitoring in the Introduction Marine bivalves, such as clams, mussels and oysters have been used worldwide as bioindicators to identify chemical pollutants in coastal environments.Indeed, these species are able to accumulate many chemicals in their tissues thanks to their sessile nature and their filter-feeding behaviour (Nasci et al. 1999, Cajaraville et al. 2000, Fournier et al. 2002, Bebianno and Serafim 2003, Sandrini-Neto et al. 2016).However, the concentration of pollutants in tissues alone is not enough to assess the deleterious effects of pollution on animal health (Solé 2000).Thus, biomarkers have been developed to detect and evaluate the effects of exposure to contaminants in the aquatic environment.Furthermore, Romeo et al. 2003(Roméo et al. 2003)underlined the importance of measuring several biomarkers at the same time in the same organisms, to assess health state of marine environment.
Exposure of aquatic organisms to pollutants can induce an increase in the production of reactive oxygen species (ROS) (Coles et al. 1994, Dyrynda et al. 1998, Camus et al. 2002, Chakraborty et al. 2013, Aguirre-Martínez and Martín-Díaz 2020) such as the hydroxyl radical ( • OH), the superoxide anion radical (O 2 •− ), the hydrogen peroxide (H 2 O 2 ) and the singlet oxygen (O 2 ).ROS are known as transient species due to their high chemical reactivity and ability to react with DNA, proteins, lipids and carbohydrates in a destructive manner (Storey 1996).To protect against the deleterious effects of ROS, cells contain antioxidant defense mechanisms which include both enzymatic and non-enzymatic components.The antioxidant system involves enzymes such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione transferase (GST) and glutathione reductase (GR).
The antioxidant systems can be induced or inhibited under stress conditions.Thereby, the assessment of antioxidant enzymes can provide information on the organism's health status and could be used as a biomarker of pollutant-induced oxidative stress in aquatic organisms (Borković et al. 2008).In recent years, the antioxidant system response has been widely studied and employed as a defense biomarker in aquatic organisms (Regoli et al. 2011).
When these defenses are not sufficient, oxidative stress can cause DNA damage, enzyme inactivation, protein degradation and lipid peroxidation (LPO) (Regoli et al. 2004, Lushchak 2011, Sureda et al. 2011).Furthermore,the measurement of Acetylcholinesterase (AChE) activity is considered a valuable biomarker of exposure to neurotoxic compounds in vertebrate and invertebrate species, such as organophosphates (OPs) and carbamates used in agriculture as pesticides and heavy metals (Akcha et al. 2000, Galgani and Bocquené 2000, Banni et al. 2005).
The aim of the present work was to investigate the hydrocarbon pollution stateof the Southern Lagoon of Tuniscaused by the anthropic activities and to assess his biochemical effectson the carpet shell clam Ruditapesdecussatusan indigenous species of the Mediterranean sea and widely recognized as a good bioindicator in monitoring and ecotoxicological studies (Bebianno et al. 2004).For this reason, Total hydrocarbons (TH) were extracted and aromatic (AH) and non-aromatic (NAH) hydrocarbons fractions were measured in the sediments and the soft tissues of clamscollected from three sites of the Lagoon: the navigation canal, the Rades harbor and the chemical industrial area.Control samples were collected from Louza site considered as a reference site (Banni et al. 2009).In addition,oxidative and neurotoxic status of clams was evaluated through the analysis of catalase, glutathione reductase, superoxide dismutase and acetylcholinesterase activities and malondialdhyde level.http://sciforum.net/conference/mol2net-07

2.
Material and methods

Sampling sites and sample collection
The Southern Lagoon of Tunis is a Mediterranean lagoon covers an area of 7 km 2 and located in the southwest of the Gulf of Tunis; on the east side of Tunis (Tunisia).This lagoon is separated from North lagoon of Tunis by the navigation canal that connects Radesharbor to the harbors of Tunis and La Goulette (Jouini et al. 2005).This lagoon is adversely affected by industrial contaminants from the industrial zone, substantial harbor activities and the untreated urban sewage from the city of Tunis and its southern suburbs (Jouini et al. 2005).Consequently, high levels of polycyclic aromatic hydrocarbons (Mzoughi and Chouba 2011) and heavy metals have been found in the lagoon sediments and clams (Hellal et al. 2011, Mansour et al. 2020).
Three sampling sites were chosen in the Lagoon of Tunis because they are geographically located near contamination sources and differently influenced by anthropogenic impact.The first sampling site (S1) is the navigation channel that connects the harbor of La Goulette to the harbor of Tunis (10°14'41.6"W,36°48'15.3"N)(Fig.1).The second site (S2) is the Rades harbor (10°16'19.1"W,36°48'12.4"N)which is the largest commercial port in Tunis.The third site (S3) is the chemical industrial area (10°16'53.1"W,36°47'59.6"N).Control clams were collected from a coastal site at Louza (SR, 35°02'00.1"N11°00'66.3"E),which has been considered as a reference site in monitoring programs along the Tunisian coasts (Banni et al. 2009).
Carpet shellclams (Ruditapes decussatus) and sediments were collected in February 2016.Upon arrival to the laboratory, clams were transferred to aquaria filled with aerated sea water collected from each sampling sites for 24 h (Hurtado et al. 2011)at 13 °C.The next day, for biochemical analysis, the entire soft body of 10 animals was separated and homogenized in TRIS buffer (TRIS 50 mM, NaCl 150 mM, DTT 1mM, protease inhibitor cocktail pH 7.4) in a 1:3 ratio (W:V) using a motor-driven glass-teflon homogenizer at 500rpm.Thehomogenateswerethencentrifugedfor25minat 9000g at 4°C and the resulting supernatants (called S9 fractions) were immediately frozen at -80°C until further use.All measurements were performed at 4°C to prevent enzyme or tissue degradation.Each biochemical measurement was carried out on 20 individual clams and each measurement was performed in triplicate.
For the hydrocarbon analysis the soft tissues of clams were extracted, washed in ultrapure water and lyophilized.Likewise, the sediment samples were lyophilized then passed through a stainless steel sieve (100 µm).http://sciforum.net/conference/mol2netFig. 1.Map of the study area and location of sampling sites in Tunis lagoon and Louza

Hydrocarbons Analyses
The extraction of the Total Hydrocarbons (TH) liquid / solid extraction following the method of amount of 50 g of sediments or 5 g of clam soft tissues were extracted in a soxhlet extractor with 250 mL of chloroform at 50 °C for sediment and with 250 mL tissues.For organic samples, the extraction was liberation from lipids.This step was carried out by adding 20 mL of KOH (0.7 M) and 30 mL of distilled water to the extractfor 2 h.After that, a liquid/liquid extraction was performed hexane.Aftercondensation by a rotary evaporator hydrocarbons (AH) and non-aromatic hydrocarbons (aliphatic/ NAH) The stationary phase used was neutral alumina/silica for organisms and silica for sediments.elution was done by using 5 mL of n NAH fractions, respectively (Zrafi were measured and the relative hydrocarbons concentration hydrocarbons concentration in the Relative hydrocarbonslevel = [hydrocarbons]

Total protein determination
Total protein concentration in S9 fractions (Bradford 1976), adapted for microplate reader at 595 nm using bovine serum albumin (BSA) as a standard.chloroform at 50 °C for sediment and with 250 mL of methanol at 30 °C for clam For organic samples, the extraction was followed by hot saponification .This step was carried out by adding 20 mL of KOH (0.7 M) and 30 mL of distilled water to the extractfor 2 h.After that, a liquid/liquid extraction was performed by a rotary evaporator, the extract was purified and separated into aromatic hydrocarbons (aliphatic/ NAH), using neutral alumina/silica for organisms and silica for sediments.elution was done by using 5 mL of n-hexane and 5 mL of n-hexane/ chloroform (2:1, v/v) for Zrafi-Nouira et al. 2008).After evaporation, the relative hydrocarbons concentrations in the contaminated site the reference site(SR) were calculated:

Antioxidant defense
Relative hydrocarbonslevel = [hydrocarbons]contaminated site / [hydrocarbons]SR Total protein determination in S9 fractions was determined according to the method microplate reader at 595 nm using bovine serum albumin (BSA) as a measured based on the method adapted fromAebi Clairbone 1985).CAT activity was determined by measuring the decrease of absorbance at 240 nm due to the presence of H 2 O 2 concentration.The enzymatic activity was mg -1 protein.
Map of the study area and location of sampling sites in Tunis lagoon and Louza (Tunisia).
and clam tissues consists of a et al. 2020).Briefly, an amount of 50 g of sediments or 5 g of clam soft tissues were extracted in a soxhlet extractor for 8 h methanol at 30 °C for clam followed by hot saponification to allow hydrocarbons .This step was carried out by adding 20 mL of KOH (0.7 M) and 30 mL of distilled water to the extractfor 2 h.After that, a liquid/liquid extraction was performed 3 times with , the extract was purified and separated into aromatic , using liquid chromatography.neutral alumina/silica for organisms and silica for sediments.The hexane/ chloroform (2:1, v/v) for AH and After evaporation, the AH and NAH fractions contaminated sites compared to / [hydrocarbons]SR.
the method of Bradford microplate reader at 595 nm using bovine serum albumin (BSA) as a Aebi (Aebi 1984) slightly CAT activity was determined by measuring the decrease of he enzymatic activity was http://sciforum.net/conference/mol2net-07 Glutathione reductase(GR) activity was determined during the reduction of oxidised glutathione (GSSG) to GSH, and measured by following the decrease in NADPH levels at 340 mn, as described by Pivaet al. (Piva et al. 2011).The enzymatic activity was expressed in nmolmin -1 mg -1 protein.
Superoxide dismutase (SOD) activity was determined following the method described by Mc Cord and Fridivich(McCord and Fridovich 1969), by measuring the decrease of absorbance of substrate cytochrome-C through xanthine oxidase/hypoxanthine system, at 550 nm.The enzymatic activity was expressed as Unit mg -1 protein.

Oxidative damage
Lipid peroxidation level was measured based on the method adapted from Buege and Aust (Buege and Aust 1978)by estimating the formation of malondialdehyde (MDA)a major decomposition product from lipid peroxidation.Absorbance was measured at 530 nm and lipid peroxidation was expressed as MDA levels in nmolmg -1 protein.

Statistical analysis
The results are expressed as mean ± standard error (SE).Data were statistically analysed by oneway analysis of variance (ANOVA) using Tukey's HSD to identify differences between groups.Statistical analyses were conducted using the software STATISTICA (Statsoft STATISTICA version 6.1.478.0) and differences were considered statistically significant when p values were lower than 0.05.Principal Component Analysis (PCA) was also performed.

Results and Discussion
In this field study, we measured a battery of biochemical biomarkers in the carpet shell clam Ruditapes decussatustissues collected from the South Lagoon of Tunis, which is influenced by the contribution of a wide variety of pollutants.CAT, GR, SOD, MDA and AChEactivities were measured in the tissues of clams collected during February 2016.In the other hand, total hydrocarbons were extracted from clam tissues and sediments and fractionated into aromatic hydrocarbons (AH) and nonaromatic hydrocarbons (NAH).

Hydrocarbon analyses
The results of relative hydrocarbon levels in clam tissues and sediments are reported in Figure 2 and Figure 3, respectively.http://sciforum.net/conference/mol2net-07 The results of petroleum hydrocarbon (AH and NAH) contents in clams tissues revealed higher levels in clams from contaminated sites than control clams(site of Louza).Moreover, spatial variation of hydrocarbon concentrations was observed among the three sampling sites.The AH level was higher in clams from S1 (3.480 ± 0.317 μg mg -1 dry weight) and S3 (3.853 ± 0.288 μg mg -1 dry weight) compared to the control site SR (0.492 ± 0.230 μg mg -1 dry weight) with significant difference in clams collected at S1 and S2 (Fig. 2A).The relativeAH level revealed 7.07-and 7.83-fold increase in S1 and S3 respectively, compared to site SR.Similarly, NAH concentration was significantly higher in clams collected at S1 (5.180 ± 0.259 μg mg -1 dry weight) and S3 (4.857 ± 0.440 μg mg -1 dry weight) compared to the control site SR (0.827 ± 0.199 μg mg -1 dry weight), with 6.26-and 5.87-fold increase, respectively (Fig. 2B).Similarly, sediment samples from contaminated site showed higher hydrocarbon levels than control site with spatial variation among the sampling sites.The AH level was significantly higher in the three site of the lagoon S1 (0.038 ± 0.004μg mg -1 dry weight), S2 (0.066 ± 0.005 μg mg -1 dry weight) and S3 (0.076 ± 0.005 μg mg -1 dry weight) compared to control site SR (0.008 ± 0.002 μg mg -1 dry weight)with 4.75-, 8.25-and 9.50-fold increase, respectively (Fig. 3A).Similarly, NAH concentration was significantly higher in the three site of the lagoon (Fig. 3B) while, the maximum value was observed in S1 (0.488 ± 0.008 μg mg -1 dry weight) compared to the control site (0.010 ± 0.002 μg mg -1 dry weight) with 48.80-fold increase.
In the present study, the results of the quantitative study showed enrichment in AH and NAH in both matrixes (sediments and clams) collected from the three sites of the South Lagoon of Tunis (S1, S2 and S3) compared to the reference site (SR).This enrichment is probably due to the shipping traffic and the urban and industrial discharges into the lagoon.These results are in agreement with previous studies which revealed a contamination of clams and sediments in the navigation channel of Tunis lagoon by petroleum hydrocarbons (Khedir-Ghenim et al. 2009, Chalghmi et al. 2016).The results of the present study confirm contamination of the Tunis lagoon by petroleum hydrocarbons (aromatic and non aromatic).
In this study, the highest levels of AH in clams and sediments were recorded at site S3, which may be due to the presence of several petrochemical industries in this area.

Antioxidant defense
The variations of the antioxidant defense enzyme CAT, SOD and GR in R.decussatus sampled at the four sites are shown in Figure4.
The CAT activity (Fig. 4A) was overall significantly higher in clams sampled from the three sites of Tunis lagoon (S1, S2 and S3) than the reference site(p < 0.05).Organisms sampled from site S1 showed thehighest CAT activity (123.9 ± 7.71 µmol min -1 mg -1 protein) compared to those from the other sites.A similar pattern of induced CAT activity was observed in previous studies carried out on the same species collected from Tunis lagoon (Banni et al. 2003, Chalghmi et al. 2016, Chalghmi et al. 2016).Increase in CAT activity means oxidative stress, often related to excessive ROSproduction during the catabolism of various organic compounds (Clairbone 1985).Indeed, an increase of ROS generation was recorded aquatic organisms exposed to several contaminants (Coles et al. 1994, Dyrynda et al. 1998, Camus et al. 2002, Chakraborty et al. 2013).Thus, our results can be explained by an increase in ROS production owing to the presence of organic contaminants in the lagoon.
Glutathione reductase is involved in the removal of hydrogen peroxide and in the conversion of oxidized glutathione (GSSG) to the reduced form (GSH).In the current study, Gr activity (Fig. 4C) was significantly higher in clams sampled from sites S1, S2 and S3 than the site SR (p < 0.05).Organisms sampled from site S1 showed the highest GR activity (121.19 ± 8.60 µmol min -1 mg -1 protein) compared to those from the other sites (72.79 ± 3.89, 107.82 ± 3.89 and 116.61 ± 12.41 nmol min -1 mg -1 prot, in SR, S2 and S3 respectively).Similar findings have been reported in the digestive glands of the fan mussel Pinna nobilis collected from Ibiza Island (Western Mediterranean) after the Don Pedro spillage (Capó et al. 2015) and the mangrove oyster Crassostreabrasiliana exposed for sublethal concentrations of diesel fuel water-accommodated fraction (Lüchmann et al. 2011).Moreover, an increase of GR concentration was recorded in the digestive glands of R.decussatus transplanted to a pesticides contaminated site in the Mar Menor lagoon (Spain) (Campillo et al. 2013).In the present study, increased Gr activity in R. decussatus collected from the lagoon are in accordance with the results of CAT activity, indicating oxidative stress in the clams harvested from the south lagoon of Tunis.The Variation of SOD activity has been widely used as a useful biomarker to assess susceptibility of organisms to oxidative stress.In the current investigation, SOD activity (Fig. 4B) was significantly higher in clams sampled from S1 (119.15±9.52U SOD mg -1 protein) than SR (70.38±4.91U SOD mg - 1 protein).These results suggest that animals from S1 had a higher efficiency to cope with oxidative stress than bivalves from S2 and S3.Matozzoet al. (Matozzo et al. 2012) explained such reduction in antioxidant enzyme activities in clam R. philippinarum by the exposure of clams to both natural and anthropogenic factors capable to affect antioxidant enzymes.The low level found at S2 and S3 might be explained by the compensatory or adaptativeantioxidative responses.This hypothesis is supported by the high CAT and Gr activities described previously at S2 and S3.

Oxidative damage
MDA level is an indicator of lipid peroxidation which has been widely used as an indicator of oxidative damages to membranes and, therefore, of oxidative stress (Banni et al. 2009, Ahmad et al. 2011).Several environmental contaminants such as heavy metals and hydrocarbons have been reported to increase MDA concentration.Capo et al. (Capó et al. 2015) highlighted an increase of MDA concentration in the digestive gland of the bivalve Pinna nobilis one month after Don Pedro oil spillage.A study performed on the clam, Ruditapesphilippinarumshowed a lowerlipid peroxidation in animal exposed for 14 days toareas near wastewater treatment plant effluent discharges in the Bay of Cádiz (Spain) (Aguirre-Martínez and Martín-Díaz 2020).In this field study, no significant difference in MDA level was observed between polluted and clean sites despite the high antioxidant enzyme activities recorded in the polluted area (Fig. 4D).A possible explanation for this is that the intensity of oxidative stress necessary to trigger a lipid peroxidation requires a different threshold of contamination.

Neurotoxicity biomarkers
The inhibition of AChE activity was frequentlyused as specific biomarkers to indicate the presence of neurotoxic compounds such as organophosphates (OPs) and carbamates used in agriculture as pesticides and heavy metals (Akcha et al. 2000, Galgani and Bocquené 2000, Banni et al. 2005).In the present study, AChE activity (Fig. 4E) was significantly lower in clams sampled from S1 (8.27 ± 0.4 nmol min -1 mg -1 protein) than SR (13.28 ± 1.41 nmol min -1 mg -1 protein).A similar inhibition of AChE activity was recorded in the same clam specie Ruditapes decussates harvested from the Lagoon of Bizerte (Tunisia) (Dellali 2001, Banni et al. 2003) which is continuously submitted to insect management and highly influenced by TinjaOued contaminated by drained pesticides from neighbouring agricultural areas (Dellali 2001).In the same way, an inhibition of AChE activity was reported in clams Ruditapes philippinarum collected from seaward and landward sites in the Lagoon of Venice (Matozzo et al. 2010, Matozzo et al. 2012).Moreover, AChE activity has been found to be modulated by other contaminants environmental such as heavy metals and hydrocarbons.For example, an inhibition of AChE activity was reported in mussels Mytilusgalloprovincialis after exposure to B(a)P (Akcha et al. 2000, Banni et al. 2010).Moreover, an inhibition of AChE activity was reported in the clam, Ruditapes philippinarum by exposing the animals to wastewater treatment plant effluent discharges in the Bay of Cádiz (Spain) (Aguirre-Martínez and Martín-Díaz 2020).The results of the present study and those available in the literature suggest a possible exposure of clams collected from the site S1 to neurotoxic compounds.http://sciforum.net/conference/mol2net-07Fig. 4. Catalase (A), Superoxide dismutase (B), Glutathione reductase (C), Acetylchlinesterase (E) activities and Malondialdehyde concentration (D) in clam Ruditapes decussatus collected from three sites of the lagoon (S1, S2 and S3) and the control site (SR).The bars represent the mean ± SE.Small letters denote significant differences between sites (ANOVA, p <0.05).

Principal component analysis
Principal component analysis (PCA) was performed to obtain an overview of the spatial distribution of the biochemical biomarker and hydrocarbon levels data.Two principal components were extracted which accounted for74.37% of the total variance (Fig. 5): PC1 explained 54.37% of the total variance was positively loaded by biochemical parameters (CAT, GR, SOD, MDA and AChE) in clams and hydrocarbon levels in both matrix sediment (S-AH and S-NAH) and clams (O-AH and O-NAH).PC2 explained 20.00% of the total variance.According to PC2 axis, the three sites S1,S2 and S3 were clearly discriminated from the control site SR.Pearson's correlation coefficients between the studied correlation coefficient higher than 0. conditions, AChE activity was positively 0.552) which was also positively O-NAH (r = 0.567).Moreover, CAT activity hydrocarbon levels (0.667, 0.606, 0.541 and 0.596 for O respectively).Furthermore, O-AH level was positively correlated 0.573) and S-NAH (r = 0.761).

Conclusion
Hydrocarbon analysis revealed a high pollution state in the south l side, our results reveal activation of antioxidant enzymes in clams sampled from the polluted area as well as neurotoxicity.These results demonstrate the alteration of the health status in these organisms  Hydrocarbon analysis revealed a high pollution state in the south lagoon of Tunis our results reveal activation of antioxidant enzymes in clams sampled from the polluted area as well as neurotoxicity.These results demonstrate the alteration of the health status in these organisms are shown in Table 1.A was considered as significant at P < 0.05.In our experimental 701) and SOD activity (r = GR activity(r = 0.756) and GR activity(r = 0.604) and NAH, S-AH and S-NAH, NAH (r = 0.922), S-AH (r = hydrocarbon levels and biomarkers.
-AH S-NAH 0.118 1 agoon of Tunis.On the other our results reveal activation of antioxidant enzymes in clams sampled from the polluted area as well as neurotoxicity.These results demonstrate the alteration of the health status in these organisms http://sciforum.net/conference/mol2net-07generated by high pollution by petroleum hydrocarbons in all sampling sites in the south lagoon of Tunis.Overall, the current study reveals the efficiency of this methodological approach to evaluate physiological responses in Ruditapes decussatus to environmental disruption caused by anthropogenic pollution and reinforces the idea that the biomarker approach can be considered complementary to the chemical characterization of sampling site.
s on Multidisciplinary Sciences 06: Molec.Diversity, Environ.Chem., and Ohio, USA, 2021 Hydrocarbon levels and biochemical biomarkers in the clam collected from Tunis lagoon (Tunisia) Saidane Mosbahi , Laboratory of Analysis, Treatment and Valorization of Pollutants of the Environment Catalase (CAT) activity was measured based on the method adapted from modified by Clairbone(Clairbone 1985 absorbance at 240 nm due to the presence of expressed in µmol of H 2 O 2 min -1 mg -5078 4 http://sciforum.net/conference/mol2net-07Map of the study area and location of sampling sites in Tunis lagoon and Louza the Total Hydrocarbons (TH) from sediments and clam tissues following the method ofMansour et al.(Mansour et al. amount of 50 g of sediments or 5 g of clam soft tissues were extracted in a soxhlet extractor

Fig. 2 .
Fig.2.Concentrations of (A) aromatic (AH) and (B) non aromatic (NAH) hydrocarbons (μg/mg dry weight) in the soft tissue of clam Ruditapes decussatus collected from three sites of the lagoon (S1, S2 and S3) and the control site (SR).The bars represent the mean ± SE.Data with different superscript expressed significant differences between sites (p < 0.05, Tukey test).

E
Fig.5.Results of PCA of the two main AH and S-NAH) and biomarkers (AChE, CAT, GR, collected from the three sites of the southern lagoon of Tunis (S1, S2 and S3) and the control site (SR).
/sciforum.net/conference/mol2net-07Results of PCA of the two main factors produced by Hydrocarbon levels and biomarkers (AChE, CAT, GR, MDA and SOD) in clams, collected from the three sites of the southern lagoon of Tunis (S1, S2 and S3) and the control site (SR).Pearson's correlation coefficients between the studied parameters are shown correlation coefficient higher than 0.5 was considered as significant at P < 0.05.In our experimental positively correlated withGR activity(r = 0.701 correlated withCAT activity (r = 0.531) GR .Moreover, CAT activitywas positively correlated withGR hydrocarbon levels (0.667, 0.606, 0.541 and 0.596 for O-AH, O-NAH, S AH level was positively correlated with O-NAH (r = 0.922), S Pearson's correlation coefficients (r) of hydrocarbon levels and biomarkers Hydrocarbon levels (O-AH, O-NAH, S-SOD) in clams, Ruditapes decussatus, collected from the three sites of the southern lagoon of Tunis (S1, S2 and S3) and the control site (SR).