Epiphytic lichens as biomonitors of atmospheric pollution in Slovenian forests

Environmental Pollution 146 (2007) 324e331 www.elsevier.com/locate/envpol

Epiphytic lichens as biomonitors of atmospheric pollution in Slovenian forests Z. Jeran a,*, T. Mrak a, R. Jac´imovic´ a, F. Batic b, D. Kastelec b, R. Mavsar c, P. Simoncic c b

a Department of Environmental Sciences, Jozef Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia Department of Agronomy, Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, 1000 Ljubljana, Slovenia c Slovenian Forestry Institute, Vecna pot 2, 1001 Ljubljana, Slovenia

Received 15 October 2005; accepted 10 March 2006

No relationship between Hypogymnia element concentrations and foliose epiphytic lichen cover was found. Abstract Two country-wide surveys using epiphytic lichens as biomonitors of atmospheric pollution carried out during 2000 and 2001 in Slovenia were compared with surveys in 1991 and 1992. In the first survey, epiphytic lichen cover was studied in more than 500 plots of the 4  4 km national grid carried out within the framework of forest decline inventories. In the second survey, the epiphytic lichen Hypogymnia physodes (L.) Nyl., was collected on a 16  16 km bioindication grid and analysed for S, N, As, Br, Ce, Cd, Cr, K, La, Mo, Rb, Sb, Th, U and Zn contents. Only ‘forested area’ sampling points were included in the present study. Lichen cover was low, with about 70% of plots with less than 10% foliose lichen cover. No relationship was found between Hypogymnia trace element, N and S concentrations and foliose epiphytic lichen cover. Ó 2006 Elsevier Ltd. All rights reserved. Keywords: Anthropogenic impacts; Hypogymnia physodes; Lichen cover; Nitrogen; Sulphur; Trace elements

1. Introduction Different bioindication methods based on epiphytic flora composition have been used in different countries (Ferry et al., 1973; Wirth, 1988) since the 1950’s when epiphytic lichens were first recognized as useful biomonitors of air pollutants. Methods varied from simply observing epiphytic lichen thallus types (Batic and Mayrhofer, 1996) to recording lichen species diversity and/or investigating phytosociological relationships between different lichen species and environmental conditions (Richardson, 1991). Mapping lichen diversity is routine in several countries (Sigal, 1988; VDI, 1995; Loppi et al., 2002; Nimis et al., 2002; Poikolainen et al., 1998a) and is especially useful where direct measurement of air

* Corresponding author. Tel.: þ386 1 5885 281; fax þ386 1 5885 346. E-mail address: [email protected] (Z. Jeran). 0269-7491/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.envpol.2006.03.032

pollutant concentrations is impossible, e.g. in forests (Poikolainen et al., 1998a; Loppi and Pirintsos, 2003), near urban areas (Loppi et al., 2002) and industrial regions. This is partly due to the ability of lichens to accumulate metals and other pollutants (e.g. nitrogen and sulphur compounds) derived from the atmosphere as dry or wet deposition. Epiphytic lichens have been widely used as monitors of metal deposition at a country level (Sloof and Wolterbeek, 1991; Reis et al., 1996; Jeran et al., 1996). A positive relationship between the nitrogen and sulphur content of bryophytes and nitrogen deposition was reported (Pitcairn et al., 1995), and a ‘traffic index’ with NOx and NH3 (Gombert et al., 2003). Slovenia is one of Europe’s most highly forested countries, forests accounting for over 60% of its territory (11 500 out of 20 273 km2). Approximately 34.3% are coniferous, 38.8% mixed and 27% deciduous broadleaf forests. Since the early eighties, a systematic monitoring programme has been developed to study forest decline at regular intervals. Epiphytic

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lichen mapping is one method used to investigate reasons for this decline. Biomonitoring surveys using lichens have been regularly carried out at five-year intervals by mapping epiphytic lichen thallus types (Batic and Mayrhofer, 1996). Around industrialized areas, or in some pristine environments, species mapping was also carried out. More detailed mapping, based on VDI protocols (VDI, 1995) or European guidelines (Asta et al., 2002) was performed to a very limited extent (Batic et al., 2003). All approaches provide only information that the air is polluted or not, none about the origin and type of pollutants. Two surveys were carried out throughout Slovenia in 1992 and 2001 using Hypogymnia physodes (L.) Nyl. to monitor trace element air pollution. The aim of this study was to investigate the relationship between foliose epiphytic lichen cover and selected trace element, N and S concentrations in H. physodes. It combines the latest mapping results from the 2000 forest inventory with H. physodes nitrogen, sulphur and metal contents obtained by analysis of samples in Slovenian forest regions collected in 2001. Latest results were also compared with similar surveys performed in 1991 and 1992. 2. Materials and methods 2.1. Determining epiphytic lichen cover A lichen survey was performed in Slovenia in 641 plots with a grid density of 4  4 km over the period from the end of August to end of September 2000 (Fig. 1). Similar observations were performed in 1991 on 543 plots. Fieldwork was carried out by a well-trained team, a forest inventory crew, under the guidance of professional staff from the Slovenian Forestry Institute. Lichen observation methods followed Jeran et al. (2002), but estimates of the three main lichen thallus types (crustose, foliose and fruticose) were only used in the 2000 survey due to some uncertainties in the calculation of lichen frequency. Whenever possible, climatozonal forest tree species, or tree species typical of forest management of the region, were selected for observation. Trees also had to meet other requirements necessary for lichen growth; at least 20 years old (on average 30e40 years) and similar light conditions. The cover of each

325

lichen thallus type at each plot was estimated for 6 neighbouring trees of the same species, using a 0e3 scale, separately for three different tree heights: 0e0.5 m, 0.5e2 m and above 2.5 m. Lichen cover scale was as follows: 0 ¼ 0%; 1 ¼ 1e10% (mean 5%); 2 ¼ 11e50% (mean 30%); 3 ¼ 51e100% (mean value of 75%). The mean foliose lichen cover per plot from the total database was calculated and expressed on the same scale (0e3). Mean lichen cover per plot was calculated as a weighted average of observed lichen cover at three tree heights on six trees. Calculated mean foliose lichen cover included all lichen species, including both those sensitive and resistant to air pollution.

2.2. Determination of trace elements in H. physodes The epiphytic lichen H. physodes (L.) Nyl. was collected at 89 sampling plots within the 16  16 km grid density (Fig. 1) from June to August 2001. H. physodes sampling was mainly carried out in open habitats or at the nearest forest glade to the plot. Sites were selected up to several hundred metres from fixed plots used in lichen mapping studies in forest decline inventories performed each time on marked trees. Healthy lichen thalli were sampled from the trunks of 3e5 neighbouring trees, belonging to the same tree species group at each site. In open habitats, samples were mostly from old orchard trees, but in forest glades from oaks (Quercus robur L., Quercus petraea (Matt.) Leibl., Quercus pubescens Willd., Quercus cerris L.) or spruce (Norway spruce (Picea abies (L.) Karsten). Samples were placed in paper bags and transported to the laboratory. Lichen material from different trees from ca 30% of sampling locations was prepared individually to establish the variation in element concentration within a sampling site; composite samples were prepared from the remainder. Samples were not washed, but moistened with distilled water to facilitate removal of loosely adhering surface particles. They were then lyophilised and homogenised by grinding in a mill with agate balls. About 100e 200 mg of dry lichen powder was used to make tablets in a hydraulic press for instrumental neutron activation analysis (INAA). Total concentrations of more than 30 elements were determined in each sample using the k0 method of INAA (De Corte et al., 1993; Smodisˇ, 1992). Lichen tablets were irradiated for 20 h in the TRIGA Mark II reactor of the ‘‘J. Stefan’’ Institute, at a thermal neutron fluence rate of 1.1  1016 n m2 s1, together with an Al-1% Au alloy disc, serving as comparator. Gamma spectrometric measurements were carried out with HPGe detectors (ORTEC, USA) connected to an EG&G ORTEC Spectrum Master high-rate multichannel analyser. Each sample was measured twice, for 3000 s after 3 days and for w20 h after 8 days cooling time. Hypermet-PC software was used (Fazekas et al., 1997; Hypermet-PC, 1997) to evaluate gamma spectra and KAYZERO/SOLCOI. (1996) program to calculate

Fig. 1. Map of inventory plots (4  4 km grid) for determination of epiphytic lichen cover (black spots) and 16  16 km grid (open squares) for collection of Hypogymnia physodes.

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element concentrations. Overall uncertainty in final element concentrations obtained by k0-method is ca 3.5%. Analytical quality of the method was checked by routine analysis of IAEA Standard Reference Material Lichen336. Elemental levels were in good agreement with certified or recommended values, with respect to the 95% confidence interval proposed by the producer (Jac´imovic´ et al., 2003). Total nitrogen (N) and sulphur (S) contents were analysed in duplicate according to ICP-Forest Manual (Anonymous, 2004) using a LECO CNS e 2000 analyser and expressed in wt %. Dry lichens were combusted at 1350  C in an oxygen atmosphere where N and S-oxides were formed and carried over several catalyst tubes and transformed to SO2 and N2. The quantity of sulphur was detected by infrared absorption in an IR cell, while nitrogen was detected by thermal conductivity in a TC cell. The standard procedure of analysis includes use of certified standards (Rye Flour Leco 502275), control of calibration and use of control charts. Measurements of nitrogen and sulphur were within tolerable limits in the 7th IC test for ash and beech leaves, 10% (for nitrogen) and 20% (for sulphur) (Fu¨rst, 2005).

2.3. Mapping and statistical analysis Maps showing mean foliose lichen cover were constructed using the inverse distance weighted interpolation method to estimate values in a grid with 1 km resolution for each observation year. Spatial distribution of element concentrations was investigated by factor analysis of 15 elements and results presented graphically to show important factors. Standard statistical methods were employed including Spearman rank coefficient, Pearson’s coefficient and the Wilcoxon signed ranks test. Mean foliose lichen cover in 2000 was compared with results of H. physodes element contents collected in 2001 in 64 sampling plots (16  16 km grid). Where lichen cover values were missing, the average value of lichen cover from the nearest plot or the average of mean lichen cover from the nearest surrounding plots of the 4  4 km grid was used.

3. Results and discussion

Fig. 2. Map of mean foliose lichen cover classes for 1991 (a) and 2000 (b).

3.1. Cover of foliose lichens Air pollution is one of several factors explaining the distribution of many lichen species. In our case, a very simple method of estimating the mean lichen cover of only foliose species on different trees, regardless of tree species, was used. Fruticose lichens are well established as being the most sensitive to air pollution, crustose the least and foliose intermediate. Since H. physodes is a foliose species and used to monitor trace element deposition, only the cover of foliose lichens was used as a measure to estimate air pollution in this study. H. physodes is a very common epiphytic lichen on trees with a slightly acidic or neutral bark, e.g. conifers, oaks, birches etc. It is rather resistant to SO2 but very sensitive to NH3 pollution. In Slovenian forests it is one of the most widespread foliose species and increasing in cover in areas polluted by SO2. Interpretation of these biomonitoring results are to a certain extent speculative since there are no monitoring devices to systematically measure air pollutant concentrations in these natural, remote forestry regions. Never-the-less, local knowledge of the geography, territory, weather conditions (average precipitation maps), prevailing wind directions and locations of major pollution sources, provides a reasonable basis for interpretation. Investigation of foliose epiphytic lichen cover vegetation during the years 1991 and 2000 at the national level (Fig. 2, Table 1) confirmed that lichen vegetation was poor,

with almost 70% of observed plots corresponding to the first two classes in both years. Plots lacking lichens in both maps/surveys (Fig. 2) were found in central, western and eastern Slovenia. One cause of lichen deserts in the central part may be because it is the most populated, with major cities, the heaviest traffic and two thermal power plants and other industrial sources. Impoverished foliose lichen vegetation in the western part with no local pollution sources may be due to transboundary air pollution, especially from NE Italy. Varied forest management across Slovenia must also be considered. Lower foliose lichen cover in eastern Slovenia appears to be due to the combined influence of local air pollution, especially from traffic, intensive agriculture and transboundary pollutants Table 1 Contingency table for the mean foliose lichen cover calculated for two surveys performed in 1991 and 2000 1991

0% 1e10% 11e50% Above 50% Total Total in %

2000 0%

1e10%

11e50%

Above 50%

Total

Total in %

60 43 12 0 115 22.5%

44 134 60 7 248 48.6%

7 48 67 12 134 26.3%

0 3 8 5 16 3.1%

111 228 147 24 510 100.0

21.8 44.7 28.8 4.7 100.0

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Table 2 Descriptive statistics for 15 elements (mean, median, median absolute deviation (MAD), min, max and no. of plots) determined in H. physodes in 1992 and in 2001 and the statistical significance for ranked sign tests comparing the medians

N S As Br Cd Ce Cr K La Mo Rb Sb Th U Zn

1992

2001

Sig. level

Mean

Median

MAD

min

max

n

Mean

Median

MAD

min

max

n

0.19 1.28 14.60 1.01 2.63 5.94 4150 1.18 0.70 15.13 0.36 0.29 0.12 91.71

0.19 1.13 13.44 0.91 2.22 4.79 3945 1.02 0.53 12.40 0.28 0.26 0.10 84.44

0.05 0.36 4.61 0.38 1.01 2.22 965 0.48 0.23 5.85 0.09 0.12 0.03 23.83

0.10 0.57 5.95 0.21 1.12 2.33 1652 0.48 0.07 3.49 0.14 0.11 0.04 47.26

0.37 2.97 32.18 5.42 6.05 21.80 8644 2.75 9.20 71.15 3.44 0.82 0.31 181.60

76 76 76 76 76 76 76 76 76 76 76 76 76 76

1.34 0.13 0.53 10.92 0.75 2.63 3.67 3878 0.76 0.26 16.54 0.24 0.19 0.07 95.33

1.28 0.14 0.49 9.79 0.63 1.48 2.89 3781 0.72 0.22 14.09 0.21 0.17 0.06 89.29

0.37 0.03 0.13 3.52 0.22 0.49 1.05 910 0.23 0.06 7.29 0.05 0.05 0.02 24.46

0.59 0.07 0.18 4.62 <0.2 0.62 1.11 2304 0.28 0.09 3.12 0.08 0.07 0.02 45.63

2.25 0.21 1.39 22.35 2.45 4.43 35.85 6188 2.07 1.87 57.10 1.51 0.56 0.17 182.52

65 65 77 77 77 77 77 77 77 77 77 77 77 77 77

imported by easterly winds. Very rich lichen vegetation with over 50% cover was restricted to isolated forest mountainous regions in the north-west and in remote southern regions. These regions, however, climatically differ from the rest of Slovenia since the Alpine region (NW) and partly also the Dinaric region have the highest precipitation in the country. Forests in the south are best preserved with little human impact. Detailed investigation of foliose lichen cover at 510 plots common to both surveys showed that 52.2% maintained the same category of mean lichen cover, 26.3% of plots showed a lower category of mean lichen cover in 2000 than in 1991 and 21.6% of plots showed improved cover since the last survey (Table 1). Higher foliose lichen cover would be expected in the recent survey, especially in central Slovenia, since atmospheric SO2 showed a gradual decrease in mean annual concentrations from 1992 onwards at all National Air-Pollution automatic monitoring stations measured in urban centres and around the main pollution sources (ARSO, 2002). Somewhat higher average levels (but below 50 mg m3 MIV maximum limit value) were recorded around the thermal power stations at Sˇosˇtanj and Trbovlje in the central part of the country. As mentioned by van Herk et al. (2003), historic SO2 levels may still influence present day lichen species distribution as lichen vegetation may not have recovered fully from the effects of SO2 during the last 30 years. Subjective judgement of personnel involved in mapping may also explain the lower cover estimated in 2000 in the same plots compared to 1991. 3.2. Elemental levels in H. physodes More than 30 elements were determined in H. physodes collected in 2001 at a national scale with a grid density of 16  16 km. Descriptive statistics for As, Br, Cd, Ce, Cr, K, La, Mo, Rb, Sb, Th, U, Zn and total sulphur and nitrogen are presented (Table 2). Because of data outliers, the median was chosen as an appropriate measure of the central tendency and median absolute deviance and range (difference between

0.0000 0.0000 0.0000 0.0004 0.0000 0.0000 0.0591 0.0000 0.0000 0.1436 0.0000 0.0000 0.0000 0.2188

maximum and minimum value) were used to assess variability of element concentrations. Results of a similar survey performed in 1992 are given for comparison. Much lower elemental levels were found in 2001 compared to in 1992. Rb and Zn were the only elements not showing significant median changes (Table 2). Decreased lichen element contents could be due to the desulphurisation equipment installed at coal-fired power plants, improved filters, the use of imported coal with lower sulphur content and gas for heating, and the considerable reduction in industrial production in Slovenia and in other parts of Europe. Similar decreasing trends over the last decade have been noted in moss biomonitoring studies (Suchara and Sucharova, 2004; Tho¨ni and Seitler, 2004). Both sulphur and nitrogen concentrations were determined in H. physodes in 2001 (in contrast to 1992 survey). Nitrogen values ranged between 0.59e2.25% with a median value of 1.28% dry weight, the first quartile at 1.05% and third quartile at 1.58% dry weight. Concentrations were similar to values obtained in the national Norwegian study (Bruteig, 1993) and other investigations (Oksanen et al., 1990; Poikolainen et al., 1998b), but lower than those measured in a French urban area (Gombert et al., 2003). Sulphur concentrations ranged between 0.07e0.21% dry weight; the first quartile was 0.11%, 0,25 0,20

S( )

Element

0 1 - 10 11- 50 50- 100 Missing value

0,15 0,10 0,05 0,00 0,0

0,5

1,0

1,5

2,0

2,5

N( ) Fig. 3. Scatter plot of total S and N concentrations (%) in H. physodes collected on a national scale in the year 2001 and arranged according to 4 different foliose lichen cover classes.

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328

a

0,30

0,25

0.3

0.2

S ( ) year 2001

S( )

0 1 - 10 11 - 50 51 - 100

0,20

0.1

0,15

0.0 8.0

0

1.0 37.0

200 400 600 800 1000 1200 1400 1600 1800

Elevation (m)

14.0

0,10

b 3.0

0,05

2.5

0

1 - 10

11 - 50

51 - 100

Mean lichen cover (year 2000)

N( )

2.0

0,00

0 1 - 10 11 - 50 51 - 100

1.5 1.0 0.5 0.0

2,5

0

200 400 600 800 1000 1200 1400 1600 1800

Elevation (m) Fig. 5. Total S (a); and N (b) concentrations in H. physodes at forest plots grouped according to 4 foliose lichen classes and according to elevation.

N ( ) year 2001

2,0

1,5 8.0 1.0 37.0

1,0

14.0

0,5

0,0 0

1 - 10

11 - 50

51 - 100

Mean lichen cover (year 2000) Fig. 4. Box plot presentation of total S and N concentrations in H. physodes collected in 2001 at forest plots belonging to 4 different lichen cover categories. Boxes present 25th and 75th percentile values and whiskers present minimum and maximum values. Median values as well as the No. of plots included in each cover class are also indicated.

the third quartile 0.16% and the median 0.14% dry weight. Nitrogen content was approximately 10 times higher than sulphur. Both elements were highly correlated (r ¼ 0.94) irrespective of foliose cover class (Fig. 3). This suggests co-deposition or similar sources for both elements. Lichen S content has been shown to correlate with sulphate deposition (Zakshek et al., 1986). NH3 emissions are readily converted in the atmosphere to (NH4)2SO4 and NH4NO3 and deposited mainly in wet precipitation, often remote from the emission source (van Herk et al., 2003). Highest nitrogen and sulphur concentrations, above the third percentile values, were found in central and eastern Slovenia and at some plots in the west. In the present study it was not possible to directly compare lichen nitrogen and sulphur results with measured atmospheric

deposition of either N or S since National Air Pollution Monitoring Network sites are located mainly in urban centres or close to the main pollution sources; lichens were collected in remote forest regions. Total sulphate deposition in 2000, expressed as total S was 0.8e2.6 g m2 in urban and industrial regions, and between 0.8e0.9 g m2 in rural regions. However, there was no major difference in total nitrogen deposition between urban and rural regions. In 2000, average cumulative de2 position was between 0.3e1.1 g m2 NO 3 and 0.5e1.1 g m Table 3 Factor loadings for 65 samples of Hypogymnia physodes after Varimax rotation with Kaiser Normalization and using Principal Component Analysis (PCA) as the extraction method Element

Factor 1

S N As Br Cd Ce Cr K La Mo Rb Sb Th U Zn Explained variance (%)

2

3

4

5

0.751

0.546 0.354

0.920 0.912 0.370

0.306 0.636

6

7

0.967 0.966 0.967 0.885 0.969 0.938 0.969 0.447 0.943 0.902 42.4

0.764

0.473 14.8

10.0

0.329 9.1

0.571 7.4

5.0

Only those loadings that were equal or greater than 0.3 are presented.

3.6

Z. Jeran et al. / Environmental Pollution 146 (2007) 324e331

NHþ 4 . Regular NO2 measurements confirmed higher average concentrations in urban areas compared to rural areas, mostly due to heavier traffic (ARSO, 2002). No significant differences in median levels of S and N between different cover classes were observed (Fig. 4) when total S and total N in H. physodes were grouped according to different foliose lichen cover classes (Fig. 4). However, a relatively large span between minimum and maximum levels in each particular class, especially for nitrogen was apparent. A significant decrease in total lichen nitrogen with increasing elevation was noticed (Pearson corr. r ¼  0.254, P ¼ 0.043) (Fig. 5b). A similar trend to lower values with increasing elevation was also observed for sulphur (r ¼  0.793, P ¼ 0.001, N ¼ 14) (Fig. 5a). Results agree with the locations of possible pollution sources of both elements (at lower altitude). Both

elements can be transported in the form of fine particles far from the source (Krupa et al., 2003). Mean foliose lichen cover did not correlate with element concentrations investigated in this study. This was not really surprising as element levels determined in H. physodes were relatively low. Even potentially toxic elements at much higher concentrations (up to several percent dry weight) need not necessarily adversely influence lichen vegetation (Purvis and Halls, 1996; Garty, 2001). Estimation of mean foliose lichen cover provides a very simple method involving observation and mapping of all foliose lichens without considering the specific sensitivity or resistance of different species to air pollution, the types of tree or tree bark chemical characteristics. All these factors influence the diversity and abundance of lichens. van Dobben et al. (2001) suggested Sb and As could harm some species.

F1

F2

F3

F4

F5

329

F6

F7

Fig. 6. Graphical distribution patterns of factors F1eF7. Increasing depth of shading represent higher factor scores as shown on the scale.

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Z. Jeran et al. / Environmental Pollution 146 (2007) 324e331

Factor analysis applied to a data set of 15 selected elements (As, Br, Cd, Ce, Cr, K, La, Mo, Rb, Sb, Th, U, Zn, S-total and N-total) identified 7 factors (Table 3) explaining 92% of the total data set variance. Although factor analysis was performed on a selected number of elements in this study, and element concentrations in the last survey were lower compared to the survey performed 9 years earlier, mapping of factor scores (Fig. 6) gave a similar spatial distribution of factors as previously published (Jeran et al., 1996, 2002). Four factors represent anthropogenically introduced chemical elements, namely the steel industry (F2: Cr, Mo, Zn), high temperature processes (F5: Sb, Zn, As, Br), a Cd source (F7) and the socalled N-S factor (F3). Sulphur and nitrogen form their own factor with a slight contribution of As (loadings of 0.37), this factor explaining 14% of the total data set variance. Although no correlation with lichen cover was observed, the geographical distribution of F3 (Fig. 6) could explain the poor lichen vegetation (Fig. 2), at least in some areas. Two other factors can be explained as non-anthropogenic or natural factors, representing soil (F1: Ce, La, Th, U) and Rb (F6). Factor 4 (Br, K, Zn) can be assigned as a combined factor, representing on the one hand a marine component due to high loadings of Br, and on the other hand anthropogenic pollution since both Br and Zn can be derived from vehicular traffic (Loppi and Pirintsos, 2003; Purvis et al., 2003).

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