Seasonal Effects of Heavy Metals on the Date Mussel Lithophaga Lithophaga (Mollusca:Bivalvia) at Eastern Harbor, Alexandria, Egypt

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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. Eastern harbor of Alexandria.

p≤ 0.01) were found between all the metals. Results

Metal analysis were carried out seasonally for Heavy metals in the soft tissues of Lithophaga seawater samples, whole soft tissues as well as lithophaga different organs of Lithophaga lithophaga collected The obtained results are shown in Table 2. The from the Eastern harbor of Alexandria. whole soft tissues of L. lithophaga accumulated heavy metals in the following sequence: Pb>Co> Cd Heavy metals in the seawater of Eastern harbor (Fig. 2a). Metals accumulation levels were variable The concentrations of Cd, Co and Pb in the among different seasons (Fig. 2b). Generally, the seawater samples collected seasonally are shown in highest accumulation level of heavy metal was Table 1. Metals concentrations levels were in the found in summer and spring, while the lowest one order: Co>Pb>Cd (Fig. 1a). The highest metal was recorded in winter and autumn. Cobalt concentration was recorded for Co (0.331±0.005), accumulation level in the soft tissues of L. and the least one was recorded for Cd (0.035± lithophaga showed no significant difference at p≤ 0.001). The respective concentrations of metals 0.01 among the four seasons, while the levels of Pb reported high values in summer and spring; whereas and Cd in the soft tissues of mussel varied their values were low in autumn and winter (Fig. significantly at p≤ 0.01 among seasons (Table 2). 1b). Statistically significant differences (ANOVA,

Table 1. The concentrations of heavy metals in seawater samples collected seasonally from Eastern harbor at Alexandria.

Metal Seasons Summer Autumn Winter Spring Mean + SD Co 0.331 ± 0.005a 0.216 ± 3.88E-05ab 0.134 ± 0.0002b 0.242 ± 0.002ab 0.231 ± 0.070 Pb 0.291 ± 4E-05a 0.142 ± 0.0002ab 0.111 ± 0.0003b 0.171 ± 1.92E-05b 0.178 ± 0.068 Cd 0.133 ± 1.28E-05a 0.055 ± 5.2E-06b 0.035 ± 5.2E-06b 0.098 ± 0.004ab 0.080 ± 0.038

Each value is the mean of 5 replicates ± SD; Values are expressed in µg/g wet weight. a with b: significant at p≤ 0.05 and a-b with ab: non-significant. 64

Abd-Ellah et al., Swed J BioSci Res 2020; 1(1): 62 - 77. DOI: https://doi.org/10.51136/sjbsr.2020.62.77

Heavy metals accumulation in different organs of Lithophaga lithophaga The digestive gland, the gonads and the remaining soft tissues of L. lithophaga were analyzed seasonally to determine the levels of metals accumulation in the various tissues and the values were recorded (Table 3). The general order of Cd, Co and Pb levels in the different tissues of the mussel can be represented as follows: digestive gland > remaining soft tissues > gonads (Fig. 3). It (a) appeared obvious that the order of metal accumulation in the digestive gland, the gonads and the remaining soft tissues of the mussel was: Pb> Co >Cd. It must be mentioned that Pb was the highest metal accumulated in the digestive gland and in the different tissues of the mussel during the four seasons (Fig. 4). Summer and spring recorded the highest concentrations for the various metals while the lowest ones were in winter and autumn (Fig. 5). The order of seasonal metals accumulation in the different tissues of L. lithophaga can be summarized in the following order: (b) Cd: Summer > Spring > Winter ≈ Autumn Fig. (2). The mean concentrations (µg/g) of the different heavy Co : Summer > Spring > Autumn > Winter metals in the whole soft tissue samples showing (a) the level of their accumulation and (b) their seasonal variations. Pb: Spring ≈ Summer > Autumn > Winter

Table 2. Heavy metals concentrations (µg/g wet weight) in the whole soft tissues of Lithophaga lithophaga collected seasonally from Eastern harbor. Metal Seasons

Summer Autumn Winter Spring Mean + SD Co 0.125 ± 0.014 0.094 ± 0.010 0.121 ± 0.017 0.121 ± 0.047 0.115 ± 0.012 a b b ab Pb 1.548 ± 0.736 0.132 ± 0.031 0.156 ± 0.052 1.128 ± 5.267 0.741± 0.615 Cd 0.120 ± 0.011a 0.034 ± 0.002b 0.039 ± 0.005b 0.098 ± 0.004ab 0.073 ± 0.037

Table 3. Seasonal variations in heavy metals concentration (µg/g wet weight) in digestive gland, gonad and remaining soft tissues of Lithophaga lithophaga collected from Eastern harbor.

Seasons Metal Tissue Summer Autumn Winter Spring Mean + SD

Digestive b b 0.216a ± 0.026 0.055 ± 0.004 0.017 ±0.0005 0.168ab ± 0.019 0.114 ± 0.080 gland Co Gonad 0.035a ± 0.002 0.007ab ± 0.0001 0.004b ± 0.0001 0.031ab ± 0.002 0.019 ± 0.014 Remaining 0.056ab ± 0.004 0.019b ± 0.0005 0.005b ± 0.0002 0.072a ± 0.001 0.038 ± 0.027 soft tissues Digestive 0.888 ± 0.970 0.472 ± 0.418 0.472 ± 0.489 1.016 ± 0.829 0.712 ± 0.244 gland Pb Gonad 0.202ab ± 0.077 0.034b ± 0.004 0.018b ± 0.001 0.274a ± 0.128 0.132 ± 0.109 Remaining 0.444 ± 0.428 0.321± 0.280 0.185 ± 0.100 0.526 ± 0.316 0.369 ± 0.128 soft tissues Digestive a b b 0.0584 ± 0.001 0.006 ± 0.0001 0.012 ± 0.001 0.043ab ± 0.001 0.030 ± 0.013 gland Cd a b b ab Gonad 0.025 ± 0.001 - - 0.01 ± 0.0002 0.009 ± 0.010 Remaining 0.048a ± 0.001 0.001b ± 1.2E-06 0.004b ± 0.0001 0.023ab ± 0.001 0.019 ± 0.019 soft tissues 65 Each value is the mean of 5 replicates ± SD; Values are expressed in µg/g wet weight. a with b: significant at p≤ 0.05 and a-b with ab: non-significant.

Abd-Ellah et al., Swed J BioSci Res 2020; 1(1): 62 - 77. DOI: https://doi.org/10.51136/sjbsr.2020.62.77

Total protein content of the soft tissues of mussels The total protein content of the soft tissues of L. lithophaga collected seasonally from Eastern harbor was shown in Table 4. The total protein content exhibited significant seasonal changes at p≤ 0.01, the lowest value being in winter (11±0.4 µg/g wet weight), it increased gradually in spring to reach 12.18±0.15 µg/g followed by 15.1±1.92 µg/g wet weight in summer and then, decreased to12.54±2.25 µg/g wet weight in autumn (Table 4). Fig. (3). The mean concentrations (µg/g wet weight) of Co, Pb The effect of seasonal variations of metals and Cd in the digestive gland (D), the gonads (G) and the concentration on protein synthesis of L. lithophaga remaining soft tissues (R) of Lithophaga lithophaga collected was evaluated by polyacrylamide gel electrophoretic from Eastern harbor of Alexandria. techniques. All samples have 7 constant protein stress-50 proteins (58 and 65 KDa), stress-90 bands belonging to four different protein families, proteins (95 KDa) and high molecular weight low molecular weight proteins (35 and 45 KDa), proteins (122 and 143 KDa).

- -

Fig. (4). The mean concentrations (µg/g wet weight) of the Fig. (5). The mean concentration (µg/g wet weight) of the different heavy metals (Cd-Co-Pd) in the digestive gland (D), different heavy metals (Cd-Co-Pd) in the digestive gland (D), the gonads (G) and the remaining soft tissue (R) of the gonads (G) and the remaining soft tissue (R) of Lithophaga lithophaga showing their seasonal variations. Lithophaga lithophaga showing their seasonal variations.

Abd-Ellah et al., Swed J BioSci Res 2020; 1(1): 62 - 77. DOI: https://doi.org/10.51136/sjbsr.2020.62.77

Table 4. Seasonal variation of the total protein content of Lithophaga lithophaga. *

Mean Seasons * concentration L Summer 15.1 ± 1.92a V Autumn 12.54 ± 2.25ab H a1 a2 Winter 11 ± 0.4b

Spring 12.18 ± 0.15b BC L Mean + SD 12.21 ± 1.873 BC

Each value is the mean of 5 replicates ± SD; Values are expressed L in µg/g wet weight. a with b: significant at p≤ 0.05 and a-b with ab: non-significant. DC DC * Mw Std 1 2 3 4 CT b c 116 66.2

45 35

Fig. (6). SDS-PAGE (12.5%) of L. lithophaga. Lane 1, 2, 3 d and 4 corresponds to summer, autumn, winter and spring, respectively. Std, standard protein marker. Plate I: Seasonal variations in the digestive gland of L. lithophaga. a) Summer, a-1. Showing atrophic digestive tubules (*) with detached epithelium in the luminal space (arrows), On the other hand, in summer five novel protein vacuoles (V) and destructed inter-tubular connective tissue (red arrow), a-2. Showing atrophic digestive tubules (*) as well as bands appeared. These protein bands belong to low detached epithelial cells in the luminal space (arrows), ruptured molecular weight protein (23, 25 and 32 KDa), stress- epithelial cells (arrow head) and hemocyte infiltration (H); b) 50 protein (53 KDa) and stress-90 protein (99KDa). In Autumn, showing basophilic cells (BC), digestive cells (DC), lumen (L) and connective tissues (CT); c) Winter, illustrating autumn, three novel protein bands belonging to low somewhat typical appearance of digestive tubules, d) Spring, molecular weight protein (27 and 30KDa) and stress- showing moderate vacuolization of digestive cells (arrow heads) and rupture of the apical part of some digestive cells (arrows). 50 protein (55KDa) have been reported (Fig.6). Note, also wide inter- tubular connective tissue (red arrow).

Histological studies of the digestive gland of L. appeared somewhat normal (Plate Ib). Each digestive lithophaga tubule is lined by regular basement membrane, two The histological structure of the digestive gland of different types of epithelial cells, digestive cells and L. lithophaga collected in summer and spring showed pyramidal basophilic cells. The digestive cells appear marked histopathological alternations when compared more vacuolated. with samples collected in winter and autumn which In winter, the digestive gland of mussels exhibited may represent more or less a normal histological a well-organized tubular structure normally seen in appearance (Plate I). Lithophaga species. The digestive tubules are well In summer degenerated and/or necrotic digestive arranged, more or less similar in size and shape with tubules were frequently observed. It appears obviously regular basement membrane, somewhat small central that the epithelial lining of the digestive tubules was lumen and very thin layer of intra-tubular connective pathologically changed drastically. The digestive tissues (Plate Ic). They have epithelium of medium tubules completely lost their form with marked tubular height, composed of digestive and basophilic cells, atrophy and intratubular hemocytic infiltration. where basophilic cells appear pyramidal in shape with Extreme vacuolization of digestive epithelial cells central nucleus that usually occupies a large area of were recorded. Mass shedding of some epithelial cells the cell. in the tubular lumen was also detected (Plate Ia). In spring, the digestive tubules of L. lithophaga The histologically examined digestive gland of appeared with marked alternations (Plate Id). The mussels collected in autumn revealed that the tubules digestive epithelial cells of the tubules were 67

Abd-Ellah et al., Swed J BioSci Res 2020; 1(1): 62 - 77. DOI: https://doi.org/10.51136/sjbsr.2020.62.77

DC HC

Ly2 EC rER Rb N BC NE R Ly2 P ER BM CT a V R Plate II: Transmission Electron Micrograph (TEM) of the digestive gland of Lithophaga lithophaga collected in winter. a) Demonstrating basophilic cell (BC) with oval nucleus (N) regular nuclear envelope (NE), nuclear pore (NP), basement membrane (BM), N connective tissue (CT), heterochromatin (HC), euchromatin (EC), rough endoplasmic reticulum (rER) and free ribosomes (R), electron- transparent vacuoles (arrows); b) Demonstrating digestive cell (DC) with nucleus (N) which display condensation of chromatin and vacuolized nuclear lymph (*), large secondary lysosomes (Ly2), L Ly mineralized lysosomes (Ly), residual bodies (Rb), vacuole (V), Ly endoplasmic reticulum (ER) and lipid droplets (L). b

characteristic with erosive disturbances and absence of concentration was found in summer for Cd, Co and their normal structure and form. Disruption in the Pb. Several previous works recorded seasonal digestive tubules, ruptured and extreme vacuolization variations in metals levels in aquatic ecosystems. of tubular epithelial cells were observed. Mirzaei et al. (2020) recorded maximum concentration of metals in seawater samples during Discussion spring, while the minimum one was during winter. In the present study the concentrations of Cd, Co and Pb in seawater samples collected seasonally from * b d ER eastern harbor showed that the highest metals DC V HC Ly22 Plate III: Transmission Electron Micrograph (TEM) of the Ly digestive gland of L. lithophaga collected in summer. a) L N Demonstrating digestive cell (DC) with nucleus (N) displayed V NE condensation of heterochromatin in the center and invagination and Ly2 M R rupture of the nuclear envelope (NE), altered mitochondria (M) were N increased in number with abnormal structure and cristae L Ly L EC fragmentation, basement membrane (BM), large secondary NE lysosomes (Ly2), note also the mineralized lysosomes (*) with a homogenous dense inner structure of heavy metals, numerous vacuoles (V), lipid droplets (L), lysis of some areas in the cytoplasm DC (arrow heads) , b) Completely destroyed digestive cell (DC) with apoptotic nucleus (N), nuclear envelope (NE), euchromatin (EC) , * heterochromatin (HC), vacuoles (V), note many membrane bound N vesicles (arrow head), endoplasmic reticulum (ER), free ribosomes DC (R), mineralized lysosomes with electron dense granules (Ly), many lipid droplets (L), c) Demonstrating digestive cell (DC) with large mineralized lysosomes (*), shrinked nucleus (N) with disorganized chromatin content and cytoplasmic vacuolization (arrowhead), d) Demonstrating abnormal digestive cell (DC) with electron lucent cytoplasm, e) Demonstrating basophilic cell (BC) with deformed BC nucleus (N) which is markedly lobulated with marginated, rER disorganized and coarse chromatin content, ruptured nuclear EC envelope (NE), numerous rough endoplasmic reticulum (rER), Rb * numerous swelled mitochondria (M) with fragmented cristae, G C residual bodies (Rb) with electron dense materials and dense M granules (G), f) Demonstrating basophilic cell (BC) suffering of lysis of the cytoplasm (C) to some extent, nucleus (N) with vacuolized BM NE nuclear lymph (*), heterochromatin (HC) predominant over HC euchromatin (EC) which gave it a peculiar aspect. N

Abd-Ellah et al., Swed J BioSci Res 2020; 1(1): 62 - 77. DOI: https://doi.org/10.51136/sjbsr.2020.62.77

Duysak & Uğurlu (2020) found that levels of all metals were significantly higher in seawater collected from Iskenderun bay, Turkey, summer when compared with other seasons. Our recorded the highest mean concentrations for all results are in agreement with those of Kefi et al. metals in summer, while the lowest ones were (2016) who revealed that metal concentrations in the recorded in winter. Nour (2012) stated that Cd, Co soft tissues of L. lithophaga showed seasonal and Pb concentrations decreased from summer to fluctuations. For Pb, maximum concentration was winter in Abo-Qir bay. The results of heavy metals registered in summer, however lower tissue level was concentrations in seawater samples were compared observed in autumn. Januar et al. (2019) reported that with the maximum permissible concentrations the accumulation of heavy metals in the soft tissue of (MPC) applied in fishery purposes (Table 6). Anadara granosa was higher on dry seasons.

Table 6. The mean concentrations of heavy metals (µg/L) in A number of explanations for seasonal variations seawater samples collected from Eastern harbor of Alexandria. of heavy metals have been proposed. Alliot and MPC Frenet–Piron (1990) suggested that each metal Metal Mean concentration ( µg/L) presents a peak of concentration every year in summer, then, the metal level decreased during winter, Co 0.231± 0.070 - going back to a normal level. Seasonal fluctuations of 210,81 µg/L Pb 0.178 ± 0.068 (USEPA, 2005) metals can be associated to the food supply and to 40 µg/L changes in run-off of metal particulate to the sea due Cd 0.080 ± 0.038 (USEPA, 2005) to high precipitation (Ferreira et al., 2005). The Maximum Permissible Concentrations( MPC) of metals for seasonal variations are also associated with local fishery purposes phytoplankton productivity. Thus, an increase in In the present study, it was apparent that the phytoplankton efficiency implies an increase in concentration of heavy metals in seawater was bivalve nutritional status which leads to increase metal considered less than the maximum permissible concentration in observed organisms. It is supposed concentrations (MPC) of metals for fishery purposes. that low rainfall in dry season leads to lower water Thus, it can be concluded that the Eastern harbor of flow, and as a consequence, higher concentration of Alexandria might be considered unpolluted with Cd, heavy metals is detected (Ali et al., 2016). Moreover, Co and Pb. dry season may primarily influence the water The whole soft tissues of L. lithophaga were temperature (Bhardwaj et al. 2017). Mollusc’s analyzed seasonally to monitor Cd, Co and Pb levels. metabolism may increase at higher temperature and Metals accumulation levels were variable among therefore, the rate of water inflow into the animal also different seasons, the highest level of each heavy increase which lead to higher amount of heavy metals metal was found in summer and spring, while the accumulation (Shakouri and Gheytasi, 2018). lowest accumulation level was recorded in winter and Seasonal fluctuations of metals can be due to the autumn. The significant effect of seasons on heavy reproductive cycle of the bivalve. During gonad metals accumulation in studied samples is similar with development, animal is submitted to various the results of other studies, for example, Kargin et al. physiological conditions which can affect its (2001) recorded seasonal variation in levels of Cd, Zn, bioaccumulation (Champeau, 2005). Fe, Pb, and Cu in tissues of two species of shrimp,

Table 7. Heavy metals concentrations in the whole soft tissues of Lithophaga lithophaga collected from Eastern harbor, Alexandria and other locations of the Mediterranean. L. lithophaga

Location Cd Co Pb Unit Reference Eastern harbor of µg/g wet weight 0.115 0.073 0.741 Present study Alexandria Bizerte Bay ND - 1.81 µg/g dry weight Kefi et al., 2016 µg/g dry weight Izmir Bay Ozsuer & Sunlu, 2.23 - 9.48 (Aegean sea) 2013. Spain (Menorca) 2.21 - 9.2 µg/g dry weight Deudero et al., 2007. Spain (Mallorca) 1.73 - 7.9 µg/g dry weight Deudero et al., 2007. MPC µg/g 1 - 1.5 EC FAO (Bivalve molluscs) 69

Abd-Ellah et al., Swed J BioSci Res 2020; 1(1): 62 - 77. DOI: https://doi.org/10.51136/sjbsr.2020.62.77

Comparison of levels recorded in L. lithophaga (2000) reported that cells of hepatopancreas tissue whole soft tissues with those found in literature seem to be involved in metal sequestering, followed (Table 7) showed that date mussel from the east by metal release from the tissue rather than in harbor of Alexandria accumulates metals much less functioning as a primary accumulating site. Znidaric than date mussel from other locations. On the other et al. (2005) reported that the hepatopancreas hand, previous works have detected high demonstrates the high toxic metals as a result of its concentrations of these metals which sometimes major role in the uptake of metals from food. Elevated exceeded the permissible levels. The present study metal concentrations in the digestive glands probably recorded concentrations of these metals below the are associated to high amounts of metallothioneins in permissible limits. No prescribed maximum the digestive glands (Amiard et al., 2006). The concentration guideline exists for Co. According to digestive glands of molluscs play an important role in the permissible limits of EC and FAO for Cd and Pb, The total protein content exhibited a significant consumption of date mussels collected from East seasonal changes being the lowest value in winter, it harbor of Alexandria does not show any health risk. gradually increased in spring followed by its The digestive gland, the gonads and the remaining maximum value in summer, then, decreased in soft tissues of L. lithophaga were analyzed seasonally autumn. Similar result was seen in the whole soft and they have shown to exhibit variations in the tissues of female M. japonicas which recorded high accumulation levels of metals in the different tissues. concentrations of protein in summer versus low Summer and spring have recorded the highest concentration in winter (Nour, 2012). Biochemical concentrations for all the metals while winter and changes of the total proteins, lipids and carbohydrates autumn have recorded the lowest metals have a key role in overcoming toxic stress. Protein is concentrations. The digestive glands of L. lithophaga one of the most important biochemical components, accumulated the highest concentrations of Cd, Co and thus it can be used as a diagnostic tool for Pb as compared to other studied soft body tissues. The determination of the physiological status of an order of metals accumulation level for Cd, Co and Pb organism under extreme conditions (Prasath and in the different tissues was as follows: digestive gland Arivoli, 2008; Suryavanshi et al., 2009). The > remaining soft tissues > gonads. This result may seasonal increase of the total protein content of confirm previous studies of several authors who molluscs in summer may be related to the increased reported that digestive gland usually accumulates gametogenic activity, oocyte maturation and spawning higher levels of heavy metals than other tissues. activity which reach its peak in summer (Li et al. Odzak et al. (2000) reported that the digestive gland 2000; Kunduz and Erkan, 2008). of shellfish (Mytilus edulis) is the preferential organ The effect of heavy metals pollution on the pattern for the accumulation of heavy metals. Yüzereroğlu et of protein synthesis of L. lithophaga has been al. (2010) found that the highest metal levels were investigated seasonally and it has been shown to be registered in the digestive gland of Patella caerulea significant. Generally, it is clear that the effect of than in gills and muscles. Jebali et al. (2014) observed heavy metals on L. lithophaga resulted in synthesis of that the digestive glands of the bivalve accumulated new polypeptides which were synthesized in the cells the highest concentrations of Cd, Cu, Pb and Zn as due to the effect of metals and were considered as compared to other studied soft body tissues. stress proteins. In summer where the soft tissues of L. Therefore, digestive gland is being proposed as lithophaga recorded the highest accumulation level of bioindicator organ for monitoring Cd, Cu, Pb and Zn Cd, Co and Pb, five novel protein bands belonging to metal pollution in freshwater ecosystem. low molecular weight proteins (23, 25 and 32 KDa), The digestive gland in molluscs is the key organ of stress-50 protein (53 KDa) and stress-90 protein metabolism serving as the main site of accumulation (99KDa) appeared . In autumn, three novel protein and biotransformation of xenobiotics, thus, resembling bands with molecular weights 27, 30 and 55 KDa the liver of vertebrates (Cajaraville et al., 1992b). appeared. These stress proteins are different in their The digestive gland represents the major site of metals molecular masses and belong to different protein accumulation in bivalves (Pipe et al., 1999). families. Sanders (1993) suggested that invertebrate Bastamante et al. (1998) reported that the digestive animals possess: (i) a heterogeneous group of low gland appears to have a key function in the molecular weight stress proteins with molecular metabolism of heavy metals. Wallace et al. (1998) masses of about 15-40 KDa; (ii) a group of stress-50 found that digestive gland with accumulated metals proteins (HSP, haperonin; molecular mass 58-60 KDa) might function as excretory compartments. Soto et al. (iii) proteins of the prominent stress-70 family (HSP

Abd-Ellah et al., Swed J BioSci Res 2020; 1(1): 62 - 77. DOI: https://doi.org/10.51136/sjbsr.2020.62.77

90; molecular mass 83-90 KDa); and (iv) high L. lithophaga collected in winter and autumn may molecular weight stress proteins (molecular mass 100- represent more or less the normal histological 110 KDa). structure. The tubules of the digestive glands had a It can be suggested that the constitutive levels of rather large lumen and epithelium of medium heights the stress proteins provide a rapid protections against composed of two major cell types: the columnar heavy metals pollution. At the biochemical level, the digestive cell and the pyramidal basophilic cell. On induction of stress proteins represents a homeostatic the other hand, the histological structure of the defense mechanism of cells in response to metabolic digestive gland of L. lithophaga collected in summer and environmental pollution (Lacoste et al., 2001). It and spring showed marked histopathological is believed that the cellular stress response protects alternations. The digestive tubules completely lost organisms from damage resulting from exposure to a their form with marked tubular atrophy, degenerated wide variety of stressors including ultraviolet light, and/or necrotic digestive tubules, extreme elevated temperatures, trace metals and xenobiotics vacuolization of digestive epithelial cells and intra- (Abdella et al., 2006; Leung et al., 2011). The tubular hemocytic infiltration. Several previous molecular stress response in invertebrates is useful as studies may clarify and support the present results. biomarkers for sub lethal effects of chemicals. Winstead (1998) reported atrophy of the digestive The present study recorded also an obvious tubules and loss of epithelia as indicators of stress. occurrence of some bands of proteins and significant Vacuolization of digestive cells of bivalves is most decrease or disappearance of other proteins. Protein commonly observed in cellular responses of aquatic pattern of L. lithophaga collected in autumn showed invertebrates to metal toxicity (Najle et al. 2000; an obvious disappearance of 28 and 80 KDa proteins. AbdAllah & Moustafa, 2002). Usheva et al. (2006) However, three polypeptides having molecular masses recorded erosive disturbances of the epithelium of the 157, 34 and 21 KDa were present in winter and spring digestive tubules of Gray’s mussel, heavy only, but they disappeared from summer and autumn. vacuolization of digestive cells and specified Reduction in the protein or disappearance of protein connective tissue by foci of cells necrosis and lysis. bands observed in the present study may be the The present study recorded mass shedding of some consequence of the stress effect of metals on the epithelial cells in tubular lumen. Kolyuchkinaa and enzymes which have a role in the biosynthesis of Ismailovb (2011) reported that the presence of cell protein. The disappearance of protein band under detritus in the lumen of the tubules may be used as a unsuitable conditions is believed as a general fact in biomarker of environmental pollution by heavy several reports. These findings are compatible with the metals. Moëzzi et al (2013) reported that the digestive results of previous publications (Mohy El-Din et gland of swan mussel Anodonta cygnea can be al. 2016; Mohy El-Din & Abdel-Kareem, 2020), considered as reliable biomarkers in biomonitoring of where unfavorable stress conditions were found to heavy metal pollution in aquatic ecosystems as they affect the protein profiles. The present study suggests recorded several histopathological effects of zinc, that stress protein response may provide a method for histopathological changes observed were: damages of quantifying adverse biological impacts of exposure to epithelium cells, atrophy of digestive tubules and metals in the environment. hemocyte aggregation in the digestive gland. Histological structure of the digestive gland of L. Hemocytic infiltration is one of the pathological lithophaga was investigated seasonally as it symptoms which has been reported in various studies accumulates metals higher than other tissues and thus (Cheng, 1996). Hemocytic infiltration may be a it can be considered as a biomarker of toxicity. symptom of disease or it may be an independent agent Histological responses to metal exposure were of mortality (Ward et al., 2004). suggested as suitable and sensitive biomarkers for Ultrastructural examination of the digestive gland detection of heavy metal exposures (Choi et al., of L. lithophaga was performed for specimens 2003). Regoli (1992) suggested that central metabolic collected in winter and summer seasons. Both, the organs such as the digestive gland of molluscs can be digestive and basophilic cells appeared somewhat with considered as biomarkers of toxicity. Cajaraville et normal histological structures. The digestive cell al. (1990 & 1992a) added that the epithelial cells appeared with moderate number of vacuoles and lining the tubules of the digestive gland are a sensitive lysosomes indicating endocytic activity. Its function is target for the damaging impact of many pollutants of the endocytosis and the intracellular digestion of food the marine environment. material and for this purpose it has a well-developed The histological structure of the digestive gland of lysosomal vacuolar system (Soto et al., 2003;

Abd-Ellah et al., Swed J BioSci Res 2020; 1(1): 62 - 77. DOI: https://doi.org/10.51136/sjbsr.2020.62.77

Dimitriadis et al., 2004). The pyramidal basophilic Increased lysosomal neutral lipid accumulation, cells were clearly identified and pronounced rough attributed possibly to lysosomal autophagy of excess endoplasmic reticulum with obvious electron dense lipid droplets, has been related to environmental stress ribosomes and numerous free ribosomes were in mussels collected from polluted sites (Lowe, 1988; obviously observed surrounding the large centrally Moore, 1988; Krishnakumar et al., 1994). On the located nucleus and scattering throughout the other hand, marked cytoplasmic vacuolization of some cytoplasm. Basophilic cells are thought to be involved digestive and basophilic cells was recorded. in the synthesis and the secretion of enzymes for Vacuolization of epithelial cells of the digestive gland extracellular digestion, thus, contain a well-developed may be used as a biomarker of environmental granular endoplasmic reticulum (Dimitriadis et al., pollution by heavy metals (Kolyuchkinaa and 2004). Furthermore, the presence of lipid droplets in Ismailovb, 2011). The pathological destructive the cytoplasm of basophilic cells suggests the changes of morphology of the digestive gland of L. involvement of these cells in the lipids metabolism. lithophaga could be apparently used as a histological In summer both digestive and basophilic cells biomarker of pollution of the marine environment. showed various signs of alternations. The most obvious alternations were concerned with the nuclei Conclusion and ranged from irregular nuclear envelope, From the present study, it was concluded that the invaginated and ruptured envelope, disorganized concentrations of Cd, Co and Pb in the date mussel L. chromatin content, poor chromatin condensation, lithophaga from the east harbor of Alexandria were vacuolized nuclear lymph. Nucleus alternation is a indicative of a relatively clean or unpolluted coastal typical sublethal symptom of toxic effects of metals environment when compared with the permissible (Hinton et al., 1992). Some digestive cells showed limits or other reported values from other regions of apoptosis. Barmo et al. (2013) considered apoptotic the Mediterranean. Consumers must also be warned cells as reliable marker of contaminant-induced about the potential risks for their health associated genotoxic effects in molluscs. In addition, with the consumption of the date mussels which mitochondrial alterations, distortion of the regular bioaccumulates heavy metals and other xenobiotics, arrangement of the rough endoplasmic reticulum and often in concentrations above recommended cisternal fragmentation were observed. Sokolova et al. thresholds levels. The present study recorded (2005) revealed that the primary target for cadmium concentrations of these metals below the permissible toxicity may be mitochondria. Fragmentation and limits. Consumption of date mussels collected from vacuolation of cisternae of the rough endoplasmic east harbor of Alexandria does not show any health reticulum of the basophilic cells are common on risk. Seasonality in the concentrations of Cd, Co and mussels under stress conditions (Carles et al., 1986; Pb was observed. Finally, the results of the present Lowe, 1988). Moreover, large number of mineralized study indicated that the digestive gland is sufficiently lysosomes with electron dense granules can be easily sensitive to reflect environmental levels of pollution, seen. The lysosomal system in the digestive cells has thus it can be used as a bioindicator of pollution. In been identified as the target site of bioaccumulation of general, our results are important as a background for heavy metals (Taϊeb, 2001). Marigómez and the estimation of the future impact of heavy metals in Baybay-Villacorta (2003) stated that lysosomal L. lithophaga. responses to environmental xenobiotics fall into essentially three categories:increased lysosomal size Conflicts of Interest ,changes in lysosomal contents and reduced membrane None of the authors have any conflict of interest to stability. Sokolova et al. (2005) reported that the declare. exposure of oysters to low sublethal amounts of cadmium resulted in accumulation of high levels of Citation: Abd-Ellah SM, El-Sherif S, El- this metal in lysosomes. Raftopoulou and Morshedy R. Seasonal effects of heavy metals on the Dimitriadis (2011) evaluated the toxic effects of date mussel Lithophaga lithophaga (Mollusca: metals on lysosomal system of the digestive cells of Bivalvia) at Eastern harbor, Alexandria, Egypt. Swed J mussels Mytilus galloprovincialis and recorded BioSci Res 2020; 1(1): 62 - 77. https://doi.org/ lysosomal enlargement in digestive cells of mussels. 10.51136/sjbsr.2020.62.77

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Abd-Ellah et al., Swed J BioSci Res 2020; 1(1): 62 - 77. DOI: https://doi.org/10.51136/sjbsr.2020.62.77

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