ANTICANCER COMPOUNDS FROM MEDICINAL PLANTS BIOLOGICA NYSSANA 3 (2)  December 2012: 53-60 Mitrović, T. et al.  Bioindication of Heavy Metal Pollution … 53 Original Article Bioindication of heavy metal pollution in the area of Southeastern Serbia by using epiphytic lichen Flavoparmelia caperata (L.) Hale Tatjana Mitrović 1 , Slaviša Stamenković 1 , Vladimir Cvetković 1 *, Tatjana Đekić 2 , Rada Baošić 3 , Jelena Mutić 3 , Tatjana Anđelković 4 , Aleksandar Bojić 4 1 University of Niš, Faculty of Sciences and Mathematics, Department of Biology and Ecology, Višegradska 33, 18000 Niš, Serbia 2 University of Niš, Faculty of Sciences and Mathematics, Department of Geography, Višegradska 33, 18000 Niš, Serbia 3 University of Belgrade, Faculty of Chemistry, Studentski trg 12-16, 11158 Belgrade, Serbia 4 University of Niš, Faculty of Sciences and Mathematics, Department of Chemistry, Višegradska 33, 18000 Niš, Serbia * E-mail: biovlada@yahoo.com Abstract: Mitrović, T., Stamenković, S., Cvetković, V., Đekić, T., Baošić, R., Mutić, J., Anđelković, T., Bojić, A.: Bioindication of heavy metal pollution in the area of Southeastern Serbia by using epiphytic lichen Flavoparmelia caperata (L.) Hale. Biologica Nyssana, 3 (2), December 2012: 53-60. The content and distribution of 21 metals in the central and peripheral parts of the foliose epiphytic lichen Flavoparmelia caperata (L.) Hale, collected in the area of Southeastern Serbia, were analysed in terms of biological monitoring. Inductively coupled plasma atomic emission spectrometry revealed higher concentrations of As, B, Ba, Cd, Ga, Pb, Se, Cr, Cu, Fe, In, Li and/or Ni in peripheral, younger, parts and Ba, K, Tl, Mg, Na and/or Zn in central, older parts of lichens. Principal component analysis and hierarchical cluster analysis were used to identify the relationship among metals in samples and their possible sources. Significant correlations were found among Ni-Cr, Cd-Ga-In-As-Se, Zn-Ba, Cu-Pb-B, suggesting a common source of pollution. Given the location of sampling, these findings probably reflect airborne metal pollution in relation to the main wind directions and vicinity of the roads and industrial complexes. The importance of this study is the evidence that the Special Nature Reserve Jelašnička Gorge is influenced by pollution sources in the area. Flavoparmelia caperata could be effective as an early indicator of environmental changes of the studied area. Key words: air pollution, bioindication, Flavoparmelia caperata, heavy metals, lichens Introduction Lichens have been the most widely accepted sensors of enviromental pollution since the 19th century (G r i n d o n , 1859). Lack of waxy cuticle and associated stomatas, parenchymal nature, slow growth and long duration of lichens result in the absorption of pollutants (metals, nonmetals, radionuclides, etc) across entire thallus surface and thus allow long-term biomonitoring of persistent pollutants in the ecosystem. Lichens depend on mineral nutrients in the form of soluble salts and particles from wet atmospheric deposition (precipitation and occult precipitation, like fog and dew) and dry atmospheric deposition (sedimentation, impaction and gaseous absorption). They are very efficient accumulators of heavy metals by: ion exchange at specific cation exchange 3 (2) • December 2012: 53-60 BIOLOGICA NYSSANA 3 (2)  December 2012: 53-60 Mitrović, T. et al.  Bioindication of Heavy Metal Pollution … 54 sites, intracellular uptake of metal solution, and entrapment of airborne, metal-rich particles (size 0.5 to 1.0 μm) (N a s h I I I , 2008). Lichens are capable of accumulating heavy metals to a concentration that vastly exceeds their physiological requirements by sequestering them extracellulary as insoluble oxalates or lichen acid complexes (B e e b y , 2001). The presence of particulate materials on the lichen surface which are trapped between the hyphae of medulla is shown by electron microprobe studies (B a r g a g l i , 1998). Metal accumulation is in correlation with the environmental levels of particulate materials (B a r i et al., 2001; B a r g a g l i et al., 2002 ; L o p p i et al., 2004) and spatial- and/or temporal- deposition patterns of trace metals are demonstrated (Z s c h a u et al., 2003). The concentration of sequestered trace metals in lichen really reflects ambient level of airborn trace metals in terms that after the withdrawal of the source of contamination from the ecosystem (closures of industries, iron-steel factories, mines, waste treatment plants, roads and railroads, etc) a dramatic decrease of trace metals concentration in lichen thalli is observed (W a l t h e r et al., 1990). N i m i s et al. (2001) discussed the importance of intra- and inter- specific variability in metal accumulation and relationship between metal content and the age of the thalli. In this study we investigated heavy metal pollution using Flavoparmelia caperata (L.) Hale as bioindicator and bioaccumulator. The study was performed at 3 different locations: northwest (near the village of Cerje), southeast (near the village of Vlase) and northeast (Jelašnička Gorge) of the biggest urban and industrial center of Southeastern Serbia - Niš (approximately 350 000 inhabitants). We would like to emphasize the importance of one location - Jelašnička Gorge. This area, situated 15 km away from Niš and 3 km away from Niška Spa, is a protected natural area having the status of a Special Nature Reserve. One of the reasons to investigate this part of Serbia is the fact that a similar study had never been undertaken previously, although the area is densely populated with large urban-industrial infrastructure, which thus has an important anthropogenic impact on surrounding nature and ecosystems. Lichen samples were analyzed for metal content. Composition data have been treated with Figure 1. Study area (Jelašnica Gorge, Vlase and Cerje in respect to the city of Niš) withwind roses for the area of Niš (data from Republic Hydrometeorological Service of Serbia) BIOLOGICA NYSSANA 3 (2)  December 2012: 53-60 Mitrović, T. et al.  Bioindication of Heavy Metal Pollution … 55 various statistical multivariate techniques, primarily principal component analysis and cluster analysis. Material and methods Study area The study was performed in the subrural and rural area of Southeastern Serbia. The collection sites were: Jelašnička Gorge (330 m altitude), Vlase (350 m altitude) and Cerje (600 m altitude) in the vicinity of roadside (2m, 500 m and 2000 m, respectively) (Figure 1). The nearest urban and industrial area is the city of Niš (approximately 350 000 inhabitants) (Figure 1). The climate is moderate continental with mean annual rainfall ranging over 543.3 mm, a mean temperature of 11.5 °C, and a mean annual relative humidity of approximately 69 %. Prevailing winds are northwesterly in winter and northeasterly and easterly in summer. Wind roses are shown in Figure 1. Lichen Material Foliose lichen Flavoparmelia caperata (L.) Hale (syn. Parmelia caperata (L.) Ach.; common name: greenshield lichen) was collected in April 2009. Lichen specimens were obtained from a height of 1.5-2 m above the ground, at the side of trunks of Prunus domestica and Salix sp. not affected by stemflow. The material from each location was sorted into two samples corresponding to peripheral and central parts of lichen. The samples were air-dried, grinded, homogenized and further analyzed. The determination of lichens was performed by using several standard keys (Boqueras, 2000; Dobson, 2005; Wirth, 1995). Lichen samples were deposited in the lichenological herbarium of the Department of Biology and Ecology, Faculty of Sciences and Mathematics, University of Niš. Reagents and Chemicals All chemicals were of analytical grade and were supplied by Merck (Darmstadt, Germany). All glassware was soaked in 4 mol/L HNO3 for a minimum of 12 hours and rinsed well with distilled water. Ultra-pure water was prepared by passing doubly de-ionized water through Milli-Q system (Millipore Simplicity 185 System incorporating dual UV filters (185 and 254 nm) to remove carbon contamination). Multi-element stock solution containing 1.000 g/L of each elements was used to prepare intermediate multi-element standard solutions. Sodium borhydride solutions were stabilized with sodium hydroxide (Merck, Germany) for hydride generation. Table 1. Instrument operating conditions for determination of heavy metals in lichen samples Spectrometar ICAP 6500 (Thermo Scientific) Nebulizer Concentric Spray chamber Cyclonic Radio frequency power (W) 1150 Principal argon flow rate (L/min) 12 Auxiliary argon flow rate (L/min) 0.5 Nebulizer flow rate (L/min) 0.5 Sample flow rate (ml/min) 1.0 Detector CID86 Table 2. Selected emission lines Element λ [nm] Ag 328.0 As 193.7 B 249.6 Ba 455.4 Cd 228.8 Co 228.6 Cr 283.5 Cu 324.7 Fe 259.9 Ga 294.3 In 230.6 K 766.4 Li 670.7 Mg 280.2 Mn 257.8 Na 588.9 Ni 231.6 Pb 220.3 Sr 215.0 Tl 276.7 Zn 213.8 Hg 253.6 Se 196.0 Instrumentation All the measurements were made with a Inductively Coupled Atomic Emission Spectrometer model 6500 Duo (Thermo Scientific, United Kingdom) equipped with a CID86 chip detector. The system is equipped with an integrated unit for BIOLOGICA NYSSANA 3 (2)  December 2012: 53-60 Mitrović, T. et al.  Bioindication of Heavy Metal Pollution … 56 hydride generation. This instrument operates sequentially with both radial and axial torch configurations. The entire system is controlled by Iteva software. Instrument operating conditions for the determination of heavy metals in lichen samples and selected emission lines are shown in Table 1 and Table 2, respectively. Microwave digestion was performed in a pressurized microwave (Ethos 1, Advanced Microwave Digestion System, Milestone, Italy) equipped with a rotor holding 10 polytetrafluoroethylene (PTFE) cuvettes. Procedure for Microwave Digestion Samples (0.5 g) were transfered into PTFE cuvette, 7 ml of concentrated HNO3 and 1 ml 30% H2O2 were added. Digestion was performed under the following programme: warmed up for 10 mins to 200° C and held for 10 mins at that temperature. After the cool off period samples were quantitatively transferred into a volumetric flask (25 ml). Statistical Analysis To identify the relationship among metals in samples and their possible sources, Pearson’s correlation coefficient analysis, principal component analysis (PCA) and cluster analysis (CA) were performed using PLS Toolbox version 5.2.2 (Eigenvector Research) for the MATLAB version 7.4.0.287 (R2007a) (MathWorks, Natick, MA, USA). The principal component analysis was performed using Varimax Normalized rotation. Cluster analysis was performed on the data sets using the between-groups linkage based on correlation coefficients (Pearson coefficient) by pair-wise deletion. Normalized data set was analyzed by Ward’s method using squared Euclidean distances as a measure of similarity between metal concentrations. Results and discussion The concentrations of 21 metals in lichen Flavoparmelia caperata, collected at 3 different localities in Southeastern Serbia (Jelašnička Gorge, Cerje and Vlase), are given in Table 3. The determination of metals was performed on both central and peripheral parts of lichens. Central parts are inner, older parts of thalli and they have therefore been exposed to pollutants longer. Peripheral parts comprise outmost 3-4 mm of the thalli, with highest physiological activity and the maximum age of 1 year (Fisher & Proctor, 1978; Loppi et al., 1997). Thus, peripheral parts represent recent changes of the environment. Our data indicated differences in the metal content of central and peripheral parts of lichens. Central parts tend to give higher concentrations of a number of metals as Table 3. Concentration of metals in central and peripheral parts of the thalli of Flavoparmelia caperata ( g/g dry wt.) Elements Flavoparmelia caperata from Jelašnička Gorge Flavoparmelia caperata from Cerje Flavoparmelia caperata from Vlase Center (sample 1) Perifery (sample 2) Center (sample 3) Perifery (sample 4) Center (sample 5) Perifery (sample 6) As 0.0037 0.0038 0.0036 0.0033 0.0031 0.0027 B 19.4303 9.5051 6.1341 6.9682 5.8576 6.7637 Ba 27.2333 21.5575 9.5615 24.5084 13.8145 15.2969 Cd 0.2558 0.1942 0.1681 0.1864 0.1310 0.1546 Co 0.1512 0.0822 1.0894 2.1865 0.0970 0.0856 Cr 1.9119 1.4572 3.7140 1.4670 1.7550 1.4879 Cu 8.3806 5.9573 15.3721 5.7307 6.1412 6.5179 Fe 476.6209 374.5863 645.2168 397.9597 493.2096 399.2086 Ga 0.1069 0.0000 0.0413 0.0000 0.0346 0.0177 In 0.0000 0.0000 0.0802 0.0341 0.0810 0.0000 K 2608.8040 2852.2720 2300.1670 2706.2540 2506.7160 3156.7050 Li 0.6932 0.5035 0.7566 0.4607 0.5617 0.4582 Mg 251.8673 315.6654 274.1127 313.7536 287.8818 373.2304 Mn 14.1271 13.5242 13.6761 12.4049 13.9091 15.6287 Na 71.1085 98.1346 89.5684 103.6953 78.8976 110.4994 Ni 1.2703 1.0977 1.5682 1.1916 1.4227 1.3011 Pb 22.3011 10.0742 12.0318 7.3955 9.8771 7.4223 Se 0.0043 0.0045 0.0042 0.0039 0.0036 0.0031 Sr 31.4610 23.8918 16.8445 11.3996 40.6104 32.6951 Tl 0.0000 0.6093 0.0000 0.9164 0.0000 1.5415 Zn 17.6053 17.0019 18.0832 19.5534 20.7641 24.2578 BIOLOGICA NYSSANA 3 (2)  December 2012: 53-60 Mitrović, T. et al.  Bioindication of Heavy Metal Pollution … 57 previously reported in the studies of Nimis et al. (2001). Pearson’s correlation coefficients for metals in lichen samples are shown in Table 4. Determined correlations between metals provided interesting information on the sources and pathways of the metals. The correlation matrix was created from the values of the variables of all 21 metals in 6 samples. The Pair-Wise method was employed for the missing values. The results showed that these metals were strongly interrelated (p<0.01), with correction coefficients ranging from -0.790 to 0.990 at the 99% confidence level. B, Cd, Ga and Pb evidently displayed significant positive correlations with each other (see: Table 3), which indicates their association in analyzed samples. Other metals, like Cr, Cu, Fe, Li and Ni, showed significant correlations, too. The exceptions were elemental pairs As-Zn and Se-Zn with significant negative correlations and As, Mn and Zn without any correlations observed. In order to better describe the relationship among metals and/or samples, principal component analysis (PCA) was performed. Analytical data were represented in a multidimensional space with variables defining the axes, and projected into a few principal components (PCs) that were linear combinations of the original variables and described the maximum variation within the data (Brereton, 2003). Obtained results clearly showed that the Co, Mn and Sr had no impact. This was in accordance with results of metals determination (Table 3) due to small differences in theirs values in different samples. Therefore, PCA analysis was done without these metals resulting in a three-component model explaining 91.78% of the data variation. The first PC comprised 49.75% of the total data variability, and the cumulative variance explained by the first two components was 81.09%. The addition of more PCs did not significantly change the classification of the analytes described below. Score values for the samples, i.e. their mutual projections, for the first two PCs are shown in Figure 2. Additionally, score values for the samples and metals, for the first two PCs are shown in Figure 3. The first PC distinguished two separate groups of samples according to metals content and age of parts of the lichen thalli sampled. Samples 1, 3 and 5 are central (older) parts of the thalli from different areas while 2, 4 and 6 are peripheral (younger) parts of the thalli. The first PC distinguished two separate groups with characteristic patterns of metals accumulation (samples 2, 4 and 6 with Ba, K, Mg, Na, Tl and Zn Figure 2. Score values of the first and the second PCs for the samples Additionally, score values for the samples and metals, for the first two PCs are shown in Figure 3. Figure 3. Biplot of the first and the second PCs for the samples and metals and samples 1, 3 and 5 with As, B, Cd, Cr, Cu, Fe, Ga, In, Li, Ni, Pb and Se). These results clearly defined the highest accumulated metals characteristic for every sample. The following metals were characteristic of sample 1: As, B, Cd, Ga, Pb and Se. Higher concentration of Ba, K and Tl were noticed in samples 2 and 4. Besides, Ba was located on the border that defined a PC and it appeared characteristic of sample 1, too. The accumulation of Cr, Cu, Fe, In, Li and Ni were characteristic for samples 3 and 5. Increased concentrations of Mg, Na and Zn were observed in sample 6. Compared to location of sampling, it BIOLOGICA NYSSANA 3 (2)  December 2012: 53-60 Mitrović, T. et al.  Bioindication of Heavy Metal Pollution … 58 BIOLOGICA NYSSANA 3 (2)  December 2012: 53-60 Mitrović, T. et al.  Bioindication of Heavy Metal Pollution … 59 seems that the Special Nature Reserve Jelašnička Gorge from which sample 1 and 2 originated, is becoming polluted due to vicinity of the road (2m) and towns (Niš and Niška Spa, 15 km and 3 km, respectively). Evidence for this could be found in the increased concentration of Cd, Pb and Se as the levels of these elements are similar to literature values for high-density traffic areas (Mendil et al., 2009). The other two locations of lichen sampling, Cerje and Vlase, with samples 3-6, showed richness in litophile elements and macronutrients such as: Cr, Cu, Fe, In, K, Li, Mg and Na. This could imply a specific composition of geological substrate, as well as the influence of vegetation, i.e. substrate from which lichen samples were collected. Moreover, there is a certain amount of anthropogenic pressure and it is manifested by pollution originating from traffic origin. Finally, metal concentration data were submitted to cluster analysis (CA) using correlation coefficient as similarity measure and weighted pair group as clustering algorithm. This method is the most appropriate for the validation of the correlation between variables and is often used in environmental studies. The obtained dendrogram is shown in Figure 4. Figure 4. Dendrogram derived from the hier- archical cluster analysis of samples Nevertheless the cluster analysis organized the metals (without macroconstituents) in such a way that within-group similarity was maximized and among-group similarity was minimized. The CA results for the heavy metals studied are shown in Figure 5 as a dendrogram. Figure 5 displays four clusters: (1) Ni-Cr; (2) Cd-Ga-In-As-Se; (3) Zn-Ba; (4) Cu-Pb-B in agreement with the PCA results. It is observed, however, that clusters 1 and 2 as well as clusters 3 and 4 join together at a relatively higher level, possibly implying a common source. Fossil fuel emits Ni, Cd, Cr, Cu, Pb, Zn during combustion (Aslan et al., 2011). Besides, the wear of car tires, degradation of parts, peeling paints and greases and metals in catalysts could be sources of pollutants (Pecheyran et al., 2000). Although vehicles with unleaded petrol prevail, the high density traffic of old vehicles using leaded petrol and diesel oil is still present in Serbian roads. Zn, as well as Cd, is well known as an atmophile element subject to long- distance transport (Loppi & Pirintsos, 2003). A part of Zn could be obtained from supporting trees since higher plants are known to release 20 % of total Zn coming from natural sources (Nriagu, 1979). Also, Flavoparmelia caperata is known for its higher capacity for Zn and Cd uptake (Nimis et al., 2001). Figure 5. Dendrogram derived from the hierar- chical cluster analysis of metals content Interestingly, cluster 4 indicates pollution caused by exhaust gases due to traffic, but having in mind the poor frequency of traffic at the roadsides in close range of the location of lichen sampling, one could wonder about the origin of Cu, Pb and B detected. This is probably due to the specific wind roses (see Figure 1) which put this area under the indirect impact of metal industry, situated 10-15 km northwesterly (in the outskirt of the city of Niš). Further research and monitoring should be performed in order to obtain the right conclusions. Conclusion Our study confirms the metal accumulation capacity of lichen Flavoparmelia caperata and its potential for biomonitoring. This lichen species could be effective as an early warning system for detecting changes in the environment. Peripheral parts of lichen samples allow annual changes to be detected. Detected accumulation of heavy metals in lichen samples is probably a result of the frequency of traffic and types of engines on the roads in this BIOLOGICA NYSSANA 3 (2)  December 2012: 53-60 Mitrović, T. et al.  Bioindication of Heavy Metal Pollution … 60 area, the activity of industrial complexes in and around the city of Niš, soil and substrate compositions and predominant wind directions. Further research and biomonitoring surveys of air pollution in the studied area should contribute to the amelioration of air quality and environment in Southeastern Serbia. Acknowledgment. This research was supported by the Ministry of Science and Education of the Republic Serbia during activities on the projects III41018, OI 171025 and TR 34008. References Aslan, A., Çiçek, A., Yazici, K., Karagöz, Y., Turan, M., Akku, F., Yildirim, O.S. 2011: The assessment of lichens as bioindicator of heavy metal pollution from motor vehicles activites. African Journal of Agricultural Research, 6 (7): 1698-1706. Bargagli, R. 1998: Trace elements in terrestrial plants: an ecophysiological approach to biomonitoring and biorecovery. Springer Verlag. Berlin. Bargagli, R., Monaci, F., Borghini, F., Bravi, F., Agnorelli, C. 2002: Mosses and lichens as biomonitors of trace metals. A comparison study on Hypnum cupressiforme and Parmelia caperata in a former mining district in Italy. Environmental Pollution, 116 (2): 279-287. Bari, A., Rosso, A., Minciardi, M.R., Troiani, F., Piervittori, R. 2001: Analysis of heavy metals in atmospheric particulates in relation to their bioaccumulation in explanted Pseudevernia furfuracea thalli. Environmental Monitoring and Assessment, 69 (3): 205-220. Beeby, A. 2001: What do sentinels stand for? Environmental Pollution, 112 (2): 285-298. Boqueras, M. 2000: Líquens epífits i fongs liquenícoles del sud de Catalunya: flora i comunitats. Institut d'Estudis Catalans. Barcelona. Brereton, R.G. 2003: Chemometrics: data analysis for the laboratory and chemical plant. John Wiley & Sons Inc. Chichester. Dobson, F.S. 2005: Lichens. The Richmond Publishing Co. Ltd. Richmond. Fisher, P., Proctor, M. 1978: Observations on a season's growth in Parmelia caperata and P. sulcata in South Devon. The Lichenologist, 10 (01): 81-89. Grindon, L.H. 1859: The Manchester Flora. White. London. Loppi, S., Pirintsos, S.A. 2003: Epiphytic lichens as sentinels for heavy metal pollution at forest ecosystems (central Italy). Environmental Pollution, 121 (3): 327-332. Loppi, S., Frati, L., Paoli, L., Bigagli, V., Rossetti, C., Bruscoli, C., Corsini, A. 2004: Biodiversity of epiphytic lichens and heavy metal contents of Flavoparmelia caperata thalli as indicators of temporal variations of air pollution in the town of Montecatini Terme (central Italy). Science of the Total Environment, 326 (1-3): 113-122. Loppi, S., Nelli, L., Ancora, S., Bargagli, R. 1997: Accumulation of trace elements in the peripheral and central parts of a foliose lichen thallus. Bryologist, 100 (2): 251-253. Mendil, D., Çelik, F., Tuzen, M., Soylak, M. 2009: Assessment of trace metal levels in some moss and lichen samples collected from near the motorway in Turkey. Journal of Hazardous Materials, 166 (2-3): 1344-1350. Nash III, T.H. 2008: Lichen biology. Cambridge University Press. Cambridge. Nimis, P., Andreussi, S., Pittao, E. 2001: The performance of two lichen species as bioaccumulators of trace metals. Science of the Total Environment, 275 (1-3): 43-51. Nriagu, J.O. 1979: Global inventory of natural and anthropogenic emissions of trace metals to the atmosphere. Nature, 279: 409-411. Pécheyran, C., Lalère, B., Donard, O.F.X. 2000: Volatile metal and metalloid species (Pb, Hg, Se) in a European urban atmosphere (Bordeaux, France). Environmental Science & Technology, 34 (1): 27-32. Walther, D.A., Ramelow, G.J., Beck, J.N., Young, J.C., Callahan, J.D., Marcon, M.F. 1990: Temporal changes in metal levels of the lichens Parmatrema praesoredious and Ramalina stenospora southwest Louisiana. Water, Air and Soil Pollution, 53: 189-200. Wirth, V. 1995: Die Flechten Baden-Württembergs, Teil 1 & 2. Eugen Ulmer & Co. GmbH. Stuttgart. Zschau, T., Getty, S., Gries, C., Ameron, Y., Zambrano, A., Nash, T. 2003: Historical and current atmospheric deposition to the epilithic lichen Xanthoparmelia in Maricopa County, Arizona. Environmental Pollution, 125 (1): 21- 30.