chlorogenic acid (+21.6% and 49.7% compared to controls, respectively). Moreover the total levels of phenols increased significantly (+44.5%). They were correlated to an increase in the activity of L- phenylalanine ammonia-lyase, that rises 1.5-fold 24 h after the fumigation and 2-fold already at the end of the fumigation. Also total content of flavonoids and anthocyanins increases (+60.6% and +1.7%, respectively). On the contrary, isoquercitrin concentration decreases (- 41.4%). No significant change has been found for hypericin, quercetin, hyperoside and amentoflavone levels. This study shows that O3 can distinctly affect the secondary metabolism of this species and therefore its medicinal value, changing the ratio amongst the active compounds. Biotechnological applications of O3 in the field of medicinal plants deserve attention.

ONE YEAR RESULTS OF SOIL NO & NO2 FLUXES MEASUREMENTS IN SUBALPINE FOREST

Fumagalli I. and Gruening C.

Joint Research Centre, Ispra, Italy. ivan.fumagalli@jrc.ec.europa.eu

In the framework of the FP7 project ECLAIRE (Effects of Climate Change on Air Pollution and Response Strategies for European Ecosystems) an automated dynamic chamber system for measure NO & NO2 fluxes from the soil has been installed during 2012 at the JRC.

In particular 5 replicate chambers measuring soil fluxes and one chamber that was closed to the ground and thus serves as a measurement blank have been located in a little forest, while the analysers, pumps, control and data acquisition systems are installed nearby in a small trailer. Air temperature and humidity probes were also placed in each chamber.

<figure><img src="image_15.png" /><figcaption>Pic. 1 and 2: Example of measuring chamber system (open and closed) used to measure NO & NO2 fluxes from soil during the experiment at the JRC Ispra forest site.</figcaption></figure>

<figure><img src="image_16.png" /></figure>

Sequentially each chamber was closed and flushed with air for approx. 55 l/min for 6 minutes. At the same time, the concentrations of NO, NO2 are measured at the outflowing air of chamber. To account for concentration changes due to the reaction NO + O3 -> NO2 + O2 in the chamber and sampling lines, the concentration of O3 is measured as well. Combining the measured concentrations, air flow and chamber size, raw soil fluxes of NO and NO2 are calculated.

The NO and NO2 soil flux data from the first year of measurements are showed and discussed. First results indicate some differences in the fluxes for every chamber probably due to slightly different soil conditions in terms of micro-organisms or illumination conditions. During the different seasons, fluxes of NO are clearly higher in summer compared to winter. This indicates that main drivers for NO emissions are soil temperature and soil water content. Regarding NO2, the situation regarding drivers is less defined.
# PHOTOSYNTHETIC PERFORMANCE OF QUERCUS ILEX L. UNDER LONG-TERM OZONE EXPOSURE PROBED BY CARBOXYLATION EFFICIENCY, MAXIMUM APPARENT QUANTUM YIELD AND MODULATED 820 NM REFLECTION.

Fusaro L.ª, Fares S.ᵇ, Gerosa G.ᶜ, Marzuoli R.ᶜ, Salvatori E.ª, Finco A.ᶜ, Quarato D.ᵇd, Monga R.ce, Manes F.ª.

Assessing ozone (O3) effects on photosynthetic machinery is a key issue to understand how the oxidative stress influences vegetation functionality and the ecosystem services provided (Manes et al., 2012). Moreover, data about the RuBisco and photosystem functionality measured during long term experiments could be important to improve the accuracy of ozone flux models to be use for risks assessment (Fares et al., 2012). Trends towards tolerance to O3 have been studied for Mediterranean tree species and contrasting results were obtained over a large number of experiments, probably due to different levels of O3 exposure and growth conditions. Moreover, only few experiment assessed the O3 effects on evergreen species in terms of dynamic response of CO2 assimilation to varying conditions of temperature and photosynthetic photon flux density (Mereu et al., 2011) and, to the best of our knowledge, no data is available about fluorescence of photosystems I (PSI), that plays a key role in response to oxidative stress. In this experiment, we have assessed long-term effects of O3 exposure on Quercus ilex, an evergreen species with great ecological relevance in Mediterranean Basin for its wide natural distribution, as well as widespread use in urban context. Two-years-old seedlings were continuously exposed for 5 months in open-top chambers facility at the Centre for Research on Effects of Pollutants on Ecosystems, in Curno, to three O3 treatments: charcoal filtered air (F, - 41% of ambient air O3), and non-filtered air supplemented with +35% (NF⁺) and +71% ppb O3 (NF⁺⁺) of ambient air O3. Interestingly, it was found that after 40 days of starting experiment, an early response to O3 leads to an increase in leaf dark respiration and photorespiration. Fluorescence parameters were also affected, and in particular the PSI functionality was involved in the first phase of oxidative stress response.

## References

Fares S., Matteucci G., Scarascia Mugnozza G., Morani A., Calfapietra C., Salvatori E., Fusaro L., Manes F., Loreto F. (2013). Testing of models of stomatal ozone fluxes with field measurements in a mixed Mediterranean forest. Atmos Environ. 67, 242-251.

Mereu S., Gerosa G., Marzuoli R., Fusaro L., Salvatori E., Finco A., Spano D., Manes F. (2011) Gas exchange and JIP-test parameters of two Mediterranean maquis species are affected by sea spray and ozone interaction. Environ Exp Bot. 73, 80-88.

Manes F., Incerti G., Salvatori E., Vitale M., Ricotta C., Costanza R. (2012). Urban ecosystem services: tree diversity and stability of tropospheric ozone removal. Ecol Appl. 22, 349-360.

<footnote>ªSapienza University of Rome, Dep. of Environmental Biology, P.le Aldo Moro 5, Rome, Italy. lina.fusaro@uniroma1.it</footnote>

<footnote>ᵇResearch Center for the Soil-Plant System, Rome, Italy.</footnote>

<footnote>ᶜCatholic University, Dep. of Mathematics and Physics, via Musei 41, Brescia, Italy.</footnote>

<footnote>ᵈUniversity of Tuscia, Dep. for Innovation in Biological, Agro-Food and Forest Systems, via S. Camillo de Lellis, Viterbo, Italy.</footnote>

<footnote>ᵉUniversity of Milan, Dep. of Agricultural and Environmental Sciences, via Celoria 2, Milan, Italy.</footnote>
EFFECTS OF OZONE AND NITROGEN DEPOSITION IN YOUNG TREES OF HORNBEAM AND OAK. RESULTS FROM THE ECLAIRE EXPERIMENTS IN ITALY

Gerosa Giacomo<fnref n="1" />, Marzuoli R.<fnref n="1" />, Monga R.<fnref n="2" />, Finco A.<fnref n="1" />

An Open-Top Chambers (OTC) experiment with ozone enrichment and increased nitrogen deposition has been performed during two consecutive years (2012 and 2013) in Northern Italy on young trees of *Quercus robur* and *Carpinus betulus*. Two hundreds and sixteen saplings of each species were potted and placed in 12 Open-Top Chambers following a split- plot design with 3 randomized blocks and two factors : ozone concentration, the main factor, at 4 different levels (CF-45%, NF, NF+35%, NF+70%), and nitrogen irrigation (NDep), the nested factor, at 2 different levels (tap water for control, tap water +NDep of 70Kg ofN*ha*y⁻<fnref n="1" />). These treatments were applied for two consecutive growing seasons. In both years stomatal conductance (gs) measurements and CO₂ assimilation response curves have been made throughout the season to assess the impacts on physiological and photosynthetical parameters. Half of the saplings was harvested at the end of 2012 season, while the remaining half was harvested at the end of 2013, in order to estimate the effects of both stresses, alone and in combination, on the total biomass production and on the root/shoot partition of the plants.

After two years of treatments, a general positive effect of nitrogen deposition on biomass production was found in both species, as it could be easily expected. This biomass increase was particularly intense in *C. betulus* (+76% of the total biomass, + 65% of roots biomass).

*Q. robur* showed a greater response to ozone than *C. betulus* in the control Ndep conditions. Oak plants showed a 10% and 18% of reduction of the total and root biomass in NF+70% treatment. This response was also found in the plants subjected increased to nitrogen deposition (-15% for total biomass, -16% for roots biomass). Hornbeam seemed stimulated by O3 when no nitrogen was added (+5% of total biomass). However, NDep treatment made hornbeam more susceptible to ozone, which caused a 30% biomass decrease in both shoots and roots. Looking at gs as a possible driver of the plants' response to ozone, we found that ozone lead to a 21% decrease of gs in oak plants when the had a total biomass reduction of 10%. This response is confirmed in NDep treatment (-18% in gs, -15% in total biomass). Thus, oak seemed to be more vulnerable to gs limitation because of the consequent reduction of CO₂ assimilation. In hornbeam, without nitrogen addition, ozone caused a slight reduction on gs leading to a decrease of stomatal dose and a small increase in total biomass (+5%).

On the contrary, nitrogen deposition caused a significant increase of gs (+23%) that lead to an increased ozone uptake, thus suggesting an overwhelming of the detoxifying defence (-30% in total biomass). Stomatal conductance, thus, reveals to be a key driver of the plant's response to ozone, but the final manifestation of the effect seems to be modulated by the detoxifying capacity of the plants.

The general decrease of gs in both species caused by ozone (also found in 2012 measurements), suggests the need to include an f(O₃) modifying function in the stomatal conductance models which will be defined for these two species. Some of biomass responses are partially in disagreement with the results of the first year, likely for the presence of a carry-over effect. This fact highlights the importance of performing long-term experiments (more than 1 year) for the investigation on ozone and nitrogen effects on biomass.

<footnote n="1">¹ Catholic University of Brescia, via dei Musei 41, Brescia (Italy). riccardo.marzuoli@unicatt.it</footnote>

<footnote n="2">² DISAA, University of Milan, via Celoria 2, Milan (Italy)</footnote>
# BIOINDICATION AND MONITORING OF ATMOSPHERIC DEPOSITION USING TREES AND SHRUBS

Gorelova S.V.<fnref n="1" />, Frontasyeva M.V.<fnref n="2" />, Gorbunov A.V.<fnref n="3" />, Lyapunov S.M.<fnref n="3" />, Okina O.I.<fnref n="3" />

Vitality and morphological changes of woody plant leaves collected in the area affected by emissions from the metallurgical enterprises was studied. It was shown that *Tilia cordata*, *Aesculus hippocastanum*, *Crataegus*, *Symphoricarpos albus* (see Figure) can be used for bioindication of the atmospheric deposition of trace elements by morphological parameters (necrosis, leaf morphology change).

<figure><img src="image_17.png" /><figcaption>*Crataegus monogyna*</figcaption></figure>

<figure><img src="image_18.png" /><figcaption>*Aesculus hippocastanum*</figcaption></figure>

<figure><img src="image_19.png" /><figcaption>*Tilia cordata*</figcaption></figure>

Figure. Necrotic and chloritic changes of shrubs and tree leaves caused by emissions from the metallurgical enterprises

Scanning electron microscopy allowed establishing the ratio of conglomerate components adsorbed on the leaves surface from aerosol emissions. Proportion of the organic components in the adsorbed particles was maximal (up to 87-95%); percentage of soil particles was 4-5%, and the amount of heavy metals was of the order of 2-4%. The dominant elements in the adsorbed particles of the technogenic origin were Fe and Mg (1.5-4.5%). Such elements as Mn, Cu, Pb, Cd, V, Mo, and Zn, which are the main components (with Fe) of industrial emissions of metallurgical enterprises in the city of Tula, were present in the range of 0.1-0.6 %. Their concentration exceeds the MPL in atmospheric air studied by means of neutron activation analysis and atomic absorption analysis of air aspirator air filters. Thus, the analysis of dust deposited on the woody plant leaves surface is an express method to assess the presence and ratio of the components of atmospheric deposition that can be used to judge on air quality exhaled by human respiratory tract.

## Acknowledgement

The authors acknowledge support of ‘The study of adaptive characteristics and buffer role of woody exotic species in the migration of toxic elements in the urban ecosystems” from RFBR Grant 13-05-97508.

<footnote n="1">¹Department of Biology abd Technology of Living Systems, Faculty of Natural Sciences, L.N. Tolstoy TSPU, Lenin Av., 125, 300026, Tula, Russia. gsvphysiology08@rambler.ru; salix35@gmail.com</footnote>

<footnote n="2">²Joint Institute for Nuclear Research, 141980 Dubna, Russia. marina@nf.jinr.ru</footnote>

<footnote n="3">³Geological Institute of RAS, Pizhevsky per.,7, Moscow, Russia. analytic@ginras.ru</footnote>
ACCUMULATION OF HEAVY METALS IN THE MOSS *PLEUROZIUM SCHREBERI* IN THE
WEST OF RUSSIA (KALININGRAD REGION)

Koroleva Yuliya, Vakhryanova Olga, Styogantsev Vasiliy, Melnikova Irina

Immanuel Kant Baltic Federal University, Universitetskaya st., 2, Kaliningrad, Russia, 236040.
yu.koroleff@yandex.ru

The moss biomonitoring technique was applied to air pollution studies in the West region of Russia (Kaliningrad region). Samples of the terrestrial moss *Pleurozium schreberi* have been collected every 5 years, from 2000 in accordance with the sampling strategy of the European moss survey program.

Moss samples were collected on regular network of 10x10 km during the August – September in 2000, 2005, 2010. Metals such as Pb, Cd, Cr, Ni, Cu, Fe, Mn were determined by AAS technique (by flame – Mn, Fe; ETA – others). In general, the concentration of Cd, Pb, Ni and Cu in mosses decreased between 2000 and 2010; the concentration of Cr increased in the same period.

The values of metals vary widely, but low values prevail. Highest concentrations were determined in the west part, lowest ones in the center of region.

<table><caption>Table 1 - Means of mediana in moss *Pleurozium schreberi* in Kaliningrad region in 2000 – 2010 year (mg/kg DW)</caption><thead><tr><th>year</th><th>Cu</th><th>Ni</th><th>Pb</th><th>Cr</th><th>Cd</th><th>Mn</th><th>Fe</th></tr></thead><tbody><tr><td>2000</td><td>5,3</td><td>4,11</td><td>13,2</td><td>0,14</td><td>0,14</td><td>256</td><td>192</td></tr><tr><td>2005</td><td>9,34</td><td>3,8</td><td>4,63</td><td>0,88</td><td>0,18</td><td>132</td><td>222</td></tr><tr><td>2010</td><td>2,84</td><td>1,21</td><td>1,91</td><td>1,41</td><td>0,05</td><td>245</td><td>168</td></tr></tbody></table>

<table><caption>Table 2 - Concentration of Heavy metals in moss *Pleurozium schreberi* in Kaliningrad region in 2010 year (mg/kg DW)</caption><thead><tr><th></th><th>Cu</th><th>Ni</th><th>Pb</th><th>Cr</th><th>Cd</th><th>Mn</th><th>Fe</th></tr></thead><tbody><tr><th>mean</th><td>4,46</td><td>1,20</td><td>2,51</td><td>2,16</td><td>0,11</td><td>346</td><td>217</td></tr><tr><th>min</th><td>1,68</td><td>0,36</td><td>0,39</td><td>0,69</td><td>0,006</td><td>73</td><td>113</td></tr><tr><th>max</th><td>19,7</td><td>2,54</td><td>9,33</td><td>12,1</td><td>1,12</td><td>960</td><td>700</td></tr></tbody></table>

Principal component factor analysis was used to identify and characterize different pollution sources and to point out the most polluted area. There were identified two factors of metals emission.

The maps of metals atmospheric deposition were created. Cartography analysis revealed spatial patterns of metals distribution. Probably highest levels of metals in moss in the west part of region (Sambia peninsula) and elevated levels in the North, North-East and South-West parts are indicating trans-boundary pollution.
FOLIAR SURFACE CLEANING TECHNIQUES FOR ANALYSIS OF PARTICULATE CHEMICAL COMPOSITION IN AIR POLLUTANTS MONITORING

Laffray, X.ª, Domanski, M.ᵇ, and Castell, J.F.ᵇ

ª. Université de Franche-Comté, Laboratoire de Chrono-Environnement, UMR CNRS 6249,
1, Place Leclerc, F-25030 Besançon cedex, France - [xavier.laffray@gmail.com](mailto:xavier.laffray@gmail.com)

ᵇ. INRA AgroParisTech, UMR Environnement et Grandes Cultures, F-78850 Thiverval-Grignon, France -
[castell@grignon.inra.fr](mailto:castell@grignon.inra.fr)

Major contributor to the adverse health effects of air pollution on the respiratory system, airborne particles are the main drivers of the increased short and long-term frequency of respiratory ailments for humans, and especially for children, with allergies, acute asthma and other respiratory diseases inducing exacerbated responses of the immune system. Moreover, accumulation of organic and inorganic pollutants in above-ground plant parts (i.e. leaves, fruits) via wet or dry deposition can menace human health after ingestion (intake through the food chain).

Acting as a protective barrier, the hydrophobic or only moderately hydrophilic epicuticular waxes of leaf surfaces of most plants can also efficiently trap airborne particles; particles remaining adsorbed or incorporated into the hydrophobic wax layer of the foliage. Over the last three decades, chemical foliar analysis was strongly privileged as a diagnostic tool of tree nutrition over chemical soil analysis, with an increasing interest as environmental indicator of air pollution. The amount of deposited particles can be modified by rainfall, which washed off the trapped particles and plant exudates, leading to an increase of element concentrations in througfall. Therefore, the wax trapping ability of plant leaves needs attention in soil/plant transfer rate analysis for air quality monitoring studies. Thus, washing sampling techniques are necessary to distinguish between absorbed and accumultated particles.

Several techniques were used to clean both dry or moistened plant tissues with various washing chemicals (i.e. distilled or tap water, chloroform, toluene, tetrahydrofuran, hydrochloric acid, EDTA) and with or without mechanical shaking. Mechanical cleaning techniques were, to a lesser extent, also used by stripping a resin film deposited on leaf surfaces.

The foliar surface cleaning methods depend on the physical and chemical properties of the studied elements trapped on the leaf surface (surface contamination risks). The protocole should be adapted to 1) plant species and 2) surface properties and age of the leaf, since they are determinant factors (washing chemical product choice, and extraction time).
# STUDY OF A TROPOSPHERIC OZONE EFFECT ON TWO CULTIVARS OF TOMATO: LYCOPERSICON LYCOPERSICUM COLGAR (L.) KARSTEN EX FARW. AND SOLANUM LYCOPERSICUM RECHAIGA II

Maamar B., Maatoug M., Dellal A., Ait Hammou M.

*Laboratory for Agro Biotechnology and Nutrition in Semi-Arid Area*
*Faculty of Natural Sciences and Life. Ibn Khaldun University, Tiaret (Algeria). Email: dellal05_aek@yahoo.fr*

Two cultivars of tomato: tomato Colgar and Rechaiga II were exposed to different concentrations of O3 (50, 80 and 100 ppb) in a fumigation chamber to evaluate their sensitivities and check some physiological parameters. The cultivar Rechaiga II (Solanum lycopersicum) appeared particularly sensitive to O3 at an early stage of growth. Typical responses chlorotic in 24 hours after exposure to a single pulse of 50 ppb for 4 h, for a period of 7 days were recorded spots. Respondents physiological parameters (stomata conductance, the membrane integrity, chlorophyll a, b, total, carotenoids and soluble sugars), have suffered alterations following exposure. The cultivar Rechaiga II was sensitive to ozone while cultivar Colgar (Lycopersicon lycopersicum (L.) Karsten ex Farw) has remained asymptomatic (resistant) throughout the entire experiment.

## Keywords

Tropospheric ozone, fumigation chamber, Solanum lycopersicum, Lycopersicon lycopersicum (L.) Karsten ex Farw, necrosis, physiological parameters.

# CRITICAL EVALUATION OF ECOSYSTEM POLLUTION

Maňkovská, B.<fnref n="1" />, Oszlányi, J.<fnref n="1" />, Izakovičová, Z.<fnref n="1" />, Frontasyeva, M. V.<fnref n="2" />,

Pollution problems in the forest ecosystems resulting from 100 year of operation of three smelter complexes in the Central Spiš are reviewed. Original data are presented with respect to temporal and spatial trends of nitrogen, sulphur, and heavy metal pollution. INAA at the IBR-2 reactor has made it possible to determine the content of 40 elements in mosses, foliage of forest tree species, lime and spruce wood, soil and wildlife in marginal Slovak hot spot - Central Spiš. Instrumental neutron activation analysis (INAA) at the IBR-2 reactor has made it possible to determine the content of 40 elements in mosses, foliage of forest tree species, lime and spruce wood, soil and wildlife in the marginal Slovak hot spot – the Central Spiš. In addition to NAA, flame atomic absorption spectrometry (AAS) was applied to determine the contents of S, Cd, Cu, Hg and Pb.

The polluted air impact on forest ecosystems is a major question at present and in the future. 1.7-fold exceeding of critical values and high concentrations of As, Fe, Hg, and N in spruce needles were found in the Central Spiš. The Low Tatras National Park served as a pristine (baseline) region. The concentration of Hg and As in humus and horizont A₀ from Central Spiš is increased markedly. Other determined elements (Cd, Co, Cu, Ni, Pb, and Zn) do not exceed the limits. In the area of Central Spiš we have found in comparison with the Norwegian limit values (the Central Norway- as relatively pristine region) exceeded levels for Al, As, Ca, Cd, Cl, Co, Fe, K, Mn, Sb, Sm, Sr, W, and Zn. In comparison with Magnitogorsk in the Ural Mountains (the most polluted area in Europe) we found in Spiš higher values for Ca, Cl, Co, K and Mn. We found the highest concentrations of Au, Br, Co, and Sb in the oldest 70–80 years old spruce wood, and the highest concentrations of Al, As, Au, Br, Cl, K, Na, Rb, Sb and Sm in the oldest 80–90 years old lime wood. We found the highest concentration of Al, As, Ba, Br, Ce, Co, Cr, Cs, Fe, Hf, Hg, K, La, Mg, Mo,

<footnote n="1">¹ Institute of landscape ecology, Slovak Academy of Science, Bratislava, Slovak Republic. bmankov@stonline.sk</footnote>

<footnote n="2">² Frank Laboratory of Neutron Physics, JINR, Dubna, Russia</footnote>
Na, Ni, Pb, Rb, Sb, Sc, Sm, Ta, Tb, Th, U, V and W in soil samples; the highest concentration of Ca, Cd, Cl and S in spruce roots; Mn and S in spruce needles; Sr in mosses and Zn in lichens. In comparison with limit values the concentration of Ba, Co, Hg, Mg, Mn, and Sr in the teeth of roe deers is increased markedly. Other determined elements (Al, As, Ca, Cd, Cu, Fe, Na, Pb and Rb) do not exceed the limits.

**Key words:** air pollution, biomonitoring, metals, foliage, mosses, soil, wood

# ATMOSPHERIC DEPOSITION OF HEAVY METAL DEPOSITION IN KOSOVO BY USING MOSS BIOMONITORING AND AAS

Albert Maxhuni<fnref n="1" />, Pranvera Lazo<fnref n="2" />

Bryophytes act as precise and sensitive bioindicators as well as bioaccumulators of metal deposition in the environment. The atmospheric deposition of Cd, Cr, Cu, Fe, Hg, Ni, Mn, Pb and Zn in Kosovo was investigated by using moss species (*Hypnum cupressiforme*) as bioindicators. This research is a part of the international programme (ICP Vegetation Programme, UNECE) investigating the impacts of air pollutants on crops and natural vegetation. Moss samples were collected from 25 sampling sites evenly distributed over the territory of Kosovo during the dry period of summer 2011. The most dominant moss species in this study area were *Hypnum cupressiforme*. The map of sampling points is shown in Fig. 1.

<figure><img src="image_20.png" /><figcaption>**Figure 1** The map of sampling points and coordinates</figcaption></figure>

Unwashed, dried samples were totally digested by using microwave digestion system and the concentrations of metal elements were determined by AAS equipped with flame and/or furnace systems. The results reflect local emission points. Descriptive statistics were made to evaluate contamination level. The data obtained in this study were compared with those obtained in the similar studies in neighboring countries and Europe in 2010. The estimated level being highest for Fe, Mn and Zn followed by Pb, Cu, Cr, Ni, Cd and Hg. Overall, Fe, Zn and Pb are responsible for causing major pollution in the studied area. Relatively high lead concentrations were also found in Bulgaria Kosovo (Harmens et al. 2013).

<figure><img src="image_21.png" /></figure>

<figure><img src="image_22.png" /></figure>

<figure><img src="image_23.png" /></figure>

<figure><img src="image_24.png" /><figcaption>**Figure 2** Cr, Ni, Pb and Zn distribution</figcaption></figure>

Statistical analyses also revealed that correlation exist between the metal content in mosses and degree of pollution in studied sites. High values of positive skewness and kurtosis were found for the elements (except Cu and Zn) by determining that the data of these elements are skewed right. EWMA chart reflected the spatial distribution of the elements, and multivariate analysis applied to the data matrix of the concentrations of the elements identified the major factors that affect the elements distribution

**Key words:** air pollution, Heavy metals; moss biomonitoring; AAS analysis, Kosovo area.

<footnote n="1">¹ Department of Chemistry, Faculty of Natural Sciences, University of Prishtina, Kosovo</footnote>

<footnote n="2">² Department of Chemistry, Faculty of Natural Sciences, University of Tirana, pranveralazo@gmail.com</footnote>
# CHANGES IN THE HEAVY METAL CONTENTS OF MOSSES DURING LONG-TERM DRY STORAGE

Poikolainen, J.

Finnish Forest Research Institute, PO Box 413, FI-90014 University of Oulu, Finland. jarmo-poikolainen@metla.fi

A heavy metal survey by means of mosses has been carried out in Finland every five years since 1985. The moss samples (*Hylocomium splendens*, *Pleurozium schreberi*) are collected from the plots of the 8th National Forest Inventory. Small amount of each moss sample is analysed soon after collection and the remainder has been stored at the Paljakka ESB. The 108 samples from same 27 plots stored in moss surveys 1985-86, 1990, 1995 and 2000 were reanalysed in 2008. Storage effects were investigated for the heavy metals Cd, Cr, Cu, Fe, Ni, Pb and Zn.

Overall, mean Fe, Pb and Cr concentrations decreased significantly. The drop was greatest on plots with initially high concentrations. The decrease of Cd, Ni and Zn contents was less pronounced. The loss of heavy metals is likely due to drying when cell membranes rupture and surface material is lost. Mean Cu contents increased during storage. Cu is the important nutrient, and probably it is moved in early stage of storage from the old shoots to new ones. Results emphasize the importance of establishing the intended use of stored moss prior to sampling, in order to select and optimize an appropriate storage technique.

## References:

Utriainen, J., Poikolainen, J., Piispanen, J. & Kubin, E. (2007). Stabilizing temperature and RH conditions in dry storage facilities. *Museum Management and Curatorship, 22*(1), 79-90.

Utriainen, J., Poikolainen, J., Kuokkanen, M., Piispanen, J. & Kubin, E. (2006). Ympäristönäytteiden pitkäaikaisen säilytyksen ja yhteiskäytön kehittäminen Suomessa. *Suomen ympäristö / The Finnish Environment 56*, 1-122. English summary: Environmental specimen banking and co-operation in Finland, 75-83.

# ATMOSPHERIC DEPOSITION OF MAJOR AND TRACE ELEMENTS IN ROMANIA STUDIED BY NAA AND AAS: MOSS SURVEY 2010/2011

I. Popescu<fnref n="1" />, C. Stihi<fnref n="1" />, A. Ene<fnref n="2" />, S. Cucu-Man<fnref n="3" />, R. Todoran<fnref n="4" />, C. Radulescu<fnref n="1" />,
I.D. Dulama<fnref n="5" />, A. Chilian<fnref n="5" />

M.V. Frontasyeva, O. Culicov, I. Zinicovscaia, My Trinh, S.F. Gundorina, S.S. Pavlov
Joint Institute for Nuclear Research, 141980 Dubna, Russian Federation

<footnote n="1">¹Valahia University of Targoviste, Faculty of Sciences and Arts, 2 Carol I str., 130024, Targoviste, Romania, E-mail: ivpopes@yahoo.com, stihi@valahia.ro</footnote>

<footnote n="2">²Dunarea de Jos University of Galati, Department of Chemistry, Physics and Environment, 47 Domneasca, 800008 Galati, Romania, E-mail: aene@ugal.ro</footnote>

<footnote n="3">³Alexandru Ioan Cuza University, Faculty of Chemistry, 11 Carol I str., 700506, Iasi, Romania</footnote>

<footnote n="4">⁴Technical University of Cluj-Napoca, North University Center, 62A Victor Babes str., 430083, Baia Mare, Romania</footnote>

<footnote n="5">⁵Valahia University of Targoviste, Multidisciplinary Research Institute for Sciences and Technologies, 13 Sinaia str., 130004, Targoviste, Romania</footnote>
*National Institute for R&D in Electrical Engineering ICPE-CA, Splaiul Unirii, Nr. 313, District 3, 030138, Bucharest Romania

**The Institute of Chemistry of the Academy of Sciences of Moldova, 3, Academiei Str., 2028 Chisinau, R. Moldova

Biomonitoring of air pollutants using mosses has been employed in Romania for the last 20 years by applying nuclear as well as non-nuclear analytical techniques. Prior to 2010/2011 moss survey, surveys in 1995 and 2000 as well as some individual projects ("NATO Science for Peace", bilateral Romania-JINR projects) covered different areas on a local scale with the network varying from 35 to 70 sampling sites. A nationwide deposition moss survey in 2010/2011 undertaken by four Romanian Universities in Targoviste, Galati, Iasi, and Baia Mare, comprised 330 sampling sites evenly distributed over 75% of the Romanian territory. A total of 34 elements (Na, Mg, Al, Cl, K, Ca, Sc, Ti, V, Cr, Mn, Fe, Co, Ni, Cu*, Zn, As, Br, Rb, Sr, Cd*, Sb, Ba, Cs, La, Ce, Sm, Tb, Hf, Ta, W, Pb*, Th, and U) were determined in a wide concentration range from 10000 mg/kg for Al and K down to 0.001 mg/kg for some rare earths by two complementary methods: instrumental epithermal neutron activation analysis (INAA) at the reactor IBR-2 in Dubna, RF, and graphite furnace/flame atomic absorption spectrometry (GFAAS/FAAS) (marked with *) in the Multidisciplinary Research Institute for Science and Technologies from Valahia University of Targoviste, Romania. Principal component (factor) analysis was used to identify the most polluted areas and to characterize different pollution sources. Four factors were revealed. Factor 1 is a mixture of light and heavy crustal components. Factor 2 is of anthropogenic origin with Zn (0.82), As (0.73), and Sb (0.92). Relevant scores point out that the main contributors to this factor are sites from Maramures and Ruschita areas. Factor 3 is a mixture of "marine elements" with Cl (0.79) and Br (0.62) while iodine is not reported as it was not determined in all samples. Ca (0.78), K (0.57), and Sr (0.45) may originate from fertilizer components. Factor 4 comprises of Pb (0.63), Cd (0.66), and Cu (0.64) which may arise from gasoline usage to the copper mining industries. From the present results it can be concluded that atmospheric deposition of trace metals is a considerable problem in the northern and western parts of Romania. This study contributes to the national monitoring system of Romania for long range transported elements of air pollutants, and along with epidemiological data it may serve for baseline human health risk assessments.

BIOMONITORING OF HEAVY METALS IN PLAYGROUNDS IN ATHENS, GREECE

Saitanis¹ C., Lappa¹ K., Ntatsi² G., Barouchas³ P., Agathokleous¹ E.

¹Agricultural University of Athens, Laboratory of Ecology and Environmental Sciences, Iera Odos 75, Votanikos, Athens, Greece.

e-mails: saitanis@aua.gr; katerinalappa@yahoo.gr; evgenios_ag@hotmail.com

²Agricultural University of Athens, Laboratory of Vegetable Production, Iera Odos 75, Votanikos, Athens, Greece. e-mail: gntatsi@aua.gr;

³Technological Educational Institute of Western Greece. Department of Agricultural Technology Lab. of Soil Science and Agricultural Chemistry. Theodoropoulou Terma T.K. 27200, Campus Amaliada, Greece. e-mail: barouchas@gmail.com

The purpose of the study was to explore the spatial pattern of heavy metals' load in playgrounds located in the greater region of Athens. To this purpose, a biomonitoring exploration of Fe, Mn, Zn, Mg and Ca was conducted, using leaves of *Platanus orientalis* L. and *Cercis siliquastrum* L. trees, grown in playgrounds.

**Methods used** : i) Sampling and samples' treatment: Leaves of the plants were collected from 16 places scattered throughout Attica, namely: Agia praskevi, Argyroupolis, Aspropyrgos, Votanikos, Dafne, Ekali, Alimos, Glyfada, Heraklion, Keratsini, Patisia, Perama, Pireas, Ilion (Tritsis' park) Chaidari, Zographou. Samples from Ekali served as control (low pollution) region. Three to five shrubs per plant species per region were sampled. The leaves of each sample were further divided to two parts; the leaves of the one part were washed with bidistilled water while the leaves of the other part were not washed. ii) Analytical procedure: For the determination of the metals a Perkin-Elmer (Model
1100B) Atomic Absorption Unit was used. iii) Statistical analysis: Pearson correlation, ANOVA and multivariate methods were used for the analysis of the data.

**Results and discussion:** Significant correlations were found between Mg_Mn (r=0.2353, p=0.000), Mg_Ca (r=0.2224, p=0.000), Fe_Zn (r=0.5053, p=0.000), Fe_Mg (r=0.1557, p=0.005), Fe_Ca (r=0.2146, p=0.000), Zn_Mg (r=0.1592, p=0.004), Zn_Ca (r=0.2037, p=0.000) and Mg_Ca (r=0.6727, p=0.000). Multivariate analysis revealed highly structured data. Statistically significance difference was observed between washed and unwashed leaves, between tree species and between regions for all the elements. The highest concentrations of Fe were observed at Aspropyrgos regions, obviously due to the metallurgical industry established there. The highest concentrations for Mn and Zn were observed at Votanikos and Keratsini respectively while for Mg and Ca at Agia Paraskevi. For all the elements the lowest concentrations were observed in the low polluted Ekali region.

**Conclusion** : Leaves of *Platanus orientalis* L. and *Cercis siliquastrum* L. can serve as useful materials for biomonitoring for heavy metals. A high spatial variability of the levels of the measured elements was observed across the playgrounds in the greater region of Athens. The study is still in progress; the samples are further analyzed for the determinations of the other elements.

**Keywords:** Heavy metals; Biomonitoring; Playgrounds; *Platanus orientalis*; *Cercis siliquastrum*

# ANALYSIS OF MOSS THUIDIUM CYMBIFOLIUM DOZ. ET MOLK TO RETRIEVE PAST ATMOSPHERIC ELEMENTAL PROFILE OF GARHWAL STATE OF IN INDIA

Dinesh Kumar Saxena

Department of Botany, Bareilly College, Bareilly, UP., India. Dinesh-botany@gmail.com

This study is the continuation of previous study on retrospective monitoring and systematic gathering of information about the atmospheric elemental profile of the environment (Pb, Fe, Zn, Cd, Fe and Ni) over the past 100 years, i.e. 1895-1999 from Garhwal region in India by analyzing moss herbarium vouchers specimens as a tool loaned from different intentional herbaria, as well as analysis of fresh moss samples of *Thuidium cymbifolium*, (Doz. et Molk.) Doz. et Molk, harvested during 1999-2013.

A significant increase in atmospheric elemental profile was observed during analysis of moss species of *Thuidium cymbifolium* (Doz. et Molk.) Doz. et Molk. collected from Garhwal states when compared with moss voucher specimens of the same species borrowed from different herbaria on loan of this region. Analyzed elemental profile seems to be a reflection of atmospheric metal load. The lowest metal concentrations were present in analyzed mosses of the voucher specimens belongs to the period, i.e. 1895 and the level increased gradually over a period of time. However, difference in increase in trend was also observed from 0.01 to 5.9 times for different metals over 1895. The maximum significant increase observed for metals Pb was 5.9 times with respect to first metal data of the year 1895 in *Thuidium cymbifolium* (Doz. et Molk.) Doz. et Molk. Major source for the same can not be ruled out from automobiles followed by generators.

For rest of the metals same could be due to overall an increase in metal load resulting from progressive commercialization and unwisely urbanization. The ratios between the content of the various metals in bryophyte tissue increased was not identical for all metals examined. An increase in metal Ni and Cd was quite low till 1999 (1.09 and 0.03 times) while same increased to 1.7 and 0.3 times in 2013. Its source can not be ruled out from Ni Cd batteries are being used in rechargeable cells. While increased values of Cu and Zn (3.1 & 3.9 times) is due to its use in agricultural production in the country.

Present study is an effort to bridge the existing gap in terms of information and analysis of the elemental data of current and past that it provides information on retrospective atmospheric elemental data and it's an increasing trend over the past 100 year from Garhwal region.
<figure><img src="image_25.png" /></figure>

Figure: Percent increase in atmospheric elemental concentrations in moss *Thuidium cymbifolium* (Doz. et Molk.) Doz. et Molk from 1895.

# IMPACT OF CLIMATE CHANGE ON STOMATAL OZONE UPTAKE

Schröder M, Grünhage L

Department of Plant Ecology, Justus-Liebig University Giessen, Heinrich-Buff-Ring 26, D-35392 Giessen, Germany, Matthias.Schroeder@bio.uni-giessen.de

The impact of increasing background O3 concentrations as well as the impact of increasing air temperatures and changes in precipitation patterns on wheat stomatal O3 uptake and AOT40 are estimated exemplarily for the Hessian air quality monitoring station Linden, Germany.

O3 concentration, air temperature and precipitation anomalies are added to the measured data of the years 1997 – 2010. Background O3 concentrations are increased linearly by 5, 10 and 15 ppb. Mean monthly anomalies of air temperature and precipitation for the time periods 2031-2060 and 2071-2100 are derived from 13 climate change projections for scenario A1B and 3 projections for scenario E1, which stabilises global air temperature change at about 2 °C.

The box-and-whisker plots below illustrate the effect of increasing background O3 concentration on AOT40 and POD6 at current climate conditions. The impact of future climate conditions will be presented and discussed.

<figure><img src="image_26.png" /></figure>
# EFFECTS OF OZONE ON BEAN
(Phaseolus vulgaris) IN CROATIA IN 2013

Zdravko Špirić Oikon Ltd. - Institute for Applied Ecology, Zagreb, Croatia [zspiric@oikon.hr](mailto:zspiric@oikon.hr)

Vladimir Kušan, Oikon Ltd. - Institute for Applied Ecology, Zagreb

Dario Kremer, Farmaceutsko-biokemijski fakultet, botanički vrt "Fran Kušan", Zagreb

Zoran Posarić, Abies d.o.o., Rasadnik Lještaki, Bukovje Križevačko Nikolina Ribarić, Osnovna škola Ljubo Babić,

Jastrebarsko Sandra Nikolić, Miomirisni otočki vrt, Mali Lošinj

This paper presents results of the study of impact of ozone on growth and yield on bean (Phaseolus vulgaris), which was implemented in Croatia during 2013 under the International Cooperative Programme on studying the effects of air pollution on natural vegetation and crops (the UNECE ICP Vegetation.

[http://icpvegetation.ceh.ac.uk/research/Ozone.html](http://icpvegetation.ceh.ac.uk/research/Ozone.html)

The research results achieved in field trials in Zagreb, Križevci, M. Lošinj and Jastrebarsko will help to provide a better understanding and resolve numerous and complex problems that result from the harmful effects of ozone on vegetation at local, regional and global scale.

**Key words:** ICP Vegetation, Croatia, biomonitoring, ozone, bean

## NITROGEN IN MOSSES IN CROATIA IN 2006/2010

Zdravko Špirić<fnref n="1" />, Trajče Stafilov<fnref n="2" />, Ivana Vučković<fnref n="2" />, Marin Glad<fnref n="3" />, Vladimir Kušan<fnref n="1" />

During 2006 moss samples were collected from 98 sampling sites evenly distributed all over the territory of Croatia. Moss sampling was repeated in 2010 when 23 additional sampling sites were added and analysed.

Kjeldahl method was used to determine the nitrogen content in all samples.

Descriptive statistics and distribution maps were prepared. Data obtained from these two surveys were compared. The median value of N content in the samples collected in 2006 is

1.60 % and varies from 0.79 % to 3.16 % while the content of N in samples collected in 2010 ranges between 0.71 % and 2.93 % with the median value of 1.49 %. High contents of N (2.32 % - 3.17 %) were found in the regions of Slavonia, Podravina, Posavina and cities Zagreb and Sisak as a result of agricultural and industry activities and traffic.

**Key words:** Croatia, nitrogen, Kjeldahl method, moss biomonitoring

<footnote n="1">¹ OIKON Ltd. - Institute for Applied Ecology, Trg senjskih uskoka 1-2, 10020 Zagreb, Croatia</footnote>

<footnote n="2">² Institute of Chemistry, Faculty of Natural Sciences and Mathematics, Ss. Cyril and Methodius University, POB 162, 1000 Skopje, Macedonia;</footnote>

<footnote n="3">³ Teaching Institute of Public Health, Primorsko-Goranska County, Rijeka, Croatia</footnote>
# BIOMONITORING OF AIR POLLUTION BY TRACE ELEMENTS USING ITALIAN RYEGRASS (LOLIUM MULTIFLORUM L. 'LEMA')

Urbaniak J.<fnref n="1" />, Budka A.<fnref n="2" />, Kayzer D.<fnref n="2" />, Borowiak K.<fnref n="1" />, Zbierska J.<fnref n="1" />, Barałkiewicz D.<fnref n="3" />, Hanć A.<fnref n="3" />,

Results of investigations and assessment of air pollution by cadmium, lead and arsenic using Italian ryegrass are presented in this paper. The experiment was carried out in the 2011 growing season in Poznan city and surroundings areas. *Lolium multiflorum* L. 'Lema' exhibits several properties useful for active biomonitoring of air pollution. Plants were exposed at sites varying in environmental characteristics. High cadmium and lead concentrations in leaves were noted in plants exposed within the city area. Canonical variate analysis illustrated variability in concentrations of elements in certain exposure series. The highest arsenic concentrations were observed in the first exposure series, while the highest lead concentrations were observed during the second series. Comparison of heavy metal concentrations at exposure sites to the control site revealed that comparable levels occurred in the city sites and the Agro-ecological Landscape Park. This was an effect of high cadmium and lead levels at city sites, and arsenic at the rural site. The lowest level of measured heavy metals was observed at an exposure site located 15 km from Poznan city in a rural area.

## Acknowledgement

Presented investigations were supported by project grant No.: N 0540/B/P01/2011/40 from The National Science Centre.

# SOME CONSIDERATONS ABOUT ¹⁵N-PATTERNS IN MOSS TISSUE AROUND A POULTRY FARM

Werner, W., & Schlimpen, K.

University Trier, Department of Geobotany, D-54386 Trier (Germany), Behring Str. 21, werner@uni-trier.de

A case study in moss monitoring close to a poultry farm in the Eifel mountains shows high accumulation of nitrogen in active exposed moss tissue of *Hypnum cupressiforme* and a parallel increase of δ¹⁵N signature. Both accumulation of nitrogen and the δ¹⁵N ratio decreased by increasing distance to the immission source (poultry farm). Most authors who investigate δ¹⁵N Patterns in Moos tissue exposed to rural (agricultural) immissions report ¹⁵N enrichment in Moss tissue. Could this be right?

Theoretically, rural N-immission should be primarily deposited as gaseous NH₃. Therefore it would be fractionated and depleted to lighter ¹⁴NH₃ molecules that are energetically favoured to be volatilised into the atmosphere than heavier ¹⁵NH₃ molecules. If only gaseous ammonia would be immitted, the δ¹⁵N signature in moss tissue would decrease and not increase.

But we and most other authors find an enrichment of ¹⁵N. The consequence is, that nitrogen would be deposited in different fraction than the gaseous. This would be if the gaseous ammonia would react with particles and during this gas to particle reaction (ammonia with sulphate or chloride in aerosol droplets to (NH₄)₂SO₄ or NH₄Cl) the heavier ¹⁵N isotope will be favoured (see Fig. 1).

<footnote n="1">¹Department of Ecology and Environmental Protection, Poznan University of Life Sciences, ul. Piątkowska 94C, 60-649 Poznań, Poland</footnote>

<footnote n="2">²Department of Mathematical and Statistical Methods, Poznan University of Life Sciences, Wojska Polskiego 28, 60-637 Poznań, Poland</footnote>

<footnote n="3">³Department of Trace Element Analysis by Spectroscopy Method, Adam Mickiewicz University, ul. Grunwaldzka 6, 60-780 Poznań, Poland</footnote>
So we will demonstrate that in exposed plant tissue, by increasing δ¹⁵N ratio, the particulate deposition predominates the gaseous deposition. Atmospheric reaction processes are important for isotope fractionation.

<figure><img src="image_27.png" /><figcaption>FIGURE 1: PATHWAYS OF N IN A LIVESTOCK FARM FROM FEEDING TO HYPOTHETICAL DEPOSITION FLUXES INFLUENCING ISOTOPIC SIGNATURE OF MOSS TRANSPLANTS. NUMBERS IN BRACKETS REFER TO Δ¹⁵N VALUES FROM LI</figcaption></figure>
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