DETERMINATION OF HEAVY METAL DEPOSITION IN THE COUNTY OF OBRENOVAC ( SERBIA ) USING MOSSES AS BIOINDICATORS II : CADMIUM ( Cd ) , COBALT ( Cd ) , AND CHROMIUM ( Cr )

In the present study, the deposition of three heavy metals (Cd, Co and Cr) in the county of Obrenovac (Ser- bia) is determined using four moss taxa (Bryum argenteum, Bryum capillare, Brachythecium sp. and Hypnum cupressi- forme) as bioindicators. Distribution of average heavy metal content in all mosses in the county of Obrenovac is present- ed in 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 in tables. Are- as of the highest contaminations are highlighted.


INTRODUCTION
Surveillance of heavy metals in mosses was originally established in the Scandinavian countries in the 1980s.However, the idea of using mosses to measure atmospheric heavy metal deposition was developed already in the late 1960s (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, this method is widely used in many countries (S c h a u g et al ., 1990;S é r g i o et al., 1993;K u i k and Wolterbeek, 1995; B e r g and S t e i n n e s, 1997a; Pottand T u r p i n, 1998; S u c h a r o v a and Suchara, 1998; G r o d z i n s k a et al., 1999;Tsakovski 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;Figueira et al., 2002; S c h i l l i n g and Lehman, 2002; S a l e m a a et al., 2004; Peñuelasand F i l e l l a , 2002; C u c u -M a n et al., 2002).Mosses have also been used to analyze contaminants spreading around thermal power plants (Tonguç, 1998;Car-balleiraand F e r n á n d e z, 2002) or oil-fired power plants (G e n o n i et al., 2000).
Moreower, some bryophytes are known to be heavy metal bioindicators of heavy metals in their environments (S a m e c k a -C y m e r m a n et al., 1997;Onianwa, 2001;N i m i s et al., 2002;C u o t o et al., 2003;Schröderand P e s c h , 2004) and are often used in environmental monitoring (R a s m u s s e n and Andersen, 1999; G i o r d a n o et al., 2004;Cuny et al., 2004; G s t o e t t n e r and F i s h e r, 1997; Zechmeister et al., 2005).
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 (Schintu et al., 2005).
Two pleurocarpous taxa (Brachythecium sp. and 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  Mosses are better than other higher plants in scanning heavy metal deposition because: they are perennial without deciduous periods; they have high cation exchange capacity that allows them to accumulate great amounts of heavy metals between the 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 the cell wall and proteins in the plasma membrane (A c e t o et al., 2003).Mosses do not possess thick and strong protective layers like cuticles.More about hyperaccumulation in plants and moss metal accumulation peculiarities 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 (Aceto et al., 2003;Vukojević et al., 2005).Also, this time-integrated way of measuring patterns of heavy metal deposition from the atmosphere to 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 (Bergand 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 were analyzed in all, only deposition and distribution of cadmium, cobalt and, chromium are treated in the present study, due to limitation of space.The presence and distribution of aluminum, arsenic and boron in the county of Obrenovac as screened by mosses were considered in an already published paper (S a b o v l j e v i ć et al., 2005).
The content of cadmium in the Earth's crust is estimated to be 0.13 g/t.Pure cadmium minerals are very rare.In nature it usually occurs in minerals together with zinc.Cadmium is obtained as a collateral product in zinc production, and this accounts for more than 95% of all its production (S t o e p p l e r , 1991).Yearly production is ca.20000 t (M e t a l l g e s e l l s c h a f t, 1993).Cadmium is widely used in ship and vehicle manufacturing to protect steel plating from corrosion, as well as in production of nickel-cadmium batteries.It is used as a neutron absorber in photocells and in nuclear technologies.Owing to its high toxicity in the environment, the use of cadmium has decreased slightly in the last decades (Treub, 1996).
Plants (cultivated or wild) accumulate cadmium in high quantities.The roots and leaves suffer most from accumulation of cadmium, which causes abnormal development, necrosis, and death (B e r g m a n n, 1988).Small quantities of cadmium are extremely toxic for humans and animals.Cadmium replaces zinc in zinc containing enzymes, which causes Itai-Itai disease with lethal consequences.The chronic presence of cadmium binds calcium out of bones, causing osteoporosis.In addition, it causes high blood pressure and has cancerogenic, mutagenic, and teratogenic effects (M e r i a n, 1984).
Emission should not exceed 0.1 mg/m 3 , and maximmum permissible values are 1.5 ng/m 3 in terrestrial ecosystems, 0.1 mg/l in water, and 0.005 mg/l in drinking water (T h ö n i and S e i t l e r, 2004).
Cobalt is present in the Earth's crust in quantities 18 g/t and together with scandium is the rarest element in its upper layers.This heavy metal is a collateral product in production of nickel, cupper and lead (G r e e n w o o d and E a r n s h a w, 1988).The yearly production is ca.24300 t (M e t a l l g e s e l l s c h a f t, 1993).Cobalt is widely used in the alloy tungsten carbide, and some 10 % of produced cobalt is employed in the making of permanent magnets.The rest of produced cobalt is used in the ceramics and paint industries (T r e u b, 1996).Cobalt is present in traces in the air.In supstrata and water emission is highly present and can form sediments.Anthropogenic emission is ca.4400 t and natural ca.7000 t (Lantzyand M a c k e n z i e , 1979).Although cobalt is essential for humans, animals, and plants, especially as a component of vitamin B12, in higher quantities it is toxic.In humans and animals cobalt dust and salt cause cancer, while in plants only very high concentrations cause abnormalities (M e r i a n, 1984; B e r g m a n n, 1988).The emission limit is 1 mg/m 3 and 0.5 mg/l in water.
Chromium is one of the most widely present elements in the Earth's crust.It is extensively used for chromization, in engineering, and in the manufacture of vehicles, airplanes, chemicals, etc.The yearly production is ca.13 million tons (M e t a l l g e s e l l s c h a f t, 1993).Emission of chromium in both air and water occurs at the highest rate in the metallurgy industry.A certain part of chromium emission (Cr(VI)) comes from the cement and concrete industry.
The toxic effect of chromium to plants is known only from ex vivo experiments (S c h e f f e r and Schachtschabel, 1984).For animals and humans, chromium is an essential element.It is known for its role in insulin effects (glucose tolerance factor), which occur due to the role of chromium in glucose exchange.The effects of Cr(III) and metallic Cr are not precisely known, but Cr(VI) complexes cause acute and chronic toxicity.Chromate and dichromate from cement can be severely toxic.Chromium dust and/or acidic deposition can cause lung cancer (M e r i a n , 1984).Emission tolerated by law is 5 mg/m 3 .The limit of chromium in soil is 50 mg/kg.In water it is 2 mg of all Cr/l [(0.1 mg Cr(VI)/l)], while in drinking water it is 0.02 mg Cr(VI)/l.

MATERIAL AND METHODS
The acrocarpous mosses Bryum argenteum and Bryum capillare were used to research long-term atmospheric deposition, while the pleurocarpous Brachythecium sp. and Hypnumcupressiforme 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 to survey heavy metal deposition survey (B u s e et al., 2003), while the other three are standard in Europe, but do not grow in this region.In estimating which other species are eligible for use in monitoring heavy metal deposition, we relied on 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), Zechmeister(1994), and R o s s (1990).
As for as possible, moss sampling was conducted according to guidelines set out in the experimental protocol for a survey performed in 2000/2001 (UNECE, 2001).Details of the procedure are given in R ü h l i n g et al. (1998).
Each sampling site was located at least 300 m from main roads and populated areas and at least 100 m from any 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 moss samples were collected from each site.Dead material and litter were removed from the samples.Green parts of mosses were used for the 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 ±10m, and GPS data (Germin) were digitalized on the maps using the following softwares: OziExplorer 3.95.3b,© D&L Software Pty.Ltd.; and WinDig 2.5 Shareware, © D.Lovy.
All material was collected during November of 2002.
Not more than one site was chosen in a 50x50 m square.Seventy-five localities were chosen out of 129 for comparison and further analyses.The selection was based on the presence of all investigated species and yearly biomass.
More than 500 samples were analyzed.
After collecting, samples were dried as soon as possible in a drying oven to constant dry weight (dw) at a constant temperature of 35°C, then stored at -20°C.
Following homogenization in porcelain, 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 volume of the samples was then normated to 50 ml.Cadmium, cobalt, and chromium were detected by AAS on a Pye Unicam SP9 atomic absorbance spectrophotometer from Philips using the flame of acetylene/nitrogen-suboxide.
For explanation of the results and their map presentation, the following statistical parameters were used: av-

RESULTS AND DISCUSSION
Since it was impossible to find all the sampled species at any precise locality, the average of all specimens is given in the map interpolation 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 ( Bryum argenteum and Bryum capillare) mosses, it can be clarly 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 their 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 by substrate solution (metals deposed from the time that is longer than their paucienial life span).
Scanning of trace metal (Cd, Co, Cr) content in the county of Obrenovac (W.Serbia) clearly shows that the northwestern, western, and central parts are the most loaded with these three metals, as a result of heavy industry and intense traffic (Fig. 1).Also cadmium is deposited in somewhat higher amounts in the town of Obrenovac, cobalt in the southeastern part of the county, and chromium in its eastern and southeastern parts.
Thus, moss analysis is shown to be a valuable method for monitoring atmospheric deposition of trace elements.
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).

Fig. 1 .
Fig. 1.Maps of the county of Obrenovac showing sampling sites (a) and extrapolated maps of average deposition of selected elements in mosses (b). 1. cadmium deposition 2. cobalt deposition 3. chromium deposition.