STUDIES ON ACCUMULATION OF HEAVY METALS FROM SUBSTRATE TO EDIBLE WILD MUSHROOMS* C. STIHI 1, C. RADULESCU1, G. BUSUIOC2, I.V. POPESCU1,3,4, A. GHEBOIANU3, A. ENE5 1
Valahia University of Targoviste, Faculty of Sciences and Arts, Sciences Department, 130024, Targoviste, Romania, E-mail:
[email protected] or
[email protected] 2 Valahia University of Targoviste, Faculty of Environmental Engineering and Biotechnologies, 130024, Targoviste, Romania 3 Valahia University of Targoviste, Multidisciplinary Research Institute for Sciences and Technologies, 130024, Targoviste, Romania 4 Academy of Romanian Scientist, Bucharest, Romania 5 Dunarea de Jos University of Galati, Faculty of Sciences, Department of Physics, Domneasca 111, 800201, Galati, Romania Received September 14, 2009
The aim of this work was to determine the heavy metal content of the fruiting bodies of Lycoperdon perlatum and Pleurotus ostreatus and their substrate collected at various distances from a metal smelter in Dambovita County, Romania. The concentrations of Mn, Fe, Cu, and Zn in the samples were determined by Energy Dispersive X-Ray Fluorescence (EDXRF) spectrometry and the concentrations of Cr, Ni, Se, Cd and Pb were determined by Atomic Absorption (AA) spectrometry. The coefficient of accumulation of heavy metals was calculated and a highest accumulation of Fe, Cu and Zn from substrate was observed for all the analyzed mushrooms samples. Furthermore, a high accumulation of Pb was observed in mushrooms growing in the vicinity of the metal smelter. Key words: mushrooms, heavy metals, accumulation, energy dispersive X-ray fluorescence, atomic absorption spectrometry.
1. INTRODUCTION
Heavy metals pollution is a serious problem in Romania, especially in areas around metal smelters. Growing up on a substrate with a high concentration of various heavy metals, edible wild mushrooms can become toxic, accumulating a large amount of heavy metals. Until now, various studies have shown that accumulation *
Paper presented at the 10th International Balkan Workshop on Applied Physics, July 6–8, 2009, Constanţa, Romania. Rom. Journ. Phys., Vol. 56, Nos. 1–2, P. 257–264, Bucharest, 2011
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of heavy metals in mushrooms is dependent on: the species of mushrooms, the mushrooms age, the source of pollution with heavy metals and distance to this source [1–3]. Many wild edible mushroom species are known to accumulate high levels of heavy metals and mainly Cd and Pb [4, 5]. The determination of heavy metal concentration in the fruiting bodies of mushrooms is essential in dietary intake studies [6, 7]. Different heavy metals are toxic, such as As, Cd, Ni, and Hg; on the other hand many elements are essential for the human metabolism, such as Fe, Zn, Mn, Cu, Cr, and Se. The aim of this work was to determine the heavy metal content of the young fruiting bodies of Lycoperdon perlatum and Pleurotus ostreatus and their substrate collected at various distances from a metal smelter in Dambovita County, Romania. The coefficient of accumulation of heavy metals was calculated and a highest accumulation of Fe, Cu and Zn from substrate was observed for all the analyzed mushrooms samples. 2. EXPERIMENTAL 2.1. SAMPLES AND PRELIMINARY SAMPLES PREPARATION
Young mushrooms species Lycoperdon perlatum and Pleurotus ostreatus were collected from different areas easy woodland of Dambovita County, Romania, at various distances (0.5 km, 4.5 km and 10.5 km respectively) from a metal smelter, in the same direction of wind. From the same collecting point were taken n = 5 samples from the young fruiting bodies of mushrooms and their substrate. The substrate from Pleurotus ostreatus was poplar bark with a pH ranged from 7.35–7.50 and from Lycoperdon perlatum was soil with a pH ranged from 6.35–7.60. The families, habitat and edibility of the mushrooms under our study are given in Table 1. The fruiting bodies of mushrooms have been washed with deionised water, dried at 60ºC between 4 and 10 hours and homogenized to fine powder and finally weighed. Substrate and soil samples have been dried at 70ºC in 24 hours. After drying the solid samples have been homogenized until to fine powder and weighed. Table 1 Families, habitat and edibility of mushrooms species No. Mushroom species 1 Lycoperdon perlatum 2 Pleurotus ostreatus
Habitat Oak, soil Tree (poplar, oak) bark
Edibility Edible while young and white Edible
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2.2. EXPERIMENTAL METHODS
2.2.1. Energy dispersive X-ray fluorescence Sample preparation Two grams of each sample (n = 5 for each species collected at various distances from a metal smelter in Dambovita County, Romania) were pressed manually, without any chemical treatment, in a plastic vial with Mylar in the bottom and then analysed. Experimental condition The concentrations of Mn, Fe, Cu and Zn in the samples were determined by Energy Dispersive X-Ray Fluorescence (EDXRF) technique [8–10], using the ElvaX spectrometer having a X-ray tube with Rh anode, operated at 50 kV and 100µA. Samples were excited for 300 s and the characteristic X-rays were detected by a multichannel spectrometer based on a solid state Si-pin-diode X-ray detector with a 140 µm Be window and a energy resolution of 200eV at 5.9 KeV. ElvaX software was used to interpret the EDXRF spectra. The accuracy of the results as evaluated by measuring a certified reference sample (NIST-1515: apple leaves). Good agreements were achieved between certified values and data obtained, with recoveries ranging from 98 to 104%. 2.2.2. Atomic absorption spectrometry Sample preparation The dried samples were digested in an acid solution using a Berghof MWS-2 microwave digestion system. Dried fungus samples (500 mg) were introduced into the digestion vessels together with 3 ml nitric acid and 5 ml hydrogen peroxide. After the digestion time (40 min) the vessels have cooled to room temperature (about 30 min.). The clear solution volume was made up to 50 ml for each sample using deionised water. Dried solid substrates (500 mg) were introduced into the digestion vessels together with 3 ml nitric acid and 9 ml hydrochloric acid (aqua regia). After digestion time (30 min) the vessels have cooled to room temperature and the clear solution volume is made up to 50 ml for each sample using deionised water. Experimental condition The concentrations of Cr, Ni, Se, Cd and Pb in the samples were determined by Atomic Absorption (AA) spectrometry [11–13] using the AVANTA GBC spectrometer with flame and hollow cathode lamps (HCL). Determination of elemental concentrations in samples of mushrooms and their substrate were performed using the method of calibration curve according to the absorber concentration. Several standard solutions of different known concentrations have been prepared and the elemental concentration in unknown sample was determined by extrapolation from the calibration curve. All samples concentrations were reported as mg/kg dry weight of material.
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3. RESULTS AND DISCUSSIONS
The heavy metal contents of the samples are given in Table 2 and Table 3. All the metal concentrations were determined on a dry weight basis. The concentrations of Mn, Fe, Cu and Zn were greater than 10 mg/kg and were determined by EDXRF technique. To determine the concentrations of Cr, Ni, Se, Cd and Pb, which were below 10 mg/kg and greater than 0.1 mg/kg, was used the AA spectrometry. The complementarity of the use of these two methods of analysis to assess the heavy metals map of soil samples and vegetation has been proven in other study of the authors [14, 15]. The content of heavy metals of the fruiting bodies of Pleurotus ostreatus ranged from 1.08–1.81, 11.8–12.4, 284–387, 1.29–1.85, 10.2–12.5, 37.9–41.3, 2.57–2.79 and 0.87–0.95 mg/kg dw. for Cr, Mn, Fe, Ni, Cu, Zn, Se and Cd, respectively. The Pb in the Pleurotus ostreatus fruiting body was detected only in the sample collected in the vicinity of the metal smelter. The content of heavy metals of the fruiting bodies of Lycoperdon perlatum ranged from 1.87–1.94, 12.6–13.9, 623–782, 1.83–1.96, 10.2-11.8. 127–134, 14.2– 15.4, 1.58–1.73 and 0.71–3.47 mg/kg d.w. for Cr, Mn, Fe, Ni, Cu, Zn, Se, Cd and Pb, respectively. The relative standard deviation (RSD) values, less than 10%, excepting the results for Fe, indicate a precise quantitative measurements in this work [16]. The relative standard deviation less than 13% for Fe can be done by the interference of Mn and Fe X-ray lines. A great attention was done to the Cd and Pb content in the analyzed mushrooms, because these elements are toxic elements included in the hygiene norms concerning the foods security. The highest Cd content determined was 1.73 mg/kg in Lycoperdon perlatum collected in the vicinity of the metal smelter. The highest Pb content was 3.47 mg/kg also in Lycoperdon perlatum collected all in the vicinity of the metal smelter. In comparison with other studies, the Cd levels determined in Pleurotus ostreatus and Lycoperdon perlatum are in the agreement with literature values. The Pb concentrations in previous studies were between 0.1 and 40 mg/kg [17, 18]. A heavy metal accumulation takes place in the analysed mushrooms species. The coefficient of accumulation of heavy metals was calculated using relation:
Ka =
Cm , Cs
were Cm is the concentration of heavy metal in mushroom and Cs is the concentration of heavy metal in mushroom substrate. In the Tables 4 and 5 are given the coefficients of accumulation of Cr, Mn, Fe, Ni, Cu, Zn, Se, Cd and Pb, respectively in the fruiting body of the analysed mushrooms.
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Table 2 Mean concentration of heavy metals in Pleurotus ostreatus fruiting body and their substrate (mg/kg d.w) Distance from metal smelter (km) 0.50
4.5
10.50
Sample Pleurotus ostreatus Substrate poplar bark Pleurotus ostreatus Substrate poplar bark Pleurotus ostreatus Substrate poplar bark
Cr*
Mn**
Fe**
Ni*
Cu**
Zn**
Se*
Cd*
Pb*
1.81
12.40
387.00
1.85
12.50
41.30
2.64
0.95
0.64
3.41
29.20
406.00
3.56
15.30
40.70
4.27
2.25
5.52
1.74
11.50
321.00
1.33
10.90
39.50
2.79
0.87
nd
3.28
27.80
352.00
2.84
12.20
38.20
5.61
2.14
2.13
1.08
11.80
284.00
1.29
10.20
37.90
2.57
0.87
nd
2.79
28.20
304.00
2.70
12.60
35.40
4.43
2.12
2.42
***
RSD % 1.1–7.5 2.5–6.4 4.8–10.2 1.1–3.7 2.8–7.5 1.3–4.5 1.8–5.3 0.7–1.26 3.2–4.6 * AA spectrometry concentrations ** EDXRF concentrations *** Relative Standard Deviation
Table 3 Mean concentration of heavy metals in Lycoperdon perlatum fruiting body and their substrate (mg/kg d.w) Distance from metal smelter (km)
Cr*
Sample
Lycoperdon 1.94 perlatum Substratesoil 18.96 Lycoperdon 1.87 perlatum 4.5 Substratesoil 11.93 Lycoperdon 1.91 10.5 perlatum Substratesoil 11.78 RSD** % 4.5–9.2 * AA spectrometry concentrations ** EDXRF concentrations *** Relative Standard Deviation 0.5
Mn**
Fe**
Ni*
Cu**
Zn**
Se*
Cd*
Pb*
13.90
782.00
1.96
10.90
134.00
14.20
1.73
3.47
168.00 6470.00
9.63
24.70
75.20
19.30
5.27
12.50
13.30
656.00
1.83
10.20
136.00
15.40
1.58
0.87
145.00 5563.00
8.42
20.90
67.80
22.50
4.52
5.64
12.60
1.94
11.80
127.00
14.80
1.64
0.71
623.00
142.00 4470.00 8.75 21.40 72.90 19.70 4.49 5.23 4.1–7.8 3.7–12.5 1.3–5.2 3.2–9.1 1.1–8.5 1.2–3.7 0.4–1.25 0.7–4.6
Table 4
Ka – accumulation coefficient of heavy metals in Pleurotus ostreatus fruiting body Distance from metal smelter (km) 0.5 4.5 10.5
Cr
Mn
Fe
Ni
Cu
Zn
Se
Cd
Pb
0.53 0.53 0.39
0.42 0.41 0.42
0.95 0.91 0.93
0.52 0.47 0.48
0.82 0.89 0.81
1.01 1.03 1.07
0.62 0.50 0.58
0.42 0.41 0.41
0.12 0.00 0.00
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Table 5 Ka – accumulation coefficient of heavy metals in Lycoperdon perlatum fruiting body Distance from metal smelter (km) 0.5 4.5 10.5
Cr
Mn
Fe
Ni
Cu
Zn
Se
Cd
Pb
0.10 0.16 0.16
0.08 0.09 0.09
0.12 0.12 0.14
0.20 0.22 0.22
0.44 0.49 0.55
1.78 2.01 1.74
0.74 0.68 0.75
0.31 0.35 0.37
0.28 0.15 0.14
The dependencies of coefficients of accumulation of Fe and Pb on distance from metal smelter are given in Fig. 1. The coefficients of accumulation of Fe, and Pb are higher inside both species in the vicinity of the metal smelter. The coefficients of accumulation of Zn and Pb are higher from Lycoperdon perlatum comparative with Pleurotus ostreatus species and ranged from 1.01–1.07 and 0.14–0.28. We conclude that Lycoperdon perlatum species is a Zn and Pb accumulator. Fe
Ka
Pb
Ka 0.30
1.20
0.28
1.00
0.25
0.95
0.93
0.91
0.20
0.80 Plurotus ostreatus
0.60
Lycoperdon perlatum
0.14
0.20 0.12
0.05
0.14
0.12
Distance (km )
0.00 0.5
4.5
10.5
(a)
Lycoperdon perlatum
0.12
0.10
0.40
Plurotus ostreatus
0.15
0.15
0.00
0.00 0.5
4.5
0.00 10.5
Distance (km )
(b)
Fig. 1. Dependency of accumulation coefficients of the analyzed heavy metals on distance from metal smelter: (a) – Fe storage; (b) Pb storage.
The heavy metals concentrations obtained in this study in the fruiting body of the mushrooms were compared with the admitted maximum level of certain contaminants in foodstuffs, established by the Commission of the European Communities (Commission Regulation [EC] No 466/2001). The admitted maximum level for Cd and Pb is set about 2 and 3 mg/kg d.w., in cultivated mushrooms. Our results reveal that only the samples of Lycoperdon perlatum collected in the vicinity of the metal smelter had Pb concentration higher than European limits for cultivated mushrooms.
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4. CONCLUSIONS
The Energy Dispersive X-Ray Fluorescence (EDXRF) spectrometry and the Atomic Absorption (AA) spectrometry are two analytical methods which can be successfully used in complementary mode to determine the heavy metal concentration of mushrooms. The combination of two different techniques, EDXRF and AAS, was well suited to this analysis. EDXRF technique enable simultaneous determination of all the elements present in the sample (Z > 13), don’t require a chemical sample preparation but, is limited by the detection limit. For this reason the elements which are a concentration less then 10 mg/kg had to be studied by the AAS technique. The studied mushrooms: Lycoperdon perlatum and Pleurotus ostreatus contain minerals required in the human diet, such as Fe, Zn, Mn, Cu, Cr and Se and also toxic elements, such as Cd, Ni and Pb. The level of toxic elements was lower than that of minerals. The concentrations obtained for heavy metals in Pleurotus ostreatus species seems to be acceptable for human consumption and nourishment value. As expected, metal uptake seems to be species dependent: Lycoperdon perlatum accumulate more Zn and Pb and Pleurotus ostreatus accumulate more Mn and Fe. In time, Lycoperdon perlatum accumulate a large amount of Pb and that fact can be one of the reasons of the non-edibility of this specie at the maturity. We must emphasize that the results obtained in this work are the first ones reported in the literature for the wild-grown edible mushrooms collected from the Dambovita County, Romania. This information can be extremely important considering that the analysed mushrooms are common mushrooms, collected in many sites from people for consumption. Acknowledgements. The work was financially supported from: National PN-II-PCE-2008-2 project nr. 978/2009 and from National PN-II-P4 project nr. 72172/2008.
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