Seasonal and Sexual Variations of Trace-Metal Elements (Cd, Pb, Cu, Zn, Fe) in Muscle and Eggs of the Deep-Water Pandalid Shrimp Plesionika edwardsii (Brandt 1851) from Northeast Algeria

The level of trace metal elements (TME) (Cd, Pd, Cu, Zn, Fe) was determined according to the seasons and the sexes in the muscle and the eggs of the deep-water pandalid shrimp Plesionika edwardsii (Brandt in Reise in den aussersten Norden und Osten Sibiriens wahrend der Jahre 1843 und 1844 mit allerhochster Genehmigung auf Veranstaltung der Kaiserlichen Akademie der Wissenschaften zu St. Petersburgausgefuhrt und in Verbindung mit vielen Gelehrten herausgegeben 2(1):77–148, 1851) of the east coast of Algeria. Using the inductively coupled atomic plasma emission spectrometry method (ICP AES), the distribution of trace metals shows a significant fluctuation according to the seasons and the sex. Throughout the annual sampling, the order of heavy metal concentrations in female and male muscle, as well as in eggs were Cd < Cu < Fe < Zn < Pb, Cd < Cu < Zn < Pb < Fe, Cd < Cu < Fe < Zn < Pb respectively. In this work the heavy metal concentrations in target tissues are below the permissible limits set by the international standard except for Pb, mainly in summer, autumn and spring, exhibiting alarming levels.


Introduction
The Mediterranean which is a semi enclosed sea, shallow and connected to the Atlantic via the Strait of Gibraltar receives a large volume of waste and pollutant: more than 500 million tons of wastewater are discharged each year, including 120,000 tons of mineral oil, 6000 tons of Pb and 3600 tons of phosphates (Laaz and Makhloufi 2011).
The pollution of coastal marine ecosystems caused by chemical pollutants, especially metallic traces elements (MTE), represents a major environmental problem with the potential risks, not only on habitats and marine resources, but also on human health (Ali and Khan 2019). The MTE are naturally found at very low concentrations in the hydrosystems, but their concentrations have increased due to anthropogenic activities. The presence of the trace metals in marine environment is the result of two main sources of contamination: natural occurring deposit and anthropogenic activities such as domestic, agricultural and industrial activities. The ingestion or exposure of the organisms to excessive amounts of these contaminants can be highly toxic and even provokes dysfunctions within the food chains by bioaccumulation and biomagnifications (Ali and Khan 2019).
The trace metal element pollution in the marine environment can be identified by measuring its concentration in water, sediment and living organisms, but due to the high solubility of heavy metals, the analysis of seawater compartment is not considered to be a reliable method for assessing the level of contamination of these chemical pollutants (Philips 1977). Studying the bioaccumulation of these elements in marine organisms exposed to them is therefore a more effective way to assess this metal pollution. Indeed, the determination of contamination levels in organism tissues reflect the concentrations of metals present in water and sediments.
According to many factors, such as: type and amount of metal introduced into the seawater, environmental context (acidity of the receiving environment, synergy phenomena between trace metals or between trace metals and other pollutants, hydrodynamic conditions and bathymetric, water temperature, etc.), processes (passive or active) of assimilation and bioassimilation of these elements and, possibly, bioconcentration/biomagnification phenomena in the food chain, regulation, tolerance and detoxification mechanisms of the organisms, pathway and duration of exposure of biota to the metal, feeding habits and ontogenetic stage of the species exposed to the contaminant (eggs, larvae or adults), these elements likely cause biological, biochemical, hematological and physiological dysfunctions in vertebrates and marine invertebrates (Chiarelli and Roccheri 2014;Authmann et al. 2015) and indirectly on human health (Ali and Khan 2019). For this reason, FAO/WHO (1984) recommends periodic monitoring of hazardous trace metals to protect ecosystems and public health.
In Algeria, the agricultural, tourist and industrial activities of the largest agglomerations are mainly concentrated in the north and in particular along the coast. Consequently, this high anthropogenic pressure can cause a potential risk on the marine environment and the fishery resource represented mainly by the small pelagic (77%) and the demersal resource, represented by fish and crustaceans (23%). The exploitation of crustaceans particularly targets the Penaeid shrimp (Aristeus antennatus, Parapenaeus longirostris and Penaeus kerathurus) and Pandalidae, in particular the deep-water pandalid shrimp Plesionika edwardsii (Brandt 1851) (locally known as ''Goubids''), which constitutes with P. longirostris, almost all the catches, especially in eastern Algeria (Derbal and Kara 2005). Plesionika edwardsii as omnivorous nectobenthic shrimp species performs vertical and seasonal migrations. This species occurs on the deepest shelf thus, reflecting the concentrations of metals from the surrounding environment. The best fishing yields for this species with the bottom trawl are obtained between 250 and 380 m (Holthuis 1980). Despite its high market value on the local market (1500-2000 DA/Kg) and its abundance in the waters of eastern Algeria, information on this species is limited to morphological description and reproduction (Oudainia and Derbal 2017).
Various atmospheric, domestic, agricultural and industrial pollutants are discharged annually directly or indirectly into the Gulf of Annaba by the outlets of the Seybouse, Mafragh and Meboudja rivers (35 tons of ore dust, 4100 tons of sulfur oxides, 6000 tons of suspended matter, 3000 tons of ammonia, 5160 tons fertilizer dust, 1281 tons of ammonium nitrate dust and 2000 tons of phosphogypsum) by the outlets of the Seybouse, Mafragh and Meboudja rivers (Mebirouk et al. 2018). Scare information is available on fishery product sanitary qualities, being widely consumed by the local population. At the toxicological level, for example, the assessment of metal concentrations in the biota along this coast was carried out in particular in mollusks (Belabed et al. 2013;Boucetta et al. 2016;Merad and Soltani 2017), teleost fish (Ouali et al. 2018a, b;Zeghdoudi et al. 2018), sponges (Bensafia and Khati 2018) and marine magnophyte Posidonia oceanica (Boutabia-Trea et al. 2017;Zeghdoudi et al. 2019). In crustaceans, the only studies on metallic contamination concern a few shrimps that colonize continental hydrosystems and coastal marine waters (Abdennour 1997;Abdennour et al. 2000;Terbeche 2006;Ghorab and Khebbeb 2012). And yet, shrimp, especially those from deep waters, are considered as biological indicators for the assessment of marine environments in terms of contaminant effects (Baboli et al. 2013;Velayatzadeh et al. 2014).
The objective of this study is to determine for the first time the accumulation of five traces Metals (Cd, Pd, Cu, Zn, Fe) in two different tissues (muscle and eggs) of deep sea shrimp P. edwardsii captured in the Gulf of Annaba, at the extreme east of the Algerian coast.

Study Area
Plesionika edwardsii samples were collected from Annaba Gulf, at extreme east of the Algerian coast, between Cap Rosa at east (8°15'E-36°68 N) and Cap de Garde at west (7°16'E -36°68 N), either an exploitable area of about 2337 km 2 . The Gulf of Annaba is considered a real receptacle of continental nutriments (Seybouse and Mafragh estuaries) (Ziouch et al. 2020), of domestic discharges (wastewater from the Annaba locality), harbor (commercial and fishing port) and industrial (Steel and metallurgical complex Arcelor-Mittal ex-ISPAT; Phosphate fertilizer complex Fertial ex-ASMIDAL). These various sources of pollution are likely to contaminate the bordering coastal ecosystems and the fishery resource with possible negative repercussions on the health of the consumer. The samples, fished in spring (April 2017), in summer (August 2017), in autumn (October 2017) and in winter (January 2018), come from trawl fishing carried out between 200-250 m depth.

Sampling and Methodology
Freshly caught samples were kept immediately in cooler box (4 °C), the brought directly to the laboratory and frozen at -25 °C until dissection. Samples were separated according to sex. Sub sampling of 60 specimens for each sex were chosen seasonally and randomly. Biometric measurements were recorded (total shrimp length and weight). Cephalothoracic lengths (CL1: distance separating the posterior part of the left eye sochet to the end of rostrum, CL2: distance from the posterior part of the left eye sochet to the end of the posterior border center of carapace) were measured for each specimen in both sexes to the nearest millimeter). The individual weight was accurately recorded to the nearest 0.1 g.
A stainless steel knife was used to dip out shells, guts, and heads to avoid any contamination with traces of any metals. Ultrapure water was used to wash and rinse samples to remove any foreign particles. Muscle tissues of males and females as well as eggs were dried at 120° C until they reached constant weight. Samples were grounded into fine powder and homogenized until processing for metal analysis. Second step consists of the mineralization, which carried out by calcinations (dry combustion). It aims at destroying organic matters to obtain mineral solutions, containing the total trace elements. For each sample 1 g of selected tissue was placed in digestion vessel with 1 ml of concentrated (65%) nitric acid (HNO 3 ) and 25 ml (30%) hydrogen peroxide (H 2 O 2 ). Glass microfiber filters GF/C (Whathman, diameter 47 mm) is used to filter solution 3 times before being conserved in labeled tubes. The obtained stock solution for each metal (1000 ppm) was digested in microwave oven.
Data (biometric measurements and metallic trace element dosages) are expressed as mean ± standard deviation (SD). Data analyses were carried out using the Excel Statistical Analysis 2013 and SPSS package 15.0 Windows software for finding out statistical differences among various parameters. The comparison between seasons concerning the distribution of the contents of each metal is carried out using the analysis of variance (ANOVA).

Results
Metal concentrations were measured in milligram per kilogram-wet basis (mg.kg −1 w.w). The mean total length and body weight of P. edwardsii shrimp samples were calculated seasonally for both sexes (Table 1). Level concentrations as well as the associated standard deviation of cadmium, lead, copper, zinc and iron in muscles and eggs of male and female P. edwardsii shrimp seasonally sampled in the Annaba Gulf are given in Table 2. The distribution of five trace metal elements in the muscle of males and females as well as in eggs according to the seasons shows that there is a very highly significant difference (p < 0.001).
In the present study, there were no significant differences in Cd concentrations in the studied tissues for both sexes, throughout four seasons. Cd levels in muscle tissues of male shrimp varied from 0.10 (in July) to 0.30 mg.kg −1 w.w.t (in October), whereas those of females were in the rage of 0.13 (± 0.04) (in April) to 0.58 mg.kg −1 w.w.t (in October). Cd concentrations in eggs are between 0.05 (in July) and 0.18 mg. kg −1 (in October). The mean concentration of Cd were 0.308, 0.179, 0.110 mg.kg −1 in muscle tissues of females, males and eggs respectively (Table 2).
Lead concentrations did not vary significantly in male and female muscle tissues. The highest values for both sexes were recorded in July and October, while the lowest levels were found in April and January. Eggs exhibit higher concentration in lead throughout seasons (max. in July: 53.49 ± 0.01 mg.kg −1 ) except in January (0.56 mg.kg −1 ). The estimated mean lead concentrations were 12.443, 17.095, 28.112 mg.kg −1 in muscle tissues of females, males and eggs respectively.
Copper was present at low concentration in the studied tissues with no significant differences between males and females in four seasons. Cu levels in muscle tissue of male shrimp range from 0.42 in April to around 0.6 mg.kg −1 w.w.t in July, October and January, whereas those of females were in the rage of 0.66 in July to 1.84 mg.kg −1 w.w.t in January. The highest concentration of Cu in eggs is recorded in October (26.25 mg. kg −1 ) and the lowest in July (0.42 mg.kg −1 ). The mean Cu concentrations in muscle tissue of males, females and eggs are estimated to be 0.597, 1.137, 7.091 g.kg −1 , respectively.
There were no significant differences in Zn concentrations of sampled tissues in both sexes. Zn levels changed significantly through seasons in the studied tissues (p > 0.05). The highest levels were observed in eggs for all seasons. Zn concentration in female muscles ranged from 3.77 in April to 10.98 mg.kg −1 in October, while the same element in males varied from 3.65 in January to 8.38 mg.kg −1 in October. The highest concentrations of Zn in eggs are recorded in October

Discussion
As non-essential metal, Cd is highly dangerous because is readily accumulated in aquatic animal cells and is not prone to bacterial detoxification (Jarup et al. 1998). The main source of Cd in many marine invertebrates is food and the ease with which these animals assimilate this contaminant from their prey (Chiarelli and Roccheri 2014). It seems that the crustaceans, in particularly, accumulate Cd from water without significant excretion, storing this element in detoxified form (Rainbow and White 1989). In addition, some environmental factors such as salinity, temperature and calcium concentration may also influence Cd toxicity (De Lisle and Roberts 1988).
In particular, crustaceans seem to be more sensitive to cadmium than fish and mollusks (Sadiq 1992). Previous studies regarding the effects of Cd on the ovarian development of crabs and shrimps showed that this metal inhibits ovarian growth, reduces the rate of fertilized eggs hatching, as well as embryonic deformity (Kang et al. 2012). Cadmium concentrations are relatively higher in muscle than in P. edwardsii eggs, thus confirming the importance of contamination through the benthic prey ingestion that colonize the homogeneous sediments (vase). Indeed, the Cd concentration in the sediment of the west coast of Gulf Annaba is estimated around at 1.5 mg.Kg -1 dry weight (Ouali et al. 2018a, b).
Since this species is fished in deep waters, between 250 and 380 m along the east coast of Algeria (Fischer et al. 1987), the concentrations of Cd obtained in the muscle assume that the dispersion of Cd in the Gulf of Annaba extends beyond from coastal area probably through the geochemical process. On the other hand, the low concentrations of Cd accumulated in the eggs which are fixed under the abdomen of the females of P. edwardsii could be explained by the molting phenomenon which allows the females to get rid of their shell after each exuviation. In Annaba Gulf, ovigerous females are observed all year except in December (Oudainia, personal data not communicated), as commonly seen the Mediterranean coasts (Colloca 2002;Possenti et al. 2007). In autumn, Abdennour et al. (2000) found in Penaeid shrimp Parapenaeus longirostris and Aristeus antennatus caught in the same study area (Annaba Gulf), concentrations of 0.61 and 0.78 µg.g −1 respectively. In Bejaia and Jijel Gulfs (East Algeria), Ghorab and Khebbeb (2012) found significantly higher cadmium concentrations in muscle tissue of males compared to females in two Peneidae (A. antennatus and P. longirostris) and a Caridae species (Palaemon serratus). In Plesionika martia and P. edwardsii sampled on the Turkish coasts between 450-500 m, Olgunoğlu (2015) found values below detectable limit. Rao et al. (2016) determine in three Caridae shrimp (Acanthephyra armata, Heterocarpus gibbosus and Plesionika spinipes) caught in the waters off the west and east coasts of India (depth: 200-1200 m), concentrations below the detectable limit. These negligible values of Cd in the muscle of these species would be explained by the absence/or low dispersion of this contaminant by seawater currents in deep waters in these study areas, hence the low accumulation of this metal in muscle tissue.
However, these low levels do not exclude the risk of bioaccumulation in these crustaceans, which are known to be bioaccumulative species. These values did not exceed the permissible limits of 0.2 mg.kg −1 WHO (1989) (Table 3).
Moreover, Cd levels in the studied samples were lower the risk international standard (0.05-2 with an average value of 0.3 mg.kg −1 wet weight) presented by FAO for Reference Dose, and do not pose any health risk to consumer. World Health Organization (WHO 2000) recommends a maximum tolerable weekly intake of cadmium (7 μg.kg −1 body weight per week).
Although Pb is a naturally occurring substance, this element is a very reactive in the environment whose the atmosphere represents its main vector towards sea and oceans. In decapod crustaceans (crabs and shrimps), the accumulation of Pd varied according to the geographical site. These invertebrate organisms can be considered as biomarkers of pollution by Pb (Mansoori et al. 2013). Using the experimental radiotracer method, Boisson et al. (2003) noted that the muscles of Palaemonetes varians shrimp could accumulate approximately 23 to 27% of Pb after ingestion of contaminated food. Therefore, the food pathway is suggested to be a significant contributor to the lead transfer to humans through ingestion of contaminated shrimp.
The high concentrations of lead in P. edwardsii indicate that the decapods are net accumulators of this element (Rainbow 1989). The high concentration of Pb in the muscle and eggs of P. edwardsii females is certainly of anthropogenic origin. Indeed, the Gulf of Annaba receives many sources of pollution: domestic (discharge of wastewater), agricultural and industrial through the water transported by Meboudja and Seybouse Rivers. With a watershed of 6471 Km 2 and an annual volume of 950.10 6 m 3 of fresh water discharged into the Gulf (Ziouch et al. 2020), the Seybouse River is one of the largest watersheds in Algeria which discharges its polluted waters in the Gulf of Annaba, without prior treatment (Belabed et al. 2013).
On the other hand, the waters of Annaba port, highly enriched in metallic contaminants, communicate directly with the waters of the Gulf. This metallic pollution is at the partly origin from terminals of the coal and iron ore and steel products from the Arcelor-Mittal industrial complex, e.g. ISPAT. In addition, the highest lead and copper contents are recorded in the two Annaba commercial and fishing ports (Ouali et al. 2008). This situation would be due to the use of anti-fouling paint in the covering of boat hulls as well as from road traffic atmospheric emissions. Generally, the lead would affect not only the water and sediment compartments (Belabed 2010), but also the biota compartment of the Gulf soft bottoms, in particular macrozoobenthic species. In reality, metallic contamination by Pb is not limited to the area bordering on the pollutant source, but can extend to all of the Gulf waters through biogeochemical, physico-chemical and biological phenomena.
According to Belabed (2010), Pb is present in the sediment of the peri-urban area of the coast at high concentrations, up to 186 mg.kg −1 dry weight near the port (Sidi Salem Beach) explaining the high concentrations of this element in the muscle and eggs of P. edwardsii. In autumn, Abdennour et al. (2000) found in Peneidae shrimp P. longirostris and A. antennatus caught in the same study area (Annaba Gulf), concentrations of 2.3 and 1.1 mg.kg −1 , respectively. On the other hand, in the Bejaia and de Jijel Gulfs, Ghorab and Khebbeb (2012) specify that P. serratus accumulates more lead than A. antennatus and P. longirostris, respectively. In the muscle of the same species A. antennatus from the Oran Gulf (West Algeria), Terbeche (2006) found a low concentration in spring (0.414 ppm) and autumn (0.290 ppm), appearing in the following decreasing order to Pb > Cd. The World Health Organization prescribes that maximum tolerable limit weekly intake for lead as 25 µg.kg −1 bw (WHO 2000). These values highly exceeded the permissible limits set by WHO (1984) (< 2 mg.kg −1 ). Moreover lead levels in the studied tissues were above the risk international standards (0.5-10 mg.kg −1 w.w.t).
Regarding copper, it is an abundant element which occurs as a natural mineral with a wide spread use. Toxicity towards marine organisms depends on the chemical form of this element and its oxidation state. In the decapod crustaceans, it represents as essential trace element, because their hemolymph contains a cooper-based respiratory pigment, called hemocyanin, serving as oxygen carrier. Everaarts and Nieuwenhuize (1995) reported that crustaceans could resist to Cu level of 140 mg.kg −1 comparatively to annelids and mollusks. Generally, decapods have the biological capacity to regulate this metal and keep it at a relatively constant threshold. In the Gulf of Annaba, even if Cu concentrations in the sediment reach values between 60 and 90 mg.kg −1 dry weight near the port (Belabed 2010), the estimated mean level of Cu in muscle tissues of females and males as well as in P. edwardsii eggs remains low, 1.137, 0.597, 0.718 mg. kg −1 , respectively.
Despite the presence of industrial activities along the Annaba coast (waste from the Fertial and Arcelor-Mittal industrial complexes) and the importance of domestic discharges and nutrient-enriched waters discharging into the Gulf through the Meboudja, Seybouse and Mafragh Rivers (Belabed 2010), Cu was present at low concentration in the studied tissues with no significant differences between males and females in four seasons. However, these low levels, especially in muscle tissue, do not exclude the risk of bioaccumulation in these crustaceans which are known to be bioaccumulative species.
In our work, the obtained values are considered lower than the International Standard set by WHO (1996). In addition these results did not exceed the permissible limits suggested by the Food and Agricultural Organization (FAO 1983), being 30 mg.Kg −1 .
After iron, and copper, zinc is the third transition metal and the most developed by human. This trace metal is essential to life of a large number of organisms since it is involved in many physiological processes as enzymatic reactions and metabolism of proteins, carbohydrates and lipids. At low concentration, it causes various tissue lesions, delays growth and disrupts reproduction particularly in aquatic invertebrates and vertebrates (Ramade 2000). Zinc concentrations in P. edwardsii shrimp are within the ranges reported in other decapod species (Abdenour et al. 2000).The anthropogenic contributions of zinc in Annaba gulf is thought to be the cause of industrial sources (ores, refinings, galvanization of iron), agricultural (animal feed, phytosanitary products use), urban (road traffic, incineration) and port activities (anti-fouling paints).
Zinc concentrations measured in the studied tissues did not exceed the risk international standards set by United Nations Food and Agriculture Organization for reference doses, which reported that Zn level should be in the range of 40-100 ppm with average value of about 50 ppm wet weight. More over Zn concentration in this study is below permissible level for marine seafood (100 ppm) (WHO 1989).
Iron is an essential bio-metal for many living organisms because it is involved in many physiological and metabolic processes in humans and animals such as oxygen transport, the synthesis of enzymes being involved in cellular respiration, as well as the synthesis of DNA and cofactors (Caiguo 2014). Iron is present in low concentration in marine water compared to fresh water; this is linked to its chemical characteristics. In fact, in seawater, iron takes the form of Fe +3 whose the solubility is very low (Morgan 1967). Toxicity of this metal to fish and crustaceans depends on pH and the deposition of ferric hydroxide on gills (Peuranen et al. 1994).The female shrimp P. edwardsii showed a high level of metallic trace elements compared to the male, practically throughout the year. In the Mediterranean Sea, ovigerous females of P. edwardsii were observed throughout the year, exhibinting a peak of spawning activity between April and July (Company and Sarda 1997;Colloca 2002). This difference in metallic accumulation is relative to the reproduction physiology of this species. During the reproduction period, gametogenesis is characterized by the increased accumulation of metallic trace elements (Zn, Cu, Fr) necessary for vitellogenesis. This period corresponds to a phase of gametogenesis being characterized by an increased accumulation of reserves and a synthesis and storage of carbohydrate, lipid and protein material.
Urban, agricultural and industrial discharges are the source of iron at sea through the Seybouse and Meboudja Rivers. High concentrations of Fe have been displayed in Meboudja River downstream from the Arcelor-Mittal steel complex discharges (liquids, dust emissions), located 10 km south of Annaba gulf. Additionally, the iron discharges also come from the open marketing of iron ore and its derivatives in the form of particles in the commercial port of Annaba. The dissemination of this metal in the marine environment is favored both by the direction of the prevailing winds (from west to east) which transport ferrous products in the form of dust, produced by the steel factory towards the sea and, hydrodynamically of the Gulf which receives the North East currents (Belabed 2010). Discharges into the sea from the Arcelor-Mittal industrial complex present many iron-rich chemical pollutants, mainly due to alloys used in industry (steelworks), being estimated at 1390 mg/L (Boughrira 2015).
The FAO/WHO (1999) regulation limits for heavy metals consumption are based on body weight. For an average adult (60-kg body weight), the FAO/WHO (1999) suggests 48 mg as the provisional tolerable daily intake for iron. Although the requirement levels of this metal from diet absorption necessary for human physiological activities are not distinctly determined.

Conclusion
The deep-water pandalid shrimp P. edwardsii could be suitable as bioindicator species to assess the quality of moderately deep waters of the Gulf of Annaba. The results showed that the level concentration of Pb, Znand Fe either in muscles or eggs were significantly affected seasonally in Annaba Gulf, except Cd and Cu being significantly very low in both sexes. Heavy metal content in Cd, Cu, Fe and Zn in four seasons indicated safe levels for human health according to the international standard limits comparatively to Pb which exhibited very high level concentrations in muscles and eggs especially during summer, autumn, and spring, consequently health risk analysis of this metal showed high carcinogenic risks to humans during these pollutant seasons. Contamination this benthic shrimp the trawl able bottoms found its explanation mainly in the intense activities of agricultural, industrial sectors surrounding the area.
Determining the contamination levels of metallic trace elements in living organisms in the marine environment of Annaba requires the establishment of a permanent and cyclic biomonitoring program which assesses health risks and knowledge of the ecological characteristics specific to the environment. In the lack of a purification and wastewater efficient treatment system, the Algerian east coasts remain submissive of marine pollution from industrial, agricultural and urban origins.