Applicability of Atomic Emission and Atomic Absorption Spectrometry for Variability Assessment of Trace and Macro-Elements Content in Moss Species from Pb-Zn Mine Environment

The aim of this study was to assess the applicability of the Inductively Coupled Plasma-Atomic Emission Spectrometry (ICP-AES) Electro Thermal Atomic Absorption Spectrometry (ETAAS), and Cold Vapour Atomic Absorption Spectrometry (CVAAS) for the analysis of total content for 23 elements. Deposition and distribution of metals in the air was determined by biomonitoring a widely prevalent moss species from a lead and zinc polluted area of the “Toranica” mine, Republic of Macedonia. Moss species Hypnum cupressiforme, Homalothecium lutescens, Campthotecium lutescens and Brachythecium glareosum were used as very specific and suitable sampling biomonitor. Moss samples were digested by the application of a closed wet digestion using a microwave digestion system. The applied instrumental techniques were useful in order to determine a wider range of content for the analyzed elements; macro contents of Ca, Mg, K and P to trace contents of As, Cd, Co, Ga, and Hg. From data processing the values for Pb and Zn were used as anthropogenic markers. Higher contents of As, Cd and Cu in moss samples from this region were also determined, confirming the impact of mining activities on anthropogenic air pollution in this area. Multivariate factoring identifies four chemical associations: F1 (As-Cd-Ca-Cu-Fe-Mn-Pb-Zn), F2 (Co-Cr-Li-V), F3 (Hg-P) and F4 (K). *Corresponding author: Trajce Stafilov, Institute of Chemistry, Faculty of Natural Sciences and Mathematics, Ss. Cyril and Methodius University, POB 162, 1000 Skopje, Macedonia, Tel: +3892-324-9906; Fax: +3892-322-6865; E-mail: trajcest@pmf.ukim.mk Received December 26, 2013; Accepted March 11, 2014; Published March 14, 2014 Citation: Angelovska S, Stafilov T, Balabanova B, Sajn R, Baceva K (2014) Applicability of Atomic Emission and Atomic Absorption Spectrometry for Variability Assessment of Trace and Macro-Elements Content in Moss Species from Pb-Zn Mine Environment. Mod Chem appl 2: 123. doi:10.4172/2329-6798.1000123 Copyright: © 2014 Angelovska S, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


Introduction
Environmental pollution has been known as one of the most important problems in modern societies. Heavy metals are a major source of environmental pollution, and determining their environmental concentrations is an important part of understanding biogeochemical processes and gauging ecosystem`s health [1]. Human activities, such as mining and tailings discharge processes are rapidly increasing environmental pollution [2,3]. Emissions into the air constitute the greatest source of the toxic metals pollution. Even the metals emitted naturally in wind-blowing dust are often of anthropogenic sources. Therefore, it is necessary to maintain a close watch on heavy metal depositions, even when local or regional areas are monitored. Specific danger arises from waste processing ore and flotation tailings in mine plants surroundings. Metals content quantifications present an ecological for the determination not only of the presence of metals but also their distribution and extent of toxicology for the environment and human health [4].
The use of mosses as biomonitors in smaller scale is a convenient way of determining levels of atmospheric deposition [5][6][7][8]. This is possible because mosses absorb/adsorb nutrients, and also contaminants, directly from the atmosphere. Compared to higher plants, mosses have several advantages which make them more suitable for this kind of study [9]. Mosses lack a root system and a well-developed cuticle, thus the substrate has little influence on the levels of contaminants in their tissues and they readily take up atmospheric contaminants. Moreover, the cationic exchange capacity in mosses is high and they possess a high surface area to volume ratio, factors which both favor the accumulation of large amounts of pollutants [10]. Bryophytes are especially suitable organisms for purposes of monitoring investigations, because procedures of sampling and chemical analyses are relatively simple and of low-cost. Bryophytes include evergreen and perennial plants, making it possible to collect them year round. Most of their species are widespread, and, thus, heavy metal concentrations of distant areas can also be compared [11].
Determination of enriched contents for certain toxic elements (As, Cd, Cr, Cu, Hg, Ni and Pb) occurring in moss species as an accumulator medium, present a challenge for analytical determination. These elements occur from trace contents to micro and macro contents in polluted areas. Thus it is necessary to find the most appropriate techniques for their quantification. Widely used instrumental techniques for multielement analyses like Inductively Coupled Plasma-Atomic Emission Spectrometry (ICP-AES) or Inductively Coupled Plasma-Mass Spectrometry (ICP-MS) are used by numerous investigations [12][13][14]. However, the elemental analyses like Electro Thermal Atomic Absorption Spectrometry (ETAAS) for ultratrace elements (As, Co) and Cold Vapour Atomic Absorption Spectrometry (CVAAS) for mercury determination are still the most efficient instrumental analytical techniques [5].
The present study is the first attempt to characterize the atmospheric deposition of 23 elements in "Toranica" lead-zinc mine vicinity by means of mosses. Therefore, the aims of this study were: 1) quantification of metals in the air in terms of determination of elements contents in mosses as well as by calculated deposition rates; 2) patterns

Investigating area description and sample collection
A total of 250 km 2 area was monitored, limited with coordinates N: 42°10' -42°20' and E: 22°17' -22°28' , located in the north-eastern part of the Republic of Macedonia (Figure 1). Lead and zinc "Toranica" mine environ was monitored as a potentially polluted area with anthropogenic introduced higher contents of certain heavy metals. The "Toranica" mine is located on the slopes of Osogovo Mountain near the Macedonian-Bulgarian border, 18 km away, and 20 km away from the town of Kriva Palanka. Mine "Toranica" is designed as a modern uderground mine with a production capacity of 700,000 tons of ore per year. Ore from the deposit "Toranica" appears in three textured types: pure ore with representation of 51%, massive ore with 29% and impregnated ore with 20%. The ore consists mainly of galena, sphalerite, chalcopyrite and pyrite [15]. The ore reserves were estimated to be 12.6 million tones, with 4.47% of Pb, 2.93% of Zn and 20 µg g -1 of Ag [16]. The flotation plant for the production of Pb and Zn concentrates is situated close to the mine. Toranica concentrator flowsheet gives the following technological results: Pb+Zn collective concentrate with average 15% of Pb and 35% of Zn and Pb selective concentrate with an average of 73% of Pb and 4,3-5% of Zn [15].
The study area region is characterized by moderate continental climate. The altitude varies between 600 and 1500 m. Most frequent winds in the region are those from the west with frequency of 199% and 2.7 m s -1 speed, and winds from the east with frequency of 124% and 2.0 m s -1 speed [17]. Climatic condition in the region allowing airdistribution of fine dust particles generated as a result of mine activities and exposure flotation tailings at open.
The study area is characterized with the presence of four moss species: Homalothecium lutescens, Hypnum cupressiforme, Brachythecium glareosum, and Campthotecium lutescens. The dominant moss species were Homalothecium lutescens (52% of 39 total collected moss species) and Hypnum cupressiforme (36% of total collected moss species), while the Brachythecium glareosum and Campthotecium lutescens were collected from two locations. Depending on the conditions and the accessibility of the locations the species that is available and typical for the region was collected according to previously adapted protocol given by Harmens et al. [18]. Random samples (in the very close vicinity of the pollution source) and samples according to sampling network (5 × 5 km) were collected as presented in figure 1.

Sample preparation
For digestion of moss samples, the microwave digestion system (CEM, model Mars) was applied. Precisely measured mass (0.5000 g) of moss samples where mixed with 5 mL concentrated HNO 3 (trace pure), and 2 mL H 2 O 2 (30%, m/V). The Teflon vessels were carefully closed and the microwave digestion method was applied. The digestion method was performed in two steps for total dissolving of moss tissue as previously established by Balabanova et al. [5]. After the digestion method was finished, digests were quantitatively transferred into 25 mL volumetric flaks. Thus, prepared digests from moss tissue were analysed for the total elements contents.

Quantifications of elements contents using ICP-AES, ETAAS and CVAAS
The analyses of 23 element contents in digested samples were performed by applying: a) atomic emission spectroscopy with inductively coupled plasma, ICP-AES (Varian, 715ES), with an application of an ultrasonic nebulizer CETAC (ICP/U-5000AT+) for better sensitivity of plants digests, for Al, B, Ba Ca, Cr, Cu, Ga, Fe, K, Li, Mg, Mn, Na, Ni, P, Pb, Sr, V, Zn; b) electrothermal atomic absorption spectroscopy, ETAAS (Varian, SpectrAA 640Z) applied for the analyses of As, Co, and Cd; c) cold vapour atomic absorption spectroscopy, CVAAS (Varian, SpectrAA) applied for the analysis of Hg. The optimization of the instrumental parameters for ICP-AES are given as follows: optimal parameters for RF Generator are given by operating frequency of 40.68 MHz, power output of 1500 W to obtain power output stability better than 0.1%. The spectrometer optimization was considered with manufacture recommendations. The variables settings of the program were set as: plasma Ar flow rate 15 L min -1 , auxiliary Ar flow rate 1.5 L min -1 , nebulizer Ar flow rate 0.75 L min -1 , pump speed of 25 rpm, stabilization time 30 s, rinse time 30 s, sample delay 30 s, number of replicates for the quantification measurements-3 (Table 1).
Optimal instrumental parameters for ETAAS determination are given in table 2. The CVAAS is the most successful and widely used technique for the determination of mercury due to its simplicity and good reproducibility. Mercury can stay in its atomic state at room temperature. The vapor pressure of mercury at 20°C is 0.16 Pa that corresponds to 14 mg m -3 mercury concentration in air. The solution of SnCl 2 (1%, m/V) was used for the reduction of Hg into elemental form in 0.1 mol L -1 HCl reaction medium [19]. Mercury vapor after volatilization is released out of the solution and carried by an inert gas to Study area Figure 1: Location of the investigating area in the Pb-Zn "Toranica" mine within territory of the Republic of Macedonia (left); moss sampling network (random sampling and 5 × 5 km organize sampling) of moss species.

Element
Wavelength the absorption cell. The mercury absorption line is has the wavelength of 253.7 nm. The magnitude of the signal shows the amount of mercury present in the samples.
The QC/QA of the applied techniques was performed by standard addition method, and it was found that the recovery for the investigated elements ranges for ICP-AES 98.5-101.2%, for ETAAS and CVAAS 96.9-103.2%. The same methods were applied for the determination of the elements in moss certificated reference materials M2 and M3 [20]. The sensitivity in regard to the lower limit of detection was done as given in table 3.

Data processing for elements contents
The obtained values for the contents of the investigated elements were statistically processed using basic descriptive statistics. The application of bivariate statistics was used for data check of elements contents correlations. For that issue the linear coefficient of correlation was used. Two-dimensional scatterplots were used to visualize relations between two data sets. Individual data points were represented by point markers in two-dimensional space, where axes represent the variables.
Multivariate statistical method (cluster and R-mode factor analyses) was used to reveal the associations of the chemical elements. The factor analysis was performed on variables standardized to zero mean and unit standard deviation [21,22]. As a measure of similarity between variables, the product-moment correlation coefficient (r) was applied. There are various rotational strategies that have been proposed [22]. The goal of all of these strategies is to obtain a clear pattern of factor scores, that is, factors that are somehow clearly marked by high loadings for some variables and low loadings for others. The elements with low communalities were excluded because of their lack of significant associations. In this study, the varimax method was used for orthogonal rotation. As before, we want to find a rotation that maximizes the variance on the new axes; put another way, we want to obtain a pattern of loadings on each factor that is as diverse as possible, lending itself to easier interpretation. Sta tistical softer Statistica 8.0. was used for data processing the obtained values for elements contents.

Results and Discussion
The descriptive statistics of analyzed elements is presented in table 4. On the basis of the normality tests and compared with histograms of distribution for the content of all analyzed elements in moss samples, the normality was assumed for the values of Al, Ca, Cr, Hg, K, Na, and Sr. High contents of Pb and Zn were assumed (average values 60 and 75 mg kg -1 , respectively) in the area very close to the pollution source. However, distribution of dust with high Pb-Zn contents was expected in the investigated area. The maximum values (420 mg kg -1 for Pb and 180 mg kg -1 for Zn) for the contents in moss occur in very close vicinity Step 4 (cleaning) Temperature, °C 2600 1800 2650 Time, s 2 2 5   of the pollution source. The median value for lead contents (3.2 mg kg -1 ) compared to the same value for the whole territory of the Republic of Macedonia (4.6 mg kg -1 ) shows no enrichments at all [14]. If close mine locations are isolated from the rest of the study area an enrichment of 10 times was obtained for 12 samples (33.4 mg kg -1 ). Fine dust from flotation tailings is distributed due to winds caring out, and deposited on moss. Almost 0.05% of these heavy metals are accumulated in the moss plant tissue, confirming how good adsorbers/absorpbers of heavy metals are. Significant skewed distributions were main problem in data processing. The rest of the potentially hazardous elements distributes in ranges for As (0.03-10.5 mg kg -1 ), Cd (0.06-1.62 mg kg -1 ), Hg (0.01-0.04 mg kg -1 ), Ni (0.45-9.43 mg kg -1 ). The lead-zinc deposits usually are characterized with Mn contents as well. Therefore, the Mn contents were ranged in 30.4-815 mg kg -1 , wherefrom it can be considered geoenrichments in the study area.
Lead and zinc were distinguished as main anthropogenic introduced elements in the mine environment. The logarithmic values were used in the interpretation of the Pb and Zn distribution. Aerial distribution of dust particles is certainly in relation to the distance at which they can reach. Therefore, a functional dependence of the content versus distance from the emission source was established. Functional interdependence relation of elements contents across the distance from the pollution source was determined. Significant higher negative correlation (r = -0.43 and -0.54, respectively for Pb and Zn) was derived from the relation given in figure 2, indicating that distancing from emission source, results in decrease of bio-accumulation of these heavy metals in moss (Table 4).
Interspecies comparison of bio-indicating ability of the collected four moss species was performed in order to determine whether there is a statistically significant difference in the accumulation of certain elements. The macro elements content does not show any significant variations in moss tissues as given in figure 3. Phosphorus is accumulated in the ranges ~0.1 % as same as Mg. Calcium contents ranges from 0.4-0.6 % in all four moss species. Sodium is the mainly accumulated macro element in ranges of 55-85 mg kg -1 .
Micro elements (Al, Mn, Fe, B, Ba, Cu, Sr, and Zn) similar to macro elements contents do not significantly vary between the moss species based on their average values ( Figure 4). However, if we consider that the higher content of certain micro elements are introduced in the mine environment, contents enrichment occurred. Such is the case with Mn and Zn contents where the maximum values of 815 and 179 mg kg -1 , respectively, are obtained from Homalothecium lutescens moss tissue sample. Hypnum cupressiforme showed higher bioaccumulation ability for Al and Ba as micro elements and Cr, Ga and Li as trace elements. Certain predominance of Homalothecium lutescens occurs in relation to the accumulation of potentially hazardous elements As, Cd, Cr and Pb, but it should be considered that this is the dominant collected species in the study area ( Figure 5). Campthotecium lutescens and Brachythecium glareosum are less dominant species but no deviation in average values for all analyzed elements were found. Considering this, no specific trend in the elements distribution pattern in mosses species were found (Figures 3-5). This evidence suggests that environmental factors influence element distribution between moss species.
By the application of multivariate statistics, three geogenic and one anthropogenic association were established on the basis of: a) visual inspection of similarities of spatial distribution of element patterns in moss samples; b) comparison of basic statistical parameters; c) the correlation coefficient matrices; d) the results of multivariate (cluster and Factor Analysis, FA) analyses ( Figure 6 and Table 5). Pearson correlation coefficient was used to cluster together variable or samples with similar characteristics. The distribution of the normal variable in FA was reduced, excluding Al, Na, Sr, Mg, Ga, Ba and B, because of their very low factor loadings.
Four factors were identified, when applying FA that includes 87% of variability of the treated elements. Factor 1 (F1) is the strongest factor representing 40% of the total variability. This factor principally associates As, Cd, Ca, Cu, Fe, Mn, Pb, and Zn, which for the most of the elements are presumed to be of anthropogenic origin. Only the Ca distribution does not relay on anthropogenic introduction, but higher content as macro-element and mechanisms of adsorption as Ca 2+ ion metal, consequented with stronger correlation with the others elements from F1. Factor 2 (F2) is the second strongest factor, with 23% of total variability. With this factor were associated elements Co, Cr, Li and V. The group also links elements that are probably naturally and anthropogenically distributed. Second geochemical association represents chemical elements that are probably naturally distributed. Factor 3 (F3) is the third strongest factor, with almost 12% of total variability. Significantly higher correlation of Hg-P was estimated for F3, due to obtained factor loadings (0.90 and -0.92, respectively). Therefore, the Hg accumulation in moss plant species probably is consequently dependent with phosphorus accumulation. The weakest expressive Factor 4 -F4, includes only K, represents 8% of the total variability. However, clustering extends the elements distribution linkage to K with Na and Ba (Figure 3). The same effect was achieved for the F3 where Ni was linked with the geochemical association Al-Li-Co-Cr-V-Ni ( Figure 6 and Table 5).
Clustering the 23 elements distributions to four synthetic variables (elements associations) was more reproductive vs. FA. The geochemical association Al-Li-Co-Cr-V confirms the dust dispersion in the investigated area, due to that element are mainly characteristic to soil particles. Moss species placed the particles with the physical absorption or adsorption from moss surface. The geochemical associations of B-Mg-Ca-Sr and Ba-K-Na associate elements primarily affected by natural factors such as lithological background with no characteristic anthropogenic influence. Implementing clustering the elements Hg, Ga, Ni and P were excluded, consequently because of no similarity linkage.
Anthropogenic impact occurs at geochemical association distribution of As-Pb-Fe-Mn-Cd-Zn-Cu. Typical elements which are consequences of air transport from flotation tailings and are not influenced by lithological background. High values are found in the close mine environ along the Kriva Reka Canyon. This factor is connected with pollution by the lead and zinc flotation tailing dam dust dispersion. Association of Fe in this factor indicates anthropogenic influence, although all southeastern countries Serbia, Bulgaria, Turkey and Macedonia reported high median values for Fe indicating a natural source of this element [14]. Normal distribution of Mn in moss species ranges 30~200 mg kg -1 , but in the present case, the maximal value was 815 mg kg -1 , indicating a significant Mn anthropogenic introduction in the investigated area. Considering this, Mn occurring in higher contents is expected phenomena, due to the lead-zinc ore deposits containing significant amounts of Mn.
Areal distribution maps were constructed in order to be determine Elements contents in mg kg -1 Figure 6: Cluster dendogram for elements contents, Pearson correlation coefficient was used to cluster together variable or samples with similar behavior. The dendrograms were performed using the linkage distance reported as Dlink/Dmax.