DETERMINATION OF HEAVY METAL DEPOSITION IN THE COUNTY OF OBRENOVAC ( SERBIA ) USING MOSSES AS BIOINDICATORS . III . COPPER ( Cu ) , IRON ( Fe ) AND MERCURY ( Hg )

In this study, the deposition of three heavy metals (Cu, Fe and Hg) in four moss taxa (Bryum argenteum, Bryum capillare, Brachythecium sp. and Hypnum cupressiforme) in the county of Obrenovac (Serbia) is presented. The distribution of average heavy metal content in all mosses in the county of Obrenovac is presented on maps, while long- term atmospheric deposition (in the mosses Bryum argenteum and B. capillare) and short-term atmospheric deposition (in the mosses Brachythecium sp. and Hypnum cupressiforme) are discussed and given in a table. Areas of the highest contaminations are highlighted.


INTRODUCTION
Surveillance of heavy metals in mosses was originally established in the Scandinavian countries in the 1980's.However, the idea of using mosses to measure atmospheric heavy metal deposition was developed already in the late 1960's (R h ü l i n g and T y l e r, 1968; T y l e r, 1970).It is based on the fact that mosses, especially the carpet-forming species, obtain most of their nutrients directly from precipitation and dry deposition.Nowadays, Nowadays, this method is widely used in many countries (S c h a u g et al.In the present investigations, we decided to use two acrocarpous moss species (Bryum argenteum Hedw.and Bryum capillare Hedw.) that can give us an idea of long-term atmospheric deposition, inasmuch as they are attached to the substrate and also accumulate metals deposed during the last few decades in the surface layers of substrate.In addition, some other Bryum species are considered from the standpoint of trace metal deposition (S c h i n t u et al., 2005).��osses are better than other higher plants in scanning heavy metal deposition because: -they are perennial without deciduous periods; -they have a high cation exchange capacity that allows them to accumulate great amounts of heavy metals between apoplast and symplast compartments without damaging vital functions of the cells (V á s q u e z et al., 1999); one of the main factors influencing cation exchange capacity is the presence of polygalacturonic acids on the external part of cell wall and proteins in the plasma membrane (A c e t o et al., 2003).
-mosses do not posses thick and strong protective layers like cuticles.��ore about hyperaccumulation of metals in plants and mosses can be found in P r a s a d and F r e i t a s (2003).Bryum argenteum has already been shown to have special metal accumulation peculiarities (A c e t o et al., 2003; V u k o j e v i ć et al., 2005).
It should also be noted that this time-integrated way of measuring patterns of heavy metal deposition from the atmosphere in terrestrial ecosystems, besides being spatially oriented, is easier and cheaper than conventional precipitation analyses, as it avoids the need for deploying large numbers of precipitation collectors.The higher trace element concentration in mosses compared to rain water makes analysis more straightforward and less prone to contamination (B e r g and S t e i n n e s, 1997b).
Use of mosses to investigate heavy metal deposition shows transboundary heavy metal pollution and can indicate the paths by which atmospheric pollutants enter from other territories or reveal their sources within the investigated area.
Although, 15 heavy metals have been analyzed in all, only deposition and distribution of Copper (Cu), Iron (Fe) and ��ercury (Hg) are treated in the present study, due to limitation of space.The presence and distribution of aluminium, arsenic, boron, cadmium, cobalt and chromium in the county of Obrenovac as screened by mosses were considered in two already published papers (S a b o v l j e v i ć et al., 2005 and V u k o j e v i ć et al., 2006).
The mean value of the copper concentration in the Earth's crust is 47 g/t.The sources of copper are 's crust is 47 g/t.The sources of copper are crust is 47 g/t.The sources of copper are its ores: chalcopyrite, cuprite and malachite (T h ö n i and S e i t l e r, 2004).Yearly, 11 million tons are produced worldwide, some 20% are coming from recycling (��etalgesellschaft, 1993).Copper is widely used as an electricity conductor, in architecture, for coins, in the paint industry, and in production of algaecides and fungicides (G r e e n w o o d and E a r n s h a w, 1988).The yearly emission of copper into the atmosphere from anthropogenic sources is ca.26000 t (P a c y n a and P a c y n a , 2001) and from natural sources ca.20000 t (L a n t z y and �� a c k e n z i e, 1979).In deposition dust in some regions, copper is 1.3-2.8times more concentrated than in the Earth's crust (T h ö n i et al., 1999).
Copper serves as building matter in many enzymes of extreme importance for plant development, but its use by plants is minimal.A deficiency of copper causes chloroses and changes in the root system of plants.The lives of humans and animals are dependent on copper.However, in higher concentrations it causes hepatitis and hemolytic anemia (T h ö n i and S e i t l e r, 2004).In still higher concentrations it can cause coma and death in humans.Legal limits in developed countries are: emission � 5 mg/m 3 ; in soil � 40 mg/kg; and in drinking water � 1.5 mg/l (T h ö n i and S e i t l e r, 2004).
Iron is very common in the Earth's crust (46.5 kg/t or 4.65%) (S c h e f f e r and S c h a c h s c h a b e l , 1984).It is produced from ores that contain 20-70% of it.Yearly production is ca.973 million tons (�� e t a l g e s e l l s c h a f t , 1993).It is often used in the production of construction material, glass, ceramics, plastics, paper, electronic devices, magnets, machines, gears, etc. Anthropogenic emission is ca.10.7 million tones vs. ca.27.8 million tons naturally emitted into the atmosphere (L a n t z y and �� a c k en z i e, 1979).The biggest emmission sources are industrial accidents and waste (�� e r i a n , 1984).Iron is for plants an essential element, vital for chlorophyll synthesis.It is usually insufficiently present in plant substrata, but high concentrations are known to be toxic and induce root illness.In humans and animals, it is essential due to its role in hemoglobin and myoglobin structuring.Acute intoxications with high concentrations of iron are rare, and many humans suffer from iron deficiency of iron especially females (�� e r i a n , 1984).In developed countries, its maximum permissible concentration in drinking water is 0.3 mg/l (T h ö n i and S e i t l e r, 2004).
��ercury is quite rare in the Earth's crust (0.08 g/t), but it is very much present in geo-chemical cycles.In nature, it is mostly present in red sulfides or cinnabar, and from this ore it is industrially produced in amounts of ca.7000 tons yearly (Tr u e b , 1996).Because of its mobility, it is widespread in the environment.Some 150000 tons enter the atmosphere from volcanoes every year (T r e u b, 1996).In 2000, its emission was ca.200 tons in Europe (P a c y n a et al., 2002).The deposition of mercury is 8-13 time highers than its value in the Earth's crust (T h ö n i et al., 1999).��ercury is essential for living organisms, but higher concentrations are toxic and in plants cause developmental problems, chloroses, and necroses (B e r g m a n n, 1988).In animals and humans, it is very toxic in small concentrations, especially its methylated forms, which cause much damage to SH groups of proteins and DNAs.��any nerve diseases are known to be induced by mercury toxicity.In developed countries, its emission limit is 0.2 mg/m 3 , while in soil its content is limited to 0.5 mg/kg and in drinking water to 0.001 mg/l (T h ö n i (T h ö n i and S e i t l e r, 2004).

��ATERIAL AND ��ETHODS
The acrocarpous mosses Bryum argenteum and Bryum capillare were used to research long-term atmospheric deposition, while the pleurocarpous Brachythecium sp. and Hypnum cupressiforme were used to scan short term atmospheric deposition in the county of Obrenovac (Serbia).Hypnum cupressiforme is one of the standard species used in Europe for heavy metal deposition surveys (B u s e et al., 2003), whereas the other three standard species used for this purpose in Europe do not grow in the Obrenovac region.In judging which other species are eligible for heavy metal deposition monitoring, the experience of T h ö n i ( 1996), H e r p i n et al. (1994), S i e w e r s and H a i r p i n (1998) Z e c h m e i s t e r (1994), and R o s s (1990) was consulted.
As far as possible, moss sampling followed the guidelines set out in the experimental protocol for the 2000/2001 survey (UNECE, 2001).The procedure is given in detail in R ü h l i n g (1998).
R ü h l i n g (1998).
Each sampling site was located at least 300 m from main roads and populated areas and at least 100 m from any other road or single house.In forests or plantations, samples were collected in small open spaces to preclude any effect of canopy drip.Sampling and sample handling were carried out using plastic gloves and bags.About three repeat moss samples were collected from each site.Dead material and litter were removed from the samples.Green parts of the mosses were used for analyses.
The county of Obrenovac was chosen for this investigation because of its industry and location.
Each sampling site was GPS-located with a precision of ±10 m, and GPS data (Garmin) were digitalized on maps with the OziExplorer 3.95.3b(© D&L Software), and WinDig 2.5 Shareware (© D.Lovy) softwares.
All material was collected during November of 2002.
Not more than one site was chosen per square measuring 50 x 50 m.Seventy-five out of 129 localities were chosen for comparison and further analyses based on all investigated species present and yearly biomass.��ore than 500 samples were analyzed.After collecting, samples were dried as soon as possible in a drying oven to a constant dry weight (dw) at a constant temperature of 35°C, then stored at -20°C.
Following homogenization in a porcelain mortar, the samples were treated with 5+1 parts of nitric acid and perchloric acid (HNO 3 :HClO 4 = 5:1) and left for 24 hours.
After that, a Kjeldatherm digesting unit was used for digestion at 150-200°C for about one hour.Digested samples were filtered on qualitative filter  paper to dispose of silicate remains, and the volume of samples was then normated to 50 ml.
Copper (Cu), Iron (Fe) and ��ercury (Hg) were detected by AAS Philips Pye Unicam SP9 instrument.The content of copper and that of iron were determined with a flame of acetylene/nitrogen-suboxide, while mercury was detected using hydride techniques.
For the explanation of the results and their map presentation, the following statistical parameters were used: average values, standard deviation, minimum and maximum values, and percent deviation.��ap making and interpolation of precise data were made with Agis v1.71 32bit (© Agis Software, 2001) software.

RESULTS AND DISCUSSION
Since it was impossible to find all the sampled species at any precise locality, the average of all specimens is given on extrapolated map to get an idea of heavy metal deposition in the county of Obrenovac (Fig. 1).However, if we separate the values of deposition obtained from pleurocarpous (Brachythecium sp. and Hypnum cupressiforme) and acrocarpous argenteum and Bryum capillare) mosses, it can be clearly seen that the first two give us an idea of short-term deposition and the last two of long-term deposition (Table 1).This can be easily explained in terms of the life forms of these mosses and thair uptake of heavy metals.Pleurocarps are not closely attached to the substrate and thus receive the bulk of deposited heavy metals directly from the atmosphere (during their pauciennial life period), while acrocarps are strictly attached to substrata and get most of deposited heavy metals with the substrate solution (metals are deposited over a period of time that is longer than their pauciennial life span).
Scanning of trace metal (Cu, Fe, Hg) content in the county of Obrenovac (W.Serbia) clearly shows that the entire area of the county is loaded with these three metals as a result of heavy industry and intense traffic (Fig. 1).The north-central region of the county is less loaded.Copper is spread over the county from a few points in the central and SW and SE regions.The pattern of iron spreading is simmi-lar, while mercury spreads from the central, northwestern, and southwestern parts of the county.
, 1990; S é r g i o et al., 1993; K u i k and Wo l t e r b e e k , 1995; B e r g and S t e i n n e s , 1997a; P o t t and Tu r p i n , 1998; S u c h a r o v a and S u c h a r a , 1998; G r o d z i n s k a, et al. 1999; Ts a k o v s k i et al., 1999; F e r n á n d e z et al., 2000, 2002; G e r d o l et al., 2000; L o p p i and B o n i n i , 2000; F i g u e i r a , et al., 2002; S c h i l l i n g and L e h m a n , 2002; S a l e m a a et al., 2004; P e ñ u e l a s and F i l e l l a , 2002; C u c u ��� a n et al., 2002).F i l e l l a , 2002; C u c u ��� a n et al., 2002).��osses have also been used to analyze contaminants spreading around thermal power plants (To n g u � , (To n g u � , 1998; C a r b a l l e i r a and F e r n á n d e z , 2002) or oil-fired power plants (G e n o n i et al., 2000).��oreover, some bryophytes are known to be heavy metal bioindicators in their environments (S a m e c k a �C y m e r m a n et al., 1997; O n i a n w a , 2001; N i m i s et al., 2002; C u o t o et al., 2004; S c h r ö d e r and P e s c h, 2004) and are often used in environmental monitoring (R a s m u s s e n and A n d e r s e n, 1999; G i o r d a n o et al., 2004; C u n y et al., 2004; G s t o e t t n e r and F i s h e r, 1997; Z e c h m e i s t e r et al., 2005).
Two pleurocarpous taxa (Brachythecium sp. and DETERMINATION OF HEAVY METAL DEPOSITION IN THE COUNTY OF OBRENOVAC (SERBIA) USING MOSSES AS BIOINDICATORS.III.COPPER (Cu), IRON (Fe) AND MERCURY (Hg) Hypnum cupressiforme Hedw.) were used to scan short-term atmospheric deposition of heavy metals, considering that these taxa are not strongly attached to the substrate and accumulate mostly from precipitation (T h ö n i et al., 1996; F a u s � K e s s l e r et al., 2001; F e r n á n d e z and C a r b a l l e i r a 2001; C u o t o et al., 2004).