Introduction

In the last several decades, heavy metal pollution in water bodies (in aquatic systems) has been a significant and growing concern in many fast-growing cities worldwide. This is because heavy metals, when present in excessive amounts in the environment, can pose significant threats to water supplies and human health by consuming fish and other aquatic organisms (Akoto et al., 2014). This problem results from various human activities and industrial processes that release heavy metals into the environment. As aquatic inhabitants, fish are highly susceptible to the harmful effects of heavy metals in aquatic environments. Because marine organisms can accumulate these contaminants from various environmental sources, including water, food, bottom sediment, and suspended particles in the water column (Ayas et al., 2007; Bat et al., 2020). However, many aquatic organisms can accumulate metals in concentrations much higher than those in the surrounding water or sediment (Usman and Mustapha, 2017). Heavy metals such as Mercury (Hg), Lead (Pb), Cadmium (Cd), and Arsenic (As) tend to accumulate in the muscle tissues and gonads of aquatic organisms, and they are generally not biodegradable, and they have long biological half-lives (Olgunoglu et al., 2015). The bioaccumulation of heavy metals in seafood can pose significant health risks to humans and other consumers when they consume contaminated seafood (Han et al., 2021).

According to the World Health Organization (WHO), managing the presence of heavy metals in food sources is crucial to ensure public safety (Olgunoglu et al., 2015; Olgunoglu, 2015). Hence, we must be sure that fish and seafood are not contaminated with heavy metals and do not exceed their acceptance limits is crucial for both human health and environmental sustainability (Sabbir et al., 2018). Several marine fish species serve as bioindicators to assess the quality of aquatic environments because they can accumulate significant amounts of certain metals in their various tissues (Farombi et al., 2007; Safahieh et al., 2011). Therefore, utilization of several fish species as bioindicators of heavy metal pollution in environmental monitoring studies has been widely recognized and emphasized in recent years by numerous investigators and researchers (Chen-Yi et al., 2001; Yılmaz, 2003; Dural et al., 2007; Yılmaz et al., 2007; Uysal et al., 2008; El-Moselhy et al., 2014; Khezri et al., 2014; Külcü et al., 2014; Oguz and Yeltekin, 2014; Gu et al., 2017; Anser and Benamal, 2018; Astani et al., 2018).

In the study, for the assessment of metal accumulation in fish, edible muscle tissue and gonads were chosen as the main organs since the metal content in muscle tissue indicates the prevailing metal levels in the surrounding waters. In contrast, the accumulation of metals in the gonads sheds light on the long-term retention of these elements within the fish (Ateş et al.,2015). Therefore, the main goal of this study is to investigate the presence and concentrations of ten metals including Aluminum (Al), Copper (Cu), Iron (Fe), Nickel (Ni), Chromium (Cr), Zinc (Zn), Manganese (Mn), Mercury (Hg), Cadmium (Cd), and Lead (Pb), in the muscle tissues and gonads of three fish species collected from the Aegean Sea in Türkiye. We assessed the heavy metal content in the muscle tissue of various fish species, which are important protein sources for human life, and examined its potential impact on public health.

Material and Methods

Collection and preparation of raw materials

The marine fish species examined in this research, which inhabit various parts of the water column and possess distinct dietary habits, were collected from the Aegean Sea in Türkiye in February 2023 (Figure 1). The Aegean Sea is situated between the western and southwestern shores of Türkiye and the eastern shores of Greece. It is an elongated embayment of the Mediterranean Sea, home to a series of islands known as the Aegean Islands (Makedonski et al., 2015).

The collected samples were immediately washed with clean seawater at the collection point to remove any external contaminants. To maintain the freshness of the samples and prevent spoilage during transportation to the laboratory, cleaned samples were placed in a container and preserved in crushed ice. In the laboratory, the samples were subject to measurements, which included determining the total size, weight, and gender of the fish. Based on the gender determination, various components, including edible tissues and gonads, were separated. After measurements and separation, the samples were placed in labeled polyethylene bags and stored at a temperature of -20ºC until they were processed for metal analysis. Fifteen specimens were collected from each fish species in the sampling area.

Determination of heavy metals

The muscle tissue and gonad samples were transported to the Accredited Industrial Services Laboratory of Türkiye in Izmir using dry ice to ensure low temperatures and preserve the sample integrity during transportation. For each fish species, 2 grams of edible muscle tissue and gonad samples were weighed and placed in a digestion vessel. To prepare the samples for analysis, 5 ml of concentrated (65%) nitric acid (HNO₃) and 2 ml of (30%) hydrogen peroxide (H₂O₂) were added to the vessel. The samples were digested in a microwave oven (NMKL, 1998). The determination of heavy metals was carried out using Inductively Coupled Plasma–Optical Emission Spectrometry (ICP-OES-Perkin Elmer Optima 8000º) instrument. The metal concentration results were expressed in micrograms per gram (μg/g) of wet weight.

Assessment of health risk

The equation associated with public health risks is presented as follows:

Equation (1): Estimated daily intake (EDI) of heavy metals (Sadeghi et al., 2020):

MC (μg/g) is the concentration of heavy metals in the fish muscle; FDC is the average food daily consumption of fish muscle (g/person/day), which is 17.81g/person/day in Türkiye (Yalçın ve Çakmak, 2023) and BW is the body weight (average 70 kg for adults). EDI was expressed as μg/kg bw/day (Sadeghi et al., 2020):

Equation (2): Estimated weekly intake (EWI) of heavy metals (Alipour et al., 2015):

EWI = EDI×7 days

Equation (3): Target hazard quotients (THQ) (Sadeghi et al., 2020):

Table 1 provides the factors, units, and values in the target hazard quotients (THQ) formula.

Equation (4): Total Target Hazard Quotient (∑THQ) (Pokorska et al., 2022):

∑THQ = THQ(Zn) + THQ(Mn) + • • • THQ(Hg)

Statistical analysis

The statistical analysis of the data was performed using SPSS 21.0 for Windows. One-way Analysis of Variance (ANOVA), Independent Samples t Test, and Post hoc test (Duncan) were employed to compare group means, with a significance level set at 0.05. The reported mean values represent the results of three experiments and are presented as X±SD.

Results and Discussion

Metal concentrations in the fish muscle and gonads

Table 2 shows the mean weights (g) and total length of female and male fish species obtained from the Aegean Sea in Türkiye.

The total length and weight differences between female and male L. piscatorius were statistically significant (p <0.05). Conversely, no noticeable variations in total length and weight were identified between males and females of B. belone and S. sphyraena (p>0.05).

The elements were categorized into two groups based on their significance in biological systems and their potential roles in environmental pollution. The first group, which includes Cu, Zn, Fe, Mn, and Cr, consists of essential elements required for various biological functions in organisms and naturally present in animal tissues, playing vital roles in maintaining health. The second group, Al, Ni, Hg, Cd, and Pb, consists of non-essential elements that can have toxic effects on living organisms (Makedonski et al., 2015). The data provided in the present study is presented in Table 3.

As shown in Table 3, for B. belone, the levels of Cu were significantly higher in the gonads of both female (♀) and male (♂) fish, with values of 1.288±0.148 µg/g and 1.238±0.142 µg/g, respectively, compared to the muscle tissue where Cu concentrations were lower at 0.346±0.040 µg/g for females and 0.454±0.052 µg/g for males (p<0.05). Similar patterns were observed for Zn, which were significantly higher in the gonads (34.71±3.77 µg/g for females and 16.85±1.83 µg/g for males) than the muscle (7.071±0.768 µg/g for females and 9.731±1.057 µg/g for males) (p<0.05). Fe concentrations also exhibited the same trend, with significantly higher levels in the gonads (79.01±8.31 µg/g for females and 48.25±5.08 µg/g for males) compared to the muscle tissue (2.354±0.248 µg/g for females and 5.044±0.531 µg/g for males) (p<0.05). Mn concentrations were below the detection limit in the muscle tissue for both females and males but were detectable in the gonads, with values of 1.568±0.339 µg/g for females and 1.138±0.149 µg/g for males (p<0.05). Cr was also below the detection limit in the muscle tissue for both genders but was present in the gonads, with values of 0.232±0.030 µg/g for females and 0.156±0.020 µg/g for males (p<0.05). Al was detected in the muscle tissue of females at a concentration of 0.287±0.029 µg/g, while it was below the detection limit in males. Al exhibited higher levels in the gonads of females, measuring 2.248±0.229 µg/g, whereas the levels were below the detection limit (<0.250 µg/g) in male gonads. Notably, while the differences in Al concentrations between gonads and muscle tissue were not statistically significant (p>0.05), they were found to be statistically significant between the genders (p<0.05). Ni concentrations in gonads were significantly higher than in muscle for both genders, with values of 0.183±0.028 µg/g for females and 0.236±0.036 µg/g for males (p<0.05). Hg levels were lower in the muscle than in the gonads for both genders. Importantly, these differences in Hg concentrations between gonads and muscle tissue were not statistically significant (p>0.05). Cd and Pb concentrations were below the detection limit in the muscle tissue for both females and males. However, they were detectable in the gonads, with Cd concentrations of 0.45±0.009 µg/g for females, 0.037±0.007 µg/g for males, and Pb concentrations of 0.058±0.011 µg/g for females and 0.026±0.005 µg/g for males. The differences in Cd and Pb concentrations between the gonads and muscle tissue were found to be statistically significant (p<0.05)

In the muscle tissue of both genders, the level of Zn was higher than other metal concentrations. The order of mean metal concentrations in the muscle of B. belone was as follows.

In females, Zn> Fe> Cu> Al> Ni> Hg

In males, Zn> Fe> Cu> Ni> Hg

Within the gonads, Fe exhibited the highest concentration, followed by others, with the order being Fe> Zn> Al>Mn> Cu> Cr>Ni>Pb> Hg> Cd for females.

In males, the order of concentration in gonadal tissues was Fe> Zn> Cu >Mn>Ni>Cr> Cd>Hg>Pb.

Except for Al, the variation in heavy metal concentrations between genders was not found to be statistically significant (p>0.05).

For S. sphyraena, Cu concentrations were found to be significantly higher in female gonads (1.040±0.120 µg/g) and male gonads (0.915±0.105 µg/g) compared to muscle tissues, where Cu concentrations were lower in females (0.309±0.036 µg/g) and males (0.263±0.030 µg/g) (p<0.05). Similarly, Zn concentrations were higher in female gonads (36.873±0.746 µg/g) and male gonads (25.77±10.40 µg/g) compared to muscle tissues (p<0.05). Fe concentrations also exhibited a similar trend, with significantly higher levels in female gonads (23.356±0.353 µg/g) and male gonads (17.54±2.90 µg/g) compared to muscle tissues (p<0.05). Mn concentrations were below the detection limit in the muscle tissues for both females and males, while they were detectable in the gonads, with values of 0.351±0.046 µg/g for females and 0.544±0.071 µg/g for males (p<0.05). Al was also below the detection limit in the muscle tissues for both sexes, while they were detectable in the gonads with values of 0.310±0.032 µg/g for females and 0.542±0.059 µg/g for males (p<0.05). In the gonads, Ni levels were higher than in muscle tissue (p<0.05). Hg levels were lower in the muscle tissues than in the gonads for both genders (p<0.05), with Hg concentrations of 0.088±0.018 µg/g for males and 0.069±0.014 µg/g for females in muscle tissues and 0.486±0.09 µg/g for females and 0.417±0.085 µg/g for males in gonads. Cr, Cd, and Pb concentrations were below the detection limit in the muscle tissues and gonads for both females and males.

The order of concentration in muscle tissues for females was Zn> Fe> Cu>Hg

For males, the order of concentration in muscle tissues is as follows: Zn> Fe> Cu> Hg

In females, the order of concentration in gonads from highest to lowest is as follows: Zn> Fe> Cu>Hg>Mn>Al> Ni

In males, Zn> Fe >Cu> Mn> Al> Hg> Ni

The variation in heavy metal concentrations between genders was not found to be statistically significant (p>0.05).

For L. piscatorius, Cu concentrations in the muscle were recorded at 0.158±0.018 µg/g for females and 0.181±0.021 µg/g for males. However, higher Cu concentrations were observed in the gonads, with values of 2.073±0.353 µg/g for females and 1.840±0.212 µg/g for males, with a statistically significant difference (p<0.05) compared to the muscle tissue. A similar pattern was also observed for Zn (p<0.05). Fe levels in the muscle were 1.025±0.250 µg/g for females and 1.244±0.131 µg/g for males. In contrast, the gonads exhibited higher Fe concentrations, with females at 7.447±0.783 µg/g and males at 13.40±3.51µg/g (p<0.05). Mn concentrations showed a similar pattern, with 0.182±0.024 µg/g in female muscle and 0.106±0.014 µg/g in male muscle, while in the gonads, females had much higher concentration with 0.368±0.048 µg/g of Mn, whereas males had 0.470±0.061 µg/g (p<0.05). Cr concentrations were below the detection limit (<0.1 µg/g) in both muscle and gonads for both genders. Al levels were also below the detection limit (<0.250 µg/g) in the muscle for both females and males. However, in the gonads, females had 1.132±0.115 µg/g of Al, while males had 0.753±0.077 µg/g(p<0.05).

Ni concentrations were below the detection limit (<0.1 µg/g) in female muscle. Ni was found to be 0.147±0.022 µg/g in male muscle. In the gonads, males had higher concentrations, with levels at 0.289±0.044 µg/g, while females had lower concentrations at 0.105±0.016 µg/g. The levels of Hg in the muscle were 1.109±0.225 µg/g for females and 0.211±0.043 µg/g for males. In the gonads, Hg values were found to be higher than muscle, with females at 1.398±0.283 µg/g and males at 0.290±0.059 µg/g. Notably, the differences in Ni and Hg concentrations between the gonads and muscle tissue were insignificant (p>0.05). However, they were statistically significant between the genders (p<0.05). Cd concentrations were below the detection limit (<0.01 µg/g) in muscle tissue for both genders. However, it was observed to be 0.087±0.018 µg/g for females and 0.076±0.013 µg/g for males in the gonads. A similar trend was observed in Pb (p<0.05).

The order of heavy metal levels in the samples was as follows:

In the muscle tissue (Female): Zn>Hg>Fe>Mn>Cu

In the muscle tissue (Males): Zn>Fe>Hg>Cu>Ni>Mn

In gonad (Female): Zn>Fe>Cu>Hg>Al>Mn>Ni> Cd>Pb

In gonad (Male): Zn>Fe>Cu>Al>Mn>Hg>Ni>Cd>Pb

Except for Ni and Hg, the variation in heavy metal concentrations between genders was not found to be statistically significant (p>0.05).

As a result, when comparing all the species examined in this study, it was determined that heavy metal levels in the gonadal tissues were higher than those in the muscle (p<0.05). Gonads are considered target organs for metal accumulation due to their metabolic activity, which can result in the accumulation of metals, sometimes at high levels (Terra et al., 2008). Therefore, this difference was attributed to the physiological activities of the gonads, as muscles are not actively involved in metal accumulation. It has also been reported that the reason for the high metal accumulation in the gonads is that the fish’s defense mechanism regulates metals and pollutants. In this process, some organic and mineral pollutants are transferred from the body to the eggs instead of being eliminated from the body (Astani et al., 2018). Several studies have reported similar findings, indicating that muscle is not an actively involved tissue in accumulating heavy metals (Olgunoglu et al., 2015; Yılmaz, 2013). For instance, in a study, Wong et al., (2001) reported that the bioaccumulation of heavy metals in muscle tissue is lower than in gonads in marine fishes. Astani et al. (2018) reported in their study that the muscle, as the target organ, exhibited the lowest concentration of metals compared to gonads. In another study, it was reported that the concentrations of heavy metals in gonads (both testis and ovary) were higher than in the other organs, as reported by Ebrahimi and Taherianfar (2010). Our findings align with those reported by researchers.

Among the species within toxic elements, Hg was predominant, while Cr, Cd, and Pb were not detected in the muscle tissues. The concentrations in gonads of Fe, Mn, Cr, and Pb in B. belone were found to be statistically more significant (p<0.05). At the same time, Hg in benthic L. piscatorius was also significantly different in muscle and gonads compared to the species examined in the study. It was reported that Hg concentrations in fish can vary depending on the habitat due to habitat-specific differences in the biogeochemical processes related to the cycling and methylation of Hg and variations in the structure of the food web (Smith et al., 2016). Based on a study conducted by Storelli and Marcotrigiano et al. (2000), Lophiidae are positioned at the highest trophic level, leading to the potential for higher body mercury accumulation as a result of mercury biomagnification. It appears that our study’s findings align with the researchers’ interpretations.

It is a fact that heavy metal accumulation can vary among species and is influenced by various factors. Furthermore, it was also observed that, when comparing males and females, heavy metal levels mostly tend to be higher in the muscles of males. It was noted that the association between metal accumulation and gender could be ascribed to differences in metabolic activity between males and females. Typically, the sex that grew faster, often females, could be expected to have lower metal concentrations (Pourang et al.,2004; Yılmaz and Yılmaz, 2007; Olgunoglu et al., 2015). Hence, the average metal concentrations in males’ tissues were higher than those in the tissue of females (p>0.05). In a study to determine the accumulation of heavy metals in little tunny (Euthynnus alletteratus) fish, the levels of some heavy metals in muscle were significantly higher in males than in females Ansel and Benamar (2018). Thus, it appears that the results obtained were consistent with previous studies.

Additionally, to provide insight into the pollution at the location where the fish are caught, recorded heavy metal levels in muscle ranged from 0.158 to 0.454 µg/g for Cu, 3.297 to 9.731 µg/g for Zn, 1.025 to 5.044 µg/g for Fe, <0.1 to 0.182 µg/g for Mn, <0.250 to 0.287 µg/g for Al, <0.1 to 0.147 µg/g for Ni, and 0.024 to 1.109 µg/g for Hg, regardless of fish species and sexes. Cr, Cd, and Pb were not detected.

In the gonads, heavy metal levels were as follows: Cu ranged from 0.915 to 2.073 µg/g, Zn from 11.75 to 36.873 µg/g, Fe from 7.447 to 79.01 µg/g, Mn from 0.368 to 1.568 µg/g, Cr from <0.1 to 0.232 µg/g, Al from <0.250 to 2.248 µg/g, Ni from 0.105 to 0.289 µg/g, Hg from 0.034 to 1.398 µg/g, Cd from <0.01 to 0.087 µg/g, and Pb from <0.01 to 0.058 µg/g.

Distribution patterns of metal concentration in both fish muscle samples and gonads follow a sequence, irrespective of fish species and sexes.

For muscle: Zn _(5.96)> Fe _(2.12)> Cu _(0.29)> Hg _(0.25)> Ni _(0.07)> Al _(0.05) = Mn _(0.05)

For gonads: Fe _(31.50)> Zn _(23.28)> Cu _(1.40)> Al _(0.83)> Mn _(0.74)> Hg _(0.45)> Ni _(0.19)> Cr _(0.06)> Cd _(0.04)> Pb _(0.02)

The graph of the ranking of heavy metal levels from highest to lowest is given in Figure 2.

Zn, as an essential element crucial for organisms’ normal growth and metabolic processes, demonstrated the highest accumulation in the tissue samples compared to other metals. Similarly, iron (Fe) and copper (Cu) are essential trace elements for animal metabolism. Studies have reported that Zn, Fe, and Cu are the most abundant elements in tissues, surpassing the levels of other metals (Pourang et al., 2005; Krishna et al., 2014). Our results are consistent with the findings reported for Zn, Cu, and Fe.

In Table 4, heavy metal concentrations in selected fish species caught from different fishing locations in the Aegean Sea were summarized, demonstrating the variability of metal levels in organisms and their reflection of contamination levels in the sampling areas for comparison.

The comparison of mean metal concentrations in muscle tissues and gonads (Table 4) reveals variations in muscle tissue metal levels among different organisms. Notably, some studies have reported significantly higher concentrations of heavy metals, including Zn, Fe, Cr, Al, Mn, Pb, and Cd, compared to the levels found in our study for all species. On the other hand, Ni concentrations were compatible with the previous studies on some fish species (Erdem et al., 2021). In the case of Hg, a higher concentration was noted in L. piscatorius. Additionally, it has been observed that the concentration of Cu, particularly in the muscles of B. belone, is partially higher when compared to the species listed in Table 4. Tecimen et al. (2023) reported higher levels of Zn, Cu, and Pb in the muscle tissue of S. sphyraena compared to those found in our study for the same species. This situation is thought to arise from the differences in sampling areas and pollution levels. When the accumulation of Zn, Cu, Pb, and Cd in the gonads was compared with the values provided in Table 4, it was evident that the Zn, Cu, and Cd levels obtained in the study were mostly by previously reported values. However, the reported quantity of Pb appeared to be higher.

In summary, prior research has demonstrated that a multitude of factors, including species variations, seasonal changes, fish length and weight, diverse ecological requirements, metabolic processes, feeding patterns, water’s physical and chemical attributes, and pollution levels, can collectively impact the accumulation of metals in fish tissues.

Health risk assessment

The permissible limits determined in the present study for edible fish muscles intended for human consumption may vary depending on the region and regulatory authority. However, while there are no specific maximum acceptable values for all elements, they were lower than the limits, including Cu, Zn, Fe, Mn, and Pb, set by national and international agencies (Table 5) such as FAO, WHO, and EC (Verep and Mutlu, 2022).

Sometimes, even though muscle tissue in marine species often has low metal concentrations, the risk it poses may vary depending on the quantity consumed (Kilercioglu et al., 2022). Therefore, EWI and THQ were calculated for each of the heavy metals in three female and male fish species to assess health risks associated with the common consumption of muscle tissue (Table 5). Also, Table 5 compares the estimated EWI to the recommended values, such as PTWI. The PTWI value calculates the quantity of a contaminant that humans can ingest throughout their lifetime without significant risk. PTWI standards are determined by the Expert Committee on Food Additives (JECFA), a collaborative body of the Food and Agriculture Organization (FAO), and the World Health Organization (WHO) for the United Nations. The quantity of food consumed influences the PTWI, the duration of consumption, and the level of contamination in the food (Alipour et al., 2015).

Therefore, in the current study, PTWI values were employed as reference standards (safe levels) for heavy metals to compare them with EWI levels, and all samples exhibited EWI values lower than the corresponding PTWI_(k) values. In general, there is no health-threatening concern due to the consumption of edible muscle of these three fish species from the Aegean Sea.

THQ is recognized as an indicator used to evaluate potential health risks to consumers. A THQ or ∑THQ below 1 indicates no adverse hazard for the exposed population. When THQ or ∑THQ equals 1, it suggests that the affected individuals may face noncarcinogenic health risks, with the likelihood of these risks increasing as the THQ value rises (Miri et al., 2017; Kilercioglu et al., 2022). In the present study, the sum of THQ values for all the related heavy metals was higher only in the muscle of male L. piscatorius than in females and other species, exceeding 1. Moreover, except for Hg in the muscle of male L. piscatorius, THQ values for all other metals were below 1, indicating an absence of health hazards for the population who consume B.belone S. Sphyraena from the Aegean Sea. These variations in THQ values have been attributed to the fact that L. piscatorius is a demersal fish species and disparities in average weight and lifespan.

Conclusions

This study evaluated heavy metal accumulation in the muscle tissues and gonad-selected fish species (B. belone, S. sphyraena, , L. Piscatorius) from the Aegean Sea to assess health risk potential. Results showed different accumulation patterns among the genders and between the species. Generally, the mean metal concentrations in the muscle of male fish were higher than in female fish species. When comparing muscle and gonads, it has been determined that gonads, especially in females, tend to accumulate heavy metals at higher concentrations than muscles (p<0.05). In the case of the edible part of the fish, primarily the muscle tissue, THQ and ∑THQ values were calculated, and the results did not exceed 1.00, except for L. piscatorius. Based on the results obtained, the primary concern for human health among the examined metals is the level of Hg in female L. piscatorius (p<0.05). In light of the potential for the accumulation of toxic levels of this metal, it is recommended to continue monitoring heavy metal concentrations in the benthic fish, L. piscatorius, caught in the Aegean Sea, Türkiye. Additionally, the following suggestions should be considered.

• Monitoring heavy metal levels in different seasons and regions as part of scientific research should be continuous

• It is recommended to conduct and monitor heavy metal studies in other aquatic species in the region.

• Studies on heavy metal monitoring should encompass water and sediment samples, as well as organisms in the Aegean Sea

• Efforts to reduce pollution from industrial and agricultural activities should be supported

• Caution should be exercised regarding the consumption of fish gonads due to potential heavy metal contamination

• Efforts should be made to raise awareness about potential risks associated with fish consumption among the public, and specific recommendations should be provided, especially for at-risk groups