Abd-Ellah et al., Swed J BioSci Res 2020; 1(1): 62 - 77. DOI: https://doi.org/10.51136/sjbsr.2020.62.77 Research Paper Seasonal effects of heavy metals on the date mussel Lithophaga lithophaga (Mollusca:Bivalvia) at Eastern harbor, Alexandria, Egypt Shahenaz M. Abd-Ellah1*, Soheir El-Sherif2, Rehab El-Morshedy2 1Department of Biological & Geological Sciences, Faculty of Education, Alexandria University, Egypt, 2Department of Zoology, Faulty of Science,, Alexandria University, Egypt. *Corresponding author: [email protected] Received: 20 October 2020 Accepted : 05 November 2020 Published online : 15 November 2020 Abstract: The edible mussel Lithophaga lithophaga is considered as one of the most important human food sources in Alexandria, Mediterranean Sea. The present study is designed to determine the seasonal bioaccumulation levels of Cd, Co and Pb in the whole soft tissues as well as different tissues of Lithophaga lithophaga. Results revealed that the seasonal bioaccumulation levels of Cd, Co and Pb in date mussel were below the permissible limits or other reported values from other regions of the Mediterranean. On the other hand, the order of metals accumulation level in different tissues was as follows: digestive gland>remaining soft tissues>gonads. The present study confirmed the role of digestive gland as a concentration center for heavy metals. Moreover, total protein content and stress protein responses of the whole soft tissues were evaluated. The total protein content was arranged in the following order: summer>autumn>spring>winter. Five novel stress proteins appeared in summer. The histological and ultrastructural studies of the digestive gland of Lithophaga lithophaga collected in summer and spring showed marked histopathological alternations. Keywords: Lithophaga lithophaga, mussel, cadmium, cobalt, lead, heavy metals, seasonal bioaccumulation. Introduction Lithophaga lithophaga is a common and widely is important in a healthy and balanced diet (WHO, distributed species, usually at shallow water of the 2003). Seafood is an important source of valuable Mediterranean, of the east Atlantic from Portugal to nutrients (Aakre et al., 2019). It contains low Morocco and in the Red Sea (Legac and Hrs- cholesterol and high percentage of n-3-poly- Brenko, 1982 ; Fischer et al., 1987; Gargominy et unsaturated fatty acids, liposoluble vitamins and al., 1999) and can also be found at a depth of 8m to essential minerals; it is rich in protein (Tahvonen et 20m or more (Simunovic and Grubelic, 1992). It is al., 2000; Adeyeye, 2002; Zalloua et al., 2007; a greatly appreciated species for human Aakre et al., 2019). Seafood can contribute to consumption as seafood and it was found in seafood human exposure to heavy metals resulting from markets and fish restaurants (Gonzalez et al., 2000). consuming contaminated aquatic organisms The over-exploitation of this species resulted in the (Hashmi et al., 2002; Carvalho et al., 2005; dramatic reduction of its population and thus, it is Francesconi, 2007; Schuwerack et al., 2007; Sioen under strict protection (Galinou-Mitsoudi and et al., 2008). Increasing anthropogenic activities Sinis, 1994; Colletti et al., 2020). It was proposed in have made heavy metals pollution in aquatic several international forums as species that should ecosystem a major cause of concern in two main be protected (Fernández-Galiano, 2000). The aspects, firstly from the public health point of view species is considered among the threatened species and secondly aquatic environmental point of view, in the Mediterranean Sea and it has been protected particularly that the pollution of the marine by the Bern and the Barcelona Conventions and the ecosystem by heavy metals is a worldwide problem. Convention on International Trade of Endangered Heavy metals are one of the serious pollutant due Species (CITES), where its collection is forbidden to their persistence, toxicity and non-degradability in by law (Kefi et al., 2016). the environment (Sharshar and Gease, 1998; Tam It is globally accepted that seafood consumption and Wong, 2000; Yuan et al.; 2004). Vymazal Copyright © 2020 Abd-Ellah et al.This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution and reproduction in any medium, provided the original author(s) and source are credited. Abd-Ellah et al., Swed J BioSci Res 2020; 1(1): 62 - 77. DOI: https://doi.org/10.51136/sjbsr.2020.62.77 (1995) stated that the most important heavy metals Mediterranean Sea through two openings: El- from the point of view of water pollution are Cd, Co, Boughaz and El-Silsila (El-Geziry et al., 2007). Cu, Cr, Fe, Pb, Hg, Ni and Zn, some of these metals (Cu, Zn, Fe and Co) are essential for living Sampling organisms but become toxic at higher In each season, 30 sexually mature mussels, concentrations, other metals are non-essential and nearly of the same size and weight, were collected toxic even at relatively low concentrations (Pb, Cd (10 for the determination of Cd, Co and Pb and Hg). The heavy metals that constitute a great concentration; 5 for biochemical studies; 5 for threat to public health include Hg, Pb and Cd which electrophoretic studies and 10 for histopathological are potent toxicants to humans. Their studies). bioaccumulation in tissues of seafood leads to intoxication causing cellular and tissue damage, Metal analysis decreased fertility, cell death and dysfunction of a Heavy metals Cd, Co and Pb were analyzed in variety of organs and death (Fatoki and water samples and mussels tissues using graphite Mathabatha, 2004; Khan et al., 2019). furnace atomic absorption spectroscopy (Perkin- The rights of all individuals to a safe and Elmer model 2380) under the recommended adequate diet were expressed at the World Health conditions of Bernhard (1976). Organization Forum in 2007 which led to the Beijing Declaration (WHO, 2007). To document Seawater analysis seafood safety, information on the status of infective Seawater samples were collected seasonally organism, drugs residues and undesired chemical during L. lithophaga sampling. The metals elements is considered necessary (Nghia et al., concentration in the seawater was determined 2009). Determination of chemical quality of aquatic according to Eaton (1976). It was expressed as organisms consumed as seafood, particularly their μg/L. content of heavy metals, are extremely important to human health. The accumulation of these metals in Tissues analysis aquatic animals should be monitored regularly to The whole soft tissues of 5 mussels as well as the check animal health in view of the quality of public digestive gland, the gonads and the remaining soft food supplies. In many African countries direct tissues of other 5 mussels were prepared individually consumption of seafood without quality control are according to the technique of Campbell and Plank frequently due to the poverty (Martin and (1998) and they were then analyzed. Heavy metals Griswold, 2009; Morais et al., 2012; Tamele and concentrations were expressed in µg/g wet weight. Loureiro , 2020). The present study is concerned with monitoring Biochemical studies the quality of L. lithophaga as safe seafood through Determination of the total protein content determination of the seasonal bioaccumulation level The whole soft tissues of mussels were of Cd, Co and Pb in the whole soft tissues. homogenized with physiological saline and the total Additionally, the following points were considered: protein was determined seasonally by the method i) the bioaccumulation levels of Cd, Co and Pb in described by Weichselbaum (1946). different tissues, ii) the determination of the biochemical impact of the previously mentioned Electrophoretic studies metals on the total protein content, iii) the The stress protein response was investigated by determination of the stress proteins response, vi) the polyacrylamide gel electrophoresis. The whole soft investigation of the effects of these metals on the tissues of 5 mussels were homogenized in a mortar histological structure of digestive gland . using 0.05M Tris-HCL buffer (pH 6.8), then the homogenates were centrifuged at 10 000 x g for 15 Materials and methods minutes. The supernatant was taken and used for gel Study area electrophoresis analysis. Sodium dodecyl sulphate The East harbor of Alexandria was chosen as a polyacrylamide gel electrophoresis (SDS-PAGE) station from which samples were collected. It was carried out using the discontinuous buffer occupies the central part of the coast of Alexandria system described by Laemmli (1970). and covers an area of about 2.8 km (Said and Maiyza, 1987). The harbor is connected to the Histological studies of the digestive gland 63 Abd-Ellah et al., Swed J BioSci Res 2020; 1(1): 62 - 77. DOI: https://doi.org/10.51136/sjbsr.2020.62.77 For light microscopic examination the specimens of the digestive gland were fixed in 10% formaldehyde for 24 hours, dehydrated in alcohol, cleared in xylene and embedded in paraffin wax. Sections of 5µ thickness were stained with haematoxylin and eosin and were critically examined. Ultrastructural studies of the digestive gland Dissection of soft tissues were performed at (a) formaline-glutraldehyde fixative (4F1G) and very small blocks (1mm3) of the digestive gland were fixed. Sectioning was performed by using a glass knife on LKB ultramicrotome. The ultrathin sections (50 nm) were either pale gold or silver interference color and were picked upon 200 mesh naked copper grids. The ultrathin sections were stained twice by seawater (µg/l) in in uranyl acetate and lead citrate and examined by Jeol 100 CX Electron Microscope. metals Mean of concentration of heavy Statistical analysis Data of metal analysis and total protein content (b) were statistically analyzed using analysis of variance and least significant difference LSD according to Fig. (1). The mean concentrations (µg/l) of the different heavy metals in the collected seawater samples showing (a) the level of Snedecor and Cochran (1981). All comparisons for their accumulation and (b) their seasonal variations at the statistical significance were made at p=0.05.