LICHENS AS INDICATORS OF HEAVY METAL POLLUTION AROUND A COAL-FIRED POWER STATION IN W. MACEDONIA, GREECE
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1 Proceedings of the 13 th International Conference of Environmental Science and Technology Athens, Greece, 5-7 September 2013 LICHENS AS INDICATORS OF HEAVY METAL POLLUTION AROUND A COAL-FIRED POWER STATION IN W. MACEDONIA, GREECE K. TSIGARIDAS 1, T. SAWIDIS 1, N. MONOKROUSOS 2, A. SIVROPOULOU 3 and E. DIAMADOPOULOS 4 1 Department of Botany, Aristotle University of Thessaloniki 2 Department of Ecology, Aristotle University of Thessaloniki 3 Department of Genetics, Development and Molecular Biology, Aristotle University of Thessaloniki 4 Department of Environmental Engineering, Technical University of Crete ABSTRACT Concentrations of five metals were determined in a wide spectrum of ten different lichen species around the CPP-A.Dimitrios and a control area. The results permit mapping of the atmospheric dispersion of metals at a local scale. Among five metal studied, chromium was accumulated in highest amount, both in lichen and soil specimens, followed by the sequence of nickel > copper > lead > cadmium. High cadmium, chromium and nickel levels were found in the North direction. The most contaminated lichen was Collema polycarpon for chromium, copper, nickel and cadmium, and Pertusaria pertusa, Neofuscelia pulla, Xanthoparmelia taractica and Lecanora muralis for lead, whereas the genus Cladonia (C. convoluta, C. rangiferina and C. rangiformis) showed the least heavy metal contamination. The high capacity of metal absorption is directly related with polysaccharides involved in the chelation of heavy metals. The SEM images revealed agglomeration of spherical and irregular particles consisting mainly of inorganic amorphous or crystalline constituents. INTRODUCTION In Greece coal is recognized as the primary source of energy and most lignite deposits are located in the Kozani Ptolemais basin, a large intensively exploited area. This region is exploited the last 60 years by opencast mining and supplies nearby lignite CPP (Stalikas et al. 1997; Sawidis et al. 2001; Tsikritzis et al. 2007). Fly ash is carried through the furnaces by the gases flowing toward the chimney. Four CPP are located in this Kozani - Ptolemais basin with a cumulative capacity of 4108 MW, the later amounts for 80% of the total lignite deriving electricity production in Greece. During the crushing process of the coal large quantities of coal dust particles (CDP) can be spread into the surrounding environment and they are deposited on the rough surfaces of the plants (Sawidis et al. 2011). The aim of the present study is to estimate the level of specific metals and their distribution pattern around the CPP - A. Dimitrios industrial area. Lichens were used as heavy metal pollution monitors in three ways: by sampling and identifying all lichen species in the area; measuring metals accumulated within the thallus of individual lichen species; and confirming the particle distribution on lichen thallus. The fluctuation of heavy metal concentrations in interrelation with the major wind directions, the distance from the CPP in the surrounding area within 10 km of its radius is also a subject of this work. MATERIALS AND METHODS The study was centered around the CPP-A. Dimitrios, the largest Greek power plant, located near the southeast side of the Kozani - Ptolemais basin (Fig. 1). Four conceivable axes (North, South, West and East) were considered as the main sampling sites around CPP - A. Dimitrios as displayed in Fig.1. There are 4 sampling sites along each axis, of
2 approximately 1000m 2, in a distance of 2.5 km. In addition to the sampling scheduled, samples from similar species from a relative clean area (Klimataki), about 80 km SW from CPP- A. Dimitrios, were also collected, in order to be used as control specimens. A wide spectrum of 10 different lichen species (terricolous, crustaceae, epiphytic, epigeic) was collected to during the summer months, after a long rainless period. All collected material was air dried in an oven at 40 C to constant weight (2-4 hours). Samples were pulverized in a Moulinex mill and kept in polyethylene beakers and stored in a desiccation in plastic beakers. 0,3g dry weight of each lichen specimen was accurately weight in an open polytetrafluoroethylene (PTFE) teflon beaker and 20ml of concentrated HNO 3 was added. The solution was left at room temperature all night and then it was heated until full evaporation, then 20ml of diluted HNO 3 (1:1) was added. Finally it was heated at 160 C for 30 minutes. The final solution was diluted to 100ml volume with distilled deionized water and analyzed for heavy metal concentrations. For the soil samples 0,2g dry weight was accurately weighted in an open PTFE beaker, 1ml HClO 4 and 20ml of concentrated HF (Merck, pro analysis) were added and heated to full evaporation. Then 20ml 6N HCl was added and heated for 30 minutes precisely. The final solution was diluted to 200ml volume with distilled deionized water. After sample preparation, metal determination was performed by means of atomic absorption spectroscopy (Analytic Jena AAS 6 Vario). For concentrations below 100 mg/l, graphite furnace was used. For higher metal concentrations, flame AAS was used. The detection limit of the spectrometer for all metals in lichen samples was adjusted over 0.4 μg/g whereas in soil samples over 2 μg/g. To confirm the presence of fly ash and coal dust particles and to determine lichen surface characteristics, segments of air dried lichen thalli, fly ash or pulverized coal particles subjected to Scanning Electron Microscopy (SEM). The samples were coated with carbon, using a Jeol JEE-4x vacuum evaporator. Both the upper and lower surface of leaf was studied and micrographs were taken at various magnifications. One-way analysis of variance (ANOVA) followed by a Bonferonni post-hoc test was performed to examine the impact of distance from the power plant and decide whether significant differences existed among the soil heavy metal concentrations in the four different sites along the four directions from the power plant. All statistical analyses used Statistica 7 for Windows (StatSoft, Tulsa, USA). RESULTS AND DISCUSSION Cadmium, chromium, copper, lead and nickel concentrations in lichen species and soil specimens are presented in Table 1 and Table 2 respectively. Fig. 1. Map of Kozani-Ptolemais region and sampling sites.
3 Table 1. Heavy metal concentrations in lichen samples (μg g -1 ) around the CPP A. Dimitrios area. Samples collected from sites 2, 3 and 4 at north (N), south (S) and east (E) direction and from the control area. Site Lichen species Cd Cr Cu Pb Ni N2 Cladonia convoluta 1.1± ± ± ± ±3.7 N2 Collema polycarpon 7.9± ± ± ± ±11.2 N2 Neofuscelia pulla 4.1± ± ± ± ±3.5 N2 Pertusaria pertusa 2.3± ± ± ± ±9.2 N2 Xanthoparmelia somloensis 2.9± ± ± ± ±7.2 Mean Value 3.7± ± ±4.6 11± ±4.6 N3 Cladonia convoluta 1.3± ± ± ± ±9.8 N3 Xanthoparmelia taractica 2.9± ± ± ± ±15.9 N3 Lecanora muralis 1.8± ± ± ± ±11.6 N3 Neofuscelia pulla 5.1± ± ± ± ±25.5 N3 Pertusaria pertusa 3.7± ± ± ± ±6.7 N3 Xanthoparmelia somloensis 2.5± ± ± ± ±5.3 Mean Value 2.9± ± ± ± ±18.4 N4 Cladonia convoluta 2.2± ± ± ± ±0.7 N4 Xanthoparmelia taractica 1.7± ± ± ± ±1.1 N4 Cladonia rangiferina 1.7± ± ± ± ±1.8 N4 Diploschichtes occelatus 5.3± ± ± ± ±3.1 N4 Xanthoparmelia 1.9± ± ± ± ±5.7 somloensis N4 Cladonia rangiformis 1.9± ± ± ± ±2.2 Mean Value 2.5± ±4 7.9± ± ±3.1 S4 Cladonia convolute 0.8± ± ± ± ±1.6 S4 Xanthoparmelia taractica 1.8± ± ± ± ±2.5 S4 Lecanora muralis 0.7± ± ± ± ±0.6 S4 Pertusaria pertusa 1.5± ± ± ± ±1.7 Mean Value 1.2± ± ± ±4 23.5±5.9 E4 Cladonia convoluta 0.9±0.1 65±4 22± ± ±0.1 E4 Xanthoparmelia taractica 0.7±0.1 67± ±0.2 15± ±0.2 E4 Lecanora muralis 0.7± ± ± ± ±0.2 Mean Value 0.8± ± ±3 19.9±4 12±1.5 Control Cladonia convoluta 0.7± ± ± ± ±8.1 Control Xanthoparmelia taractica 0.7± ± ± ± ±9.5 Control Cladonia rangiferina 0.8± ± ± ± ±0.8 Control Collema polycarpon 0.9± ± ± ± ±2.3 Control Lecanora muralis 0.8± ± ± ± ±3.2 Control Neofuscelia pulla 1.1± ± ± ± ±4.1 Control Pertusaria pertusa 0.8± ± ± ± ±5.1 Control Xanthoparmelia somloensis 0.7± ± ± ± ±5.1 Control Cladonia rangiformis 0.7± ± ± ± ±4.2 Mean Value 0.8± ± ±2.8 5± ±3.3 M.V. 1.9±0.3 70± ± ± ±5.3
4 Table 2. Mean values (±SE) of heavy metal concentrations in soil samples (μg g -1 ) in the Kozani-Ptolemais basin region and differences between the sampling areas as revealed by analysis of ANOVA. Data collected at four distance points from the power station at four directions (N is north, S is south, W is west and E is east) and from the control area. Site Cd Cr Cu Pb Ni N1 2.01± ±20.2 a 46.8±4.9 a 2.12± ±40.2 a N2 2.01± ±20.4 b 63.7±5.3 b 2.06± ±58.8 b N3 2.02± ±55.7 c 60.5±8.2 b 2.11± ±21.8 c N4 2.03± ±12.3 d 90.1±8.5 c 2.01± ±11.2 d Control 2.01± ±8.2 e 17.1±1.5 d 2.01± ±8.2 e Bonferonni ns *** ** ns *** S1 2.04± ±42.3 a 34.3±4.6 a 2.15± ±14.4 a S2 2.51± ±20.2 b 33.5±4.6 a 2.11± ±13.2 b S3 2.11± ±19.2 b 25.2±1.5 b 2.01± ±8.3 c S4 2.08± ±6.9 c 21.2±1.9 bc 2.09± ±4.6 d Control 2.01± ±8.2 e 17.1±1.5 c 2.01± ±8.2 e Bonferonni ns *** * ns *** W1 2.12± ±11.2 a 120.3±7.7 a 2.16± ±16.3 a W2 2.03± ±15.5 a 120±5.3 a 2.11± ±20.3 b W3 2.06± ±9.1 a 125±6.3 a 2.08± ±17.7 b W4 2.14± ±20.4 b 140.4±6.9 b 2.39± ±11.6 a Control 2.01± ±8.2 c 17.1±1.5 c 2.01± ±8.2 c Bonferonni ns *** *** ns *** E1 2.07± ±46.2 a 95.5±5.3 a 2.12± ±8.2 a E2 2.46± ±14.5 a 104.1±5.7 a 2.49± ±21.2 a E3 2.13± ±31.4 b 120.3±7.3 b 2.51± ±33.4 b E4 2.09± ±16.3 b 56.8±4.1 c 2.49± ±51.2 c Control 2.01± ±8.2 c 17.1±1.5 d 2.01± ±8.2 a Bonferonni ns *** *** ns *** M. V. 2.11± ± ± ± ±19.3 Fig. 2 demonstrates metal concentrations versus distance in different axes. One way ANOVA was applied to the average soil heavy metal concentrations of the four directions as well as the control samples. The results showed that for those heavy metals that presented variation along the four directions chromium and nickel the North while copper the West directions showed high soil metal concentrations. The control area had the lowest values compared to all other directions (Fig. 7). Lead and cadmium did not differ statistically among the sampling areas. The similarity of the lichen communities was assessed by calculating the Sørensen similarity index. Τen lichen species were found in total in all three directions and the control area. The N2 sampling site presented five, the N3 six and the N4 six dominant lichen species, while in the south and east areas only four and three dominant species were found respectively. Values of the similarity indexes ranged from 0.44 to 0.86 (Table 3). Along the North axis, the lichen communities of the N2 and N3 areas have a high similarity value (0.73), while their lichen composition seems to differ than the N4 as the similarity value decreases (0.55 and 0.5 respectively). Regarding sampling stations 10 km away from the CPP (N4, S4, E4 and W4) the E4 sampling station presented the lowest number of lichen species (Cladonia convoluta, Cladonia rangiformis and Lecanora muralis), nevertheless, all three lichen species were in common with those in the S4 area and presented the highest lichen composition similarity recorded in our sampling sites. Moreover, despite the fact that the N4 area was the most rich in lichen species, only Cladonia convolute and Cladonia rangiformis were found to be common with the other two areas. N3 site presented the highest similarity with the control area (0.8), while E4 the lowest (0.5).
5 Table 3. Sørensen similarity indices of lichen communities between the sampling sites and the control area. N2 N3 N4 S4 E4 Control N2 1 N N S E Control In Fig. 8 the ordination of lichen samples, based on their heavy metal concentrations in a PCA biplot is presented. The first two axes of the analysis (factor 1 and factor 2) explained almost 77% of data variability (61.32 and 15.31% respectively). In relation to the first axis (factor 1), Collema polycarpon was ordinated towards the left part of the graph, while Cladonia rangiferina, Cladonia rangiformis and Cladonia convoluta were ordinated towards the right one. The ordination of heavy metal variables in the same biplot indicated that the ordination of Collema polycarpon samples was driven by the high concentrations of Ni, Cd, Cu and Cr found on the specific lichen species, while that of Cladonia rangiferina, Cladonia rangiformis and Cladonia convoluta samples was related to the low concentrations of the above metals. Along the second axis, Neofuscelia pulla, Pertusaria pertusa, Xanthoparmelia taractica and Lecanora muralis samples with high Pb concentrations were ordinated towards the lower part, while those of Collema polycarpon with low Pb concentration towards the upper part. It is also worth mentioning, that the ordination of Xanthoparmelia somloensis and Diploschistes ocellatus, as well as that of Cladonia convoluta, Cladonia rangiferina and Cladonia rangiformis is almost identical, showing similar heavy metal uptake. Fig. 7. Mean values of soil metal (chromium, nickel and copper) variables of each direction and the control area and differences among them as revealed by ANOVA. Different letters correspond to statistically significant differences among the sites for each heavy metal variable. In lichen samples close to CPP, up to 20% of the thalli surface was covered by fly ash or coal dust particles. The SEM images revealed agglomeration of spherical and irregular particles consisting mainly of inorganic amorphous or crystalline constituents. On the upper surface of the lichen thalli the absence of cuticula combined with the presence of apothecia or aerolia, enables the trapping and strong adhering of bigger size flying particles although spatial distribution of particles showed significant variation among various thalli surfaces. Locally, symptoms of injury to lichen thalli i.e. discoloration and fungi parasitism, were observed at a few sites near the CPP (Fig. 9).
6 The diversity of lichens influenced by the spatial distribution of sampling sites in the study area. The North direction showed maximum diversity of lichens represented by nine species followed by four species in South and three in East, whereas in West no lichen species were found. In Sørensen similarity analysis (Table 3) E4 and S4 sampling sites present the highest similarity rate (0.86) indicating same lichen species growth. Elevation plays an important role in the lichen species similarity rate between E4 and S4, probably owing to the similar amount of precipitation, and deposition of more soluble metals (e.g. cadmium). Among five metal studied, chromium was accumulated in highest concentration, especially in North direction (Figs. 3, 7), both in lichen and soil specimens, followed by the sequence of nickel > copper > lead > cadmium. The direction in which pollutants are transported by the wind is most surely fundamental in determining their main fallout point (Loppi et al. 1994). The dispersion of metals depends on the gravity of a particular metal along with speed and direction of wind (Triantafyllou 2001). The results of one way ANOVA analysis for soil samples indicated that chromium and nickel showed higher values to the North direction in front of Mt. Vermion. This mountainside probably works as a huge natural wall delaying the air masses for longer time. In case of copper the West direction showed higher values influenced probably from the operation of CPP-Cardia in this direction. On the contrary, cadmium and lead did not differ statistically among the four directions with, a slightly preference in the East direction. The ability of lichens to intercept, retain and accumulate soluble and particulate airborne elements is species-specific and may be altered by climatic and environmental conditions and influenced by the duration of the exposure (Wolterbeek 2002). Different morphological types of lichens exhibit differing levels of sensitivity to pollution as a result of combined factors that are not well understood. In general sensitivity increases in the following series: crustose < foliose < fructicose. In our case study the most contaminated lichen was Collema polycarpon for chromium, copper, nickel and cadmium, and Neofuscelia pulla, Pertusaria pertusa, Xanthoparmelia taractica and Lecanora muralis samples for lead, whereas in general, the members of genus Cladonia (C. convoluta, C. rangiferina and C. rangiformis) showed the least metal contamination (Fig. 8). Lichens with crustose morphology are capable of accumulating considerable amounts of heavy metals, sometimes reaching several percents of their surface but crustose species are seldom considered for environmental studies, mainly because insufficient biomass is available for analytical measurements. Surface structure, adhesiveness and waterholding capacity also affect metal accumulation in lichen thalli. Morphological peculiarities of the lichen upper surface contribute particle trapping under moist or dry conditions. Lichens possess a number of characteristics that make them suitable biomonitors for air pollution. They are usually very long lived and their morphology does not vary with the seasons, thus accumulation of pollutants can occur throughout the year. Many lichen species have large geographical ranges, allowing study of air pollution gradients over long distances and measurements can be compared with the national and international guideline values.
7 Fig. 8. Results of PCA (Principal Components Analysis) based on the metal concentrations of lichen species in 10 km distance from the CPP. Ordination of the lichen samples and the metal variables on a PCA biplot. Error bars indicate standard errors. It is also worth mentioning, that the ordination of Xanthoparmelia somloensis and Diploschistes ocellatus, as well as that of Cladonia convoluta, Cladonia rangiferina and Cladonia rangiformis is almost identical, showing similar heavy metal uptake. Fig. 9. Fly ash and coal dust particles deposited on apothecia of Diploschistes ocellatus (left) and on lobes of foliose thali of Xanthoparmelia somloensis (right) (Fungal parasite on lichen thalli increases the particle trapping) collected from the surrounding area of CPP-A. Dimitrios.
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