Distribution and safety assessment of heavy metals in fresh meat from Zhejiang, China

There are increasing concerns on heavy metals in animal derived foods. We analyzed the levels of As, Cd, Cr, Cu, Hg, Ni, and Pb in 1066 fresh meat samples including pork, beef, mutton, chicken and duck from Zhejiang province, southeast China. The average levels of As, Cd, Cr, Cu, Hg, Ni, and Pb were 0.018, 0.002, 0.061, 0.801, 0.0038, 0.055, and 0.029 mg/kg wet weight respectively. There are significant positive correlations among Cd, Hg and Pb (P < 0.05) and negative correlations for Cu–Pb or Cu–Cd (P < 0.05). The exposure assessment showed that the health risk to humans by consuming these meat products was relatively low. However, regular monitoring of heavy metals in meat products is still recommended considering their intensive industrial activities.

www.nature.com/scientificreports/ Chemical analysis. The concentrations of arsenic (As), cadmium (Cd), chromium (Cr), copper (Cu), mecury (Hg), nickel (Ni), and lead (Pb) were tested according to the Chinese standard analysis method of GB 5009.268-2016 22 . Briefly, samples (0.5-1.0 g) were digested in acid-clean Teflon vessels containing 6 mL HNO 3 in a Mars-6 microwave digestion system (CEM, Charlotte, NC, USA). The samples in closed vessels were heated at 190 °C for 20 min. After digestion, the residue was heated at 150 °C till nearly dry. For the Hg test, the digested sample was directly diluted without heating for removing residual acid. Then, it was diluted to 20 mL by deionized water for instrumental analysis. As, Cd, Cr, Cu, Hg, Ni, and Pb in all samples were tested using inductively coupled plasma mass spectrometry (ICP-MS) (NexION 300, Perkin Elmer, Inc., Shelton, CT USA). For quality assurance and quality control purposes, sample blanks, certified reference materials (CRMs), and duplicates of the samples (10% of the load) were applied in each batch of treated samples.
Method validation. The analytical procedures were verified by analysis of appropriate certified reference materials (CRMs) using the same digestion and analytical methods. Two CRMs (Table 1) were purchased from National Research Center for Certified Reference Materials, China (NRCCRM). Quantitative results (no more than 10% of the certified value) were obtained for targeted elements of CRMs. Limits of detection (LODs) were  Health risk assessment. According to the recommendation of the report Reliable Evaluation of Low-Level Contaminations of Food issued by WHO, half of LOD was assigned to all results of element levels below the LOD, where the proportion of data below the LOD is not more than 60% 24 . The targeted hazard quotient (THQ) and hazard index (HI) were used to estimate health risk according to US EPA's IRIS database 25 . We adopted the mean and 97.5th percentile (P97.5) of obtained element level to represent the consumers with normal and high exposure, respectively 21 . The sum of all THQs for each element was referred as the HI. The formulas were as follows: Ci is the average or P97.5 concentration of the element in meat samples (mg/kg wet weight); Di is the daily intake of livestock and poultry meat (112.9 g/capita/day) 23 ; Ed is the average exposure duration (e.g., 70 years) 20 ; Bw is the average weight (e.g., 60 kg) 20 ; At is the average lifetime (e.g., 70 years) 11 . RfD is the recommended reference dose (RfD) 26 ; According to US EPA guidelines for assessing conservative risk, HI were calculated by sum of the THQ. When HI < 1, no health risk is expected to occur; If HI ≥ 1, there is moderate or high risk for adverse human effects.
Statistical analysis. Data analysis and statistical analysis were performed using Excel (2017 edition) and SPSS16 (Tried edition). The difference was considered as significant by single factor analysis (one-way ANOVA) when P < 0.05. The correlation between each factor was analyzed by Pearson correlation analysis.

Results and discussion
Heavy metals in meats. Total 1066 meat samples including 511 pork samples, 250 chicken, 184 beef, 74 duck, and 47 mutton purchased from local markets of Zhejiang were analyzed in this study. As shown in Tables 2 and 3, average levels of As, Cd, Cr, Cu, Hg, Ni, and Pb were 0.018, 0.002, 0.061, 0.801, 0.0038, 0.055, and 0.029 mg/kg wet weight respectively. Based on the Chinese National Food Safety standard 27 , the maximum allowable concentrations (MAC) of As, Cd, Cr, Hg and Pb in meat in China were 0.5, 0.1, 1, 0.05 and 0.2 mg/kg. The number of sample exceeding the MAC is 1 for As, 2 for Hg and 10 for Pb. Our results were similar with those found in Beijing China, where there were Cr (0.573 mg/kg), Cd (0.015 mg/kg), Pb (0.167 mg/kg), As (0.053 mg/ kg), Hg (0.018 mg/kg) in meats (pork, beef, mutton, chicken) 28 and the results from Taiwan, China 29 . In some potential polluted areas, average levels of heavy metals, such Cd and Pb were more than 0.2 mg/kg in meat product 30,31 . It shows that levels of these metals in animals change with different area, where may have diverse sources of the contaminant.
Targeted Hazard Quotient Table 2. The concentration of heavy metals in meat samples from Zhejiang province (mg/kg fresh weight). a Target analytes with concentrations lower than LOD were treated as one-half of LOD when calculating the mean values. b Maximum allowable concentrations of contaminants in foods. www.nature.com/scientificreports/ Different animal species may have different bio-accumulation ability to heavy metals. The average levels of As, Cd, Cr, Cu, Hg, Ni, and Pb in different meat samples were shown in Fig. 2. By comparison with beef, chicken, duck and pork, mutton had relative lower levels of As, Cd, and Cr (P < 0.05). Mutton accumulated lower levels of As, Cd, and Cr, which may be caused by the grassy feed and less mineral supplement. High Cu concentration (average 3.1 mg/kg) was found in duck meat. Considering the nearly 80% water content in duck meat, our result was similar with the report of Aendo et al. 32 , who found duck meat with 15.28 mg/kg dry weight for Cu in Thailand. For Hg, Ni, and Pb, there was no significant difference among five targeted meats (P < 0.05). Furthermore, 4 of 248 chicken muscle samples contained Pb with levels above the safety threshold of 0.2 mg/kg (fresh weight) 27 . The ratio of over-limit was lower than that reported in Guangzhou, China where 2 of 63 muscle samples had Pb contents exceeding this limit 33 . But, the mean level (0.058 mg/kg) was higher than reported in Korea (0.005 mg/ kg) 34 . The Cd (0.002 mg/kg) in beef was lower than report of Hashemi 35 who found 0.28 mg/kg Cd in Iran. Feeds and mineral supplement products may be one of many sources of heavy metal for these animals.
Pearson correlation analysis (Table 4) showed that there were significant positive correlations for Cd-Hg (r = 0.9141, P < 0.05), Pb-Hg (r = 0.98837, P < 0.05) and Cd-Pb (r = 0.9504, P < 0.05) in meat samples. Negative correlations in Cu-Cd (r = − 0.6515, P < 0.05) and Cu-Pb (r = − 0.6101, P < 0.05) were found in our results. We suppose that two groups of Cd-Hg-Pb and Cu were accumulated by different sources. Actually, most of livestock and poultry in Zhejiang were farming with artificial feeds which may be the main source of heavy metals. The contamination incidence rates of harmful elements, such as Cd, Hg, Pb, and As in feedstuffs and feeds were high, and the feeds were usually contaminated with contaminated with Cr, followed by As, Cd, and Hg 9 . The mean As contents of chicken feeds collected in Jiangsu province of southern China was reported to be 0.13 mg/kg 36 while the total contents of As in poultry feeds in northeastern China varied from 0.02 to 6.42 mg/kg 37 . Studies found that level of Cu was 2 to 8 times higher than the required ones in poultry and livestock feeds in China 36,37 . As we known, compounds including Cu element were commonly used as a growth promoter in diets of poultry, especial duck 38 .

Exposure assessment and health risk.
According to the data of food consumption survey 23 , the estimated livestock and poultry meat intake was 112.9 g/day/person. The recommended reference doses (RfDs) or safe values were based on previous reports 31, 33 . The mean and high exposure was presented by the average and P97.5 elements levels, respectively. As shown in Table 4, mean exposure doses of As, Cd, Cr, Cu, Hg, Ni, and Pb by meat consumption were0.034, 0.004, 0.115, 1.507, 0.007, 0.103, and 0.055 μg/kg bw/day. And high exposure values were 0.207, 0.024, 0.598, 7.696, 0.047, 0.790, and 0.339 μg/kg bw/day. Our mean exposure data (As, Cd, Cr, Hg and Pb) were lower than those reported in Beijing, China 28 .
To appraise the health risk associated with these metals, targeted hazard quotient (THQ) was calculated by dividing daily intake of elements by their reference doses. Hazard index (HI) combined all THQs was adopted to assess the total health risk 39,40. An HI more than 1 is considered as not safe for human health. As shown in Table 5, all THQs were less than 1. Both mean and P97.5 HIs were no more than 1. HI for P97.5 level presented as the high exposure was 0.768. It indicated that there was low health risk to exposure of common toxic elements by intake of these meats. However, it should be noticed that other potential exposure pathways for foods, such as vegetables, cereals, fruits, and fish might be considered except for livestock and poultry meats.

Conclusion
The present study revealed the levels of As, Cd, Cr, Cu, Hg, Ni, and Pb in livestock and poultry meats from Zhejiang of southeast China, which showed samples with 0.09% (As), 0.19% (Hg) and 0.94% (Pb) were exceeding the maximum allowable concentrations set by Chinese legislation. Obvious positive correlations among Cd, Hg and Pb and negative correlations for Cu-Pb and Cu-Cd were found in analyzed samples. Dietary exposure assessment showed that there is relatively low health risk to these elements for general people in Zhejiang province of southeast China. However, it should be noted that the detailed information for animal species, feeding pattern cultivation, and feedstuffs was not involved in this study. Moreover, different heavy metal speciation showed diverse toxicity, such as organic and inorganic mercury. Our future survey will focus on the levels of heavy metal speciation in different animal products and feeds.