Bioaccumulation of metals in eels taken from Lakes Köyceğiz (Turkey) and Võrtsjärv (Estonia) and 1 health risk assessments 2

Monitoring heavy metal contaminants in fish is important for the assessment of environmental quality 12 as well as food safety. In this study, European eel samples were collected from Lake Köyceğiz and Lake 13 Võrtsjärv in 2017 and 2018. The concentrations of Mn, Cd, Zn, Pb, and Cu metals were measured by using GF- 14 AAS in four selected tissues of eel, including liver, gill, skin, and muscle in both lakes. The pollution indexes 15 (P i , MPI) values were calculated for both lakes and the health risk for consumers was assessed for both adults 16 and children in Turkey and Estonia. The estimated weekly intake (EWI), hazard index (HI), and lifetime cancer 17 risk values (CRs) for the metals were calculated for both lakes. According to the results of this study, a 18 significant difference was determined the between the metal concentrations (especially Cu, Cd and Pb) in the 19 tissues of the eel samples taken from the two lakes. These results show that besides the pollution levels in the 20 aquatic environment, physiological needs, and metabolic activities in different habitats have a significant effect 21 on metal accumulation in eels. In addition, HI was found to be for both adult and child consumers in both 22 lakes, which indicates that consumers would not experience non-carcinogenic health effects. However, the 23 values of CR for Pb and Cd were found negligible in Lake Köyceğiz, while the CR value for Pb was found to be 24 very close to the danger limits in Lake Võrtsjärv.


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Fish is one of the main food sources containing a large number of essential proteins and nutrients in high 28 concentrations that can be easily absorbed and digested by humans. Although, protein and essential nutrients are 29 extremely necessary for the human body, fish is considered one of the inexpensive sources of protein and 30 essential nutrients for consumption (FAO 2008). However, fish, which are at the top of the food chain in aquatic 31 ecosystems, are highly affected by heavy metal (HM) pollution from many different sources and as a result 32 heavy metals can accumulate in their bodies in the aquatic environments. Therefore, fish can be considered as HMs are known as the most important environmental pollution indicators. They tend to bioaccumulate and remain in food for a long time (Ahmed et al. 2019).
sector in Turkey and LV is one of the most important inland commercial/reacreational fisheries zone and the second-largest lake in Estonia. LK is located in the Köyceğiz-Dalyan Lagoon System, which is declared a Special Environmental Protection Area due to its ecological importance and natural beauties (Özdemir 1998).

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The eels in LK are naturally found and can migrate by maintaining their completely natural life. While being 69 restocked, eels perform migrations from LV as well. Besides the annual seaward silver eel migration through the 70 Narva River Basin, eels also use rivers to move between lakes of the aforementioned river basin district. The aims of this study, which is carried out considering these different life cycles of the same species, are; Fig. 1 The map of the study area All of the canals and marshy areas connecting LK to the sea are called the Köyceğiz-Dalyan Lagoon System and the lake forms the main part of this system. It is a meromictic lake with two different layers of water 104 as oxygen-poor, hydrogen sulfide-scented, salty water in the bottom and oxygen-rich, freshwater at the surface 105 (Kazancı et al. 2003). The physico-chemical structure of the lake is constantly changing and these changes occur chambers (mesh size 10 mm in cod end) connected by 100 m leader nets (height 1.8 m, mesh size 10 mm) 123 forming a rectangle in the water body. 24 small fyke nets (2 funnels connected with 8 m leaders with mesh size 124 11 mm in the cod end) are set in chains inside the rectangle. In LK, eel samples were taken by the fishermen of 125 DALKO with fyke nets consisting of 7 circles whose mouth width narrowed from 40 cm to 15 cm. Fyke nets 126 were thrown into the water after sunset and eels were caught by pulling before sunrise.
homogenate tissue was transferred into a teflon vessel and then digested using closed-vessel microwave digestion (CEM, Mars 6) with 9 mL nitric acid (65%) and 1 mL hydrogen peroxide (30%) mixture. The resulting solutions were diluted to 50 mL with deionized water. Digestion blanks were prepared similarly. The microwave was programmed to ramp to 210 o C for 20 min, hold for 15 min, and then cool for 20 min. Cd, Mn, Zn, Pb, and for the metals were run in triplicate for the samples and standard solutions and the results were reported as the mean. Also, the accuracy of the method was checked with standard reference material (DOLT-5 fish liver).

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To analyze the normality of the data, the Shapiro-Wilk test was applied. To detect the difference in metal 146 concentration among the countries and tissues of the European eel, non-parametric test Kruskal Wallis and Post 147 hoc test that using the criterium Fisher's least significant difference with Bonferroni Correction method were 148 performed by using Kruskal function with the R package 'agricolae' (Mendiburu 2020). Statistical differences 149 were illustrated on the box plot graphs. In addition, spearman correlation analysis was applied to assess the 150 statistical assosiations between metal in different organs, fish length and weight. R project 4.0.2 was used for statistical procedures (R Core Team 2020).

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The monomial metal pollution index (P i ) was used in this study to evaluate the pollution status of metals 154 in the eels. P i was calculated by using the following Eq. 1 (Zhu et al. 2015;Miao et al. 2020): Where: P i : monomial metal pollution index; C i : content of metal in the fish; C si : threshold value of metal 157 in the fish. In this study, the tolerable limit values of the Pb and Cd as prescribed by some international 158 regulatory bodies were used as the threshold values of Pb and Cd (Table 1).
where C n is the concentration of metal n in fish.

Estimation of daily intake (EDI)
To

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Humans are often exposed to multiple pollutants that have combined or interactive effects. When exposed 210 to different elements, HI is calculated according to the Eq. 5 given below to evaluate the additive effects from 211 the elements.

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HI values being > 1 refers that consumers may be exposed to possible non-carcinogenic health risk effects.

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The lifetime carcinogenic risk (CR)

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The lifetime CR identifies an increased probability of cancer in a lifelong period due to exposure to a 216 major carcinogen. The acceptable limit value for CR exposure is 10 −6 to 10 −4 . CR values> 10 −4 affect (increase) 217 the possibility of carcinogenic risk. CR was calculated according to the Eq. 6 given below.

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In equation 6, CSF represents the oral carcinogenic slope factor from the Integrated Risk Information

Results and discussion
Presence and comparison of the metals found in European eel in two lakes

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Many fish species are negatively affected by increasing anthropogenic pressures, climate changes due to 224 global warming, water pollution, overfhishing and recreational fisheries and habitat degradation (Bernotas et al. waters and is connected to the Mediterranean by a natural channel system. Therefore, eel can freely move in and 232 out, thus restocking the eel is not considered in LK.

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The concentrations of Cu, Mn, and Zn as micronutrients and Cd and Pb as toxic metals were measured in 234 the liver, muscle, gill, and skin of eels caught from LK and LV in 2017-2018. The average weight and length of 235 the measured eels in LK and LV l were 366 ± 113 g, 548 ± 72 mm, and 247 ± 97 g, 505 ± 73 mm, respectively.
While the mean length of the eels in both lakes was close to each other, the weight of the eels taken from LK was determined heavier than from LV. On the other hand, in this study, significant differences were observed between the average HM concentrations determined for eels caught from LK and LV.   both lakes (Fig. 2). On the other hand, it was determined that Mn concentrations showed a significant difference 270 between the two lakes in the liver and skin, but not in gill and muscle tissues (p>0.05). However, it was 271 determined that the concentration of Zn in all tissues did not show a significant difference between the two lakes. skin, and gill, and the highest Cu in the liver (Table 3). The liver is one of the organs that represent the main sites 281 Table 3 shows the distribution of HM concentrations in tissues of the eels determined in both lakes.
282 Table 3 Ranking of HM concentrations in tissues of the eel

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Mn is an essential micronutrient for fish, but it can be a very toxic metal for fish at concentrations above 295 the optimum threshold level. However, it is an element that has not been investigated sufficiently in aquatic    The MPI was used to compare the total metals accumulation level in the muscle of eels of both countries.
about metal bioavailability, bio-concentration, and metal penetration into the environment. The MPI is a reliable 361 and sensitive method for monitoring metal contamination in food samples (Zakir et al. 2019). In this study, the 362 MPI value in LV was found to be slightly higher than the MPI value in LK. This result may indicate that there is 363 slightly more metal contamination in LV than in LK.

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Potential risks on human health 365

Metal concentrations in muscle
Since the most important accumulation mechanism of HMs is the food chain, there are many studies in 367 the literature regarding the measurement of HM concentrations in the muscle tissues of fish (Table 6).

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Especially the detection of toxic metals whose limit values have been determined in the national and international codec is very important in terms of species that are connected by food chains. In this study, Cd and with the limit values and the results obtained from other studies (Table 6). All values measured in this study were found as dry weight. region, which has been declared an environmental protection zone.

Health risk assessment
As fish is a vital source of nutrients, the accumulation of HMs in muscle tissue is of great importance 391 from human health perspective. HM concentrations in the muscle tissue of eels from both lakes were measured to evaluate the health risks of eel consumption for adults and children. The daily consumption of HMs was evaluated based on the HM concentrations in the muscle tissue of eels caught. Consumption rate limit values are given in Table 7. The mean EDIs of the HMs were higher in the eels from LV than those from LK. EDI values   is very close to the danger limits. However, it is a known fact that eel is not consumed every day of the week.

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For this reason, the values obtained in the study are below the hazard limits.  Although it was determined that there would be no health risks for eel consumers in both lakes, HI and CR values in LV were found higher than in LK.
It was determined that the concentrations of the metals except Zn were different from each other in the 445 tissues of eel taken from the two lakes with completely different ecological structures.

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High positive and negative correlations were found between metal concentrations and fish length and weight.

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Ethics approval and consent to participate Not applicable (this paper does not contain studies involving