DETERMINATION OF HEAVY METAL DEPOSITION IN THE COUNTY OF OBRENOVAC (SERBIA) USING MOSSES AS BIOINDICATORS. IV. MANGANESE (MN), MOLYBDENUM (MO), AND NICKEL (NI)

In this study, the deposition of three heavy metals (Mn, Mo, and Ni) in the county of Obrenovac (Serbia) in four moss taxa (Bryum argenteum, Bryum capillare, Brachythecium sp., and Hypnum cupressiforme) is presented. The distribution of average heavy metal content in all mosses in the county of Obrenovac is presented on maps, while the 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 tabular form. Areas of the highest contaminations are highlighted.


INTRODUCTION
This study represents the fourth part in a series (Sabovljević et al., 2005(Sabovljević et al., , 2007;;Vukojević et al., 2006) on surveillance of heavy metals in mosses in the county of Obrenovac (W.Serbia).
The idea of using mosses to measure atmospheric heavy metal deposition was developed already in the late 1960's (Rhüling and Tyler, 1968;Tyler, 1970) and became well established in the Scandinavian countries in the 1980's.
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

DETERMINATION OF HEAVY METAL DEPOSITION IN THE COUNTY OF OBRENOVAC (SERBIA) USING MOSSES AS BIOINDICATORS. IV. MANGANESE (MN), MOLYBDENUM (MO), AND NICKEL (NI)
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).The metals studied were chosen according to the significance of their presence in their environments (Lantzy and Mackenzie, 1979).
Mosses 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 (Vásquez 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 (Aceto et al., 2003).
-mosses do not possess thick and strong protective layers like cuticles.
More about hyperaccumulation of metals in plants and mosses can be found in Prasad and Freitas (2003).Bryum argenteum has already been shown to have special metal-accumulation properties (Aceto et al., 2003;Vukojević et al., 2004Vukojević 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 (Berg and Steinnes, 1997b).
Use of mosses to investigate heavy metal deposition shows trans-boundary 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 Mn, Mo, and Ni Mn, Mo, and Ni are treated in the present study, due to limitation of space.The presence and distribution of aluminium, arsenic, boron, cadmium, cobalt, chromium, copper, iron, and mercury in the county of Obrenovac as screened by mosses were considered in three already published papers (Sabovljević et al., 2005(Sabovljević et al., , 2007;;Vukojević et al., 2006).

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 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 (Buse 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 Thöni (1996), Herpin et al. (1994Herpin et al. ( ), (1994)), Siewers andHairpin (1998), Zechmeister (1994), and Ross (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 Rühling (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.
More 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 (HNO3:HClO4 = 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.Nickel (Ni), manganese (Mn), and molybdenum (Mo) were detected by a Philips Pye Unicam SP9 AAS instrument.The content of manganese and nickel were determined with a flame of acetylene/ air, while molybdenum was detected using a flame of acetylene/nitrogen-suboxide.
For 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.Map making and interpolation of precise data were done 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 an 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 (Bryum 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 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 with the substrate solution (metals are deposited over a period of time that is longer than their pauciennial life span).
The north-central urban region of the county with green surfaces is less loaded with the studied trace metals.The central and southwest part of the county are the most loaded with manganese.
Manganese is represented in the Earth's crust with some 900 ppm (Scheffer and Schachtschabel, 1984;Kaim and Schwederewski, 2005).It is an important micro-element for plants and animals, being included in enzymatic systems and involved in production of vitamin B1 and insulin (Kaim and Schwederewski, 2005).In plants, it is an important transporter of electrons in photosystem II.The best known manganese-containing polypeptides are arginase, the diphteria toxin, and Mn-containing superoxide dismutase (Mn-SOD), which is an enzyme typically present in eukaryotic mitochondria, and also in many bacteria (this fact is in keeping with the bacterial-origin theory of mitochondria).The Mn-SOD enzyme is probably one of the most ancient.Nearly all organisms living in the presence of oxygen use it to deal with the toxic effects of superoxide, formed from the 1-electron reduction of dioxygen.
Manganese is extracted from pyrolusite (MnO 2 ).Due to its resemblance to iron, it is used in production of alloys, primary steel production, in the chemical industry, and in the manufacture of fertilizers and paints (Williams, 1988).The EU uses manganese in 1-and 2-Euro coins, due to its cheapness and availability.
Manganese compounds are less toxic than those of other widespread metals.Exposure to manganese dusts and fumes should not exceed a ceiling value of 5 mg/m 3 .Manganese poisoning has been linked to impaired motor skills (e.g., Parkinson's desease) and cognitive disorders.
Scanning of trace metal contents in the county of Obrenovac (W.Serbia) clearly shows that the entire area of the county is loaded with manganese as a  result of heavy industry and intense traffic (the position of the heavily loaded area on Fig. 1 corresponds to industrial and traffic sites).
Molybdenum is spread equally over the region examined and does not exceed 0.0009 mg/g of moss mass.Molybdenum is present in the Earth's crust with an average of 1.1 g/t (Scheffer and Schachtschabel, 1984;Thöni and Seitler, 2004).The The most important mineral containing molybdenum is molybdenite (MoS 2 ) (Greenwood and Earnshaw, 1988).However, it is a collateral product in produc-However, it is a collateral product in production of copper.The yearly production of molybdenum is about 0.111 million tons (Metallgesellschaft, 1993).Some 85% of molybdenum is used in the steel and iron industry.The rest of molybdenum usage goes to production of catalysts, alloys, etc. in the organic chemical, petrochemical, and electronics industries.Also, it is used in small quantities as an additive to animal foods, vitamin preparations, and fertilizers (Trueb, 1996).The emission of molybdenum decreased from the 1970's and in 1995 it was ca.530 tons yearly in Europe (Thöni and Seitler, 2004).Molybdenum is a micro-element that in plants takes part in many enzymatic processes.Although it is important for normal plant growth, the plant's requirements for molybdenum is very small.Tolerance of molybdenum toxicity is high and damage to the plant is rare and expressed as yellow-orange chlorosis on leaf surfaces (Merian, 1984;Bergmann, 1988).Molybdenum is essential for animal and human life.To date, there has been small risk of molybdenum poisoning.It becomes very toxic only in very high concentrations.Only some cases of heavy poisoning of oxen near industrial complexes are known to date (Merian, 1984).The legal limit in developed countries is 5 mg/kg of dry soil (Thöni and Seitler, 2004).
Nickel is spread over the county unequally, with highest concentrations along the roads and industrial complexes.Nickel is one of the major elements in the Earth's crust with some 58 g/t (Scheffer and Schachtschabel, 1984;Thöni and Seitler, 2004).Almost 90% of nickel comes from the iron-nickel sulfide mineral pentlandite (Merian, 1984).Its production is ca.800000 tons a year (Metallgesellschaft, 1993).Nickel is a part of more than 3000 alloys, of which the most important is chromium-nickel steel.It is widely used in the construction, chemical, food, and transportation (cars and planes) industries, as well as in housekeeping.Coins are made of coppernickel alloys, and nickel hydride batteries are used in remote-control equipment (Treub, 1996).The annual values for emission in Europe are around 20000 tons, with a declining tendency due to greater concern for the environment and modern technological usage (Pacyna and Pacyna, 2001).Bergmann (1988) states that it is not clear whether nickel is essential for plants.However, it is known to be toxic in acid and sand substrates, and it is species-specific.For animals and humans, nickel is probably essential, and toxicity from natural nickel is minimal.However, allergies from modern jewelry are frequent (Trueb, 1996).Nickel in constant and higher concentrations can act as a cancerogen (Merian, 1984).The legal limits in emission of stationary sources are 1 mg/m 3 , 50 mg/kg in dried soil, 0.01 mg/l in drinking water, and 2 mg/l in waste waters (Thöni and Seitler, 2004).
In the county of Obrenovac, its deposition is localized at specific sites, and these sites are highlighted as potentially threatening zones for humans, animals, and plants.Thöni, L. (1996).Vergleich der Elementkonzentrazionen in drei Biomonitormoosen untereinander und mit Depositionfrachten im Bulksammler nach Bergerhoff, 89 pp.Bern.Bern.
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