Health risk assessment of heavy metals (Zn, Pb, Cd, and Hg) in water and muscle tissue of farmed carp species in North Iran

This cross-sectional study was conducted to determine and compare the concentrations of heavy metals (Zn, Pb, Cd, and Hg) in carp-farming water and muscle of various carp species including common carp (Cyprinus carpio), bighead carp (Hypophthalmichthys nobilis), silver carp (Hypophthalmichthys molitrix), and grass carp (Ctenopharyngodon idella) collected from three major warm-water fish farms in Mazandaran Province (Iran) during March 2018 to March 2019. In addition, bioaccumulation of heavy metals (BCFs) and carcinogenic and non-carcinogenic risk assessments of consumers exposed to heavy metals through fish consumption were estimated. The water concentration of all metals in this study was lower than permissible limits. The concentration of Zn in the water (10.21–17.11 μg L−1) was higher than that of other metals in all sites, followed by Pb > Cd > Hg. In fish muscle, Zn concentration in silver carp was the highest, and the lowest concentrations were related to Hg and Cd in common carp and grass carp, respectively. The target hazard quotients (THQ) indicated that the non-carcinogenic health risk to humans was relatively low by consuming four farmed carp species products. The carcinogenic risk of inorganic Pb was 1.24E-04 (common carp) to 2.11E-04 (grass carp) for adults, which is within the acceptable range. The values of BCFs for all metals demonstrated that farmed carp muscle could not be considered a bioaccumulative tissue for heavy metals. The results indicated that the concentrations of heavy metals in the farmed carp species in North Iran were relatively low and did not cause considerable human health risks.


Introduction
Nowadays, one of the global issues raising scientific concern is the pollution of the aquatic environment with heavy metals, because these metals are environmentally resistant and most of them have toxic effects on organisms (Islam et al. 2017). Heavy metals are defined as metals having an elemental density greater than 5 g cm −3 and an atomic number greater than 20 (Ali & Khan 2018b). The contamination of heavy metals in aquatic ecosystems can have adverse health effects (Hu et al. 2021;Mohan et al. 2012). The heavy metals can accumulate in the water, sediments, and fish through the aquatic food chain; therefore, their toxic effects spread throughout the food chain (Bibi et al. 2016;Hu et al. 2021). Humans, as part of the food chain, can also be affected by transferring and accumulating heavy metals in their tissues through long-term food consumption. Fish can be one of the main sources of heavy metals for their consumers ). In freshwater fish, an important human food, bioaccumulation of toxic heavy metals raised public health concerns (Ali and Khan 2017). Consumption of Hg-contaminated fish in Japan in the 1950s, which caused Minamata disease, is considered one of the major environmental chemical disasters of the twentieth century (Ali and Khan 2017). The heavy metals in human bodies can cause toxic effects on the deoxyribonucleic acid (genotoxicity), central nervous system (neurotoxicity), liver (hepatotoxicity), and kidney (nephrotoxicity) Khan 2018a, Mahurpawar 2015;Sharma et al. 2014). A great affinity of most heavy metals for sulfur atoms caused these metals to make bonds with sulfur in enzymes and disrupt their function (Gul et al. 2022;Waseem and Arshad 2016). The Cd, Pb, Zn, and Hg are of the greatest concern among common heavy metals, because they may interact with the metabolism of nutritionally essential metals (Goyer 1997;Stanković et al. 2014). The toxicity level of heavy metals in the human body are depending on several criteria such as the dose of metal exposure, their exposure route, and the chemical properties, along with the properties of individuals exposed to these metals, including gender, genetic behaviors, age, and the nutritional deficiency (Tchounwou et al. 2012). A direct exposure route for the majority of people could be the ingestion of metal elements through a dietary substance (Loutfy et al. 2006).
There is a standard limitation in traditional risk assessments to compare pollutant levels in the water, air, and food. Traditional risk assessments cannot provide a perfect interpretation of environmental quality. In this type of risk assessment, fewer clinical signs were observed; therefore, they have no data about chronic exposure to lower levels of standard values and adverse health effects (Dadar et al. 2017;Shahsavani et al. 2017). Hence, for a better interpretation of pollutants in the environment, several risk assessment indices, such as target hazard quotient (THQ) and hazard index (HI), were developed (Shahsavani et al. 2017).
During the last decade, there is an increasing trend in the global production of aquaculture that predicted aquaculture will provide the most reliable supply of seafood in the future (Hixson 2014). Carp species could be the best selection for aquaculture in almost all places in the world due to rapid growth, easiness of breeding, and food efficiency (Tokur et al. 2006). The carp species are counted as the important farmed species in Iran, which as now are included in about 50% of fish in warm freshwater fishponds (Afkhami et al. 2011). However, there is a great lack of knowledge on the pollutant levels such as heavy metals in these fishes, a risk factor for human health. Therefore, the aims of this study were (1) determination of heavy metal concentrations (Pb, Cu, Zn, and Cd) in the muscle tissue of farmed carp species including common carp (Cyprinus carpio), bighead carp (Hypophthalmichthys nobilis), silver carp (Hypophthalmichthys molitrix), and grass carp (Ctenopharyngodon idella)) collected from two economically important aquaculture locations of Babol and Galugah in Iran and (2) evaluation of the human health and ecological risks posed by these heavy metals, which could help us expand our knowledge about the health of this important species.

Sample collection
In this cross-sectional study, water (total n = 120, n = 5 for each station) and fish including common carp (Cyprinus carpio), bighead carp (Hypophthalmichthys nobilis), silver carp (Hypophthalmichthys molitrix), and grass carp (Ctenopharyngodon idella) (total n = 360; n = 120 for each station) samples were collected from three major warm-water fish farms in Babol and three in Galugah in Mazandaran Province (Iran) during March 2018 to March 2019 ( Fig. 1). Water physico-chemical properties including pH, dissolved oxygen (DO), and total carbon (TC) were measured based on previous standard protocols (Ehiagbonare and Ogunrinde 2010). Water samples were collected in 500-mL polyethylene bottles and transferred to the laboratory at 4 °C. Then, fish samples were collected by fishnet and transported alive to the laboratory by plastic packing box.

Sample preparation and analysis
The water samples were filtered through a 0.45-mm filter, and the pH was adjusted to < 2 with 10% nitric acid. The fish were sacrificed and dissected immediately. The skin of fish samples was removed, and the epaxial muscle located at the dorsal part of the fish body was cut, washed with distilled water, and completely crushed with a meat grinder. Then, 10-20 g of samples were incubated at 105 °C temperature for 4 h. After that, the samples were digested in 65% HNO 3 and filtered according to the previous method (Yi et al. 2011). Briefly, Whatman filter papers (No. 1) were used to filter water samples. The filtered water samples (stored at 4 °C) were digested in 5 mL of ultra-pure nitric acid for 30-40 min at 200 °C.
The measurement of heavy metals (Zn, Hg, Cd, and Pb) concentrations was carried out by graphite furnace atomic absorption spectrophotometer (Thermo Scientific's iCE 3300, Waltham, MA, USA) (Table 1). Cold vapor atomic absorption spectroscopy (CVAAS) was used to measure Hg. The results for each metal were reported as μg g −1 dried weight.

Quality assurance/quality control
The blank experiments, matrix spike experiments, blank spike experiments, and parallel experiments (n = 3) were done to assess the reliability of the preparation methods for fish samples. Each of the metals was added to the fish samples in triplicate, at concentration levels of 0.025 µg g −1 , 0.25 µg g −1 , and 0.5 µg g −1 to ensure the accuracy of the analysis method, and recovery percentages were calculated as the following:

Target hazard quotient (THQ) and hazard index (HI)
For the estimation of non-carcinogenic health risk due to heavy metal exposure, the target hazard quotient (THQ) was Recovery (%) = (metal amount found∕metal amount spiked) × 100 used based on the previously suggested equation (Chien et al. 2002): In this equation, E F is the exposure frequency (365 days/ year), E D is the exposure period (70 years), F IR is per capita fish consumption (g/person/day) (based on EPA (2000) assumptions, the average weights of the adults were noted as 70 kg), C is the heavy metal concentration in fish (µg g −1 b w), R FD is the oral reference dose (µg kg −1 b w day −1 ), W AB is the average of body weight, and T A is the average exposure time for non-carcinogens (365 days/year for 70 years). The oral reference dose (R FD ) for Zn, Cd, Pb, and Hg was 300, 1, 3.6, and 0.1 µg kg −1 b w day −1 , respectively (EPA 2000). THQ < 1 indicates unlikely adverse health effects, but THQ ≥ 1 indicates the possibility of high adverse effects (Lei et al. 2015). All the THQ values of the studied fish were added together, and the hazard index (HI) was calculated. A value of HI > 10 is indicated as the highest non-carcinogenic risk for the exposure group of people (Council 2014).

Bioconcentration factor
The bioconcentration of an element means that the concentration of that element in an aquatic organism is greater than  its concentration in the aquatic environment after exposure to that element (Hashizume et al. 2014). The bioconcentration (BCF) was calculated as the ratio of the mean heavy metal concentration in water (C water ) to the mean heavy metal concentration in fish muscle tissue (C fish ) (Zafarzadeh et al. 2018):

Estimated daily intake (EDI)
The estimated daily intake (EDI) of heavy metals in fish was measured by the equation described by Shaheen et al. (2016): where FIR is the fish ingestion rate (on average an adult FIR is 9.1 to 10.2 kg/n-year for adults) (FCCI 2015), C is the heavy metal concentration in fish (µg/g −1 ), and BW is the average body weight (70 kg for an adult person) (Shaheen et al. 2016).

Carcinogenic risk (CR) analysis
Carcinogenic health risks (CR) indicate an incremental probability of heavy metals developing cancer over longtime exposure. The cancer slope factor (CSF), provided by EPA, was used to estimate cancer risk over a lifetime of exposure to Pb (EPA 2000). Carcinogenic risk (CR) was determined by considering the following equation: where CSF is the carcinogenic slope factor of 0.0085 (mg/ kg/day) −1 for Pb USPEA (EPA 2000). EDI is the estimated daily intake of heavy metals. Acceptable risk levels for carcinogens range from 10 −4 to 10 −6 . When CR is more than 10 -4 , carcinogenic risk impact could increase.

Statistical analysis
The data analysis was done statistically using the SPSS software (ver. 24), and the graphs were prepared using MS Excel 2019 and R software 4.1.0. The normality of data was confirmed using the Kolmogorov-Smirnov test. For statistical comparisons among sites, one-way ANOVA and twoway ANOVA following Tukey's post-test were used. The significance level was set at p < 0.05, and all values were reported as mean (s) ± standard deviation (SD). To evaluate relationships between a pair of variables, Pearson's correlation analysis was applied.

Occurrence of heavy metals in the water
There was no significant difference in pH, TC, and DO among all water sample sites (p > 0.05). The total concentrations of Zn, Pb, Cd, and Hg in the water samples are shown in Fig. 2A. The average heavy metal concentrations in all sites were below the permissible limits for aquatic life. All concentrations of all heavy metals in this study were lower than the allowable recommended limits for drinking water by US EPA (EPA 2000; Martins et al. 2019). The concentration of Zn in the water (10.21-17.11 μg L −1 ) was higher than that of other metals in all sites, followed by Pb > Cd > Hg. It seems that the main source of Zn was leaching from agricultural soils to surface water used for aquaculture (Bonten et al. 2008). There was no significant difference in the concentrations of all heavy metals between water samples of 6 farms (p > 0.05). The concentration of Zn and Cd in water samples of six farms in the current study was higher than those reported by Heshmati et al. (2019) in Hamadan Province, Iran, and by Dadar et al. (2016) in the Haraz River, Northern Iran, whereas the Pb and Hg concentrations were lower than those reported in these studies (Dadar et al. 2016;Heshmati et al. 2019). The concentrations of Zn, Cd, and Pb in the water of 16 carp farming systems from central and eastern North China were higher than those concentrations reported in the present study (Zhong et al. 2018). Pearson correlation analysis (Fig. 2B) indicated that there were significant positive correlations among Zn, Pb, and Cd in the water samples, implying that these metals may be from similar sources. There was a significant negative correlation between Pb and Hg.

Comparison of heavy metals, non-carcinogenic risk assessment, and their estimated daily intake in different fish species
In this study, Zn was the most abundant metal in fish species. However, the Zn concentration among different fish species showed no significant difference (p > 0.05) ( Table 2). The highest Zn content was found to be 4.11 µg g −1 in Silver carp, and the lowest was 2.4 µg g −1 in bighead carp. The concentration of Zn in carp muscle in this study was much lower than Zn concentration in the muscle of wild crucian carp reported by Yanqiang et al. (2012) and in common carp (Cyprinus carpio) reported by Zafarzadeh et al. (2018) (Yanqiang et al. 2012;Zafarzadeh et al. 2018). However, the concentration of Zn in this study was higher than Zn concentration in wild and farmed carp in the study reported by Heshmati et al. (Heshmati et al. 2017). In the present study, the maximum Zn concentration in the collected samples was lower than the guideline level established by the joint FAO/ WHO/Expert Committee on Food, indicating this Zn concentration is not a threat to human health through the consumption of fish (JECFA 1993). The findings of this study, based on the calculated value for THQ (Table 3), showed that Zn concentration in fish muscle samples can be considered safe for a human (body weight of at least 70 kg) if the consumption of products is 25.2 g daily −1 . In this study, the daily intake of Zn was estimated in the range of 4.05E-01 µg kg −1 bw day −1 which was about 2.70E + 00% of the daily Zn requirements (15 mg) for adult humans (Salgueiro et al. 2000). Since the Zn contributes to most human metabolic pathways, the deficiency of this element can lead to loss of 0.10 ± 0.02 a 0.14 ± 0.04 b 0.13 ± 0.01 b 0.17 ± 0.08 c 0.001 2 0.5 1.7 1 Cd 0.004 ± 0.001 a 0.004 ± 0.00 a 0.002 ± 0.00 b 0.002 ± 0.0001 b 0 0.5 0.5 4 0.5 Hg 0.002 ± 0.000 a 0.011 ± 0.001 b 0.017 ± 0.001 c 0.016 ± 0.001 c 0.01 -0.5 0.5-1 0.5-1 appetite, growth retardation, skin alterations, and immunological abnormalities in humans (Salgueiro et al. 2000). The range of Pb concentration among fish collected was 0.09-0.18 µg g −1 , which is less than the limit allowed by the joint FAO/WHO/Expert Committee on Food Additives (JECFA 1993). These concentrations were higher than the Pb concentration reported in different carp species (Wei et al. 2014) and lower than the Pb concentration reported in common carp (Cyprinus carpio) in Alagol wetland in the Golestan, Iran (Zafarzadeh et al. 2018). The Pb concentration was the lowest in normal carp muscle and highest in grass carp muscle (Table 2). According to the average Pb concentration in the fish samples, the most daily intake of Pb was observed through the consumption of grass carp (2.48E-02) (Table3) which was approximately 6.88E-01% of the RfD index for a 70-kg adult (EPA 2000). Therefore, based on the results of this study, Pb exposure may not be important in farmed carp consumers in Iran. In the human body, Pb exposure can lead to decreased intelligence quotient (IQ) in children and occurrences of anemia (García-Lestón et al. 2010).
In the present study, Cd concentration in common carp and bighead carp was significantly higher than in silver carp and grass carp (p < 0.05) ( Table 2). Moreover, Cd concentration in common carp and bighead carp was significantly higher than in silver carp and grass carp (p < 0.05) ( Table 2). As shown in Table 2, the Cd concentration was lower than the guideline level established by the joint FAO/ WHO/Expert Committee on Food Additives (JECFA 1993). Therefore, the Cd toxicity through farmed carp consumption, based on THQ calculated in this study, is not a threat to human health. The mean concentration of Cd in fish samples detected in the present study was lower than that reported by Zafarzadeh et al. (2018) for common carp (Cyprinus carpio), Zohra and Habib (2016) in fish from the Mediterranean Sea (Zohra and Habib 2016), and Ahmed et al. (2020) for farmed carp fish (Ahmed et al. 2020). However, the concentration of Cd in carp from Beysehir Lake in Turkey was below the detection limit (Tekin-Özan and Kir 2008). Although the concentration of Cd in farmed carp from most different areas may be safe for humans, the accumulation of this heavy metal over time can induce nephrotoxic and hepatotoxic effects (Ardeshir et al. 2017;Byber et al. 2016).
A significant difference was found in the Hg contents of different farmed carp samples (p < 0.05). The highest concentration of Hg was found in silver carp and grass carp, and the lowest concentration was observed in bighead carp samples ( Table 2). The presence of Hg in the fish body is considered a threat to fish and humans as their consumers (Moallem et al. 2010). The Hg concentration of more than 5 µg g −1 can induce adverse effects such as reduced coordination, loss of appetite, emaciation, and mortality (Bernhoft 2012). In this study, Hg concentration in fish samples was not a threat to the fish themselves and lower than the guideline level established by the joint FAO/WHO/Expert Committee on Food Additives (JECFA 1993). The calculation of EDI and THQ confirmed the safety of the Hg concentration in farmed carp in this study (Table 3). The Hg concentration in this study was lower than that reported by Heshmati et al. (2017) in wild and farmed carp (Cyprinus carpio) and Caspian kutum (Rutilus frisii kutum) in Iran (Heshmati et al. 2017) and by Nasrollahzadeh Saravi et al. (2013) in carp obtained from the Southern Iranian Caspian Sea Coast (Nasrollahzadeh Saravi et al. 2013). It seems that the different concentrations of Hg in water are different between wild and farmed fish. In most organisms, particularly humans, Hg can be induced neurotoxic effects (Rice et al. 2014).

The BCFs and carcinogenic risk assessment in different fish species
The natural logarithm of BCF was calculated for the heavy metals (Fig. 3). In fish samples, especially in silver carp and bighead carp, Zn and Pb displayed the highest log BCFs, which were 2.34 and 2.21 L/kg, respectively. The mean log BCFs of each heavy metal in this research followed the order: Zn > Pb > Cd > Hg. The highest log BCF of Zn in fish samples may be originated from the fact that Zn is an essential element and fish prefer to intake this element actively (Roney 2005). In general, the log BCFs of the heavy metals in the fish in previous studies were in the range of reported values (Pandey et al. 2017;Vu et al. 2017). For example, the log BCFs of Zn and Pb in this study were higher than those reported in central and eastern North China (Zhong et al. 2018), the freshwater fish in northern méxico (Luna-Porres et al. 2014) and lower than those in Cyprinus carpio in Pakistan river (Iqbal and Shah 2014;Yousafzai et al. 2012).
Estimation of lead (Pb) carcinogenic risk value in all fish samples showed a range of 1.24E-04 (in common carp) to 2.11E-04 (in grass carp) for adults (Fig. 3). According to EPA's (2016) recommendation, a carcinogenic risk within the range of 10 −6 -10 −4 is an acceptable and negligible cancer-causing hazard risk (EPA 2016).

Conclusion
Contamination of food items, like fish to heavy metals, is one of the most concerning challenges for health hazards. Keeping the alarming situation in mind, the present study was designed on the four farmed carp species from Mazandaran Province, Iran, to highlight the distribution of the metals in water, fish, and the pattern of their risk to an adult human after consumption. The results showed that all concentrations of Zn, Pb, Cd, and Hg in water (18.2 ± 3.3, 0.78 ± 0.27, 0.13 ± 0.08, and 0.09 ± 0.03 μg L −1 , respectively) and fish muscle (3.04 ± 0.1, 0.13 ± 0.05, 0.003 ± 0.0001, and 0.011 ± 0.001 µg g −1 dw, respectively) were below the permitted limits. The analysis of heavy metal risk assessment indicated that common carp, silver carp, bighead carp, and grass carp had no health risk to fish consumers and no carcinogenic risk was observed due to the lower CR value of the lead. The accumulation of heavy metals (BCF) was lower than 1000; therefore, the muscle of farmed carp species could not be considered a bioaccumulative tissue for heavy metals.