Analysis of heavy metals content in the soil and in the macromycetes species growing on mine waste dumps

This study aims at an ecological reconstruction, by means of macromycetes species, of the soils degraded by mining activities. To this end, samples of both soils and macromycetes from altitude mining waste dumpsresulted from the exploitation of iron and sulphur ores have been collected and analyzed. The metal contents were determined by atomic spectrometry and the results were performed with Microsoft Excel, Origin and SPSS programs. The statistical study of the distribution of the metal content data among soil, substrate and macromycetes indicates an adequate correlation.

In the areas of mining ores extraction, soils are affected by structural and functional degradation, being frequently contaminated by various pollutants, such as: nitrites, sulphites, heavy metals, radioactive residues, carbohydrates, etc [1][2][3].
Together with other categories of microorganisms (bacteria, algae, filamentous micromycetes), the macromycetes play an important part in the detoxification of the contaminated media, through the so-called mycoremediation process [4][5][6].
Some macromycetes species may be employed as key organisms, capable of releasing basic nutrients in the environment, thus stimulating plant growing and development of complex biological communities.Essentially, biological succession may be oriented by means of only one species or by several complexes formed of various macromycetes species, which may contribute to the ecological recovery of the mining waste dumps of altitude [7][8][9][10][11][12].
Researches drove by the authors of these papers confirmed the toxicological bioindicator role of fungi in those areas where the chemical pollution of the soils is on high levels.The results showed that metal accumulations may be influenced by species, growth phase, environment conditions and soil types.Also, they observed that the presence of metals in soils influence the synusial structure of fungi by reducing the density of the spore bodies and the diversity of the saprophytic and mycorrhizal species [13][14][15].
The direct advantages of the application of macromycetes for the ecological recovery of the soils degraded by mining activities might be the following: i) in situ application, at low costs; ii) possible application in the treatment of large-sized contaminated surfaces; iii) accumulation of pollutants in the mycelium, to be subsequently recycled and eliminated off the environment; iv) absence of any perturbation of the surroundings; v) a higher porosity and aggregation of the soil, thus accelerating the circulation of nutrients.
The aim of the present study was to evaluate the distribution of heavy metals in the (mine waste dumps) soil -macromycetes circuit.The contents of metals was determined by atomic spectrometry and the results were performed with Microsoft Excel, Origin and SPSS programs

Experimental part
The content of heavy metals (Zn, Pb, Hg, Cu, Fe, Cr, Ni, Mn and Cd) has been determined by flame atomic absorption spectrometry (FAAS).All the FAAS standard solutions used for calibration method and to prepare standard mixtures solution were used as supplied by Merck Chemical Company.For the present analyses both samples type have been treated as following: However, such analytical procedure have been applied into similar studies for other research group interested to analyzed the heavy metal into similar natural matrix [16][17][18][19].

I. Mechanical sample preparation:
a) Soils.The dried soils were gentile disaggregated and particles greater than 2 mm sieved out.The < 2 mm fraction was then reduced to fine powered by means of swing-mill using a agate mortars to avoid any trace metal contamination of samples.b) Macromycetes samples.This type it must be milled to pass a 1mm aperture before analysis.To achieve this has been used a small knife mill with carbon steel construction.

II. Sample digestion method.
All the reagents were used as supplied by Merck Chemical Company.a) Soils.For the present study has been used nitric acidperchloric acid method digestion.
a2. Procedure used: 1-2 g of each solid sample has been weighted into clean dry glass tube.Additional into clean dry glass tubes leaved empty have been added 5 mL standard mixtures solution, that contain 1 ppm of all of analyzed elements herein and 10 ppm lead only, distributed after each three real samples.The role of those was to ensure the level of accuracy and the precision of samples (table 1).After in, have been added 4 mL of nitric acid and 1 mL of perchloric acid in all glass tubes.The new mixtures have been introduced into the heating block and the temperature has been raised up to 150 ± 5 o C for the next 3 h.All of them have been leaved at this temperature until the copious evolution of fumes ceases.After that all tubes have been allowed to be cooled.In the next step 2mL hydrochloric acid has been added into each tube and the temperature of heating the block system has risen up to 60 0 C and leave to this value for one hour.After that all solutions have been leaved to be cooled and then have been quantitatively transferred into 25 mL quoted flasks with distilled water.After four hours (time enough to allow residue to settle) all the samples have been analyzed using a Perkin Elmer Atomic Absorption Spectrometer 3300.Only for iron an additional dilution (100 dilution factor) has been done for attaining the optimum concentration level for analysis.
b) Macromycetes samples.For the present study has been used nitric acid-perchloric acid method digestion b1.Reagents involved were: 1. Nitric acid (70%); 2. Perchloric acid (60%); 3. Hydrochloric acid (6M) (diluted hydrochloric acid (516 mL, 36%) to 1 L with distillated water).b2.Procedure used: 3-5 g of each solid sample (dried at 105 0 C has been weighted into clean dry conical flask.To ensure the level of accuracy and the precision of samples, two clean dry conical flask have been filled with 40 ml standard mixture solution that contain 1 ppm of all of analyzed elements herein, and 10 ppm for led only distributed after each three real samples (table 1).After in, have been added 40 mL of nitric acid to each flask, and covered with watch glass and set aside in a fume cupboard overnight, then, all covered flasks have been placed on a hotplate and warm gently until frothing ceased.After that all tubes have been allowed to be cooled.In the next step have been replaced the flasks on the hotplate and 3mL perchloric acid has been added into each tube.The new solutions have been heated cautiously just until dryness.After that the flasks have been leaved to be cooled and 2 mL of hydrochloric acid and 3 mL deionised water have been added and warmed gently to dissolve the residue.At this stage all the samples have been quantitatively transferred to quote flasks and analyzed by using a Perkin Elmer Atomic Absorption Spectrometer 3300.
Prior to readings of the atomic absorption device, the optimum working conditions were established, according to the recommendations of the producer (which involves settlement of the wavelength characteristics to the metal subjected to analysis and adjustment of the two gases (air, acetylene) flows.The standard conditions that have been used in present work are in the table 1.
There follows preparation of a set of standard solutions, over the linear domain of concentration indicated by the manufacturer.
Checking on whether reading, on the device, of the most diluted calibration sample follows, after which the device is calibrated through successive readings of the standard solutions, in increasing order of their concentration values, and the samples are read, after their preparation, as described above.
The results were performed using Microsoft Excel, Origin and SPSS programs.
As can be observed in table 1 the analytical control qualitative results indicated that the applied procedure are reasonable good with respect to additional losses of elements during the all involved analytical steps.In the other hand is accepted today that a value of 100±10% for recovery factor indicated a quantitative recovery and no matrix effects under experimental conditions used herein [20].However, depending of analyses aim, values of 100±30% can be considered satisfactorily [21].
All the results presented in this work have been corrected considering the recovery factors.

Results and discussion
The soil and, respectively, macromycetes samples, have been collected from waste dumps of the former mining exploitation of Calimani (47 06' 42.0" N; 25 13' 08.6" E; 1750 m altitude), an area in which the chemical composition conditions act as restrictive factors for the vegetation growing on the sterile dumps, especially in the sulphur pit.
In the pit area, the exploitation works of the sulphur ore included digging of the existing soil layer, dislocation of the rocks, sorting according to the sulphur content, processing of the ore with higher sulphur content and depositing of the resulting waste in dumps.
This working method explains the general aspect of the pit and dumps and also its deepening southwards, where the sulphur content in higher.The zone of the former peak (Negoiu Romanesc) remained relatively not exploited, as due to the lower sulphur content and to the presence of the iron.
At the level of such antropogenous forms of relief, the occurrence of an edaphic cover as such is excluded, so that the external layer from the surface of the waste dumps may be included, from a taxonomic perspective, to the Entiantrosol type, rudic sub-type of soil, with skeletal material.
In the opinion of the authors, this is a soil now under development, formed on anthropogenous parental materials, with a minimum thickness of 50 cm, evidencing no diagnosing horizons or showing an incipient A horizon, the presence of which may be explained by the harsh climatic conditions and, especially, by the young age of the waste dumps in the area under investigation.
The ecological reconstruction of this soil unit should take into consideration the nature of the deposited materials, their thickness and chemical composition, their reaction, the presence of some polluting substances, etc.

Determination of heavy metals concentration.
Analysis of the material concentration in the soilmacromycetes circuit involved determination of the Zn, Pb, Hg, Cu, Fe, Cr, Ni, Mn and Cd content, by atomic absorption spectroscopy, a working method based -as generally known -on the absorption -by the sample's atomic vapors -of a radiation with a wavelength characteristic for the element subjected to analysis.The most frequently applied procedure for shifting the sampling solution into an atomic state involves its introduction into a flame, at a sufficiently high temperature, assuring its complete transformation into atoms, on considering the necessary reduction -up to minimum values -of the number of either thermally-excited or ionized atoms, known as affecting the results of the analysis.
Metal distribution, varying from one region to another, depends on the depth from which the samples had been collected.Distributions of analyzed metals are in concordance with similar results obtained of other authors from another mine waste dump [3].
In the analysis to follow, the metal content will be described as a function of the place from which the soil samples, the samples from the substrate of the macromycetes species and from the macromycetes, respectively, had been taken over.
No cadmium and mercury has been found in the soil samples under analysis, although, in some macromycetes samples, some very small amounts of cadmium were evidenced.
Most of the species of macromycetes have been taken over from waste dumps, so that a corresponding number of soil samples were subjected to analysis.Table 2 lists the metal content found in the analyzed samples.
Unlike the soil samples, the macromycetes species contain cadmium and chromium, determined in the initial samples under the detection limit.Instead, it seems that the macromycetes do not concentrate the lead, while the iron amount is more than 20 times lower, which is quite normal (table 4).
A comparative analysis of the metal content in the substrate and in the macromycetes evidences the following aspects: the Lactarius rufus species concentrate the zinc (present in amounts 3 times higher than in the substrate) and the chromium, while the contents of copper and manganese are slightly lower than in the substrate, the iron content -much lower, while lead and nickel are totally absent; in the Paxillus involutus species, the zinc and the chromium are concentrated, the metals being present in lower ratios.
Metal distribution in the soil waste dumpsmacromycetes substrate -macromycetes circuits is plotted graphically in figure 1.The metal content found in the soil samples from the waste dumps are taken as the average value of their concentrations.
Statistical processing of dataDispersion analysis is a statistical procedure, known as Anova, which may be applied in the analysis of a variable variation, comparatively with the factors influencing it [22].From an Anova perspective, there exist two situations: i) Significant differences appear between the means of the concentrations of the chemical elements subjected to investigation, which means that, statistically speaking, the macromycetes species has a considerable influence on the concentration values of the 6 elements for the soil samples from the basis of the macromycetes (substrates), yet with the restriction imposed by the test of variance equalities, namely that the "macromycetes species" grouping factor cannot be interpreted, with Anova, to the coefficient of risk accepted in this former case (0.05), but to a different value of this parameter, that might be determined in a subsequent stage.
ii) Significant differences do appear between the mean concentration values of the chemical elements under analysis; therefore, from a statistical point of view, the macromycetes species influences to a considerable extent the concentrations of the 6 elements for the samples taken over from the macromycetes body, on observing the test of variance equality, for the risk coefficient accepted in the latter case (0.10) [23].

Analysis of Pearson correlation
Analysis of Pearson correlation among the macromycetes species from the waste dumps as a function of the metal ion concentration and of the determination place [24].a).Analysis of Pearson correlation among the places of the determination of ions metals concentrations for each of the 5 macromycetes species, as a function of the metal ions concentrations, separately for substrate samples and for samples taken over from inside the macromycetes.Pearson coefficients of correlation considered for the Pearson waste dump.In the present study, the Pearson correlation expresses the intensity of the link established between 2 variables.Under analysis here are 5 variables, i.e., 5 types of substrate, corresponding to the 5 macromycetes species, presented in the following order: 1. Lactarius rufus; 2. Paxillus involutus; 3. Hypholoma fasciculare; 4. Gyromitra infula; 5. Hebeloma subsaponaceum, all of them previously tested as to their content in 6 elements, namely: Mn, Zn, Pb, Fe, Ni, Mn, (table 5).
The optimum correlation found among the fungi species, as to the concentration of metallic ions determined from the substrate, has been registered between the Lactarius rufus and Hebeloma subsaponaceum species, with a Pearson coefficient of 0.908 and a probability of 98.9%, while the weakest correlation among the macromycetes species, as to the concentrations of metallic ions determined in the macromycetes substrates, appears between the Gyromitra infula and Hebeloma subsaponaceum species, with a Pearson coefficient of 0.431 and a probability of 60.6%.
An important observation to be made is that the best correlation among the fungi was found out between the Gyromitra infula and Hebeloma subsaponaceum species, with a Pearson coefficient of 0.901 and a probability of 99.8%, while the weakest correlation among the macromycetes species as to the ion metal concentrations determined in the macromycetes body was between Lactarius rufus and Hebeloma subsaponaceum, with a Pearson coefficient of 0.338 and a probability of 58.8%.b) Analysis of Pearson correlation among the places in which the ion metal concentrations were determined for each of the 5 macromycetes species from the waste dumps, as a function of the metal ion concentrations, on comparing the substrate samples with samples from the body of the macromycetes.
The Pearson correlation expresses the intensity of the link manifested between 2 variables.The present investigation considers 5 variables, respectively 5 macromycetes species, defined in the following order: 1. Lactarius rufus; 2. Paxillus involutus; 3. Hypholoma fasciculare; 4. Gyromitra infula; 5. Hebeloma subsaponaceum, all of them tested as to their content in 6 elements, namely: Mn, Zn, Pb, Fe, Ni, Mn; more exactly, the correlations taken by two (determinations in the substrate versus determinations in the body of the macromycetes), within each of the 5 species.The correlations coefficients show reduced values, either positive (direct correlation) or negative (reverse correlation), the exception represented by the Gyromitra infula species (recording to a coefficient of 0.950) being practically annulled by the significance level of this extremely reduced result (0.013).
The assertion may be therefore made that, practically, no correlation exists -as to the content of metals from these macromycetes -in the substrates and those from the fungus body.

Table 5 PEARSON CORRELATION COEFICIENTS FOR THE METALS CONTENT OF THE SUBSTRATES
Exploring factorial analysis for reducing the number of variables Analysis of the main components For the substrate samples, factorial analysis has been performed on the main components, on the basis of the correlations observed among the concentrations of the 6 determined metallic ions [25,26].
There has been extracted only one factor with an "Eigenvalue" coefficient higher or equal to 1, whose variance represents alone 73.514% of the total variance.No orthogonal rotation was possible.Consequently, the 5 soil samples corresponding to the respective macromycetes species, from the viewpoint of the concentrations of the microelements from the samples of the soil beneath them should be all considered as a unitary whole, no significant differentiation among them being possible as to the concentrations of the 6 metallic ions determined (fig.2).
of the correlations observed among the concentrations of the 6 determined metallic ions.There has been extracted only one factor with an "Eigenvalue" coefficient higher or equal to 1, whose variance represents alone 78.924% of total variance.No orthogonal rotation was possible.Consequently, the 5 soil samples corresponding to the respective macromycetes species should be all considered, from the viewpoint of the concentrations in the microelements from the samples of the soil beneath them, as a unitary whole, no significant differentiation among them being possible as to the concentrations of the 6 metallic ions determined (fig.3).
The samples taken over from the body of the Gyromitra infula and Paxillus involutus macromycetes are essential for their characterization.
Cluster analysis for reducing the number of variables, by their grouping according to dispersion [27,28] The analysis is meant at reducing the 5 variables, represented by the 5 macromycetes species, by their grouping into clusters on the basis of the dispersion of the concentration values of the determined metallic ions.
For the substrate samples, there have been employed: The Lactarius rufus and the Paxillus involutus substrate samples show the closest dispersion values, so that they may be grouped into a first cluster, characterized by a dispersion quite different from that of the Gyromitra infulasubstrate samples, thus leading to a new cluster which, in its turn, shows quite a different dispersion from that of the The correlation reached at is that the Lactarius rufuscsubstrate, Paxillus involutus -substrate samples are essential for a correct characterization of the substrate samples corresponding to the fungus species.
For macromycetes samples, the factorial analysis has been performed on the main components, on the basis of the correlations established among the correlations of the 6 previously determined metallic ions.Factorial analysis has been performed on the main components, on the basis

The samples taken over from the macromycetes body
The Hypholoma fasciculare and Gyromitra infula samples form a cluster with approximately the same dispersion of the concentration values of the metallic ions determined with a second cluster, formed of sample Lactarius rufus and Paxillus involutus.The Hebeloma subsaponaceum species form a cluster by itself, three clusters thus resulting in the end (fig.5).

Conclusions
The atomic absorption spectroscopic analyses followed the distribution of metals in the waste dumps soilmacromycetes -macromycetes substrate circuit.
The data obtained show that each species of macromycetes in part concentrate heavy metals in a different manner.
In the case of soil samples, the maximum Pearson correlation coefficient between any two of the 5 variables (samples) is of 0.996, with a probability around 100%; the two samples found in this situation are samples no. 3 and no. 4. The minimum Pearson correlation coefficient between any 2 of the 5 variables (samples) is of 0.921, with a probability of 99.7-samples no. 2 and no. 5. Pearson analysis indicates that no correlation exists as to the content of metals form macromycetes, substrates and those from the macromycetes body.
Exploring factorial analysis indicates that the Lactarius rufus and Paxillus involutus-substrate samples are essential for a correct characterization of the substrate samples, corresponding to the macromycetes species, however the samples taken over from the body of the Gyromitra infula and Paxillus involutus macromycetes are essential in the characterization of the macromycetes.

Fig. 3 .Fig. 2 .Fig. 4 .Fig. 5 .
Fig. 3. Screen-plot diagram for the "Eigenvalue" values of the factors of the factorial analysis on the main components, in the case of the substrate samples

Table 1
THE FAAS STANDARD CONDITIONS USED IN PRESENT WORK

Table 2
METAL CONTENT IN SOIL SAMPLES*

Table 3
METAL CONTENT IN SAMPLES FROM THE SUBSTRATE OF THE MACROMYCETES SPECIES

Table 4
METAL CONTENT IN SAMPLES OF THE MACROMYCETES SPECIES