Assessment of Toxic and Essential Elements in Muscles of Sparus Aurata With Special Reference to Impacts on Human Health

The goal of this investigation was to determine the contamination of toxic (Pb, Hg, Cd) and essential (Cu, Fe, and Zn) elements in the comestible esh of economically substantial sh gilthead seabream (Sparus aurata) and to assess the dangers of seafood consumption to human health. Fish samples for metal analysis were collected from different sh markets in Sinop and Samsun, respectively. Inductively Coupled Plasma Mass Spectrometry (ICP-MS) was used to examine for toxic and important components in gilthead seabream muscles. The results of this investigation demonstrated that metal concentrations in tissues were under the maximum allowable limit set by the European Commission and the Turkish Food Codex. The measured values were compared to international and national food and human health standards in this study. The estimated daily intake (EDI) amounts for per diem mean consumption and hazard index (HI) in gilthead seabream samples were found to be below human safety levels. To evaluate their standing in zonal surroundings, comparison research was conducted among the accessible current data on elements in S. aurata from different parts of the Turkish seas and those of the current study. Increased levels of heavy metals in sh in various locations could be attributable to an increase in farm inow water, domestic sewage, and a variety of other anthropogenic sources, all of which should be investigated further. To protect this sh from metal contamination and to decrease the risk to human health, necessary precautions should be taken.


Introduction
One of the most serious environmental issues of the century has been the contamination of the marine environment (Alam et al. 2021) with chemical contaminants, in particular toxic elements. Consequently, many marine ecosystems are space or time worrying, because of the transfer of elements from different industries into the environment Bhuyan and Islam 2017a,b;. Non-essential elements are not biologically degradable and persist as toxic over a long time (Bhuyan et al. 2019). Essential elements are decomposable and swiftly break down into nonharmful or less harmful forms Bhuyan et al. 2019). Concerns are growing around the world that such toxins are being utilized carelessly, putting marine creatures at danger of contamination and poisoning (Bat 2017;Ahmed et al. 2018;. Therefore, environmental damage, as well as the presence of contaminants, may impact livelihood and human health (Bhuyan and Islam 2016;Bhuyan et al. 2016a).
Many in situ metal accumulation experiments in sh living in contaminated environments have shown that signi cant levels of different metals can be accumulated in sh tissues without inducing mortality (Bhuyan et al. 2016b). In advanced species, toxic elements accumulate through the bio-amplifying process in the food web. As a result, they can enter the human body and developing chronic toxicity in body cells. The known cause of cancer is chronic assimilation of components. A framework for developing marine plans for achieving Good Ecological Status (GES) in the ocean ecosystem is set by the Marine Strategy Framework Directive (MSFD) using eleven qualitative descriptors. According to MSFD, the concentration and effects of pollutants including trace elements in the ocean ecosystem should be evaluated because of the effects and threats on the ecosystem. European Food Safety Authority (EFSA) pointed out that sh is a nutritious food because of the great biological grade proteins, suitable lipid pro le, important mineral contents, and vitamins it contains. Its lipid fraction, which is high in omega-3 polyunsaturated fatty acids (PUFA), particularly docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), and low in cholesterol, making it a major component of human meals (EFSA NDA Panel 2014). As a result, determining elements amounts in trading sh is critical to appraise the likely harm of sh consumption to human health (Bat 2019). EFSA has stated that the consumption of approximately one-two portions of sh weekly, and up to three-four portions of sh weekly for the time of pregnancy was linked to higher neurodevelopment results among children compared to not consumed (EFSA NDA Panel 2014). These quantities are likewise related to cause to fall possibility of coronary heart trouble in grown-up persons and comparable with existing intakes and guidelines in nearly all the European countries. At greater intakes, there could be no extra advantage to neurodevelopmental outcomes and no reduction to the risk of dying of coronary heart disease (James 2013; EFSA NDA Panel 2014).
Turkey's total sh catch has been reported as 836 524 t in 2019, where 463 168 t of this total production came from shing (431 572 t of marine and 31 596 t of freshwater sh) and 373 356 t from aquaculture (BSGM 2020). However, the sh production in 2020 is 785 811 tons, of which 364 400 t were provided by and muddy environments with seagrass, which are generally distributed in tropical, subtropical, and temperate zones. Although they are found in all Turkish seas, they are rare in the Black Sea. Their tolerance to salinity is very high. It is among the most valuable species in terms of aquaculture and shing. It is one of the two most cultivated sh in the Mediterranean basin. The success in aquaculture has been possible by the fact that the species can be obtained alive in large numbers at the entrance and exit of the sh and brackish waters. This species is a carnivorous form and especially adult individuals feed on species belonging to Crustacea and Mollusca families (Bat et al. 20011).
More information is needed on the levels of contaminants in Turkish sh. Especially commercial sh is a valuable source of protein, which necessitates thorough research and regulation of their contamination levels. This study offers information on muscle tissue elements concentrations of S. aurata regularly consumed on Turkish Black Sea coasts and compares the results to allowed values both at international and national levels and those reported from past studies. These elements are studied well due to the accumulative nature of the sh species and, if consumed enough, are known to produce detrimental health effects. To establish the substantial health hazards associated with the ingestion of contaminated sh, we determined estimated daily index (EDI) amounts for per diem mean consumption and hazard index (HI) in sh samples for infants, children, and adults.

Materials And Methods
A total of 32 random gilthead seabream were obtained from different markets in the provinces of Sinop and Samsun, the crucial shing cities of the Southern Black Sea Region to assess poisonous (Pb, Hg, Cd) and vital elements (Cu, Fe, and Zn) levels in 2019 and 2020. The sh were subsequently transported in an icebox to the Hydrobiology laboratory. Fish was kept at a temperature of around 4°C in the laboratory. Bernhard's (1976) technique was used to prepare the sh's muscle for investigation. Individual sh llets were prepared separately stored at -21°C in a freezer until metal analysis. Aqueous solutions were stored in plastic bottles. They were made acidic (pH: 1-2) to minimize chemisorption and hydrolysis of metal ions.
Following pressure digestion by a certi ed environmental food analysis laboratory, toxic and essential components in gilthead seabream muscles were analyzed using Inductively Coupled Plasma Mass Spectrometry (ICP-MS). Trace elements were determined using acid, wet digestion, and standard reference material according to the European Standard method EN 15763. Samples were weighed and 10 ml in cells of the microwave approximately 0.5 g by two parallels and 10 ml 65% (v/v) nitric acid was added on the samples. All glass vessels were washed, rinsed, and then soaked in 10-20% (v/v) nitric acid for 24 hours. Plastic vessels were soaked in 1-5 % (v/v) nitric acid. Finally, before using all vessels were rinsed in high purity deionized water. All values are in milligrams per kilogram of wet weight.
Health assessment of elements in aquacultured gilthead seabream A hazardous index (HI) may be used to quantify the risk from metals consumption through the intake as the proportion of the projected element dose (EDI mg/kg/day of bw) and the dosage reference (Rf. D. mg/kg/year). The HI was determined using the following formula.
In HI > 1.0, a certain component's EDI is greater than Rf. D., which indicates that this element has a possible risk. The EDI is based on the level of element and the quantity of aqua-cultured S. aurata consumed. The metals EDI was calculated with the following equation: EDI = C e ×W / body wt.
Where: C e is the level of elements in S. aurata; W denotes the per diem average consumption of S. aurata. Annual sh consumption is 5, 10, and 15 kg for infants, children, and adults, respectively (UNSCEAR 2010) and it has been calculated as 13, 27, and 41 g/day for infants, children, and adults, respectively; body wt. is the body weight of 10 kg for infants, 30 kg for children and 70 kg for adults.
For Pb and its compounds, there is no Rf. D. value. Oral slope factor (SF), on the other hand, is only indicated for Pb and its' contents as 0.0085 mg/kg-day (RAIS 2021). The following formula was used to compute the risk index (RI):

RI= EDI x SF
The RI is taken into account as insigni cant if the RI<10 -6 , permissible or acceptable if RI is 10 -6 <RI<10 -4 , and likewise regarded substantial if the RI > 10 -4 .
As seen in the following equation, the Total Hazard Index (THI) is the sum of the HIs: THI= HI (Pb) + HI (Hg) + HI (Cd) + HI (Cu) + HI (Fe) + HI (Zn)

Results And Discussion
The concentrations (Mean ± S.D) of toxic and vital metals in the muscles of Sparus aurata are given in Figure 1. All metal levels were found in muscle samples, according to the ndings of this investigation.
Among studied elements, Fe had the highest mean concentration in gilthead seabream muscles collected from sh markets in Sinop and Samsun, followed by Zn and Cu. These essential elements are critical trace nutrients required by sh at little levels. These metals' impacts on marine animals can be lethal, either directly or indirectly. Essential elements were found to be higher than non-essential toxic elements levels. To assess the risk of element levels for consumers, the sh edible tissues were analyzed. Although there is no maximum allowable level for Fe, the acceptable levels for Cu and Zn are 20 and 50 mg/kg wet wt. (O cial Gazette 1995) These values are well below the values we obtained from our current study (Table 1). As a result of the comparison between the mean element concentrations determined in muscle and international recommended limits, none of the sh samples had exceeded the limits for the examined elements.
Muscles are not an active site for element bioaccumulation, implying that the element concentrations in S. aurata may be regarded benign and acceptable for people. The study reveals that S. aurata produced through sh farming practices, being contaminated with elements are safe to consume.
There was no statistical difference between the metal levels detected in Sparus aurata purchased from the markets in the cities of Sinop and Samsun (p>0.05). Though the metal amounts in the sh taken in 2019 were found to be higher than those taken in 2020, and there is a statistical difference (p<0.05). This may Page 6/15 be because, after March 2020, many industrial activities were reduced, human activities were greatly restricted, and much fewer contaminants entered the surrounding water where the culture cages are located due to the Coronavirus Disease (COVID-19).
Elements levels in gilthead seabream tissue vary signi cantly between investigations, according to the research (Table 1). Fe and Zn were found at the highest values, followed by the essential element Cu. As expected in general, toxic Cd, Hg, and Pb were found to be lower. When the previous studies are examined, it is seen that these toxic elements are very high in the Gulf of Iskendurun. Higher levels of heavy metals in sh in different places could be due to an increase in farm in ow water, residential sewage, and several anthropogenic sources, all of which should be looked into more. The ndings of this research reveal that the build-up of components harms gilthead seabream sold in Sinop, and Samsun is quite low. The results of the present study compared with other study carried out by various scientists (Table 1). The daily consumption of Pb, Hg, Cd, Cu, Fe and Zn in gilthead seabream from Sinop and Samsun sh markets in 2019 and 2020 ranged from 2.16E-05 to 6.24E-05, 9.95E-06 to 3.64E-05, 5.27E-06 to 2.08E-05, 2.57E-04 to 6.63E-04, 5.35E-03 to 1.50E-02 and 3.65E-03 to 1.11E-02 µg/day/person, respectively. The average daily intake of elements through gilthead seabream consumption can be ordered as follows: Fe>Zn>Cu>Pb>Hg>Cd. These values are considerably lower than the recommended values of Commission Regulation (2006) and Turkish Food Codex (O cial Gazette 2011).
Hazard Index (HI) The HI, which is a ratio of the estimated exposure to the oral reference dose, is used to estimates the potential non-carcinogenic risk of the consumers of the discerned contaminated seafood like sh. The estimated HIs of the elements reveals that the elements in the muscles of Sparus aurata do not appear to represent any hazard to the people, as seen in Table 3, where the THIs of all the investigated elements are less than one. Except for Pb, which was not given a reference dose, the THI of all other elements was well below 1 (Table   3). Therefore, it can be said that the consumption of gilthead seabream from Sinop and Samsun sh markets does not pose any health risk when it comes to the toxic and essential elements examined.

Risk Index (RI)
Similarly, since the RI for Pb was less than 10 -6 in both 2019 and 2020 from the cities of Sinop and Samsun, it was calculated as permissible or acceptable for infants, children, and adults (Table 4), and the risk index was considered insigni cant. The ndings indicated that all metals are within permitted limits, and customers are free to utilize them. Thus, gilthead seabream is regarded to have a high level of food safety. This assumes that the person obtains all their sh stores from the Sinop and Samsun sh markets and that food processing has no effects on the levels of toxic and essential elements present in the muscles of gilthead seabream.  1995,2011). Being aware of the element levels in farmed sh is critical for the region's environmental health, aquatic life's well-being, and human sh consumption. The ndings of the current S. aurata concentration testing showed that both toxic and essential elements levels were below the required requirements. It should be noted that the intake of these gilthead seabreams from the researched area as foods during the time of the study may not pose health risks for people. Gilthead seabream is a popular seafood in many countries across the world, and it's a crucial technique for determining the impacts of multiple pollutants on marine ecosystems. In addition, the results from this study would contribute to the consolidation of the limited baseline data for evaluating the distribution of elements in S. aurata. Using these data, it can now be con rmed that S. aurata, which is very abundant in Turkish waters, is considered safe for consumption. In Turkey, all seafood, including sh, is inspected for food safety before being consumed by humans. The ndings of this study can be used as a baseline for future safety monitoring of S. aurata and other species cultivated in the area.

Declarations
Funding: No funding was received for the present study. The authors would like to thank all colleagues for help and support this research and wish to acknowledge the Department of Hydrobiology, Fisheries Faculty, University of Sinop for providing laboratory facilities during the study.
Con icts of interest/Competing interests: The authors declare that they have no known competing nancial interests or personal relationships that could have appeared to in uence the work reported in this article.
Availability of data and material: All data generated or analysed during this study were included in this published article.
Code availability: Not applicable.
Authors' contributions: LB conceptualized the problem, performed the sampling wrote original the manuscript. EA made tables, literature collection. SB reviewed and edited the manuscript. AÖ helped Figure 1 Toxic and essential elements in muscles of Sparus aurata (mg/kg wet wt.) from different sh markets in Sinop and Samsun in 2019 and 2020. Vertically, letters a and b show a statistically signi cant relationship among elements at p<0.05