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Introduction
Various types of pollutants in sewage drainage or marine wastewater continuously flow into coastal waters from land. Among them, heavy metals flow into sediments or are adsorbed and accumulated in aquatic organisms because of their physicochemical properties. These characteristics, enable the monitoring of heavy metals in marine organisms to directly determine the pollution level of marine ecosystems and predict the effects on the human body. Thus, several studies have been conducted on this in each country. Korea and Japan are countries that consume a lot of seafood. In Korea, the annual intake of seafood per person is 58.4 kg/year, which is relatively higher than that of Norway (53.3 kg/year), Japan (50.2 kg/year), and China (39.5 kg/year) (FAO, 2016). In particular, shellfish are adherent organisms that rarely migrate, they have a wide distribution and long lifespan. Therefore, it is a useful indicator for identifying the origin and actual condition of pollutants through the identification of heavy metals with strong concentrations (Viarengo & Canesi, 1991). In addition, in Korea, heavy metals are highly likely to accumulate in the body because of shellfish consumption; thus, it is essential to evaluate human exposure to heavy metals and risk assessment.
In general, heavy metals are associated with mercury (Hg), cadmium (Cd), lead (Pb), and arsenic (As). They are well known as harmful contaminants. According to the Risk Assessment of Cadmium by MFDS (2016), the exposure of Cd through food was 0.292 μg/kg b.w./day. It was also found that marine products (0.141 μg/kg b.w./day) contributed the most. Joint FAO/WHO Expert Committee on Food Additives (JECFA, 2020) strengthens human exposure safety standards for major heavy metals such as lead, Cd, As, and Hg, and strengthens international standards for heavy metals in food.
In this study, copper (Cu) and zinc (Zn), which are essential heavy metals for the human body, and Hg, Pb, Cd, As, chromium (Cr), silver (Ag), and nickel (Ni), which are non-essential heavy metals, were analyzed in shellfish. Heavy metals are classified into two heavy metals due to their toxicity. Heavy metals were classified into two groups based on their toxicity, essential and non-essential heavy metal. Essential heavy metals are harmless or relatively less harmless at low concentration (Zn, Cu, Iron [Fe], and Cobalt [Co]). Non-essential metals are highly toxic even at low concentration (such as Cd, Hg, As, and Cr) (Kim et al., 2019). Cu and Zn, which are essential heavy metals, have metabolic effects when consumed in an appropriate amount. However, Hg, Pb, and Cd are classified as toxic substances and cause carcinogenesis when accumulated in the human body. In addition, As and Cr cause disease when ingested above the limit value.
Korea is currently conducting heavy metal safety management that focuses on exported shellfish. However, heavy metal safety management of shellfish for domestic use produced in coastal areas of the country is also required.
In this study, nine types of heavy metal monitoring were performed on the different species of shellfish. In addition, a risk assessment was done on shellfish in which heavy metals were detected. This research data is considered to help establish guidelines and reference data for safe seafood consumption.
Materials and Methods
Samples were collected from January to July 2018 by selecting representative species according to regional characteristics in 11 regions, including mussel (26), short neck clam (19), oysters (17), abalone (12), horned turban (11), scallop (6), surf clam (6), ark shell clam (4), comb pen clam (2), and sea squirt (8). The sampling sites are shown in Fig. 1. The collected samples were washed with distilled water according to the sample treatment manual for heavy metal experiment in seafood (MFDS, 2014). After washing, the intestines were removed from the large shellfish and only the muscles and small shellfish were separated and rewashed. The separated samples were frozen at –20°C until analysis. The frozen samples were used for analyses after freeze-drying using a freeze-dryer (OPR-FDT8612, Operon, Gimpo, Korea). In addition, the moisture content of the wet and dry samples was measured according to the food code (MFDS, 2019).
Nitric acid was used as the reagent for sample pretreatment (Suprapur, 65%, Merck, Darmstadt, Germany). Distilled water was deionized using the Milli-Q water purification system (Millipore, Bedford, MA, USA) to obtain 18.2 mΩ. The species analyzed in this study are Hg, lead, Cd, As, Cr, Cu, Ni, Zn, and Ag. Multi-Element Calibration Standard 3 (PerkinElmer, Waltham, MA, USA) was used as the calibration standard, and the certified reference material (CRM) was 1566b (oyster tissue; NIST, Gaithersburg, MD, USA) to verified the recovery (%). CRM did not contain Cr therefore it was prepared by adding the Cr standard (Fisher Chemical, Waltham, MA, USA). The Hg-certified reference standard (Fisher Chemical) was the calibration standard.
Samples for heavy metal analysis excluding Hg, were used for food code (MFDS, 2019) to ensure the reproducibility and accuracy of the experimental results. The microwave method was applied for pretreatment. In other words, 10 mL of nitric acid was added to 0.5 g of a sample, digested by a microwave digestion system (START D, Milestone, Sorisole, BG, Italy), transferred to a volumetric flask, and used as a sample solution. An inductively coupled plasma mass spectrometer (inductively coupled plasma-mass spectrometer, 7700, Agilent Technologie, Santa Clara, CA, USA) was used for heavy metal analysis. Then, Hg was analyzed using the amalgam heat vaporization method (Hydra-C, Teledyne Instruments, OH, USA), and the sample weight was 0.02 g. Table 1 presents the conditions of each analytical instrument.
The analysis results of this study validated recovery, limit of detection (LOD), limit of quantification (LOQ), and linearity of the standard calibration curve using blank, duplicated, and spiked samples according to Environmental Protection Agency (EPA) Method 3052 (US EPA, 2014).
The recovery (%) was verified using CRM (NIST, 1566b, oyster tissue). However, Cr was not contained in CRM and was confirmed using the spiked sample. Consequently, seven samples were prepared at a concentration near LOQ. In addition, the value obtained by multiplying the standard deviation of the concentration using the test method by 3.14 was determined as LOD, and the value obtained by multiplying by 10 was determined as LOQ (NIER, 2011).
The risk assessment was performed by calculating the provisional tolerable weekly intake (PTWI) from the heavy metal concentration (mg/kg, ww) of shellfish and shellfish ingestion rate (g/person/day) detected in this study, and comparing it with health based guidance value of JECFA (Table 2).
The PTWI of heavy metals for Korean people through shellfish consumption was calculated using the following formula and expressed as µg per kg of body weight per day (µg/kg b.w./day):
where C is the heavy metal concentration in shellfishes (mg/g, ww); IR is the shellfish ingestion rate (g/person/day) as used in national food and nutrition statistics (KHIDI, 2016) (Table 3); ED is the exposure duration (seven days); ABW is the average body weight (64.2 kg for adults) as used in Korean exposure factor (NIER, 2020).
Results and Discussion
In this study, the recovery rate was excluded from CRM (NIST, 1566b, oyster tissue) and Cr, consisting the same matrix as shellfish. The results of the spiked samples, showed that the recovery rate ranged from 83.8% to 102% (Table 4). The LOD and LOQ of the test method were less than 0.001 mg/kg (Table 5).
| Pb | Cd | As | Cr | Cu | Ni | Zn | Ag | Hg | |
|---|---|---|---|---|---|---|---|---|---|
| LOD (mg/kg) | 0.0002 | 0.0002 | 0.0003 | 0.0001 | 0.0002 | 0.0003 | 0.0003 | 0.0003 | 0.0003 |
| LOQ (mg/kg) | 0.0005 | 0.0008 | 0.0008 | 0.0005 | 0.0006 | 0.0009 | 0.0010 | 0.0010 | 0.0008 |
The coefficient of determination (R2) of the standard calibration curve obtained by analyzing the standard solution for each step using the US EPA Method 6020B (US EPA, 2014) was 0.99 or higher. Furthermore, to the standard calibration curve, the intermediate concentration was analyzed once for every ten samples immediately after the test curve through the environmental test/inspection QA/QC handbook. The concentration satisfied the range from 90% to 110%.
A blank sample was prepared using the US EPA Method 3052 (US EPA, 1996) and analyzed following the same procedure as the sample. As a result of identifying sample contamination in the experimental procedure, there was no effect on the experiment. Moreover, reproducibility (%) was calculated from a duplicate sample analysis of one sample for every 20 samples, and it was 0.25% to 16.8%. Moreover, the accuracy (%) of the experimental process was obtained by analyzing one spiked sample for every 20 samples, and it was 75.9% to 119%.
Moisture was measured to calculate the content of heavy metals in the sample by wet weight. The moisture content of shellfish samples was abalone (71.5%–76.7%), ark shell clam (69.4%–74.9%), comb pen clam (69.0%–75.1%), horned turban (72.5%–77.9%), mussel (74.3%–81.9%), oyster (68.7%–78.8%), scallop (75.8%–82.6%), sea squirt (78.6%–82.1%), short neck clam (71.2%–83.0%), and surf clam (74.9%–79.2%).
The results of heavy metal concentration by shellfish are shown in Table 6 and Fig. 2. In the food code, the maximum levels of heavy metal concentrations for shellfish in seafood are 2, 2, and 0.5 mg/kg for Pb, Cd, and Hg, respectively. The heavy metal concentrations in all the shellfish samples analyzed were lower than the maximum levels. Among heavy metals without a maximum level, Cu (maximum of 143 mg/kg) and Zn (maximum of 466 mg/kg) in oysters were relatively high.
In particular, the concentrations of Zn and Cu are detected higher than other heavy metals. This is because these substances accumulate relatively more than other heavy metals in the human body. Biswas et al. (2013) measured heavy metals in oysters and reported that Zn or Cu, along with Fe, may accumulate preferentially in the body compared to other heavy metals.
Comparing the results of this study with the results of other studies (Table 7), Zn and Cu showed higher concentration levels than other species. The concentrations of Zn (6.69 to 466 mg/kg) and Cu (0.0636 to 143 mg/kg) were similar to or higher than those of other studies (Zn: 3.34 to 217 mg/kg; Cu: 0.9 to 137 mg/kg). Furthermore, As (0.460 to 15.0 mg/kg) was higher than the Korean results (0.095 to 3.545 mg/kg). The samples with a high concentration of As are shellfish that mainly inhabit the bottom of the sea, such as horned turnban, ark shell clam, and short neck clam, which are considered to be influenced by the environment. However, it showed a lower concentration range than the foreign results (0.216 to 22.1 mg/kg). Moreover, Cd, Cr, Ni, Pb, and Hg were similar to or lower than the Korean and foreign results. The concentration of heavy metals was compared by the region where shellfishes were collected (Table 8). Cu and Zn, which have higher concentrations than other heavy metals, showed relatively higher concentrations in Jeonnam than in other regions. Also, high concentrations of Cu and Zn were detected in Gyeonggi and Incheon. All samples with high concentrations of Cu and Zn were oyster samples. In addition, heavy metals, except Cu and Zn, were detected in a similar range; thus, there is no significant difference by region.
| Country | Heavy metal (mg/kg, ww) | Reference | |||||||
|---|---|---|---|---|---|---|---|---|---|
| Pb | Cd | As | Cr | Cu | Ni | Zn | Hg | ||
| Korea | 0.761–1.708 | 0.168–0.247 | – | 0.682–1.645 | 3.914–6.484 | 0.785–1.998 | 40.36–77.98 | – | Hwang et al., 2001 |
| Korea | 0.763–1.306 | 0.128–0.298 | – | 0.648–1.346 | 8.89–27.48 | 0.810–1.856 | 97.9–217.0 | – | Hwang et al., 2002 |
| Korea | 0.045–0.210 | 0.198–2.654 | – | ND | 1.385–9.985 | – | – | – | Kim et al., 2002 |
| Korea | Trace–0.984 | 0.030–0.617 | – | Trace–0.849 | – | – | 0.002–0.020 | Kim et al., 2003 | |
| Korea | ND–1.38 | ND–1.85 | – | – | – | – | – | ND–0.19 | Ham, 2002 |
| Korea | 0.5471–1.1571 | 0.1748–0.3529 | – | 0.4280–0.9300 | – | – | – | 0.0057–0.0203 | Ha & Song, 2004 |
| Korea | 0.061–0.246 | 0.010–0.256 | 0.095–3.545 | – | – | – | – | 0.002–0.052 | KCA, 2011 |
| India | ND | 0.1 | – | 0.9 | 6.5 | 0.8 | 15.7 | ND | Sankar et al., 2006 |
| Greece | 0.68 | 0.34 | – | 0.26 | 1.4 | 0.34 | 40 | 0.024 | Copat et al., 2013 |
| China | 0.033–0.243 | 0.027–0.329 | 0.216–0.807 | – | 1.31–9.11 | – | 3.34–17.90 | 0.006–0.022 | Huang et al., 2007 |
| Malaysia | 0.18–0.88 | 0.04–5.45 | 0.95–22.10 | 0.47–3.39 | – | 0.37–2.23 | – | – | Sharif et al., 2016 |
| Egypt | 0.2–17 | 0.04–1.7 | – | – | 0.9–137 | 7.8–41 | 41–192 | ND–0.2 | Nemr et al., 2016 |
We compared Korean and foreign maximum levels with the results of this study to evaluate the safety of shellfish produced along the Korean coast. The maximum concentration of Pb detected in shellfish in this study was 1.02 mg/kg, which was lower than that of Korea (2.0 mg/kg), EU (1.5 mg/kg), and Australian (2.0 mg/kg) levels. It was also higher than the Chinese maximum level of 1.0 mg/kg. Cd was detected at a maximum of 1.56 mg/kg, which was lower than the Korean, Chinese, and Australian maximum levels of 2.0 mg/kg; however, it exceeded the EU level of 1.0 mg/kg. Hg was detected at a maximum of 0.174 mg/kg, which was lower than all national and international standards. Although all national standards were satisfied with this result, it may have been detected at a higher concentration than some foreign standards, and continuous monitoring will be required in the future.
Human exposure was calculated for a total person average for all surveyors in the National Health and Nutrition Examination Survey (KHIDI, 2016) and an ingestion person average for only the ingestion person. The results are presented in Table 9. Since the human body exposure to all surveyors is an average value including the case of not ingesting, the human body exposure to only the ingestion person was further calculated to investigate the effect on the actual ingestion person. The total amount of human body exposure for all surveyors in the National Health and Nutrition Examination Survey (KHIDI, 2016) is shown for each heavy metal substance. However, when only the ingestion person is targeted, the populations differ, and the total amount of human body exposure is calculated. Table 2 presents the health based guidance value of JECFA. Among the heavy metals investigated, PTWI of JECFA was suggested for Cd, Cu, Zn, Hg, and As. Moreover, there were no standards for other heavy metal species. Therefore, the risk assessment through comparison with PTWI could not be performed. Health based guidance value are expressed as provisional tolerable daily, weekly, or monthly intake, which were converted to weekly (Table 2) in this study. The human exposure to heavy metals calculated in this study was lower than the health based guidance value of JECFA. This means that the exposure of the human body to Cd, Cu, Zn, Hg, and As by ingestion of shellfish in Korean coastal area does not risk.
Conclusion
In this study, we analyzed heavy metals in the Korean coastal shellfish. We used this result for a safety evaluation. The heavy metals investigated in this study were Hg, Pb, Cd, As, Cr, Cu, Ni, Zn, and Ag.
Based on the analysis results, Zn and Cu were detected at an average of 56.7 mg/kg (6.70 to 466 mg/kg) and 13.2 mg/kg (0.064 to 143 mg/kg), respectively, compared with other heavy metal. Pb (average of 0.208 mg/kg, 0.000750 to 1.02 mg/kg), Cd (average of 0.454 mg/kg, 0.0388 to 1.56 mg/ kg), and Hg (average of 0.0266 mg/kg, 0.00548 to 0.174 mg/kg) were detected. Additionally, As, Cr, Ni, and Ag with average concentrations of 4.02 (0.460 to 15.0 mg/kg), 0.167 (< LOQ to 0.820 mg/kg), 0.281 (< LOQ to 1.46 mg/kg), and 0.158 mg/kg (< LOQ to 1.15 mg/kg) were detected. Region-specific, shellfish, except that Cu and Zn were detected relatively high in oysters. No significant difference was found.
Most heavy metal materials were detected with a concentration tendency similar to that of other studies. The heavy metal results of Korean coastal shellfish met all Korean maximum levels. However, Pb and Cd exceed some foreign standards as the maximum detection concentration standard, and continuous monitoring in the future is considered necessary. The human body exposure calculated using the congestion intake and heavy metal detection concentrations was less than the JECFA human safety standard, and the risk of heavy metals due to shellfish intake was at an acceptable level. The results of this survey will be used as basic data for heavy metal pollution in the Korean coastal shellfish. It can also be used as policy data for the safe seafood supply.
