Assessment of oxidative stress, genotoxicity and histopathological responses in the digestive gland of Ruditapes decussatus collected from northern Tunisian lagoons

Safa Bejaoui 1, Imen Rabeh 1, Khaoula Telahigue 1,#, Mariem Tir 1,#, Imene Chetoui 1, Chaima Fouzai 1, Salwa Nechi 2, Emna Chelbi 2, Mhamed EL Cafsi 1, Nejla Soudani 1

1 Laboratory of Ecology, Biology and Physiology of aquatic organisms, Tunis Faculty of Science, University of Tunis El Manar, 2092, Tunis, Tunisia.
(SB) (Corresponding author) E-mail: safa.BEJAOUI@fst.utm.tn. ORCID iD: https://orcid.org/0000-0002-7946-2763
(IR)E-mail: rabehimen@yahoo.fr. ORCID iD: https://orcid.org/0000-0002-0307-473X
(KT)E-mail: k_telahigue@yahoo.fr.ORCID iD: https://orcid.org/0000-0001-8841-9911
(MT)E-mail: tirmariem@yahoo.fr. ORCID iD: https://orcid.org/0000-0002-2477-4815
(IC) E-mail: chetouiimene@gmail.com. ORCID iD: https://orcid.org/0000-0002-2259-5397
(CF)E-mail: fouzai.chaima93@gmail.com. ORCID iD: https://orcid.org/0000-0001-7588-1859
(MEC)E-mail: mhamed.elcafsi@gmail.com. ORCID iD: https://orcid.org/0000-0002-9771-1110
(NS)E-mail: nejla.soudani@tunet.tn. ORCID iD: https://orcid.org/0000-0002-7652-9678
2 Anatomy and Cytology Service, Mohamed TaherMaamouri hospital, Road Mrezka 8000, Nabeul, Tunisia.
(SN)E-mail: salwanechi@gmail.com. ORCID iD: https://orcid.org/0000-0003-2477-5954
(EC)E-mail: emnachalbi@gmail.com. ORCID iD: https://orcid.org/0000-0002-1562-1788

#Dr Khaoula Telahigue and Dr Mariem Tir contributed equally to this work.

Summary: The aim of the present study was to investigate the combined effects of seasonality and anthropogenic pressure on a battery of oxidative stress, lipid peroxidation, protein oxidation, DNA damage and histological alterations in the native clam Ruditapes decussatus collected from a less contaminated area (LCA), Ghar El Melh, a moderately contaminated area (MCA), the North Lake, and a highly contaminated area (HCA), the South Lake, all located in the southern Mediterranean Sea. The accumulation of cadmium, lead, copper, iron and zinc was higher in the digestive glandsof clams collected from the MCA and the HCAthan in those from the LCA, particularly during the warm season. Our results reveal that metallothionein, lipid peroxidation, protein oxidation levels and antioxidant defence systems were higher, while acetylcholinesterase activity was lower, in clams from the MCAand HCAthan in those from the LCA. The results also indicate that clams from the MCA and the HCAare characterized by histological alterations and DNA damage. In conclusion, the evident changes of antioxidant defence systems and macromolecules between the studied lagoons reveal the perturbation of the physiological states of clams from polluted sites thatcope with seasonal changes and trace element accumulations.

Keywords: Ruditapes decussatus; digestive gland; trace element accumulations; redox status; macromolecule injuries; histoarchitecture alteration.

Evaluación del estrés oxidativo, genotoxicidad y respuestas histopatológicas en la glándula digestiva Ruditapes decussatus recolectada de las lagunas del norte de Túnez

Resumen: El objetivo del presente estudio es investigar los efectos combinados de la estacionalidad y la presión antropogénica en una batería de estrés oxidativo, peroxidación lipídica, oxidación de proteínas, daños en el DNA y alteraciones histológicas en la almeja nativa Ruditapes decussata recolectada de un área menos contaminada (Ghar El Melh «LCA») y de dos sitios con diferentes niveles de contaminación (la laguna norte «MCA» y la laguna sur «HCA» de Túnez) en el sur del mar Mediterráneo. La acumulación de cadmio, plomo, cobre, hierro y zinc fue mayor en la glándula digestiva de las almejas recolectadas de la MCA y la HCA en comparación con las de la LCA, particularmente durante la estación cálida. Nuestros resultados revelan que la metalotioneína, la peroxidación lipídica, los niveles de oxidación de proteínas y los sistemas de defensa antioxidante aumentaron, mientras que la actividad de la acetilcolinesterasa disminuyó en las almejas del área moderadamente y altamente contaminada en comparación con la menos contaminada. Los resultados también indican que las almejas del MCA y el HCA se caracterizan por varias alteraciones histológicas y daños en el ADN. En conclusión, los cambios evidentes de los sistemas de defensa antioxidante y las macromoléculas entre las lagunas estudiadas revelan la perturbación de los estados fisiológicos de las almejas de los sitios contaminados que hacen frente a los cambios estacionales y las acumulaciones de metales.

Palabras clave: Ruditapes decussatus; glándula digestiva; acumulaciones de metales; estado redox; lesiones de macromoléculas; alteración de la histoarquitectura.

Citation/Como citar este artículo: Bejaoui S., Rabeh I., Telahigue K., Tir M., Chetoui I., Fouzai C., Nechi S., Chelbi E., EL Cafsi M., Soudani N. 2020. Assessment of oxidative stress, genotoxicity and histopathological responses in the digestive gland of Ruditapes decussatus collected from northern Tunisian lagoons. Sci. Mar. 84(4): 403-420. https://doi.org/10.3989/scimar.05054.23A

Editor: C. Porte.

Received: March 23, 2020. Accepted: October 5, 2020. Published: November 4, 2020.

Copyright: © 2020 CSIC. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International (CC BY 4.0) License.

Contents

Summary
Resumen
Introduction
Materials and methods
Results
Discussion
Conclusion
Acknowledgements
References

INTRODUCTIONTop

Marine ecosystems, particularly lagoons, are of great ecological and economic importance becausethey support vital habitats for numerous marine organisms. However, they sustain several anthropogenic pressures (Barhoumi et al. 2014Barhoumi B., Le Menach K., Clerandeau C., et al. 2014. Assessment of pollution in the Bizerte lagoon (Tunisia) by the combined use of chemical and biochemical markers in mussels, Mytillus galloprovincialis. Mar. Pollut. Bull. 84: 379-390. ). In fact, increasing industrialization has led to a massive release of pollutants into these enclosed ecosystems (Di Salvatore et al. 2013Di Salvatore P., Calcagno J.A., Ortiz N., et al. 2013. Effect of seasonality on oxidative stress responses and metal accumulation in soft tissues of Aula comyaatra, a mussel from the South Atlantic Patagonian coast. Mar. Environ. Res. 92: 244-252.). During the last few decades, the aquatic environment in Tunisia, especially the north coast, has been contaminated by trace element (TE) pollutants as a result of rapid industrialization and urbanization (Mzoughi and Chouba 2012Mzoughi N., Chouba L. 2012. Heavy metals and PAH assessment based on mussel caging in the North coast of Tunisia (Mediterranean Sea). Int. J. Environ. Res. 11: 109-118., Daldoul et al. 2015Daldoul G., Souissi F., Jemmaili N. et al. 2015. Assessment and mobility of heavy metals in carbonated soils contaminated by old mine tailings in North Tunisia. Afr. Earth Sci. 110: 150-159.). In recent years, the discharge of industrial wastewater containing a high level of TEs has increased (Ghribi et al. 2020Ghribi F., Richir J., Bejaoui S., et al. 2020. Trace elements and oxidative stress in the Ark shell Arca noae from a Mediterranean coastal lagoon (Bizerte lagoon, Tunisia): are there health risks associated with their consumption? Environ. Sci. Pollut. Res. 27: 15607–15623.). The TE content in sediments from the Tunisian gulfhas been observed to reach a considerable level that could be released into the water by flow changes or benthic agitation, causinga sustained impact on aquatic organisms and eventually on human beings through the food chain (Ennouri et al. 2016Ennouri R., Zaaboub N., Fertouna-Bellakhal M., et al. 2016. Assessing trace metal pollution through high spatial resolution of surface sediments along the Tunis Gulf coast (Southwestern Mediterranean). Environ. Sci. Pollut. Res. 23: 5322-5334.).

Among these contaminants, metals and metalloids are a particular concern because of their toxic effects and their persistence through bioaccumulation and biomagnification along the food chain (Leung et al. 2016Leung H.M., Leung A.O.W., Wang H.S., et al. 2016. Assessment of heavy metals/metalloid (As, Pb, Cd, Ni, Cr, Cu, Mn) concentrations in edible fish species tissue in the Pearl River Delta (PRD), China. Mar. Pollut. Bull. 78: 235-245., Faggio et al. 2018Faggio C., Tsarpali V., Dailianis S. 2018. Mussel digestive gland as a model for assessing xenobiotics: an overview. Sci. Total Environ. 613: 220-229.). From an ecotoxicological point of view, it has been demonstrated that TEs can adversely affect aquatic organisms mainly through an excessive generation of reactive oxygen species (ROS) (Telahigue et al. 2018Telahigue K., Rabeh I., Bejaoui S., et al. 2018. Mercury disrupts redox status, up-regulates metallothionein and induces genotoxicity in respiratory tree of sea cucumber (Holothuria forskali). Drug Chem. Toxicol. 43: 287-297., Yang et al. 2018Yang D., Guo X., Xie T., et al. 2018. Reactive oxygen species may play an essential role in driving biological evolution: The Cambrian Explosion as an example. J. Environ. Sci. 63: 218-226.). Indeed, ROS are proven to cause metabolic and oxidative homeostasis imbalances because of their high reactivity towards cellular components such as lipids, proteins and nucleic acids (Bejaoui et al. 2020Bejaoui S., Michan C., Telahigue K., et al. 2020. Metal body burden and tissue oxidative status in the bivalve Venerupis decussata from Tunisian coastal lagoons. Mar. Environ. Res. 159: 105000.). To protect cells against ROS, some specific antioxidants such as superoxide dismutase, catalase (CAT), glutathione peroxidase (GPx), thiols as glutathione (GSH) and ascorbic acid (Vit C) are also elevated in the detoxification of free radicals (Barhoumi et al. 2014Barhoumi B., Le Menach K., Clerandeau C., et al. 2014. Assessment of pollution in the Bizerte lagoon (Tunisia) by the combined use of chemical and biochemical markers in mussels, Mytillus galloprovincialis. Mar. Pollut. Bull. 84: 379-390. ). Oxidative stress can also be reduced through the complication of free TEs by metallothionein (MT). The latter is an oxyradicalscavenger playing an important role inthe homeostasis of essential TEs and in the detoxification of non-essential ones (Gagné et al. 2008Gagné F., André C., Blaise C. 2008. The Dual Nature of Metallothioneins in the Metabolism of Heavy Metals and Reactive Oxygen Species in Aquatic Organisms: Implications of Use as a Biomarker of Heavy-Metal Effects in Field Investigations. Biochem Insights ). Histopathology is also a powerful tool for monitoring anthropogenic contamination (El-Shenawy et al. 2009El-Shenawy N.S., Moawad T.I.S., Mohallal M., et al. 2009. Histopathologic Biomarker Response of Clam, Ruditapes decussatus, to Organophosphorous Pesticides Reldan and Roundup: A Laboratory Study. Ocean Sci. 44: 27-34.). According to Bejaoui et al (2020)Bejaoui S., Michan C., Telahigue K., et al. 2020. Metal body burden and tissue oxidative status in the bivalve Venerupis decussata from Tunisian coastal lagoons. Mar. Environ. Res. 159: 105000., the histological study could provide information on the adaptive response to environmental pollution and cell damage. In marine ecosystems, histopathological alterations have been associated either with deterioration of environmental conditions or with pollution (Fanta et al. 2003Fanta E., Rios F.S.A., Romão S., et al. 2003. Histopathology of the fish Corydoras paleatus contaminated with sublethal levels of organophosphorus in water and food. Ecotoxicol. Environ. Saf. 54: 119-130., Marchand et al. 2009Marchand M.J., van Dyk J.C., Pieterse G.M., et al. 2009. Histopathological alterations in the liver of the sharptooth catfish Clarias gariepinus from polluted aquatic systems in South Africa. Environm. Toxicol. 24: 133-147.).

During the last few decades, marine invertebrates, especially bivalve molluscs, have been widely used as sentinel species for aquatic pollutants associated with ROS generation (Tsangaris et al. 2016Tsangaris C., Moschino V., Steogyloudi E., et al. 2016. Biochemical biomarker responses to pollution in selected sentinel organisms across the Eastern Mediterranean and the Black Sea. Environ Sci. Pollut. Res. 23: 1789-1804., Uluturhan et al. 2019Uluturhan E., Darilmaz E., Kontas A., et al. 2019. Seasonal variations of multi-biomarker responses to metals and pesticides pollution in M. galloprovincialis and T. decussatus from Homa Lagoon, Eastern Aegean Sea. Mar. Pollut. Res. 141: 176-186., Bejaoui et al. 2020Bejaoui S., Michan C., Telahigue K., et al. 2020. Metal body burden and tissue oxidative status in the bivalve Venerupis decussata from Tunisian coastal lagoons. Mar. Environ. Res. 159: 105000.). These sessile filter-feeder organisms are commonly used to assess the biological effects of contaminated ecosystems (Cravo et al. 2012Cravo C., Pereira T., Gomes C., et al. 2012. Multibiomarker approach in the clam Ruditapes decussatus to assess the impact of pollution in the Ria Formosa lagoon, South Coast of Portugal. Mar. Environ. Res. 75: 23-34.). The clam Ruditapes decussatus is among the most common marine molluscs in Tunisia and around the world. It is an important worldwide economic resource and has been widely used as a bioindicator for monitoring water quality in numerous environmental investigations (Campillo et al. 2013Campillo J.A., Albentosa M.N., Valdes J., et al. 2013.Impact assessment of agricultural inputs into a Mediterranean coastal lagoon (Mar Menor, SE Spain) on transplanted clams (Ruditapes decussatus) by biochemical and physiological responses. Aquat. Toxicol. 142-143: 365-379., Mansour et al. 2020Mansour C., Guibbolini M., Hacene O.R, et al. 2020. Oxidative Stress and Damage Biomarkers in Clam Ruditapes decussatus Exposed to a Polluted Site: The Reliable Biomonitoring Tools in Hot and Cold Seasons. Arch. Environ. Contam. Toxicol. 78: 478-494., Bejaoui et al. 2020Bejaoui S., Michan C., Telahigue K., et al. 2020. Metal body burden and tissue oxidative status in the bivalve Venerupis decussata from Tunisian coastal lagoons. Mar. Environ. Res. 159: 105000.).

There are numerous studies focusing on TEs contamination in sediments and marine organisms from the northern coast of Tunisia (including the Tunisian Gulf and the Bizerte lagoon) (Chalghmi et al. 2016Chalghmi H., Bourdineaud J.P., Haouas Z., et al. 2016. Transcriptomic, Biochemical, and Histopathological Responses of the Clam Ruditapes decussatus from a Metal-Contaminated Tunis Lagoon. Arch. Environ. Contam. Toxicol. 70: 241-256., Ghannem et al. 2016Ghannem S., Khazri A., Sellami B. et al. 2016. Assessment of heavy metal contamination in soil and Chlaenius (Chlaeniellus) olivieri (Coleoptera, Carabidae) in the vicinity of a textile factory near Ras Jbel (Bizerte, Tunisia). Environ. Earth Sci. 75: 442.). However, investigation of the effect of TE contamination in bivalves from Ghar el Melh and the Northern and Southern Lagoons of Tunisia has so far been insufficient. Previous studies have integrated the toxicity responses provided by the clam R. decussatus and contamination to assess the quality of some Tunisian lagoons (Chalghmi et al. 2016Chalghmi H., Bourdineaud J.P., Haouas Z., et al. 2016. Transcriptomic, Biochemical, and Histopathological Responses of the Clam Ruditapes decussatus from a Metal-Contaminated Tunis Lagoon. Arch. Environ. Contam. Toxicol. 70: 241-256.). The current study is aimed at comparing the environmental quality of three lagoons with different contamination levels, a less contaminated area (LCA), a moderately contaminated area (MCA) and a highly contaminated area (HCA) on the northern Tunisian coastline by integrating water parameters, TE accumulation, biological responses, macromolecules and tissue injuries using the digestive gland of R. decussatus.

MATERIALS AND METHODSTop

Study areas

Three sites with different anthropic pressure levels were chosen to conduct our study (Fig. 1):

– Ghar El Melh (37°11′26.25″N 10°18′73.49″E) is considered one of the most important wetlands in Tunisia. It is situated in the extreme north of the Gulf of Tunis (Fig. 1) at the downstream end of the lower valley of the Mejerda River and is linked to the Mediterranean Sea through a dredged inlet (Ayache et al. 2009Ayache F., Thompson J.R., Flower R.J., et al. 2009. Environmental characteristics, landscape history and pressures on three coastal lagoons in the Southern Mediterranean Region: MerjaZerga (Morocco), GharElMelh (Tunisia) and Lake Manzala (Egypt). Hydrobiology 622: 15-43.). This lagoon has an elliptical shape, covers an area of approximately 28.5 km2 and has an average depth of 0.8 m (Moussa et al. 2005Moussa M., Baccar L., Ben Khemis R. 2005. La lagune de Ghar El Melh: Diagnostic écologique et perspectives d’aménagement hydraulique. Rev. Sci. Eau 18: 13-26.). The water column of Ghar el Melh lagoon residues are well homogenized throughout the year thanks to wind-induced mixing. A narrow channel enables a restricted water exchange with the open sea (water residence time in the lagoon: 35 days; Rasmussen et al. 2009Rasmussen E.K., Petersen O.S., Thompson J.R. 2009. Model analyses of the future water quality of the eutrophicated Ghar El Melh lagoon (Northern Tunisia). Hydrobiologia 622: 173-193.). Ghar El Melh Lagoon receives various discharges from the Mejerda River, which delivers 17 million t of sediment annually (Oueslati et al. 2010Oueslati W., Added A., Abdeljaoued S. 2010. Geochemical and statistical approaches to evaluation of metal contamination in a changed sedimentary environment: Ghar El Melh lagoon, Tunisia. Chem. Spec. Bioavailab. 22: 227-240.). The lagoon ecosystem has suffered progressive deterioration and is considered hypereutrophic (Rasmussen et al. 2009Rasmussen E.K., Petersen O.S., Thompson J.R. 2009. Model analyses of the future water quality of the eutrophicated Ghar El Melh lagoon (Northern Tunisia). Hydrobiologia 622: 173-193.) because of human activities within the lagoon itself and in the surrounding area (such as discharges of domestic wastewater, industrial waste from the drainage system and fishing activity) (Moussa et al. 2005Moussa M., Baccar L., Ben Khemis R. 2005. La lagune de Ghar El Melh: Diagnostic écologique et perspectives d’aménagement hydraulique. Rev. Sci. Eau 18: 13-26.). Furthermore, natural disturbances may cause alterations to the hydrodynamics and sediments resulting in a hyper-eutrophication of the environment. According to Nourisson et al. (2013)Nourisson D.H., Scapini F., Massi L., et al. 2013. Optical characterization of coastal lagoons in Tunisia: Ecological assessment to underpin conservation. Ecol. Inf. 14: 79-83., Ghar El Melh is considered a moderately polluted site, because it is one of the world’s wetlands awarded the Wetland City Label accredited by the international Ramsar Convention for the protection of wetlands (Ben Haj 2012Ben Haj S. 2012. Etude du plan de gestion et d’amenagement integres du site ramsar de la zone humide de ghar el melh site ramsar n°1706. Thetis Ecologue Conseil, WWF.). Thus, it was selected as the LCA site in the current study on the basis of other published works (Oueslati et al. 2010Oueslati W., Added A., Abdeljaoued S. 2010. Geochemical and statistical approaches to evaluation of metal contamination in a changed sedimentary environment: Ghar El Melh lagoon, Tunisia. Chem. Spec. Bioavailab. 22: 227-240., Bejaoui et al. 2018Bejaoui S., Telahigue K., Chetoui I., et al. 2018. Integrated effect of metal accumulation, oxidative stress responses and DNA damage in Venerupis decussata gills collected from two coast Tunisian lagoons. J. Chem. Environ. Biol. Eng. 2: 44-51.).

– The Northern Lagoon of Tunis is a shallow submarine environment located at the bottom of the Gulf of Tunis, on the eastern side of Tunis City (36°37′N 10°11′E). It communicates with the sea via the Kheireddine channel (Fig. 1) and receives various influxes of anthropogenic contaminants. The length and the maximum breadth are 10 and 3 km, respectively, for a total surface area of 24 km2 and an average depth of 1.5 m (Ben Maiz 1997Ben Maiz N. 1997. Le lac Nord de Tunis: un milieu en mutation. In: Karem A., Maamouri F., et al. (eds), Gestion et conservation des zonzs humides tunisiennes. Acts de séminaire, DGF, WWF, 77-84.). The hydrodynamics of this lagoon is semidiurnal, principally controlled by wind and tide (Jouini et al. 2005Jouini Z., Ben Charrada R., Moussa M. 2005. Caractéristiques du Lac Sud de Tunis après sa restauration. Mar. Life 15: 3-11.). Furthermore, the average water residence time in the lagoon varies from 6.6 to 8.2 days, with 2.57 million m3 day–1 of total water exchange with the sea (Jouini 2003Jouini Z. 2003. Le Fonctionnement Hydrodynamique et Écologique du lac sud de Tunis après les Aménagements, DEA, ENIT, Springfield, VA, USA.). The zones close to this lagoon are characterized by the presence of activities such as harbours, tourism, agriculture, urban developments and industries (Chalghmi et al. 2016Chalghmi H., Bourdineaud J.P., Haouas Z., et al. 2016. Transcriptomic, Biochemical, and Histopathological Responses of the Clam Ruditapes decussatus from a Metal-Contaminated Tunis Lagoon. Arch. Environ. Contam. Toxicol. 70: 241-256.). The Northern Lagoon of Tunis is part of the wetlands partially separated from the marine environment by a coastline, meeting both marine salty waters and continental fresh water (Ben Mosbah et al. 2010Ben Mosbah Z.C.H. Ben Isail L.K., Gueddari M., et al. 2010. Evolution bio sédimentaire du dépôt quaternaire de la lagune de l’Ariana, Tunisie (une zone humide du Maghreb Nord). Quaternarie 21: 281-292.). Also, this lagoon is the largest water body of the Tunis conurbation. It is divided into two parts named the North Lake and the South Lake, separated by a navigation channel. In the current study, two remote sites from the two different parts of the lagoon were chosen to determine the effects of pollution on R. decussatus. The MCA (36°49′4.31″N 10°13′5.49″E) is located in the North Lake near the protected zone (Chikly Island). This site is characterized by a sandy substrate and is supposedly less polluted after the restoration works in 1985 to 1988, as reported by Barthel (2006)Barthel P.A. 2006. Aménager la lagune de Tunis : un modèle d’urbanisme et de développement durable? Autrepart 39: 129-146. and Chouba et al. (2010)Chouba L., AjjabiChebil L., Herry S. 2010. Etudesaisonniere de la contamination metallique des macroalgues de la lagune nord de Tunis. Bull. Inst. Natl. Sci. Tech. Mer de Salammbô 37: 123-131.. The HCA is located in the South Lake (36°47′12.40″N 10°13′12.88″E), in a highly polluted area with higher anthropic pressure than the other sampling sites (Chalghmi et al. 2016Chalghmi H., Bourdineaud J.P., Haouas Z., et al. 2016. Transcriptomic, Biochemical, and Histopathological Responses of the Clam Ruditapes decussatus from a Metal-Contaminated Tunis Lagoon. Arch. Environ. Contam. Toxicol. 70: 241-256.). Indeed, this site is characterized by black sticky mud and water stagnation, with a strong smell of hydrogen sulphide (H2S), and is very rich in seaweeds with some fragments of shells (Kochlef 2003Kochlef M. 2003.Contribution à L’étude du Fonctionnement Hydrodynamique du lac Sud Tunis après les Travaux D’aménagement, DEA, National Agronomy Institute of Tunisia, Carthage University, Tunis, Tunisia., Bejaoui et al. 2019Bejaoui S., Boussefa D., Telahigue K., et al. 2019. Geographic variation in fatty acid composition and food source of the commercial clam (Ruditapes decussatus, Linnaeus, 1758), from Tunisian coasts: Trophic links. Grasas y Aceites 70: e289.). Previous studies have shown that R. decussatus is abundant in this lagoon (Tlig-Zouari and Maamouri 2008Tlig-Zouari S., Maamouri M.F. 2008. Macrozoobenthic species composition and distribution in the Northern lagoon of Tunis. Trans. Waters Bull. 2: 1-15., Zamouri-Langar 2010Zamouri-Langar N. 2010. Analyse et modélisation des paramètres d’exploitation des stocks du bivalve Ruditapes decussatus des cotes Tunisiennes. Univ. Inst. Nat. Agronom. Tunis (Tunisie), 240 pp., Fradi 2012Fradi J. 2012. Etude comparée de la reproduction et de la croissance de la palourde Ruditapes decussatus dans deux lagunes tunisiennes: Lagune nord de Tunis et Lagune de Boughrara. pp. 145.). These studies showed that R. decussatus has a maturation period in spring and a partial spawning period commencing in summer, the reproductive cycle finishing with an inactive stage during winter after degeneration during the autumn. The growth curves of these bivalves from the MCA were fitted to the von Bertalanffy equation as follows: Lt =57, 75(1-e -0, 22(t+1934)) (Zamouri-Langar 2010Zamouri-Langar N. 2010. Analyse et modélisation des paramètres d’exploitation des stocks du bivalve Ruditapes decussatus des cotes Tunisiennes. Univ. Inst. Nat. Agronom. Tunis (Tunisie), 240 pp., Fradi 2012Fradi J. 2012. Etude comparée de la reproduction et de la croissance de la palourde Ruditapes decussatus dans deux lagunes tunisiennes: Lagune nord de Tunis et Lagune de Boughrara. pp. 145.).

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Fig. 1. – The location of sampling sites at Ghar El Melh (LCA) and the North Lake (MCA) and South Lake (HCA) of the northern Tunis lagoon.

Clams and water sampling

A total of 300 clam individuals with a similar size were sampled at low tide from the LCA, the MCA and the HCA on about the 15th day of each month from March 2014 to February 2015. The clams were collected by hand or by scuba divers at depths greater than 1m. The samples were immediately placed in iced boxes and transported to the laboratory within two hours. Upon arrival, the specimens were dissected and the organs were removed. Then, ten digestive glands per month were analysed individually to obtain 30 samples per season. For each season, 30 (n=30 independent replicates) digestive glands were homogenized with Tris HCl buffer (20 mM, pH 7.4) and stored at liquid nitrogen until the biochemical analysis. Twenty digestive glands per season were pooled into six independent replicates (n=6) and frozen in nitrogen liquid for TE analysis. Also, nine independent digestive glands (n=9) were conserved in ethanol (70°) for DNA analysis and ten (n=10) others were fixed in buffered formalin (10%) for histological study. The choice of the digestive gland as a model organ for assessing the three study areas was based on the fact that this organ has the ability to accumulate and detoxify various xenobiotic substances, reflecting the state of an ecosystem, as reported previously for many bivalve species (Usheva et al. 2006Usheva L.N., Vaschenko M.A., Durkina V.B. 2006. Histopathology of the digestive gland of the bivalve mollusk Crenomytilus grayanus (Dunker, 1853) from southwestern Peter the Great Bay, Sea of Japan. Rus. J. Mar. Biol. 32:166-172. , Faggio et al. 2018Faggio C., Tsarpali V., Dailianis S. 2018. Mussel digestive gland as a model for assessing xenobiotics: an overview. Sci. Total Environ. 613: 220-229.). Also, the digestive gland is known as the major site of oxyradical-generating biotransformation enzymes (Livingstone et al. 1992Livingstone D.R., Lips F., Garcia M.P., et al. 1992. Antioxidant enzymes in the digestive gland of the common mussel Mytilus edulis. Mar Biol. 112: 265-276.). The water parameters (temperature, salinity and pH) were measured in situ using a WTW portable multi-parameter probe (model WTW LF.325). Suspended matter and chlorophyll a were determined according to the method of Aminot and Chaussepied (1983)Aminot A., Chaussepied C. 1983. Manuel des analyses chimiques en milieu marin. CNEXO, Brest, 395 pp. (see Supplementary Material Table S1).

Trace element analysis

Concentrations of zinc (Zn), copper (Cu), lead (Pb), iron (Fe) and cadmium (Cd) were measured in the digestive gland tissues of R. decussatus according to Carvalho et al. (2000)Carvalho G.P., De Cavalcante P.R.S., Castro A.C.I., et al. 2000. Preliminary assessment of heavy metal levels in Mytella falcata (Bivalvia, Mytilidae) from Bcanga river estuary, SAO LUIS, state of Maranjao, Northeastern Brazil. Rev. Bras Biol. 60: 11-16.. Firstly, the digestive glands were cleaned in order to remove all the epibiotic material and to prevent the interference of TE presence in the clams related to inorganic particles. Secondly, all the digestive glands were lyophilized and the dried samples of 0.5 g were finely ground in a porcelain mortar and digested with nitric acid (HNO3; 60%) and hydrogen peroxide (H2O2; 37%) at 80°C. The mineralized solution was gauged with water at a volume of 50 mL until analysis. The TE content was determined with inductively coupled plasma mass spectrometry (ICP-MS). The ICP-MS was equipped with a graphite furnace using a dynamic reaction cell and had a detection limit of 0.002 ppm. Blank samples and certified reference materials (Mussel Tissue Standard Reference Material SRM 2976, National Institute of Standards and Technology) were processed to check the analytical accuracy. TE contents obtained for standard reference materials were continually within the 95% confidence interval of certified values.

Condition and gonad index analysis

The condition index (CI) was considered an indicator of the clams’ physiological condition. The CI for the population of R. decussatus collected from the LCA, MCA and HCA was determined each season on a group of 40 individuals. After complete dehydration of the soft tissues and shells in the oven for 24 hours at a temperature of 105°C, the dry weight was determined using a 0.01 g precision balance. CI was estimated according to the Walne (1979)Walne P.R.1979. Experiments on the culture in the sea of the butterfish Venerupis decussata L. Aquaculture 8: 371-381. formula:

CI=(dry weight of the organ or individual/weight of dry shell) × 100

The digestive gland index was estimated using the formula established by Campillo et al. (2013)Campillo J.A., Albentosa M.N., Valdes J., et al. 2013. Impact assessment of agricultural inputs into a Mediterranean coastal lagoon (Mar Menor, SE Spain) on transplanted clams (Ruditapes decussatus) by biochemical and physiological responses. Aquat. Toxicol. 142-143: 365-379.:

DGI = (dry weight of digestive gland/weight of dry shell) × 100

Biochemical analysis

Metallothionein level measurement

Metallothioneins (MTs) are involved in the maintaining of the essential TE in homoeostasis and the detoxification of the non-essential one (Amiard et al. 2006Amiard J.C., Amiard-Triquet C., Barka S., et al. 2006. Metallothioneins in aquatic invertebrates: their role in metal detoxification and their use as biomarkers. Aquat. Toxicol. 76: 160-202.). MT concentrations were determined in the digestive gland of R. decussatus according to the method developed by Viarengo et al. (1985)Viarengo A., Palmero S., Zanicchi G., et al. 1985. Role of metallothioneins in Cu and Cd accumulation and elimination in the gill and digestive gland cells of Mytilus galloprovincialis lam. Mar. Environ. Res. 16: 23-26. One mL of digestive gland supernatant was added to 1 mL of cold absolute ethanol and 80 µL of chloroform and centrifuged at 6000×g for 10 min. The resulting supernatant was mixed with absolute ethanol (3V) and incubated at –20°C for 1 h. After incubation, the mixture was centrifuged at 6000×g for 10 min and the pellet was cleaned with 87% ethanol and 1% chloroform. The pellet containing MTs was resuspended in 150 µL NaCl (0.25 M) and 150 µL HCl (1 N) containing EDTA (4 mM). Before centrifugation at 3000 g for 5 min, 4.2 mL of NaCl (2 M) containing DTNB (0.43 Mm) buffered with Na-phosphate (0.2 M; pH=8) was added to each pellet at room temperature. MT absorbance was read at 412 nm and the expression was presented as nmol of GSH/mg protein using glutathione (GSH) as a standard.

Malondialdehyde level measurement

Malondialdehyde (MDA) is a convenient index that is widely used to monitor the lipid peroxidation status in the body (Jamil 2001Jamil K. 2001. Bioindicators and biomarkers of environmental pollution and risk assessment. Sci. Publish., USA.). MDA was determined spectrophotometrically according to the method of Draper and Hadley (1990)Draper H.H., Hadley M. 1990. Malondialdehyde determination as index of lipid peroxidation. Meth. Enzymol. 86: 421-431.. An aliquot of 500 µL was mixed with 500 µL of trichloroacetic acid (TCA 30%). After centrifugation at 3500 g for 10 min at cold, 1 mL of TBA mixture (0.67 %; pH: 7.4) was added to 1 mL of supernatant and then incubated for 15 min at 90°C and cooled. The absorbance of the TBA-MDA complex was measured at 532 nm using a spectrophotometer. 1,1,3,3-tetraethoxypropane (TEP Sigma) was used as a standard and the MDA amount was expressed as nmol/mg protein.

Advanced oxidation protein product level measurement

The advanced oxidation protein product (AOPP) has been considered a reliable marker of oxidant-mediated protein damage (Wu 2015Wu Q. 2015. Advanced oxidation protein products as a novel marker of oxidative stress in postmenopausal osteoporosis. Med. Sci. Monit. 21: 2428-2432.). AOPP levels were quantified according to Kayali et al. (2006)Kayali H., Young V.E., Barket K. 2006. Empire to Nation: Historical Perspective on the Making of the Modern Word, Series: World Social Change, Rowmanet Littlefield Publ., 440 pp.. Briefly, 400 µL of the digestive gland supernatant was mixed with 0.8 mL of phosphate buffer (0.1 M; pH 7.4). After 2 min, 0.1 mL of 1.16 M potassium iodide (KI) was treated with the previous solution followed by 0.2 mL of acetic acid. The absorbance of the reaction mixture was registered at 340 nm. The AOPP level for each sample was calculated using the extinction coefficient of 261 and the results were expressed as nmol/mg protein.

Antioxidant assays

Glutathione is a crucial component of the antioxidant defence mechanism and it functions as a direct reactive free radical scavenger (Romao et al. 2006Romao P.R.T., Tovar J., Fonseca S.G., et al. 2006. Glutathione and the redox control system trypanothione/trypanothione reductase are involved in the protection of Leishmania spp. against nitrosothiol-induced cytotoxicity. Braz. J. Med. Biol. Res. 39: 355-363.). Total GSH concentration was quantified by the reduced glutathione recycling assay (Ellman 1959Ellman G.L. 1959. Tissue sulfhydryl groups. Arch. Biochem. Bioph. 82: 70-77.). An aliquot of 500 µL of digestive gland homogenate was added to 3 mL of sulfosalicylic acid (4%) and then centrifuged at 1.600×g for 15 min. Five hundred mL of supernatant was taken and added to Ellman’s reagent. The absorbance was measured spectrophotometrically at 412 nm after DTNB addition (10 mM). The level of GSH was calculated by a standard concentration and expressed as µg/mg protein.

Non-protein thiols react significantly faster with oxidizing species than other amino acid side-chains and thus contribute to antioxidant defence (Hansen et al. 2009Hansen R.E., Roth D., Winther J.R. 2009. Quantifying the global cellular thioldisulfide status. Procs. Natl. Acad. Sci. U.S.A. 106: 422-427). NPSH levels were determined by the method of Ellman (1959)Ellman G.L. 1959. Tissue sulfhydryl groups. Arch. Biochem. Bioph. 82: 70-77.. A 500 mL aliquot of the homogenate was mixed with trichloroacetic acid (10%). After centrifugation, the –SH groups were determined in a pure supernatant. An aliquot of supernatant was added to potassium phosphate buffer (pH=7.4; 0.1 M) and DTNB (10 mM) 5,5-dithio-bis (2-nitrobenzoic acid). The absorbance of colorimetric reaction was measured at 412 nm and NPSH content was expressed as µmol of GSH/mg protein.

Vitamin C is known to directly scavenge ROS (Halliwell and Gutteridge 2001Halliwell B., Gutteridge J.M.C. 2001. Free Radicals in Biology and Medicine. Oxford University Press, New York.). Vit C was determined according to the method of Jacques-Silva et al. (2001)Jacques-Silva M.C., Nogueira C.W., Broch L.C. 2001. Diphenyldiselenide and ascorbic acid changes deposition of selenium and ascorbic acid in liver and brain of mice. Pharmacol. Toxicol. 88: 119-125.. Protein was precipitated in a cold trichloroacetic acid solution (4 %), centrifuged for 10 min and incubated at 85°C for 30 min with DNPH (4.5mg/ mL) and CuSo4 (0.075mg/mL). The reaction was measured at 540 nm and expressed as nmol/mg protein.

Glutathione peroxidase participates in the detoxification of lipid hydroperoxides using glutathione (GSH) and consequently reducing the cellular pool of GSH (Winston and Digiulio, 1991Winston G.W., Digiulio R.T. 1991. Prooxidant and antioxidant mechanisms in aquatic organisms. Aquat. Toxicol. 19: 137-161.). GPx was measured following the procedure of Flohe and Gunzler (1984)Flohe L., Gunzler W.A. 1984. Assays of gluthathione peroxidase. Methods Enzymol. 105: 114-121.. A 200 µL aliquot of digestive gland extract was mixed with 100 µL of phosphate buffer (pH=7.4) and 200 µL of glutathione (4 mM). This mixture was incubated for 10 min at 37°C and then 500 µL of H2O2 (5 mM) and 1 mL of TCA (5%) were added. The reaction was detected after the DTNB (10 mM) addition to the 100 µL of the mixture using spectrophotometric absorbance at 340 nm. GPx was expressed as nmol of GSH/min/mg protein.

Catalase activity is used as a marker involved in the primary defence against oxidative damage (Gutteridge 1995Gutteridge J.M.C. 1995. Lipid peroxidation and antioxidants as biomarkers of tissue damage. Clin. Chem. 4: 1819-1828.). The CAT activity was determined by the method of Aebi (1984)Aebi H. 1984.Catalase in Vitro. Methods. Enzymol. 105: 121-126. using H2O2 (0.5 M) as a substrate. The reaction was started by adding an aliquot of 20 µL of the homogenized digestive gland and the substrate (H2O2) to a concentration of 0.5 M in a medium containing 100 mM phosphate buffer (pH 7.4). The H2O2 decomposition level was followed by monitoring absorption at 240 nm. One unit of CAT was defined as µmol/min/mg of protein.

Acetylcholinesterase (AChE) is a key enzyme of the nervous system and one of the most commonly used biomarkers of neurotoxicity (Durieux et al. 2011Durieux E.D.H., Farver T.B., Fitzgerald P., et al. 2011. Natural factors to consider when using acetylcholinesterase activity as neurotoxicity biomarker in young-of-year striped bass (Morone saxatilis). Fish Physiol. Biochem. 37: 21-29.). AChE activity was measured using a spectrophotometric method (Ellman et al. 1961Ellman G.L., Courtney K.D., Anders V., et al. 1961. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 7: 88-95.). Acetylthiocholine iodide was used as a substrate in a concentration of 8.25 mM. The activity of AChE was expressed as nmol of substrate/min/mg protein and measured spectrophotometrically at 412 nm.

DNA degradation analysis

DNA extraction from the digestive gland was determined according to the Clarke (2002)Clarke J.D. 2002. Cetyltrimethyl Ammonium Bromide (CTAB) DNA Miniprep for Plant DNA isolation. Cold Spring Harb Protoc. method. The DNA was separated from the digestive gland with cetyltrimethylammonium bromide buffer, which was added to the sample and incubated for 1 hour at 55°C. Subsequently, 400 µL of the chloroform-alcohol mixture was added to the above solution, which was centrifuged for 15 minutes at 10000×g. The purified DNA obtained undergoes migration on an agarose gel (1%), which was observed in a dark room under an ultraviolet lamp and photographed. The molecular weight size marker (3 kb DNA ladder) was loaded. The DNA change was examined by a wavelength based on the method of Sambrook and Russell (2001)Sambrook J., Russell D.W. 2001. Molecular cloning: a laboratory manual. 3rd edn. CSHL Press, New York, NY, pp. 577-581. at 260 nm. Results were expressed as µg/g of tissue.

Histophatological analysis

For the histological study, tissues from digestive glands were fixed in formaldehyde 37% at ambient temperature. The dehydration was done using increasing ethanol concentrations and toluene. Histological sections of 5 µm were cut with a rotary manual microtome (Micros, Austria). Before coloration, sections were dewaxed, mounted on a glass slide with albuminous water and coloured with haematoxylin and eosin (Reactifral: 33650 Martillac, France) (Martoja and Martoja-Pierson 1967Martoja R., Martoja-Pierson M. 1967. Initiation aux techniques de l’histologie animale. Masson et Cie Editeurs, France, 345 pp.) to visualize morphological structures and degraded tissues with a light microscope coupled to a CCD camera.

Data analysis

Index calculation

To order and to compare TE according to the overall spatial variability of the environmental levels along the Tunisian lagoons throughout the studied seasons, the Trace Element Spatial Variation Index (TESVI) was determined to compare each studied TE as described by Richir and Gobert (2014)Richir J., Gobert S. 2014. A reassessment of the use of Posidonia oceanica and Mytilus galloprovincialis to biomonitor the coastal pollution of trace elements: New tools and tips. Mar. Pollut. Bull. 89: 390-406..

TESVI = [(Xmax / Xmin) / (Σ(Xmax / Xi) / n)] × SD

Xmax is the maximum mean concentrations recorded among the sites; Xmin the minimum mean concentrations recorded among the sites; Xi the mean concentrations recorded at each site; n the number of sites; and SD the standard deviation from the mean ratio Σ(Xmax/Xi)/n.

The Target hazard quotient ratio (THQ) was determined to express the risk of non-carcinogenic effects of all the tested TEs. This index has been proposed for the estimation of the potential risks to human health caused by toxic TEs by the USEPA (2000)United States Environmental Protection Agency (USEPA). 2000. Risk-based concentration table. United States Environmental Protection Agency, Washington, DC..

THQ=[(EF × ED × FIR × Cm) / (Wm × EF ×RfD)] × 10–3

EF is the exposure frequency (365 days/year).
ED is the exposure duration, equivalent to an average lifetime of a Tunisian person (i.e. 60 years).
FIR is the average level of bivalve consumers (17.86 g/person/day) (Jović and Stanković 2014).
Cm is the TE concentration in bivalves (mg/kg dry weight basis).
Wm is the average body weight of an adult person (60 kg).
RfD is the oral reference dose based on 1 10–3 (Cd), 0.04 (Cu), 0.004 (Pb), 0.3 (Zn) and 45 (Fe), estimating the probable daily oral consumption of TEs in a human population relative to those frequently consumed over a lifetime without a considerable danger of harmful effects (USEPA 2000United States Environmental Protection Agency (USEPA). 2000. Risk-based concentration table. United States Environmental Protection Agency, Washington, DC.).

A THQ value above 1 means that contaminated food intake likely has some noticeable harmful effects on the exposed population.

A THQ value below 1 means that food intake is safe and appropriate for human consumption.

Statistical analysis

The results are expressed as means±SD (standard deviation) for each site. The level of significance was ascertained at 0.05. The results were first tested for normality using the Kolmogorov-Smirnov test and two-way ANOVA analysis was performed to assess significant effects for spatial and seasonal variations on the tested parameters. All statistics and the principal component analysis (PCA) were performed with R software version 2.15.2 (R Core Team 2017R Core Team. 2017. R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. https://www.R-project.org/) using larger data sets. Herein, PCA is the tool most used to explore similarities and hidden patterns among samples and clarify the relationship between data and grouping.

RESULTSTop

Physicochemical parameters

The environmental parameters of the water in the study areas are summarized in Supplementary Material Table S1. Our data showed no significant variations in salinity and pH, whereas temperature, suspended matter and chlorophyll a, which showed significant differences between the sites (p<0.01; two-way ANOVA). Temperature and suspended matter were significantly higher in the MCA and the HCA (p<0.01; two-way ANOVA), while chlorophyll a concentration was far higher in the LCA (p<0.001; two-way ANOVA).

Biometric parameters

Results of the biometric parameters (weight (w), length (L), CI and gonadic index (GI)) are illustrated in Supplementary Material Table S2. Our results revealed a similar variation of R. decussatus weight and length in the three studied lagoons. As shown in Supplementary Material Table S1, CI was significantly lower in R. decussatus from the MCA and the HCA than in those from the LCA in each season (p<0.05, two-way ANOVA). The lowest values were recorded for R. decussatus collected from the HCA during the summer season (12.477%). Similar variations were observed for GI (see Supplementary Material Table S2). GI was significantly lower in clams collected from the MCA (36%) and the HCA (42%) than in those collected from the LCA, particularly in summer (p<0.001, two-way ANOVA). In summer, the CI and GI of R. decussatus from the LCA, MCA and HCA were significantly lower (p<0.01, two-way ANOVA).

Trace element concentrations

Site and seasonal variations of TE concentrations in R. decussatus digestive glands are reported in Table 1. Our results showed that the essential TE (Fe, Zn) exhibited higher concentrations than the non-essential ones (Cd, Pb and Cu). The mean Cd, Cu and Zn concentrations were lower than the certified reference materials recorded in bivalves; however, Fe and Pb concentrations were higher in R. decussatus digestive gland from the MCA and the HCA than the certified reference materials (Table 1).

Table 1. – Comparison of trace element concentrations (in mg kg–1 of dry weight) in R. decussatus from Tunisian coasts with other bivalves from different Mediterranean coastal areas. Concentrations are also compared with maximum levels of TE admissible in shellfish flesh, as set by international organizations. LCA, Ghar el Melh; MCA, North Lake; HCA, South Lake. Values are presented as mean±SD (n=6 repetitions). Significant differences are determined at 0.05 using two-way ANOVA: *p<0.05, **p<0.01; ***p<0.001. Metal determination in shellfish tissue based on IAEA452 (scallop tissue, standard reference material, dispersed by International Atomic Energy Agency reference materials); WHO/FAO (1984)WHO/FAO. 1984. List of Maximum Levels Recommended for Contaminants by the Joint FAO/WHO Codex Alimentarius Commission. Second Series. 3. CAC/FAL, Rome, pp. 1–8. (Food and Agriculture Organization of the United Nations); SRM 2976 (muscle tissue, National institute of Standards and Technology). Values of IAEA 452 and SRM 2976 are presented as means and standard deviation, while WHO/FAO (1984)WHO/FAO. 1984. List of Maximum Levels Recommended for Contaminants by the Joint FAO/WHO Codex Alimentarius Commission. Second Series. 3. CAC/FAL, Rome, pp. 1–8. values are presented as a range. NB:Zn is not declared in IAEA 452 and WHO/FAO (1984)WHO/FAO. 1984. List of Maximum Levels Recommended for Contaminants by the Joint FAO/WHO Codex Alimentarius Commission. Second Series. 3. CAC/FAL, Rome, pp. 1–8..

Cd Pb Cu Zn Fe
Spring LCA 0.34±0.08 0.08±0.01 1.69±0.31 43.47±7.93*** 105.49±13.33
MCA 0.69±0.13*** 0.12±0.05** 3.16±0.38*** 61.92±0.50*** 191.25±3.69***
HCA 0.80±0.10 *** 0.18±0.09*** 4.46±0.22*** 63.92±6.72*** 252.75±11.80***
Summer LCA 0.32±0.04 0.13±0.01** 1.69±0.22*** 47.47±6.24*** 107.74±0.60
MCA 0.50±0.08*** 1.29±0.21*** 4.16±0.21 *** 51.78±4.82*** 617.75±21.56 ***
HCA 0.75±0.10 *** 1.34±0.22 *** 5.20±0.56*** 63.43±6.67*** 740.96±24.01 ***
Autumn LCA 0.29±0.01** 0.10±0.01 1.34±0.14 46.47±1.49*** 119.19±2.35
MCA 0.61±0.06*** 1.66±0.32*** 2.16±0.22*** 58.00±1.04*** 692.97±19.70 ***
HCA 0.67±0.12*** 2.07±0.49*** 5.37±0.27*** 65.32±6.09*** 772.27±56.08 ***
Winter LCA 0.52±0.06*** 0.13±0.01** 1.24±0.08*** 48.97±3.46 *** 122.18±1.18
MCA 0.72±0.12*** 0.69±0.26*** 1.74±0.08 *** 53.70±0.48*** 463.06±10.78 ***
HCA 0.81±0.10*** 0.89±0.06*** 1.79±0.16*** 55.12±2.89*** 640.42±66.55 ***
IAEA 452 29.6±3.7 0.37±0.01 10.80±1.30 166±21 ND
WHO/FAO (1984)WHO/FAO. 1984. List of Maximum Levels Recommended for Contaminants by the Joint FAO/WHO Codex Alimentarius Commission. Second Series. 3. CAC/FAL, Rome, pp. 1–8. 2 1-6 10-30 40-100 ND
SRM 2976 0.82±0.16 1.19±0.18 4.02±0.33 137±13 171±4.9
Figueira and Freitas (2013)Figueira E., Freitas R. 2013. Consumption of Ruditapes philippinarum and Ruditapes decussatus: comparison of element accumulation and health risk. Environ. Sci. Pollut. Res. 20: 5682-5691. 0.4 0.19-0.34 0.5-0.7 8 -10 ND
Chalghmi et al. (2016) Chalghmi H., Bourdineaud J.P., Haouas Z., et al. 2016. Transcriptomic, Biochemical, and Histopathological Responses of the Clam Ruditapes decussatus from a Metal-Contaminated Tunis Lagoon. Arch. Environ. Contam. Toxicol. 70: 241-256. 0.16±0.01-0.96±0.6 3.6±0.8-239±22 2.2±0.5-18±1.5 45±7-432±35 ND
Gabr et al. (2020) Gabr G.A.F., Masood M.F., Radwan E.H., et al. 2020. Potential Effects of Heavy Metals Bioaccumulation on Oxidative stress Enzymes of Mediterranean clam Ruditapes decussatus. Catrina 21: 75-82. 1.5-3.0 3.0-4.3 1.6-2.7 2.9-4.2 ND
Bejaoui et al. (2020)Bejaoui S., Michan C., Telahigue K., et al. 2020. Metal body burden and tissue oxidative status in the bivalve Venerupis decussata from Tunisian coastal lagoons. Mar. Environ. Res. 159: 105000. 0.87±0.10-1.87±0.38 1.13±0.33-2.33±0.31 5.39±0.97-8.42±1.06 36.08±7.49-50.44±6.36 395±15.03-435.22±13.07

Clams from the MCA and the HCA had higher concentrations of Cd, Pb, Cu, Fe and Zn than those from the LCA (p<0.01, two-way ANOVA). These significant concentrations were distributed among the seasons. In spring, MCA and HCA showed significantly higher accumulations of Cd (98% and 181%, respectively), Cu (86% and 162%, respectively), Zn (42% and 47%, respectively) and Fe (81% and 140%, respectively) than the LCA. In summer, Pb, Fe and Cu concentrations in the digestive gland of specimens from the MCA and the HCA were significantly higher than those in the LCA (p<0.01, two-way ANOVA). However, only in individuals from the HCA were Cd and Zn concentrations significantly higher than in individuals from the LCA (p<0.01, two-way ANOVA). In autumn, clams from the HCA and the MCA had significantly higher Cd, Pb, Cu, Zn and Fe levels in their digestive gland than those from the LCA (p<0.01, two-way ANOVA). In winter, clams from the MCA and the HCA showed higher Pb, Cu and Fe than those from the LCA. Likewise, TE concentrations obtained for each sampled lagoon were significantly higher in summer and autumn (p<0.05, two-way ANOVA).

Table 2 represents the TESVI and THQ values of the studied TEs. Regarding the overall spatial variability of the three study sites and the five studied TEs, Zn concentration displayed the lowest variability of TESVI, with 0.010 (in summer). However, the highest value among seasons was recorded for Pb. The highest levels of the THQ index among seasons were recorded for Cd and the lowest for Fe.

Table 2. – Trace Element Spatial Variation Index (TESVI) and target hazard quotient (THQ) values calculated from trace element mean concentrations in R. decussatus from the Tunisian coast: Ghar el Melh, the North Lake and the South Lake. Numbers indicate the highest value of each TE per season. Numbers in bold indicate the TEs with the highest values.

TESVI THQ
Spring Cd 0.027 0.461
Pb 0.074 0.023
Cu 0.047 0.053
Fe 0.047 0.002
Zn 0.023 0.134
Summer Cd 0.018 0.417
Pb 0.063 0.091
Cu 0.014 0.065
Fe 0.045 0.007
Zn 0.010 0.125
Autumn Cd 0.042 0.375
Pb 0.076 0.222
Cu 0.036 0.051
Fe 0.023 0.008
Zn 0.020 0.130
Winter Cd 0.021 0.460
Pb 0.060 0.099
Cu 0.026 0.048
Fe 0.031 0.006
Zn 0.022 0.119

Metallothionein levels

Site and seasonal variations of MTs levels in R. decussatus digestive gland are given in Table 3. Our results revealed a significantly higher MT level in R. decussatus collected from the HCA and the MCA than in those collected from the LCA (p<0.01, two-way ANOVA).

Table 3. – Mean (±SD) biomarker responses in R. decussatus from Tunisian coast: Ghar el Melh (LCA), the North Lake (MCA) and the South Lake (HCA). MTs, metallothioneins; GPx, glutathione peroxidase; CAT, catalase; GSH, glutathione; NPSH, non-protein SH; Vit C, vitamin C. Values are expressed as means±SD (n=30). Significant differences are determined at 0.05 using two-way ANOVA: *p<0.05, **p<0.01; ***p<0.001; a, nmol of GSH/ mg protein; b, nmol of GSH/min/mg protein; c, µmol/min/mg of protein; d, µg/mg protein; e, µmol of GSH/mg protein; f: nmol/mg protein.

MTs a GPx b CATc GSHd NPSHe Vit Cf
Spring LCA 0.09±0.01 0.28±0.03 0.34±0.01 0.50±0.02 0.04±0.002 11.93±0.45
MCA 0.12±0.02*** 0.48±0.08*** 0.48±0.01 *** 0.51±0.21*** 0.06±0.02*** 13.51±2.12***
HCA 0.20±0.04*** 1.17±0.14*** 1.02±0.26 *** 2.71±0.57*** 0.14±0.02*** 18.85±3.68***
Summer LCA 0.09±0.006 0.50±0.01*** 0.30±0.01*** 0.709±0.03 0.04±0.001* 6.24±0.39***
MCA 0.18±0.03*** 1.10±0.33*** 0.40±0.10 1.04±0.30** 0.06±0.02*** 7.00±0.25***
HCA 0.35±0.06*** 2.98±0.32*** 0.96±0.05*** 4.63±0.21*** 0.19±0.04*** 13.73±2.55***
Autumn LCA 0.08±0.002 0.59±0.05 0.40±0.04** 0.50±0.01 0.03±0.006 10.84±0.67
MCA 0.15±0.01*** 0.94±0.19*** 0.68±0.03 *** 0.57±0.06** 0.05±0.01*** 12.38±2.00***
HCA 0.188±0.05*** 1.59±0.25*** 1.40±0.43*** 1.39±0.18*** 0.07±0.01*** 16.71±2.57***
Winter LCA 0.08±0.005 0.42±0.10*** 0.56±0.08*** 0.33±0.009*** 0.03±0.002 5.19±0.25***
MCA 0.09±0.002 0.80±0.26*** 0.91±0.24 0.42±0.07 0.05±0.05*** 6.50±1.98***
HCA 0.11±0.03*** 0.93±0.34*** 1.02±0.16*** 1.11±0.33*** 0.08±0.02*** 7.00±1.82***

MT levels in digestive glands from the HCA were significantly higher in spring (139%), summer (285%) and autumn (98%) than in those from the LCA (p<0.001, two-way ANOVA). However, only in summer did clams from the MCA exhibit a significantly higher MT level than those from the LCA (p<0.05, two-way ANOVA). Nonetheless, no significant differences were registered in winter (p>0.05, two-way ANOVA). The greatest difference in MT levels was recorded in summer, when levels in digestive glands collected from the MCA and the HCA were significantly higher (p<0.001, two-way ANOVA).

Malondialdehyde levels

Site and seasonal variations in MDA levels in digestive glands from the three sites are presented in Figure 2A. Higher MDA levels in clams from the MCA and the HCA than in clams from the LCA were observed in summer (106% and 496%, respectively) and winter (84% and 170%, respectively) (p<0.001, two-way ANOVA).

figure2

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Fig. 2. – Seasonal variation of (A) malondhyaldehide (MDA) and (B) advanced oxidation of proteins product (AOPP) levels in the digestive gland of R. decussatus sampled from three coastal Tunisian lagoons: Ghar el Melh (LCA), the North Lake (MCA) and the South Lake (HCA). Results are presented by mean±SD. Significant differences are determined at 0.05 using two-way ANOVA: *p<0.05, **p<0.01; ***p<0.001.

However, only clams from the HCA exhibited significantly higher MDA levels in spring and autumn than those from the MCA (120% and 45%, respectively) and the LCA (133% and 135%, respectively) (p<0.01, two-way ANOVA).

Advanced oxidation protein products levels

Site and seasonal variations of AOPP levels in the digestive glands are shown in Figure 2B. AOPP levels were significantly higher in clams from the HCA than in those from the LCA (p<0.01, two-way ANOVA). As shown in Figure 2B, AOPP levels appeared to be significantly higher in digestive glands from the HCA than in those from the LCA in spring (348%), summer (230%) and autumn (85%). However, similar variations in AOPP levels were observed in digestive glands collected from the LCA and the MCA.

Glutathione levels

Sites and seasonal variations in GSH levels are summarized in Table 3. R. decussatus collected from the HCA displayed higher levels of GSH than those from the LCA and the MCA (p<0.01, two-way ANOVA). Clams from the HCA showed significantly higher levels than clams from the MCA and the LCA in spring (169% and 339%, respectively), summer (345% and 553%, respectively), autumn (143% and 178%, respectively) and winter (164% and 236%, respectively). GSH levels were significantly higher in clams from the MCA than in clams from the LCA only in summer (p<0.01, two-way ANOVA).

Non-protein SH levels

Site and seasonal variations of non-protein-SH levels in R. decussatus digestive glands are summarized in Table 3. NPSH levels were significantly higher in digestive glands of clams from the MCA and the HCA than in those from the LCA (p<0.05, two-way ANOVA). Among seasons, R. decussatus from the HCA showed significantly higher NPSH levels in spring (250%), summer (375%), autumn (133%) and winter (166%) than those from the LCA (p<0.05, two-way ANOVA). A similar trend was observed in clams from the MCA, which in all seasons showed significantly higher NPSH levels (≤50%) than those from the LCA (p<0.05, two-way ANOVA).

Vitamin C levels

Site and seasonal variations in vitamin C level were slightly modified over the study period (Table 3). Clams from the LCA and the MCA showed significantly lower Vit C levels than those from the HCA in spring (58% and 50%, respectively), summer (120% and 96%, respectively) and autumn (54% and 54%, respectively).

Glutathione peroxidase activity

Site and seasonal variations of GPx activities in R. decussatus from the sites are summarized in Table 3. GPx activities in clams sampled from the MCA and the HCA were significantly higher in spring (70% and 315%, respectively), summer (119% and 492%, respectively) and autumn (58% and 166%, respectively). However, no significant difference was observed in the GPx activities among sites in winter (p>0.05, two-way ANOVA). GPx activity was more pronounced in summer and autumn in clams collected from all three sites (p<0.01, two-way ANOVA).

Catalase activity

Site and seasonal variations in CAT activities in R. decussatus digestive glands from the LCA, the MCA and the HCA are shown in Table 3. CAT activity was significantly higher in digestive glands of clams from the MCA and the HCA than in those from the LCA (p<0.01, two-way ANOVA). The differences were of the order of ≤41% in all seasons (p<0.01, two-way ANOVA).

Acetylcholinesterase activity

Site and seasonal variations in AChE activity are documented in Figure 3. Significantly lower AChE activity was observed in digestive glands from the MCA and the HCA than in those from the LCA (p<0.05, two-way ANOVA). The lowest levels were observed in clams from the HCA in spring (69%), summer (62%) autumn (30%) and winter (25%). However, similar activity was recorded in clams from the MCA and the LCA in all seasons (p>0.05, two-way ANOVA), except in summer, when activity was 27% lower in clams from the MCA.

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Fig. 3. – Seasonal variation of the acetylcholinesterase (AChE) activity in the digestive gland of R. decussatus sampled from three coastal Tunisian lagoons (Ghar el Melh: LCA, North lagoon: MCA and South lagoon: HCA). Results are presented as mean±SD. Significant differences are determined at 0.05 using two-way ANOVA: *p<0.05; **p<0.01; ***p<0.001.

Principal component analysis

PCA is shown in Figure 4, which allowed us to retain the first two factorial axes that explained 68% of the total variance. The first one is the axis which is preserved by projection, for maximum dispersion of the initial cloud point (53.1% of the total dispersion): MDA, AOPP, GPx, GSH, NPSH, Vit C, GSH, MTs, Cd, Cu and Fe contributed negatively with this first axis (Fig. 4). Nevertheless, only AChE activity correlated positively with the first axis. The second axis was described by 14.9% of the total dispersion. This axis was characterized by a negative correlation with T°C, pH and chlorophyll a, while, Zn and CAT correlated positively with it.

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Fig. 4. – Principal analysis component results represented by two factors and produced by abiotic factor, biochemical and chemical variables measured in the digestive gland of R. decussatus sampled from three costal Tunisian lagoons.

Correlation matrix

Multivariate statistical analyses were performed to establish correlations between the environmental conditions (T°C, salinity, chlorophyll a, carotenoids and suspended matter), TE accumulations (Pb, Cu, Cd, Fe, and Zn) and the tested parameters of R. decussatus digestive glands (see Supplementary Material Table S4). Significant correlations were found between TE concentrations and almost all of the antioxidant responses and biometric parameters. The Pearson correlation matrix indicated that digestive gland AChE activity showed a significant negative correlation with the environmental parameters and the trace element concentrations. Also, chlorophyll a showed a significant and negative correlation with all the parameters tested, with a high value observed in MTs (p<0.05, r=–0.535) (see Supplementary Material Table S4). The results indicated that all tested parameters were positively correlated with temperature (p<0.05), salinity (p<0.05) and MES (p<0.01), but pH did not correlate with the biometric and biochemical parameters (p>0.05). Important positive relationships were observed between trace element concentrations in R. decussatus digestive glands and the tested parameters (including abiotic, biotic and biochemical) (p<0.01). Both enzymatic and non-enzymatic activities showed a strikingly positive relationship with Cd (p<0.001, r≈0.373-0.771), Pb (p<0.001, r≈0.338-0.733), Cu (p<0.001, r≈0.236-0.853), Fe (p<0.01, r≈0.264-0.806) and Zn (p<0.01, r≈0.406-0.813).

DNA analysis

Site and seasonal variations in DNA damage in R. decussatus digestive glands from the LCA, the MCA and the HCA are summarized in Figure 5. In spring, summer and autumn, results showed significantly higher DNA damage contents in digestive glands of R. decussatus from the MCA and the HCA than in those from the LCA (p<0.001, two-way ANOVA). However, similar contents were observed in winter for R. decussatus digestive glands collected from all the studied lagoons. Nevertheless, among seasons, clams from the LCA showed similar contents of DNA damage (in the order of ≈1200 µg g–1), however, MCA and HCA showed comparable contents of DNA damage in spring, summer and autumn, which decreased significantly in winter (p<0.01, two-way ANOVA). On the other hand, clams collected from the HCA were characterized by a significant reduction of DNA degradation in autumn and winter (p<0.01, two-way ANOVA).

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Fig. 5. – Seasonal deterioration among seasons of the DNA structure isolated from R. decussatus collected from Tunisian coastal lagoons: Ghar el Melh (LCA), the North Lake (MCA) and the South Lake (HCA). Results are presented as mean±SD. Significant differences are determined at 0.05 using two-way ANOVA: *p<0.05, **p<0.01; ***p<0.001.

Histological analysis

Site and seasonal variations in the histopathological alterations in the digestive gland of R. decussatus from the north lagoon of Tunisia are summarized in Figure 6. All the digestive glands of R. decussatus from the LCA were characterized by a normal digestive tubule with epithelial cells, a narrow or almost occluded tubule lumen and interstitial tissue between them (Fig. 6). However, clams from the MCA and the HCA were characterized by an infiltration of their haemocytes, lumen occlusion and degradation at the level of the epithelial cells. We also noted the presence of atrophic tubules, melanized haemocyte aggregate, epithelial cell degradation, tubule infiltration and fibrosis in spring, summer and autumn, revealing serious damage in these two populations. A neoplastic haemocyte intruding into a necrotic focus was observed in summer (Fig. 6). Nevertheless, similar digestive gland structures were observed in winter for clams from all sampling sites, showing less damage than in the other seasons (Fig. 6).

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Fig. 6. – Histological sections of R. decussatus digestive glands from three coastal Tunisian lagoons: Ghar el Melh (LCA), the North Lake (MCA) and the South Lake (HCA). A, atrophy; ct, connective tissue; ec, epithelial cells; ecd, epithelial cell degradation; F, fibrosis; hi, hemocyte infiltration; L, lumen; LO, lumen occlusion; M, melanin; np, neoplasic hemocytes; TD, digestive tubules. Optic microscopy: haematoxylin-eosin ×100.

DISCUSSIONTop

Owing to their sedentary nature and filter-feeding behaviour, bivalves are particularly exposed to anthropogenic contaminants that enter coastal environments. These organisms have a great ability to accumulate TEs in their tissues and some species may even thrive in highly polluted environments (Cantillo 1998Cantillo A.Y. 1998. Comparison of results of Mussel Watch Programs of the United States and France with worldwide mussel watch studies. Mar. Pollut. Bull. 36: 712-717.). In the current study, the recorded levels of TEs (Zn, Cu, Pb, Fe and Cd) in R. decussatus digestive glands collected from three sites impacted by different human activities provide evidence for the ability/capacity of this organ to accumulate TEs. The inter-relationship for essential (Fe and Zn) and non-essential (Pb, Cd, and Cu) TEs accumulated in R. decussatus digestive gland strongly indicates a similar pathway of TE uptake. The results obtained showed that TE concentrations in R. decussatus were significantly higher in clams collected from the MCA and the HCA. Bivalves can absorb TEs directly from the surrounding environment, both by absorption and/or adsorption from interstitial water and by direct ingestion from sediment, and these TEs may influence their metabolism and other species via the food chain (Chen et al. 2007Chen C.W., Kao C.M., Chen C.F., et al. 2007. Distribution and accumulation of heavy metals in the sediments of Kaohsiung Harbor, Taiwan. Chemosphere 66: 1431-1440.).

Several investigations have demonstrated that sediments from the HCA are highly impacted by TEs. Their accumulation rate in R. decussatus digestive gland must depend on their filtration, ingestion rates, gut fluid quality and detoxification strategies (Chouba et al. 2010Chouba L., AjjabiChebil L., Herry S. 2010. Etudesaisonniere de la contamination metallique des macroalgues de la lagune nord de Tunis. Bull. Inst. Natl. Sci. Tech. Mer de Salammbô 37: 123-131., Bejaoui et al. 2020Bejaoui S., Michan C., Telahigue K., et al. 2020. Metal body burden and tissue oxidative status in the bivalve Venerupis decussata from Tunisian coastal lagoons. Mar. Environ. Res. 159: 105000.). Our data match well with previous studies that reported a major accumulation of TEs, especially Pb, in the sediments of the central area of the Gulf of Tunis (Ennouri et al. 2010Ennouri R., Chouba L., Magni P. et al. 2010. Spatial distribution of trace metals (Cd, Pb, Hg, Cu, Zn, Fe and Mn) and oligo-elements (Mg, Ca, Na and K) in surface sediments of the Gulf of Tunis (Northern Tunisia). Environ. Monit. Assess. 163: 229., Chalghmi et al. 2016Chalghmi H., Bourdineaud J.P., Haouas Z., et al. 2016. Transcriptomic, Biochemical, and Histopathological Responses of the Clam Ruditapes decussatus from a Metal-Contaminated Tunis Lagoon. Arch. Environ. Contam. Toxicol. 70: 241-256.) and in others organisms such as green macroalgae (Chouba et al. 2010Chouba L., AjjabiChebil L., Herry S. 2010. Etudesaisonniere de la contamination metallique des macroalgues de la lagune nord de Tunis. Bull. Inst. Natl. Sci. Tech. Mer de Salammbô 37: 123-131.) and mollusks (Brahim et al. 2015Brahim M., Atoui A., Sammari C., et al. 2015. Surface sediment dynamics along with hydrodynamics along the shores of Tunis Gulf (north-eastern Mediterranean). J. Afr. Earth Sci. 103: 30-41., Lahbib et al. 2016Lahbib Y., Mleiki A., Trigui-El-Menif N. 2016. Bioaccumulation of trace metals in Hexaplex trunculus: spatial and temporal trends from 2004 to 2011 along the Tunisian coast. Environ. Sci. Pollut. Res. 23: 16259-16271., Bejaoui et al. 2017Bejaoui S., Boussoufa D., Tir M., et al. 2017. DNA damage and oxidative stress in digestive gland of Ruditapes decussatus collected from two contrasting habitats in the southern Tunisian coast: biochemical and histopathological studies. Cah. Biol. Mar. 58: 123-135.) following the discharge of TEs from several anthropogenic activities such as harbours, industrial and electrical discharges (Bejaoui et al. 2020Bejaoui S., Michan C., Telahigue K., et al. 2020. Metal body burden and tissue oxidative status in the bivalve Venerupis decussata from Tunisian coastal lagoons. Mar. Environ. Res. 159: 105000.).Similar results have demonstrated that TE accumulations in R. decussatus is associated with the anthropogenic pollution of the Tunisian coasts (Chalghmi et al. 2016Chalghmi H., Bourdineaud J.P., Haouas Z., et al. 2016. Transcriptomic, Biochemical, and Histopathological Responses of the Clam Ruditapes decussatus from a Metal-Contaminated Tunis Lagoon. Arch. Environ. Contam. Toxicol. 70: 241-256., Bejaoui et al. 2020Bejaoui S., Michan C., Telahigue K., et al. 2020. Metal body burden and tissue oxidative status in the bivalve Venerupis decussata from Tunisian coastal lagoons. Mar. Environ. Res. 159: 105000.) and other Mediterranean areas (Figueira and Freitas 2013Figueira E., Freitas R. 2013. Consumption of Ruditapes philippinarum and Ruditapes decussatus: comparison of element accumulation and health risk. Environ. Sci. Pollut. Res. 20: 5682-5691., Gabr et al. 2020Gabr G.A.F., Masood M.F., Radwan E.H., et al. 2020. Potential Effects of Heavy Metals Bioaccumulation on Oxidative stress Enzymes of Mediterranean clam Ruditapes decussatus. Catrina 21: 75-82.).

The current study has also revealed that TE concentrations in the digestive glands of R. decussatus from the MCA and the HCA vary considerably depending on seasons, showing higher trends of Cd, Pb, Cu, Zn and Fe in summer and winter. Such findings could be explained by the fact that in summer the low hydrodynamics of these lagoons and the high evaporation rate might contribute to the concentration of TEs in the water column and their deposition on the surface sediments and consequently on the bivalve tissues (Ghribi et al. 2020Ghribi F., Richir J., Bejaoui S., et al. 2020. Trace elements and oxidative stress in the Ark shell Arca noae from a Mediterranean coastal lagoon (Bizerte lagoon, Tunisia): are there health risks associated with their consumption? Environ. Sci. Pollut. Res. 27: 15607–15623.). However, in winter the current and wave activity agitates the sediments and transports the TEs in the aqueous phase, leading to their bioavailability and their uptake by the bivalves. This increase in winter could be due to the possible discharge of TE concentrations into the waters of the MCA and the HCA or to runoff from adjacent farmland, because these sampling areas are located near the conurbation of Tunis city (Chouba et al. 2010Chouba L., AjjabiChebil L., Herry S. 2010. Etudesaisonniere de la contamination metallique des macroalgues de la lagune nord de Tunis. Bull. Inst. Natl. Sci. Tech. Mer de Salammbô 37: 123-131., Chalghmi et al. 2016Chalghmi H., Bourdineaud J.P., Haouas Z., et al. 2016. Transcriptomic, Biochemical, and Histopathological Responses of the Clam Ruditapes decussatus from a Metal-Contaminated Tunis Lagoon. Arch. Environ. Contam. Toxicol. 70: 241-256.). Our results were confirmed by the characteristics of the lagoon, which is a laminar medium with an average bathymetry not exceeding one metre and prevailing drastic conditions, such as abrupt and very great spatiotemporal fluctuations of abiotic parameters. In addition, exchanges with the sea are weak and the hydrodynamics are highly attenuated, especially in the coves, where the waters are almost stagnant (Ben Mosbah et al. 2010Ben Mosbah Z.C.H. Ben Isail L.K., Gueddari M., et al. 2010. Evolution bio sédimentaire du dépôt quaternaire de la lagune de l’Ariana, Tunisie (une zone humide du Maghreb Nord). Quaternarie 21: 281-292.). Furthermore, recent anthropogenic activities such as industrial wastewater discharges (e.g. CHOTRANA) and sediment excavation can also alter the hydraulic loading and TE source at these two sites (Ben Mosbah et al. 2010Ben Mosbah Z.C.H. Ben Isail L.K., Gueddari M., et al. 2010. Evolution bio sédimentaire du dépôt quaternaire de la lagune de l’Ariana, Tunisie (une zone humide du Maghreb Nord). Quaternarie 21: 281-292.).

In addition, the environmental conditions such as temperature, salinity, pH and chlorophyll a are considered the main factors influencing TE concentrations in bivalves, affecting their speciation, solubility and reaction rates in the water column (Frías-Espericueta et al. 1999Frías-Espericueta M.G., Osuna-López J.I., Sandoval-Salazar G., et al. 1999. Distribution of trace metals in different tissues in the rock oyster Crassostrea iridescens: seasonal variation. Bull. Environ. Contam. Toxicol. 63: 73-79., Ghribi et al. 2020Ghribi F., Richir J., Bejaoui S., et al. 2020. Trace elements and oxidative stress in the Ark shell Arca noae from a Mediterranean coastal lagoon (Bizerte lagoon, Tunisia): are there health risks associated with their consumption? Environ. Sci. Pollut. Res. 27: 15607–15623.). As shown in our present work, TE showed positive correlations with T°C, salinity and suspended particulate matter (p<0.01; r≤0.451) (see Supplementary Material Table S4). Similar works have reported on Mytilus galloprovinciallis from Algerian coasts (Rouane Hacene et al. 2015Rouane Hacene O., Boutiba Z., Belhaouaria B., et al. 2015. Seasonal assessment of biological indices, bioaccumulation and bioavailability of heavy metals in mussels Mytilus galloprovincialis from Algerian west coast, applied to environmental monitoring. Oceanologia 57: 362-374.) and Bizerte lagoon (Kefi et al. 2015Kefi FJ., Mleiki A., Maâtoug-Béjaoui J., et al. 2015. Seasonal variations of trace metal concentrations in the soft tissue of Lithophaga lithophaga collected from the Bizerte Bay (northern Tunisia, Mediterranean Sea). J. Aqua Res. Dev. 7: 432.) and on Arca noae from Bizerte lagoon (Ghribi et al. 2020Ghribi F., Richir J., Bejaoui S., et al. 2020. Trace elements and oxidative stress in the Ark shell Arca noae from a Mediterranean coastal lagoon (Bizerte lagoon, Tunisia): are there health risks associated with their consumption? Environ. Sci. Pollut. Res. 27: 15607–15623.). The seasonal variations in TE concentrations could also be related to the physiological status of bivalves and their reproductive cycle (Bordin et al. 1992Bordin G., McCourt J., Rodriguez A. 1992. Trace metals in the marine bivalve Macoma balthica in the Westerschelde estuary, the Netherland. Part 1: analysis of total copper, cadmium, zinc and iron- locational and seasonal variations. Sci. Total. Environ. 127: 225-280., Regoli and Orlando 1994Regoli F., Orlando E. 1994. Seasonal variation of trace metal concentrations in the digestive gland of the Mediterranean mussel Mytilus galloprovincialis: Comparison between a polluted and a non-polluted site. Arch. Environ. Contam. Toxicol. 27: 36-43.). According to several studies, bivalves accumulate high TE contents during maturation and less during the spawning period. The reproductive cycle of R. Decussatus is characterized by a maturation phase followed by an extended spawning phase during summer (Fradi 2012Fradi J. 2012. Etude comparée de la reproduction et de la croissance de la palourde Ruditapes decussatus dans deux lagunes tunisiennes: Lagune nord de Tunis et Lagune de Boughrara. pp. 145.). A new ripening phase takes place in winter, especially for the clams from the HCA, followed by a short massive spawning in late November and early December (Hmida 2004Hmida L. 2004. Reproduction de la palourde Ruditapes decussatus, en milieu naturel (sud Tunisie) et en milieu contrôlé (écloserie expérimentale): relation avec le système immunitaire. Ph. D. thesis. Univ. Bretagne Occidentale, 97 pp.). These dynamisms of clam maturation may explain the higher accumulation of TEs during the summer and winter, especially for these collected from the HCA.

The bioaccumulation of TEs in bivalve species depends not only on direct exposure to the contaminant and its environment, but also on different physiological and biochemical activities through which a specific organism treats micronutrients (Nicholson and Lam 2005Nicholson S., Lam P.K.S. 2005. Pollution monitoring in Southeast Asia using biomarkers in the mytilid mussel Perna viridis (Mytilidae: Bivalvia). Environ. Int. 31: 121-132. ). Likewise, bivalves have seasonal changes in their biometric parameters, especially during the warm season. Previous findings on bivalves demonstrate that the decline of biometric indices, such as the CI, is a consequence of anthropogenic activities in the ecosystem (Nicholson and Lam 2005Nicholson S., Lam P.K.S. 2005. Pollution monitoring in Southeast Asia using biomarkers in the mytilid mussel Perna viridis (Mytilidae: Bivalvia). Environ. Int. 31: 121-132., Bejaoui et al. 2017Bejaoui S., Boussoufa D., Tir M., et al. 2017. DNA damage and oxidative stress in digestive gland of Ruditapes decussatus collected from two contrasting habitats in the southern Tunisian coast: biochemical and histopathological studies. Cah. Biol. Mar. 58: 123-135.). It appears that TE uptake could be one of several factors influencing the CI and GI of clams sampled from the MCA and the LCA. Our results agree with several studies carried out on TE accumulation in bivalve tissues (Sabatini et al. 2011Sabatini S.E., Rocchetta I., Nahabedian D.E., et al. 2011. Oxidative stress and histological alterations produced by dietary copper in the fresh water bivalve Diplodon chilensis. Comp. Biochem. Physiol. Part C 154: 391-398., Marques et al. 2016Marques A., Piló D., Araújo O., et al. 2016. Propensity to metal accumulation and oxidative stress responses of two benthic species (Cerastoderma edule and Nephtys hombergii): are tolerance processes limiting their responsiveness? Ecotoxicology 25: 664-676.).

The application of the TESVI and THQ could possibly demonstrate suitable tools for the monitoring programmes, in order to define the anthropogenic pollution sources of the study sites. In fact, the TESVI has been determined in R. decussatus for the first time, providing an operational system that highlights the most environmentally challenging elements and allows comparisons of TEs based on the overall spatial variability of their environmental levels throughout the study areas (Richir and Gobert 2014Richir J., Gobert S. 2014. A reassessment of the use of Posidonia oceanica and Mytilus galloprovincialis to biomonitor the coastal pollution of trace elements: New tools and tips. Mar. Pollut. Bull. 89: 390-406.). Our results indicate that the high TESVI values recorded for Pb correlate with our previous data, showing that it has a larger spatial variation than the other TEs. Regarding the THQ, our data indicate that all R. decussatus investigated were safe for human consumption regarding the levels of Zn, Cu, Fe, Cd and Pb (which were below 1), as already stated by Vieira et al. (2011)Vieira C., Morais S., Ramos S., et al. 2011. Mercury, cadmium, lead and arsenic levels in three pelagic fish species from the Atlantic Ocean: intra-and inter-specific variability and human health risks for consumption. Food Chem. Toxicol. 49: 923-932..

It has been widely reported that TEs in bivalves are capable of stimulating ROS production and inducing MT production (Cipro et al. 2017Cipro C.V.Z., Cherel Y., Bocher P., et al. 2017. Trace elements in invertebrates and fish from Kerguelen waters, Southern Indian Ocean. Pol. Biol. 41: 175.). MTs are low molecular weight peptides rich in cysteine, which work against the toxicity caused by TEs and are involved in detoxification processes (Viarengo et al. 1985Viarengo A., Palmero S., Zanicchi G., et al. 1985. Role of metallothioneins in Cu and Cd accumulation and elimination in the gill and digestive gland cells of Mytilus galloprovincialis lam. Mar. Environ. Res. 16: 23-26.). These metallo-proteins are known for their protective free radical scavenging activity, playing an active role in the capture of harmful oxidant radical species (Kumari et al. 1998Kumari M.V.R., Hiramatsu M., Ebadi M. 1998. Free radical scavenging actions of metallothionein isoforms I and II. Free Radic. Res. 29: 93-101., Telahigue et al. 2018Telahigue K., Rabeh I., Bejaoui S., et al. 2018. Mercury disrupts redox status, up-regulates metallothionein and induces genotoxicity in respiratory tree of sea cucumber (Holothuria forskali). Drug Chem. Toxicol. 43: 287-297.). The observed induction of MTs in R. decussatus when exposed to environmental pollution probably shows an adaptive response of the digestive gland to the toxicological manifestation induced by TEs, especially Cd and Pb. Our results are in agreement with previous studies carried out by Rabei et al. (2018)Rabei A., Hichami A., Beldi H., et al. 2018. Fatty acid composition, enzyme activities and metallothioneins in Donax trunculus (Mollusca, Bivalvia) from polluted and reference sites in the Gulf of Annaba (Algeria): Pattern of recovery during transplantation. Environ. Pollut. 237: 900-907. on Donax trunculus collected seasonally from polluted sites in the gulf of Annaba and Bejaoui et al. (2020)Bejaoui S., Michan C., Telahigue K., et al. 2020. Metal body burden and tissue oxidative status in the bivalve Venerupis decussata from Tunisian coastal lagoons. Mar. Environ. Res. 159: 105000. on R. decussatus collected from Tunisian lagoons.

Stimulation of MTs is related to the protection against oxidative stress and macromolecule damage (Baltaci et al. 2017Baltaci A.K., Yuce K., Mogulkoc R. 2017. Zinc metabolism and metallothioneins. Biol. Trace. Elem. Res. 183: 22–31.). TE accumulation generates an intracellular imbalance and stimulates ROS, leading to the appearance of lipids, proteins and DNA damage through the generation of hydroxyl and free radicals from H2O2 via the Fenton reaction (Thomas et al. 2013Thomas C., Melissa M., Amy A. 2013. Hydroxyl radical is produced via the Fenton reaction in sub-mitochondrial particles under oxidative stress: implications for diseases associated with iron accumulation. Redox Rep. 14: 102-108.). As a result of massive free radical generation, MDA performs as second toxic messengers and can form during lipid peroxidation (Cravo et al. 2012Cravo C., Pereira T., Gomes C., et al. 2012. Multibiomarker approach in the clam Ruditapes decussatus to assess the impact of pollution in the Ria Formosa lagoon, South Coast of Portugal. Mar. Environ. Res. 75: 23-34., Telahigue et al. 2018Telahigue K., Rabeh I., Bejaoui S., et al. 2018. Mercury disrupts redox status, up-regulates metallothionein and induces genotoxicity in respiratory tree of sea cucumber (Holothuria forskali). Drug Chem. Toxicol. 43: 287-297.). Our results showed an increase in the MDA level in the digestive gland of R. decussatus collected from the HCA and the MCA, particularly during the warm season. This increase appears to be due to TE uptake, which accelerates the ability to scavenge ROS through the extreme production of lipid peroxides in the digestive gland. In earlier studies, similar increases in MDA levels have been reported in Fulvia fragilis from the Bizerte lagoon (Mahmoud et al. 2010Mahmoud N., Dellali M., El Bour M., et al. 2010. The use of Fulvia fragilis (Mollusca: Cardiidae) in the biomonitoring of Bizerta lagoon: A multimarkers approach. Ecol. Ind. 10: 696-702.), R. decussatus from the Ria Formosa lagoon (Cravo et al. 2012Cravo C., Pereira T., Gomes C., et al. 2012. Multibiomarker approach in the clam Ruditapes decussatus to assess the impact of pollution in the Ria Formosa lagoon, South Coast of Portugal. Mar. Environ. Res. 75: 23-34.) and Aulacomya atra from the South Atlantic Patagonian coast (Di Salvatore et al. 2013Di Salvatore P., Calcagno J.A., Ortiz N., et al. 2013. Effect of seasonality on oxidative stress responses and metal accumulation in soft tissues of Aula comyaatra, a mussel from the South Atlantic Patagonian coast. Mar. Environ. Res. 92: 244-252.). Furthermore, many studies have established a clear link between lipid peroxidation and genotoxic diseases (Marnett 2002Marnett L.J. 2002. Oxy radicals, lipid peroxidation and DNA damage. Toxicology 27: 219-222., Javed et al. 2016Javed M. Ahmad I. Usmani N. et al. 2016. Studies on biomarkers of oxidative stress and associated genotoxicity and histopathology in Channa punctatus from heavy metal polluted canal. Chemosphere 151: 210-219., Bejaoui et al. 2020Bejaoui S., Michan C., Telahigue K., et al. 2020. Metal body burden and tissue oxidative status in the bivalve Venerupis decussata from Tunisian coastal lagoons. Mar. Environ. Res. 159: 105000.). In fact, MDA appears to be an important contributor to DNA damage and mutations, as reported by Ayala et al. (2014)Ayala A., Mynoz M.F., Arguelles S. 2014. Lipid Peroxidation: Production, Metabolism and Signaling Mechanisms of Malondialdehyde and 4-Hydroxy-2-Nonenal. Oxid. Med. Cell. Longev. 2014: 360438.. In our study, the increase in MDA levels may be considered a significant endogenous source of DNA damage. This finding was confirmed by a significant degradation of the DNA contents in the R. decussatus digestive glands collected from the MCA and the HCA. The alteration of the DNA in clams’ digestive glands from the HCA indicates how bivalves are experiencing pollution stress, in view of the fact that this area has long been receiving untreated waters and harbour discharges (Ennouri et al. 2016Ennouri R., Zaaboub N., Fertouna-Bellakhal M., et al. 2016. Assessing trace metal pollution through high spatial resolution of surface sediments along the Tunis Gulf coast (Southwestern Mediterranean). Environ. Sci. Pollut. Res. 23: 5322-5334.). Similarly, high DNA alteration rates in aquatic organisms collected from different contaminated sites have been observed (Bejaoui et al. 2018Bejaoui S., Telahigue K., Chetoui I., et al. 2018. Integrated effect of metal accumulation, oxidative stress responses and DNA damage in Venerupis decussata gills collected from two coast Tunisian lagoons. J. Chem. Environ. Biol. Eng. 2: 44-51.).

The potential of TE accumulation causing adverse effects in the environment and its constituents has been well reported for the tissues of organisms (Mahmoud et al. 2010Mahmoud N., Dellali M., El Bour M., et al. 2010. The use of Fulvia fragilis (Mollusca: Cardiidae) in the biomonitoring of Bizerta lagoon: A multimarkers approach. Ecol. Ind. 10: 696-702., Bejaoui et al. 2018Bejaoui S., Telahigue K., Chetoui I., et al. 2018. Integrated effect of metal accumulation, oxidative stress responses and DNA damage in Venerupis decussata gills collected from two coast Tunisian lagoons. J. Chem. Environ. Biol. Eng. 2: 44-51. ). Proteins are one of the main targets of the effects of TEs (Bejaoui et al. 2020Bejaoui S., Michan C., Telahigue K., et al. 2020. Metal body burden and tissue oxidative status in the bivalve Venerupis decussata from Tunisian coastal lagoons. Mar. Environ. Res. 159: 105000.). Oxidation is the major consequence of protein damage both externally and within cells. The occurrence of protein oxidation in the digestive gland of R. decussatus was also confirmed by the level of AOPP, which reflects an excess of free radical generation (Prevodnik et al. 2007Prevodnik A., Gardestrom J., Lilja K., et al. 2007. Oxidative stress in response to xenobiotics in the blue mussel Mytillus edulis: Evidence for variation along a natural salinity gradient of the Baltic Sea. Aquat. Toxicol. 82: 63-71.). The comparison between sites showed higher AOPP levels in the digestive gland of clams from the MCA and the HCA than in those from the LCA, suggesting the important role of TEs in the exertion of protein oxidation (Rabei et al. 2018Rabei A., Hichami A., Beldi H., et al. 2018. Fatty acid composition, enzyme activities and metallothioneins in Donax trunculus (Mollusca, Bivalvia) from polluted and reference sites in the Gulf of Annaba (Algeria): Pattern of recovery during transplantation. Environ. Pollut. 237: 900-907.).

Protein oxidation can lead to amino acid changes leading to the formation of carbonyl and other oxidized moieties, as well as to the failure of sulfhydryl groups (Bainy et al. 1996Bainy A.C.D., Saito E., Carvello P.S.M., et al. 1996. Oxidative stress in gill, erythrocytes, liver and kidney of Nile tilapia (Oreochromis niloticus) from a polluted site. Aquat. Toxicol. 34: 151.). These groups, including thiols, can react with free radicals and with products of lipid peroxidation to protect cells against the development of oxidative stress (Tandon et al. 2002Tandon S.K., Prasad S., Singh S., et al. 2002. Influence of age on lead-induced stress in rat. Biol. Trace Elem. Res. 88: 59-69., Carmeli et al. 2008Carmeli E., Bachar A., Barchad S., et al. 2008. Antioxidant status in the serum of persons with intellectual disability and hypothyroidism: A pilot study. Res. Dev. Disabil. 29: 431-438.). Among them, the glutathione status is one of the first lines of defence including GSH and NPSH. These antioxidants are the most abundant thiols in the cell and are considered the main cellular redox barrier. In our study, specimens from the MCA and the HCA exhibited higher GSH and NPSH levels over the year than those from the LCA. This increase can be explained by the new synthesis of GSH and NPSH as an adaptive response in addition to ROS generation. Our results are in accordance with those of previous studies carried out on bivalves and show the increase of the GSH and NPSH levels in relation to seasonal change and TE exposure (Mahmoud et al. 2010Mahmoud N., Dellali M., El Bour M., et al. 2010. The use of Fulvia fragilis (Mollusca: Cardiidae) in the biomonitoring of Bizerta lagoon: A multimarkers approach. Ecol. Ind. 10: 696-702., Augustyniak et al. 2009Augustyniak M., Babczynska A., Augustyniak M. 2009. Does the grasshopper Chorthippus brunneus adapt to metal polluted habitats? A study of glutathione-dependent enzymes in grasshopper nymphs. Insect Sci. 16: 33-42.).

Vitamin C is a powerful reducing agent and an important hydrophilic vitamin for animals and humans. It plays an important protective role in the damage induced by free radicals scavenging hydroxyls, radicals and singlet oxygen (Tandon et al. 2002Tandon S.K., Prasad S., Singh S., et al. 2002. Influence of age on lead-induced stress in rat. Biol. Trace Elem. Res. 88: 59-69.). The increase in Vit C levels in the digestive gland of R. decussatus from the MCA and the HCA could be due to the crucial role of this antioxidant in protecting the physical status of organisms exposed to TE pollution (Chetoui et al. 2019Chetoui I., Bejaoui S., Trabelsi W., et al. 2019. Exposure of Mactra corallina to acute doses of lead: effects on redox status, fatty acid composition and histomorphological aspect. Drugs Chem.). In line with our data, Bejaoui et al (2020)Bejaoui S., Michan C., Telahigue K., et al. 2020. Metal body burden and tissue oxidative status in the bivalve Venerupis decussata from Tunisian coastal lagoons. Mar. Environ. Res. 159: 105000. showed a similar variation in Vit C levels in R. decussatus sampled from the HCA. Conversely, Jena et al. (2009)Jena K.B., Verlecar X.N., Chainy G.B.N. 2009. Application of oxidative stress indices in natural populations of Perna viridis as biomarker of environmental pollution. Mar. Pollut. Bull. 58: 107-113. showed a decrease in the Vit C level in P. viridis digestive gland collected from a polluted site. In fact, these authors explained the decrease in Vit C by the availability of glutathione to reduce the dehydroascorbate into ascorbate as a result of the high ROS generation.

To mitigate damage to the lipid membrane, bivalves are capable of enhancing their antioxidant defence systems, such as CAT and GPx enzymes (Regoli and Giuliani 2014Regoli F., Giuliani M.E. 2014. Oxidative pathways of chemical toxicity and oxidative stress biomarkers in marine organisms. Mar. Environ. Res. 93: 106-117.). Several studies have demonstrated that antioxidant enzymes could protect bivalves from the toxicity of environmental pollutants. In the present work, clams collected from the MCA and the HCA exhibited increases in GPx and CAT activities. These findings highlight the ability of R. decussatus to quench ROS overproduction and to alleviate cellular injuries. Our results are in total agreement with the increase in CAT and GPx activities in Tapes philippinarum from the Adriatic Sea (Bocchetti et al. 2008Bocchetti R., Lamberti C.V., Pisanelli B., et al. 2008. Seasonal variation of exposure biomarkers, oxidative stress responses and cell damage in the clams, Tapes philippinarum, and mussels, Mytilus galloprovincialis, from Adriatic sea. Mar. Environ. Res. 66: 24-26.), in Aulacomya atra from the Nuevo Gulf in Northern Patagonia (Giarratano et al. 2014Giarratano E., Gil M.N., Malanga G. 2014. Biomarkers of environmental stress in gills of ribbed mussel Aulacomya atra atra (Nuevo Gulf, Northern Patagonia). Ecotoxicol. Environ. Saf. 107: 111-119. ) and in Fulvia fragilis in the Bizerte lagoon (Mahmoud et al. 2010Mahmoud N., Dellali M., El Bour M., et al. 2010. The use of Fulvia fragilis (Mollusca: Cardiidae) in the biomonitoring of Bizerta lagoon: A multimarkers approach. Ecol. Ind. 10: 696-702.) in relation to high TE concentrations and chemical pollutants.

The effect of environmental pollution on digestive gland mechanisms was also assessed through the AChE activity. In fact, AChE is present in the neuromuscular junctions and cholinergic synapses of the central nervous system and terminates the signal transmission by hydrolysing acetylcholine (Ach), a neurotransmitter that conducts nerve impulses across neuromuscular junctions (Lodish et al. 2000Lodish H., Berk A., Zipursky S.L., et al. 2000. Molecular Cell Biology. W.H. Freeman, New York.). TEs show a noticeable affinity with the sulphur donor, which binds to the thiol residues of proteins (Viarengo 1985Viarengo A., Palmero S., Zanicchi G., et al. 1985. Role of metallothioneins in Cu and Cd accumulation and elimination in the gill and digestive gland cells of Mytilus galloprovincialis lam. Mar. Environ. Res. 16: 23-26.). Our results confirm that the metallic pollution found in the MCA and the HCA affected the cholinergic system, as demonstrated by a significant inhibition of AChE in the digestive gland of R. decussatus. Tankoua et al (2012)Tankoua O.F., Triquet C.A., Denis F., et al. 2012. Physiological status and intersex in the endobenthic bivalve Scorbicularia plana from thirteen estuaries in northwest France. Environ. Pollut. 167: 70-77. suggested that the decrease in AChE activities could be induced by the accumulation of TEs (such as Cd, Cu and Zn) in Scrobicularia plana on the French coast.

Oxidative stress responses in R. decussatus digestive glands resulting from the combination of TE accumulation and abiotic factors may cause injuries in tissues. The study of histological injuries induced by prolonged exposure to environmental conditions is considered a successful approach for the assessment of the environmental quality of an area (Cappello et al. 2013Cappello T., Mauceri A., Corsaro C., et al. 2013. Impact of environmental pollution on caged mussels Mytilus galloprovincialis using NMR-based metabolomics. Mar. Pollut. Bull. 77: 132-139., Vajargah et al. 2018Vajargah M.F., Yalsuyi A.M., Hedayati A., et al. 2018. Histopathological lesions and toxicity in common carp (Cyprinus carpio L. 1758) induced by copper nanoparticles. Micros. Res. Tech. 81: 724-729.). It is well established that the enhancement of antioxidant responses in the digestive gland may trigger a cascade of events, eventually leading to cell death. Clams collected from each of the three sampling areas exhibited pathological changes. The occurrence and severity of abnormalities showed fluctuations throughout the sampling seasons. The investigation of the interaction between the location and pollution type showed that the rate of histopathological lesions in the digestive glands was highest in clams from the HCA moderate in clams from the MCA and lowest in clams from the LCA.

The two affected clam populations were characterized by the presence of haemocyte infiltration and epithelial cell degradation during winter and autumn. This phenomenon has been well described as one of the responses to diverse environmental stressors (Pirrone et al. 2018Pirrone C., Rossi F., Cappello S., et al. 2018. Evaluation of biomarkers in Mytilus galloprovincialis as an integrated measure of biofilm-membrane bioreactor (BF-MBR) system efficiency in mitigating the impact of oily wastewater discharge to marine environment: a microcosm approach. Aquat. Toxicol. 189: 49-62.). It has previously been established that haemocyte infiltration is the cause of the inflammatory process due to a rapid release of pro-inflammatory and vasoactive mediators (Bouallegui et al. 2018Bouallegui Y., Ben Younes R., Bellamine H., et al. 2018. Histopathological indices and inflammatory response in the digestive gland of the mussel Mytilus galloprovincialis as biomarker of immunotoxicity to silver nanoparticles. Biomarkers 23: 277-287.). Furthermore, atrophic tubules were observed in the same seasons and were characterized by a decrease in the density of the epithelia followed by the extension of the digestive lumen. Also, necrosis and fibrosis were observed, confirming the cell membrane rupture and the inadequate DNA degradation in the digestive gland of clams from the HCA. These abnormalities are known to be induced by exposure to a variety of pollutants (such as polycyclic aromatic hydrocarbons and TEs) and untreated municipal wastewater (Schlacher et al. 2007Schlacher T.A., Mondon J.A., Connolly R.M. 2007. Estuarine fish health assessment: Evidence of wastewater impacts based on nitrogen isotopes and histopathology. Mar. Pollut. Bull. 54: 1762-1776., Sonawane 2015Sonawane S.M. 2015. Effect of Heavy metals on gills of fresh water bivalve Lamellidens marginalis. J. Environ. Sci. Toxicol. Food Technol. 9: 5-11., Osman et al. 2017Osman G., Galal M, Abul-Ezz A., et al. 2017. Polycyclic aromatic hydrocarbons (PAHS) accumulation and histopathological biomarkers in gills and mantle of Lanistes carinatus (Molluscs, Ampullariidae) to assess crude oil toxicity. Punjab Univ. J. Zool. 32: 39-50.). These abnormalities could be correlated to TE uptake but also to seasonal variability of water parameters (Kolyuchkina et al. 2017Kolyuchkina G.A., Budko D.F., Chasovnikov V.K., et al. 2017. Influence of the Bottom sediment characteristics on the bivalve Mollusk Anadara kagoshimensis Histopathology’s variability in the northeastern coast of the black sea. Oceanology 57: 828-840.). Our results were confirmed by the existence of several probable sources of contaminants within the HCA, including the watershed, a wastewater treatment plant, harbour activities and industrial and domestic waste (Ben Ayed et al. 2012Ben Ayed L., Yang W., Widmer G., et al. 2012. Survey and genetic characterization of wastewater in Tunisia for Cryptosporidium spp., Giardia duodenalis, Enterocytozoon bieneusi, Cyclospora cayetanensis and Eimeria spp. J. Water Health 10: 431-444. , Ennouri et al. 2016Ennouri R., Zaaboub N., Fertouna-Bellakhal M., et al. 2016. Assessing trace metal pollution through high spatial resolution of surface sediments along the Tunis Gulf coast (Southwestern Mediterranean). Environ. Sci. Pollut. Res. 23: 5322-5334.). In addition, our results were similar to those of previous reports showing that the histological abnormalities can be harmful when the non-essential TE levels exceed those required for the normal metabolic function (Yee-Duarte et al. 2018Yee-Duarte J.A., Ceballos-Vazquez B. P., Arellano-Martinez M., et al. 2018. Histopathological alterations in the gonad of Megapitaria squalida (Mollusca: Bivalvia) inhabiting a heavy metals polluted environment. J. Aquat. Anim. Health 30: 144-154.). Similar studies reported changes in the histological structure of some bivalves in relation to TE pollution and seasonal variation (Costa et al. 2013Costa P.M., Carreira S., Costa M. H., et al. 2013. Development of histopathological indices in a commercial marine bivalve (Ruditapes decussatus) to determine environmental quality. Aquat. Toxicol. 126: 442-454., Bejaoui et al. 2020Bejaoui S., Michan C., Telahigue K., et al. 2020. Metal body burden and tissue oxidative status in the bivalve Venerupis decussata from Tunisian coastal lagoons. Mar. Environ. Res. 159: 105000.).

The general analyses of data by PCA indicate a significant separation among sampling periods and lagoons. This study established a clear seasonality of the abiotic factors, TEs and biological parameters tested as biomarkers in clams from different lagoons. Indeed, clams’ digestive glands from the HCA were characterized by elevated concentrations of TE, confirming their metabolic responses in relation to TE accumulation. Homogeneity in response to TE may also be due to a combination of geomorphologic and hydrologic factors characterizing the HCA. Indeed, this lagoon is a semi-enclosed basin with a high input of anthropic discharges, backwater and relatively high water mixing between sites (Chouari 2015Chouari W. 2015. The contribution of cartography in reconstructing past human impacts on coastal wetlands: the case the Tunis Lagoon in the 20th century. J. Mediterr. 125: 75-84., Ennouri et al. 2016Ennouri R., Zaaboub N., Fertouna-Bellakhal M., et al. 2016. Assessing trace metal pollution through high spatial resolution of surface sediments along the Tunis Gulf coast (Southwestern Mediterranean). Environ. Sci. Pollut. Res. 23: 5322-5334.). Previous reports have described that the circulation of water in the HCA area is strongly limited by the shallow depth and by the very irregular shape of the banks (Chouari 2015Chouari W. 2015. The contribution of cartography in reconstructing past human impacts on coastal wetlands: the case the Tunis Lagoon in the 20th century. J. Mediterr. 125: 75-84.). Furthermore, these important oxidative responses to TEs in the clam digestive glands from the HCA could be associated with the filtration rate of the sediment particles, which have a high level of TEs, brought from the Tunis Gulf by currents (Ennouri et al. 2010Ennouri R., Chouba L., Magni P. et al. 2010. Spatial distribution of trace metals (Cd, Pb, Hg, Cu, Zn, Fe and Mn) and oligo-elements (Mg, Ca, Na and K) in surface sediments of the Gulf of Tunis (Northern Tunisia). Environ. Monit. Assess. 163: 229., Zaaboub et al. 2014Zaaboub N., Oueslati W., Helali M.A., et al. 2014. Trace elements in different marine sediment fractions of the Gulf of Tunis (Central Mediterranean Sea). Chem. Speciat. Bioavailab. 26: 1-12.). In addition, a marked effect of seasonality was detected for clams from the HCA, especially during the summer season. These results indicate that R. decussatus was more influenced by the seasonal variations in environmental parameters such as temperature, salinity and food availability (suspended matter, etc.). Indeed, the HCA was characterized by a black sticky mud with a strong smell of hydrogen sulphide and rich seaweeds with some fragments of shells and water stagnation (Kochlef 2003Kochlef M. 2003. Contribution à L’étude du Fonctionnement Hydrodynamique du lac Sud Tunis après les Travaux D’aménagement, DEA, National Agronomy Institute of Tunisia, Carthage University, Tunis, Tunisia.).

CONCLUSIONTop

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

ACKNOWLEDGEMENTSTop

This work was supported by the Physiology and Aquatic Environment Laboratory, Faculty of Sciences, University of Tunis El Manar. We would like to thank all the team of this laboratory, and in particular Mr. Hssan Mejri, for their technical assistance. This research did not receive any specific grant from funding agencies in the public, commercial or not-for-profit sectors.

Conflict of interest. The authors declare no conflict of interest.

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Supplementary material

The following supplementary material is available through the online version of this article and at the following link:
http://scimar.icm.csic.es/scimar/supplm/sm05054esm.pdf

Table S1. – Mean (±SD) environment parameters recorded during four seasonsinthe three Tunisian lagoons: Ghar el Melh (LCA), the North Lake (MCA) and the South Lake (HCA). Values are presented as mean ±SD (n=3 repetitions). Significant differences are determined at 0.05 using two-way ANOVA: *p<0.05, **p<0.01; ***p<0.001.

Table S2. – Mean (±SD) weight (W), length (L), condition (CI) and gonad (GI) indices of R. decussatus from Tunisian lagoons: Ghar el Melh (LCA), the North Lake (MCA) and the South Lake (HCA). Values are expressed as means ±SD (n=40 replications). Significant differences are determined at 0.05 using two-way ANOVA: *p<0.05, **p<0.01; ***p<0.001.

Table S3. – Levels of trace elements in the water column and sediments of Tunisian lagoons: Ghar el Melh (LCA), the North Lake (MCA) and the South Lake (HCA).

Table S4. – Correlation matrix of non-parametric Spearman’s rank correlation coefficients between R. decussatus biomarkers, trace element digestive gland tissue levels (Cd, Pb, Cu, Zn, and Fe) and environmental parameters measured in this study. AChE, acetylcholinesterase; CAT, catalase; AOPP, advanced oxidation proteins products; GPx, glutathione peroxidase; GSH, gluthatione; NPSH, non-protein SH; MDA, malondialdehyde; Vit C, vitamin C; MTs, metallothioniens; T, temperature; S, salinity; pH, hydrogen potential; SPM, suspended matter; Ch a, Chlorophyll a. The positive and significant correlation is presented in red and the negative and significant correlation is presented in green.