Health risk assessment and bioaccumulation of heavy metals in Procambarus clarkii from six provinces of China

Contamination with heavy metals in wild red swamp crayfish (Procambarus clarkii) from 7 different geographical areas in six provinces of China (Hubei, Hunan, Jiangxi, Anhui, Jiangsu, and Shandong) was evaluated. Concentrations of chromium (Cr), arsenic (As), lead (Pb), cadmium (Cd), and mercury (Hg) in the abdominal muscle, gonad, and hepatopancreas were determined by inductively coupled plasma mass spectrometry (ICP-MS) and atomic fluorescence spectrometer (AFS). Except for the Cd content in the hepatopancreas, the contents of selected heavy metals in three different tissues were significantly lower than the proposed limits provided by United States Environmental Protection Agency (USEPA). The maximum accumulations of Cd and Pb were in the hepatopancreas, while the maximum accumulation of As was in the gonad, and the maximum accumulations of Hg and Cr were in the abdominal muscle. The highest contents of Cr, Hg, and Pb were all detected in Dongting Lake, Hunan, which was consistent with the trend of the metal pollution index (MPI). Risk value of the target hazard quotient (THQ) was below 1.0, suggesting that the intake of selected heavy metals through crayfish consumption would not pose a significant health risk to consumers.


Introduction
With the rapid development of industrialization and urbanization, heavy metal pollution has become increasingly serious and received widespread attention in China (Huang et al. 2018). The majority of the known heavy metals can be readily absorbed and bio-accumulated in organisms via anthropogenic and natural emissions, and even those considered as essential can be toxic if present in excess (Jaishankar et al. 2014).
Potential health risks of heavy metal exposure have been documented in the past few decades. In particular, recent studies have demonstrated that heavy metals have carcinogenic or non-carcinogenic risks to human beings (Peng et al. 2016a;Jia et al. 2017). An increased risk of stomach cancer and lung cancer after ingesting chromium (Cr VI) in drinking water in Liaoning Province of China was reported (Beaumont et al. 2008). Arsenic (As), which is more toxic in its inorganic form than in its organic form, was established as a group 1 carcinogen by the International Agency for Research on Cancer (IARC). Inhalation of inorganic As caused lung cancer in smelter workers (Enterline et al. 1987). Inorganic As might be associated with fetal loss, low birth weight, cognitive deficit, skin impairment, and cardiovascular disease (Smith and Steinmaus 2009). Cadmium (Cd) can cause acute kidney damage, spontaneous abortion, bone damage, and cancer through absorption of the stomach, intestine, skin, and lung (Godt et al. 2006). Lead (Pb) is reported to be an accumulative metabolic poison, which affects the nervous, cardiovascular, renal, hematopoietic, and reproductive systems in human. It can also Aijie Mo and Yangyang Huang contributed  cause mental retardation and hyperactivity in children (Demayo et al. 1982). Elemental mercury (Hg) was harmful for the central nervous system, while inorganic Hg compounds primarily affected the kidney. Particularly, methylmercury (MeHg) was a potent neurotoxin (Beckers and Rinklebe 2017). Even low dose Hg was reported to decrease performance in motor function and memory not only in children but also in adults (Zahir et al. 2005). Therefore, it is very important to evaluate the potential risks to human health caused by food intake contaminated by heavy metals. Red swamp crayfish (Procambarus clarkii) have been farmed extensively and become one of the most economically important farmed aquatic species in China since the 1990s. Nowadays, China is the world's leading crayfish producer (Wang et al. 2005;Yi et al. 2018;Mo et al. 2019). The geographic conditions in Hubei, Hunan, Jiangxi, Anhui, Jiangsu, and Shandong make them be the main production areas of edible crayfish in China. Wild crayfish was most commonly found in warm fresh water, such as lakes, ponds, rivers, and wetlands. Chaohu Lake, Poyang Lake, Taihu Lake, Dongting Lake, and Weishan Lake are main sources of freshwater and aquatic products for local residents in these provinces. Evidences from previous studies suggested that the seven lakes mentioned above are endangered with various pollutants, especially heavy metals (Chi et al. 2007;Yang et al. 2009;Li et al. 2013;Wei et al. 2014).
Crayfish camps on the special benthic lifestyle, and serves as a high-level consumer of benthic animals in natural water bodies. It can not only accumulate heavy metals in its own body through the food chain, but also can survive and multiply under the stress of heavy metals (Alcorlo et al. 2006;Kuklina et al. 2014). Risk to human health via crayfish consumption has been reported based on a small-scale survey in Shanghai (Wu et al. 2010). The consumption of crayfish in the peak summer season reached more than 100 metric tons per day in Nanjing, Wuhan, Hefei, and other central cities in these six provinces (Mu et al. 2007). Moreover, wild crayfish account for a part of the entire crayfish consumption and consumers tend to think that large wild fish, foraging for natural food, are more nutritious than pond-raised fish (Xiong et al. 2020). However, Xiong et al. (2020) found that the average heavy metal concentrations in cultured crayfish were lower than those in wild crayfish. Given the abovementioned heavy metal pollution and wide population exposure, the health risk of crayfish consumption in these regions deserves attention. However, limited studies focus on contamination with heavy metals and health risk in the main production and consumption region of crayfish.
In order to provide information about heavy metal pollution in wild crayfish and further conduct risk assessment, this study investigated the contents of five heavy metals (Cr, As, Hg, Cd, and Pb) in three edible parts of field-collected crayfish from six provinces. Metal pollution index (MPI) and target hazard quotient (THQ) were used to evaluate the health risk of heavy metals after consumption of crayfish.

Sample collection and preparation
The wild red swamp crayfish (Procambarus clarkii) were obtained from the lakes in six provinces from August to October 2018, including Hubei, Hunan, Jiangxi, Shandong, Anhui, and Jiangsu Provinces (Fig. 1), which have higher crayfish consumption levels. A total of 177 specimens (about 25 specimens every sampling sites) were collected. The crayfish were weighed (Table 1), killed, and dissected. And then the gonad, hepatopancreas, and abdominal muscle were sampled. The same tissues from 9 crayfish in each site were mixed as a mixed sample after separation. The hepatopancreas was a metabolically very active organ that could absorb and sequester heavy metals for detoxification (Chavez-Crooker et al. 2003). The dissected gonad, hepatopancreas, and abdominal muscle were washed with deionized water, dried at 60°C for 24 h to determine dry weight, grounded with a pestle and mortar, and then stored at −20°C in sealed polyethylene bags until analysis.

Heavy metal analysis
The digestion of crayfish samples was performed according to the methods described by Suami et al. (2018) with some modifications. Briefly, approximately 0.3 g of the tissue sample was weighed into a microwave digestion inner tank. They were digested with 10 mL of a suprapur HNO3 (Nitric acid 65% Suprapur®)-HClO4 (perchloric acid 70%) mixture (3:1). Then the microwave digestion tank was covered, placed for 24 h, and performed by a microwave digestion system (MARS-6, China Everbest Machinery Industry Co., Ltd., Shenzhen, China), according to the following program: 5 min to 120°C , 5min at 120°C, 5 min to 150°C, 10 min at 150°C, 5 min to 190°C, and 20 min at 190°C. After cooling, the digestion solution was diluted to 50 mL for later use. The contents of As, Cr, Pb, and Cd based on dry weight (d.w.) were determined by inductively coupled plasma mass spectrometry (ICP-MS, Agilent 7700x, Tokyo, Japan). Sc, Ge, Rh, and Lu were applied for instrument calibration. The concentrations of Hg were measured by atomic fluorescence spectrometer (AFS, Beijing Titan Instruments Co., Ltd., Beijing, China). All reagents used for digestion achieved trace metal grade, and no metals were detected in the blank sample which was prepared following the integral pretreatment procedure only with the digestion reagents. The reporting limits for Pb, Cd, Hg, As, and Cr were 5.0, 0.5, 0.3, 0.5, and 2.0 μg/kg, respectively. The contents of Pb, Cd, Hg, As, and Cr in crayfish tissues, rice, straws, and soils were all normalized to dry sample weights. To ensure analytical accuracy, the blanks and the duplicates were tested after every five samples. Toxic elements standards were spiked and digested to check for recovery. The recoveries of Pb, Cd, Hg, As, and Cr in the standard reference materials were 89%, 93%, 91%, 85% and 90%, respectively. Relative standard deviations (RSDs) of repeated measurements were < 10%. By calculating the moisture content of different samples, the contents of heavy metals in different tissues based on wet weight (w.w.) were converted to the contents of heavy metals based on dry weight.

Health risk assessment
Total heavy metal accumulation in various tissues at different sampling sites was examined by the metal pollution index (MPI). The MPI was described by the following equation (Usero et al. 1997): where C Cr , C As , C Hg , C Cd , and C Pb (mg/kg w.w.) are the contents of Cr, As, Hg, Cd, and Pb based on wet weight in different tissue of crayfish sampled at each sampling sites. Muscle and hepatopancreas are the main tissues of crayfish consumed by local residents. Therefore, this study evaluated the health risks of muscle and hepatopancreas consumption. Firstly, the daily heavy metal intake from crayfish consumption was estimated. Estimated daily intake (EDI) (μg/BW kg day) was calculated through the following formula: where C i (mg/kg w.w.) is the content of heavy metal based on wet weight, DIR (g/day) is the average daily intake of crayfish by local residents, and BW is the average weight of consumers. The DIR for inhabitants is 2.0 g/day/person for the abdominal muscle and 1.0 g/day/person for the hepatopancreas based on annual total crayfish production minus total export in middle and lower reaches of Yangtze River (MLYR) region. MLYR is the main area of crayfish production and consumption in China. The BW for inhabitants is 60 kg (Peng et al. 2016a). The target hazard quotients (THQ) index was applied to assess the potential non-cancer risk associated with consumption of the different species of marine organisms sampled. The THQ value was calculated on the base of the metals concentrations recorded in the edible parts of the organisms. THQ values exceeding one unit indicate a potential health risk to the consumers (USEPA 1989). Then the potential health risks from exposure to heavy metal through crayfish consumption were assessed using the target hazard quotient (THQ) regulated by USEPA (1989), described as follows: where ED is the exposure duration (year), EF is the exposure frequency (day/year), and AT is the average exposure time (day). In this study, the AT, ED, and EF were set as 25,550 days, 70 years, and 365 days/year, respectively. RfD i (mg/kg/ day) is the chronic oral reference dose for heavy metal regulated by USEPA (2019). The values of RfD i for heavy metals is 3.0×10 −3 mg/kg/day for Cr, 3×10 −4 mg/kg/day for As, 1×10 −4 mg/kg/day for Hg, 1.0×10 −3 mg/kg/day for Cd, and 2×10 −2 mg/kg/day for Pb, respectively (Wei et al. 2014;USEPA 2019).

Results and discussion
Heavy metal contents in three different tissues of field-collected crayfish The contents of the selected heavy metals (Cr, As, Hg, Cd, and Pb) in the hepatopancreas, abdominal muscle, and gonad of crayfish collected from 7 sampling sites were summarized in Table 2. Hexavalent chromium was a group 1 carcinogen with multiple complex mechanisms by which it triggers cancer development. Increased levels of DNA adduct formation, chromosome breaks, and oxidative stress were the main mechanisms of Cr VI causing cellular damage (DesMarais and Costa 2019). The highest content of Cr was 59.59±0.26 (×10 −2 mg/kg d.w.) in the abdominal muscle of crayfish from Dongting Lake. This result was consistent with these observations that the average of Cr content in sediment from Dongting Lake (88.29 mg/kg) was higher than Cr contents from Poyang Lake (70.77 mg/kg), Taihu Lake (41.5 mg/kg), and Chaohu Lake (63.36 mg/kg) (Yuan et al. 2011;Jiang et al. 2012;Tang et al. 2010;Li et al. 2013). The lowest content of Cr was 0.29±0.29 (×10 −2 mg/kg d.w.) in gonad from Anqing Area. The contents of Cr in the abdominal muscle were significantly higher than those in other tissues collected from 5 sampling sites (Chaohu Lake, Waishan Lake, Dongting Lake, Jianli Area, and Anqing Area). Kuklina et al. (2014) also found that Cr was more concentrated in the abdominal muscle. However, the content of Cr in the abdominal muscle was far lower than those in the exoskeleton, hepatopancreas, and gills of crayfish after exposure to waterborne Cr (Bollinger et al. 1997). For fish, Cr was investigated to enrich in the bladder and liver (Wei et al. 2014). Possible reason was that the enrichment of Cr might be specific and change according to the surrounding environment. Organic As was non-toxic whereas inorganic As was toxic. Trivalent form (As 2 O 3 ) and pentavalent form (As 2 O 5 ) are two oxidation states of inorganic As. However, As (III) is 60 times more toxic than As (V) (Ratnaike 2003). In this study, the highest content of As was 273.79±3.06 (×10 −2 mg/kg d.w.) in the hepatopancreas of crayfish collected from Taihu Lake, while the lowest content of As (14.11±0. 35 (×10 −2 mg/kg d.w.)) was observed in the abdominal muscle collected from Chaohu Lake. It was reported that the content of As in crayfish muscle collected from Chaohu Lake (14.02-24.10 (×10 −2 mg/kg d.w.) was higher than those in crayfish muscle collected from Poyang Lake (0.010-0.080 mg/kg d.w.) and Xiang River (0.037-0.141mg/kg d.w.) (Wei et al. 2014;Jia et al. 2017). These results may indicate that As is more easily enriched in crayfish than other aquatic invertebrates. Results showed that As contents in the hepatopancreas and gonad were markedly higher than those in the muscle. Because the allowable values of As in fish are very different values and the few portions of total As (about the 3%) are inorganic As, which is more toxic, it cannot be assessed with certainty (Okati et al. 2021). In this research, there were no conditions for measuring the inorganic arsenic concentration of crayfish. However, according to the US FDA (1993), the content of inorganic As could be estimated as 10% of total As. Based on this parameter, the contents of inorganic arsenic in all the samples did not exceed the safety limit set by the China National Standards Management Department (CNSMD).
Since the Minamata incident in Japan in 1950s, methylmercury in the aquatic environment and aquatic organisms has raised global concerns (Harada 1995). Peng et al. (2016a) reported that MeHg constituted 92-99% of mercury in crayfish muscle. The highest content of Hg was in the muscle from Dongting Lake (6.13±0.22 (×10 −2 mg/kg d.w.)) and the lowest content of Hg was in the gonad from Taihu Lake (0.65 ±0.03 (×10 −2 mg/kg d.w.)). These results were consistent with the content of Hg in the muscle of crayfish collected from 23 cities in China (58.1±19.2 μg/kg). Notably, unlike the other four heavy metals, the total contents of Hg in the hepatopancreas and gonad were significantly lower than that in muscle. The same results were also observed in previous researches (Stinson and Eaton 1983;Goldstein et al. 1996;Wei et al. 2014).
Cadmium was one of the global health problems that affected many organs, and in some cases, it could even cause deaths. Long-term exposure to cadmium through food, soil, water, and air might cause cancer and organ system toxicity such as cardiovascular, skeletal, reproductive, urinary, respiratory systems, and central and peripheral nervous (Rahimzadeh et al. 2017). The highest Cd content was detected in the hepatopancreas of crayfish collected from Poyang Lake (456.71±4.07 (×10 −2 mg/kg d.w.)). The contents of Cd in the hepatopancreas of crayfish collected from Poyang Lake, Taihu Lake, Dongting Lake, Anqing Area, and Jianli Area exceeded the threshold values in the national food safety standards of China. The contents of Cd in sediment were 0.13-1.49 mg/kg in Poyang Lake, 1.71 mg/kg in Dongting Lake, and 0.20-2.88 mg/kg in Taihu Lake, which exceeded the content of class three (1 mg/kg) from the Chinese Environmental Quality Standard for sediment Qin et al. 2012;Hu et al. 2015). These evidences indicated that cadmium pollution was serious in these areas. The hepatopancreas is the main organ of cadmium accumulation and detoxification in crayfish (Kouba et al. 2010). The contents of Cd in the abdominal muscle and gonad (<0.89 ×10 −2 mg/kg d.w.) were far below the Cd contents in the hepatopancreas in this study. The contents of Cd in the hepatopancreas exhibited an apparent positive correlation between accumulation time and exposure contents while the muscle did not ). This might be due to the existence of metallothionein proteins in the hepatopancreas which could bind Cd for detoxification (Ploetz et al. 2007). The present study showed that the contents of Cd in muscle were lower than those reported in other studies, including 1.2-60.6 μg/kg d.w. Cd from 12 provinces in China (Peng et al. 2016b), and 0.08 mg/kg d.w. Cd from Lake Washington (Stinson and Eaton 1983). Table 2 Heavy contents (Cr, As, Hg, Cd, and Pb) (mg/kg w.w.) of crayfish (hepatopancreas, muscle, and gonad) sampled from 7 locations in China (mean ± SD)

Location
Tissues Heavy metal content (× 10 -2 mg/kg w.w.) Lead toxicity is a major public health problem in developed and developing countries. Both acute and chronic exposure to lead have the potential to cause many deleterious systematic effects, including immune imbalances, frank anemia, hypertension, cognitive deficits, vitamin D deficiency, infertility, gastrointestinal effects, and delayed skeletal and deciduous dental development (Mitra et al. 2017). The highest content of Pb was observed in the hepatopancreas collected from Dongting Lake (21.41±0.20 (×10 −2 mg/kg d.w.)). The contents of Pb in the abdominal muscle ranged from 0.0062 mg/kg d.w. to 3.73 ×10 −2 mg/kg d.w., which was consistent with the values (mean 0.023 mg/kg d.w.) reported by Peng et al. (2016b). The contents of Pb in gonad were not detected, except those in Dongting Lake. The contents of Pb in the hepatopancreas were slightly higher when compared with those in the abdominal muscle and gonad. The same results were also reported by previous studies (Wei et al. 2014;Jia et al. 2017). Roldan and Shivers (1987) indicated that Pb could store in metal-containing vacuoles of hepatopancreatic cells.
Metals showed different affinity to organs, which might be due to the different functions of organs and metabolic roles of metals (Ashraf 2005). As presented in Table 2, it was concluded that the hepatopancreas was the primary organ for Cd, As, and Pb deposition, the abdominal muscle was the ideal organ for Cr and Hg deposition, and the gonad was the primary organs for As deposition. The maximum limit required of these heavy metals for crayfish in the national food safety standards of China was 2 mg/kg w.w. (Cr), 0.5 mg/kg w.w. (Inorganic As), 0.5 mg/kg w.w. (MeHg), 0.5 mg/kg w.w. (Cd), and 0.5 mg/kg w.w. (Pb), respectively (GB 2762(GB 2017. The values of Cr, As, Hg, and Pb contents in these three different tissues of crayfish all met national food safety standards of China. However, the content of Cd in the hepatopancreas exceeded the lowest limit. Hence, it is necessary to conduct health risk assessment of the edible part of crayfish. Health risk assessment Fig. 2 presented the MPI values for heavy metals in three tissues collected from seven sampling points. Considering the MPI in different tissues, the distribution of the heavy metals was in the ascending order of abdominal muscle < gonad < hepatopancreas for the seven sites. Generally, the muscle is weak to accumulate heavy metals. The liver and gill, as metabolically active organs, have a great tendency to store high levels of heavy metals (Monikh et al. 2013). Considering the MPI in the same tissues, the order of MPI in the hepatopancreas was as follows: Jianli Area > Poyang Lake > Dongting Lake > Taihu Lake > Anqing Area > Weishan Lake > Chaohu Lake; the order of MPI in the abdominal muscle was as follows: Dongting Lake > Chaohu Lake > Jianli Area > Anqing Area > Poyang Lake > Weishan Lake > Taihu Lake; the order of MPI in the gonad was as follows: Dongting Lake > Chaohu Lake > Poyang Lake > Taihu Lake > Jianli Area > Anqing Area.
The THQ provided an indication of the risk level associated with pollutant exposure. This method of risk estimation had recently been used by many researchers and had been shown to be valid and useful (Yi et al. 2011). In this study, the index of THQ was introduced to estimate potential health risk of chronic exposure to heavy metals in crayfish. As shown in Fig. 3, the average THQ of individual heavy metal in the abdominal muscle followed the order 1 > Hg > Cr > As > Cd > Pb for all sampling sites. It was worth noting that the hepatopancreas was the favorite food of crayfish for Chinese, while the heavy metals tended to be enriched in the hepatopancreas, so it could not be ignored when evaluated. The Fig. 2 The MPI in different tissues and sampling sites Fig. 3 The THQ of heavy metals through abdominal muscle consumption of crayfish collected from 7 sampling sites average THQ of individual heavy metal in the hepatopancreas followed the order 1 > Cd > As > Hg > Cr > Pb (Fig. 4), suggesting that risk of heavy metals exposure via crayfish consumption in these locations is extremely low.

Conclusions
Heavy metal pollution has become a crucial environmental problem in China. The aim of this paper was to investigate the contamination of five heavy metals (Cr, As, Hg, Cd, and Pb) of wild crayfish in China. The results showed that the contents of these five heavy metals in crayfish in the main production and consumption area were mostly below the national safety standards, suggesting heavy metal pollution in these regions has been controlled. Health risk assessment indicated that exposure to selected heavy metals from crayfish consumption had no noncarcinogenic health risk to local inhabitants. However, given the importance of crayfish in the consumption of aquatic products by Chinese people, more research should be conducted on the relationship between harmful pollutants and human health and environmental sanitation.
Funding This work was supported by the National Natural Science Foundation of China (31770553) and the Fundamental Research Funds for the Central Universities (2662019FW008).
Data availability Data and material access are not available.

Declarations
Ethical approval and consent to participate Not applicable.

Consent for publication Not applicable.
Competing interests The authors declare no competing interests.