Assessment of single and combined effects of bisphenol-A and its analogue bisphenol-S on biochemical and histopathological responses of sea cucumber Holothuria poli.

Bisphenols (BPs) are among emerging pollutants that have been frequently detected in different compartments of marine ecosystems and elicited great concern due to their potential toxicity to marine organisms. This work aimed to investigate the toxicity of bisphenol A (BPA) and bisphenol S (BPS) on oxidative stress markers, neurotoxicity and histopathological alterations in sea cucumbers (Holothuria poli). The results showed that exposure to 200 µg/L of BPA and BPS produced oxidative stress, neurotoxicity in the digestive tract and respiratory tree, and several types of histopathological lesions in tissues of the respiratory tree of the sea cucumber, posing a health hazard to this aquatic organism. In addition, BPA has greater effects than BPS on the generation of oxidative stress marked by the inductions of catalase (CAT), glutathione S-transferase (GST) and malondialdehyde (MDA) levels and neurotoxicity shown by the decrease in acetylcholinesterase activity (AChE). The respiratory tree of sea cucumbers might be an appropriate tissue for assessing CAT, MDA and AChE activity levels, which are reliable biomarkers that may be useful in marine biomonitoring studies. Evaluation of histopathological lesions of the respiratory tree suggests that BPA and BPS and their mixture cause various tissue alterations that may be associated with oxidative stress damage and neurotoxicity. In conclusion, this study showed that oxidative stress (CAT and MDA) and neurotoxicity (AChE) markers, as well as respiratory tree lesions, are sensitive biomarkers for the assessment of BPA and BPS toxicity in sea cucumbers.

Does thermal stress modulate the biochemical and physiological responses of Ruditapes decussatus exposed to the progestin levonorgestrel?

In this study, we investigated the effects of 1000 ng/l levonorgestrel (LNG) alone or combined with increased temperature of 20, 24, and 28 °C on the biochemical and physiological responses of the clam (Ruditapes decussatus) for 28 days. Our results revealed that female clams treated with levonorgestrel (LNG) alone showed enhancement of the antioxidant defense against oxidative stress related to the inductions of catalase (CAT), gluthatione -S -transferase (GST), and protein sulfhydryl (PSH), while the elevated temperatures of 20, 24, and 28 °C diminished most of the specific responses to LNG and was the main factor in the determining the responses to combine exposures. The responses of lysosomal membrane stability, alkaline phosphatase, and NADP+-dependent isocitrate dehydrogenase detected were the most common signs of an adverse effect in all exposures. Female clams' testosterone and estradiol responses to LNG were the most particular manifestations depending on the exposure. Overall, these findings showed clearly that chronic warming stress caused disruption in physiological, biochemical parameters of the female clam R. decussatus, and this may have implications for the whole organism and populations.

Application of the Paracentrotus lividus sea-urchin embryo-larval bioassay to the marine pollution biomonitoring program in the Tunisian coast.

The pollution of the marine environment by treated and untreated effluents has increased due to human activities. Monitoring the marine ecosystem is nowadays a global concern. In this work, we evaluated the effect of contaminated and uncontaminated seawater, from different Tunisian coastal areas, on the fertilization, gastrulation, and embryo-larval development events of sea urchins (Paracentrotus lividus). The station of Salakta (SA) is considered as a control station, while the stations of Hamdoun Wadi (HW), Port of Monastir (PM), Karaia Monastir (KM), Teboulba (TE), and Khniss Lagoon (KL) are considered to be contaminated stations. The analysis of seawater physicochemical characteristics showed that levels of the total suspended matter (TSM), chemical oxygen demand (COD), biochemical oxygen demand (BOD), total organic carbon (TOC), and nitrate (NO3-) were lower in the seawater of the reference site Salakta (SA) when compared to those of the contaminated seawater sites. In addition, a very strong variation in the levels of trace metals in seawaters sampled in the studied sites was noted. In fact, the highest concentrations of Pb and Cu were observed in Hamdoun Wadi (HW), port of Monastir (PM), and Karaia Monastir (KM), while the highest concentration of Zn was noted in the Teboulba lagoon (TE) and Khniss (LK). Alterations in physicochemical characteristics as well as elevated trace metal levels in the studied seawater samples were correlated with reduced fertility rate, gastrulation rate, and the frequency of normal sea urchin larvae. The total absence of normal sea urchin pluteus larvae in the sea waters of heavily polluted sites proves the great sensitivity of the larval frequency to mixed pollution. This work recommends the utility of urchin fertilization and gastrulation rates and normal pluteus larval frequencies as useful bioassays to monitor the exposure of marine ecosystems to mixed pollution.

Effect of melatonin and folic acid supplementation on the growth performance, antioxidant status, and liver histology of the farmed gilthead sea bream (Sparus aurata L.) under standard rearing conditions.

The present study aimed to investigate the effect of dietary of melatonin (MLT) and folic acid (FA) administrations on growth performance, antioxidant status, and liver histological structure of juvenile gilthead sea bream, Sparus aurata L. under standard rearing conditions. Four diets were considered: a basal diet considered a control and three diets supplemented with 40 mg/kg of melatonin (MLT), 2 mg/kg of folic acid (FA), and with the mixture of melatonin and folic acid (MLT + FA). Each diet was randomly allocated to triplicate groups of fish (mean initial weight was 2.99 ± 0.55 g) for 41 days. The obtained results clearly indicated that the melatonin-supplemented diet decreased significantly the growth performance parameters (final body weight, weight gain rate, and specific growth rate) and IGF-1 level of the gilthead sea bream, while the folic acid-supplemented diet has no significant effect on these parameters. The mixture supplementation of melatonin and folic acid has no significant effect on the growth parameters due to the possible interaction between melatonin and folic acid effects. Furthermore, fish fed with all experimental diets showed significantly higher superoxide dismutase activity (SOD) and protein sulfhydryl level (PSH) and lower lipid peroxidation level (TBARS) and catalase activity (CAT) which confirm their powerful antioxidant role. The acetylcholinesterase activity (ACHE) decreased in fish fed with all experimental diets. The underlying mechanisms of driving melatonin and folic acid to reduce acetylcholinesterase activity require further studies. The histological structure of liver of control S. aurata fish shows severe hepatic lipid accumulation in large vacuoles that diminished after dietary individual or mixture folic acid and melatonin supplementations over 41 days. This work proved that 2 mg/kg of dietary folic acid has a positive effect on the growth performance, oxidative stress defense, and hepatic lipid accumulation reduction in the gilthead sea bream fish. Under our experimental conditions, melatonin failed to improve the growth indexes WGR, SGR, and IGF-I. This study recommends the diet supplementation with a dose lower than 2 mg/kg of food due to the observed effects on tissue ACHE activity.

Incidence of morphometry variation, growth alteration, and reproduction performance of the annular sea bream (Diplodus annularis) as effective tools to assess marine contamination: how useful is a multi-biotimarkers approach?

A wide variety of contaminants in the aquatic environment causes several deleterious effects on aquatic organism traits ranging from molecular to individual and population levels. This in situ study investigated morphometry, growth performance, and reproduction success of a teleost fish, Diplodus annularis, from a polluted site "Sayada" and a reference site "Salakta" in the eastern Tunisian coastline. Morphometric indexes, generalized Procruste analysis, thin plate, and otolith contour methods were used to assess the pollution effects on morphological traits. The growth performance of fish from contaminated and control sites were studied using the Von Bertalanffy equation. Gonadosomatic (GSI) and Hepatosomatic (HSI) Indexes and absolute and relative fecundity were used to assess the impact of pollution on the reproduction success of this species. The obtained results showed that the fish of the contaminated zone had significant alteration of the morphology, slower growth, significant increase in the HSI, significant decrease in female GSI, and a decrease in absolute and relative fecundity compared to specimens from the unpolluted zone. Based on these results, the biological traits of Diplodus annularis can be used as biological biomarkers in the monitoring and protection programs of the marine contamination in the Mediterranean Sea.

Assessment of the individual and mixture toxicity of cadmium, copper and oxytetracycline, on the embryo-larval development of the sea urchin Paracentrotus lividus.

Multiple pollutions by trace metals and pharmaceuticals have become one of the most important problems in marine coastal areas because of its excessive toxicity on organisms living in this area. This study aimed to assess the individual and mixture toxicity of Cu, Cd, and oxytetracycline frequently existing in the contaminated marine areas and the embryo-larval development of the sea urchin Paracentrotus lividus. The individual contamination of the spermatozoid for 1 h with the increasing concentrations of Cd, Cu, and OTC decreases the fertility rate and increases larvae anomalies in the order Cu > Cd > OTC. Moreover, the normal larva frequency and the length of spicules were more sensitive than the fertilization rate and normal gastrula frequency endpoints. The mixture toxicity assessed by multiple experimental designs showed clearly that concentrations of Cd, Cu, and OTC superior to 338 µg/L, 0.56 µg/L, and 0.83 mg/L, respectively, cause significant larva malformations.

Using environmental proteomics to assess pollutant response of Carcinus maenas along the Tunisian coast.

Biochemical responses to pollutants were studied at four Tunisia littoral sites using Carcinus maenas as a bioindicator. Proteomic analysis was used to assess the global impact of complex pollution mixtures, and to provide new biomarkers and basic insights into pollutant toxicity. Metal contents and metallothionein levels followed a gradient based on sampling sites: Bizerte ≫ Teboulba > Gargour~Mahres. Approximately 900 and 700 spots were resolved in digestive glands and gills, respectively. Gills from Bizerte animals had the maximum number of altered spots, mostly upregulated. In other locations, the number of altered spots in gills decreased in parallel to total metals in in the following order: Teboulba > Gargour > Mahres (mostly downregulated). Out of the 39 spots excised, ten proteins were identified in digestive glands and eight in gills. Digestive glands of Bizerte crabs had higher levels of ferritin, three vitellogenin forms and mannose-binding protein, while Gargour crabs had higher levels of four cryptocyanin forms. Gills of Bizerte crabs had higher levels of ferritin, three vitellogenins forms, lectin 4C, actin, and collagenolytic serine protease. Proteins with altered expression in crabs from Tunisia littoral are related to molting, oxidative stress and inflammation, innate immune response, and proteolysis.

Assessment of toxic interactions between deltamethrin and copper on the fertility and developmental events in the Mediterranean sea urchin, Paracentrotus lividus.

Deltamethrin pesticide and copper have intensively been used in agriculture and industrial activities and can finally reach the marine ecosystem at high concentrations affecting the health of organisms. In this study, we assessed under laboratory conditions the toxic interactions between deltamethrin and copper and their effects on the fertility rate, cell mitotic division rate, and embryo developmental events of the sea urchin (Paracentrotus lividus). The exposure of sperm to increasing concentrations of deltamethrin (6.10(-5) and 6.10(-2) µg/L) and copper (50 and 100 µg/L) or to their mixture (6.10(-5) µg/L of deltamethrin and 50 µg/L of CuSO4) caused a significant alteration on the fertilizing capability of spermatozoids. Concentration-dependent toxic effects on the early cleavage in P. lividus were observed in groups treated with copper, deltamethrin, and their mixture. The kinetics of early divisions was accelerated and the average size of pluteus larvae was decreased under pollutant treatments. Several developmental anomalies were identified in pluteus, including crossed skeletal tips at the hood apex, joined or atrophied arms, and alteration of general larva shape. In conclusion, the sea urchin represents a suitable and sensitive model for testing the toxicity and the effects of deltamethrin pesticide and copper in sea water. In addition, the sensitivity of various end points to studied contaminants, proved their utility in the infield biomonitoring studies.

Comparative study of the bioaccumulation and elimination of trace metals (Cd, Pb, Zn, Mn and Fe) in the digestive gland, gills and muscle of bivalve Pinna nobilis during a field transplant experiment.

The purpose of this study was to investigate the long-term bioaccumulation and elimination of Cd, Pb, Mn, Zn and Fe by Pinna nobilis tissues after their 90 day-transplantation period at Téboulba fishing harbor. During the transplantation period, the Cd, Pb, Mn, Zn and Fe concentrations in the different tissues of the mussels were measured before and after exposure period. Metal (Cd, Pb, Mn, Zn and Fe) accumulation in P. nobilis mussels varied depending on the analyzed tissue and the caging times. Notable differences in Cd, Pb, Mn, Zn and Fe accumulation patterns within the digestive gland, gills and muscle were found and may be due to the ability of each tissue to accumulate metals. During the depuration phase, the elimination of Cd, Pb, Mn, Zn and Fe depended on the target tissue and the metal speciation. Cd, Pb, Mn and Fe were eliminated rapidly from one organ and increased in other when compared to those of 90 day transplanted mussels. The increase of metal loads during the elimination phase is not clear and particularly what kind of processes is responsible for such response. However, it is reasonable to assume that metals increase is related to the existence of an accumulation/detoxification mechanism, which involves the transport of metals from an organ to another. The data obtained indicate that because of the significantly high quantities of Cd, Pb, Mn, Zn and Fe accumulated during the exposure phase, the transplanted mussels are suitable bioindicators for monitoring trace metals in marine ecosystem.

Proteomic analysis in caged Mediterranean crab (Carcinus maenas) and chemical contaminant exposure in Téboulba Harbour, Tunisia.

This study uses proteomics approach to assess the toxic effects of contaminants in the Mediterranean crab (Carcinus maenas) after transplantation into Téboulba fishing harbour. High levels of aliphatic and aromatic hydrocarbons were detected in sediments. Although their effects on vertebrates are well described, little is known about their early biological effects in marine invertebrates under realistic conditions. Protein expression profiles of crabs caged for 15, 30 and 60 days were compared to unexposed animals. Nineteen proteins with significant expression differences were identified by capLC-µESI-IT MS/MS and homology search on databases. Differentially expressed proteins were assigned to five different categories of biological function including: (1) chitin catabolism, (2) proteolysis, (3) exoskeleton biosynthesis, (4) protein folding and stress response, and (5) transport. The proteins showing major expression changes in C. maenas after different caging times may be considered as novel molecular biomarkers for effectively biomonitoring aquatic environment contamination.

Metals bioaccumulation and histopathological biomarkers in Carcinus maenas crab from Bizerta lagoon, Tunisia.

Metals concentrations and histolopathological lesions of gills and digestive gland were investigated in Carcinus maenas crabs sampled from Bizerta Lagoon and Kuriat Island (Tunisia) as control site. The concentrations of trace metals varied between tissues, sites and sampling time. The highest levels of the analysed metals in gills and digestive gland were noted in Menzel Bourguiba and Cimentery sites at both sampling times (February and July). The higher metals loads were associated with severe and various tissues alterations in contaminated crabs. We particularly noted in the gills a haemocytic infiltration, distension and enlargement of the lamellae, lifting of lamellar epithelium, necrotic lesions and fusion of lamellae in the most polluted sites (Menzel Bourguiba and Cimentery). Moreover, others pathological alterations were observed in digestive gland of crabs collected from polluted sites and with a severity site dependent. We observed necrotic tubules containing tissue debris in the lumen with more intensity in crabs collected from Cimentery site in both sampling times. The thickened basal laminae and the walling off of the tubules by haemocytes around the thickened basal laminae were more abundant at Menzel Bourguiba than at others sites. The coagulation in the thickened basal laminae was observed only at Cimentery in February. Tissues histopathological lesions were sensitive to discriminate crabs of different sites and demonstrated its usefulness in this biomonitoring study. We recommend the association of histopatholocial lesions to biochemical biomarkers in future biomonitoring studies.

Biochemical effects in crabs (Carcinus maenas) and contamination levels in the Bizerta Lagoon: an integrated approach in biomonitoring of marine complex pollution.

The biochemical effects in Carcinus maenas and contamination levels in seawater and sediments of Bizerta Lagoon (northeast of Tunisia) were investigated. The levels of metals and hydrocarbons were higher in seawater and sediments in Menzel Bourguiba and Cimentery in February and July than in the other sampling sites. Differences among sites for glutathione S-transferase, catalase, acetylcholinesterase activities, and the content of lipid peroxidation and metallothioneins in two important organs which accumulated contaminants (the gills and the digestive gland) of the C. maenas were found and possibly related to differences in metal and hydrocarbon levels. The seasonal variation of biomarkers was possibly associated with chemical contamination and also with the high fluctuation of physico-chemical characteristics of the sites. The integrated biomarker response values found in the five sites is in good agreement with hydrocarbon and trace metal concentrations detected in the water and sediments of the stressful places where crabs are living.

Biochemical responses in seabream (Sparus aurata) caged in-field or exposed to benzo(a)pyrene and paraquat. Characterization of glutathione S-transferases.

Gilthead seabream (Sparus aurata) specimens were caged in-field at the Téboulba harbour or exposed to benzo(a)pyrene [B(a)P] or to paraquat [PQ] plus B(a)P, and several biochemical biomarker responses were investigated. Antioxidant enzymes, such as glutathione peroxidase, catalase and glutathione reductase, significantly increased in the in-field and B(a)P+PQ exposures, but were only moderately affected by B(a)P alone. Glucose-6-phosphate and 6-phosphogluconate dehydrogenases significantly diminished after in-field exposure. Different responses with biotransformation enzymes were observed: the P4501A-associated EROD activity was highly induced in response to B(a)P and B(a)P+PQ exposures, while total activity of the glutathione S-transferase (GST) was similar to control. However, after purification of the GST proteins by affinity chromatograpy and analysis by two-dimensional electrophoresis, nineteen highly reproducible isoforms were resolved. In addition, some of reproducible isoforms showed different and specific expression patterns in response to contaminants. Thus, proteomic analysis of the purified GST subunits is a reliable tool for ecotoxicological research, useful in polluted marine ecosystem as an effective biomarker of contamination.

Multiple biomarkers of pollution effects in Solea solea fish on the Tunisia coastline.

This field study investigates the morphological indices (condition index, hepatosomatic index) and biochemical (catalase (CAT), glutathione S-transferase (GST), acetylcholinesterase (AChE), metallothionein (MT), lipid peroxidation) parameters in liver, gills and kidney of common sole (Solea solea) originating from different sites of the Tunisian coast area impacted by different anthropogenic activities. Differences among sites and tissues for AChE, GST, CAT, MT and TBARS were found and possibly related to known sources of domestic and industrial discharges in the studied sites. Liver, gills and kidney CAT, liver and kidney MT and brain AChE were key biomarkers to discriminate fish of different sites. So, we suggest using these biomarkers in future biomonitoring.

Biochemical responses and metals levels in Ruditapes decussatus after exposure to treated municipal effluents.

This study assessed the responses of biochemical biomarkers and metals levels in Ruditapes decussatus exposed to the increasing concentrations of treated municipal effluents (TME) discharged into the Tunisian coastal area. Clams were exposed to 0%, 1%, 3% and 10% for 7 and 14 day and the following biochemical responses were measured: (1) catalase activity and lipid peroxidation levels (TBARS) as oxidative stress biomarkers, (2) gluthathione S-transferase (GST) activity as a phase II conjugation enzyme; (3) cholinesterase activity (ChE) as biomarker of neurotoxicity, and (4) metallothioneins as a proteins highly induced by heavy metals. A significant uptake of Cu, Cd and Zn in digestive gland and serious biochemical alterations were observed. Thus, exposure of clams to croissant concentration of TME have the potential to increase the oxidative stress biomarkers (TBARS, CAT activity) and MT levels; and decrease ChE activity in both gills and digestive gland. Current experimental results suggest that CAT, GST, ChE activities and MT and TBARs levels in gills and digestive gland of clam R. decussatus are sensitive and suitable responses for assessing the effects of anthropogenic contaminants on the aquatic ecosystems, particularly effluent complex mixtures.

Mixture toxicity assessment of nickel and chlorpyrifos in the sea bass Dicentrarchus labrax.

The present research work was designed to study Dicentrarchus labrax biotransformation and detoxification responses to acute exposure to nickel (Ni) and chlorpyrifos (CHP). Sexually immature sea bass were treated by intraperitoneal injection of nickel chloride (500 µg kg⁻¹), chlorpyrifos (10 mg kg⁻¹), and their binary mixture for 1, 3, and 7 days. Ni and CHP accumulation was quantified in liver after the exposure periods. The following biological responses were measured: (1) NADPH cytochrome P450 reductase (NCR) activity, as phase I biotransformation parameter; (2) gluthathione S-transferase (GST) activity as a phase II conjugation enzyme, acetylcholinesterase activity, and metallothionein (MT) content. Ni bioaccumulation in the liver resulted in an increasing uptake up to 15.48 µg g⁻¹ wet weight (Ni-treated animals) and 16.73 µg g⁻¹ wet weight (mixture-treated animals) after 7 days of exposure. CHP accumulation showed a distinct pattern in animals exposed to the mixture of chemicals in comparison with CHP-treated animals. NCR activity exhibited a marked activation in CHP and mixture-treated animals. GST activity was significantly increased starting from 1 day exposure in CHP-treated animals and after 3 days in Ni-treated animals. MT accumulation increased in all conditions, with a marked synergetic effect after 7 days of exposure. These data should be carefully considered in view of the biological effects of mixture pollutants, particularly in fish farming conditions.

Integrated assessment of biochemical responses in Mediterranean crab (Carcinus maenas) collected from Monastir Bay, Tunisia.

The biochemical response of Mediteranean Crab (Carcinus maenas) collected at five stations of Monastir Bay and from Kuriat station as control was studied using a set of complementary biomarkers. The catalase, glutathione S-transferase, lactate dehydrogenase, acetycholinesterase activities; and metallothionein and malonediladehyde levels in gills were evaluated. Results revealed differences among sites in relation to each specific biomarker. Hence, a suite of biomarkers can be used to discriminate sampling sites according to types of pollution, reflecting differing conditions of anthropogenic impact. Based on Integrated Biomarker Response, the highest values and critical biochemical alteration were observed at Khniss and Ksibat in response to urban and industrial discharges and the lowest IBR value was found at reference site. The current study has shown clearly that a biomarker-based index is usefulness tool in the monitoring Tunisian coast using C. maenas as sentinel specie. Further studies in progress to investigate the seasonal variations of IBR levels and its relationship to pollutants concentrations in the sediment, gills and digestive gland of Carcinus maenas from Monastir Bay.

Uptake and biochemical responses of mussels Mytilus galloprovincialis exposed to sublethal nickel concentrations.

In the present study, mussel (Mytilus galloprovincialis) digestive gland oxidative stress biomarkers and detoxification responses to acute exposure to nickel (Ni) were investigated. Mussels were exposed to two sublethal concentrations of Ni (135 µg/L per animal (2.5 µM) and 770 µg/L per animal (13 µM)) for 24, 48, 72, 96 h and 8 days. Following biological responses were measured: (1) glutathione S-transferase (GST) activity as a phase II conjugation enzyme, (2) catalase activity as antioxidant response, (3) malondialdehyde accumulation (MDA) as lipid peroxydation marker and metallothionein as specific response to metals exposure. The cholinergic system was evaluated using the acetylcholinesterase activity (AChE). Moreover, Ni uptakes during the exposure periods were assessed and the uptake rate constant determined. A correlation matrix (CM) between the investigated biomarkers and a principal component analysis (PCA) were achieved for the two tested concentrations. The Ni-uptake constant was higher in animals exposed to the lowest concentration. The CM and the PCA showed a time-dependent effect of the Ni exposure on the investigated biomarkers being more pronounced in animals exposed to the highest Ni concentration. While AChE showed a significant increase after 48 h and a further return to control values in the lowest concentration, it was drastically maintained inhibited in the highest concentration. Our data provided clues about the occurrence of different toxicokinetics and toxicodynamics of two Ni sublethal concentrations in an ecologically relevant organism.

Multimarker approach analysis in common carp Cyprinus carpio sampled from three freshwater sites.

The aim of this study is to assess the response of a multimarker approach in common carp Cyprinus carpio sampled from three Tunisian dam lakes selected according to different environmental and ecological characteristics. Glutathione-S-transferase (GST) activity was analyzed in carp liver and used as a phase II detoxification enzyme, hepatic metallothionein content (MTs) was used as a metallic stress indicator, and cholinesterase activities were analyzed in muscle and brain and used as neurotoxicity biomarker. Micronucleus frequency test (MN) as a genotoxicity marker. GST and MT levels showed an increase in fish from the Bir Mcherga site and a decrease in Sidi Saâd site with respect to fish from Nebhana site. Results showed a strong inhibition of cholinesterase activities in fish from Bir Mcherga and Sidi Saâd sites compared to Nebhana site. Relatively high level of MN is reported specially in fish blood from the Bir Mcherga site.

Monitoring pollution in Tunisian coasts using a scale of classification based on biochemical markers in worms Nereis (Hediste) diversicolor.

This paper aims to assess the marine environment quality along the Tunisian coasts using a statistical approach based on biomarkers response in the polychaete worms Nereis (Hediste) diversicolor. Worms were collected from six sites: Bizerta Lagoon, Gargour, Nakta, Mahres, Skhira and from Teboulba considered as a reference site. The biomarkers selected in this work were (1) the activities of cytochrome P450-dependent NADPH cytochrome c reductase (NADPH red) as phase I enzyme, (2) glutathione S-transferase as phase II enzyme and (3) the acetylcholinesterase activity as neurotoxicity marker. Oxidative stress was evaluated using catalase activity and malondialdehyde accumulation. For each biomarker, a discriminatory factor was calculated and a response index was allocated. For each site, a multi-marker pollution index was calculated as the sum of the response index of each of the five more discriminating biomarkers. The results show differences between sites compared with the reference samples. The multi-marker approach confirms that worms from Bizerta and Mahress have been submitted to highly polluted environment. Mahress shows the highest multi-marker pollution index, indicating a highly contamination status.