Metal content of Armillaria mellea in the Tumen River Basin

ABSTRACT

Food and environmental safety concerns as well as ecotoxicology considerations require investigation of the metal content of certain types of edible fungi. Thus, we conducted inductively coupled plasma optical emission spectrometry to obtain the metal content profiles of Cr, Mn, Fe, Ni, Cu, Zn, Pb, and Cd in Armillaria mellea (Vahl ex Fr.) Quel from nine zones in the Tumen River Basin. Statistical analysis showed that the characteristics of Yanji (Zone 1) differed from those of the other eight zones. Compared with the levels in the reference site (0.20 ± 0.01, 0.07 ± 0.02 µg/g for Pb and Cd), the Cd and Pb levels in three zones (1.2 ± 0.1, 8.5 ± 0.4 µg/g for Yanji; 0.35 ± 0.01, 0.73 ± 0.03 µg/g for Longjing; and 6.2 ± 0.4, 0.039 ± 0.000 µg/g for Hunchun) were higher than those listed in the Chinese Industrial Standards (WM/T2-2004; Pb ≤ 5.0 µg/g, Cd ≤ 0.3 µg/g). The present study can contribute data related to pollution control and food safety in the Tumen River Basin.

KEYWORDS:

• Metal content
• Armillaria mellea (Vahl ex Fr.) Quel
• Pollution control
• Food safety
• Tumen River Basin

Introduction

In recent years, the Chinese express delivery industry has grown faster than the gross domestic product of the country. No regional boundaries are set for food circulation. This development has implications on food quality, safety, and pollution control, which are issues that affect human lives. Since the survival of mankind depends on food and the natural environment, bioindicator selection is one of the ways to monitor and evaluate their quality.^[1,2] Attempts to utilize Artemisia capillaries,^[3] Rhodiola angusta, and Rhodiola sachalinensis A. Bor.^[4] as bioindicators are inadequate. Limited distribution prevents these species from becoming potential bioindicators. According to previous studies, mushrooms continue to be considered as an important research object. The quality of mushrooms have a direct effect on human life^[5–7] because mushrooms are edible and have nutritional and medicinal properties. Armillaria mellea (Vahl ex Fr.) Quel, which is commonly known as honey fungus^[8,9] is widely distributed in temperate regions of the northern hemisphere. A. mellea (Vahl ex Fr.) Quel has significant research and development value because its body, mycelium, and rhizomorph can be used in medicine production. Growing attention to A. mellea (Vahl ex Fr.) Quel has led to the production of medicinal and healthcare products that are sold in various markets.^[10] Many cultured products of A. mellea (Vahl ex Fr.) Quel have been used to develop commercial medicines.^[11,12] Called Zhen Mo in Chinese, A. mellea (Vahl ex Fr.) Quel is a widely grown species in the Tumen River Basin; it is popular among mushroom enthusiasts because it is also one of the unique mountain delicacies in this region.^[13–15] However, industrial development in China and North Korea has caused pollution in the Tumen River Basin, thereby leading to the accumulation of heavy metals in plants, such as mushrooms, and causing food safety problems. Some poisoning incidents have been reported^[11] as a result of consumption of wild mushrooms. Thus, market survey and risk assessment by identifying trace metals in edible fungi have been proposed.^[12] Investigating the conditions of mushroom growth in various zones is necessary to ensure pollution control and food safety, particularly because A. mellea (Vahl ex Fr.) Quel is a favorable bioindicator candidate.

Mn, Fe, Ni, Cu, and Zn are essential elements, whereas As, Pb, and Cd are potentially toxic metals. As, Cd, and Pb are among the most hazardous pollutants that can enter the component of human diet,^[16,17] edible mushrooms tend to accumulate metal content. The high metal concentrations in mushrooms indicate a potentially significant toxicological hazard. Therefore, the metal levels in mushrooms must be assessed, and potential contaminations that pose health risks must be studied. Previous research has been conducted on the use of algae to monitor and evaluate water pollution^[18] and the characteristics of riparian heavy metal pollution in the Tumen River.^[19]

The metal content of mushrooms is determined through methods such as atomic absorption spectrometry,^[20] inductively coupled plasma mass spectrometry,^[21,22] inductively coupled plasma optical emission spectroscopy,^[23] X-ray fluorescence methods, energy-dispersive X-ray fluorescence analysis,^[24] flame atomic absorption spectroscopy,^[25] and inductively coupled plasma atomic emission spectroscopy. Among these methods, inductively coupled plasma optical emission spectrometry (ICP-OES) has become the standard analytical technique for the rapid multi-element analysis of biological samples. To the best of our knowledge, the metal contents of A. mellea (Vahl ex Fr.) Quel collected from the Tumen River Basin have not been detected with ICP-OES.

Materials and methods

Aerial parts of A. mellea (Vahl ex Fr.) Quel^[26] were collected in 2014. Mushroom samples were confirmed taxonomically by Prof. Lv Hui-zi of Yanbian University College of Pharmacy. A voucher specimen (number YB-SYC-1410) was deposited at the College of Pharmacy, Yanbian University. A total of 270 samples of A. mellea (Vahl ex Fr.) Quel corresponding to 9 different zones (30 samples from each zone) were analyzed to obtain their metal content profiles. The habitat of A. mellea (Vahl ex Fr.) Quel is illustrated in Fig. 1. As mentioned, the mushroom typically grows on hardwoods, but may be found also on other living and dead wood as well as in open areas. In our study, 100 g of A. mellea (Vahl ex Fr.) Quel were collected from each of the 10 locations (three samples from one location) in different zones. All mushroom samples possessed species-typical color, flavor, smell, and appearance, and exhibited no alien invaders, molds, and moths that were visible to the naked eye.

Figure 1. Sampling sites in the Tumen River Basin.

Then, the samples were dried in an oven at 105°C for 24 h. The plasticware and glassware used were soaked overnight in 10% nitric acid solution and then rinsed with ultrapure water. Metal content was analyzed with a PerkinElmer Optima 7300DV ICP-OES. Carrier gas is argon. The wavelengths (nm) used to determine the contents of Fe, Zn, Cd, Pb, Cr, Mn, Ni, and Cu were 238.20, 213.86, 214.44, 220.35, 267.72, 257.61, 221.65, and 324.75, respectively. The following operational parameters were used: applied power, 1.3 kw; nebulization gas flow, 0.8 L/min; sample flow, 1.0 mL/min; auxiliary gas flow, 0.2 L/min; plasma gas flow, 15 L/min; pumping flow, 1.0 mL/min; injector tube diameter, 2.0 mm; radially viewed height, 14 mm; and interface, shear gas.^[27]

A standard multi-element 1000 mg L⁻¹ solution containing all the elements studied was used. Nitric acid (65%) and hydrogen peroxide (30%) were obtained from Merck. All working standard solutions were prepared daily from the stock solutions by simple dilution with ultrapure water obtained with a Millipore purification system (Millipore, Milford, MA, USA).

Quantitative analysis was conducted through 5-point linear calibration. Blank samples were processed together with the mushroom samples, and limits of detection were estimated as the average signal of the blanks plus five times the standard deviation of the signal of the blanks according to Chinese national standards (GB/T 27415-2013). Statistical analyses of the data were conducted by using SPSS version 19. Minitab Statistical Program Release 13.20 was used to determine the mean and RSD values. The data showed homogeneity of variance and were normally distributed.

To ensure accuracy and precision of the method, standard reference materials (SRM 1547 Peach Leaves, National Institute of Standards and Technology) were analyzed for corresponding elements. A control sample was digested and analyzed with each analytical batch of samples to validate the effectiveness of the digestion procedure. Each time the concentration deviated by more than 10% from the certified value, the calibration curve was reconstructed (Table 1).

Table 1. Metal values measured using certification standards (µg/g).

Metals	Peach leaves (spiked)	Determined	RSD/%	Recovery (%)
Fe	431	436 ± 29	2.87	101.4
Mn	75.4	75.9 ± 5.4	1.64	96.9
Zn	22.8	21.5 ± 2.5	1.86	98.5
Cu	10.4	10.5 ± 1.6	3.11	98.1
Pb	0.90	1.02 ± 0.01	2.59	98.0
Cr	0.90	0.96 ± 0.04	3.85	97.7
Ni	0.25	0.25 ± 0.02	2.26	95.9
Cd	0.010	0.021 ± 0.001	3.37	102.3
Cr	0.90	0.96 ± 0.04	3.85	97.7

LODs: Cd: 0.02 µg/g; Pb: 0.03 µg/g; Zn: 0.003 µg/g; Cr: 0.004 µg/g; Mn: 0.0005 µg/g; Cu: 0.002 µg/g; Fe: 0.002 µg/g; Ni: 0.009 µg/g.

Results and discussion

Researchers have investigated the metal elements in wild mushroom species along with the level of risk elements.^[28–31] However, data on the metal contents of A. mellea (Vahl ex Fr.) Quel from the Tumen River Basin are too limited and fragmentary to enable the assessment of ecological and food quality.^[32] Previous research has shown the advantages of microwave digestion;^[33] thus, this process was used to determine the metal contents (Cu, Fe, Cd, Cr, Ni, Pb, Mn, and Zn) of A. mellea (Vahl ex Fr.) Quel collected from the nine zones of the Tumen River Basin. Average of 30 samples was collected from each zone during onsite sampling in reliable growing areas. Subsequently, standard laboratory analysis^[29] was conducted on the samples to ensure that the data were representative and reliable. Table 2 shows the mean content of the metals.

Table 2. Mean contents of heavy metals in mushroom samples (µg/g).

Zones	Habitat	Fe	Mn	Zn	Cu	Pb	Ni	Cd	Cr
1.	Yanji	46 ± 4	10 ± 0.6	0.21 ± 0.01	5.2 ± 0.3	8.5 ± 0.4	0.17 ± 0.02	1.2 ± 0.1	205 ± 9
2.	Tumen	56 ± 2.6	0.88 ± 0.24	0.11 ± 0.01	5.04 ± 0.03	0.64 ± 0.09	n.d.	n.d.	2.83 ± 0.02
3.	Helong	35 ± 1.8	5.28 ± 0.03	29.2 ± 0. 1	3.13 ± 0.02	0.52 ± 0.05	n.d.	n.d.	108 ± 6
4.	Antu	56 ± 2.6	0.42 ± 0.01	0.30 ± 0.01	0.091 ± 0.000	0.23 ± 0.03	n.d.	n.d.	68 ± 3.5
5.	Dunhua	64 ± 5	3.4 ± 0.1	0.23 ± 0.01	0.98 ± 0.04	0.44 ± 0.01	n.d.	0.053 ± 0.000	109 ± 5
6.	Hunchun	47 ± 4	7.1 ± 0.7	0.20 ± 0.01	3.3 ± 0.2	6.2 ± 0.4	0.12 ± 0.02	0.039 ± 0.000	200 ± 12
7.	Longjing	66 ± 6	0.56 ± 0.06	0.25 ± 0.01	0.12 ± 0.01	0.35 ± 0.01	n.d.	0.73 ± 0.03	141 ± 7
8.	Wangqing	50 ± 4	0.081 ± 0.002	0.22 ± 0.01	n.d.	n.d.	n.d.	n.d.	930 ± 50
9.	Hamgyong	65 ± 5	1.4 ± 0.2	0.14 ± 0.01	0.78 ± 0.04	0.36 ± 0.02	0.78 ± 0.09	n.d.	220 ± 46
10.	Reference site	61 ± 2	1.32 ± 0.01	0.34 ± 0.01	0.56 ± 0.03	0.20 ± 0.01	0.06 ± 0.02	0.07 ± 0.02	1.95 ± 0.02

n.d.: undetected.

According to SPSS, the average coefficient of variation for eight elements (Fe, Mn, Zn, Cu, Pb, Ni, Cd, and Cr) was 75.94%, and the variation coefficient of Fe was 19.27%. The obtained data were also processed by analysis of variance (ANOVA); the data are presented in the form of a bar chart (the data figure is not presented in this article). This result implies that among the various metals, only Fe was stable in the nine areas and the Fe content was consistent with that in the reference site.

The variation coefficients of the other seven elements ranged from 108.65 to 300.08% (the data table and processing procedure are not presented in this article). Statistical results were converted into graphics (Fig. 2). The populations were divided into groups 2, 3, 4, 5, and 6. The metal distributions in Zones 2, 4, 5, and 7 differed from those in Zones 1, 3, 6, 8, and 9. Thus, 5 zones (1, 3, 6, 8, and 9) were assigned to the same group. If all of the 9 zones were divided into 2 groups, then Zone 1 would represent a single group. When the data from the 9 zones and the reference site were compared (Table 2), 8 types of elements were detected in Zone 1. Notably, the Cd, Mn, Cu, and Pb contents in this zone were higher than those in the other zones.

Figure 2. Dendrogram with average linkage (among groups).

Kalač and Svoboda^[34,35] reported that the Cu levels in mushrooms usually range from 100 to 300 µg/g of dry matter. In the present study, the detected Cu contents ranged from undetected to 5.2 µg/g, and the amounts detected in Zones 1, 2, 3, and 6 were higher than those in the other regions. Cu levels in mushrooms were lower than those reported previously.

Zn content was minimal (0.11 µg/g) in Zone 2 and high (29.2 µg/g) in Zone 3; the concentrations of this metal in these two zones were higher than those in the other zones.^[36] Fe content ranged from 35 µg/g (Zone 3) to 66 µg/g (Zone 7); however, previous research does not provide information on the Fe content of A. mellea (Vahl ex Fr.) Quel for comparison purposes. A very high Ni concentration (0.78 µg/g) was detected in Zone 9; however, this element was not identified in Zones 2, 3, 4, 5, 7, and 8. No reliable differences in Ni concentrations were observed.^[37] The Mn content of the mushrooms detected in the present study ranged from 0.081 µg/g in Zone 8 to 10 µg/g in Zone 1. In Zone 6, the Mn content was 7.1 µg/g. Among the nine zones and eight metals, the Cr content was higher than 100 µg/g except in Zones 2 and 4. In Zone 8, the Cr content was 930 µg/g. The concentrations of Cr were highest in A. mellea (Vahl ex Fr.) Quel in this study.^[37]

High contents of potentially toxic metals (Cd and Pb) were detected in Zones 1, 6, and 7.^[20] These high Pb and Cd contents were higher than the accepted values listed in the Chinese Green Standards of Medicinal Plants and Preparations for Foreign Trade and Economy (WM/T2-2004; Pb ≤ 5.0 µg/g, Cd ≤ 0.3 µg/g).^[13] Compared with the levels in the reference site, the concentrations of Cd and Pb were beyond the standards in WM/T2-2004, in accordance with the following findings: higher non-point source pollution load of organic nitrogen and organic phosphorus for Yanji areas, as well as obviously changed content of Cu and Pb in the riparian soil along the Tumen River. The results of quantitative analyses of metals indicate potential health hazards for humans. Thus, the consumption of these species should be restricted. The pollution in Zones 1 and 7 likely originate from vehicles because Yanji (Zone 1) is a busy transportation hub; thus, automobile exhaust may have contributed to the metal content of plants. Furthermore, the pollution in Zone 6 suggests the influence of industrial pollution discharged from paper mills and an iron mine.

Statistically significant correlation coefficients were established between metal concentrations. The correlation coefficients between different types of metals are presented in Table 3. Significantly positive correlations were observed between Cu and Mn, Ni and Mn, Cd and Mn, and Cu and Ni. Significantly negative correlations were observed between Fe and Zn, Fe and Cu, and Fe and Mn. Data showed that A. mellea (Vahl ex Fr.) Quel contained trace metals with varying levels in different zones, and no significant position correlations were observed between trace metal concentrations in mushrooms.

Table 3. Correlation among metals in different locations.

	Fe	Mn	Zn	Cu	Ni	Cd	Cr	Pb
Fe	1.000
Mn	–0.577	1.000
Zn	–0.682	0.218	1.000
Cu	–0.521	0.680	0.184	1.000
Ni	–0.403	0.893	–0.167	0.648	1.000
Cd	–0.010	0.515	–0.192	0.313	0.625	1.000
Cr	–0.142	–0.196	–0.153	–0.370	–0.082	–0.087	1.000
Pb	0.305	0.025	–0.177	–0.075	0.065	–0.035	–0.010	1.000

The concentrations of Cd, Cu, Mn, Cr, Ni, Pb, Fe, and Zn in the tested fungi ranged from not detected (nd) to 1.2 µg/g, nd to 1.62 µg/g, 0.081 to 10 µg/g, 1.95 to 930µg/g, nd to 0.78 µg/g, nd to 8.5 µg/g, 35 to 65 µg/g, and 0.11 to 29.2 µg/g, respectively.

This article provides information on metal concentrations in edible fungi collected from the Tumen River Basin, which is useful for consumers and regulators who need to know the metal content levels in edible fungi. In addition, statistics show differences in the data obtained on the basis of geographical location. The findings of this study show that A. mellea (Vahl ex Fr.) Quel is a suitable regional bioindicator of pollution control and food safety.

Conclusion

The concentrations of eight metals (Cu, Zn, Fe, Mn, Cd, Cr, Ni, and Pb) in A. mellea (Vahl ex Fr.) Quel collected from the Tumen River Basin were determined by ICP-OES after microwave digestion. The levels of Cd and Pb in several zones were found to be higher than standard limits. The metal content levels in wild edible mushrooms obtained from this region should be analyzed regularly to evaluate the potential risk to human health. By presenting information on the trace metal concentrations in edible fungi collected from the Tumen River Basin, this article provides a useful reference for both consumers and regulators.

Acknowledgments

The authors would like to thank Professor Senior Engineer Wu Xin-Zi (Product Quality Supervision and Testing Institute, Yanbian Korean Autonomous Prefecture, Yanji, China) for providing QA/QC technical assistance and samples from the reference site.

Funding

This work was supported by the National Natural Science Foundation of China (No. 21365023).

Additional information

Funding

This work was supported by the National Natural Science Foundation of China (No. 21365023).